Different NK cell surface phenotypes defined by the DX9 antibody are due to KIR3DL1 gene polymorphism

KIR3DL1 and KIR3DL2 are NK cell receptors for polymorphic HLA-B and -A determinants. The proportion of NK cells that bind anti-KIR3DL1-specific Ab DX9 and their level of binding vary between individuals. To determine whether these differences are due to KIR polymorphism, we assessed KIR3D gene diversity in unrelated individuals and families. Both KIR3DL1 and KIR3DL2 are highly polymorphic genes, with KIR3DS1 segregating like an allele of KIR3DL1. A KIR haplotype lacking KIR3DL1 and KIR3DS1 was defined. The two KIR3DL1 alleles of a heterozygous donor were expressed by different, but overlapping, subsets of NK cell clones. Sequence variation in KIR3DL1 and KIR3DL2 appear distinct; recombination is more evident in KIR3DL1, and point mutation is more evident in KIR3DL2. The KIR3DL1 genotype correlates well with levels of DX9 binding by NK cells, but not with the frequency of DX9-binding cells. Different KIR3DL1 alleles determine high, low, and no binding of DX9 Ab. Consequently, heterozygotes for high and low binding KIR3DL1 alleles have distinct subpopulations of NK cells that bind DX9 at high and low levels, giving characteristic bimodal distributions in flow cytometry. The Z27 Ab gave binding patterns similar to those of DX9. Four KIR3DL1 alleles producing high DX9 binding phenotypes were distinguished from four alleles producing low or no binding phenotypes by substitution at one or more of four positions in the encoded protein: 182 and 283 in the extracellular Ig-like domains, 320 in the transmembrane region, and 373 in the cytoplasmic tail.     

Synthesis and inotropic activity of hydroindene derivatives

A synthetic approach to hydroindenic inotropic agents has been developed, starting from enantiopure Hajos-Parrish (1). Hajos-Wiechert (2), and related diketones. Their transformation into C-1 formyl derivatives and other subsequent synthetic targets is described. The results of the thermodynamic equilibration between both epimers of each formyl derivative are analysed. The inotropic activities of selected compounds on right and left atrial preparations are also evaluated and discussed.     

pPAC-ResQ: A yeast-bacterial shuttle vector for capturing inserts from P1 and PAC clones by recombinogenic targeted cloning

We have developed a method to capture inserts from P1 and P1 artificial chromosome (PAC) clones into a yeast-bacteria shuttle vector by using recombinogenic targeting. We have engineered a vector, pPAC-ResQ, a derivative of pClasper, which was previously used to capture inserts from yeast artificial chromosome clones. pPAC-ResQ contains DNA fragments flanking the inserts in P1 and PAC vectors as recombinogenic ends. When linearized pPAC-ResQ vector and P1 or PAC DNA are cotransformed into yeast, recombination between the two leads to the transfer of inserts into pPAC-ResQ. pPAC-ResQ clones thus obtained can be further modified in yeast for functional analysis and shuttled to Escherichia coli to produce large quantities of cloned DNA. This approach provides a rapid method to modify P1/PAC clones for functional analysis.     

Inhibition of hepatoma cell growth in vitro by arylating and non-arylating K vitamin analogs. Significance of protein tyrosine phosphatase inhibition

We recently found that a thioether analog of K vitamin (Cpd 5) inhibited the activity of protein-tyrosine phosphatases (PTPases) and induced protein-tyrosine phosphorylation in a human hepatoma cell line (Hep3B). We have now examined the structural requirements for induction of protein-tyrosine phosphorylation and PTPase inhibition by several K vitamin analogs. Thioether analogs with sulfhydryl arylation capacity, especially those with a hydroxy (Cpd 5) or a methoxy group at the end of the side chain, induced protein-tyrosine phosphorylation, but non-arylating analogs, such as those with an all-carbon or O-ether side chain, did not. Among the receptor-tyrosine kinases, epidermal growth factor receptors were tyrosine-phosphorylated by treatment with thioether analogs, whereas insulin and hepatocyte growth factor receptors were not. An increase in tyrosine-phosphorylated ERK2 mitogen-activated protein kinase was also observed. The activity of purified T cell PTPase was inhibited only by the thioether analogs, but not by non-arylating analogs. Furthermore, the epidermal growth factor receptor dephosphorylation activity of Hep3B cell lysates was inhibited by Cpd 5 treatment. A similar induction of protein-tyrosine phosphorylation by Cpd 5 was seen in other human hepatoma cell lines together with growth inhibition. However, one cell line (HepG2), which was relatively resistant to growth inhibition by Cpd 5, did not increase its phosphorylation levels upon Cpd 5 treatment. These results suggest that cell growth inhibition by thioether analogs is closely associated with inhibition of PTPases by sulfhydryl arylation and with tyrosine phosphorylation of selected proteins.     

Multiple-antibiotic resistance of Enterococcus spp. isolated from commercial poultry production environments

The potential impact of food animals in the production environment on the bacterial population as a result of antimicrobial drug use for growth enhancement continues to be a cause for concern. Enterococci from 82 farms within a poultry production region on the eastern seaboard were isolated to establish a baseline of susceptibility profiles for a number of antimicrobials used in production as well as clinical environments. Of the 541 isolates recovered, Enterococcus faecalis (53%) and E. faecium (31%) were the predominant species, while multiresistant antimicrobial phenotypes were observed among all species. The prevalence of resistance among isolates of E. faecalis was comparatively higher among lincosamide, macrolide, and tetracycline antimicrobials, while isolates of E. faecium were observed to be more frequently resistant to fluoroquinolones and penicillins. Notably, 63% of the E. faecium isolates were resistant to the streptogramin quinupristin-dalfopristin, while high-level gentamicin resistance was observed only among the E. faecalis population, of which 7% of the isolates were resistant. The primary observations are that enterococci can be frequently isolated from the poultry production environment and can be multiresistant to antimicrobials used in human medicine. The high frequency with which resistant enterococci are isolated from this environment suggests that these organisms might be useful as sentinels to monitor the development of resistance resulting from the usage of antimicrobial agents in animal production.     

The effect of flight, fasting and p,p'-DDT on thyroid hormones and corticosterone in Gambel's white-crowned sparrow, Zonotrichia leucophrys gambelli

This study examined the effects of p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT), fasting and flight on thyroid hormones and corticosterone in Gambel's White-crowned Sparrows (Zonotrichia leucophrys gambelli). Female sparrows were dosed daily with either 5 mg p,p'-DDT per kg body mass or corn oil vehicle over 3 days. On the fifth day the sparrows were divided into 3 groups: (1) unstressed - non-stressed control sparrows; (2) fasted - sparrows fasted for intervals ranging from 20 min to 9 h; or (3) flown - sparrows flown in a wind tunnel for intervals between 20 min and 2.5 h while fasting. Half the sparrows from each group received DDT (DDT-dosed sparrows) and the other half corn oil vehicle only (vehicle sparrows). Trunk blood plasma was analyzed for thyroxine, triiodothyronine and corticosterone using radioimmunoassay. In the flown group, corticosterone was elevated (DDT-dosed 35.52 ng/ml, P < or = 0.05), and thyroxine was depressed (DDT-dosed 4.09 ng/ml, P < or = 0.05; vehicle 4.33 ng/ml, P < or = 0.05). Elevated corticosterone likely decreased thyroid hormone production through a negative feedback mechanism originating at the hypothalamus. Mean triiodothyronine concentrations did not differ among any of the test groups. Relative to time fasted and flown, thyroxine decreased in flown birds dosed with DDT (P < 0.001) and triiodothyronine decreased in fasted birds dosed with DDT (P = 0.004). The increased rate of hormone diminution may be a result of the ability of DDT to induce microsomal enzyme production.