Differential expression of the human CD8beta splice variants and regulation of the M-2 isoform by ubiquitination

The CD8alphabeta heterodimer functions as a coreceptor with the TCR, influencing the outcome of CD8(+) T cell responses to pathogen-infected and tumor cells. In contrast to the murine CD8B gene, the human gene encodes alternatively spliced variants with different cytoplasmic tails (M-1, M-2, M-3, and M-4). At present, little is known about the expression patterns and functional significance of such variants. We used quantitative RT-PCR to demonstrate differential mRNA expression patterns of these splice variants in thymocytes and in resting, memory, and activated primary human CD8(+) T cells. In total CD8(+) T cells, mRNA levels of the M-1 variant were the most predominant and levels of M-3 were the least detected. The M-4 isoform was predominant in effector memory CD8(+) T cells. Upon stimulation of CD8(+) T cells, the M-2 variant mRNA levels were elevated 10-20-fold relative to resting cells in contrast to the other isoforms. Curiously, the M-2 isoform was not expressed on the cell surface in transfected cell lines. Using fluorescent chimeras of the extracellular domain of mouse CD8beta fused to the cytoplasmic tails of each isoform, the M-2 isoform was localized in a lysosomal compartment regulated by ubiquitination of a lysine residue (K215) in its cytoplasmic tail. In contrast, upon short-term stimulation, the M-2 protein localized to the cell surface with the TCR complex. The relatively recent evolution of CD8B gene splice variants in the chimpanzee/human lineage is most likely important for fine-tuning the CD8(+) T cell responses.     

Effects of High Fat Feeding and Diabetes on Regression of Atherosclerosis Induced by Low-Density Lipoprotein Receptor Gene Therapy in LDL Receptor-Deficient Mice

We tested whether a high fat diet (HFD) containing the inflammatory dietary fatty acid palmitate or insulin deficient diabetes altered the remodeling of atherosclerotic plaques in LDL receptor knockout (Ldlr^-/-) mice. Cholesterol reduction was achieved by using a helper-dependent adenovirus (HDAd) carrying the gene for the low-density lipoprotein receptor (Ldlr; HDAd-LDLR). After injection of the HDAd-LDLR, mice consuming either HFD, which led to insulin resistance but not hyperglycemia, or low fat diet (LFD), showed regression compared to baseline. However there was no difference between the two groups in terms of atherosclerotic lesion size, or CD68+ cell and lipid content. Because of the lack of effects of these two diets, we then tested whether viral-mediated cholesterol reduction would lead to defective regression in mice with greater hyperglycemia. In both normoglycemic and streptozotocin (STZ)-treated hyperglycemic mice, HDAd-LDLR significantly reduced plasma cholesterol levels, decreased atherosclerotic lesion size, reduced macrophage area and lipid content, and increased collagen content of plaque in the aortic sinus. However, reductions in anti-inflammatory and ER stress-related genes were less pronounced in STZ-diabetic mice compared to non-diabetic mice. In conclusion, HDAd-mediated Ldlr gene therapy is an effective and simple method to induce atherosclerosis regression in Ldlr^-/- mice in different metabolic states.     

In love and war: The morphometric and phylogenetic basis of ornamentation,and the evolution of male display behavior,in the livebearer genus Poecilia

Exaggerated male traits under sexual selection are often used for both competition and courtship, raising the question of whether ornaments evolved simultaneously for both functions, or if use in one context preceded use in another. Here, we apply a phylogenetic approach to study the evolution of ornamental dorsal fins in male poeciliid fish of the subgenera Mollienesia and Limia, which exhibit convergent development of an enlarged dorsal fin, and often direct erect‐fin displays to male and female conspecifics. Unlike prior categorical assessments of poeciliid adornments, we measure dorsal fin exaggeration with a continuous index of ornamentation. Phylogenetic logistic and generalized least squares regression analyses indicate that high index values are significantly associated with the use of two component postures of courtship and aggressive displays, dorsal fin erection and body curvature, but not with the presence of sexual dichromatism. Male displays initially evolved for male–male aggression in the common ancestor of Mollienesia and Limia, suggesting that this signal originated for competition, then became co‐opted for courtship. These results support the armament‐ornament hypothesis for evolution of exaggerated male traits, and are consistent with an evolutionary shift in the predominant mechanisms of sexual selection from intra‐ to intersexual.     

A case-control study estimating accident risk for alcohol, medicines and illegal drugs

Background

The aim of the present study was to assess the risk of having a traffic accident after using alcohol, single drugs, or a combination, and to determine the concentrations at which this risk is significantly increased.

Methods

A population-based case-control study was carried out, collecting whole blood samples of both cases and controls, in which a number of drugs were detected. The risk of having an accident when under the influence of drugs was estimated using logistic regression adjusting for gender, age and time period of accident (cases)/sampling (controls). The main outcome measures were odds ratio (OR) for accident risk associated with single and multiple drug use. In total, 337 cases (negative: 176; positive: 161) and 2726 controls (negative: 2425; positive: 301) were included in the study.

Results

Main findings were that 1) alcohol in general (all the concentrations together) caused an elevated crash risk; 2) cannabis in general also caused an increase in accident risk; at a cut-off of 2 ng/mL THC the risk of having an accident was four times the risk associated with the lowest THC concentrations; 3) when ranking the adjusted OR from lowest to highest risk, alcohol alone or in combination with other drugs was related to a very elevated crash risk, with the highest risk for stimulants combined with sedatives.

Conclusion

The study demonstrated a concentration-dependent crash risk for THC positive drivers. Alcohol and alcohol-drug combinations are by far the most prevalent substances in drivers and subsequently pose the largest risk in traffic, both in terms of risk and scope.     

A chimaeric tryptophan decarboxylase gene as a novel selectable marker in plant cells

A cDNA clone, pMA1949, detects two mRNA species in wheat seedling tissue that are late embryogenesis-abundant (LEA) and dehydration stress-inducible. Sequence analysis of the pMA1949 clone shows it to be a 991 bp partial cDNA encoding a polypeptide of 317 amino acids with homology to two group 3 LEA proteins, carrot (DC8) and a soybean protein encoded by pGmPM2 cDNA. Molecular analysis of the deduced protein reveals a 33 kDa acidic and extremely hydrophilic protein with potential amphiphilic -helical regions. In addition, the protein contains eleven similar, contiguous repeats of 11 amino acids, which are separated by 118 amino acids from two additional and unique repeats of 36 residues each at the carboxyl end of the protein. Comparisons of sequences of reported group 3 LEA proteins revealed that there are two types, separable by sequence similarity of the 11 amino acid repeating motifs and by the presence or absence of a certain amino acid stretch at the carboxyl terminus. Based on resuls from these comparisons, we propose a second type of group 3 LEA proteins, called group 3 LEA (II).     

Protein tyrosine phosphatases regulate arachidonic acid release, StAR induction and steroidogenesis acting on a hormone-dependent arachidonic acid-preferring acyl-CoA synthetase

The activation of the rate-limiting step in steroid biosynthesis, that is the transport of cholesterol into the mitochondria, is dependent on PKA-mediated events triggered by hormones like ACTH and LH. Two of such events are the protein tyrosine dephosphorylation mediated by protein tyrosine phosphatases (PTPs) and the release of arachidonic acid (AA) mediated by two enzymes, ACS4 (acyl-CoA synthetase 4) and Acot2 (mitochondrial thioesterase). ACTH and LH regulate the activity of PTPs and Acot2 and promote the induction of ACS4. Here we analyzed the involvement of PTPs on the expression of ACS4. We found that two PTP inhibitors, acting through different mechanisms, are both able to abrogate the hormonal effect on ACS4 induction. PTP inhibitors also reduce the effect of cAMP on steroidogenesis and on the level of StAR protein, which facilitates the access of cholesterol into the mitochondria. Moreover, our results indicate that exogenous AA is able to overcome the inhibition produced by PTP inhibitors on StAR protein level and steroidogenesis. Then, here we describe a link between PTP activity and AA release, since ACS4 induction is under the control of PTP activity, being a key event for AA release, StAR induction and steroidogenesis.     

Effect of hormones on growth rates of malignant and nonmalignant human mammary epithelia in cell culture

Summary The individual effects of seven hormones on the in vitro growth rate of different classifications of human mammary epithelium were compared. Hormones used were: 17β-estradiol, estriol, progesterone, hydrocortisone, testosterone, prolactin, and growth hormone. Cell cultures included three established breast cell lines and primary monolayer cultures established form breast fluids and excised mammary tissue from 40 women and 4 men. Specimens comprised three classifications: normal, nonmalignant atypical, and malignant. Growth was quantitated in situ and expressed as population doubling time. Principal findings were: (a) estrogens, prolactin, and growth hormone stimulated growth of normal cells more frequently than growth of malignant cells, whereas testosterone and hydrocortisone stimulated growth of malignant cells more frequently than growth of normal cells; (b) cells cultured from nonmalignant atypias generally showed hormone response profiles intermediate between those of normal and malignant cells; (c) progesterone stimulated the growth of cells from malignant specimens but not the growth of cells from normal and nonmalignant atypical samples. This research was supported by NIAID Research Training Grant 5-TO1-A1-00332-06.     

Expression of transglutaminase substrate activity on Candida albicans germ tubes through a coiled, disulfide-bonded N-terminal domain of Hwp1 requires C-terminal glycosylphosphatidylinositol modification

By serving as a microbial substrate for epithelial cell transglutaminase, Hwp1 (Hyphal wall protein 1) of Candida albicans participates in cross-links with proteins on the mammalian mucosa. Biophysical properties of the transglutaminase substrate domain were explored using a recombinant protein representative of the N-terminal domain of Hwp1 and were similar to other transglutaminase substrates, the small proline-rich proteins of cornified envelopes found in stratified squamous epithelia. Recombinant Hwp1 lacks alpha and beta structures by circular dichroism and likely exists as a disulfide-cross-linked coiled-coil. The transglutaminase substrate property prompted a unique approach for investigating the features of surface Hwp1 on germ tubes. A lysine analog, 5-(biotinamido)pentylamine, was cross-linked to germ tubes catalyzed by transglutaminase 2 prior to cell fractionation, immunoprecipitation, and detection with streptavidin conjugates. The majority of the transglutaminase-modifiable Hwp1 was covalently attached to the beta-glucan of hyphae by the C terminus of Hwp1 via a glycosylphosphatidylinositol remnant anchor. A putative precursor of cell wall forms of Hwp1 was identified in the cell extract and in the culture medium. Hwp1 was modified by relatively short N-linked glycans, and the molecular size of the protein was reduced by hypomannosylation when expressed in O-glycosylation mutant strains. Hwp1 combines features of mammalian transglutaminase substrate proteins with characteristics of fungal cell wall proteins to form an unconventional adhesin at the hyphal wall of C. albicans.     

Genetic control of the mitochondrial form of superoxide dismutase in hexaploid wheat

Extracts of mature grains of a large number of aneuploid derivatives of Triticum aestivum cv. Chinese Spring and of the members of five wheat-alien chromosome addition series were subjected to isoelectric focusing in polyacrylamide gels in order to study the genetic control of superoxide dismutase (SOD). Evidence was obtained that homologous structural genes for the mitochondrial form of SOD are located in the long arms of the homoeologous group 2 chromosomes of Chinese Spring and in chromosome 2R of Secale cereale cv. Imperial. The SOD gene loci located in chromosomes 2A, 2B, 2D, and 2R were designated Sod-A1, Sod-B1, Sod-D1, and Sod-R1, respectively. Chromosome-arm pairing data indicate that 2DL is not homoeologous to either 2AS or 2BL. The results of this study suggest, however, that 2BL is partially homoeologous to both 2AL and 2DL.Technical article No. 21074 of the Texas Agricultural Experiment Station. This work was supported by USDA Grant 83-CRCR-1-1322 to GEH.