Minimum neuron density for synchronized bursts in a rat cortical culture on multi-electrode arrays.

To investigate the minimum neuron and neurite densities required for synchronized bursts, we cultured rat cortical neurons on planar multi-electrode arrays (MEAs) at five plating densities (2500, 1000, 500, 250, and 100 cells/mm(2)) using two culture media: Neuron Culture Medium and Dulbecco's Modified Eagle Medium supplemented with serum (DMEM/serum). Long-term recording of spontaneous electrical activity clarified that the cultures exhibiting synchronized bursts required an initial plating density of at least 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum. Immediately after electrical recording, immunocytochemistry of microtubule-associated protein 2 (MAP2) and Neurofilament 200 kD (NF200) was performed directly on MEAs to investigate the actual densities of neurons and neurites forming the networks. Immunofluorescence observation revealed that the construction of complicated neuronal networks required the same initial plating density as for synchronized bursts, and that overly sparse cultures showed significant decreases of neurons and neurites. We also found that the final densities of surviving neurons at 1 month decreased greatly compared with the initial plating densities and became saturated in denser cultures. In addition, the area of neurites and the number of nuclei were saturated in denser cultures. By comparing both the results of electrophysiological recording and immunocytochemical observation, we revealed that there is a minimum threshold of neuron densities that must be met for the exhibition of synchronized bursts. Interestingly, these minimum densities of MAP2-positive final neurons did not differ between the two culture media; the density was approximately 50 neurons/mm(2). This value was obtained in the cultures with the initial plating densities of 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum.

The influence of climatic oscillations during the Quaternary Era on the genetic structure of Asian black bears in Japan.

The Asian black bear (Ursus thibetanus) inhabits two of the main islands, Honshu and Shikoku, in Japan. To determine how climatic oscillations during the Quaternary Era affected the genetic structure of the black bear populations in Japan, we examined their phylogeographic relationships and compared their genetic structure. We analysed an approximately 700-bp sequence in the D-loop region of mitochondrial DNA collected from 589 bears in this study with 108 bears from a previous study. We observed a total of 57 haplotypes and categorized them into three clusters (Eastern, Western and Southern) based on the spatial distribution of the haplotypes. All but 2 of the 41 haplotypes in the Eastern cluster were distributed locally. Genetic diversity was generally low in northern Japan and high in central Japan. Demographic tests rejected the expansion model in northern populations. Haplotypes of the Western and Southern clusters were unique to local populations. We conclude that the extant genetic structure of the Asian black bear populations arose as follows: first, populations became small and genetic drift decreased genetic diversity in the northern area during the last glacial period, whereas large continuous populations existed in the southern part of central Japan. These patterns were essentially maintained until the present time. In western and southern Japan, the effects of climatic oscillations were smaller, and thus, local structure was maintained.

Bottlenecks, drift and differentiation: the population structure and demographic history of sika deer (Cervus nippon) in the Japanese archipelago.

We assessed genetic differentiation and diversity in 14 populations of sika deer (Cervus nippon) from Japan and four populations of sika deer introduced to the UK, using nine microsatellite loci. We observed extreme levels of differentiation and significant differences in diversity between populations. Our results do not support morphological subspecies designations, but are consistent with previous mitochondrial DNA analyses which suggest the existence of two genetically distinct lineages of sika deer in Japan. The source of sika introduced to the UK was identified as Kyushu. The underlying structure of Japanese populations probably derives from drift in separate glacial refugia and male dispersal limited by distance. This structure has been perturbed by bottlenecks and habitat fragmentation, resulting from human activity from the mid-nineteenth century. Most current genetic differentiation and differences in diversity among populations probably result from recent drift. Coalescent model analysis suggests sika on each of the main Japanese islands have experienced different recent population histories. Hokkaido, which has large areas of continuous habitat, has maintained high levels of gene flow. In Honshu the population is highly fragmented and is likely to have been evolving by drift alone. In Kyushu there has been a balance between gene flow and drift but all the populations have experienced high levels of drift. Habitat fragment size was not significantly associated with genetic diversity in populations but there was a significant correlation between habitat fragment size and effective population size.

Genetic Variations Revealed by Microsatellite Markers ina Small Population of the Sika Deer (Cervus nippon)on Kinkazan Island, Northern Japan.

Genetic variations within a population of the Japanese sika deer, Cervus nippon, on Kinkazan Island were studied by microsatellite analysis. Seventeen pairs of polymerase chain reaction primers designed for several species of ungulates successfully amplified polymorphic microsatellite DNA in sika deer. About 20% of the Kinkazan population was sampled and genotyped for nine diagnostic microsatellite loci. Alleles at those loci in the Kinkazan population were found to be under the Hardy-Weinberg equilibrium. To determine whether the Kinkazan deer have a reduced level of genetic variability, an average heterozygosity in the population was calculated and compared with the values determined for other populations from Hyogo, Yamaguchi, Shimane, Tsushima, and Nagasaki. Neither the observed nor the expected heterozygosity in the Kinkazan deer significantly differed from that in the other populations. Our result indicated that, despite its small population size, the Kinkazan deer preserve extensive microsatellite variations.

Pedigree Analysis of the Sika Deer (Cervus nippon) using Microsatellite Markers.

The usefulness of microsatellite markers in pedigree analysis of the sika deer (Cervus nippon) was tested in a herd in which the maternal lineages were recorded. Eighteen sets of microsatellite primers originally designed for bovine, ovine, and cervine loci successfully amplified polymorphic DNA in the deer. The numbers of alleles per locus ranged from two to seven, and the observed heterozygosity ranged from 0.350 to 0.900. The resolution power of the markers in paternity testing was then determined by calculating exclusion probabilities and paternity indices. Parentages of the study population were efficiently discriminated by genotyping 17 microsatellite loci. The microsatellite data were also used to calculate the genetic relatedness between individuals, which significantly correlated with coancestry coefficients for the pairs. Our results demonstrate that the microsatellite markers are efficient tools in studying the social structure and behavior of the sika deer, as well as in monitoring the inbreeding status.

Partial nuclear localization of a bovine phosphoprotein, BCNT, that includes a region derived from a LINE repetitive sequence in Ruminantia.

BCNT, named after Bucentaur, is a protein that contains a 324-amino-acid region derived from part of a long interspersed DNA sequence element (LINE) in Ruminantia. However, the unique portion is completely missing in human and mouse BCNTs. Since no significant information on their function has been obtained by homology search, we at first examined cellular localization and biochemical characteristics of bovine BCNT to get a hint on its function. Subcellular fractionation and immunohistochemical analyses using a normal bovine epithelial cell line and bovine brain revealed that a significant amount of bovine BCNT is localized in the nuclei, while the major portion is present in the cytosol. Furthermore, it was shown that bovine BCNT is a phosphoprotein and that both bovine and human BCNTs are phosphorylated by casein kinase II in vitro. These results show that BCNTs consist of a unique family, probably a substrate of casein kinase II, which may contribute further to the understanding of gene evolution.

Two genetically distinct lineages of the sika deer, Cervus nippon, in Japanese islands: comparison of mitochondrial D-loop region sequences.

To investigate genetic diversity among populations of the sika deer, Cervus nippon, nucleotide sequences (705-824 bases) of the mitochondrial D-loop regions were determined in animals from 13 localities in the Japanese islands. Phylogenetic trees constructed by the sequences indicated that the Japanese sika deer is separated into two distinct lineages: the northern Japan group (the Hokkaido island and most of the Honshu mainland) and the southern Japan group (a part of the southern Honshu mainland, the Kyushu island, and small islands around the Kyushu island). All sika deer examined in this study shared four to seven units of repetitive sequences (37 to 40 bases each) within the D-loop sequences. The number of tandem repeats was different among the populations, and it was specific to each population. Six or seven repeats occurred in populations of the northern Japan group, while four or five repeats occurred in populations of the southern Japan group. Each repeat unit included several nucleotide substitutions, compared with others, and 26 types were identified from 31 animals. Sequences of the first, second, and third units in arrays were clearly different between the northern and the southern groups. Based on these D-loop data, colonization and separation of the sika deer populations in the Japanese islands were estimated to have occurred less than 0.5 million years before present. Our results provide an invaluable insight into better understanding the evolutionary history, phylogeny, taxonomy, and population genetics of the sika deer.

Assessment of genetic variations within populations of Sika deer in Japan by analysis of randomly amplified polymorphic DNA (RAPD).

Random amplified polymorphic DNA (RAPD) was exploited as a genetic marker to assess the level of genetic variation in populations of Sika deer, Cervus nippon, in Japan. DNA samples were collected from three local populations in Japan, namely, Kinkazan, Goyozan and Ashoro populations. Four arbitrary primers, when used individually, amplified an average of five RAPD fragments in the polymerase chain reaction (PCR). The number of polymorphic bands was scored to calculate band-sharing coefficients within populations. Average band-sharing coefficients revealed a higher degree of homogeneity in the Kinkazan population. Samples collected from larger populations, namely, Ashoro and Goyozan, revealed greater polymorphism than samples from the Kinkazan deer. Our data suggest that RAPD is useful as a marker for detecting genetic variations in populations of Sika deer with reduced levels of genetic diversity.

Mitochondrial DNA polymorphism in subspecies of the Japanese Sika deer, Cervus nippon.

Variations in the mitochondrial DNA (mtDNA) of Sika deer (Cervus nippon) from seven localities in Japan were examined to assess the degree of genetic differentiation among subspecies. The entire mtDNA of C. n. centralis, which is about 16,600 base pairs in length, was cloned into plasmids. Using various clones as hybridization probes, we constructed restriction maps of mtDNA for 59 deer and identified six different mtDNA genotypes. The genotypes observed in the subspecies centralis from Honshu Island (the mainland) were distinct from that of the same subspecies from the Tsushima Islands. By contrast, the subspecies yesoensis, which is distributed on Hokkaido Island, shared at least two mtDNA genotypes with the mainland centralis. Analysis of genetic distances showed that Sika deer on the Japanese islands consist of two distinct clusters, namely, the Hokkaido-Honshu (yesoensis-mainland centralis) and the Yakushima-Tsushima (yakushimae-Tsushima centralis) populations. The molecular phylogenetic data obtained for these subspecies do not reflect the present taxonomic status of these subspecies that is based on comparative morphometry. Genetic differentiation of Sika deer in the Japanese islands is a possible example of vicariance.

Overproduction and translational regulation of rp49 ribosomal protein mRNA in transgenic Drosophila carrying extra copies of the gene.

During Drosophila early development the translation of ribosomal protein mRNAs is regulated specifically and coordinately. In this study we assayed for changes of ribosomal protein rp49 gene expression in flies transformed with extra copies of the gene. RNA blot analysis revealed that the rp49 transcript was overrepresented in most of the transformed fly lines: flies carrying three times more genes than the wild type contained up to seven times more mRNA. The abundance of other ribosomal protein mRNAs was not affected. Despite the large differences in rp49 mRNA content, the proportion of the rp49 mRNA which was associated with polysomes during oogenesis and early embryogenesis did not differ significantly from the wild type, implying that rp49 protein is overproduced in the transgenic flies. The results indicate that the basis for coordinate r-protein gene expression lies in the intrinsic properties of r-protein genes, rather than in a dynamic system that separately modulates the expression of individual genes.

Levels of tyrosinase and its mRNA in coat-color mutants of C57BL/10J congenic mice: effects of genic substitution at the agouti, brown, albino, dilute, and pink-eyed dilution loci.

The levels of tyrosinase activities, tyrosinase cross-reacting material (TY-CRM), and tyrosinase mRNA were determined for skins from five congenic strains of mice (Mus musculus) with different coat colors. The dopa oxidase activity and melanin formation activity were directly proportional to the abundance of TY-CRM in all of the mutants. The levels of the enzyme activities and TY-CRM were increased in brown (b/b) and dilute (d/d) mice, while they were reduced in agouti (A/A) and pink-eyed dilution (p/p) animals. In albino (c/c) mice, the tyrosinase activities and melanin formation activity were scarcely detectable, and no TY-CRM was observed. Contrary to these, the levels of tyrosinase mRNA in agouti and albino mutants were almost the same as that in black mice, and those in brown and dilution mutants were 20-30% higher than that in black mice. These results indicate that the mutations at the brown and dilute loci exert their influence on the tyrosinase mRNA level, presumably by affecting the transcription of the tyrosinase gene, and that the mutations at the agouti and albino loci exert their influence on the TY-CRM level by regulating translation of tyrosinase mRNA and/or post-translation modification of the enzyme.

Distribution of myofiber types in the hip and thigh musculature of sheep.

The composition of muscles by myofiber type is associated with their locomotory or postural functions. In the present study the composition of the hip and thigh musculature of sheep by myofiber types and the differences in their distribution were examined. Myofibers were classified into type I, IIA, and IIB myofibers by differences in myosin ATPase and NADH tetrazolium reductase (NADH-TR) activity. The vastus intermedius muscle consisted only of type I myofibers, which exhibit weak alkali-stable myosin ATPase and strong NADH-TR activity. The gluteus accessorius and profundus muscles had more than 50% type I myofibers. The other muscles had less than 50% type I myofibers as a whole. Type I myofibers were concentrated in the deep portions of the gluteus and quadriceps femoris muscles, which extend the hip and stifle joints, and of the pectineus muscle. They were scattered evenly in the caudally situated locomotory muscles in the thigh. Type IIA myofibers, characterized by strong alkali-stable myosin ATPase and NADH-TR activity, showed little difference in distribution in the hip and thigh muscles. Type IIB myofibers, characterized by strong alkali-stable myosin ATPase and weak NADH-TR activity, were distributed more in the cranial, caudolateral, and caudomedial portions than in the middle portions of the thigh. The distribution of type IIB myofibers is suited to powerful flexion and extension of the thigh and leg. In the hip and thigh musculature, it appears that type I myofibers are effectively distributed to maintain a standing posture without diminishing the propulsive force of the hindlimb.

[Epidemiologic and therapeutic studies on gonorrheal infections--use of AT-2266--Sapporo Clinical Research Group for STD].

From August of 1982 through February of 1983, the Sapporo Clinical Research Group for STD treated 131 cases of male gonorrheal urethritis at its affiliated clinical facilities in Sapporo City. The therapeutic efficacy of AT-2266 was investigated, together with an epidemiological study on the cases and bacteriological studies on the isolated strains of gonococcus. In addition, a few cases of female gonorrheal cervicitis were treated, and the therapeutic results for 3 of these cases evaluated. AT-2266 was administered at a daily dosage of 600 mg in one dose to 14 patients, 2 doses to 10 patients and 3 doses to 98 patients. At the end of 3 days of this therapy, the gonococci had been eliminated in all of the cases, but there was variation in the status of disappearance of the secretion. The "excellent" efficacy rates for these three regimens were thus 7.7%, 50% and 57%, respectively. These results showed that administration of the total dosage in two or more divided doses maintained higher minimum concentrations of the antibiotic in the blood and the urine, and were thus more clinically beneficial than when the dosage was given as one daily dose. Of the patients who were treated with 600 mg/day of AT-2266 in 3 divided doses, 93 were evaluated for the therapeutic efficacy. At the end of 3 days 57.0% were "excellent" cases, 39.8% were "good" cases and 3.2% were "fair" cases, and at the end of 7 days of therapy, 67 patients were 74.6% "excellent" cases, 23.9% were "good" cases and 1.5% were "fair" cases. The clinical efficacy rate was thus quite high. Those cases in which the secretion and leukocytes had not disappeared from the urine even at the end of the 7 days of therapy were probably cases of mixed infection involving Chlamydia, etc. Eight of the 77 gonococcal isolates (10.4%) were beta-lactamase producers. The MIC of AT-2266 hardly differed with the size of the bacterial inoculum. With an inoculum of 10(6)CFU/ml, the MIC distribution showed two peaks, i.e., at 0.05-0.2 micrograms/ml and 3.16-12.5 micrograms/ml. Only 11.7% of the strains were found to have high MICs. The distribution of these high MICs was found to be unrelated to the ability to produce beta-lactamase. Mild side effects of AT-2266 were seen in 2 out of 128 patients. (1.6%), an extremely low incidence.

[Therapeutic studies on male non-gonorrheal urethritis--use of AT-2266--Sapporo Clinical Research Group for STD].

AT-2266 tablets were administered orally in a daily dosage of 600 mg for 7 consecutive days as treatment of non-gonorrheal urethritis in 106 cases. The therapeutic results in 90 cases were investigated, and a detailed analysis of various background factors was also performed. Most of the patients were between 15 and 35 years old; 11.3% of the patients were between 15 and 19 years old. The source of infection was a "professional" woman such as a prostitute in 37 cases, and a non-professional woman in 53 cases. This is noteworthy because the source is more often a "professional" women in the case of gonorrhea. Bacterial isolates were obtained by culture of the urethral secretions. Most were gram-positive cocci, especially S. epidermidis, but, in 51.7% of the patients, no bacteria could be cultured. The urethral secretion was purulent in 51.9% of the patients, and serous in the remaining 48.1%. The efficacy rates obtained when AT-2266 was administered in a daily dosage of 600 mg (in 3 divided oral doses) for 7 days were as follows. In the purulent secretion group, the 3-day and 7-day therapeutic efficacy (elimination of the secretion) rates were 24.2% and 61.2%. In the serous secretion group, the corresponding efficacy rates were 36% and 71.4%. Thus the therapeutic efficacy was slightly inferior in the purulent secretion group. Oral administration of AT-2266 resulted in side effects in 5 of the patients (5.5%). These side effects consisted primarily of mild gastrointestinal disturbances, which did not require discontinued drug administration.

[Therapeutic studies on chronic prostatitis--use of AT-2266--Sapporo Clinical Research Group for STD].

AT-2266 is a new antibiotic of the pyridone carboxylic acid class which possesses a broad, low-MIC antibacterial spectrum. Therapeutic studies were carried out on the use of this drug in the treatment of chronic prostatitis, and at the same time a pathological analysis was performed on chronic prostatitis. The subjects were 97 chronic prostatitis patients for whom the prostate fluid had been confirmed to contain at least 30 leukocytes per 400-power magnification field. An analysis of the background factors revealed that 71% of these patients had a past history of gonorrheal or non-gonorrheal urethritis. Culture of the prostate fluid yielded gram-positive cocci (S. epidermidis in most cases) in 44.1% of the patients. E. coli was detected in 3.2% of the patients, while the remaining cases gave negative cultures. In 53.9% of the patients who had not been receiving therapy prior to inclusion in this study, the subjective symptoms consisted of urethral irritation or irritation upon urination. In the other patients, the relationship of the complaints to the disease could not be clearly established. In the patients who had been receiving therapy, the majority did not complain of subjective symptoms. AT-2266 was administered in a daily dosage of 600 mg (in 3 divided doses) for 14 days. The therapeutic efficacy was evaluated. At the end of 7 days of AT-2266 therapy, 15.5% of the previously-untreated group and 8% of the previously-treated group were "excellent" cases, and the efficacy rate was 32.8% and 36%, respectively, when the "good" cases were also included. At the end of the full 14 days of therapy, the corresponding efficacy rates were 21.7% and 17.4%, and 54.3% and 56.5%. Considerable improvement was achieved in the subjective symptoms of urethral irritation and irritation upon urination at the end of 7 days of therapy, and the improvement was even greater following the next 7 days of treatment. With regard to the complaints for which the relationship to the disease could not be clearly established, however, the improvement was not very good: there was not much difference between the results on the 7th and 14th days, and the elimination rate even after 14 days was slightly below 30%. In the previously-untreated patients, improvement in leukocyte count in the prostate fluid to 10 or fewer cells per microscopic field was achieved in 15.6% at 7 days and 21.7% at 14 days. As side effects of AT-2266, mild symptoms were observed to occur in only 1.8% of the patients.

Effects of the lethal yellow (Ay) and recessive yellow (e) genes on the population of epidermal melanocytes in newborn mice.

Very few melanocytes can be detected by the DOPA reaction in the dorsal epidermis of newborn lethal yellow mice (Ay/a). Nevertheless, the epidermis contains a considerable number of melanoblasts (cells positive for the combined DOPA-premelanin reaction). On the other hand, numerous melanocytes as well as melanoblasts are found in the dorsal epidermis of black mice (a/a). The number of epidermal melanoblasts is smaller in (Ay/a than in a/a mice even though the same number of melanocytes is found in the dermis of these animals. It seems probable that the product of the A y gene suppresses either the differentiation or the proliferation of epidermal melanoblasts. The number of melanoblasts plus melanocytes in day-17 embryos from a cross between Ay/a and a/a mice shows a bimodal distribution. It seems possible that half of the embryos were Ay/a and possessed a reduced number of melanoblasts and melanocytes. This result seems to suggest that the Ay gene is active at this embryonic stage. In contrast to the case for the epidermis from Ay/a mice, numerous DOPA-positive melanocytes were detected in the epidermis from e/e mice. However, the total number of melanoblasts plus melanocytes in e/e epidermis did not differ from that in Ay/a epidermis, suggesting that the mode of action of the e gene in the epidermis is different from that of the Ay gene.

The occurrence of paranuclear vacuoles in sheep ruminal epithelium treated with distilled water in vitro and from sheep injected with a large quantity of water into the rumen.

In the histological preparation taken after animal death, paranuclear vacuoles (PV) in sheep ruminal epithelium were already present in high percentage (7.5-20.3%). With the incubation of the ruminal mucosae in distilled water (10-15 degrees C) up to 120 min, PV occurrence did not alter. With incubation in NaCl solution, however, PV value decreased with time proportional to NaCl concentration (0.5, 0.85 or 1.5%). In the preparation taken by biopsy, on the other hand, PV were rare in ruminal epithelium (less than 0.3%). With the injection of warm water (10-141/animal), however, PV occurrence in the ruminal epithelium was not affected. PV as cytoplasmic processes of the ruminal Langerhans cells are probably formed by the reaction of these cells to environmental changes in the tissue caused by animal death.

[Epidemiological and therapeutic studies of gonorrheal infection--clinical efficacy of sultamicillin].

We conducted an epidemiological study including analyses of background factors of 192 male and 13 female patients with gonorrheal infection in the Sapporo area and at the same time, investigated the therapeutic efficacy of sultamicillin, an ester linked prodrug of ampicillin and beta-lactamase inhibitor sulbactam in the treatment of these patients. The percentage of infections in Sapporo was rather high in the young generation, being as high as 13.5% in teen-age boys and 30.8% in teen-age girls, which were higher than the 6.1% and 6.3% of corresponding groups in Honshu island. The source of infections was so-called special public bath-ouse which accounted for about 31.8% of all cases which however, was lower than the 50% in Honshu island. By contrast, the percentage of their friends or so-called pick-up friends as a source of infection in Sapporo was as high as 46.9% which was significantly higher than the 19.9% in Honshu. Juveniles who had nonprostitutes of the other sex as a source of infection are a characteristic of the patients in Sapporo. The isolation rate of PPNG was 13.8%. The MIC (10(6) CFU/ml) of sultamicillin ranged from 0.05 to 0.39 micrograms/ml in beta-lactamase non-producing strains and from 0.20 to 1.56 micrograms/ml in beta-lactamase producing strains showing no trend of higher MIC against beta-lactamase producing strains. There was almost no difference in the efficacy of sultamicillin between a daily dose of 750 mg (2 tablets) and 1125 mg (3 tablets) nor in side effects. The eradication rate (efficacy rate) of gonococcus following a 3-day therapy was 96.2% (38.9% excellent cure rate) in male patients and 83.3% (8.3%) in female patients. In 31% of the male patients who underwent a 7-day therapy, residual serous secretion was found though some inaccuracy is involved in this data since dropouts were not counted. This suggests the need of concurrent therapy with other appropriate drugs in consideration of possible mixed infection involving Chlamydia trachomatis or other microorganisms.

[Epidemiologic and therapeutic study on gonorrheal infections--one shot therapy by aztreonam].

A clinical study of a new monocyclic beta-lactam antibiotic, AZTREONAM (hereafter referred to as AZT) for gonorrheal infections as well as epidemiologic study of gonorrheal infections were made Epidemiology: There was a reflection of the increasing sexual activity of the younger generation; both male and female patients in their twenties were most frequent (male 49.5%, female 43.7%) and the percent of teen age patients was 15.1% (male) and 34.4% (female). Forty two strains (17.2%) out of 244 clinically isolated gonococci were PPNG. Residue of serious secretion was observed in a little less than 20% suggesting a complication by Chlamydia trachomatis. Clinical Result: One shot (1-2 g) therapy by AZT was given to 244 gonorrheal infection cases (212 urethritis cases of males. 32 cervicitis cases of females) with the following highly effective rate. Although beta-lactamase producing MIC of AZT at 10(6) CFU/ml showed a peak of 0.025 microgram/ml and ranged between less than 0.0125 microgram/ml to 0.2 microgram/ml. The time required for the elimination of gonococci was studied by the administration of 1 g and 2 g AZT. Gonococci became extinct in 1-8 hours or 4-4.5 hours on average. The difference between n 1 g and 2 g was scarcely observed. Clinical effect of 1 g one shot and 2 g one shot AZT was examined on the 3rd treatment day for 244 male and females cases. The effective rate was high; 90.7% by 1 g, 97.1% by 2 g for male urethritis, 100% by 1 g also by 2 g for female cervicitis. This therapeutic efficacy was kept even in PPNG, isolated cases. There were two side effects (0.8%), one case each of numbness and, redness and swelling of both hands, out of 244 cases, but both of them were minor ones without clinical complication.