The protein kinase YakA regulates g-protein-linked signaling responses during growth and development of Dictyostelium.

A genetic screen for Dictyostelium mutants that phenotypically resemble cells lacking the G-protein beta-subunit yielded the protein kinase YakA. Like gbeta-null cells, yakA-null cells fail to enter development and display slow growth on bacterial lawns. We created a temperature-sensitive yakA mutant and showed that YakA activity is required not only at the onset but also during development. The yakA-null cells have strong defects in folic acid-induced responses, such as actin polymerization and cGMP accumulation, indicating that they play a role in G-protein-mediated signaling responses. We propose that YakA acts downstream of G-proteins, because cAMP receptors still couple to G-proteins in the yakA mutant. In addition, the previously observed growth arrest induced by overexpression of YakA also occurs in gbeta mutants. We localized YakA-GFP to the cytosol suggesting that YakA may be a functional homolog of its mammalian counterparts Dyrk2 and Dyrk3, a subclass of dual-specificity Yak-related kinases (Dyrk) with unknown function.     

Hypertension after bilateral kidney irradiation in young and adult rats

The mechanism of a rise in blood pressure after kidney irradiation is unclear but most likely of renal origin. We have investigated the role of the renin-angiotensin system and dietary salt restriction in the development of systolic hypertension after bilateral kidney irradiation in young and adult rats. Three to 12 months after a single X-ray dose of 7.5 or 12.5 Gy to both kidneys of young and adult rats, the systolic blood pressure (SBP) and plasma renin concentration (PRC) were measured regularly. A single X-ray dose of 12.5 Gy caused a moderate rise in SBP and a slight reduction in PRC in both young and adult rats. A dose of 7.5 Gy did not significantly alter the SBP or PRC during the follow-up period of 1 year. In a second experiment, the kidneys of young rats received an X-ray dose of 20 Gy. Subsequently, rats were kept on a standard diet (110 mmol sodium/kg) or a sodium-poor diet (10 mmol sodium/kg). On both diets, SBP started to rise rapidly 3 months after kidney irradiation. Sodium balance studies carried out at that time revealed an increased sodium retention in the irradiated rats compared to controls on the same diet. In rats on a low sodium intake, there was neither a delay nor an alleviation in the development of hypertension. Compared to controls, the PRC tended to be lower in irradiated rats up to 4 months after irradiation. Subsequently, malignant hypertension developed in all 20 Gy rats, resulting in pressure natriuresis, stimulating the renin-angiotensin system. Our findings indicated that hypertension after bilateral kidney irradiation was not primarily the result of an activation of the renin-angiotensin system. Although there were some indications that sodium retention played a role, dietary sodium restriction did not influence the development of hypertension.     

Multiple regulation of testicular steroidogenesis

One single injection of ethylene dimethane sulfonate (EDS) to mature rats causes specific degeneration of testicular Leydig cells which is complete after 3 days. At this time no steroidogenic activities can be detected, indicating that Leydig cells are the source of steroids. The mechanism of this cytotoxic effect of EDS has been investigated with isolated cells. Extensive protein alkylation has been shown to occur in Leydig cells, Sertoli cells and hepatocytes. Steroid production by Leydig cells is always inhibited by EDS, but cytotoxic effects of EDS could only be demonstrated in Leydig cells from mature rats or tumour tissue and not in Leydig cells from immature rats. A new population of Leydig cells develops during the next 2-5 weeks after EDS treatment. In hypophysectomized rats this repopulation only occurs when hCG is given daily. FSH has no effects. The proliferative activity in the interstitial tissue increases within 2 days after administration of hCG or EDS and there are indications that LH and locally produced factors are involved in the proliferation of Leydig cells or Leydig cell precursor cells. Inhibition of cAMP production with inhibitors of adenylate cyclase results in an enhancement of the LH-stimulated steroid production similar to that observed with an LHRH agonist and phospholipase C (PLC). Since the effects of LHRH and PLC on protein phosphorylation and steroid production are similar and different from LH or active phorbol esters, it is proposed that LHRH and PLC may stimulate steroid production via liberation of calcium from a specific intracellular pool. Sterol carrier protein2 (SCP2) which is specifically localized in Leydig cells and regulated by LH probably plays a role in the delivery of cholesterol to the mitochondria although the mechanism of this carrier function is not clear. The results indicate that regulation of Leydig cell development and the steroidogenic activities by gonadotrophins and locally produced factors occur via different transducing systems and regulatory pathways.     

The role of MHC class I heterodimer expression in mouse ankylosing enthesopathy

 Ankylosing enthesopathy (ANKENT) is a spontaneous mouse joint disease with strikingly similar pathology to human HLA-B27-associated enthesopathies such as ankylosing spondylitis. In C57Bl/10 mice, transgenic HLA-B^*2702 as well as H2 genes have been shown to be relative risk factors for ANKENT. To investigate the role of major histocompatibility complex (MHC) class I expression in disease pathogenesis, ANKENT occurrence was compared among β₂-microglobulin (β₂m) knockout littermates with or without transgenes for HLA-B^*2702 and human β₂m. In the knockout phenotype lacking β₂m, ANKENT occurrence is significantly reduced (P = 0.016). In the absence of β₂m, B^*2702 is not detected on the cell membrane, nor does it increase the risk for ANKENT. This means that the previous finding that HLA-B^*2702 increases susceptibility to ANKENT in C57Bl/10 mice cannot be ascribed to a transgene insertion effect. Rather, in order to increase disease susceptibility, B^*2702 must be coexpressed with mouse β₂m (mo-β₂m). In contrast, when HLA-B^*2702 is expressed with β₂m of human origin, disease susceptibility is not affected. Thus, both H2^b-derived class I heterodimers and HLA-B^*2702/mo-β₂m heterodimers contribute to ANKENT susceptibility.     

A strictly anaerobic betaproteobacterium Georgfuchsia toluolica gen. nov., sp. nov. degrades aromatic compounds with Fe(III), Mn(IV) or nitrate as an electron acceptor

A bacterium (strain G5G6) that grows anaerobically with toluene was isolated from a polluted aquifer (Banisveld, the Netherlands). The bacterium uses Fe(III), Mn(IV) and nitrate as terminal electron acceptors for growth on aromatic compounds. The bacterium does not grow on sugars, lactate or acetate. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain G5G6 belonged to the Betaproteobacteria . Its closest, but only distantly related, cultured relative is Sterolibacterium denitrificans Chol-1S^T (94.6% similarity of the 16S rRNA genes), a cholesterol-oxidizing, denitrifying bacterium. Strain G5G6 possesses the benzylsuccinate synthase A ( bssA ) gene encoding the α-subunit of Bss, which catalyzes the first step in anaerobic toluene degradation. The deduced BssA amino acid sequence is closely related to those of Azoarcus and Thauera species, which also belong to the Betaproteobacteria . Strain G5G6 is the first toluene-degrading, iron-reducing bacterium that does not belong to the Geobacteraceae within the Deltaproteobacteria . Based on phylogenetic and physiological comparison, strain G5G6 could not be assigned to a described species. Therefore, strain G5G6 (DSMZ 19032^T=JCM 14632^T) is a novel taxon of the Betaproteobacteria . We propose the name Georgfuchsia toluolica gen. nov., sp. nov.     

A model for the architecture of the hemocyanin from the arthropod Squilla mantis (Crustacea, Stomatopoda)

Squilla mantis hemocyanin is composed of two hexameric subunits but has electron microscopic profiles different from other bis-hexameric hemocyanins, e.g. Astacus and Homarus. We distinguished three different electron microscopic profiles of S. mantis hemocyanin: two sideviews and a topview. These profiles were studied using computer image alignment and correspondence analysis [Van Heel, M. and Frank, J. (1981) Ultramicroscopy 6, 187 - 194]. With the results of this analysis we were able to build a three-dimensional model for the quaternary structure of this hemocyanin. In this model the two hexamers are stacked in such a way that their hexagonal surfaces overlap to about 60% of their width. In the overlap area four subunits are arranged in two different interhexameric pairs, each forming a bridging area between the two hexamers.     

Mutagenic and clastogenic activity of nitracrine analogues in cultured V79 Chinese hamster cells

Nitracrine is used clinically as an antitumour agent, and analogues are actively being developed in some laboratories. The mutagenic activity of 9-[(3-dimethylaminopropyl)amino]-acridine and its 1-nitro (nitracrine), 2-, 3- and 4-nitro derivatives was evaluated at the 6-thioguanine and ouabain resistance loci in cultured Chinese hamster fibroblasts (V79-171b cell line). The des-nitro, 2- and 3-nitro caused no statistically significant mutagenic activity at either locus. Each of these 3 compounds weakly increased (approximately 2-fold) the incidence of micronuclei in the same cell line when tested at cytotoxic doses. Both the 1- and 4-nitro compounds increased the incidence of 6-thioguanine resistant cells from around 1 in 10(-6) to approximately 1 in 10(-4). The former compound significantly increased the frequency of ouabain-resistant cells. Both of these compounds were potent inducers of micronuclei in V79-171b cells, indicating high clastogenic activity. It would appear prudent to regard both of these compounds as potential human carcinogens.     

Monitoring the Metabolic State of Fungal Hyphae and the Presence of Melanin by Nonlinear Spectral Imaging

Label-free nonlinear spectral imaging microscopy (NLSM) records two-photon-excited fluorescence emission spectra of endogenous fluorophores within the specimen. Here, NLSM is introduced as a novel, minimally invasive method to analyze the metabolic state of fungal hyphae by monitoring the autofluorescence of NAD(P)H and flavin adenine dinucleotide (FAD). Moreover, the presence of melanin was analyzed by NLSM. NAD(P)H, FAD, and melanin were used as biomarkers for freshness of mushrooms of Agaricus bisporus (white button mushroom) that had been stored at 4°C for 0 to 17 days. During this period, the mushrooms did not show changes in morphology or color detectable by eye. In contrast, FAD/NAD(P)H and melanin/NAD(P)H ratios increased over time. For instance, these ratios increased from 0.92 to 2.02 and from 0.76 to 1.53, respectively, at the surface of mushroom caps that had been harvested by cutting the stem. These ratios were lower under the skin than at the surface of fresh mushrooms (0.78 versus 0.92 and 0.41 versus 0.76, respectively), indicative of higher metabolism and lower pigment formation within the fruiting body. Signals were different not only between tissues of the mushroom but also between neighboring hyphae. These data show that NLSM can be used to determine the freshness of mushrooms and to monitor the postharvest browning process at an early stage. Moreover, these data demonstrate the potential of NLSM to address a broad range of fundamental and applied microbiological processes.