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)     

Isolation of a cDNA clone for the alpha subunit of the human GABA-A receptor

A cDNA clone of an alpha subunit of the human GABA-A receptor has been isolated. The human clone (pCLL800) contains 1055 nucleotides in an open reading frame and 260 nucleotides in the 5' non-coding region. The 351 amino acid sequence of this human alpha subunit shows 97% homology with its bovine counterpart. Hybridization of pCLL800 to Northern blots shows a 3.9/4.3 Kb RNA doublet in human cortex, rat whole brain, cortex, hippocampus, midbrain, olfactory bulb and cerebellum. Developmental studies show that the levels of the rat alpha mRNA increase between one and three weeks of age in a manner similar to the development of the benzodiazepine binding sites.     

Participation of microsomal aldehyde reductase in long-chain fatty alcohol synthesis in the rat brain

The participation of microsomal aldehyde reductase in long-chain fatty alcohol synthesis in the rat brain was examined. A reaction mixture of [1-14C]hexadecanoic acid with brain microsomes and NADPH formed two radioactive products having the same mobilities as pure hexadecanal (RF 0.61) and hexadecanol (RF 0.22), respectively, on TLC plates. The product of the RF 0.61 spot was further identified as hexadecanal using gas-liquid chromatography after methylation and TLC of its reduced product with LiAlH4 and semicarbazide. The ratio of hexadecanal to hexadecanol varied from 0.4 to 1.2 under the present experimental conditions. When solubilized rat brain microsomes were applied to a Sepharose 4B column coupled with the rabbit antibody raised against rat liver microsomal NADPH-cytochrome-c reductase, which reacts with aldehyde reductase from rat brain, the eluted fraction ceased to form [14C]hexadecanol but continued to form [14C]hexadecanal from [14C]hexadecanoic acid. These results strongly indicate that hexadecanal is the intermediate in the synthesis of hexadecanol from hexadecanoic acid in rat brain microsomes with the participation of microsomal aldehyde reductase.     

Acute reversible cataract due to nitrocompounds in Japanese quail (Coturnix coturnix japonica)

The present study was designed to examine the usefulness of the Japanese quail as an experimental model of cataractogenic activity. Chemicals, 2, 6-dibromo-4-nitro-phenol (2, 6-D), 2, 4-dinitroanisole (2, 4-DA), and 2, 4-dinitrophenol (2, 4-D; for the positive control), were administered singly through an oral route to 2-week old male Japanese quails to investigate the reversibility of cataracts. A single administration of 2, 4-D (36 and 43 mg/kg) produced reversible cataract in 14 of 16 animals (87.5%). This cataract was seen 1 or 2 hours after treatment and continued for 1 to 12 hours. Treatment with 2, 6-D (20 and 25 mg/kg) and 2, 4-DA (120 and 150 mg/kg) caused cataracts in 7 of 11 (63.6%) and 8 of 8 surviving animals (100%), respectively. Cataracts produced by 2, 6-D and 2, 4-DA, which were observed from 1 and 2 to 4 hours after the treatment, continued for 6 to 15 and 1 to 13 hours, respectively. Mortalities in the 25 mg/kg group of 2, 6-D, 120 mg/kg and 150 mg/kg group of 2, 4-DA were found in 2 of 5 animals, 1 of 5 animals and 5 of 9 animals, respectively. These results indicate that the Japanese quail is useful as an animal model to evaluate toxicity to the eye and cataractogenesis.     

Characterization of interleukin 2-expanded human peripheral blood lymphocytes: not only NKH-1+-NK cells but also NKH-1+ and NKH-1- T cells are LAK effectors

In vitro culture of human peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the expansion of lymphocytes including lymphokine-activated killer (LAK) cells. Using flow cytometry, studies were undertaken to determine the phenotype and LAK activity of each subset of lymphocytes expanded in vitro as a result of incubation for 2 weeks with 2500 U/ml of recombinant IL-2. Such expanded PBMC, when examined by two-color staining with various combinations of anti-CD3, 4, 8, 16, and NKH-1 monoclonal antibodies, consisted of the following six subgroups of cells: (1) CD3+4+8-, (2) CD3+4-8+, (3) CD3+4-8-, (4) CD3-16+NKH-1+, (5) CD3-16-NKH-1+, and (6) CD3-16-NKH-1-. Of the six subgroups, all five subgroups that could be tested, i.e., CD3+ T cells (CD3+4+8-, CD3+4-8+, CD3+4-8-), CD16+ natural killer (NK) cells (CD3-16+NKH-1+), and CD3-16-NKH-1- non-T non-NK cells, possessed LAK activity. Both NKH-1- as well as NKH-1+ T and non-T cells possessed LAK activity.     

The effects of monensin on membrane lipids of cultured human skin fibroblasts

We have investigated the effects of monensin, a monovalent cationophore, on the metabolism of neutral lipids, fatty acids, ceramide and phospholipids in cultured human skin fibroblasts. Treatment with 1 microM monensin for 18 h reduced the cellular cholesterol ester content to less than one-third of untreated cells, and incorporation of [3H]acetate into cholesterol ester was also reduced, to less than one-fifth. Concomitantly, a greater conversion of [3H]acetate into free cholesterol occurred. There was a moderate increase in free fatty acids, but no change in triacylglycerol content, although the content of the latter appeared to increase in the presence of fetal calf serum in the culture medium. Phosphatidylcholine decreased in content and phosphatidylserine increased among the phosphatides, but ceramide remained unchanged after monensin treatment. These findings suggest that monensin influences the metabolic interrelationships of structural lipids in fibroblasts.     

Analyses of constituents of feces and the effect of a vitamin B12 fortified diet on coprophagy in the mouse

In order to investigate coprophagy from the viewpoint of nutrition, fecal constituents were analyzed in freeze-dried samples. Feces were collected from 7:00 to 11:00 and from 19:00 to 23:00. Inorganic elements and crude fibers per unit weight were 3-4 times more concentrated in feces than in basal diet, whereas, crude proteins, crude fats and nitrogen-free extract showed various degrees of reduction. There were no differences in these tendencies with sampling time. As for some B vitamins, feces collected from 7:00 to 11:00 contained 22-92% more vitamins than feces collected from 19:00 to 23:00. In comparison with the dietary concentration, vitamin B12 was increased by 124-197 times (520-730 micrograms/100 g) in feces collected between 7:00 and 11:00. Folic acid in feces collected between 7:00 and 11:00 was 10 times greater than that in the diet. On the basis of the findings on vitamins, the effect of a vitamin B12 fortified diet (1,350 micrograms/100 g) on coprophagy was examined. Mean frequency of coprophagy per animal per day was 9.6 when animals were fed on the basal diet, whereas the frequency was immediately and significantly (p less than 0.05 approximately p less than 0.01) reduced to 4.7 after the diet had been replaced by the fortified one. However, coprophagy was not completely inhibited by vitamin B12 fortification. This indicates that some nutrient(s) in feces other than vitamin B12 might be of use to the host, and that otherwise, coprophagy might be a basically habitual form of behavior. Furthermore, under the fortified diet, the frequency of coprophagy increased gradually.(ABSTRACT TRUNCATED AT 250 WORDS)