Biomedical subjects
Publications and source records attributed to S M Gray.
Plant viruses transmitted by invertebrate vectors either reversibly bind to vector mouthparts or are internalized by the vector and later secreted. Viral proteins mediate the binding of plant viruses to vector mouthparts and the transport of virus across vector-cell membranes. Both mechanisms probably involve conformational changes of virus proteins during their association with the vector.
Purified particles of barley yellow dwarf luteovirus (BYDV) contain a major 22-kDa protein and a minor protein of approximately 58 kDa. The 22-kDa capsid protein is encoded by open reading frame (ORF) 3. ORF 5 is immediately downstream and in frame with ORF 3 and a 72-kDa protein can be translated via a readthrough suppression of the ORF 3 termination codon. Antibodies were produced against two Escherichia coli expressed polypeptides that represent the amino- and carboxyl-terminal halves of a putative 50-kDa protein encoded by ORF 5. Immunological analyses indicated that the 58-kDa protein associated with purified virions contained sequences encoded by ORF 3 and ORF 5. The carboxyl terminal portion of the full-length (72 kDa) readthrough protein was absent from the 58-kDa protein. The full-length readthrough protein was detected in infected oat protoplasts and plant tissue, but was not associated with virus particles purified from plants. The carboxyl-terminal portion of the 72-kDa readthrough protein was not required for aphid transmission; however, virus was transmitted more efficiently from protoplast extracts containing virions and soluble 72-kDa readthrough protein than from mock-inoculated protoplast extracts to which plant purified virus was added. The full-length readthrough protein, although not required for transmission, may increase the transmission efficiency of BYDV by aphids.
Hexokinase catalyzes the first step in cerebral glucose utilization and is a rate-limiting enzyme in glycolysis. Glucose utilization is tightly coupled to cerebral blood flow so that during ischemia the brain has a decreased supply of glucose, as well as oxygen. We studied hexokinase enzymatic activity in a newborn piglet model of ischemia-reperfusion to determine if any changes in the activity or mitochondrial binding of the enzyme occurred. We observed that mitochondrial binding of cortical HK increased from 55 to 71% with ischemia and returned toward control levels, but did not completely recover, after 2 h of reperfusion.
We assessed whether audit might reduce the time taken to give thrombolysis and aspirin in patients with acute myocardial infarction (N = 116). A retrospective analysis was performed of the sources of delay in giving the drugs (N-60) and the data were presented to clinical staff accompanied by guidelines aimed at eliminating delays. A prospective survey was undertaken (N = 56) after these interventions. Audit resulted in an overall 31% reduction (P = 0.013) in the time to administer thrombolysis (median 55 minutes [range 21-148] v 38 [15-155]): there was a 57% fall (P < 0.0001) in the time to record an electrocardiogram (14 minutes [4-34] v 6 [1-19]) and a 33% decrease (P = 0.047) in the time taken to begin thrombolysis in the coronary care unit (15 minutes [0-110) v 10 [5-70]). The time taken to give aspirin was also reduced (P = 0.001) from 58 minutes (15-400) to 15 (3-235). The time taken to administer thrombolysis and aspirin to patients admitted with acute myocardial infarction can be reduced by audit.
We describe a method for localizing protein domains situated at the surface of virus particles. A cDNA clone of the New York PAV isolate (NY-PAV) of barley yellow dwarf virus (BYDV) containing the capsid protein gene was generated and sequenced. A defined set of overlapping cDNA fragments specific to the capsid protein ORF of NY-PAV was subcloned into the pGEX expression vectors. Cells of Escherichia coli carrying these plasmids synthesize recombinant glutathione S-transferase/capsid proteins. These proteins were used in immunoblot experiments to localize the epitopes of three PAV-BYDV-directed monoclonal antibodies to a 20 amino acid-long segment of the NY-PAV capsid protein. All three monoclonal antibodies reacted with NY-PAV virions in a triple antibody sandwich enzyme-linked immunosorbent assay, indicating that their epitopes are located at the virion surface.
A general diagnostic assay for a number of distinct luteoviruses was developed using the polymerase chain reaction (PCR) and restriction enzyme analysis. Two minimally degenerate, group-specific primers were derived from previously published RNA sequences of three luteoviruses. This primer pair generated specific PCR fragments of about 530 bp from extracts of plants infected with potato leafroll virus, beet western yellows virus, or New York barley yellow dwarf virus (BYDV) serotypes MAV, PAV, RMV, RPV and SGV, which span much of the respective viral coat protein gene. Each virus was easily distinguished from the others by restriction enzyme analysis of the amplified DNA products. Samples from BYDV-infected oat and wheat collected in Nebraska were identified as containing PAV-like serotypes; micro-heterogeneity was detected in several samples. This method provides a rapid, sensitive and relatively inexpensive means of luteovirus detection and identification. It is the first test capable of simultaneously detecting all five BYDV serotypes.
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The formation of rabbit oviduct fluid was monitored continuously by using an in situ vascular perfusion technique. Oviduct fluid was secreted linearly for at least 3 h at a mean rate of 20.8 +/- 1.5 microliter/h in estrous does. The rate more than doubled on Day 1 following mating, was similar to the value at estrus on Day 2, and dropped to 8.3 microliter on Day 3. Dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP, 1 mM) added to the vascular medium abolished fluid secretion. The same response was obtained, after a lag period, following the addition of cholera toxin (1 mM), forskolin (1 mM), theophylline (1 mM), phorbol dibutyrate (40 microM), A23187 (2 micrograms/ml), 4-acetamido-4'-isothiocyonatostilbene-2,2'-disulphonic acid (SITS, 1 mM), and bumetanide (10 microM) to the vascular medium. N-ethylmaleimide (1 mM), which inhibits adenylate cyclase, stimulated oviduct fluid formation. The transmural potential difference (p.d.) across the oviduct was 5.46 +/- 1.01 mV. This was increased after cAMP addition to 8.7 +/- 1.22 mV. The p.d. in oviducts taken 3 days post-ovulation was 7.6 +/- 1.75 mV, and was increased by cAMP to 12.7 +/- 0.53 mV. Exposure to cholera toxin and forskolin almost doubled the cAMP content of the oviduct. The undirectional flux of chloride ions from the vascular compartment into the lumen was reduced by about 75% after the addition of cAMP, SITS, and bumetanide. A tentative model to account for the formation and regulation of rabbit oviduct fluid in terms of ion fluxes and cAMP and calcium ion concentrations is presented.
An in situ preparation for the combined vascular and luminal perfusion of the rabbit oviduct has been developed. Medium 199, gassed with 5% CO2 in O2 and supplemented with heparin, antibiotics, and 2.5% wt/vol dialyzed bovine serum albumin was infused into the ovarian artery at a rate of 1 ml/min. Krebs Ringer bicarbonate medium was recirculated through the lumen at a rate of 50 microliter/min. The ovary was perfused together with the oviduct, and the preparation is viable for up to 3 h. Equal concentrations of pyruvate, lactate, glucose, and sucrose added to the vascular medium were transported at different rates into the lumen, as was a physiological mixture of amino acids. A proportion of the lactate entering the lumen was synthesized within the oviduct from vascular glucose. When glucose and pyruvate were omitted from the vascular medium, their appearance and that of lactate in the lumen was barely detectable, suggesting that these oviduct fluid components are mainly derived from the blood. The oviduct maintained a steady transmural potential difference of 5.9 mV (lumen negative). With vascular perfusion alone, oviduct fluid entered the oviduct lumen at a rate of 16.8 microliter/h. In oviducts taken from rabbits 3 days postovulation, there was a general decrease in the vascular to lumen flux of all nutrients measured. Preliminary work has shown that the preparation may be used to study ovulation, ovum pickup and transport, and fertilization.
Solid-phase first antibody (SP), liquid-phase double-antibody (DA), and pre-precipitated double-antibody (PPT) separation methods have been compared in a radioimmunoassay (RIA) for cortisol in unextracted serum. Both double-antibody methods gave values for pools close to target values assigned by gas chromatography-mass spectrometry (GCMS) whereas the SP assay had a significant negative bias, p less than 0.001 (mean biases: SP -8%, DA -2%, PPT -3%). The SP and DA assays gave average values on patients' samples 12% and 4% lower than values by PPT. These differences could be attributed to different recoveries in the three methods. Between-assay precision of the DA and PPT systems was better than SP (mean CV:SP 11%, DA 7%, PPT 5%). This allowed sensitivity in the PPT assay comparable to that of SP and DA to be achieved, despite the less steep slope of the calibration curve. The DA and PPT assays have several practical advantages over SP. In addition, the PPT assay requires only a 1-hour incubation. It is concluded that the PPT separation system is the method of choice in terms of precision and practical convenience.
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