Colicin K greatly decreased the incorporation of 32P-labeled inorganic orthophosphate into nucleotides and nucleic acids, causing a concomitant increase in the formation of 32P-labeled sugar phosphates in sensitive cells of Escherichia coli. These sugar phosphates were formed in aerobically growing cells, as well as in cells under stringent control of ribonucleic acid synthesis. The main 32P-labeled product was identified as sedoheptulose 7-phosphate in two strains (B1 and K-12 MK-1) and fructose 1,6-diphosphate in one strain (K-12 CP78). The formation of sugar phosphates induced by colicin K was inhibited by carbonyl cyanide m-chlorophenylhydrazone. It was also not observed in N,N'-dicyclohexylcarbodiimide-treated cells or Mg2+-(Ca2+)-adenosine triphosphatase-less mutant (strain K-12 AN120) cells. Thus, the formation of sugar phosphates in colicin K-treated cells is dependent on the formation of adenosine 5'-triphosphate by oxidative phosphorylation.
Whole cardiac myosin induced experimental autoimmune myocarditis (EAM) in animals. The identification of the epitope causing EAM is expected to elucidate the mechanism leading to the onset of this autoimmune disease. Until now such studies have been done mostly with synthetic oligo-peptides. We employed recombinant technology to produce the immunogenic fragment of the self-cardiac myosin heavy chain (CMHC) for EAM in Lewis rats, and successfully induced severe myocarditis with short recombinant peptide fragment CMHC residues 1107 to 1186. The immunogenicity was completely abolished from the fragment with further excision of 12 amino acids from residues 1131 to 1143. This recombinant technology clearly has advantages in the ease of manipulation and the purity for the creation of immunogenic epitopes.
The past two decades can be characterized by a revolution in our understanding of living organism. The major driving force in this revolution has been recombinant DNA techniques. The technique is characterized specially by its ease in application. The most frequently used technique is the gel electrophoresis, followed by blotting of the gel separated samples to a transfer membrane and the subsequent hybridization of the membrane with a probe.In this article, I reviewed some of the practical problems associated with the use of transfer membranes in the laboratories.When radioactive tracers are used for detection by X-ray film, the strength of the signals above the minimun threshold of the X-ray film detection determine its usability. It was necessary to maximize the amount of the samples absorbed on the membrane, and to optimize the fixation method to get the signal strength required for satisfactory results. Among the materials used for transfer membrane, Nylon gained the popularity. When Bioimage-analyzer from Fuji Film Company was introduced, the machine dramatically changed the scope of the technique because of its high sensitivity, the accuracy in quantitation, and the flexibility for data processing by the ample power of the computer in the analyzer. The deciding factor for sensitivity in detection is now low background noise which increases the signal-to-noise ratio (S/N). Cellulose nitrate membrane and PVDF membrane are the material more preferred over Nylon in this regard.More recently, non-radioactive detection systems are gaining popularity. The systems using Chemi-luminescence are begining to be used widely, and CCD cameras are getting better allowing the method to get the benefit of computer power. The biggest problem with this method is in the inaccuracy in quatitation due to the nature of the membrane ; e.g. the transfer membranes have thickness which is important to increase the capacity of binding samples but inhibits the light signals from within the membranes due to blockage of light by the membrane matrix. Another non-radioactive method using fluorescent probes suffers from the natural fluorescence of the membrane matrix causing the high background. This problem is additional to the light permeability problem. It is now highly desirable to develop new transfer membranes suitable for optical methods used in the non-radioactive detection systems.
Mutants of Escherichia coli lacking in the highly penicillin-sensitive enzyme activities of D-carboxy-peptidase, transpeptidase, and endopeptidase, and with the concomitant absence of penicillin-binding protein 4 of B.G. Spratt and A.B. Pardee [(1975) Nature 254, 516-517] were isolated. The defect of these mutants is ascribed to the lack of an enzyme, D-alanine carboxypeptidase Ib. Genetic mapping studies show the mutation (dacB) to be located at 68 min on the E. coli chromosome map. The dacB mutation results in the simultaneous loss of D-alanine carboxypeptidase and penicillin-binding protein 4. The mutants grew normally under a wide range of growth conditions. We conclude that the enzyme is not a necessary component for normal peptidoglycan biosynthesis in E. coli.
