MPTP, MPP+ and mitochondrial function

1-Methyl-4-phenylpyridinium (MPP+), the putative toxic metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), inhibited NAD(H)-linked mitochondrial oxidation at the level of Complex I of the electron transport system. MPTP and MPP+ inhibited aerobic glycolysis in mouse striatal slices, as measured by increased lactate production; MPTP-induced effects were prevented by inhibition of monoamine oxidase B activity. Several neurotoxic analogs of MPTP also form pyridinium metabolites via MAO; these MPP+ analogs were all inhibitors of NAD(H)-linked oxidation by isolated mitochondria. 2'-Methyl-MPTP, a more potent neurotoxin in mice than MPTP, was also more potent than MPTP in inducing lactate accumulation in mouse brain striatal slices. Overall, the studies support the hypothesis that compromise of mitochondrial oxidative capacity is an important factor in the mechanisms underlying the toxicity of MPTP and similar compounds.     

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

The structure of toxic protein, the mistletoe lectin, at different pH: the study using intrinsic fluorescence method

The effects of pH on the conformation of mistletoe lectin I and its isolated A- and B-subunits has been investigated by using the methods of intrinsic fluorescence. By the denaturating action of guanidine hydrochloride and the influence of the quenchers (I-, Cs+, acrylamide) the structural stability of the native protein and its isolated subunits was estimated. Treatment of the protein with the denaturant and quenchers revealed its different structure at pH 7.0 and 4.0. At pH 4.0 tryptophan residues become more accessible to quenchers, positive charge of the surrounding area increases and the protein becomes more stable to the action of denaturant. The structure of the isolated A- and B-chains of mistletoe lectin I differs considerably from that of the whole protein: a) its stability to the action of guanidine hydrochloride is lower; b) it depends on the ionic strength of the solvent; c) it is characterized by increased accessibility of tryptophan residues to quenchers (for B-chain). Differences between the conformations of the isolated chains at pH 7.0 and 4.0 are marked more strongly; moreover, at pH 4.5 the B-chain undergoes structural transition. The possible relationship between structural peculiarities of mistletoe lectin I and the mechanism of its transmembrane transfer is discussed.     

A variable region gene subfamily encoding T cell receptor beta-chains is selectively conserved among mammals

Studies of murine T cell receptor genes indicate that the VT beta repertoire, in contrast to the large VT alpha, VH, and VK repertoires, is limited to approximately 21 genes. Large differences between the various VT beta sequences allow classification into distinct subfamilies consisting of one or few members. The VT beta sequence of a gene, RTB92, expressed in a rabbit T cell line is most closely related to a VT beta gene expressed in the human cell line Molt-4. Genomic blots with RTB92 VT beta region used as probe indicated conservation of related VT beta gene subfamilies in man and pig, but no related sequences were seen in rat, mouse, or hamster. The relationship among these VT beta genes mirrors similarities and differences in MHC genes of the compared species and suggests coordinate evolution of these functionally related gene families. The constant region of RTB92 was shown to be rabbit CT beta 2 by reference to genomic clones. Comparisons with constant region sequences of human and murine CT beta 1 and CT beta 2 chains reveal no similarities in amino acid or nucleic acid sequence in the coding region characteristic of the respective isotypes. Intraspecies homologies between CT beta 1 and CT beta 2 sequences were much greater than those between CT beta 1 or CT beta 2 from other species. By contrast, comparisons of JT beta and 3' untranslated region sequences showed significant interspecies conservation of sequences in the beta 1 and in the beta 2 regions.     

Localization of C4 genes within the HLA complex by molecular genotyping

Segregation of the complement component, C4, was analyzed in six families that each included an individual who inherited an HLA haplotype where a crossover event had occurred in the region between HLA-B and HLA-DR. Two cDNA clones corresponding to the C4 gene were utilized as probes in Southern blot analysis of DNA from members of each family. Restriction fragment length polymorphisms (RFLP) were observed and were assigned to haplotypes. In one family RFLP, hybridizing with the C4 probes, segregrated with HLA-B, and in four families RFLP segregated with HLA-DR; one family was not informative in this respect. These analyses have made it possible to localize the genes for C4 between HLA-B and HLA-DR by molecular genotyping and to characterize three different genomic configurations of C4 genes by limited restriction mapping.Abbreviations RFLP restriction fragment length polymorphisms - LCL lymphoblastoid cell lines     

Molecular genotyping of human T-cell antigen receptor variable gene segments

The gene complex encoding the chain of the T-cell antigen receptor (Tcr) in man was previously reported to contain a restriction fragment length polymorphism (RFLP) involving a single Bgl II site adjacent to the second constant region gene. This RFLP allowed assignment of Tcr genotypes in certain human families. In the present study, two different RFLP in a V gene family were detected using the murine probe V8.1 in genomic DNA samples digested with the restriction endonucleases Hind III and Bam HI. Use of these RFLP to mark the V gene complex allowed complete haplotype assignment in four of seven families studied and provided support for linkage of the V gene complex to the constant region genes. Different combinations of the C and two V region markers can result in eight possible distinct haplotypes. The observation of all but one of the eight possible haplotypes in parents of the families studied suggests that recombination events occur between the C and V region and among members of the V region subfamily marked by the V8.1 probe. These markers can be used for mapping studies of the V gene complex in man and will allow an appraisal of possible associations between Tcr genes and disease susceptibility.Abbreviations used in this paper: Tcr T-cell antigen receptor - RFLP restriction fragment length polymorphism - C2 second Tcr constant region gene - V Variable - C constant - J joining - D diversity     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.