Purification and some properties of cytotoxin produced by Clostridium difficile

The cytotoxin produced by Clostridium difficile was highly purified by using ammonium sulfate fractionation and successive column chromatographies of DEAE-Sephadex A-25, hydroxyapatite, Bio-Gel A-0.5m, Phenyl-Sepharose CL-4B, and Mono Q. The purified cytotoxin gave a single band on conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol. Its molecular weight was estimated to be 260,000 and 50,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of dithiothreitol, respectively. Thus it was supposed that the toxin consists of 5 subunits having molecular weight of approximately 50,000. It had an isoelectric point of 6.6. The toxin was heat-labile (60 C for 10 min) and inactivated by treatment with trypsin and pronase, or at pH below 4 or over 10. The minimum cytotoxic dose of the cytotoxin against Chinese hamster ovary cells was 3 ng. It was also demonstrated that the toxin is antigenically different from enterotoxin of C. difficile.     

Taq I-generated HLA-DQ αpolymorphism in Japanese patients with narcolepsy

Taq I-generated HLA-DQrestriction fragment length polymorphism was examined in Japanese patients with narcolepsy. All patients were DR2 positive and shared a 6.0 kb fragment, although this fragment was found only in 54 % of the healthy DR2-positive Japanese. This finding added the DQ gene to the list of candidates for the possible narcolepsy-susceptibility gene. In contrast, there was no complete association between narcolepsy and DXrestriction fragment length polymorphism. These findings suggest that a narcolepsy-susceptibility gene is located closer to the DQ locus than to the DX locus.     

An assessment of nasal functions in control of breathing

Breathing pattern and steady-state CO2 ventilatory response during mouth breathing were compared with those during nose breathing in nine healthy adults. In addition, the effect of warming and humidification of the inspired air on the ventilatory response was observed during breathing through a mouthpiece. We found the following. 1) Dead space and airway resistance were significantly greater during nose than during mouth breathing. 2) The slope of CO2 ventilatory responses did not differ appreciably during the two types of breathing, but CO2 occlusion pressure response was significantly enhanced during nose breathing. 3) Inhalation of warm and humid air through a mouthpiece significantly depressed CO2 ventilation and occlusion pressure responses. These results fit our observation that end-tidal PCO2 was significantly higher during nose than during mouth breathing. It is suggested that a loss of nasal functions, such as during nasal obstruction, may result in lowering of CO2, fostering apneic spells during sleep.     

Somatomedin-C in the kidney of streptozotocin diabetic rats

To evaluate the possible role of somatomedin-C, insulin-like growth factor I, in renal hypertrophy in early diabetes, kidney tissue SmC concentrations were measured in streptozotocin-induced (80 mg/kg ip) diabetic rats. Body weight, liver weight, plasma SmC concentration, and SmC concentration in the liver of diabetic rats were significantly lower than those of controls. Seven days after induction of diabetes, the kidney weight (898 +/- 95 mg) in diabetic rats was significantly greater than that in controls (755 +/- 69 mg), while SmC concentration in the kidney of diabetic rats (1.7 +/- 0.3 U/g kidney) was significantly lower than that of control rats (5.4 +/- 0.6 U/g kidney). These results suggest that renal SmC may not have an important role in renal hypertrophy in early stages of diabetes and that renal production of SmC may be impaired by insulin deficiency in rats.     

Asymmetric Reduction of 4-Oxoisophorone Using Immobilized Thermophilic Bacteria Under Conditions of Controlled Cell Growth

Asymmetric reduction of 2,6,6,-trimethyl-2-cyclohexene-l,4-dione (4-oxoisophrone) to (6R)-2,2,6-trimethyl-1,4-cyclohexane-dione((3R)-dihydro-4-oxoisophorone) was catalysed by immobilized thermophilic bacteria, Thermomonospora curvata JTS 321. Because of leakage of entrapped cells from gel beads during reactions using culture medium, we optimized the medium to allow the microbial conversion under conditions of controlled cell growth. Of the media screened, liver infusion medium was found to be the most suitable and microbial conversion was achieved without cell leakage from the immobilized gels. Immobilized T. curvata cells were repeatedly used for the asymmetric reduction of 4-oxoisophorone, more than 15 times, with an extent of conversion of 50%.     

Structural and functional features of cis-acting sequences in the basic replicon of plasmid ColIb-P9.

We have structurally and functionally analyzed the cis-elements essential for ColIb-P9 plasmid DNA replication. The putative oriV region encompassed a region of 172 base pairs (bp) located 152 bp downstream of the repZ gene. A typical dnaA box found in this region proved nonessential for the DNA replication of ColIb-P9. The ssi signal of ColIb-P9 is a homologue of the G-sites of R1 and R100 plasmids. Deletion of the G-site led to 1.5-fold reduction of the copy number, suggesting that although this G-site is not essential, it is important for efficient ColIb-P9 DNA replication. In addition, the ColIb-P9 replicon is highly and extensively homologous with the P307 (RepFIC) replicon, and highly homologous with the R100 (RepFIIA) replicon around the G-site region. These facts imply a common ancestry from which the plasmids have evolved.     

An interspecific linkage map of mouse chromosome 15 positioned with respect to the centromere

We have used an interspecific backcross between C57BL/6J and Mus spretus to derive a molecular genetic linkage map of chromosome 15 that includes 25 molecular markers and spans 93% of the estimated length of chromosome 15. Using a second interspecific backcross that was analyzed with a centromere-specific marker, we were also able to position our map with respect to the chromosome 15 centromere. This map provides molecular access to many discrete regions on chromosome 15, thus providing a framework for establishing relationships between cloned DNA markers and known mouse mutations and for identifying homologous genes in mice and humans that may be involved in disease.     

A molecular genetic linkage map of mouse chromosome 18 reveals extensive linkage conservation with human chromosomes 5 and 18

An interspecific backcross between C57BL/6J and Mus spretus was used to generate a molecular genetic linkage map of mouse chromosome 18 that includes 23 molecular markers and spans approximately 86% of the estimated length of the chromosome. The Apc, Camk2a, D18Fcr1, D18Fcr2, D18Leh1, D18Leh2, Dcc, Emb-rs3, Fgfa, Fim-2/Csfmr, Gnal, Grl-1, Grp, Hk-1rs1, Ii, Kns, Lmnb, Mbp, Mcc, Mtv-38, Palb, Pdgfrb, and Tpl-2 genes were mapped relative to each other in one interspecific backcross. A second interspecific backcross and a centromere-specific DNA satellite probe were used to determine the distance of the most proximal chromosome 18 marker to the centromere. The interspecific map extends the known regions of linkage homology between mouse chromosome 18 and human chromosomes 5 and 18 and identifies a new homology segment with human chromosome 10p. It also provides molecular access to many regions of mouse chromosome 18 for the first time.