The Physical Environment Affects Cyanophage Communities in British Columbia Inlets

Little is known about the natural distribution of viruses that infect the photosynthetically important group of marine prokaryotes, the cyanobacteria. The current investigation reveals that the structure of cyanophage communities is dependent on water column structure. PCR was used to amplify a fragment of the cyanomyovirus gene (g) 20, which codes for the portal vertex protein. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified g20 gene fragments was used to examine variations in cyanophage community structure in three inlets in British Columbia, Canada. Qualitative examination of denaturing gradient gels revealed cyanophage community patterns that reflected the physical structure of the water column as indicated by temperature and salinity. Based on mobility of PCR fragments in the DGGE gels, some cyanophages appeared to be widespread, while others were observed only at specific depths. Cyanophage communities within Salmon Inlet were more related to one another than to communities from either Malaspina Inlet or Pendrell Sound. As well, surface communities in Malaspina Inlet and Pendrell Sound were different when compared to communities at depth. In the same two locations, distinct differences in community composition were observed in communities that coincided with depths of high chlorophyll fluorescence. The observed community shifts over small distances (only a few meters in depth or inlets separated by less than 100 km) support the idea that cyanophage communities separated by small spatial scales develop independently of each other as a result isolation by water column stratification or land mass separation, which may ultimately lead to changes in the distribution or composition of the host community. Present address (S.M. Short): Ocean Sciences Department, University of California, Santa Cruz 1156 High Street Santa Cruz, CA 95064; sshort@es.ucsc.edu     

Yeast orotidine-5'-phosphate decarboxylase: steady-state and pre-steady-state analysis of the kinetic mechanism of substrate decarboxylation

The catalytically active form of monofunctional yeast orotidine-5'-phosphate decarboxylase was a dimer (E(2)). The dimer equilibrium dissociation constant was 0.25 microM in 0.01 M MOPS Na(+) at pH 7.2. The bimolecular rate constant for dimer formation was 1.56 microM(-1) s(-1). The dimeric form of the enzyme was stabilized by NaCl such that the enzyme was E(2) in 100 mM NaCl at all concentrations of enzyme tested. The kinetics of binding of OMP to E(2) was governed by two ionizations (pK(1) = 6.1 and pK(2) = 7.7). From studies with substrate analogues, the higher pK was assigned to a group on the enzyme that interacted with the pyrimidinyl moiety. The value of the lower pK was dependent on the substrate analogue, which suggested that it was not exclusively the result of ionization of the phosphoryl moiety. During the decarboxylation of OMP, the fluorescence of E(2) was quenched over 20%. The enzymatic species with reduced fluorescence was a catalytically competent intermediate that had kinetic properties consistent with it being the initial enzyme-substrate complex. The stoichiometry for binding of OMP to E(2) was one OMP per enzyme monomer. The value of the first-order rate constant for conversion of the enzyme-substrate complex to free enzyme (36 s(-1)) calculated from a single turnover experiment ([E] > [S]) was slightly greater than the value of k(cat), 20 s(-1) (corrected for stoichiometry), calculated from steady-state data. In the single turnover experiments, the enzyme was E(2)*S, whereas in the steady-state turnover the experiment enzyme was E(2)*S(2). The similarity of these values suggested that the subunits were catalytically independent such that E(2)*S(2) could be treated as E*S and that conversion of the enzyme-substrate complex to E was k(cat). Kinetic data for the approach to the steady-state with OMP and E(2) yield a bimolecular association rate complex of 62 microM(-1) s(-1)and a dissociation rate constant for E*S of 60 s(-1). The commitment to catalysis was 0.25. By monitoring the effect of carbonic anhydrase on [H(+)] changes during a single turnover experiment, the initial product of the decarboxylation reaction was shown to be CO(2) not HCO(3-). UMP was released from the enzyme concomitantly with CO(2) during the conversion of E*S to E. Furthermore, the enzyme removed an enzyme equivalent of H(+) from solvent during this step of the reaction. The bimolecular rate constants for association of 6-AzaUMP and 8-AzaXMP, substrate analogues with markedly different nucleobases, had association rate constants of 112 and 130 microM(-1) s(-1), respectively. These results suggested that the nucleobase did not contribute significantly to the success of formation of the initial enzyme-substrate complex.     

Selection of peptidic mimics of digoxin from phage-displayed peptide libraries by anti-digoxin antibodies

Since the initial report of the development of methodology to generate high-affinity digitalis-specific (digoxin) antibodies, these antibodies have proven extremely useful tools to monitor digoxin levels in digitalized patients and, as Fab fragments, to reverse toxic digoxin effects in life-threatening digoxin overdoses. These antibodies (both digoxin-specific and ouabain-specific) have been used extensively by investigators for the identification and characterization of putative endogenous digitalis-like factors. In this study, we used two well-characterized mouse anti-digoxin monoclonal antibodies (mAbs), designated 26-10 and 45-20, as binding templates with which to select short bacteriophage-displayed (pIII protein inserted) peptides that are capable of binding to these mAbs and mimicking the conformational structure of digoxin. Selective enrichment from two phage-displayed random peptide libraries enabled us to isolate and identify distinct 15 and 26 amino acid residue peptide inserts that bind with high avidity and idiotypic specificity to the selecting mAbs. Among these displayed inserts a subset was identified whose mAb binding is inhibited by digoxin and whose corresponding synthetic peptides inhibit phage binding. They, therefore, appear to bind at the mAbs digoxin-binding sites. These data provide the first clear evidence that short polypeptides can serve as surrogates for the low molecular mass hapten digoxin.     

Quantifying the Effects of Green Crab Damage to Eelgrass Transplants

Mesocosm experiments were conducted in the summer of 1996 to quantify the effect of bioturbation by Carcinus maenas (the introduced European green crab) on survival of transplanted Zostera marina (eelgrass). The research grew out of a successful 2.52 ha eelgrass transplant project in the Great Bay Estuary of New Hampshire. At several subtidal sites, green crabs were found to damage transplanted eelgrass by cutting the shoots to the extent that some sites demonstrated poor survival. In three separate experiments, eight replicate mesocosm tanks were transplanted with 36 shoots of eelgrass, and different crab densities were introduced into the tanks. The number of shoots damaged by crabs was significantly higher in tanks with moderate (4.0 crabs/m²), high (7.0 crabs/m²), or very high (15.0 crabs/m²) crab densities than in tanks with low (1.0 crabs/m²) crab densities. Up to 39% of viable shoots were lost within one week of exposure to green crab activities. The mesocosm results demonstrated that green crabs were not directly attracted to eelgrass but that they significantly decreased transplant survival through their activity. Field densities of green crabs were found to exceed the density at which most damage occurred in the experiments, suggesting that this introduced species can be a major determinant of eelgrass transplant survival. The results underscore the major influence that biological components of transplant sites can have on transplant survival, and the need for their consideration in the site selection process.     

Eelgrass recovery after nutrient enrichment reversal

Mumford Cove, a 48 ha Connecticut embayment on Long Island Sound, has a history of excessive nutrient inputs and corresponding eutrophic conditions with concomitant eelgrass (Zostera marina L.) loss. From 1945 to 1987, a municipal wastewater treatment facility discharged into the cove. In 1987, when the wastewater outfall was diverted to another location, the cove supported a near monoculture of the green algae Ulva lactuca L., covering 74% of the bottom. By 1988, macroalgal areal cover in Mumford Cove had declined to 9%. When we first sampled the cove in 1992, Z. marina and Ruppia maritima L. were present. By 1999, areal distribution of Z. marina and R. maritima had expanded to cover a third of the bottom. Periodic summertime surveys from 2002 through 2004 indicated that Z. marina was present in approximately half of the cove; R. maritima was sparse. Fifteen years after termination of nutrient enrichment, this cove had recovered from 40 years of point source anthropogenic nutrient input, returning from an Ulva-dominated to a Zostera-dominated state.     

Oral health and access to dental care: a qualitative investigation among older people in the community

doi:10.1111/j.1741‐2358.2009.00320.x
Oral health and access to dental care: a qualitative investigation among older people in the community Objective: The aim of this study was to explore older persons’ beliefs and attitudes towards oral health and access to and use of dental care services. Background: As the proportion of dentate older people increases, the need and demand for dental services will rise (J Public Health Dent, 60, 2000, 276). Design: Focus groups and semi‐structured interviews were used for data collection. Setting and subjects: The study participants included 63 older people in Perth, WA. Results: Five major themes emerged from the interviews – the need for information and knowledge; accessibility of services; cost and affordability of oral care; fear and anxiety regarding dental visits and relationships with dentists. Attitudes and behaviours were slow to change in this group. Conclusion: This investigation provided important perspectives regarding oral health and dental access for older people residing in the community and demonstrated the importance of understanding this group when considering provision and use of services.     

Solution structure of the MID1 B-box2 CHC(D/C)C(2)H(2) zinc-binding domain: insights into an evolutionarily conserved RING fold

The B-box type 2 domain is a prominent feature of a large and growing family of RING, B-box, coiled-coil (RBCC) domain-containing proteins and is also present in more than 1500 additional proteins. Most proteins usually contain a single B-box2 domain, although some proteins contain tandem domains consisting of both type 1 and type 2 B-boxes, which actually share little sequence similarity. Recently, we determined the solution structure of B-box1 from MID1, a putative E3 ubiquitin ligase that is mutated in X-linked Opitz G/BBB syndrome, and showed that it adopted a betabetaalpha RING-like fold. Here, we report the tertiary structure of the B-box2 (CHC(D/C)C(2)H(2)) domain from MID1 using multidimensional NMR spectroscopy. This MID1 B-box2 domain consists of a short alpha-helix and a structured loop with two short anti-parallel beta-strands and adopts a tertiary structure similar to the B-box1 and RING structures, even though there is minimal primary sequence similarity between these domains. By mutagenesis, ESI-FTICR and ICP mass spectrometry, we show that the B-box2 domain coordinates two zinc atoms with a 'cross-brace' pattern: one by Cys175, His178, Cys195 and Cys198 and the other by Cys187, Asp190, His204, and His207. Interestingly, this is the first case that an aspartic acid is involved in zinc atom coordination in a zinc-finger domain, although aspartic acid has been shown to coordinate non-catalytic zinc in matrix metalloproteinases. In addition, the finding of a Cys195Phe substitution identified in a patient with X-linked Opitz GBBB syndrome supports the importance of proper zinc coordination for the function of the MID1 B-box2 domain. Notably, however, our structure differs from the only other published B-box2 structure, that from XNF7, which was shown to coordinate one zinc atom. Finally, the similarity in tertiary structures of the B-box2, B-box1 and RING domains suggests these domains have evolved from a common ancestor.     

A simple centrifugal dehydration force method to characterize water compartments in fresh and post-mortem fish muscle

A centrifugal dehydration force (CDF) method to quantify changes in tissue hydration in fresh and in post-mortem muscular fish tail tissue is presented. The data obtained were used to assess fluid flow rate from tissues and the size of hydration compartments expressed in g water/g dry mass (DM). Curve fit analysis demonstrated that muscle tissue has three detectable water compartments. Application of the method to the fresh fish indicated the presence of a large non-bulk water compartment (3.14 g water/g DM) with a much smaller (0.11 g water/g DM) inner non-bulk water sub-compartment in addition to a comparatively small bulk water compartment (0.99 g water/g DM). At 10 min and at 4h post-mortem, no significant change in size or flow rate of the water compartments was observed. At 24h post-mortem the muscular fish tissue, stored in water, swelled with statistically significant increase in total water and in the bulk water compartment but no significant change in the size of the non-bulk water compartments. The water flow rate from the non-bulk water compartment was, however, increased significantly in the 24h dead tissue. This simple CDF method has application for quantization of bulk and non-bulk water compartments in other biological and non-biological systems.     

Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse Yolk Sac: a comparative study between <Emphasis Type="Italic">in situ</Emphasis> electroporation and retroviral transduction

Background  

Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.