Sequence analysis of replication origin of plasmid pLS11 of Bacillus subtilis IFO 3022.

The structure of a 1.6-kb SphI-HindIII DNA sequence necessary and sufficient for the replication of a 8.6-kb plasmid pLS11 of Bacillus subtilis IFO 3022, which is responsible for gamma-polyglutamate production, has been characterized by using a trimethoprim (Tmp)-resistance gene derived form B. subtilis TTK24 chromosomal DNA as a selective marker. The 1.6-kb DNA sequence contains a rep gene encoding the protein (333 amino acids) essential for initiation of replication and a possible origin of replication. The predicted REP protein of pLS11 has an overall homology with the REP proteins of pUH1 (74.8% identity), pBAA1 (92.8%), and pFTB14 (78.7%) in Bacillus spp., pLP1 (42.1%) and pLAB1000 (36.3%) in Lactobacillus spp., and pUB110 (35.3%) and pC194 (37.4%) in Staphylococcus aureus, but has not any similarity with the REP protein of the staphylococcal plasmid pT181.     

Acute pulmonary alveolar hypoxia increases lung and plasma endothelin-1 levels in conscious rats.

To investigate the effect of pulmonary alveolar hypoxia on the synthesis and release of endothelin (ET)-1, ET-1-like immunoreactivity (-LI) levels of the lung and plasma were measured in conscious unrestrained rats under hypoxic conditions. Sixty-min exposure to alveolar hypoxia (10% O2 or 5% O2) increased the ET-1-LI level in the lung. The plasma ET-1-LI level in hypoxic rats also increased significantly. The increase of plasma and lung ET-1-LI levels were parallel to the severity of hypoxia. These results demonstrates that acute pulmonary alveolar hypoxia increases lung and plasma ET-1-LI levels in conscious unrestrained rats, suggesting a possible physiological or pathophysiological significance of ET in alveolar hypoxia.     

Purification and characterization of dipeptidyl peptidase IV from Streptococcus salivarius HHT.

Dipeptidyl peptidase IV (DAP IV) was purified from Streptococcus salivarius HHT by anion-exchange chromatography, gel filtration and affinity chromatography after lysis of cell walls with N-acetylmuramidase. DAP IV was purified 114-fold with a yield of 16.6% from total activity of the crude extract. The purified enzyme was shown to be homogeneous by disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 109,000 by gel filtration and 47,000 by sodium dodecylsulphate SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a dimeric form. The optimum pH for the reaction was 8.7 in Gly-NaOH buffer, and the isoelectric point of the enzyme was pH 4.2. The enzyme hydrolysed specifically N-terminal X-Pro from X-Pro-p-nitroanilides. The enzyme activity was hardly affected by various cations, sulphydryl-blocking reagents and metal chelators. The enzyme activity was markedly inhibited by 1 mM diisopropylfluoride, and the desialysed enzyme was attacked by proteinases.     

Microsurgical toenail transfer to the hand

Free nonvascularized toenail grafts have been used to reconstruct congenital or traumatic nail defects of the thumb or finger. Unfortunately, these transfers often result in deformity or atrophy. To avoid these undesirable results, microsurgical free vascularized toenail transfer was performed in 10 patients, 3 for congenital nail absence and 7 for traumatic nail defects. Patient age averaged 17 years (range 2 to 32 years). In contrast with previous reports, the whole big or second toenail complex without pulp was used in reconstruction. All 10 nails were successfully transferred with complete survival. No digits required reexploration. There were no donor- or recipient-site problems. Follow-up averaged 3 years, with a range of 14 months to 5 years and 4 months. Appropriate nail growth occurred in the congenital patients. No atrophy of the nail complex was found as long as sufficient bony support was present (9 of 10 cases). Whole free vascularized toenail transfers for reconstruction of congenital and traumatic nailbed defects achieve excellent aesthetic results while maintaining normal hand function.     

An establishment of ELISA for murine IL-6

Summary Murine interleukin-6 (mIL-6) was expressed inEscherichia coli as human growth hormone (hGH) fusion protein. The products were cleaved by thrombin to liberate mIL-6. Monoclonal and polyclonal antibodies specific to mIL-6 were prepared by immunizing rats with mIL-6 thus obtained. ELISA for the quantitation of mIL-6 was also established, which could detect mIL-6 in a quantity as low as 2 ng/ml.     

Structural and functional analysis of a polyoma-related mammalian plasmid (L factor): the enhancer activity and plasmid establishment.

L factor is a unique plasmid DNA which was originally discovered in a subclone (B822) of mouse L cells at a high copy number (more than 5,000 copies/cell). The presence of L factor caused no detectable abnormalities to the plasmid-bearing cells. We determined the total DNA sequence of the L factor I (and a part of L factor II) and compared it with that of polyoma DNA. Both DNA are common to the general construction of DNA frames such as early, late and noncoding regions, suggesting the two to be closely related. On the other hand, the L factor DNA sequences differ substantially from that of polyoma in the DNA sequences corresponding to the polyoma large T antigen, capsid proteins and a portion of the enhancer region. In order to investigate the mechanism of plasmid establishment of L factor, we compared the enhancer activity, capacity of DNA replication and efficiency of plasmid establishment of L factor with those of polyoma. The results indicate that L factor enhancer activity and DNA replication capacity were considerably lower than those of polyoma, suggesting that these altered (lowered) activities associated with L factor contribute to the plasmidal establishment and stable maintenance of L factor.     

Two monoclonal antibodies against small-cell lung cancer show existence of synergism in binding

Summary Murine IgG1 monoclonal antibodies (mAbs), ITK-2 and ITK-3, were generated against a small-cell lung cancer (SCLC) cell line. Enzyme-linked immunosorbent assay using a variety of established cell lines as substrates, immunoperoxidase staining of freshly frozen tissue sections, and fluorescence-activated cell sorter analysis of peripheral blood leukocytes showed that these mAbs recognize a part of the SCLC-associated cluster 1 antigen. In immunoprecipitation studies, both ITK-2 and ITK-3 bound to a 145-kDa glycoprotein of SCLC cell membrane extracts, as did MOC-1 and NKH-1, which both recognize the cluster 1 antigen. However, because the binding of¹²⁵I-labeled ITK-2 to SCLC cells was not inhibited by MOC-1 or NKH-1, the binding site of ITK-2 on SCLC cells appeared to be different from that of either MOC-1 or NKH-1. Unexpectedly, binding of¹²⁵I-labeled ITK-2 to SCLC cells increased in the presence of ITK-3. This ITK-3-induced increase in ITK-2 binding was due partly to an increase in the number of binding sites for ITK-2 on SCLC cells. Addition of ITK-3 may, therefore, improve the effectiveness of ITK-2-based tumor detection or therapy.     

Inhibitor of interferon-induced double-stranded RNA-dependent protein kinase and its relevance to alteration of cellular protein kinase activity level in response to external stimuli.

In FL cells, interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) was found to be present in a form complexed with a potent inhibitor of its dsRNA-dependent activation. The inhibitor was readily dissociated from PK-I by DEAE-cellulose chromatography to yield a dsRNA-responsive PK-I. The inhibitor was also dissociated easily from PK-I by gel filtration through Sephacryl S-200. The apparent molecular mass of the inhibitor as estimated by gel filtration was more than 160 kilodaltons. Activity of the inhibitor was decreased on IFN treatment for 8.5 hr or on Sindbis virus infection with concomitant increase in the amount of dsRNA-activatable form of PK-I. This result implies that the inhibitor may be one of the regulatory factors of cellular PK-I activity. Longer IFN treatment (24 hr) led to recovery of the inhibitor activity, but it was overridden by an extensive net synthesis of the PK-I protein.     

Suppression of postprandial glucose, insulin, C-peptide, and glucose-dependent insulinotropic peptide (GIP) in man by oral administration of a prostaglandin analogue (enprostil)

Enprostil, a long-acting, orally active dehydroprostaglandin E2 with cytoprotective and gastric antisecretory properties, is a potent inhibitor of meal-stimulated gastrin release. Recent data have suggested suppression of additional other gastrointestinal peptide hormones following single doses of enprostil. The current investigation was conducted to further clarify the effects of enprostil administration on gastrointestinal hormones and glucose metabolism under physiologic conditions and to determine whether these effects were present following multiple doses of the agent. Enprostil 70 mcg/d and its placebo were each administered for 7 1/2 days to eight normal male subjects in a study of crossover design, each treatment period lasting 7 1/2 days and separated by a 7 day washout period. Subjects received a test meal on days 1 and 8 and an oral glucose challenge on day 3 of each treatment period following enprostil or its placebo. Following the test meal, there was a delay and suppression of the maximum measured serum glucose levels. Mean overall peak glucose concentrations were lower during the enprostil phase compared to placebo (112 vs. 121 mg/dd, P = 0.025) with a trend toward delay in the time to achievement of peak glucose concentrations. Mean overall peak levels for insulin, C-peptide, and glucose-dependent insulinotropic peptide (GIP) were significantly suppressed by 36%, 16% and 60%, respectively by enprostil when compared to placebo. The overall integrated postprandial responses for insulin, C-peptide, and GIP were significantly reduced by 42%, 39% and 90%, respectively while that for glucose above baseline was reduced by 44% (P = 0.098). Similar effects were present following the oral glucose challenge.(ABSTRACT TRUNCATED AT 250 WORDS)