Ribosomal DNA Intergenic Spacer of the Swimming Crab, Charybdis japonica

We have determined the full sequence of the ribosomal DNA intergenic spacer (IGS) of the swimming crab, Charybdis japonica, by long PCR for the first time in crustacean decapods. The IGS is 5376 bp long and contains two nonrepetitive regions separated by one long repetitive region, which is composed mainly of four subrepeats (subrepeats I, II, III, and IV). Subrepeat I contains nine copies of a 60-bp repeat unit, in which two similar repeat types (60 bp-a and 60 bp-b) occur alternatively. Subrepeat II consists of nine successive repeat units with a consensus sequence length of 142 bp. Subrepeat III consists of seven copies of another 60-bp repeat unit (60 bp-c) whose sequence is complementary to that of subrepeat I. Immediately downstream of subrepeat III is subrepeat IV, consisting of three copies of a 391-bp repeat unit. Based on comparative analysis among the subrepeats and repeat units, a possible evolutionary process responsible for the formation of the repetitive region is inferred, which involves the duplication of a 60-bp subrepeat unit (60 bp-c) as a prototype. Received: 13 April 1999 / Accepted: 2 August 1999     

Ala99ser mutation in RIα Regulatory Subunit of protein kinase A causes reduced kinase activation by cAMP and arrest of hormone-dependent breast cancer cell growth

Expression of the RI regulatory subunit of protein kinase A type I is increased in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. Ala99 (the pseudophosphorylation site) of human RI was replaced with Ser (RI-p) for the structure-function analysis of RI. MCF-7 hormone- dependent breast cancer cells were transfected with an expression vector for the wild-type RI or mutant RI-p. Overexpression of RI-P resulted in suppression of protein kinase A type II, the isozyme of type I kinase, production of kinase exhibiting reduced cAMP activation, and inhibition of cell growth showing an increase in G0/G1 phase of the cell cycle and apoptosis. The wild-type RI overexpression had no effect on protein kinase A isozyme distribution or cell growth. Overexpression of protein kinase A type II regulatory subunit, RII, suppressed RI and protein kinase A type I and inhibited cell growth. These results show that the growth of hormone-dependent breast cancer cells is dependent on the functional protein kinase A type I.     

Cloning of the Human Monocarboxylate Transporter MCT3 Gene: Localization to Chromosome 22q12.3–q13.2

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon–intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3–q13.2.     

Role of nuclear chromogranin B in inositol 1,4,5-trisphosphate-mediated nuclear Ca2+ mobilization

Recently, secretory granule Ca(2+) storage protein chromogranin B (CGB) was shown to be present in the nucleoplasm proper in a complex structure that consists of the inositol 1,4,5-trisphosphate receptor (IP(3)R)/Ca(2+) channels and the phospholipids. Further, the amounts of IP(3)Rs present in the nucleus of bovine chromaffin cells were shown to be comparable to that of the endoplasmic reticulum. Therefore, we investigated here the potential contribution of nuclear CGB on the IP(3)-dependent Ca(2+) mobilization in the nucleus, using both neuroendocrine PC12 and nonneuroendocrine NIH3T3 cells. Chromogranin A (CGA) expression in the NIH3T3 cells, which do not contain intrinsic chromogranins, increased the IP(3)-induced Ca(2+) releases in the nucleus by 45%, while CGB expression in the same cells increased the IP(3)-induced Ca(2+) releases in the nucleus by 80%. Microinjection of IP(3) into the nucleus of CGB-expressing NIH3T3 cells increased the IP(3)-dependent nuclear Ca(2+) mobilization approximately 3-fold, whereas in CGA-expressing cells it remained the same as that of control cells. In contrast, inhibition of CGA expression in PC12 cells by siRNA treatment decreased the IP(3)-induced Ca(2+) releases in the nucleus by 17%, while inhibition of CGB expression decreased the IP(3)-induced Ca(2+) releases in the nucleus by 55%. Microinjection of IP(3) into the nucleus of siCGB-treated PC12 cells decreased the IP(3)-dependent nuclear Ca(2+) mobilization by approximately 75%, whereas in siCGA-treated cells it remained the same as that of control cells. Given the presence of CGB in the nucleus, these results further highlight the critical contribution of nuclear CGB in the IP(3)-induced Ca(2+) release in the nucleus.     

Deinococcus swuensis sp. nov., a gamma-radiation-resistant bacterium isolated from soil

Strain DY59^T, a Gram-positive non-motile bacterium, was isolated from soil in South Korea, and was characterized to determine its taxonomic position. Phylogenetic analysis based on the 16S rRNA gene sequence of strain DY59^T revealed that the strain DY59^T belonged to the family Deinococcaceae in the class Deinococci. The highest degree of sequence similarities of strain DY59^T were found with Deinococcus radiopugnans KACC 11999^T (99.0%), Deinococcus marmoris KACC 12218^T (97.9%), Deinococcus saxicola KACC 12240^T (97.0%), Deinococcus aerolatus KACC 12745^T (96.2%), and Deinococcus frigens KACC 12220^T (96.1%). Chemotaxonomic data revealed that the predominant fatty acids were iso-C_15:0 (19.0%), C_16:1 ω7c (17.7%), C_15:1 ω6c (12.6%), iso-C_17:0 (10.3%), and iso-C_17:1 ω9c (10.3%). A complex polar lipid profile consisted of a major unknown phosphoglycolipid. The predominant respiratory quinone is MK-8. The cell wall peptidoglycan contained D-alanine, L-glutamic acid, glycine, and L-ornithine (di-amino acid). The novel strain showed resistance to gamma radiation, with a D₁₀ value (i.e. the dose required to reduce the bacterial population by 10-fold) in excess of 5 kGy. Based on the phylogenetic, chemotaxonomic, and phenotypic data, strain DY59^T (=KCTC 33033^T =JCM 18581^T) should be classified as a type strain of a novel species, for which the name Deinococcus swuensis sp. nov. is proposed.     

Zwitterionic chitosan derivative, a new biocompatible pharmaceutical excipient, prevents endotoxin-mediated cytokine release

Chitosan is a cationic polymer of natural origin and has been widely explored as a pharmaceutical excipient for a broad range of biomedical applications. While generally considered safe and biocompatible, chitosan has the ability to induce inflammatory reactions, which varies with the physical and chemical properties. We hypothesized that the previously reported zwitterionic chitosan (ZWC) derivative had relatively low pro-inflammatory potential because of the aqueous solubility and reduced amine content. To test this, we compared various chitosans with different aqueous solubilities or primary amine contents with respect to the intraperitoneal (IP) biocompatibility and the propensity to induce pro-inflammatory cytokine production from macrophages. ZWC was relatively well tolerated in ICR mice after IP administration and had no pro-inflammatory effect on naïve macrophages. Comparison with other chitosans indicates that these properties are mainly due to the aqueous solubility at neutral pH and relatively low molecular weight of ZWC. Interestingly, ZWC had a unique ability to suppress cytokine/chemokine production in macrophages challenged with lipopolysaccharide (LPS). This effect is likely due to the strong affinity of ZWC to LPS, which inactivates the pro-inflammatory function of LPS, and appears to be related to the reduced amine content. Our finding warrants further investigation of ZWC as a functional biomaterial.     

Cigarette Smoking Increases Abdominal and Visceral Obesity but Not Overall Fatness: An Observational Study

Background

Cigarette smoking and obesity are leading public health concerns. Both increase the risk for cardiovascular disease, cancer, and metabolic abnormalities. This study was conducted to assess the association between cigarette smoking and different types of obesity.

Methodology/Principal Findings

Two hundred eighty-three visitors to university hospitals located in four main provinces of South Korea were participated. All participants were classified as either current/past or never smokers and were divided into quartiles according to the total pack-years. Body mass index, waist circumference, total body fat percentage, and area of visceral and abdominal subcutaneous fat were measured. These results of each groups were compared. Waist circumference, and visceral fat area showed a J- or U-shaped association with total smoking amount during a lifetime. After restricting the analyses to past/current smokers, we found significant dose-dependent associations of smoking pack-years with abdominal and visceral obesity. Overall obesity measured by body mass index and total body fat percentage did not show such associations. Although current smokers clearly showed significant associations, we could not demonstrate these in past smokers, possibly because of the limited sample size.

Conclusions/Significance

Although smokers did not show significant difference in mean body mass index than those who never smoked, they showed more metabolically adverse fat distributions with increasing smoking amounts. This finding suggests that smoking is not beneficial for weight control. Therefore, smoking cessation and avoidance of smoking commencement should be addressed as important public health issues in preventing obesity and related complications.     

Both E6 and E7 oncoproteins of human papillomavirus 16 inhibit IL-18-induced IFN-gamma production in human peripheral blood mononuclear and NK cells.

Cervical carcinoma is the predominant cancer among malignancies in women throughout the world, and human papillomavirus (HPV) 16 is the most common agent linked to human cervical carcinoma. The present study was performed to investigate the mechanisms of immune escape in HPV-induced cervical cancer cells. The presence of HPV oncoproteins E6 and E7 in the extracellular fluids of HPV-containing cervical cancer cell lines SiHa and CaSki was demonstrated by ELISA. The effect of HPV 16 oncoproteins E6 and E7 on the production of IFN-gamma by IL-18 was assessed. E6 and E7 proteins reduced IL-18-induced IFN-gamma production in both primary PBMCs and the NK0 cell line. FACS analysis revealed that the viral oncoproteins reduced the binding of IL-18 to its cellular surface receptors on NK0 cells, whereas there was no effect of oncoproteins on IL-1 binding to its surface IL-1 receptors on D10S, a subclone of the murine Th cell D10.G4.1. In vitro pull-down assays also revealed that the viral oncoproteins and IL-18 bound to IL-18R alpha-chain competitively. These results suggest that the extracellular HPV 16 E6 and E7 proteins may inhibit IL-18-induced IFN-gamma production locally in HPV lesions through inhibition of IL-18 binding to its alpha-chain receptor. Down-modulation of IL-18-induced immune responses by HPV oncoproteins may contribute to viral pathogenesis or carcinogenesis.