Mutants with Altered Ca2+-Channel Properties in PARAMECIUM TETRAURELIA: Isolation, Characterization and Genetic Analysis

Dancers are a group of mutants in Paramecium tetraurelia whose Ca²⁺ current inactivates poorly and are likely to be defective in the structure of their Ca²⁺ channels. These mutants show prolonged backward swimming in response to K⁺ and Ba ²⁺ in the medium and were selected by this property in a galvanotactic trough. The dancer mutants are semidominant, and all isolated mutants belong to one complementation group; they are not allelic to any of the previously isolated behavioral mutants of P. tetraurelia. The phenotypic change from the homozygous parent to heterozygous F₁ generation takes three to five fissions. There is no evidence of a cytoplasmic factor capable of converting the dancer to the wild-type phenotype, as has been demonstrated in the mutants pawn and cnr. We suggest that the dancer locus is a structural gene for the Ca²⁺ channel.     

Production of Methyl Ketones from Secondary Alcohols by Cell Suspensions of C2 to C4n-Alkane-Grown Bacteria

Nineteen new C₂ to C₄n-alkane-grown cultures were isolated from lake water from Warinanco Park, Linden, N.J., and from lake and soil samples from Bayway Refinery, Linden, N.J. Fifteen known liquid alkane-utilizing cultures were also found to be able to grow on C₂ to C₄n-alkanes. Cell suspensions of these C₂ to C₄n-alkane-grown bacteria oxidized 2-alcohols (2-propanol, 2-butanol, 2-pentanol, and 2-hexanol) to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Cells grown on 1-propanol or 2-propanol oxidized both primary and secondary alcohols. In addition, the activity for production of methyl ketones from secondary alcohols was found in cells grown on either alkanes, alcohols, or alkylamines, indicating that the enzyme(s) responsible for this reaction is constitutive. The optimum conditions for in vivo methyl ketone formation from secondary alcohols were compared among selected strains: Brevibacterium sp. strain CRL56, Nocardia paraffinica ATCC 21198, and Pseudomonas fluorescens NRRL B-1244. The rates for the oxidation of secondary alcohols were linear for the first 3 h of incubation. Among secondary alcohols, 2-propanol and 2-butanol were oxidized at the highest rate. A pH around 8.0 to 9.0 was found to be the optimum for acetone or 2-butanone formation from 2-alcohols. The temperature optimum for the production of acetone or 2-butanone from 2-propanol or 2-butanol was rather high at 60°C, indicating that the enzyme involved in the reaction is relatively thermally stable. Metal-chelating agents inhibit the production of methyl ketones, suggesting the involvement of a metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble fraction; this activity requires a cofactor, specifically NAD. Propane monooxygenase activity was also found in the cell-free soluble fraction. It is a nonspecific enzyme catalyzing both terminal and subterminal oxidation of n-alkanes.     

Natural variants of the human papillomavirus type 16 E6 protein differ in their abilities to alter keratinocyte differentiation and to induce p53 degradation.

Three naturally occurring variant human papillomavirus type 16 (HPV-16) E6 proteins, which contained amino acid substitutions predominantly near the N terminus, exhibited significant differences in their abilities to abrogate keratinocyte differentiation in response to serum and calcium and to induce the degradation of p53 in vitro. One variant surpassed the reference E6 protein in its ability to abrogate keratinocyte differentiation responses, whereas another showed a reduction in this activity. Interestingly, the biological activities of the HPV-16 E6 proteins and their abilities to induce p53 degradation in vitro were directly correlated. These results demonstrate that naturally occurring variants of HPV-16 differ in biological and biochemical properties which might result in differences in pathogenicity.     

Canine erythrocyte pyruvate kinase. II. Properties of the abnormal enzyme associated with hemolytic anemia in the basenji dog

We have compared the solubility, kinetic, immunological, and electrophoretic properties of erythrocyte pyruvate kinase from normal dogs and Basenji dogs with congenital hemolytic anemia due to pyruvate kinase deficiency. Differences can be detected between the two enzymes by all methods. The enzyme from the affected animals has a greater solubility in ammonium sulfate. It has a lower K _m for phosphoenolpyruvate, while the K _m for ADP is increased. This enzyme is not inhibited by ATP or activated by fructose 1,6-diphosphate. The enzyme from the affected animals has none of the allosteric properties characteristic of the normal canine enzyme. No difference can be detected by enzyme inactivation with rabbit antiserum against the human erythrocyte enzyme, but a slight spur is observed on comparison of the two enzymes by Ouchterlony immunodiffusion. The enzymes also differ in their electrophoretic mobilities on starch gel electrophoresis.     

Characterization of humoral responses in mice immunized with plasmid DNAs encoding SARS-CoV spike gene fragments

The immunological characteristics of SARS-CoV spike protein were investigated by administering mice with plasmids encoding various S gene fragments. We showed that the secreting forms of S1, S2 subunits and the N-terminus of S1 subunit (residues 18-495) were capable of eliciting SARS-CoV specific antibodies and the region immediate to N-terminus of matured S1 protein contained an important immunogenic determinant for elicitation of SARS-CoV specific antibodies. In addition, mice immunized with plasmids encoding S1 fragment developed a Th1-mediated antibody isotype switching. Another interesting finding was that mouse antibodies elicited separately by plasmids encoding S1 and S2 subunits cooperatively neutralized SARS-CoV but neither the S1 nor S2 specific antibodies did, suggesting the possible role of both S1 and S2 subunits in host cell docking and entry. These results provide insights into understanding the immunological characteristics of spike protein and the development of subunit vaccines against SARS-CoV.     

SNP frequency, haplotype structure and linkage disequilibrium in elite maize inbred lines

Background  

Recent studies of ancestral maize populations indicate that linkage disequilibrium tends to dissipate rapidly, sometimes within 100 bp. We set out to examine the linkage disequilibrium and diversity in maize elite inbred lines, which have been subject to population bottlenecks and intense selection by breeders. Such population events are expected to increase the amount of linkage disequilibrium, but reduce diversity. The results of this study will inform the design of genetic association studies.     

Resistin induces monocyte-endothelial cell adhesion by increasing ICAM-1 and VCAM-1 expression in endothelial cells via p38MAPK-dependent pathway

Resistin, firstly reported as an adipocyte-specific hormone, is suggested to be an important link between obesity and diabetes. Recent studies have suggested an association between resistin and atherogenic processes. The adhesion of circulating monocytes to endothelial cells is a critical step in the early stages of atherosclerosis. The purpose of the present study was to investigate the effect of resistin on the adhesion of THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. Our results showed that resistin caused a significant increase in monocyte adhesion. In exploring the underlying mechanisms of resistin action, we found that resistin-induced monocyte adhesion was blocked by inhibition of p38MAPK activation using SB203580 and SB202190. Furthermore, resistin increased the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by HUVECs and these effects were also p38MAPK-dependent. Resistin-induced monocyte adhesion was also blocked by monoclonal antibodies against ICAM-1 and VCAM-1. Taken together, these results show that resistin increases both the expression of ICAM-1 and VCAM-1 by endothelial cells and monocyte adhesion to HUVECs via p38MAPK-dependent pathways.     

A PHO80-like cyclin and a B-type cyclin control the cell cycle of the procyclic form of Trypanosoma brucei

Cyclins bind and activate cyclin-dependent kinases that regulate cell cycle progression in eukaryotes. Cell cycle control in Trypanosoma brucei was analyzed in the present study. Genes encoding four PHO80 cyclin homologues and three B-type cyclin homologues but no G1 cyclin homologues were identified in this organism. Through knocking down expression of the seven cyclin genes with the RNA interference technique in the procyclic form of T. brucei, we demonstrated that one PHO80 homologue (CycE1/CYC2) and a B-type cyclin homologue (CycB2) are the essential cyclins regulating G1/S and G2/M transitions, respectively. This lack of overlapping cyclin function differs significantly from that observed in the other eukaryotes. Also, PHO80 cyclin is known for its involvement only in phosphate signaling in yeast with no known function in cell cycle control. Both observations thus suggest the presence of simple and novel cell cycle regulators in trypanosomes. T. brucei cells deficient in CycE1/CYC2 displayed a long slender morphology, whereas those lacking CycB2 assumed a fat stumpy form. These cells apparently still can undergo cytokinesis generating small numbers of anucleated daughter cells, each containing a single kinetoplast known as a zoid. Two different types of zoids were identified, the slender zoid derived from reduced CycE1/CYC2 expression and the stumpy zoid from CycB2 deficiency. This observation indicates an uncoupling between the kinetoplast and the nuclear cycle, resulting in cell division driven by kinetoplast segregation with neither a priori S phase nor mitosis in the trypanosome.