Bacterial Consortium of Millepora dichotoma Exhibiting Unusual Multifocal Lesion Event in the Gulf of Eilat, Red Sea

Colonies of the hydrocoral Millepora dichotoma along the Gulf of Eilat are exhibiting unusual tissue lesions in the form of white spots. The emergence and rapid establishment of these multifocal tissue lesions was the first of its kind reported in this region. A characterization of this morphological anomaly revealed bleached tissues with a significant presence of bacteria in the tissue lesion area. To ascertain possible differences in microbial biota between the lesion area and non-affected tissues, we characterized the bacterial diversity in the two areas of these hydrocorals. Both culture-independent (molecular) and culture-dependent assays showed a shift in bacterial community structure between the healthy and affected tissues. Several 16S rRNA gene sequences retrieved from the affected tissues matched sequences of bacterial clones belonging to Alphaproteobacteria and Bacteroidetes members previously associated with various diseases in scleractinian corals.     

Catalytic and structural effects of amino acid substitution at histidine 30 in human manganese superoxide dismutase: insertion of valine C gamma into the substrate access channel

Catalysis of the disproportionation of superoxide by human manganese superoxide dismutase (MnSOD) is characterized by an initial burst of catalysis followed by a much slower region that is zero order in superoxide and due to a product inhibition by peroxide anion. We have prepared site-specific mutants with replacements at His30, the side chain of which lies along the substrate access channel and is about 5.8 A from the metal. Using pulse radiolysis to generate superoxide, we have determined that kcat/K(m) was decreased and product inhibition increased for H30V MnSOD, both by 1-2 orders of magnitude, compared with wild type, H30N, and H30Q MnSOD. These effects are not attributed to the redox potentials, which are similar for all of these variants. An investigation of the crystal structure of H30V Mn(III)SOD compared with wild type, H30Q, and H30N Mn(III)SOD showed the positions of two gamma carbons of Val30 in the active site; Cgamma1 overlaps Cgamma of His30 in wild type, and Cgamma2 extends into the substrate access channel and occupies the approximate position of a water molecule in the wild type. The data suggest that Cgamma2 of the Val side chain has significantly interrupted catalysis by this overlap into the access channel with possible overlap with the substrate-product binding site. This is supported by comparison of the crystal structure of H30V MnSOD with that of azide bound to Mn(III)SOD from Thermus thermophilus and by visible absorption spectra showing that azide binding to the metal in H30V Mn(III)SOD is abolished. Moreover, the presence of Val30 caused a 100-fold decrease in the rate constant for dissociation of the product-inhibited complex compared with wild type.     

The First Nucleotide Binding Domain of Cystic Fibrosis Transmembrane Conductance Regulator Is a Site of Stable Nucleotide Interaction, whereas the Second Is a Site of Rapid Turnover

As in other adenine nucleotide binding cassette (ABC) proteins the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR) bind and hydrolyze ATP and in some manner regulate CFTR ion channel gating. Unlike some other ABC proteins, however, there are preliminary indications that the two domains of CFTR are nonequivalent in their nucleotide interactions (Szabo, K., Szakacs, G., Hegeds, T., and Sarkadi, B. (1999) J. Biol. Chem. 274, 12209-12212; Aleksandrov, L., Mengos, A., Chang, X., Aleksandrov, A., and Riordan, J. R. (2001) J. Biol. Chem. 276, 12918-12923). We have now characterized the interactions of the 8-azido-photoactive analogues of ATP, ADP, and 5'-adenyl-beta,gamma-imidodiphosphate (AMP-PNP) with the two domains of functional membrane-bound CFTR. The results show that the two domains appear to act independently in the binding and hydrolysis of 8-azido-ATP. At NBD1 binding does not require a divalent cation. This binding is followed by minimal Mg(2+)-dependent hydrolysis and retention of the hydrolysis product, 8-azido-ADP, but not as a vanadate stabilized post-hydrolysis transition state complex. In contrast, at NBD2, MgN(3)ATP is hydrolyzed as rapidly as it is bound and the nucleoside diphosphate hydrolysis product dissociates immediately. Confirming this characterization of NBD1 as a site of more stable nucleotide interaction and NBD2 as a site of fast turnover, the non-hydrolyzable N(3)AMP-PNP bound preferentially to NBD1. This demonstration of NBD2 as the rapid nucleotide turnover site is consistent with the strong effect on channel gating kinetics of inactivation of this domain by mutagenesis.     

The NMDA receptor is coupled to the ERK pathway by a direct interaction between NR2B and RasGRF1

The NMDA subtype of glutamate receptors (NMDAR) at excitatory neuronal synapses plays a key role in synaptic plasticity. The extracellular signal-regulated kinase (ERK1,2 or ERK) pathway is an essential component of NMDAR signal transduction controlling the neuroplasticity underlying memory processes, neuronal development, and refinement of synaptic connections. Here we show that NR2B, but not NR2A or NR1 subunits of the NMDAR, interacts in vivo and in vitro with RasGRF1, a Ca(2+)/calmodulin-dependent Ras-guanine-nucleotide-releasing factor. Specific disruption of this interaction in living neurons abrogates NMDAR-dependent ERK activation. Thus, RasGRF1 serves as NMDAR-dependent regulator of the ERK kinase pathway. The specific association of RasGRF1 with the NR2B subunit and study of ERK activation in neurons with varied content of NR2B suggests that NR2B-containing channels are the dominant activators of the NMDA-dependent ERK pathway.     

Autosomal-Recessive Hypophosphatemic Rickets Is Associated with an Inactivation Mutation in the ENPP1 Gene

Human disorders of phosphate (Pi) handling and hypophosphatemic rickets have been shown to result from mutations in PHEX, FGF23, and DMP1, presenting as X-linked recessive, autosomal-dominant, and autosomal-recessive patterns, respectively. We present the identification of an inactivating mutation in the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene causing autosomal-recessive hypophosphatemic rickets (ARHR) with phosphaturia by positional cloning. ENPP1 generates inorganic pyrophosphate (PPi), an essential physiologic inhibitor of calcification, and previously described inactivating mutations in this gene were shown to cause aberrant ectopic calcification disorders, whereas no aberrant calcifications were present in our patients. Our surprising result suggests a different pathway involved in the generation of ARHR and possible additional functions for ENPP1.     

Carrier rates of the ancestral Indian W24X mutation in GJB2 in the general Gypsy population and individual subisolates

Mutations in the GJB2 gene are the most common cause of autosomal recessive nonsyndromic hearing loss and occur in approximately 20% of all cases of prelingual deafness. Previous studies of Roma/Gypsies in Slovakia, the Czech Republic, and Spain have shown that W24X, the most common GJB2 mutation in India, is also the prevalent molecular defect in the Gypsy population. The reported W24X frequencies vary broadly from 23% to 93% of Gypsy mutant alleles, likely reflecting local founder effects, drift, and differential admixture in the subisolates of this genetically structured population. Our goal was to provide more representative data on W24X carrier rates in European Gypsies, which can inform individual diagnostic investigations and public health initiatives across countries. Mutation testing in 603 control subjects of Gypsy ethnicity, representing 8 traditional subisolates in southeastern Europe and 4 additional European regions revealed that W24X is spread across subisolates, as expected for an ancestral founder mutation. While variation between subisolates does exist, the average carrier rates, overall and in the major linguistic/migrational categories of Balkan Gypsies, Vlax Roma, and west European Gypsies, are consistently in the 4%-5% range. The results place W24X among the three most common founder mutations in the Gypsies, and classify them as one of the high-risk populations for prelingual deafness. Higher demands on language acquisition in this bilingual population, together with poorer quality of health care compared to autochthonous Europeans, make the consequences of congenital deafness even more damaging than is usually the case. Neonatal screening for W24X among Gypsies would be a justified and cost-effective public health intervention.     

Intersubunit complementation of sugar signal transduction in VirA heterodimers and posttranslational regulation of VirA activity in Agrobacterium tumefaciens

The VirA/VirG two-component regulatory system of Agrobacterium tumefaciens regulates expression of the virulence (vir) genes that control the infection process leading to crown gall tumor disease on susceptible plants. VirA, a membrane-bound homodimer, initiates vir gene induction by communicating the presence of molecular signals found at the site of a plant wound through phosphorylation of VirG. Inducing signals include phenols, monosaccharides, and acidic pH. While sugars are not essential for gene induction, their presence greatly increases vir gene expression when levels of the essential phenolic signal are low. Reception of the sugar signal depends on a direct interaction between ChvE, a sugar-binding protein, and VirA. Here we show that the sugar signal received in the periplasmic region of one subunit within a VirA heterodimer can enhance the kinase function of the second subunit. However, sugar enhancement of vir gene expression was vector dependent. virA alleles expressed from pSa-derived vectors inhibited signal transduction by endogenous VirA. Inhibition was conditional, depending on the induction medium and the virA allele tested. Moreover, constitutive expression of virG overcame the inhibitory effect of some but not all virA alleles, suggesting that there may be more than one inhibitory mechanism.     

Genetic evidence for a distinct subtype of schizophrenia characterized by pervasive cognitive deficit

A novel phenotyping strategy in schizophrenia, targeting different neurocognitive domains, neurobehavioral features, and selected personality traits, has allowed us to identify a homogeneous familial subtype of the disease, characterized by pervasive neurocognitive deficit. Our genome scan data indicate that this subtype, which accounts for up to 50% of our sample, has a distinct genetic basis and explains linkage to chromosome 6p24 reported previously. If representative of other populations, the ratio of schizophrenia subtypes observed in our families could have a profound impact on sample heterogeneity and on the power of genetic studies to detect linkage and association. Our proposed abbreviated battery of tests should facilitate phenotype characterization for future genetic analyses and allow a focus on a crisply defined schizophrenia subtype, thus promoting a more informed search for susceptibility genes.