Arg-14 loop of site 3 anemone toxins: effects of glycine replacement on toxin affinity

Anthopleurin B (ApB) is a high-affinity sea anemone neurotoxin that interacts with voltage-sensitive sodium (Na(V)) channels, causing a delay in channel inactivation. The solution structures of all known anemone toxins having this activity include a poorly defined region encompassing ApB residues 8-17, which we call the Arg-14 loop. We propose that the inherent mobility of the Arg-14 loop is necessary for the toxins' ability to maintain a high-affinity channel complex throughout the continual conformational transitions experienced by the channel during its functional cycle. We have previously shown that Arg-12, located in this loop, and Leu-18, which is adjacent, are important for ApB activity. Here, we characterized the role of two glycines located within the loop (Gly-10 and Gly-15) and an additional glycine positioned immediately C-terminal to it (Gly-20). We used site-directed replacement by alanine to assess the functional contribution to toxin binding of each of these residues singly and in combination. Gly-20 was found to be an essential toxin folding determinant; Gly-10 and Gly-15 were important for determining toxin affinity. Compared to wild-type toxin, the G10A and G15A toxins displayed significantly higher K(D) values for both cardiac (Na(V)1.5) and neuronal (Na(V)1.2) channels, although both demonstrated greater isoform discrimination for Na(V)1.5 than did wild-type ApB. For both G10A and G15A, significant Na(V) isoform differences were evident for on- and off-rates, with the most dramatic effect of a single mutation being the 467-fold reduction in the on-rate for G10A binding to Na(V)1.2, suggestive of a more accommodating binding site on Na(V)1.5 as compared to Na(V)1.2. Because alanine replacement of glycines is known to be associated with reduced backbone freedom, these results suggest an essential role for Arg-14 loop flexibility in toxin function, although a direct steric effect of the mutant methyl group cannot be excluded.     

A Genomewide Screen for Generalized Vitiligo: Confirmation of AIS1 on Chromosome 1p31 and Evidence for Additional Susceptibility Loci

Generalized vitiligo is a common autoimmune disorder characterized by the development of white patches of skin and overlying hair due to loss of pigment-forming melanocytes from the involved areas. Family clustering of cases is not uncommon, in a pattern suggestive of multifactorial, polygenic inheritance, and there is strong association between vitiligo and other autoimmune diseases. To map genetic loci that confer susceptibility to generalized vitiligo and perhaps other autoimmune diseases, we performed a genomewide linkage scan in 71 white multiplex families with vitiligo from North America and the United Kingdom. Linkage was assessed by multipoint nonparametric linkage analyses. One linkage signal, AIS1, located at 1p31, met genomewide criteria for highly significant linkage (nonparametric LOD 5.56; P=.000000282), establishing its importance as a major vitiligo susceptibility locus. An additional seven signals, on chromosomes 1, 7, 8, 11, 19, and 22, met genomewide criteria for "suggestive linkage," and will thus be of particular importance for follow-up studies.     

Specificity of coenzyme Q inhibition of an aging-related cell surface NADH oxidase (ECTO-NOX) that generates superoxide

Our laboratories have described a novel class of ectoproteins at the cell surface with both NADH or hydroquinone oxidase (NOX) and protein disulfide-thiol interchange activities (ECTO-NOX proteins). The two activities exhibited by these proteins alternate to generate characteristic patterns of oscillations where the period length is independent of temperature. The period length for the constitutive ECTO-NOX is 24 min. Here we describe a distinctive age-related ECTO-NOX (arNOX) whose activity is blocked by coenzyme Q10. arNOX occurs exclusively in aged cells and tissues. The period length of the oscillations is 26 min. Rather than reducing 1/2 O2 to H2O, electrons are transferred to O2 to form superoxide. Superoxide formation was demonstrated by superoxide dismutase-sensitive reduction of ferricytochrome c and by reduction of a superoxide-specific tetrazolium salt. Quinone inhibition was given by coenzymes Q8, 9 and Q10 but not by Q0, Q2, Q4, Q6 or 7. The arNOX provides a mechanism to propagate reactive oxygen species generated at the cell surface to surrounding cells and circulating lipoproteins of importance to atherogenesis. Inhibition of arNOX by dietary coenzyme Q10 provides a rational basis for dietary coenzyme 10 use to retard aging-related arterial lesions.     

Latent phenoloxidase activity and N-terminal amino acid sequence of hemocyanin from Bathynomus giganteus,a primitive crustacean

N-terminal amino acid sequences for the two hemocyanin subunits from the deep-sea crustacean Bathynomus giganteus have been determined by Edman degradation, providing the first sequence information for a hemocyanin from an isopod. In addition, purified hemocyanin from B. giganteus exhibited phenoloxidase activity in the presence of sodium dodecyl sulfate. Although a natural activator has not yet been identified, a preliminary study of the enzyme indicated a K(m) of 5mM for dopamine and an initial rate of 0.1 micromol per min per mg protein, values consistent with a significant role for this enzyme in the innate immune system of B. giganteus. Moreover, after separation of hemolymph by alkaline polyacrylamide gel electrophoresis, the only detectable phenoloxidase activity coincided with the two hemocyanin subunits. The hemocyanin of this primitive crustacean may fulfill dual functions, both as oxygen carrier and as the phenoloxidase crucial for host defense.     

3q29 microdeletion syndrome: clinical and molecular characterization of a new syndrome

We report the identification of six patients with 3q29 microdeletion syndrome. The clinical phenotype is variable despite an almost identical deletion size. The phenotype includes mild-to-moderate mental retardation, with only slightly dysmorphic facial features that are similar in most patients: a long and narrow face, short philtrum, and high nasal bridge. Autism, gait ataxia, chest-wall deformity, and long and tapering fingers were noted in at least two of six patients. Additional features--including microcephaly, cleft lip and palate, horseshoe kidney and hypospadias, ligamentous laxity, recurrent middle ear infections, and abnormal pigmentation--were observed, but each feature was only found once, in a single patient. The microdeletion is approximately 1.5 Mb in length, with molecular boundaries mapping within the same or adjacent bacterial artificial chromosome (BAC) clones at either end of the deletion in all patients. The deletion encompasses 22 genes, including PAK2 and DLG1, which are autosomal homologues of two known X-linked mental retardation genes, PAK3 and DLG3. The presence of two nearly identical low-copy repeat sequences in BAC clones on each side of the deletion breakpoint suggests that nonallelic homologous recombination is the likely mechanism of disease causation in this syndrome.     

DNA binding properties of the yeast Msh2-Msh6 and Mlh1-Pms1 heterodimers

We describe here our recent studies of the DNA binding properties of Msh2-Msh6 and Mlh1-Pms1, two protein complexes required to repair mismatches generated during DNA replication. Mismatched DNA binding by Msh2-Msh6 was probed by mutagenesis based on the crystal structure of the homologous bacterial MutS homodimer bound to DNA. The results suggest that several amino acid side chains inferred to interact with the DNA backbone near the mismatch are critical for repair activity. These contacts, which are different in Msh2 and Msh6, likely facilitate stacking and hydrogen bonding interactions between side chains in Msh6 and the mismatched base, thus stabilizing a kinked DNA conformation that permits subsequent repair steps coordinated by the Mlh1-Pms1 heterodimer. Mlh1-Pms1 also binds to DNA, but independently of a mismatch. Mlh1-Pms1 binds short DNA substrates with low affinity and with a slight preference for single-stranded DNA. It also binds longer duplex DNA molecules, but with a higher affinity indicative of cooperative binding. Indeed, imaging by atomic force microscopy reveals cooperative DNA binding and simultaneous interaction with two DNA duplexes. The novel DNA binding properties of Mlh1-Pms1 may be relevant to signal transduction during DNA mismatch repair and to recombination, meiosis and cellular responses to DNA damage.     

In vivo whole body and appendicular bone mineral density in rats: a dual energy X-ray absorptiometry study

Bone mineral density (BMD) of the whole body and hind limb of young adult rats, with and without a sham-operated stifle joint was studied, using dual energy x-ray absorptiometry (DEXA) at three time points. Data from the whole body scan were used for analyses of BMD, bone mineral content (BMC), fat, lean, body weight (BW), percentage of BMC (%BMC), percentage of fat (%fat), and percentage of lean (%lean), none of which were significantly different between the groups at any time point. Significant (P < 0.05) differences in BMD, BMC, %BMC, BW, fat, %fat, and %lean were apparent at the second and third scans, compared with the initial scan, within both groups. Changes in whole body BMD, BMC, and %BMC as well as BW were highly correlated with time in both groups. In the hind limb scans, regions of interest (ROIs) were created to obtain values of BMD and BMC from the whole femur, whole tibia including the fibula, distal portion of the femur, and proximal portion of the tibia. Significant differences were not found between the groups for any ROIs. However, significant BMD and BMC increases were evident in all ROIs at the second and third scans, compared with the initial scan. Similar to those in the whole body scan, BMD and BMC obtained from ROIs were highly correlated with time. The positioning technique for the whole body and appendicular scans was analyzed by calculating percentage of the coefficient of variation (%CV) at the beginning of the study. The %CV was low and acceptable in ROIs for the hind limb and for all parameters of the whole body scan, except fat. The results suggest that in vivo DEXA scanning of the rat whole body and appendicular skeleton is highly reproducible and useful to study the whole skeleton, as well as a region of a long bone of the rat. Values for the sham-operated rats were not significantly different from those for the untreated controls, which suggests that soft tissue damage around the stifle joint did not alter BMD in the subchondral bone of the distal portion of the femur and proximal portion of the tibia.     

Effects of chronic exposure to simulated microgravity on skeletal muscle cell proliferation and differentiation

Cell culture models that mimic long-term exposure to microgravity provide important insights into the cellular biological adaptations of human skeletal muscle to long-term residence in space. We developed insert scaffolding for the NASA-designed rotating cell culture system (RCCS) in order to study the effects of time-averaged microgravity on the proliferation and differentiation of anchorage-dependent skeletal muscle myocytes. We hypothesized that prolonged microgravity exposure would result in the retardation of myocyte differentiation. Microgravity exposure in the RCCS resulted in increased cellular proliferation. Despite shifting to media conditions promoting cellular differentiation, 5 d later, there was an increase in cell number of approximately 62%, increases in total cellular protein (52%), and cellular proliferating cell nuclear antigen (PCNA) content (2.7 times control), and only a modest (insignificant) decrease (10%) in sarcomeric myosin protein expression. We grew cells in an inverted orientation on membrane inserts. Changes in cell number and PCNA content were the converse to those observed for cells in the RCCS. We also grew cells on inserts at unit gravity with constant mixing. Mixing accounted for part, but not all, of the effects of microgravity exposure on skeletal muscle cell cultures (53% of the RCCS effect on PCNA at 4-6 d). In summary, the mechanical effects of simulated microgravity exposure in the RCCS resulted in the maintenance of cellular proliferation, manifested as increases in cell number and expression of PCNA relative to control conditions, with only a modest reciprocal inhibition of cellular differentiation. Therefore, this model provides conditions wherein cellular differentiation and proliferation appear to be uncoupled.