Intracellular pH measurement in frog muscle by means of 31P-nuclear magnetic resonance.

The 31P-NMR technique was used for the monitoring of intracellular pH and studying its heterogeneity in the femoral biceps muscle of Rana catesbiana under anaerobic conditions. The value of intracellular pH of fresh muscle calculated from the chemical shift of intracellular inorganic phosphate (P1) was 7.3 on average and the line width of P1 was about 0.2 ppm. As the line width determined by the relaxation mechanism was 0.099 ppm, the P1 signal in fresh muscle was concluded to consist of overlapped narrow components, which indicated the heterogeneity of muscular pH (about 0.2 pH unit). Living muscle showed gradual acidification due to glycolysis and the decrease in heterogeneity. When glycolysis was suppressed by iodoacetic acid, slight alkalization due to the breakdown of creatine phosphate was observed. When the Lohmann reaction was suppressed by 2, 4-dinitro-1-fluorobenzene, rapid acidification accompanied by the appearance of a new acidic component was observed with the onset of ATP decrease. This new component was not detected in the muscle pretreated with glycerol to disrupt the transverse tubules. Therefore, it is likely that this new acidic component originates in the intracellular compartment, and not in the cellular difference.     

IgE antibody responses induced by transplantation of the nematode Nippostrongylus brasiliensis in rats: a possible role of nematode excretory-secretory product in IgE production.

In order to examine the effective site of sensitization for IgE responses, we transplanted 2000 adult-stage worms of the nematode Nippostrongylus brasiliensis into the duodenum or the peritoneal cavity of naive rats. Total serum IgE began to increase 1 week after the nematode inoculations and reached a peak at week 2. Living worms inoculated into the duodenum induced the highest serum IgE, this being 800 times the level in control animals. Intraperitoneal inoculations of living and dead worms resulted in increases of the serum IgE levels to 120 and 13 times the control level, respectively. The intraduodenal inoculation of living adult worms also induced a significant increase in specific IgE against the excretory-secretory (ES) product of adult N. brasiliensis 1 week later than the rise in total IgE, whereas intraperitoneal inoculations did not induce such an increase. These results suggest that sensitization through the intestinal mucosa with adult N. brasiliensis might be important for the effective induction of both specific and non-specific IgE responses. Since these findings also indicated that factors secreted by living worms play an important role in the induction of total IgE response, the ES product was injected to naive rats for 6 consecutive days (total 2.7-4.4 mg). Intraperitoneal injection of the ES product alone induced a 14.7-fold increase in total IgE without any specific IgE response. This indicates that some constituents of the ES product have the potential to trigger a non-specific IgE response.     

Detection of antibody to Staphylococcus aureus teichoic acid by enzyme-linked immunosorbent assay.

A sensitive, specific, and rapid enzyme-linked immunosorbent assay has been developed for the detection of immunoglobulin G to Staphylococcus aureus teichoic acid in human sera. Detection of S. aureus teichoic acid antibody is at least 800 times more sensitive than a double diffusion in gel assay, and positive titers of 1:25,600 and greater were observed with this assay. Results with the enzyme-linked immunosorbent assay can be obtained within 3.5 h by using antigen-coated cuvettes. Quantitation of S. aureus teichoic acid antibody by this enzyme-linked immunosorbent assay may be useful in the initial as well as the follow-up diagnosis of serious S. aureus infections.     

Mucopolysaccharidosis type II (Hunter disease): Identification and characterization of eight point mutations in the iduronate-2-sulfatase gene in Japanese patients

Mucopolysaccharidosis type II (Hunter disease) is a lysosomal storage disorder caused by a deficiency of the enzyme iduronate-2-sulfatase. Varied clinical phenotypes of this disease have been described. To identify mutations in individual patients and to examine possible correlations between mutations and clinical phenotypes, we analyzed the iduronate-2-sulfatase gene in Japanese patients with different clinical phenotypes. Five missense mutations, S333L (severe), R468Q (severe), R468L (severe), W337R (intermediate), R48P (mild), and three nonsense mutations, W345X (severe), R443X (intermediate), Q531X (mild), were identified by the RT-PCR method. Transient expression in the enzyme-deficient fibroblasts revealed that all five missense mutant enzymes were synthesized as the normal-size precursor (73 kD), and the nonsense mutant enzymes were synthesized as truncated ones (W345X:54 kD, R443X:59 kD, and Q531X:69 kD), although stable mature enzymes (45–56 kD) were not detected by Western blot analysis. Further more, expression of the eight mutant cDNAs resulted in severe reductions of iduronate-2-sulfatase enzyme activity in comparison with a normal cDNA. © 1995 Wiley-Liss, Inc.     

Significant role of interleukin-8 in pathogenesis of pulmonary disease due to Mycoplasma pneumoniae infection

We found elevated levels of interleukin-8 in pleural fluid samples from patients with pleural effusion and with a sustained fibrotic change of the lung due to Mycoplasma pneumoniae infection. This result suggests a critical role of interleukin-8 in the pathogenesis of a certain type of pulmonary disease caused by M. pneumoniae.     

Isolation, characterization, and disruption of dnr1, the areA/nit-2-like nitrogen regulatory gene of the zoophilic dermatophyte, Microsporum canis.

A homolog of the major nitrogen regulatory genes areA from Aspergillus nidulans and nit-2 from Neurospora crassa was isolated from the zoophilic dermatophyte, Microsporum canis. This gene, dnr1, encodes a polypeptide of 761 amino acid residues containing a single zinc-finger DNA-binding domain, which is almost identical in amino acid sequence to the zinc-finger domains of AREA and NIT-2. The functional equivalence of dnr1 to areA was demonstrated by complementation of an areA loss-of-function mutant of A. nidulans with dnr1 cDNA. To further characterize this gene, dnr1 was disrupted by gene replacement based on homologous recombination. Of 100 transformants analyzed, two showed the results expected for replacement of dnr1. The growth properties of the two dnr1(-) mutant strains on various nitrogen sources were examined. Unlike the A. nidulansareA(-) mutant, these dnr1(-) mutants showed significantly reduced growth on ammonia, a preferred nitrogen source for fungi. These mutant strains were also able to utilize various amino acids for growth. In comparison with wild-type M. canis, the two dnr1(-) mutants showed reduced growth on medium containing keratin as the sole nitrogen source. This is the first report describing successful production of targeted gene-disrupted mutants by homologous recombination and their phenotypic analysis in dermatophytes.