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.     

Isolation and structure determination of a new lasso peptide specialicin based on genome mining

Based on genome mining, a new lasso peptide specialicin was isolated from the extract of Streptomyces specialis. The structure of specialicin was established by ESI-MS and NMR analyses to be a lasso peptide with the length of 21 amino acids, containing an isopeptide bond and two disulfide bonds in the molecule. The stereochemistries of the constituent amino acids except for Trp were determined to be L and the stereochemistry of Trp at C-terminus was determined to be D. Three dimensional structure of specialicin was determined based on NOE experimental data, which indicated that specialicin possessed the similar conformational structure with siamycin I. Specialicin showed the antibacterial activity against Micrococcus luteus and the moderate anti-HIV activity against HIV-1 NL4-3. The biosynthetic gene cluster of specialicin was proposed from the genome sequence data of S. specialis.     

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; )     

Conditions for Production of Microbial Cell Flocculant by Aspergillus sojae AJ7002

Cultivation is reported on Aspergillus sojae AJ7002 which synthesized an extracellular bio-flocculant. Growth studies in shaking flasks and fermentors were conducted to obtain higher flocculant production. The highest level of polymer accumulation was attained after 48–72 hr cultivation at 30–34°C. The favorable substrates for polymer formation were casein, yeast extract, polypepton and amino acids, such as glutamic acid and alanine. The addition of saccharides to the medium was found to reduce the pH of the culture broths, and hence inhibit the accumulation of flocculant in the culture broth. The finding that the product was a single substance from the early stage of fermentation suggested that the polymer was not a product of cell autolysis. The components of the polymer which were produced by Asp. sojae did not vary even if the medium composition or culture condition changed. The addition of 2-ketogluconic acid, which is one of the constituents of the polymer increased the flocculating activity of the culture medium.     

Soluble-type small-molecule CD4 mimics as HIV entry inhibitors

Several small molecule CD4 mimics have been reported previously as HIV-1 entry inhibitors, which block the interaction between the Phe43 cavity of HIV-1 gp120 and the host CD4. Known CD4 mimics such as NBD-556 possess significant anti-HIV activity but are less soluble in water, perhaps due to their hydrophobic aromatic ring-containing structures. Compounds with a pyridinyl group in place of the phenyl group in these molecules have been designed and synthesized in an attempt to increase the hydrophilicity. Some of these new CD4 mimics, containing a tetramethylpiperidine ring show significantly higher water solubility than NBD-556 and have high anti-HIV activity and synergistic anti-HIV activity with a neutralizing antibody. The CD4 mimic that has a cyclohexylpiperidine ring and a 6-fluoropyridin-3-yl ring has high anti-HIV activity and no significant cytotoxicity. The present results will be useful in the future design and development of novel soluble-type molecule CD4 mimics.     

Detection and Early Phase Assessment of Radiation-Induced Lung Injury in Mice Using Micro-CT

Radiation therapy is an important therapeutic modality for thoracic malignancies. However, radiation-induced pulmonary injuries such as radiation pneumonitis and fibrosis are major dose-limiting factors. Previous research shows that micro-computed tomography (micro-CT) can detect radiation-induced lung injuries a few months following irradiation, but studies to assess the early response of lung tissue are lacking. The aim of this study was to determine if micro-CT could be used to detect and assess early-phase radiation–induced lung injury in mice. Twenty-one animals were divided into three groups: normal (n = 7), one day after x-ray exposure (n = 7), and at four days after x-ray exposure (n = 7). The x-ray-exposed groups received a single dose of 20 Gy, to the whole lung. Histology showed enlargements of the air space (Lm: mean chord length) following irradiation. 40.5±3.8 µm and 60.0±6.9 µm were observed after one and four days, respectively, compared to 26.5±3.1 µm in normal mice. Three-dimensional micro-CT images were constructed and histograms of radiodensity - Hounsfield Units (HU) - were used to assess changes in mouse lungs. Radiation-induced lung injury was observed in irradiated mice, by the use of two parameters which were defined as shifts in peak HU between −200 to −800 HU (Peak_HU) and increase in the number of pixels at −1000 HU (Number_-1000). These parameters were correlated with histological changes. The results demonstrate that micro-CT can be used for the early detection and assessment of structural and histopathological changes resulting from radiation-induced lung injury in mice. Micro-CT has the advantage, over traditional histological techniques, of allowing longitudinal studies of lung disease progression and assessment of the entire lung, while reducing the number of animals required for such studies.     

Quantitative expression profile of distinct functional regions in the adult mouse brain

The adult mammalian brain is composed of distinct regions with specialized roles including regulation of circadian clocks, feeding, sleep/awake, and seasonal rhythms. To find quantitative differences of expression among such various brain regions, we conducted the BrainStars (B*) project, in which we profiled the genome-wide expression of ～50 small brain regions, including sensory centers, and centers for motion, time, memory, fear, and feeding. To avoid confounds from temporal differences in gene expression, we sampled each region every 4 hours for 24 hours, and pooled the samples for DNA-microarray assays. Therefore, we focused on spatial differences in gene expression. We used informatics to identify candidate genes with expression changes showing high or low expression in specific regions. We also identified candidate genes with stable expression across brain regions that can be used as new internal control genes, and ligand-receptor interactions of neurohormones and neurotransmitters. Through these analyses, we found 8,159 multi-state genes, 2,212 regional marker gene candidates for 44 small brain regions, 915 internal control gene candidates, and 23,864 inferred ligand-receptor interactions. We also found that these sets include well-known genes as well as novel candidate genes that might be related to specific functions in brain regions. We used our findings to develop an integrated database (http://brainstars.org/) for exploring genome-wide expression in the adult mouse brain, and have made this database openly accessible. These new resources will help accelerate the functional analysis of the mammalian brain and the elucidation of its regulatory network systems.     

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.