Molecular genetic analysis of Dichelobacter nodosus proteases AprV2/B2, AprV5/B5 and BprV/B in clinical material from European sheep flocks

Dichelobacter nodosus, the etiological agent of ovine footrot, exists both as virulent and as benign strains, which differ in virulence mainly due to subtle differences in the three subtilisin-like proteases AprV2, AprV5 and BprV found in virulent, and AprB2, AprB5 and BprB in benign strains of D. nodosus. Our objective was a molecular genetic epidemiological analysis of the genes of these proteases by direct sequence analysis from clinical material of sheep from herds with and without history of footrot from 4 different European countries. The data reveal the two proteases known as virulent AprV2 and benign AprB2 to correlate fully to the clinical status of the individuals or the footrot history of the herd. In samples taken from affected herds, the aprV2 gene was found as a single allele whereas in samples from unaffected herds several alleles with minor modifications of the aprB2 gene were detected. The different alleles of aprB2 were related to the herds. The aprV5 and aprB5 genes were found in the form of several alleles scattered without distinction between affected and non-affected herds. However, all different alleles of aprV5 and aprB5 encode the same amino acid sequences, indicating the existence of a single protease isoenzyme 5 in both benign and virulent strains. The genes of the basic proteases BprV and BprB also exist as various alleles. However, differences found in samples from affected versus non-affected herds do not reflect the currently known epitopes that are attributed to differences in biochemical activity. The data of the study confirm the prominent role of AprV2 in the virulence of D. nodosus and shed a new light on the presence of the other protease genes and their allelic variants in clinical samples.

Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17alpha-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase.

Human adrenal corticocarcinoma NCI-H295R cells produce more androgens than NCI-H295A cells and differ in 3beta-hydroxysteroid dehydrogenase type 2 and 17,20 lyase activities

The human adrenal cortex produces mineralocorticoids, glucocorticoids, and androgens in a species-specific, hormonally regulated, zone-specific, and developmentally characteristic fashion. Most molecular studies of adrenal steroidogenesis use human adrenocortical NCI-H295A and NCI-H295R cells as a model because appropriate animal models do not exist. NCI-H295A and NCI-H295R cells originate from the same adrenocortical carcinoma which produced predominantly androgens but also smaller amounts of mineralocorticoids and glucocorticoids. Research data obtained from either NCI-H295A or NCI-H295R cells are generally compared, although for the same experiments no direct comparison between the two cell lines has been performed. Therefore, we compared the steroid profile and the expression pattern of important genes involved in steroidogenesis in both cell lines. We found that steroidogenesis differs profoundly. NCI-H295A cells produce more mineralocorticoids, whereas NCI-H295R cells produce more androgens. Expression of the 3beta-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5, and sulfonyltransferase genes is higher in NCI-H295A cells, whereas expression of the cytochrome P450c17 (CYP17), 21-hydroxylase (CYP21), and P450 oxidoreductase genes does not differ between the cell lines. We found lower 3beta-hydroxysteroid dehydrogenase type 2 but higher 17,20-lyase activity in NCI-H295R cells explaining the 'androgenic' steroid profile for these cells and resembling the zona reticularis of the human adrenal cortex. Both cell lines were found to express the ACTH receptor at low levels consistent with low stimulation by ACTH. By contrast, both cell lines were readily stimulated by 8Br-cAMP. The angiotensin type 1 receptor was highly expressed in NCI-H295R than NCI-H295A cells and angiotensin II stimulated steroidogenesis in NCI-H295R but not NCI-H295A cells. Our data suggest that comparative studies between NCI-H295A and NCI-H295R cells may help find important regulators of mineralocorticoid or androgen biosynthesis.

Личните и географските имена в българския и сръбския превод на Стишния пролог (по материал от месец март)

The paper presents a comparative study of the personal and geographic names in the two complete Slavic translations of the Byzantine Versified Synaxarion, namely the Bulgarian and Serbian Prolog Stišnoj, which appeared around the first half of the 14 th century. On the basis of the March texts in seven South Slavic manuscripts, the differences in the rendering of the personal names are analysed on the level of phonetics, orthography, morphology and word formation. The data allow the following conclusions: 1) The differences in the forms of these names in the Bulgarian and Serbian Prolog Stišnoj give further arguments supporting their independent origin; 2) Several specific tendencies are noted which more or less differentiate them. The Bulgarian translation reproduces more accurately the graphics of the original names, allows dativus possessivus, often replaces the Greek anthroponyms and toponyms with adjectives, presents many local names in the plural, and sometimes retains Greek nominative endings in masculine personal names. The Serbian translation, on the other hand, follows more often the Byzantine pronunciation of the names, complies more strictly with their grammatical characteristics (case, number), separates more often the ending -ς from the stem, and incorporates the accusative ending -ν into the stem of certain anthroponyms several times. 3) The tendency towards Slavicisation of the personal names is nearly the same in both translations and cannot be viewed as peculiar of either of them.

Interaction of Plectin with Keratins 5 and 14: Dependence on Several Plectin Domains and Keratin Quaternary Structure.

Plectin, a cytolinker of the plakin family, anchors the intermediate filament (IF) network formed by keratins 5 and 14 (K5/K14) to hemidesmosomes, junctional adhesion complexes in basal keratinocytes. Genetic alterations of these proteins cause epidermolysis bullosa simplex (EBS) characterized by disturbed cytoarchitecture and cell fragility. The mechanisms through which mutations located after the documented plectin IF-binding site, composed of the plakin-repeat domain (PRD) B5 and the linker, as well as mutations in K5 or K14, lead to EBS remain unclear. We investigated the interaction of plectin C terminus, encompassing four domains, the PRD B5, the linker, the PRD C, and the C extremity, with K5/K14 using different approaches, including a rapid and sensitive fluorescent protein-binding assay, based on enhanced green fluorescent protein-tagged proteins (FluoBACE). Our results demonstrate that all four plectin C-terminal domains contribute to its association with K5/K14 and act synergistically to ensure efficient IF binding. The plectin C terminus predominantly interacted with the K5/K14 coil 1 domain and bound more extensively to K5/K14 filaments compared with monomeric keratins or IF assembly intermediates. These findings indicate a multimodular association of plectin with K5/K14 filaments and give insights into the molecular basis of EBS associated with pathogenic mutations in plectin, K5, or K14 genes.Journal of Investigative Dermatology advance online publication, 10 July 2014; doi:10.1038/jid.2014.255.

Steroidogenesis of the testis - new genes and pathways

Defects of androgen biosynthesis cause 46,XY disorder of sexual development (DSD). All steroids are produced from cholesterol and the early steps of steroidogenesis are common to mineralocorticoid, glucocorticoid and sex steroid production. Genetic mutations in enzymes and proteins supporting the early biosynthesis pathways cause adrenal insufficiency (AI), DSD and gonadal insufficiency. The classic androgen biosynthesis defects with AI are lipoid CAH, CYP11A1 and HSD3B2 deficiencies. Deficiency of CYP17A1 rarely causes AI, and HSD17B3 or SRD5A2 deficiencies only cause 46,XY DSD and gonadal insufficiency. All androgen biosynthesis depends on 17,20 lyase activity of CYP17A1 which is supported by P450 oxidoreductase (POR) and cytochrome b5 (CYB5). Therefore 46,XY DSD with apparent 17,20 lyase deficiency may be due to mutations in CYP17A1, POR or CYB5. Illustrated by patients harboring mutations in SRD5A2, normal development of the male external genitalia depends largely on dihydrotestosterone (DHT) which is converted from circulating testicular testosterone (T) through SRD5A2 in the genital skin. In the classic androgen biosynthetic pathway, T is produced from DHEA and androstenedione/-diol in the testis. However, recently found mutations in AKR1C2/4 genes in undervirilized 46,XY individuals have established a role for a novel, alternative, backdoor pathway for fetal testicular DHT synthesis. In this pathway, which has been first elucidated for the tammar wallaby pouch young, 17-hydroxyprogesterone is converted directly to DHT by 5α-3α reductive steps without going through the androgens of the classic pathway. Enzymes AKR1C2/4 catalyse the critical 3αHSD reductive reaction which feeds 17OH-DHP into the backdoor pathway. In conclusion, androgen production in the fetal testis seems to utilize two pathways but their exact interplay remains to be elucidated.

ABCB5 Identifies Immunoregulatory Dermal Cells

Cell-based strategies represent a new frontier in the treatment of immune-mediated disorders. However, the paucity of markers for isolation of molecularly defined immunomodulatory cell populations poses a barrier to this field. Here, we show that ATP-binding cassette member B5 (ABCB5) identifies dermal immunoregulatory cells (DIRCs) capable of exerting therapeutic immunoregulatory functions through engagement of programmed cell death 1 (PD-1). Purified Abcb5(+) DIRCs suppressed T cell proliferation, evaded immune rejection, homed to recipient immune tissues, and induced Tregs in vivo. In fully major-histocompatibility-complex-mismatched cardiac allotransplantation models, allogeneic DIRCs significantly prolonged allograft survival. Blockade of DIRC-expressed PD-1 reversed the inhibitory effects of DIRCs on T cell activation, inhibited DIRC-dependent Treg induction, and attenuated DIRC-induced prolongation of cardiac allograft survival, indicating that DIRC immunoregulatory function is mediated, at least in part, through PD-1. Our results identify ABCB5(+) DIRCs as a distinct immunoregulatory cell population and suggest promising roles of this expandable cell subset in cellular immunotherapy.