Pre-treatment of Caco-2 cells with zinc during the differentiation phase alters the kinetics of zinc uptake and transport(2)

The Caco-2 cell model was used to study the efficiency of absorption and endogenous excretion of zinc (Zn) regulated by dietary Zn concentration. Cells were seeded onto high pore-density membranes and maintained in medium supplemented with 10% FBS. After confluence, cells were treated with 5 or 25 μmol Zn/L for 7 d, and Zn uptake and transport were measured in both apical (AP) and basolateral (BL) directions by using ⁶⁵Zn. Similar cells were labeled with ⁶⁵Zn and the release of Zn to the AP and BL sides was measured. The AP uptake of Zn in cells exposed to 25 μmol Zn/L was slower (p < 0.05) than that in cells exposed to 5 μmol Zn/L. The AP to BL transport rate in the 25 μmol Zn/L group was only 40% (p < 0.05) of that in the 5 μM group. In contrast, the rate of BL Zn uptake was 4-fold higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). The BL to AP transport rate was 2-fold higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). Basolateral uptake was 6 to 25 times greater (p < 0.05) than AP uptake for cells treated with 5 and 25 μmol Zn/L, respectively. The rate of Zn release was enhanced about 4-fold (p < 0.05) by 25 μmol Zn/L treatment. Release to the BL side was 10 times greater than to the AP side. Zn-induced metallothionein (MT), thought to down-regulate AP to BL Zn transport, was 4-fold higher (p < 0.001) in the 25 μmol Zn/L group than in the 5 μM group, but the rate of BL Zn release was higher in cells treated with 25 μmol Zn/L than in those treated with 5 μmol Zn/L (p < 0.05). Induced changes in transport rates by media Zn concentrations could involve the up- and/or down-regulation of Zn influx and efflux proteins such as the ZIP and ZnT families of Zn transporters.     

Synthesis and biological activity of selective pipecolic acid-based TNF-alpha converting enzyme (TACE) inhibitors

A series of novel, selective TNF-alpha converting enzyme inhibitors based on 4-hydroxy and 5-hydroxy pipecolate hydroxamic acid scaffolds is described. The potency and selectivity of TACE inhibition is dramatically influenced by the nature of the sulfonamide group which interacts with the S1' site of the enzyme. Substituted 4-benzyloxybenzenesulfonamides exhibit excellent TACE potency with >100x selectivity over inhibition of matrix metalloprotease-1 (MMP-1). Alkyl substituents on the ortho position of the benzyl ether moiety give the most potent inhibition of TNF-alpha release in LPS-treated human whole blood.     

The O-antigen gene cluster of Escherichia coli O55:H7 and identification of a new UDP-GlcNAc C4 epimerase gene

Escherichia coli O55 is an important antigen which is often associated with enteropathogenic E. coli clones. We sequenced the genes responsible for its synthesis and identified genes for O-antigen polymerase, O-antigen flippase, four enzymes involved in GDP-colitose synthesis, and three glycosyltransferases, all by comparison with known genes. Upstream of the normal O-antigen region there is a gne gene, which encodes a UDP-GlcNAc epimerase for converting UDP-GlcNAc to UDP-GalNAc and is essential for O55 antigen synthesis. The O55 gne product has only 20 and 26% identity to the gne genes of Pseudomonas aeruginosa and E. coli O113, respectively. We also found evidence for the O55 gene cluster's having evolved from another gene cluster by gain and loss of genes. Only three of the GDP-colitose pathway genes are in the usual location, the other two being separated, although nearby. It is thought that the E. coli O157:H7 clone evolved from the O55:H7 clone in part by transfer of the O157 gene cluster into an O55 lineage. Comparison of genes flanking the O-antigen gene clusters of the O55:H7 and O157:H7 clones revealed one recombination site within the galF gene and located the other between the hisG and amn genes. Genes outside the recombination sites are 99.6 to 100% identical in the two clones, while most genes thought to have transferred with the O157 gene cluster are 95 to 98% identical.     

Influence of simulated microgravity on human skeletal muscle architecture and function

Ten male volunteers underwent a period of prolonged bed rest. Four subjects performed exercise countermeasures 2-3 times per week, while 6 subjects received no countermeasures. After bed rest plantarflexor force declined significantly (P < 0.001) in both exercise (-42%) and control (-55%) groups. The internal architecture of the gastrocnemius medialis (GM) muscle was significantly altered. This was associated with a reduction in fascicle shortening during isometric contraction. Exercise countermeasures partially mitigated the loss of muscle force and function following 90 days of bed rest.     

Selective expansion of fibroblast subpopulations from pulmonary artery adventitia in response to hypoxia

Proliferation of fibroblasts contributes to the adventitial thickening observed during the development of hypoxia-induced pulmonary hypertension. However, whether all or only specific subpopulations of fibroblasts proliferate during this process is unknown. Because lung, skin, and gingiva contain multiple fibroblast subpopulations, we hypothesized that the pulmonary artery (PA) adventitia of neonatal calves is composed of multiple fibroblast subpopulations and that only selective subpopulations expand under chronic hypoxic conditions. Fibroblast subpopulations were isolated from PA adventitia of control calves using limited dilution cloning techniques. These subpopulations exhibited marked differences in morphology, actin expression, and serum-stimulated growth. Only select fibroblast subpopulations demonstrated the ability to proliferate in response to hypoxia. Fibroblast subpopulations were similarly isolated from calves exposed to hypoxia (14 days). With regard to morphology, actin expression, and serum-stimulated growth of subpopulations, there were no obvious differences in fibroblast subpopulations between the hypoxic and the control calves. However, the number of fibroblast subpopulations with about a twofold increase in hypoxia-induced DNA synthesis was significantly greater in the hypoxic calves (26%) compared with control calves (10%). We conclude that the bovine PA adventitia comprises numerous phenotypically and biochemically distinct fibroblast subpopulations and that select subpopulations expand in response to chronic hypoxia.     

Temporal flux in the morphological and molecular diversity of UK barley

Genetic-diversity assessments, using both phenotypic and molecular-marker data, were made on a collection of 134 barley varieties (both winter and spring types), chosen on the basis of their representation on the NIAB "Recommended List" over the period 1925-1995. Genotypic (AFLP and SSR) and phenotypic (UPOV characters) data were analysed to determine short- and long-term temporal trends in diversity over the period. A consistent pattern emerged demonstrating that only a minor proportion of the overall variance appears to be the result of any temporal drift, although there were strong indications of qualitative shifts in diversity, probably related to the changing relative acreage of winter and spring barleys over the study period. Our overall conclusions are that systematic plant breeding does not inevitably lead to a reduction in the genetic diversity of agricultural crops, and that diverse breeding programmes and the variety delivery systems in place in the UK have generally been successful in maintaining sufficient genetic diversity to allow the steady rise in genetic potential that has been a feature of 20th century crop breeding. The concentration of breeding effort into a smaller number of independent programmes is likely to be prejudicial to the maintenance of the genetic diversity of a crop.     

Immunization against luteinizing hormone-releasing hormone fusion proteins does not decrease prostate cancer in the transgenic adenocarcinoma mouse prostate model

This study was undertaken to test the effect of immunization against luteinizing hormone-releasing hormone (LHRH) fusion proteins on the development and progression of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model. Two LHRH fusion proteins, ovalbumin with seven LHRH peptides (OV-LHRH-7), and thioredoxin with seven LHRH peptides (TH-LHRH-7) were used in a cocktail vaccine. Two groups of male TRAMP mice were immunized with the cocktail. Primary immunizations were at either 4 or 8 weeks of age. LHRH immunized mice (n=19) were compared with castrated (n=19) and intact mice (n=18) for testosterone concentration, tumor weight, and lifespan. Immunization against LHRH in the TRAMP mice resulted in significant production of antibodies to LHRH compared with surgically castrated and intact control mice. Testicular weight was significantly reduced in the LHRH immunized groups compared with intact control mice. Serum testosterone was reduced (P<0.05) in the immunized mice compared with intact control mice and was not different from that of castrated mice (P>0.05). Tumor weight was variable and inconsistent throughout all treatment groups. Lifespan was not increased by immunization against LHRH or castration. Intact control mice (lived the longest (227+/-11 days), whereas immunized mice lived 206+/-11 days and castrated mice lived 213+/-13 days. Tumors from immunized TRAMP mice appeared more aggressive than tumors of castrated and intact mice, as demonstrated by 35% expression of gross lung tumors in the immunized mice whereas none were observed in the castrated or intact TRAMP mice. Prostate cancer is initially dependent upon androgens for growth and development, but cells have the ability to escape androgen dependence and progress to an androgen independent state, which was evident in this study. The TRAMP mouse model immunized against LHRH may have utility in future studies and treatments of the androgen independent prostate cancer.     

Synthesis and biological activity of piperazine-based dual MMP-13 and TNF-alpha converting enzyme inhibitors

A series of novel MMP-13 and TNF-alpha converting enzyme inhibitors based on piperazine 2-hydroxamic acid scaffolds are described. The TACE, MMP-1 and MMP-13 activity of these inhibitors as well as the effect of substitution of the piperazine nitrogen and the P-1' benzyloxy tailpiece is discussed. Moderate in vivo activity is observed with several members of this group.