Modulation of [3H]Diazepam Binding in Rat Cortical Membranes by GABAA Agonists

GABAA receptor agonists modulate [3H]diazepam binding in rat cortical membranes with different efficacies. At 23 degrees C, the relative potencies for enhancement of [3H]diazepam binding by agonists parallel their potencies in inhibiting [3H]gamma-aminobutyric acid [( 3H]GABA) binding. The agonist concentrations needed for enhancement of [3H]diazepam binding are up to 35 times higher than for [3H]GABA binding and correspond closely to the concentrations required for displacement of [3H]bicuculline methochloride (BMC) binding. The maximum enhancement of [3H]diazepam varied among agonists: muscimol = GABA greater than isoguvacine greater than 3-aminopropane sulphonic acid (3APS) = imidazoleacetic acid (IAA) greater than 4,5,6,7-tetrahydroisoxazolo (4,5,6)-pyridin-3-ol (THIP) = taurine greater than piperidine 4-sulphonic acid (P4S). At 37 degrees C, the potencies of agonists remained unchanged, but isoguvacine, 3 APS, and THIP acquired efficacies similar to GABA, whereas IAA, taurine, and P4S maintained their partial agonist profiles. At both temperatures the agonist-induced enhancement of [3H]diazepam binding was reversible by bicuculline methobromide and by the steroid GABA antagonist RU 5135. These results stress the importance of studying receptor-receptor interaction under near-physiological conditions and offer an in vitro assay that may predict the agonist status of putative GABA receptor ligands.     

Identification of hepatitis B virus polypeptides encoded by the entire pre-s open reading frame.

The open reading frame (ORF) that encodes the 226-amino-acid coat protein (hepatitis B virus surface antigen [HBsAg]) of hepatitis B virus has the potential to encode a 400-amino-acid polypeptide. The entire ORF would direct the synthesis of a polypeptide whose C-terminal amino acids represent HBsAg with an additional 174 amino acids at the N terminus (pre-s). Recently, virus particles have been shown to contain a polypeptide that corresponds to HBsAg with an additional 55 amino acids at the N terminus encoded by the DNA sequence immediately upstream of the HBsAg gene. A novel ORF expression vector containing the TAC promoter, the first eight codons of the gene for beta-galactosidase, and the entire coding sequence for chloramphenicol acetyltransferase was used in bacteria to express determinants of the 174 amino acids predicted from the pre-s portion of the ORF. The resulting tribrid protein containing 108 amino acids encoded by pre-s was expressed as one of the major proteins of bacteria harboring the recombinant plasmid. Single-step purification of the tribrid fusion protein was achieved by fractionation on a chloramphenicol affinity resin. Polyclonal antiserum generated to the fusion protein was capable of detecting 42- and 46-kilodalton polypeptides from virus particles; both polypeptides were also shown to contain HBsAg determinants. The ability of the polyclonal antiserum to identify polypeptides with these characteristics from virus particles presents compelling evidence that the DNA sequence of the entire ORF is expressed as a contiguous polypeptide containing HBsAg. The presence of multiple promoters and primary translation products from this single ORF argues that the function and potential interaction of the encoded polypeptides play a crucial role in the life cycle of the virus. Furthermore, the procedure and vector described in this report can be applied to other systems to facilitate the generation of antibodies to defined determinants and should allow the characterization of the epitope specificity of existing antibodies.     

In vitro replication of human mitochondrial DNA: accurate initiation at the origin of light-strand synthesis

Synthesis of human light-strand mitochondrial DNA was accomplished in vitro using DNA primase, DNA polymerase, and other accessory proteins isolated from human mitochondria. Replication begins with the synthesis of primer RNA on a T-rich sequence in the origin stem-loop structure of the template DNA and absolutely requires ATP. A transition from RNA synthesis to DNA synthesis occurs near the base of the stem-loop structure and a potential recognition site for signaling that transition has been identified. The start sites of the in vitro products were mapped at the nucleotide level and were found to be in excellent agreement with those of in vivo nascent light-strand DNA. Isolated human mitochondrial enzymes recognize and utilize the bovine, but not the mouse, origin of light-strand replication.     

Correlation between limitation of growth ofPachysolen tannophilus on D-xylose with the formation of ethanol and other products

The formation of ethanol, xylitol, ribitol, arabitol and acetic acid from D-xylose byPachysolen tannophilus correlated with the limitation of growth. The correlation was consistent with these products being secondary metabolites.Issued as NRCC Publication Number 24259.     

In vitro development of the hamster and chick secondary palate

A series of experiments were undertaken to compare the in vitro behaviour of the medial edge epithelium (MEE) of hamster, in which palatal shelves normally fuse, and chick, in which they do not fuse. Homotypic pairs of hamster and chick embryo palatal processes, single palatal processes, and heterotypic palatal shelves of both animals were grown in vitro. The results indicated that contact between palatal shelves may not be crucial for MEE differentiation in mammals. The ability to acquire pre-fusion characteristics may be present in mammalian palatal tissue from their early development and may be expressed by cessation of DNA synthesis in the MEE, elevation of cAMP, and MEE cell death. Isolated chick palatal shelf cultured under identical conditions did not express these mammalian pre-fusion characteristics. When MEE of hamster and chick palatal shelves were placed in contact with one another, the intervening epithelia underwent cytolysis. This could be due to either the destruction of chick MEE by lysosomal enzymes liberated from adjacent degenerating hamster MEE cells, or by induction of cell death in chick MEE by hamster mesenchyme. Heterotypic palatal tissue combinations also suggest that release of lysosomal enzymes in the hamster MEE, which leads to its dissolution, may be the terminal event in epithelial differentiation prior to the establishment of mesenchymal continuity. It is suggested that an inverse relationship exists between DNA synthesis and cAMP levels during palatogenesis: when palate closes (as in mammals) the MEE is eliminated by increasing cAMP levels, whereas when palate remains open (as in birds) low level of cAMP preserve the integrity of MEE by supporting DNA synthesis.     

Differential ability of a T-antigen transport-defective mutant of simian virus 40 to transform primary and established rodent cells.

The transforming potential and oncogenicity of a simian virus 40 (SV40) mutant affecting T-antigen (T-ag), SV40(cT)-3, was examined in an effort to dissect T-ag functions in transformation. SV40(cT)-3 has a point mutation at nucleotide 4434 that abolishes the transport of T-ag to the nucleus but does not affect its association with the cell surface. Transfection-transformation assays were performed with primary cells and established cell lines of mouse and rat origin. The efficiency of transformation for established cell lines by SV40(cT)-3 was comparable to that of wild-type SV40, indicating that transformation of established cell lines can occur in the absence of detectable amounts of nuclear T-ag. Transformation of primary mouse embryo fibroblasts by SV40(cT)-3 was markedly influenced by culture conditions; the relative transforming frequency was dramatically reduced in assays involving focus formation in low serum concentrations or anchorage-independent growth. Immunofluorescence tests revealed that the transformed mouse embryo fibroblasts partially transport the mutant cT-ag to the cell nucleus. Transformed cell lines induced by SV40(cT)-3 did not differ in growth properties from wild-type transformants. SV40(cT)-3 was completely defective for the transformation of primary baby rat kidney cells, a primary cell type unable to transport the mutant T-ag to the nucleus. The intracellular localization of cellular protein p53 was found to mimic T-ag distribution in all the transformants analyzed. The mutant virus was weakly oncogenic in vivo: the induction of tumors in newborn hamsters by SV40(cT)-3 was reduced in incidence and delayed in appearance in comparison to wild-type SV40. These observations suggest that cellular transformation is regulated by both nuclear and surface-associated forms of SV40 T-ag.     

Abortion due to infection with Chlamydia psittaci in a sheep farmer's wife.

A farmer''s wife who had helped with lambing aborted spontaneously in March after a short febrile illness in the 28th week of her pregnancy. She developed disseminated intravascular coagulation post partum with acute renal failure and pulmonary oedema. Recovery was complete after two weeks of hospital care. A strain of Chlamydia psittaci, probably of ovine origin, was isolated from the placenta and fetus. The patient''s serum showed rising titres of antibody against chlamydia group antigen; the placental and fetal isolates; and a known ovine abortion, but not a known avian, strain of C psittaci. IgG against both ovine abortion and enteric strains of C psittaci was detected, but IgM against only an abortion strain was detected. Histological examination showed pronounced intervillus placentitis with chlamydial inclusions in the trophoblast but no evidence of fetal infection or amnionitis. Laboratory evidence of chlamydial infection was found in an aborting ewe on the farm in January and in remaining sheep and lambs in July. Doctors should recognise the possible risk to pregnant women in rural areas where chlamydial infections in farm animals are widespread.     

成人腹泻轮状病毒的提纯及其高价免疫血清的制备

本文采用交叉免疫电泳技术,首先提纯了成人腹泻轮状病毒(Adult DiarrhoeaRotavirus简称:ADRV),并经电镜确认,用作免疫抗原,首次制备了高价兔与豚鼠抗ADRV血清和小鼠抗ADRV腹水,其特异性经交叉免疫电泳和对流免疫电泳鉴定,其效价经酶联免疫吸附试验(ELISA)检测均在1:2,000以上。     

Chorismate mutase/prephenate dehydrogenase from Escherichia coli K12: purification, characterization, and identification of a reactive cysteine

The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase/prephenate dehydrogenase, has been isolated from extracts of a regulatory mutant of Escherichia coli K12. The pure enzyme is a homodimer of total molecular weight 78 000 and displays Michaelis-Menten kinetics for both activities. Fingerprinting and amino acid sequencing of tryptic and thermolytic peptides of the S-[14C]carboxymethylated enzyme allowed the identification of three unique cysteine-containing sequences per subunit. Chemical modification of the native enzyme with 5,5'-dithiobis(2-nitrobenzoate) or iodoacetamide showed that one sulfhydryl group per subunit was particularly reactive, and the integrity of this group was essential for both enzymic activities. This work supports previous proposals for a close spatial relationship between the active sites.