A new mouse TCR V gamma gene that shows remarkable evolutionary conservation.

We have identified a new mouse T-cell receptor V gamma gene segment, V gamma 4.4, which frequently undergoes rearrangements in AKR thymomas, and at a lower frequency in fetal thymocytes. V gamma 4.4 is the fourth and the most 5' V gene segment in the gamma 4 cluster, being 7.3 kb from V gamma 4.3. Surprisingly, V gamma 4.4 is more homologous to eight human V gamma genes than to the other mouse V gamma genes. It has only a 38% nucleotide and 21% amino acid sequence homology to the most homologous mouse V gamma gene (V gamma 4.1), whereas these homologies to the human V gamma 8 gene are as high as 68% and 48% respectively.     

Pyrazole is different from acetone and ethanol as an inducer of the polysubstrate monooxygenase system in mice: evidence that pyrazole-inducible P450Coh is distinct from acetone-inducible P450ac

The induction of liver microsomal monooxygenase activities elicited by pyrazole, ethanol, and acetone, all shown to be inducers of rat P450j and rabbit P450LM3a, has been compared in inbred strains of DBA/2N, AKR/J, and Balb/c mouse. Pyrazole strongly increases coumarin 7-hydroxylase (COH) activity in DBA/2N but much less in other strains. The effect of pyrazole on aniline p-hydroxylase and ethanol oxidase activities is also strain dependent: an increase was seen only in the DBA/2N strain. Ethanol and acetone were unable to induce COH, whereas aniline p-hydroxylase and ethanol oxidase were elevated about 1.4- to 3.3-fold in all strains. No strain difference could be detected in aniline p-hydroxylase or ethanol oxidase inducibility. There was a strong correlation between aniline p-hydroxylase and ethanol oxidase activities in every strain, whereas no positive correlation could be found between COH and aniline p-hydroxylase activities. Immunoinhibition experiments showed that a polyclonal antibody against purified pyrazole-inducible COH (P450Coh) blocked about 90% of COH activity, but only about 10% of aniline p-hydroxylase or ethanol oxidase in mouse liver microsomes. Monoclonal antibody 1-91-3 (raised against rat acetone-inducible P450ac) did not inhibit COH, whereas aniline p-hydroxylase was blocked 46-76% and ethanol oxidase 25-70%, depending on the source of microsomes. In immunoblots, anti-P450Coh recognized only its own antigen but not the P450ac, whereas monoclonal antibody 1-98-1 against P450ac detected P450ac and a corresponding form in the D2 mouse liver, but not the P450Coh. The purified P450ac and P450Coh had molecular masses of 52 and 50 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These antigens were expressed differentially in response to pyrazole, ethanol, and acetone: P450Coh was increased only after pyrazole treatment, but 1-98-1-detectable protein was elevated in D2 mouse liver microsomes by ethanol and acetone, but not by pyrazole. We conclude that mouse P450Coh and rat P450ac are not corresponding forms of the same isozyme, and that a P450ac-like protein, responsible for most of aniline p-hydroxylation and ethanol oxidation, is present in the D2 mouse liver. These two P450 isozymes are also dissimilarly expressed in the mouse liver in response to inducer administration.     

Isotype concentrations of human antibodies to group A meningococcal polysaccharide

Antibodies to meningococcal group A polysaccharide (MenA) in the sera of 34 vaccinated adults were quantitated by an isotype-resolving solid-phase RIA (IgA, IgM, IgG1, IgG2, IgG3, and IgG4). All individuals had antibodies before vaccination. The geometric mean concentration was 2.9 micrograms/ml. Two weeks after vaccination the mean antibody concentration had trebled. Average proportions of the three isotypes were then as follows: IgA 15%, IgM 48%, IgG 37%. No differences were found between individuals who had been immunized with the polysaccharide 7 to 8 yr earlier and "primary responders." The subclass composition of IgG antibodies was determined in the 24 postvaccination samples with a definite IgG response (greater than 2-fold increase). IgG1 was the predominant subclass in antibodies of some sera and IgG2 in others, but the average proportions of both subclasses were nearly the same. IgG3 and IgG4 were only found in occasional sera, but when present, each subclass accounted for up to 6%. Although the ratio of kappa and lambda chains could not be determined, there was evidence to suggest that it was higher in anti-MenA antibodies than in antibodies to protein antigens.     

Enzyme-inducing and cytotoxic effects of wood-based materials used as bedding for laboratory animals. Comparison by a cell culture study

Enzyme-inducing and cytotoxic effects of wood-based materials used as bedding for laboratory animals were studied in a cell culture system. Mouse hepatoma cell line, Hepa-1, was exposed to acetone extracts of hardwoods (alder and aspen), softwoods (pine and a mixture of pine and spruce) and cellulose materials. Cytotoxicity and induction of cytochrome P450IA1 (aryl hydrocarbon hydroxylase) and aldehyde dehydrogenase were measured. Both softwood and hardwood extracts were shown to contain inducers of these enzymes. Pine appeared to be the most potent inducer and softwoods more potent than hardwoods. The softwoods and alder were clearly more cytotoxic than aspen. The two bleached cellulose materials were found to contain inducers of aryl hydrocarbon hydroxylase. Unlike the wood beddings, the extracts of the cellulose materials were not found to be toxic to the cells. Hepa-1 cell culture system was found to be a rapid and sensitive method for screening and comparative purposes.     

A Genetic Polymorphism in Coumarin 7-Hydroxylation: Sequence of the Human CYP2A Gnes and Identification of Variant CYP2A6 Alleles

A group of human cytochrome P450 genes encompassing the CYP2A, CYP2B, and CYP2F subfamilies were cloned and assembled into a 350-kb contig localized on the long arm of chromosome 19. Three complete CYP2A genes—CYP2A6, CYP2A7, and CYP2A13—plus two pseudogenes truncated after exon 5, were identified and sequenced. A variant CYP2A6 allele that differed from the corresponding CYP2A6 and CYP2A7 cDNAs previously sequenced was found and was designated CYP2A6ν2. Sequence differences in the CYP2A6ν2 gene are restricted to regions encompassing exons 3, 6, and 8, which bear sequence relatedness with the corresponding exons of the CYP2A7 gene, located downstream and centromeric of CYP2A6ν2, suggesting recent gene-conversion events. The sequencing of all the CYP2A genes allowed the design of a PCR diagnostic test for the normal CYP2A6 allele, the CYP2A6ν2 allele, and a variant—designated CYP2A6ν1—that encodes an enzyme with a single inactivating amino acid change. These variant alleles were found in individuals who were deficient in their ability to metabolize the CYP2A6 probe drug coumarin. The allelic frequencies of CYP2A6ν1 and CYP2A6ν2 differed significantly between Caucasian, Asian, and African-American populations. These studies establish the existence of a new cytochrome P450 genetic polymorphism.     

Unusual patterns of benzo[a]pyrene metabolites and DNA-benzo[a]pyrene adducts produced by human placental microsomes in vitro

Human placental microsomes were incubated with [³H]benzo[a]pyrene (BP) and Salmon sperm DNA and the resulting metabolite-nucleoside complexes resolved by Sephadex LH-20 chromatography. The metabolite pattern was analyzed by high-pressure liquid chromatography (HPLC). The incubates were also co-chromatographed with extracts obtained from incubates with rat liver microsomes and [¹⁴C]BP. Phenols, quinones and 7,8-dihydrodiol were detected in the placental incubates. Both 9,10- and 4,5-dihydrodiols were very low as compared with control rat liver samples. Placental microsomes catalyzed the binding of BP metabolites to DNA in vitro, giving rise to two main complexes which co-chromatographed with rat liver-produced peaks attributable to 7,8-diol-9,10-epoxide and 7,8-oxide and/or quinones when metabolized further. The nucleoside metabolite peaks attributable to 4,5-oxide and 9-phenol-4,5-oxide were lacking when compared with the binding pattern catalyzed by rat liver. Both the total binding and specific metabolite-nucleoside adducts in the placenta correlated with fluorometrically measured aryl hydrocarbon hydroxylase (AHH) activity and with the amount of dihydrodiol formed. The results demonstrate that both the metabolite pattern and the nucleoside-metabolite complexes formed by the placental microsomes in vitro differed greatly from those produced by rat liver microsomes. These studies also suggest that it is not possible to predict specific patterns of DNA binding from AHH measurements or even from BP metabolite patterns, especially when comparing different tissues and species.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Effects of weaning age and formic acid-based feed additives on pigs from weaning to slaughter

Two hundred and forty piglets were used in a 2 x 6 factorial experiment to study the effects of weaning age (26 or 36 d) and four formic acid-based feed additives on the performance of pigs from weaning to slaughter. Either formic acid (F) or a mixture of formic acid, propionic acid, and potassium sorbate (FPS) or a mixture of formic acid, propionic acid, and sodium benzoate (FPB) or formic acid in a diatomaceous earth carrier (FD) were added to the diets of weaned piglets (from weaning to 60 d of age) and growing (18-46 kg) and finishing pigs (46-107 kg) to provide 8, 6, and 6 g acid per kg feed, respectively. The negative control treatment's (C) diets contained no growth promoters, whereas the positive control treatment's weaner and grower diets were supplemented with 40 mg/kg of avilamycin (A). The piglets weaned at the age of 26 and 36 d weighed 7.6 and 10.7 kg at weaning (p < 0.001), and 18.5 and 17.9 kg at the age of 60 d (p > 0.05), respectively. There was a weaning age x feed additive interaction in the weight gain of piglets after weaning (p < 0.05). The weight gain of piglets weaned on day 26 was enhanced by A, FPS, and FD (p < 0.05), and that of piglets weaned on day 36 by A and FPB (p < 0.05). The feed conversion ratio was not affected by weaning ages but was decreased in groups A, F, FBS, and FPB (p < 0.05). The severity of post-weaning diarrhoea was less in groups A, F, FPS, and FD than in C (p < 0.05). In piglets weaned on day 26, faecal water content and the total Escherichia coli count were highest 9 d after weaning. The total E. coli count was reduced only by FD (p < 0.05). Increased faecal water content was characterized by increased faecal Na+ and decreased K+ concentrations. Weaning age did not influence performance or carcass quality in the growing-finishing pigs. Feed additives did not affect weight gain in the growing pigs, but FPS and FPB enhanced weight gain during finishing period and total fattening (p < 0.05). In summary, the pigs' growth performance from weaning to slaughter was not affected by weaning age but it was enhanced by mixtures of formic and propionic acids with small amounts of sorbate or benzoate.     

Extracellular glycosaminoglycans modify cellular trafficking of lipoplexes and polyplexes

It has been shown that extracellular glycosaminoglycans (GAGs) limit the gene transfer by cationic lipids and polymers. The purpose of this study was to clarify how interactions with anionic GAGs (hyaluronic acid and heparan sulfate) modify the cellular uptake and distribution of lipoplexes and polyplexes. Experiments on cellular DNA uptake and GFP reporter gene expression showed that decreased gene expression can rarely be explained by lower cellular uptake. In most cases, the cellular uptake is not changed by GAG binding to the lipoplexes or polyplexes. Reporter gene expression is decreased or blocked by heparan sulfate, but it is increased by hyaluronic acid; this suggests that intracellular factors are involved. Confocal microscopy experiments demonstrated that extracellular heparan sulfate and hyaluronic acid are taken into cells both with free and DNA-associated carriers. We conclude that extracellular GAGs may alter both the cellular uptake and the intracellular behavior of the DNA complexes.