Genomic and functional analysis of Vibrio phage SIO-2 reveals novel insights into ecology and evolution of marine siphoviruses.

We report on a genomic and functional analysis of a novel marine siphovirus, the Vibrio phage SIO-2. This phage is lytic for related Vibrio species of great ecological interest including the broadly antagonistic bacterium Vibrio sp. SWAT3 as well as notable members of the Harveyi clade (V.harveyi ATTC BAA-1116 and V.campbellii ATCC 25920). Vibrio phage SIO-2 has a circularly permuted genome of 80598 bp, which displays unusual features. This genome is larger than that of most known siphoviruses and only 38 of the 116 predicted proteins had homologues in databases. Another divergence is manifest by the origin of core genes, most of which share robust similarities with unrelated viruses and bacteria spanning a wide range of phyla. These core genes are arranged in the same order as in most bacteriophages but they are unusually interspaced at two places with insertions of DNA comprising a high density of uncharacterized genes. The acquisition of these DNA inserts is associated with morphological variation of SIO-2 capsid, which assembles as a large (80 nm) shell with a novel T=12 symmetry. These atypical structural features confer on SIO-2 a remarkable stability to a variety of physical, chemical and environmental factors. Given this high level of functional and genomic novelty, SIO-2 emerges as a model of considerable interest in ecological and evolutionary studies.

In vitro, animal, and human characterization of OPTISON infusions for myocardial contrast echocardiography.

UNLABELLED: Traditionally, performing myocardial contrast echocardiography with OPTISON required maximal bolus dosing. However, sustained and consistent opacification of the myocardium would be preferable for perfusion imaging. METHODS: Images of 5 anesthetized dogs and 6 human volunteers were obtained with a second harmonic ultrasound system during bolus administration of OPTISON and 2 infusion techniques. One infusion technique used diluted OPTISON, and the other used the buoyant properties of OPTISON microspheres by placing the contrast agent between an infusion source and the intravenous site in a vertically oriented extension line (ELT). Myocardial intensities and in vitro microsphere characteristics were analyzed to assess the consistency of microsphere delivery over time. RESULTS: In addition to providing higher myocardial opacification intensity than diluted infusions, ELT infusions provided consistent microsphere concentration, phantom enhancement, and near-peak bolus-level myocardial opacification for 7 to 15 minutes. The myocardial intensity at 3 and 5 minutes in human subjects during ELT infusions (30 mL/h; 2.5 mL) was lower (220 arbitrary units [au] and 165 au, respectively) but not significantly different (P =.3 and.1, respectively) than the peak myocardial intensity (265 au) after bolus administration. CONCLUSION: This new ELT infusion method provides an acceptable alternative to bolus administration of OPTISON for prolonged myocardial opacification.

Physical and biochemical stability of Optison, an injectable ultrasound contrast agent.

Optison(R) is an ultrasound contrast agent, consisting of gas-filled microspheres surrounded by a solid shell of heat-denatured human albumin. Size-distribution measurements of these microspheres are a critical stability indicating factor, because loss of encapsulated gas eliminates ultrasound contrast activity. Composition of the encapsulated gas is also critical, because air-filled microspheres do not persist nearly as long in vivo as microspheres filled with less soluble gases. Optison(R) stability has been tested during exposure to chemical substances expected to dissolve microsphere shells. In addition, size-distribution and gas-composition measurements were used to evaluate the effects of external gas composition, elevated temperature, mixing, needle shear and pressure on product stability. Optison(R) microsphere shells dissolve only when exposed to relatively extreme chemical conditions, such as low pH (<4.0), detergents or chaotropic salts. The shells are highly gas-permeable, and microspheres lose encapsulated gas rapidly and irreversibly when exposed to gas-deficient liquids. Pressure, impact stress, and the application of ultrasound energy all cause liquids to become gas-deficient, and also cause irreversible gas loss. Pressure sensitivity differs dramatically between mixed and unmixed microspheres, further supporting the conclusion that gas diffusion is the major cause of Optison(R) instability. To preserve the efficacy of Optison(R) as an ultrasound contrast agent, it is necessary to devote special attention to minimizing opportunities for gas exchange, mixing and exposure to gas-deficient liquids, so that the size distribution and gas composition of the original product are maintained during handling.

Euglycemic control of gestational diabetes mellitus by specific dietary manipulation: a case study presentation.

Gestational diabetes is the most common complication of pregnancy. If maternal hyperglycemia is not well controlled, excess glucose is transmitted to the fetus, which can lead to fetal macrosomia and maternal and fetal complications. Dietary treatment for gestational diabetes varies among practitioners. A case review is presented of a 32-year-old white woman with gestational diabetes whose condition was complicated by her blood glucose intolerance to lactose in milk. By following a carefully monitored regimen using specific dietary manipulation to maintain normoglycemia, the woman was able to deliver a normal, healthy baby by spontaneous vaginal delivery.

Comparison of solution hybridization efficiencies using alkaline phosphatase-labelled and 32P-labelled oligodeoxynucleotide probes.

The hybridization efficiencies of oligonucleotide probes directly labelled with alkaline phosphatase and probes labelled with 32P were compared by quantitating the enzyme activity or radioactivity associated with hybridization targets over time. The targets tested included both synthetic oligonucleotides (53 bases in length) and single-stranded and double-stranded cloned M13 DNA (7350 bases long). Hybrid molecules were separated from unhybridized probes using size exclusion FPLC. This system allowed quantitative analysis of the time course and efficiency of hybridization for both probes and targets in complex hybridization media containing protein blocking agents, formamide, and carrier DNA. Similar maximum hybridization efficiencies were attained for probes labelled with either radioactivity or alkaline phosphatase as a marker. The reaction rate constant for oligonucleotide hybridization to long M13 targets was 3.6 x 10(5) mol-1 s-1 for a probe labelled with alkaline phosphatase, and 5.8 x 10(5) mol-1 s-1 for the same probe labelled with 32P.

The sequence of chicken c-yes and p61c-yes.

We have deduced the sequence of the protein encoded by the chicken c-yes gene from overlapping cDNA clones. The predicted protein, p61c-yes, contains 541 amino acids and has a molecular weight of 60,911 with the amino terminal methionine residue. Chicken p61c-yes differs from Y73 virus p90gag/v-yes in three respects. First, the carboxy-terminal eight amino acids of p61c-yes are replaced by three amino acids in p90gag/v-yes, which are encoded by the avian leukemia virus env gene. This alteration changes the position and context of a tyrosine residue in p61c-yes. Second, nucleotides which are present as 5' non-translated sequence in the p61c-yes mRNA, are translated in the p90gag/v-yes mRNA. Third, there are fourteen dispersed nucleotide differences in Y73 v-yes which result in six amino differences between the body of p90gag/v-yes and p61c-yes. Chicken p61c-yes differs from human p61c-yes at 43 residues, and from chicken pp60c-src at 122 residues.

Chicken proto-oncogene c-ros cDNA clones: identification of a c-ros RNA transcript and deduction of the amino acid sequence of the carboxyl terminus of the c-ros product.

Three overlapping cDNA clones that hybridized to a v-ros probe were isolated from a cDNA library constructed from chicken kidney mRNA. Sequence analysis of these clones showed that they were all derived from c-ros mRNA. Using hybridization probes synthesized from the cDNA clones, a c-ros mRNA transcript of approximately 8.3 kb was detected in chicken kidney RNA, but not in chick embryo fibroblast RNA. The amino acid sequence predicted from the cDNA sequence indicates that the carboxyl terminus of the chicken c-ros protein contains 58 amino acids which are not present in v-ros. The predicted amino acid sequence of the chicken c-ros protein differs by 25% from that of its closest known human counterparts within the tyrosine protein kinase catalytic domain, and by 66% downstream of this domain. Despite these differences, both the chicken and human amino acid sequences share a potential site of tyrosine phosphorylation near their carboxyl termini that is absent from v-ros.

Purification of the Mr 80,000 and Mr 210,000 proteins of the sea urchin sperm plasma membrane. Evidence that the Mr 210,000 protein interacts with egg jelly.

Two immunologically cross-reactive plasma membrane proteins, of Mr 80,000 and Mr 210,000, have been purified to apparent homogeneity from sperm of the sea urchin Strongylocentrotus purpuratus. Purification includes a combination of antibody and wheat germ agglutinin affinity chromatography. The two proteins have similar but not identical amino acid compositions; however, their carbohydrate composition differs substantially. After purification, the Mr 210,000 protein binds to both living eggs and isolated egg jelly in a species-specific manner, but the Mr 80,000 protein does not. The inactivity of the Mr 80,000 protein could be due to denaturation during purification. The data are consistent with a model in which the Mr 210,000 protein acts as a jelly receptor in the sperm membrane, promoting the ion movements necessary to initiate the sperm acrosome reaction.

Inhibition of sea urchin sperm acrosome reaction by antibodies directed against two sperm membrane proteins. Characterization and mechanism of action.

The egg jelly-induced acrosome reaction is inhibited by polyclonal antibodies raised against either of two S. purpuratus sperm-membrane proteins, of Mr 80 and 210 kD. Although the two antigens used have dissimilar CNBr peptide maps, antisera produced against each of them cross-react with both proteins. Inhibition of the egg jelly-induced acrosome reaction by the antisera is bypassed by a combination of the ionophores monensin and A23187. This result, along with data showing that the antisera inhibit egg jelly-induced uptake of 45Ca2+, suggests that the antisera may block both Ca2+ uptake and Na+/H+ exchange in the sperm. The acrosome reaction blockage appears to be caused by the same component of the polyclonal sera responsible for cross-reaction; consequently, these antisera cannot be used to determine whether one or both of the cross-reacting proteins modulate a critical step in the acrosome reaction.

Wheat germ agglutinin blocks the acrosome reaction in Strongylocentrotus purpuratus sperm by binding a 210,000-mol-wt membrane protein.

Wheat germ agglutinin (WGA) binds to the entire surface of Strongylocentrotus purpuratus sperm, and inhibits the egg jelly-induced acrosome reaction. The binding was found to be species dependent and was completely inhibited by 5 mM N-acetyl-D-glucosamine. Blockage of the acrosome reaction by WGA was bypassed by a combination of the ionophores A23187 and monensin, although neither ionophore was effective individually. These experiments suggest that WGA blocks both Ca2+ uptake and Na+/H+ exchange in these sperm, which was confirmed by direct measurements of 45Ca2+ uptake and H+ efflux. The target of WGA in S. purpuratus sperm appears to be a membrane glycoprotein of Mr = 210,000. Treatment of this protein with neuraminidase or endo-beta-N-acetylglucosaminidase F abolished WGA binding.

Isolation and characterization of a plasma membrane fraction from sea urchin sperm exhibiting species specific recognition of the egg surface.

A method is described for isolating preparative quantities of plasma membranes from sea urchin sperm. The final membrane fraction is homogeneous by sucrose density sedimentation and is enriched in adenylate cyclase as well as in the four glycoproteins accessible to radioiodination of intact sperm. The electrophoretic profiles of sperm membranes from three sea urchin species are very similar. The membrane preparation consists primarily of sealed vesicles which release carboxyfluorescein when exposed to detergents or distilled water. Ninety-two percent of the 125I-labeled vesicle material binds to wheat germ lectin columns, suggesting a right-side-out orientation. The isolated sperm membrane vesicles exhibit species specific adhesion to the surfaces of sea urchin eggs; this adhesion is blocked by pretreatment of the vesicles with trypsin or egg jelly. This method will be useful for isolating biologically active sperm membrane components involved in sperm-egg recognition during fertilization.

Effectiveness of hypertension screening in an urban optometric clinic.

461 randomly selected eyecare patients were screened for systemic hypertension at the clinic of the University Optometric Center, State College of Optometry, State University of New York. The results of this survey showed 52 screening failures, 30 of whom had previously been identified as having hypertension. The study showed that the screening and follow-up added $2.00 per patient to the chair cost. The authors concluded that the information provided to our patients, students and practitioners warrants this cost.