Siphonops annulatus possesses a single line ("Zahnzeile") of teeth in the upper jaw as well as in the palate paralleling the curvature of the jaws. An undivided continuous dental lamina provides the 2 nasopremaxillas and both maxillaries of the maxillopalatines with teeth, whereas each vomer and each (maxillo)-palatine is toothed by a separate dental lamina. The teeth are monocuspid, pedicellate and ancylosed to the bones in an extremely pleural condition. The results are compared to the conditions observed in the palate and upper jaw of urodeles.
Giraffes are ruminants feeding on fresh browse and twigs in the wild, but in zoos, their diet is mainly based on alfalfa hay, grains, and pellets occasionally supplemented by twigs and foliage. These diets, which differ in composition and digestibility, affect the behavior of the animals, tooth wear patterns, and chewing efficiency. We quantified several parameters of the rumination process in ten zoo housed giraffes of different sexes and ages fed either with alfalfa hay, fresh browse, or a combination of both. Chewing during rumination was highly ritualized and specimens showed an even distribution of chewing directions during this process, which prevents uneven tooth wear and use of chewing muscles. During rumination of alfalfa hay, chewing cycles of the giraffes took on average 49 s and included 54 jaw movements compared to 37 s and 42 jaw movements during rumination of browse, respectively. Single jaw movements (measured as basic chewing rates) were on average significantly slower during rumination of alfalfa hay (alfalfa: 1.10 chews per second, browse: 1.17 chews per second) and intercycle times between two chewing cycles took significantly longer (alfalfa: 7.77 s, browse: 7.46 s). Our results clearly indicate that several rumination parameters are influenced by the type of diet.
The earthworm Enchytraeus is able to survive in cadmium (Cd)-polluted environments. Upon Cd exposure, the worms express a gene encoding the putative non-metallothionein 25-kDacysteine-rich protein (CRP), which contains eight repeats with highly conserved cysteines in Cys-X-Cys and Cys-Cys arrangements exhibiting 36–53% identities to the 6–7-kDa metallothioneins of different organisms. Here, we demonstrate that the CRP protein confers a highly Cd-resistant phenotype to a Cd-hypersensitive yeast strain. Cd resistance increases with increasing numbers of expressed CRP repeats, but even one 3-kDa CRP repeat still mediates Cd resistance. Site-directed mutagenesis reveals that each single cysteine within a given repeat is important for Cd resistance, though to a different extent. However, replacement of other conserved amino acids such as Pro136 and Asp196 at the CRP repeat junctions does not affect Cd resistance. Our data indicate (i) that the non-metallothionein CRP protein is able to detoxify Cd and (ii) that this is dependent on the availability of sulfhydryl groups of the conserved cysteines. The earthworm Enchytraeus is able to survive in cadmium (Cd)-polluted environments. Upon Cd exposure, the worms express a gene encoding the putative non-metallothionein 25-kDacysteine-rich protein (CRP), which contains eight repeats with highly conserved cysteines in Cys-X-Cys and Cys-Cys arrangements exhibiting 36–53% identities to the 6–7-kDa metallothioneins of different organisms. Here, we demonstrate that the CRP protein confers a highly Cd-resistant phenotype to a Cd-hypersensitive yeast strain. Cd resistance increases with increasing numbers of expressed CRP repeats, but even one 3-kDa CRP repeat still mediates Cd resistance. Site-directed mutagenesis reveals that each single cysteine within a given repeat is important for Cd resistance, though to a different extent. However, replacement of other conserved amino acids such as Pro136 and Asp196 at the CRP repeat junctions does not affect Cd resistance. Our data indicate (i) that the non-metallothionein CRP protein is able to detoxify Cd and (ii) that this is dependent on the availability of sulfhydryl groups of the conserved cysteines. Anthropogenic pollution of the environment by heavy metals has been recognized as an increasingly threatening hazard for animals, plants, and even human health. Cadmium (Cd), 1The abbreviations used are:CdcadmiumMTmetallothioneinsCRPcysteine-rich proteinCIP2cadmium-induced proteinYCF1yeast cadmium factor 1 for example, has been utilized eight times more during the last 40 years by mankind than in its entire history; the Cd2+ input into biosphere is estimated to be about 30,000 tons/year (1Nriagu J.O. Pacyna J.M. Nature. 1988; 333: 134-139Crossref PubMed Scopus (3515) Google Scholar, 2Friberg L. Environmental Health Criteria 134. World Health Organization, Geneva, Switzerland1992Google Scholar, 3Dobson S. Environmental Health Criteria 135. World Health Organization, Geneva, Switzerland1992Google Scholar). The situation is deteriorating due to the acid rain that mobilizes the soil-bound Cd thus increasing the bioavailability of Cd (4Stöppler M. Merian E. Metals and Their Compounds in the Environment. VCH, Weinheim, Germany1991: 803-851Google Scholar). Cd is highly toxic because of its strong affinity to purines, pyrimidines, phosphates, porphyrins, and the cysteine and histidine residues of proteins (5Vallee B.L. Ulmer D.D. Annu. Rev. Biochem. 1972; 41: 91-128Crossref PubMed Scopus (1253) Google Scholar, 6Jacobson K.B. Turner J.E. Toxicology. 1980; 16: 1-37Crossref PubMed Scopus (296) Google Scholar). However, organisms are able, at least in part, to cope with the toxic Cd. This is primarily ascribed to metallothioneins (MTs), cysteine-rich 6–7-kDa proteins ubiquitously expressed among eukaryotes as well as in some prokaryotes (7Kägi J.H.R. Suzuki K.Y. Imura N. Kimura M. Metallothionein III. Birkhauser Verlag, Basel1993: 29-55Google Scholar, 8Klaassen C.D. Liu J. Choudhuri S. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 267-294Crossref PubMed Scopus (1001) Google Scholar, 9Vasak M. Hasler D.W. Curr. Opin. Chem. Biol. 2000; 4: 177-183Crossref PubMed Scopus (378) Google Scholar). MTs contain an α-domain and a β-domain, each with characteristic sequence arrangements of cysteines. The constitutively expressed MTs are obviously able to detoxify Cd (10Ghoshal K. Jacob S.T. Prog. Nucleic Acids Res. Mol. Biol. 2000; 66: 357-384Crossref Google Scholar). However, their actual primary role does not seem to be the detoxification of Cd. Rather, MTs appear to control cellular zinc distribution, translocation, and availability (11Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8428-8430Crossref PubMed Scopus (605) Google Scholar), although, besides other functions, they may also act as general anti-stress factors (12Miles A.T. Hawksworth G.M. Beattie J.H. Rodilla V. Crit. Rev. Biochem. Mol. Biol. 2000; 35: 35-70Crossref PubMed Scopus (405) Google Scholar). cadmium metallothioneins cysteine-rich protein cadmium-induced protein yeast cadmium factor 1 Scarce information is available suggesting that, besides MTs, also larger non-MT proteins are involved in Cd detoxification (13Stone H. Overnell J. Comp. Biochem. Physiol. C Comp. Pharmacol. Toxicol. Endocrinol. 1985; 80: 9-14Crossref PubMed Scopus (80) Google Scholar). However, the investigation of these non-MTs has been oddly neglected to date. Especially some invertebrates have been reported to contain Cd-binding non-MTs but without any evidence for their role in Cd detoxification or molecular characterization (14Bauer-Hilty A. Dallinger R. Berger B. Comp. Biochem. Physiol. C Comp. Pharmacol. Toxicol. Endocrinol. 1989; 94: 373-379Crossref Scopus (31) Google Scholar, 15Morgan J.E. Norey C.G. Morgan A.J. Kay J. Comp. Biochem. Physiol. C Comp. Pharmacol. Toxicol. Endocrinol. 1989; 92: 15-21Crossref Scopus (74) Google Scholar, 16Nejmeddine A. Sautiere P. Dhainuat-Courtois N. Baert J.-L. Comp. Biochem. Physiol. C Comp. Pharmacol. Toxicol. Endocrinol. 1992; 101: 601-605Crossref PubMed Scopus (19) Google Scholar, 17Ruffin P. Demuynck S. Hilbert J.L. Dhainaut A. Biochimie (Paris). 1994; 76: 423-427Crossref PubMed Scopus (16) Google Scholar). We have identified a putative Cd-binding non-MT protein as a cDNA in the earthwormEnchytraeus using differential screening of cDNA library (18Willuhn J. Schmitt-Wrede H.-P. Greven H. Wunderlich F. Ecotoxicol. Environ. Saf. 1994; 29: 93-100Crossref PubMed Scopus (12) Google Scholar, 19Willuhn J. Schmitt-Wrede H.-P. Greven H. Wunderlich F. J. Biol. Chem. 1994; 269: 24688-24691Abstract Full Text PDF PubMed Google Scholar). These small oligochaete worms of high ecological relevance due to their function in soil formation and preservation are capable of surviving in acidic soils highly contaminated with Cd (20Didden W.A.M. Pedobiologia. 1993; 37: 2-29Google Scholar). The identified cDNA of Enchytraeus encodes a putativecysteine-rich non-MT 25-kDaprotein, termed CRP, with eight tandemly arranged repeats exhibiting a characteristic conserved arrangement of cysteines (19Willuhn J. Schmitt-Wrede H.-P. Greven H. Wunderlich F. J. Biol. Chem. 1994; 269: 24688-24691Abstract Full Text PDF PubMed Google Scholar). The crp gene is induced by Cd, and its transcript level positively correlates with Cd accumulation of worms (21Willuhn J. Otto A. Schmitt-Wrede H.-P. Wunderlich F. Biochem. Biophys. Res. Commun. 1996; 220: 581-585Crossref PubMed Scopus (12) Google Scholar). However, Cd inducibility of crp-mRNA does not necessarily mean that the CRP protein is directly involved in Cd detoxification. Indeed, a Cd-inducible mRNA encoding the non-MT CIP2 (cadmium-induced protein) has been recently detected in the fungus Candida sp. (22Park K.S. Kwon J. Choi S.Y. FEMS Microbiol. Lett. 1998; 162: 325-330Crossref PubMed Google Scholar). However, the only 4 cysteines containing CIP2 is presumably not a Cd-binding protein, and it is not directly involved in Cd detoxification. Rather it is assumed to be involved in coping with the oxidative stress induced by Cd. Here, we provide experimental evidence for the actual role of the non-MT CRP protein in Cd detoxification. Transformation of crp-cDNA into Cd-hypersensitive yeast dramatically increases their Cd resistance, the extent of which is dependent on the number of CRP repeats and on the position of the different cysteines in a given CRP repeat. Yeast expression vector pRS425 (23Mumberg D. Müller R. Funk M. Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (803) Google Scholar), kindly provided by Dr. G. Jansen (Institute of Microbiology, Heinrich-Heine University, Düsseldorf, Germany), was modified by inserting a 6× c-Myc tag as N-terminal fusion. Different arrangements of crp repeats were constructed by amplifying corresponding regions of the 1474-bp crp-cDNA (GenBankTM accession number X79344) and inserting the PCR products in the SalI restriction site of pRS425. Constructs of crp repeat 4 were cloned in pRS425 vector without a 6× c-Myc tag. This was performed with the “Altered Sites II in Vitro Mutagenesis” system (Promega, Madison, WI). In brief, PCR-generated fragments of the complete coding region of the crp-cDNA (753 bp) and the crp repeat 4 (93 bp) were cloned in the SalI site of vector pALTER-1. Oligonucleotides used for mutation of the nine cysteines of repeat 4 and the conserved residues at the repeat junctions 4/5 and 6/7, respectively, were as follows: Cys1(5′-GAGTCGACAATGAGCTCCTGTGGTT); Cys3(5′-ACAATGTGCTCCAGTGGTTCAGGA); Cys7(5′-TGTGGTTCAGGAAGTAAGTGTGAGA); Cys9(5′-TCAGGATGTAAGAGTGAGAAGGGA); Cys14(5′-GAGAAGGGAGAGAGTAAGCCAGGTT); Cys18(5′-GTAAGCCAGGTAGTACCAAGCGA); Cys22(5′-GTACCAAGCGAAGCTGTGGTACTA); Cys23(5′-ACCAAGCGATGCAGTGGTACTAAA); Cys27(5′-GTGGTACTAAAAGTGGAGTTGAA G); r4/5 (5′-TTGAAGATTGCCTATGTGGTCGTCCAAG); r6/7 (5′-TGTGGAATGCAGAACTGCCCGTGTG). The reactions of mutagenesis were performed according to the manufacturer's guidelines. Mutated crp inserts were finally cloned into the SalI site of yeast vector pRS425. Clones were sequenced using the LICOR 4000 laser fluorescent sequencing system as described recently (24Krücken J. Stamm O. Schmitt-Wrede H.-P. Mincheva A. Lichter P. Wunderlich F. J. Biol. Chem. 1999; 274: 24383-24391Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Sequences were analyzed using the programs FASTA, BLITZ, and BLAST (25Smith T.F. Waterman M.S. J. Mol. Biol. 1981; 147: 195-197Crossref PubMed Scopus (7200) Google Scholar, 26Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9393) Google Scholar, 27Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar) and aligned using LALIGN and ClustalW (28Huang X. Miller W. Adv. Appl. Math. 1991; 12: 337-357Crossref Scopus (844) Google Scholar, 29Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar). The isogenic yeast strains DTY165 (strain SEY6210; MATα ura3-52, leu2-3,-112, his3-D200, trp1-D901, lys2-801, suc2-D9) and DTY167 (strain JW53F;MATα ura3-52, leu2-3,-112, his3-D200, trp1-D901, lys2-801, suc2-D9, ycf1::hisG) were used for the experiments (30Li Z.-S., Lu, Y.-P. Zhen R.-G. Sczcypka M. Thiele D.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 42-47Crossref PubMed Scopus (507) Google Scholar). Yeast cells transformed with expression vector pRS425 were selected on minimal SD medium (0.67% yeast nitrogen base, 2% glucose) without leucine and methionine. For the different assays of Cd resistance, 5 ml of yeast pre-cultures were grown for 24 h in minimal SD before inoculating SD medium containing CdCl2(initial A 600 = 0.005). Those assays with yeast cells expressing the different CRP fragments (Fig. 3) were performed in a total volume of 7 ml in 10-ml test tubes, those assays with the mutagenic CRP-r4 fragments in 20 ml in 100-ml Erlenmeyer flasks. Optical density of the cultures was determined after 72 h of growth at different Cd concentrations. Generation times were determined at the subtoxic Cd concentrations of 30 and 40 μm, respectively. Each experiment was reproduced at least three times. Data are shown with means ± S.D. Proteins extracted from yeast cells were separated by SDS-PAGE (31Lämmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), electroblotted onto nitrocellulose, and processed as detailed previously (24Krücken J. Stamm O. Schmitt-Wrede H.-P. Mincheva A. Lichter P. Wunderlich F. J. Biol. Chem. 1999; 274: 24383-24391Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The filters were blocked with Rotiblock (Roth, Karlsruhe, Germany) and then incubated with a monoclonal mouse anti-c-Myc antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and with peroxidase-conjugated rabbit anti-mouse IgG (1:25,000) (Jackson ImmunoResearch Laboratory, West Grove, CT) as a secondary antibody before detection with ECL (Amersham Biosciences). Protein loading on filters was examined with a goat anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Exponentially growing DTY167 cells transformed with c-Myc-tagged crp constructs (2A 600 units) were fixed with 5% paraformaldehyde at 30 °C for 4 h. Spheroplasts were prepared using 100 units of lyticase (Sigma), 10 mm dithiothreitol, and 3000 units of β-glucuronidase (type HP-2S) (Sigma), permeabilized with 0.1% Triton X-100 at room temperature for 5 min and treated with 0.1 mg/ml RNase A (Roche Molecular Biochemicals) (32Pringle J.R. Adams A.E.M. Drubin D.G. Haarer B.K. Methods Enzymol. 1991; 194: 565-602Crossref PubMed Scopus (601) Google Scholar). Aliquots were allowed to adhere onto polylysine-coated glass coverslips. The c-Myc-tagged CRP proteins were detected with a polyclonal rabbit anti-c-Myc antibody (1 μg/ml) (Santa Cruz Biotechnology, Santa Cruz, CA) and fluorescein isothiocyanate-coupled goat anti-rabbit IgG (1:100 working dilution) as secondary antibody (Sigma). Coverslips were finally mounted on slides in a 1:1 (v/v) mixture of glycerol and vectashield (Serva, Heidelberg, Germany) containing 2% (w/v) 1,4-diazobicyclo-[2.2.2]octane (Merck) and 5 μg/ml propidium iodide. Fluorescence was analyzed with the confocal laser scanning microsopy Leica TCS version 1.5.451 (Leica Lasertechnik, Heidelberg, Germany) as detailed recently (33Benten W.P.M. Lieberherr M. Giese G. Wrehlke C. Stamm O. Sekeris C.E. Mossmann H. Wunderlich F. FASEB J. 1999; 13: 123-133Crossref PubMed Scopus (264) Google Scholar, 34Benten W.P.M. Lieberherr M. Stamm O. Wrehlke C. Guo Z. Wunderlich F. Mol. Biol. Cell. 1999; 10: 123-133Crossref Scopus (221) Google Scholar). The crp gene of Enchytraeus encodes the putative non-MT 25-kDa CRP protein consisting of eight tandemly arranged repeats (Fig. 1 A). The repeats 1–7 contain 31 amino acids, whereas the repeat 8 is truncated at the C terminus (19Willuhn J. Schmitt-Wrede H.-P. Greven H. Wunderlich F. J. Biol. Chem. 1994; 269: 24688-24691Abstract Full Text PDF PubMed Google Scholar). The repeats exhibit a characteristic arrangement of nine cysteines in Cys-X-Cys and Cys-Cys segments as it is shown for the consensus sequence of the CRP repeats (Fig. 1 A). Data bank analysis does not reveal the existence of any other protein similar to CRP. Only the much smaller 6–7-kDa MTs exhibit sequence similarities. The MTs from oligochaetes such asLumbricus terrestris, Lumbricus rubellus, andEisenia foetida display the highest identities of more than 50% to the same CRP region comprising repeats 2–4 in highly scored alignments. The MT of the free living nematode Caenorhabditis elegans surprisingly aligns with only 37% identity to CRP repeats 6–8. Incidentally, the MT of C. elegans also exhibits lower identities in comparison with other invertebrate MTs (35Dallinger R. Comp. Biochem. Physiol. C Comp. Pharmacol. Toxicol. Endocrinol. 1996; 113: 125-133PubMed Google Scholar). The MT ofDrosophila melanogaster exhibits 46% identities to CRP repeat 3. The mouse MT-I and MT-II align best to CRP repeat 3 as those MTs of the other oligochaetes, although with lower identities. Like MT-I of C. elegans, mouse MT-IV is highly identical to CRP repeats 6–8, whereas mouse MT-III fits best to CRP repeat 2. Human MT-I, -II, and -IV align predominantly to repeat CRP regions 4–6, whereas the human and the mouse MT-III show highest identities of about 37% to CRP repeats 2 and 3. The multiple sequence alignment in Fig.1 B shows that there is a conspicuous conserved distribution of the cysteines in the CRP repeats and in the different MTs, besides some other amino acids such as glycine, lysine, and serine. For expression in yeast, diverse crp constructs were generated by PCR (Fig. 2 A). All crp constructs, with the exception of crp-r4, were N-terminally tagged with a 8-kDa 6× c-Myc by cloning in yeast expression vector pRS425 under the control of a MET25 promoter (23Mumberg D. Müller R. Funk M. Nucleic Acids Res. 1994; 22: 5767-5768Crossref PubMed Scopus (803) Google Scholar). The crp-r4 construct was cloned in pRS425 without a c-Myc tag to avoid possible steric hindrance by the c-Myc polypeptide. All constructs were transformed in Cd-hypersensitive Saccharomyces cerevisiaestrain DTY167 (30Li Z.-S., Lu, Y.-P. Zhen R.-G. Sczcypka M. Thiele D.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 42-47Crossref PubMed Scopus (507) Google Scholar), which harbors an inactivated gene for theyeast Cd factor (ycf1), normally required for Cd resistance (36Szczypka M. Wemmie J.A. Moye-Rowley W.S. Thiele D.J. J. Biol. Chem. 1994; 269: 22853-22857Abstract Full Text PDF PubMed Google Scholar). The crp constructs are expressed in approximately equal amounts in yeast (Fig. 2 B). The expression rate is low, because the CRP polypeptides are not detectable in Coomassie-stained SDS gels but only by Western blot analysis using anti-c-Myc antibody. Confocal laser scanning microscopy reveals that the 25-kDa CRP is exclusively localized in the cytoplasm but not in the vacuole or nuclei of yeast cells (Fig.2 C). In order to investigate the possible role of CRP in mediating Cd resistance, the DTY167 cells expressing the 25-kDa CRP (strain designated as DTY167-CRP), the isogenic wild-type DTY165 cells, and the Cd-hypersensitive DTY167-pRS425 cells transformed with empty vector were exposed for 72 h to Cd2+ concentrations up to 500 μm (Fig. 3 A). The DTY167-CRP cells do not only restore the Cd resistance but even exhibit a dramatically increased Cd resistance in comparison with wild-type DTY165 cells. At 100 μm Cd2+, DTY167-CRP reaches about 90% of growth that can be observed for control cultures without Cd. By contrast, wild-type DTY165 cells only reach a level of about 60% at 100 μm Cd2+. There is no growth observed for DTY165 at 300 μmCd2+ and higher concentrations. However, strain DTY167-CRP even tolerates 500 μm Cd2+, because it still reaches 20% of growth level of the untreated control (Fig.3 A). The hypersensitive DTY167-pRS425 cells are unable to grow at the used Cd2+ concentrations. The levels of Cd resistance restored in DTY167 positively correlate with the increasing number of CRP repeats (Fig. 3 B). All transformants expressing CRP repeats, except for DTY167-CRP-r4, show comparable growth at 100 μm Cd2+ after 72 h. Deletion of the N-terminal region and repeat 1 (DTY167-CRPΔr1) has no significant influence on the Cd resistance compared with DTY167-CRP (Fig. 3 B). The five repeats expressing transformants DTY167-CRP-r12345 and DTY167-CRP-r12678 have identical growth rates, but both display much lower resistance at 300 μm Cd2+ and higher Cd2+concentrations compared with DTY167-CRP. Growth of these strains stops at 400 μm Cd2+. A further reduced Cd resistance is observed for those yeasts expressing only three repeats such as DTY167-CRP-r345 and DTY167-CRP-r678. They have equivalent levels of Cd resistance, although the three repeats expressed in the cells are different. Growth of both strains is completely inhibited at 300 μm Cd2+. DTY167-CRP-r4 cells still exhibit a slight Cd resistance at 100 μmCd2+, because they reach 15% of the growth of the untreated control cells. Depending on the number of expressed repeats, generation times span between 3.17 ± 0.23 h for DTY167-CRP and 4.60 ± 0.64 h for DTY167-CRP-r4 at 40 μmCd2+, whereas hypersensitive DTY167-pRS425 cells double only after 13.65 ± 1.73 h at this Cd concentration. In order to investigate the importance of the individual Cys residues within a given CRP repeat for Cd resistance, Cys → Ser replacements were introduced in crp-r4 by oligonucleotide-directed site-specific mutagenesis. The nine mutated crp-r4 constructs cloned in vector pRS425 were transformed in hypersensitive strain DTY167 and exposed to 100 μm Cd2+ for 72 h. All Cys mutants reveal a dramatic decrease in Cd resistance compared with wild-type CRP-r4 expressing DTY167. Incidentally, the latter cells exhibit different growth depending on the used experimental conditions (cf.Figs. 3 and 4). The extent of the decreased Cd resistance depends solely on the mutated Cys position in crp-r4 (Fig.4). Cys3, Cys14, Cys22, and Cys23 are obviously of particular importance, because Cd resistance of the corresponding mutants decreases almost to the level of hypersensitive DTY167-pRS425 cells. Cys9 and Cys18 mutants confer a slightly better resistance since reaching about 10% of the growth of DTY167-CRP-r4. Mutations of Cys1, Cys7, or Cys27are less influential on Cd resistance than the other Cys positions, as the growth of these mutants is reduced by only 60% compared with DTY167-CRP-r4. Moreover, determination of generation times at a sub-lethal concentration of 30 μm Cd2+confirms the relevance of Cys3. Mutation of that position at least doubles the generation time to 7.9 ± 0.4 h, whereas the generation times of the other mutants span between 3.26 ± 0.08 h (Cys27) and 3.78 ± 0.28 h (Cys22). However, wild-type DTY167-CRP-r4 has a doubling time of 3.08 ± 0.1 h at the same Cd concentration. Besides the conserved Cys residues in the CRP repeats, there are also other conserved amino acids such as Gly, Pro, Val, and Asp at the repeat junctions (Fig. 5 A). In order to investigate their role in functioning of CRP in Cd resistance, oligonucleotide-directed site-specific mutagenesis was used to generate amino acid replacements at two repeat junctions in the 25-kDa CRP (Fig. 5 A). A Pro136 → Leu replacement was introduced in repeat 5 at the junction between repeat 4 → 5, and the mutated cDNA, cloned in pRS425, was expressed in strain DTY167 (designated as DTY167-CRPm4/5). Furthermore, a mutant CRP with an Asp196 → Asn replacement in repeat 6 at repeat junction 6 → 7 was constructed (DTY167-CRPm5/6). Comparative determination of the generation times at sub-lethal 40 μm Cd2+and the growth in increasing Cd2+ concentrations up to 750 μm reveal no significant difference in Cd resistance among native DTY167-CRP, DTY167-CRPm4/5, and DTY167-CRPm6/7 (Fig.5 B). Our data provide evidence that, besides MTs, also larger non-MT cysteine-rich proteins are able to detoxify Cd. Indeed, the non-MT 25-kDa CRP protein of the terrestric earthworm Enchytraeusmediates Cd resistance to the Cd-hypersensitive yeast strain DTY167 when transformed by crp. Remarkably, Cd resistance is not only restored but rather dramatically increased in comparison to isogenic wild-type yeast. The CRP protein is unique to date, i.e. data bank analysis does not reveal any other similar non-MT protein in any other organism. The best fitting alignments can be obtained with MT of different sources. MT of other earthworms such as Lumbricus (37Stürzenbaum S.R. Kille P. Morgan A.J. FEBS Lett. 1998; 431: 437-442Crossref PubMed Scopus (123) Google Scholar) orEisenia (38Gruber C. Stürzenbaum S. Gehrig P. Sack R. Hunziker P. Berger B. Dallinger R. Eur. J. Biochem. 2000; 267: 573-582Crossref PubMed Scopus (76) Google Scholar) display the best identities of about 50–53% to distinct regions of CRP, and even the more distant and diverse MT types of mouse and human exhibit identities of 37–44%. Besides the conserved cysteines, both MT and CRP contain still other conserved amino acids such as lysines, glycines, and serines. This structural similarity suggests a role of CRP in Cd detoxification similar to that of MT. Accordingly, mammalian MTs has been shown to mediate Cd resistance in yeast (39Cody C.W. Huang P.C. Biochem. Biophys. Res. Commun. 1994; 202: 954-959Crossref PubMed Scopus (21) Google Scholar). In yeast, Cd resistance is normally regulated by complex mechanisms. These involve GSH, the GSH-derived phytochelatins, and diverse membrane transporters (40Perego P. Howell S.B. Toxicol. Appl. Pharmacol. 1997; 147: 312-318Crossref PubMed Scopus (89) Google Scholar). The hypersensitive strain we used in our study harbors an inactive ycf1 (yeast cadmium factor) gene (30Li Z.-S., Lu, Y.-P. Zhen R.-G. Sczcypka M. Thiele D.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 42-47Crossref PubMed Scopus (507) Google Scholar). The ATP-binding cassette YCF1 protein is localized in the vacuolar membrane and serves as a pump, which transports Cd as a glutathioneS-conjugate into the yeast vacuole (36Szczypka M. Wemmie J.A. Moye-Rowley W.S. Thiele D.J. J. Biol. Chem. 1994; 269: 22853-22857Abstract Full Text PDF PubMed Google Scholar, 41Li Z.-S. Sczcypka M., Lu, Y.-P. Thiele D.J. Rea P.A. J. Biol. Chem. 1996; 271: 6509-6517Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). A similar pump mechanism is mediated in yeast by the transporter MRP1, the human multidrug-associated protein (42Tommasini R. Evers R. Vogt E. Mornet C. Zaman G.J.R. Schinke A.H. Borst P. Martinoia E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6743-6748Crossref PubMed Scopus (155) Google Scholar). CRP does not simply replace the lost function of YCF1 in Cd resistance of yeast, but rather CRP works by a different mechanism. This view is substantiated by our finding that CRP is uniformly distributed among the cytoplasm and is not detectable in the vacuolar membrane, although the CRP contains a putative transmembrane domain at the N terminus (19Willuhn J. Schmitt-Wrede H.-P. Greven H. Wunderlich F. J. Biol. Chem. 1994; 269: 24688-24691Abstract Full Text PDF PubMed Google Scholar). Currently, two mechanisms of MT in Cd detoxification are considered as follows: (i) chelation of Cd through coordinate covalent bonds to –SH groups of cysteines and/or (ii) scavenging of free radicals originating during Cd-induced stress (8Klaassen C.D. Liu J. Choudhuri S. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 267-294Crossref PubMed Scopus (1001) Google Scholar). CRP presumably functions by the same mechanisms. This view is supported by our finding that (i) Cd resistance of yeasts increases with increasing numbers of expressed CRP repeats and that (ii) mutations of distinct cysteines in a given CRP repeat result in a dramatic decrease or even loss of Cd resistance. However, our data also indicate differences in the mode of action of CRP and MT in Cd detoxification in yeast. First, each cysteine of a given CRP repeat is important for Cd resistance. Mutations of the cysteines in the CRP repeat at positions 1, 7, 9, and 27 result in a dramatic reduction of Cd resistance by at least 60%, whereas mutations in Cys3, Cys14, Cys18, Cys22, and Cys23 even result in a complete loss of the capability of mediating Cd resistance. By contrast, mammalian MTs expressed in yeast contain at least some cysteines without any effect on Cd resistance at all (43Chernaik M.L. Huang P.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3024-3028Crossref PubMed Scopus (39) Google Scholar). Second, S. cerevisiaeis known to contain only one MT, i.e. the Cu-MT encoded by the CUP1 gene (44Winge D.R. Nielson K.B. Gray W.R. Hamer D.H. J. Biol. Chem. 1985; 260: 14464-14470Abstract Full Text PDF PubMed Google Scholar, 45Ecker D.J. Butt T.R. Sternberg E.J. Neeper M.P. Debouck C. Gorman J.A. Crooke S.T. J. Biol. Chem. 1986; 261: 16895-16900Abstract Full Text PDF PubMed Google Scholar). This gene is specifically induced by copper but not by Cd, and its physiological role is the detoxification of copper and not that of Cd (44Winge D.R. Nielson K.B. Gray W.R. Hamer D.H. J. Biol. Chem. 1985; 260: 14464-14470Abstract Full Text PDF PubMed Google Scholar). However, when expressed under a constitutive promoter, Cu-MT is then also able to confer resistance to Cd (45Ecker D.J. Butt T.R. Sternberg E.J. Neeper M.P. Debouck C. Gorman J.A. Crooke S.T. J. Biol. Chem. 1986; 261: 16895-16900Abstract Full Text PDF PubMed Google Scholar). By contrast, the crp gene is not inducible by copper. However, if the gene is specifically induced by Cd, the copper toxicity is significantly decreased inEnchytraeus (21Willuhn J. Otto A. Schmitt-Wrede H.-P. Wunderlich F. Biochem. Biophys. Res. Commun. 1996; 220: 581-585Crossref PubMed Scopus (12) Google Scholar, 46Willuhn J. Otto A. Koewius H. Wunderlich F. Chemosphere. 1996; 32: 2205-2210Crossref Scopus (23) Google Scholar). Third, replacement of conserved amino acids other than the cysteines in MT results in a reduced Cd resistance of yeast hosts, whereas in CRP, substitution of conserved amino acids including the α-helix incompatible proline has no effect on CRP-mediated Cd resistance. The differences revealed between CRP and MT in yeast hosts support previous findings also revealing differences of CRP and MT at the RNA level. MT genes are constitutively expressed and respond to various stressors (47Jacob S.T. Ghoshal K. Sheridan J.F. Gene Expr. 1999; 7: 301-310PubMed Google Scholar). However, the crp gene is not constitutively expressed; it is specifically induced by Cd but not by other stressors such as lead, mercury, copper, or H2O2 (21Willuhn J. Otto A. Schmitt-Wrede H.-P. Wunderlich F. Biochem. Biophys. Res. Commun. 1996; 220: 581-585Crossref PubMed Scopus (12) Google Scholar). Collectively, our data indicate (i) that the non-MT CRP protein is able to detoxify Cd and (ii) that this capability is dependent on the availability of –SH groups similar to MTs. Although our results in yeast cannot yet be considered as applying to the situation in the earthworm Enchytraeus without further studies, the differences in the regulation of expression of the crp and MT genes reported to date suggest different actual physiological roles of both proteins. We thank Dr. D. J. Thiele for providing the yeast strains DTY165 and DTY167.
Members of the taxon Zenarchopteridae (Beloniformes) possess external paired olfactory organs each consisting of a small cone-like papillae also called nasal barbel. We examined the structure of these barbels in the viviparous halfbeak Dermogenys pusilla using scanning (SEM) and transmission electron microscopy (TEM). Nasal barbels are covered by a typical epidermis characterized by ridged surface cells. Further, the epidermis contains goblet cells and small taste buds. The epidermis is interspersed with small depressions or pits (sensory islets) which contain the olfactory epithelium. Typically, taste buds consist of spindle-shaped dark cells with numerous apical microvilli, light cells with a thick microvillus each, basal cells and a rich nerve fiber plexus between receptor and basal cells. The olfactory epithelium at least contains two types of receptor cells, i.e. ciliated cells with a strikingly variable microtubular pattern and microvillous cells, and supporting and basal cells. An olfactory organ with an open groove and an elongated papilla is considered as synapomorphy of the Beloniformes (does not hold for the Adrianichthyoidei). Comparison of these olfactory organs suggests that D. pusilla and very probably all Zenarchopteridae may have the most uniform and least elaborated olfactory organs.
1. Dentition, tooth structure and course of dental laminae of adult and subadult Sirenidae (Siren intermedia, S. lacertina, Pseudobranchus striatus) have been studied by light microscopy and scanning electron microscopy. 2. Splenials, vomers und palatines bear monocuspid unbladed teeth, arranged in a polystichous pattern, whereas praemaxillaries and maxillaries (so far as present) are edentate. Teeth in S. intermedia show a rough basis, which is more prominent in S. lacertina and which has break-throughs in P. striatus. This zone perhaps is homologous to a developing dividing zone typical for teeth in many “Lissamphibia”. 3. With respect to structure and organisation of dentigerous bones and teeth Sirenidae obviously possess a mosaicism of differently developed larval characters in their mouth cavity. 4. The dentition in the recent forms investigated is compared to that of other paedomorphic Urodela und the ancient Habrosaurus dilatus (Sirenidae).
Epidermal papillomas of alpine newts (Ichthyosaura alpestris) collected in the field (Germany, Austria) were studied by histology (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Papillomas were found on the head, the trunk and the tail, with the most and largest on the head of males. They protruded beyond the body surface exhibiting an appearance like a cauliflower. The head of one specimen studied by SEM had a large papilloma and was densely populated with bacteria, fungi and sessile ciliates. The surface of papillomas was covered either by stratum corneum cells, or by deeper cell layers that may be exposed by injuries. Histology revealed that papillomas consisted of compact bulbous extensions that were deeply embedded into the dermis and separated from each other by small septa (papillae) of connective tissue. Bulbs were distinctly demarcated by a thin basal lamella that was continuous with the basal lamella of the adjacent non-altered epidermis. An invasion of papilloma-cells through the basal lamella in the underlying connective tissue could not be unequivocally demonstrated; only once we found an area by TEM, which could be interpreted in this way. Bulbs may have two types of cavities or cysts. One type contained masses of keratinized cell layers, the other appeared either largely empty, or contained cellular debris and/or PAS-positive substances discharged by secretory cells lining the cyst. Tumor cells within a bulb are offen arranged in clusters or nests. Generally, cells appeared relatively undifferentiated having large euchromatic or heterochromatic nuclei, prominent nucleoli, and a moderate amount of cell organelles. Also the amount of tonofilamets and number and size desmosomes (macuale adhaerentes) seemed to be reduced. Virus-like particles were found neither in the cytoplasm nor in the nucleus. Compared to the unaltered epidermis, in which no mitoses were seen, mitotic cells occurred in all papillomas examined. In addition, the neoplastic tissue always contained macrophages and further ‘leucocytes’, but necrotic areas were rare. Dermal papillae separating the bulbs from each other and the dermal tissue immediately beneath the basal lamina of papillomas contained a high number of cells (e.g., fibroblasts and cells of the immune system).
'/%3e%3cpath id='l' d='M-26.25 0h52.5v-12h-40.5v-16h33v-12h-33v-11H25v-12h-51.25z'/%3e%3cpath id='k' d='M-31.5 0h12v-48l14 48h11l14-48V0h12v-70H14L0-22l-14-48h-17.5z'/%3e%3cpath id='b' fill-rule='evenodd' d='M0 0a31.5 35 0 0 0 0-70A31.5 35 0 0 0 0 0m0-13a18.5 22 0 0 0 0-44 18.5 22 0 0 0 0 44'/%3e%3cpath id='d' fill-rule='evenodd' d='M-31.5 0h13v-26h28a22 22 0 0 0 0-44h-40zm13-39h27a9 9 0 0 0 0-18h-27z'/%3e%3cpath id='n' d='M-15.75-22C-15.75-15-9-11.5 1-11.5s14.74-3.25 14.75-7.75c0-14.25-46.75-5.25-46.5-30.25C-30.5-71-6-70 3-70s26 4 25.75 21.25H13.5c0-7.5-7-10.25-15-10.25-7.75 0-13.25 1.25-13.25 8.5-.25 11.75 46.25 4 46.25 28.75C31.5-3.5 13.5 0 0 0c-11.5 0-31.55-4.5-31.5-22z'/%3e%3cuse xlink:href='%23a' id='o' transform='scale(31.5)'/%3e%3cuse xlink:href='%23a' id='p' transform='scale(26.25)'/%3e%3cuse xlink:href='%23a' id='r' transform='scale(21)'/%3e%3cuse xlink:href='%23a' id='q' transform='scale(15)'/%3e%3cuse xlink:href='%23a' id='s' transform='scale(10.5)'/%3e%3cg id='m'%3e%3cclipPath id='c'%3e%3cpath d='M-31.5 0v-70h63V0zM0-47v12h31.5v-12z'/%3e%3c/clipPath%3e%3cuse xlink:href='%23b' clip-path='url(%23c)'/%3e%3cpath d='M5-35h26.5v10H5z'/%3e%3cpath d='M21.5-35h10V0h-10z'/%3e%3c/g%3e%3cg id='h'%3e%3cuse xlink:href='%23d'/%3e%3cpath d='M28 0c0-10 0-32-15-32H-6c22 0 22 22 22 32'/%3e%3c/g%3e%3cg id='a' fill='%23fff'%3e%3cg id='f'%3e%3cpath id='e' d='M0-1v1h.5' transform='rotate(18 0 -1)'/%3e%3cuse xlink:href='%23e' transform='scale(-1 1)'/%3e%3c/g%3e%3cuse xlink:href='%23f' transform='rotate(72)'/%3e%3cuse xlink:href='%23f' transform='rotate(-72)'/%3e%3cuse xlink:href='%23f' transform='rotate(144)'/%3e%3cuse xlink:href='%23f' transform='rotate(216)'/%3e%3c/g%3e%3c/defs%3e%3crect width='100%25' height='100%25' x='-50%25' y='-50%25' fill='%23009b3a'/%3e%3cpath fill='%23fedf00' d='M-1743 0 0 1113 1743 0 0-1113z'/%3e%3ccircle r='735' fill='%23002776'/%3e%3cclipPath id='g'%3e%3ccircle r='735'/%3e%3c/clipPath%3e%3cpath fill='%23fff' d='M-2205 1470a1785 1785 0 0 1 3570 0h-105a1680 1680 0 1 0-3360 0z' clip-path='url(%23g)'/%3e%3cg fill='%23009b3a' transform='translate(-420 1470)'%3e%3cuse xlink:href='%23b' y='-1697.5' transform='rotate(-7)'/%3e%3cuse xlink:href='%23h' y='-1697.5' transform='rotate(-4)'/%3e%3cuse xlink:href='%23i' y='-1697.5' transform='rotate(-1)'/%3e%3cuse xlink:href='%23j' y='-1697.5' transform='rotate(2)'/%3e%3cuse xlink:href='%23k' y='-1697.5' transform='rotate(5)'/%3e%3cuse xlink:href='%23l' y='-1697.5' transform='rotate(9.75)'/%3e%3cuse xlink:href='%23d' y='-1697.5' transform='rotate(14.5)'/%3e%3cuse xlink:href='%23h' y='-1697.5' transform='rotate(17.5)'/%3e%3cuse xlink:href='%23b' y='-1697.5' transform='rotate(20.5)'/%3e%3cuse xlink:href='%23m' y='-1697.5' transform='rotate(23.5)'/%3e%3cuse xlink:href='%23h' y='-1697.5' transform='rotate(26.5)'/%3e%3cuse xlink:href='%23j' y='-1697.5' transform='rotate(29.5)'/%3e%3cuse xlink:href='%23n' y='-1697.5' transform='rotate(32.5)'/%3e%3cuse xlink:href='%23n' y='-1697.5' transform='rotate(35.5)'/%3e%3cuse xlink:href='%23b' y='-1697.5' transform='rotate(38.5)'/%3e%3c/g%3e%3cuse xlink:href='%23o' x='-600' y='-132'/%3e%3cuse xlink:href='%23o' x='-535' y='177'/%3e%3cuse xlink:href='%23p' x='-625' y='243'/%3e%3cuse xlink:href='%23q' x='-463' y='132'/%3e%3cuse xlink:href='%23p' x='-382' y='250'/%3e%3cuse xlink:href='%23r' x='-404' y='323'/%3e%3cuse xlink:href='%23o' x='228' y='-228'/%3e%3cuse xlink:href='%23o' x='515' y='258'/%3e%3cuse xlink:href='%23r' x='617' y='265'/%3e%3cuse xlink:href='%23p' x='545' y='323'/%3e%3cuse xlink:href='%23p' x='368' y='477'/%3e%3cuse xlink:href='%23r' x='367' y='551'/%3e%3cuse xlink:href='%23r' x='441' y='419'/%3e%3cuse xlink:href='%23p' x='500' y='382'/%3e%3cuse xlink:href='%23r' x='365' y='405'/%3e%3cuse xlink:href='%23p' x='-280' y='30'/%3e%3cuse xlink:href='%23r' x='200' y='-37'/%3e%3cuse xlink:href='%23o' y='330'/%3e%3cuse xlink:href='%23p' x='85' y='184'/%3e%3cuse xlink:href='%23p' y='118'/%3e%3cuse xlink:href='%23r' x='-74' y='184'/%3e%3cuse xlink:href='%23q' x='-37' y='235'/%3e%3cuse xlink:href='%23p' x='220' y='495'/%3e%3cuse xlink:href='%23r' x='283' y='430'/%3e%3cuse xlink:href='%23r' x='162' y='412'/%3e%3cuse xlink:href='%23o' x='-295' y='390'/%3e%3cuse xlink:href='%23s' y='575'/%3e%3c/svg%3e)
