Reliable measurement of MUC5AC in human tears is essential for elucidation of the pathophysiological role of MUC5AC in dry eye disease. The purpose of this study was to develop a sensitive and reliable method for measurement of MUC5AC in human tear samples extracted from Schirmer strips by modifying a commercially available ELISA.MUC5AC was extracted from Schirmer strips containing human tears by PBS with various concentrations of polysorbate 20. The extracts were treated with neuraminidase A to cleave the sialic acids in MUC5AC. An ELISA plate was blocked to prevent nonspecific binding. The rate of extraction of MUC5AC from Schirmer strips, linearity of dilution, limit of quantification, calibration range, and intra-assay and inter-assay reproducibility were examined.MUC5AC was extracted using polysorbate 20 in a concentration-dependent manner. Extraction was more efficient at 37°C than at 25°C. The signal-to-noise ratio of the assay was dramatically increased by treatment with neuraminidase A. Treatment with a blocking reagent before incubation produced good linearity of dilution. The inter-assay and intra-assay coefficients of variation were ≤16.6%. The relative error was within 13%.We developed an efficient method for extraction of MUC5AC from Schirmer strips and a highly sensitive, reliable assay for MUC5AC in human tear samples using a commercially available ELISA kit. This method will aid in our understanding of the pathophysiology of dry eye, assessment of the effects of treatment in daily practice, and selection of appropriate therapeutic agents for patients.
A biosurfactant termed arthrofactin produced by Arthrobacter species strain MIS38 was purified and chemically characterized as 3-hydroxydecanoyl-D-leucyl-D-asparagyl-D-threonyl-D- leucyl-D-leucyl-D-seryl-L-leucyl-D-seryl-L-isoleucyl-L-isoleucyl-L-as paragyl lactone. Surface activity of arthrofactin was examined, with surfactin as a control. Critical micelle concentration values of arthrofactin and surfactin were around 1.0 x 10(-5) M and 7.0 x 10(-5) M at 25 degrees C, respectively. Arthrofactin was found to be five to seven times more effective than surfactin. The minimum surface tension value of arthrofactin was 24 mN/m at a concentration higher than the critical micelle concentration. According to the oil displacement assay, arthrofactin was a better oil remover than synthetic surfactants, such as Triton X-100 and sodium dodecyl sulfate. Arthrofactin is one of the most effective lipopeptide biosurfactants.
Here we describe recent progress on our studies on the hyperthermophilic archaeon, Thermococcus kodakaraensis. The complete genome sequence has been determined, revealing the presence of 2,306 genes on the chromosome. We have also developed a system that allows us to disrupt genes in a specific manner, which is the first of its kind in hyperthermophiles. The system has been improved, and it is now possible to isolate strains with multiple gene disruptions. We have utilized this technology to examine gene function in vivo. In one case, between two proteins that displayed frucose 1,6-bisphosphatase in vitro, we have been able to determine the physiologically relevant protein in T. kodakaraensis. In another study, we were able to examine a mutant strain deprived of the reverse gyrase gene, the one and only hyperthermophile-specific gene.
We aimed to evaluate the feasibility of using a modified Schirmer test to determine the increase in tear volume after administration of 3% diquafosol ophthalmic solution (diquafosol 3%) in dry eye patients.A randomized, multicenter, prospective, double-blind clinical study recruited 50 qualified subjects. They received diquafosol 3% in one eye and artificial tears in the other eye. The study protocol comprised a screening and treatment procedure completed within 1 day. The Schirmer test was performed on closed eyes three times a day. The primary efficacy end points were the second Schirmer test scores 10 minutes after the single dose. Secondary end points were the third Schirmer test scores 3 hours and 40 minutes after the single dose and the symptom scores prior to the second and third Schirmer tests.According to the Schirmer test, 10 minutes after administration, diquafosol 3% significantly increased tear volume compared to artificial tears. Diquafosol 3% and artificial tears both showed significant improvements in the symptom scores compared to baseline. However, there was no significant difference in the symptoms score between diquafosol 3% and artificial tears.The modified Schirmer test can detect a minute change in tear volume in dry eye patients. These findings will be useful in the diagnosis of dry eye, assessment of treatment benefits in daily clinical practice, and the development of possible tear-secreting compounds for dry eye.
We report a rare case of prostate cancer that apparently metastasized to the bladder and formed a pedunculated mass that caused a ball valve-like obstruction in urination. A 57-year-old man with metastatic prostate cancer was referred to the emergency department for acute urinary retention. Cystoscopy and magnetic resonance imaging revealed a pedunculated mass measuring approximately 2 cm in size in the bladder neck that appeared to cause urinary obstruction in a ball valve-like manner. Transurethral resection of the bladder tumor was performed that resolved his symptom, and histopathological findings confirmed a diagnosis of poorly differentiated prostate adenocarcinoma.
The nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene and its flanking regions was determined. An open reading frame was found, comprising a total of 1,647 base pairs (549 amino acids) and starting from a GUG codon as methionine. It was shown by NH2-terminal amino acid sequence analysis that the extracellular amylase consisted of 515 amino acid residues, which corresponded to a molecular weight of 58,779. Thus the NH2-terminal portion of the gene encodes 34 amino acid residues as a signal peptide. The amino acid sequence deduced from the alpha-amylase gene was fairly homologous (61%) with that of another thermostable amylase from Bacillus amyloliquefaciens.
One in five couples who wish to conceive is infertile, and half of these couples have male infertility. However, the causes of male infertility are still largely unknown. Creatine is stored in the body as an energy buffer, and the testes are its second-largest reservoir after muscles. Further, even though intratesticular creatine levels have long been known to decrease in male patients with infertility, its role in the testis is unknown. We investigated the intratesticular role of creatine, specifically in the context of the creatine synthesizing enzyme Gamt, and the creatine transporter Slc6a8. The Slc6a8 knockout mice showed no abnormalities in spermatogenesis. While Gamt knockout mice formed spermatozoa, they demonstrated reduced sperm count and decreased sperm motility and fertilization rate. Additionally, intratesticular creatine in Gamt knockout mice was significantly decreased, resulting in the disruption of tight junctions, which could be rectified by creatine supplementation, as was evidenced by the improved sperm count and fertilization rate in these mice. In conclusion, we identified creatine as being required for the maintenance of the tight junction in the testis.
