Genomic and Phylogenetic Analysis of the Human CD1 and HLA Class I Multicopy Genes

The human CD1 proteins belong to a lipid-glycolipid antigen-presenting gene family and are related in structure and function to the MHC class I molecules. Previous mapping and DNA hybridization studies have shown that five linked genes located within a cluster on human chromosome 1q22-23 encode the CD1 protein family. We have analyzed the complete genomic sequence of the human CD1 gene cluster and found that the five active genes are distributed over 175,600 nucleotides and separated by four expanded intervening genomic regions (IGRs) ranging in length between 20 and 68 kb. The IGRs are composed mostly of retroelements including five full-length L1 PA sequences and various pseudogenes. Some L1 sequences have acted as receptors for other subtypes or families of retroelements. Alu molecular clocks that have evolved during primate history are found distributed within the HLA class I duplicated segments (duplicons) but not within the duplicons of CD1. Phylogeny of the alpha3 domain of the class I-like superfamily of proteins shows that the CD1 cluster is well separated from HLA class I by a number of superfamily members including MIC (PERB11), HFE, Zn-alpha2-GP, FcRn, and MR1. Phylogenetically, the human CD1 sequences are interspersed by CD1 sequences from other mammalian species, whereas the human HLA class I sequences cluster together and are separated from the other mammalian sequences. Genomic and phylogenetic analyses support the view that the human CD1 gene copies were duplicated prior to the evolution of primates and the bulk of the HLA class I genes found in humans. In contrast to the HLA class I genomic structure, the human CD1 duplicons are smaller in size, they lack Alu clocks, and they are interrupted by IGRs at least 4 to 14 times longer than the CD1 genes themselves. The IGRs seem to have been created as "buffer zones" to protect the CD1 genes from disruption by transposable elements.     

Immunohistochemical properties of nerve fibres supplying accessory male genital glands in the pig. A colocalisation study

 Immunohistochemical studies have been performed to investigate the occurrence and coexistence of two catecholamine-synthesising enzymes, tyrosine hydroxylase and dopamine-β-hydroxylase, and several neuropeptides, including neuropeptide Y, vasoactive intestinal polypeptide, Leu⁵-enkephalin, somatostatin, calcitonin gene-related peptide and substance P, in nerve fibres supplying porcine accessory genital glands, the seminal vesicles, prostate (body and the disseminated part) and bulbourethral glands. Three major populations of nerve fibres supplying non-vascular elements of the glands have been distinguished (from the largest to the smallest one): (1) noradrenergic fibres, the majority of which contain Leu⁵-enkephalin, neuropeptide Y or, to a lesser extent, somatostatin, (2) non-noradrenergic, putative cholinergic fibres containing vasoactive intestinal polypeptide, neuropeptide Y and/or somatostatin and, (3) non-noradrenergic, presumably sensory fibres, containing calcitonin gene-related peptide and substance P. Whilst the coexistence patterns within nerves supplying particular glands are similar, the density of innervation varies between the organs. The innervation of the seminal vesicles and prostatic body is more developed than that of the disseminated part of the prostate and bulbourethral glands. The majority of noradrenergic fibres related to blood vessels contain neuropeptide Y only, while the non-noradrenergic nerves contain mainly vasoactive intestinal polypeptide. The possible function and origin of particular nerve fibre populations are discussed. Accepted: 16 November 1998     

Dark reoxidation of the plastoquinone-pool is mediated by the low-potential form of cytochrome b-559 in spinach thylakoids

We have found that in isolated spinach thylakoids, plastoquinone-pool (PQ-pool), after its photoreduction, undergoes dark-reoxidation with the half-time of _1/2 = 43 ± 3 s. To explain the observed rates of PQ-pool reoxidation, a nonenzymatic plastoquinol (PQH₂) autoxidation under molecular oxygen and an enzymatic oxidation by the low-potential form of cytochrome b-559 (cyt. b-559LP), as the postulated PQ-oxidase in chlororespiration, were investigated. It was found that the autoxidation rate of PQH₂ in organic solvents and liposomes was too low to account for the observed oxidation rate of PQH₂ in thylakoids. The rate of cyt. b-559LP autoxidation in isolated Photosystem II was found to be similar (_1/2 = 26 ± 5 s) to that of the PQ-pool. This suggests that the LP form of cyt. b-559 is probably responsible for the PQ-oxidase activity observed during chlororespiration.     

Complexity of phenotypes and symbiotic behaviour of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> exopolysaccharide mutants

Quantitative in vitro antibacterial activities, i.e., minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs), of 12 -lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 were examined, in order to identify antibiotics effective in eliminating the bacteria in Agrobacterium-mediated plant genetic transformation. The antibacterial activities of -lactams tested against strain EHA101 were equal to or less than those tested against strain LBA4404. Cefotaxime, cefbuperazone, and meropenem had high activities against strain LBA4404 (MBC <1 mg l^–1). Against strain EHA101, however, only meropenem showed activity comparable to that against strain LBA4404. The production of -lactamase was observed only in strain EHA101.Abbreviations CFU Colony-forming unit - MBC Minimum bactericidal concentration - MIC Minimum inhibitory concentration - PBP Penicillin-binding protein     

Lipoxygenase interactions with natural flavonoid, quercetin, reveal a complex with protocatechuic acid in its X-ray structure at 2.1 A resolution

PUFA metabolites have a profound effect on inflammatory diseases and cancer progression. Blocking their production by inhibiting PUFA metabolizing enzymes (dioxygenases: cyclooxygenases and LOXs) might be a successful way to control and relieve such problems, if we learn to better understand their actions at a molecular level. Compounds with strong antioxidative and free radical scavenging properties, such as polyphenols, could be effective in blocking PUFA activities, and natural flavonoids possess such qualities. Quercetin belongs to the group of natural catecholic compounds and is known as a potent, competitive inhibitor of LOX. Structural analysis reveals that quercetin entrapped within LOX undergoes degradation, and the resulting compound has been identified by X-ray analysis as protocatechuic acid (3,4-dihydroxybenzoic acid) positioned near the iron site. Its C3-OH group points toward His523, C4-OH forms a hydrogen bond with O=C from the enzyme's C-terminus, and the carboxylic group is incorporated into the hydrogen bonding network of the active-site neighborhood via Gln514. This unexpected result, together with our previous observations concerning other polyphenols, yields new evidence about the metabolism of natural flavonoids. These compounds might be vulnerable to the co-oxidase activity of LOX, leading to enzyme-stimulated oxidative degradation, which results in an inhibitor of a lower molecular weight.     

Down-regulation by nuclear factor kappaB of human 25-hydroxyvitamin D3 1alpha-hydroxylase promoter

1,25-(OH)(2) vitamin D(3) is important for calcium homeostasis and cell differentiation. The key enzyme for the activation of liver-derived 25(OH) vitamin D(3) is 25-hydroxyvitamin D(3) 1alpha-hydroxylase. It is expressed mainly in the kidney but also in peripheral tissues. A 1413-bp fragment of the 1alpha-hydroxylase promoter was cloned into luciferase vectors pGL2basic and pGL3basic. Sequence analyses revealed four base exchanges and three base deletions compared with the published sequence which were identically found in five control persons. In silico promoter analyses revealed 17 putative nuclear factor (NF)kappaB sites, 10 of which were found to bind NFkappaB in EMSA experiments. Cotransfection of NFkappaB p50 and p65 subunits resulted in dramatic reduction of the promoter activity of the full-length construct as well as a series of 5'-deletion constructs. Deletion of the farmost 3'-situated NFkappaB-responsive element almost abolished NFkappaB responsiveness. Treatment of human embryonic kidney 293 cells with sulfasalazine, a NFkappaB inhibitor, resulted in enhanced 1alpha-hydroxylase mRNA production. Down-regulation of 1alpha-hydroxylase promoter through NFkappaB signaling may contribute to the pathogenesis of inflammation-associated osteopenia/osteoporosis.     

Effect of restricted feed intake and addition of vitamins B2 and B6 and folic acid on the liver concentration of zinc and copper in rats

The aim of the present study was to investigate the influence of nutritional deficiency and dietary addition of vitamins (B₂, B₆, and folate) on hepatic concentration of zinc and copper in rats. The experiment was performed on 260 growing male Wistar rats divided into 13 groups. Animals of 11 groups were fed isocaloric diets (14.7 MJ/kg) in which the 20% of energy was derived from protein. Another two groups of rats were offered diets with 9% or 4.5% of energy originating from protein. Animals of both mentioned groups and of the control group (20% of energy from protein) were offered diets ad libitum. The other 10 groups were offered 50% and 30% of the amount consumed in the control group. Eight groups, from those 10 restricted ones, were differentiated by dietary addition of vitamins B₂ and B₆ and folate (300% addition). Restricted feed intake did not affect the liver zinc concentration but significantly increased the copper concentration. The addition of vitamin B₆ decreased the liver Zn concentration. The highest liver Cu concentration was noted in rats offered restricted diets to only 30% of intake in the control group and high in vitamin B₂ and in rats supplemented with all of studied vitamins together. It suggests that vitamin B₂ had the strongest impact on liver Cu concentration in rats fed restricted diets.     

Localization of human chorionic gonadotropin beta subunit transcripts in ovarian cancer tissue

Recent studies demonstrated that besides placenta and malignant trophoblastic tumors, hCG and especially its beta-subunit is secreted by a varieties of tumors of different origin. The aim of the present investigation was to determine the expression pattern of human chorionic gonadotropin gene in ovarian cancer tissue. The study included 8 patients with epithelial ovarian carcinoma. The expression and distribution of hCGbeta mRNA was assessed by in situ RT-PCR method. The semi-quantitative assessment was performed using computer image analysis. Transformation of the images into the pseudocolour scale showed a clear difference in fluorescence intensity among individual cancer cells. The intensity of ISRT-PCR products corresponding with expression level of hCGbeta demonstrated that its production by individual cancer cells is different. In all studied specimens of the ovarian carcinoma tissue, cancer cells characterized by the presence of active hCGbeta gene were found, whereas noncancerous tissue demonstrated lack of the gene expression. Thus, the study clearly shows that the expression of hCGbeta is the feature of ovarian cancer tissue.