Identification of amino acid residues critical for LD78beta, a variant of human macrophage inflammatory protein-1alpha, binding to CCR5 and inhibition of R5 human immunodeficiency virus type 1 replication.

In an attempt to determine which amino acid(s) of LD78beta, a variant of human macrophage inflammatory protein-1alpha, plays a critical role in the interaction with CCR5, we generated six LD78beta variants with an amino acid substituted to Ala at the NH(2) terminus of LD78beta. There was no significant difference in eliciting Ca(2+) flux and chemotaxis among the variants with the exception of LD78beta(T9A) showing a substantially reduced activity. The comparative order for human immunodeficiency virus type 1 (HIV-1) replication inhibition was: LD78beta(P8A) > LD78beta(D6A) > LD78beta(WT), LD78beta(L3A) > LD78beta(T7A), LD78beta(P2A) > LD78beta(T9A). In binding inhibition assays of LD78beta variants using 2D7 monoclonal antibody and (125)I-labeled macrophage inflammatory protein-1alpha, the comparative order was: LD78beta(P8A), LD78beta(D6A) > LD78beta(WT) > LD78beta(L3A) > LD78beta(T7A) > LD78beta(T9A), LD78betaP2A). The order for CCR5 down-regulation induction was comparable to that for binding inhibition. The present data suggest that Pro-2, Asp-6, Pro-8, and Thr-9 are critical for LD78beta binding to CCR5 and HIV-1 replication inhibition, and that LD78beta binding to CCR5, regardless of affinity, is sufficient for the initial signal transduction of LD78beta, whereas the greater anti-HIV-1 activity requires the greater magnitude of binding. The data also suggest that LD78beta variants with appropriate amino acid substitution(s) such as LD78beta(D6A) and LD78beta(P8A) may represent effective chemokine-based anti-HIV-1 therapeutics while preserving LD78beta-CCR5 interactions.     

Polysaccharide composition of the cell wall of baker's yeast with special reference to cell walls obtained from large- and small-sized cells

In order to obtain further information on the mode of cell wallformation during the growth process, the compositions of cellwall polysaccharides were compared in detail using cell wallsamples prepared from large and small cells obtained by fractionationof baker's yeast cells. Gas liquid chromatography of the methanolyzed specimen gavestable, reproducible results, when the sample contained bothglucose and mannose. Much mannan was liberated from the cellwall during its preparation and this must be taken into consideration. Total glucose and mannose accounted for about 40% each of dryweight of cell walls obtained from large and small cells. Glucanswere tentatively divided into alkali-soluble and alkali, acid-insolubleones. Alkaline extraction caused considerable degradation ofpolysaccharides. Nevertheless, a distinct difference existedbetween the glucan contents of the two cell walls. The cellwall sample of large cells contained a higher amount of insolubleglucan, whereas that of small cells contained a higher amountof alkali-soluble glucan. The mode of formation of cell wall polysaccharides during growthwas discussed on the basis of a small-to-large cell process. ¹Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka 558, Japan. (Received April 13, 1976; )     

recE-Dependent Gene Amplification Induced by a Tunicamycin-resistant Mutation (tmr A7) in Bacillus subtilis

A recombinant Bacillus subtilis phage, ρ11-AA248, contains the tmr A7-amy R2-amy E⁺-tmr B⁺-aroI⁺ region of the B. subtilis N7 chromosome on a 22.4 kb DNA fragment. The amy E⁺-tmr B⁺ gene region in the phage genome of the B. subtilis 207-21 transductants by ρ11-AA248 was amplified to approximately 10 copies after cultivation in the presence of tunicamycin (10 μg/ml) and to two copies without tunicamycin. The amplification of the gene region caused hyper-production of extracellular α-amylase. In contrast, no amplification of the gene region was detected in the transductants of B. subtilis 207-25, a recE-deficient derivative of 207-21 strain.     

CD4 mimics as HIV entry inhibitors: Lead optimization studies of the aromatic substituents

Several CD4 mimics have been reported as HIV-1 entry inhibitors that can intervene in the interaction between a viral envelope glycoprotein gp120 and a cell surface protein CD4. Our previous SAR studies led to a finding of a highly potent analogue 3 with bulky hydrophobic groups on a piperidine moiety. In the present study, the aromatic ring of 3 was modified systematically in an attempt to improve its antiviral activity and CD4 mimicry which induces the conformational changes in gp120 that can render the envelope more sensitive to neutralizing antibodies. Biological assays of the synthetic compounds revealed that the introduction of a fluorine group as a meta-substituent of the aromatic ring caused an increase of anti-HIV activity and an enhancement of a CD4 mimicry, and led to a novel compound 13a that showed twice as potent anti-HIV activity compared to 3 and a substantial increase in a CD4 mimicry even at lower concentrations.     

Follicular dendritic cell of the knock-in mouse provides a new bioassay for human prions

Infectious prion diseases initiate infection within lymphoid organs where prion infectivity accumulates during the early stages of peripheral infection. In a mouse-adapted prion infection, an abnormal isoform (PrP(Sc)) of prion protein (PrP) accumulates in follicular dendritic cells within lymphoid organs. Human prions, however, did not cause an accumulation of PrP(Sc) in the wild type mice. Here, we report that knock-in mouse expressing humanized chimeric PrP demonstrated PrP(Sc) accumulations in follicular dendritic cells following human prion infections, including variant Creutzfeldt-Jakob disease. The accumulated PrP(Sc) consisted of recombinant PrP, but not of the inoculated human PrP. These accumulations were detectable in the spleens of all mice examined 30 days post-inoculation. Infectivity of the spleen was also evident. Conversion of humanized PrP in the spleen provides a rapid and sensitive bioassay method to uncover the infectivity of human prions. This model should facilitate the prevention of infectious prion diseases.     

Enhanced amyloidogenic metabolism of the amyloid beta-protein precursor in the X11L-deficient mouse brain

X11L, a neuronal adaptor protein, associates with the cytoplasmic domain of APP and suppresses APP cellular metabolism. APP is the precursor of Abeta, whose metabolism is strongly implicated in Alzheimer disease pathogenesis. To examine the roles of X11L function in APP metabolism, including the generation of Abeta in the brain, we produced X11L-deficient mutant mice on the C57BL/6 background. The mutant mice did not exhibit histopathological alterations or compensatory changes in the expression of other X11 family proteins, X11 and X11L2. The expression level and distribution of APP in the brain of mutant mice were also identical to those in wild-type mice. However, in the hippocampus, where substantial levels of X11L and APP are expressed, the mutant mice exhibited a significant increase in the level of the C-terminal fragments of APP produced by cleavage with beta-secretase but not alpha-secretase. The levels of Abeta were increased in the hippocampus of aged mutant mice as compared with age-matched controls. These observations clearly indicate that X11L suppresses the amyloidogenic but not amyloidolytic processing of APP in regions of the brain such as the hippocampus, which express significant levels of X11L.     

Regional variation in mitochondrial DNA copy number in mouse brain

Mitochondria have their own DNA (mitochondrial DNA [mtDNA]). Although mtDNA copy number is dependent on tissues and its decrease is associated with various neuromuscular diseases, detailed distribution of mtDNA copies in the brain remains uncertain. Using real-time quantitative PCR assay, we examined regional variation in mtDNA copy number in 39 brain regions of male mice. A significant regional difference in mtDNA copy number was observed (P<4.8×10(-35)). High levels of mtDNA copies were found in the ventral tegmental area and substantia nigra, two major nuclei containing dopaminergic neurons. In contrast, cerebellar vermis and lobes had significantly lower copy numbers than other regions. Hippocampal dentate gyrus also had a relatively low mtDNA copy number. This study is the first quantitative analysis of regional variation in mtDNA copy number in mouse brain. Our findings are important for the physiological and pathophysiological studies of mtDNA in the brain.     

Screening,Isolation, and Some Properties of Microbial Cell Flocculants

Nineteen out of 214 strains of microorganisms including moulds, bacteria, actinomycetes and yeasts, which were selected from the culture collection of our laboratory were found to produce the substances which flocculate Saccharomyces cerevisiae AJ4005 (baker’s yeast). Among them are Aspergillus sojae, Anixiella reticulata, Geotrichum candidum, Eupenicillium crustaceus, Circinella sydowi, Monascus anka, Sordaria fimicola, Pseudomonas fluorescens, Staphylococcus aureus, Corynebacterium brevicale, Brevibacterium insectiphilum, Streptomyces vinaceus.

The flocculants in the culture broth of these microorganisms precipitated readily by addition of acetone, and flocculated various microorganisms nonspecifically. The flocculant produced by Asp. sojae AJ7002 sedimented activated sludge well.     

A molecular level study of selective cation capture by a host–guest mechanism for 25,26,27,28-tetramethoxycalix[4]arene in MClO4 solution (M = Na,K)

25,26,27,28-Tetramethoxycalix[4] arene selectively captures cations by changing the conformation. In this study, a hybrid approach of ab initio molecular orbital calculation and statistical mechanics for molecular liquid was utilised to understand the capture mechanism in electrolyte solution phase at molecular level. The association free energy and solvation structure were evaluated on the basis of statistical mechanics for molecular liquids. The selectivity is correctly reproduced by the computation, namely cone conformer captures Na⁺ while K⁺ is recognised by the partial-cone conformer.