Induction of adult-type nicotinic acetylcholine receptor gene expression in noninnervated regenerating muscle

Expression of adult-type nicotinic acetylcholine receptors at the neuromuscular junction is thought to result from selective induction of their genes in endplate-associated nuclei due to local neurotrophic control. However, denervation studies indicate that endplate-specific expression can be maintained in the absence of the nerve. We investigated the role played by the basal lamina in this expression by assaying for the adult-type-specific epsilon RNA in noninnervated regenerating muscle. We found that this RNA is locally expressed beneath the old endplates after 10 days of regeneration. At earlier times epsilon RNA is also found in areas other than the endplate region. These results indicate that in adult muscle the basal lamina contains all the components necessary to direct nicotinic acetylcholine receptor gene expression to the endplate.     

Current status of antisense DNA methods in behavioral studies

The antisense DNA method has been used successfully to block the expression of specific genes in vivo in neuronal systems. An increasing number of studies in the last few years have shown that antisense DNA administered directly into the brain can modify various kinds of behaviors. These findings strongly suggest that the antisense DNA method can be used as a powerful tool to study causal relationships between molecular processes in the brain and behavior. In this article we review the current status of the antisense method in behavioral studies and discuss its potentials and problems by focusing on the following four aspects; (i) optimal application paradigms of antisense DNA methods in behavioral studies; (ii) efficiencies of different administration methods of antisense DNA used in behavioral studies; (iii) determination of specificity of behavioral effects of antisense DNA; and (iv) discrepancies between antisense DNA effects on behaviors and those on protein levels of the targeted gene.      

Plasma proteome analysis in HTLV-1-associated myelopathy/tropical spastic paraparesis

Background

A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans.

Results

Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression.

Conclusions

Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.     

Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Observations on the use of diazo-I-H-tetrazole and phenylglyoxal in protein histochemistry as applied to human gingiva

Synopsis A solution of diazo-1-H-tetrazole, freshly prepared by the diazotization of 5-amino-1-H-tetrazole under conditions to avoid explosion, was adjusted to pH 8.8, diluted (11 or 19) with 0.67 M bicarbonate buffer, pH 8.8, and used immediately as a histochemical reagent for demonstrating histidine, tryptophan, and tyrosine residues in deparaffinized sections of frozen-dried human gingiva, rat abdominal skin, and mouse larynx fixed in modified Newcomer's solution. Diazo-1-H-tetrazole reacted histochemically like other diazonium coupling reagents in common use, except that in sections pretreated with bromoacetic acid at pH 7, diazo-1-H-tetrazole staining was increased, rather than decreased as expected. Pretreatment with bromoacetic acid also increased staining in gingival sections exposed to an acetic anhydride-pyridine mixture and then reacted with diazo-1-H-tetrazole. Similarly, pretreatment with bromoacetic acid increased the intensity of Millon's reaction in gingival sections. Sections of human gingiva or mouse larynx pretreated with diazo-1-H-tetrazole stained less intensely with Biebrich Scarlet used respectively at pH 2.62 and 6.50.In test-tube experiments to check the specificity of diazo-1-H-tetrazole for amino acids, only histidine, tryptophan, and tyrosine gave solutions with colours visually distinguishable from the buffer blank. In similar tests a solution of ribonuclease A gave a colour like that given by histidine and tyrosine. Whereas pyridine failed to yield a colour with undiluted diazo-1-H-tetrazole reagent in test-tube experiments, gingival sections exposed to pyridine for 24 hr stained more intensely with diazo-1-H-tetrazole, but diazo-1-H-tetrazole staining of abdominal skin sections was not altered by prior treatment with pyridine.Phenylglyoxal, used as a 1.5% w/v solution inN-ethylmorpholine-acetate buffer (0.2 M acetate) pH 8, blocked the Sakaguchi reaction in human gingival sections. Pretreatment with phenylglyoxal also led to a reduction in their staining by Biebrich Scarlet at pH 2.62, dinitrofluorobenzene, or diazo-1-H-tetrazole. In addition the dimethylaminobenzaldehyde nitrite reaction for tryptophan was reduced. Phenylglyoxal blockade of arginine residues in gingival sections was labile to 1% acetic acid containing 0.05 M choline chloride after 60 min; but in test-tube experiments extending over 320 min, di(phenylglyoxal)-l-arginine hydrochloride was more stable in this acetic acid-choline solution than in water. It is suggested that after treatment of gingival sections with bromoacetic acid at pH 7.0, additional tyrosine residues become available for reaction with diazo-1-H-tetrazole. Pyridine is thought to remove bound lipid from gingival epithelium, thereby exposing protein residues reactive with diazo-1-H-tetrazole. The use of diazo-1-H-tetrazole and phenylglyoxal for characterizing amino acid residues of gingival proteins responsible for anionic dye binding is discussed.