Virustat, a device for continuous production of viruses

Methods for continuous production of viruses, and operation of the virustat, an apparatus in which such production was accomplished, were studied. Continuous production requires a separate continuous host growth chamber, such as the chemostat, and a multiunit virus growth chamber into which the virus-inoculated host cells are led. Successful continuous output of MS-2 and ϕX174 viruses, the latter in lysates, over periods of several days and at titers approximating those of batch lysates, was observed. Design problems include chamber sizes and flow rates, growth of resistant mutants within both virus and host growth chambers, clogging by lysis debris, and the phenomenon of self-inoculation. The latter represents virus growth in the first section of the chamber in excess of the washout rate, leading to lack of need for virus inoculation after an initial period. Use of the virustat for production and research purposes will require some attention to the formation of resistant bacterial colonies at pockets and surface sites of limited washout. With the virustat as a continuous virus production device, continuous purification methods are desirable. Research use of the virustat in continuous mutagenic population studies would require suppression of self-inoculation by use of many sections in the chamber, and improved servo control of host populations at low concentrations.     

Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs

Our objective was to examine the ability of nucleate and anucleate fragments of artificially activated mouse eggs to transform sperm nucleus into male pronucleus. To this end, zona-free oocytes in metaphase II were activated by ethanol and bisected into halves (one with the spindle, the other anucleate) either within 10 to 20 min (series A) or 3 or 5 hr later (series B). In series A, the fragments were inseminated 3,5, and 8 h after activation, and in series B. 3 and 5 h after activation. Both nucleate and anucleate fragments lose the capability of transforming sperm nucleus into fully formed pronucleus sometime between 3 and 5 h after activation. In 8 h old parthenogenetic fragments, the majority of sperm nuclei remain unchanged or begin decondensation but never reach the stage of an early pronucleus. In over 1/3 of anucleate fragments of this age group, sperm nuclei develop defectively: chromatin decondenses inside the persisting nuclear envelope. In other experimental groups, the incidence of these abnormal sperm nuclei varies between 0 and 10%. In general, the anuclcate fragments retain the capability to transform sperm nuclei (fully or partially) longer than their nuclear counterparts. This difference may be accounted for by a different level of substances required for pronuclcar growth (extrachromosomal constituents of the germinal vesicle and nuclear lamins): high and constant in the cytoplasm of anucleate egg halves and low and progressively decreasing in the nucleate halves because of their putative uptake by the female pronucleus. However, the cytoplasmic factors responsible for the initial stages of transformation (nuclear envelope breakdown, chromatin decondensation) become eventually inactivated both in the presence and in the absence of a female pronucleus.     

Effect of pentoxiphylline on oxygen transport during hypothermia

At least two investigators have demonstrated a reduction in O2 extraction during induced hypothermia (Cain and Bradley, J. Appl. Physiol. 55: 1713-1717, 1983; Schumacker et al., J. Appl. Physiol. 63: 1246-1252, 1987). We hypothesized that administration of pentoxiphylline (PTX), a theobromine that lowers blood viscosity and has vasodilator effects, would increase O2 extraction during hypothermia. To test this hypothesis, we studied O2 transport in anesthetized, paralyzed, mechanically ventilated beagles exposed to hypoxic hypoxia during either 1) normothermia (38 degrees C), 2) hypothermia (30 degrees C), or 3) hypothermia + PTX (30 degrees C and PTX, 20 mg.kg-1.h-1). Measurements included arterial and mixed venous PO2, hemoglobin concentration and saturation, cardiac output, systemic vascular resistance (SVR), blood viscosity, and O2 consumption (VO2). Critical levels of O2 delivery (DO2, the product of arterial O2 content and cardiac output) were determined by a system of linear regression. Hypothermia significantly decreased base line cardiac output (-35%), DO2 (-37%), and VO2 (-45%), while increasing SVR and blood viscosity. Addition of PTX increased cardiac output (35%) and VO2 (14%), and returned SVR and blood viscosity to normothermic levels. Hypothermia alone failed to significantly reduce the critical level of DO2, but addition of PTX did [normothermia, 11.4 +/- 4.2 (SD) ml.kg-1.min-1; hypothermia, 9.3 +/- 3.6; hypothermia + PTX, 6.6 +/- 1.3; P less than 0.05, analysis of variance]. The O2 extraction ratio (VO2/DO2) at the critical level of DO2 was decreased during hypothermia alone (normothermia, 0.60 +/- 0.13; hypothermia, 0.42 +/- 0.16; hypothermia + PTX, 0.62 +/- 0.19; P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for two distinct conformations of the Escherichia coli mannitol permease that are important for its transport and phosphorylation functions

Column chromatography of the Escherichia coli mannitol permease (mannitol-specific enzyme II of the phosphotransferase system) in the presence of deoxycholate has revealed that the active permease can exist in at least two association states with apparent molecular weights consistent with a monomer and a dimer. The monomeric conformation is favored by the presence of mannitol and by the phosphoenolpyruvate (PEP)-dependent phosphorylation of the protein. The dimer is stabilized by inorganic phosphate (Pi), which also stimulates phospho-exchange between mannitol and mannitol 1-phosphate (a partial reaction in the overall PEP-dependent phosphorylation of mannitol). Kinetic analysis of the phospho-exchange reaction revealed that Pi stimulates phospho-exchange by increasing the Vmax of the reaction. A kinetic model for mannitol permease function is presented involving both conformations of the permease. The monomer (or a less-stable conformation of the dimer) is hypothesized to be involved in the initial mannitol-binding and PEP-dependent phosphorylation steps, while the stably associated dimer is suggested to participate in later steps involving direct phosphotransfer between the permease, mannitol and mannitol 1-phosphate.     

The specification of heart mesoderm occurs during gastrulation in Xenopus laevis

The establishment of heart mesoderm during Xenopus development has been examined using an assay for heart differentiation in explants and explant combinations in culture. Previous studies using urodele embryos have shown that the heart mesoderm is induced by the prospective pharyngeal endoderm during neurula and postneurula stages. In this study, we find that the specification of heart mesoderm must begin well before the end of gastrulation in Xenopus embryos. Explants of prospective heart mesoderm isolated from mid- or late neurula stages were capable of heart formation in nearly 100% of cases, indicating that the specification of heart mesoderm is complete by midneurula stages. Moreover, inclusion of pharyngeal endoderm had no statistically significant effect upon either the frequency of heart formation or the timing of the initiation of heartbeat in explants of prospective heart mesoderm isolated after the end of gastrulation. When the superficial pharyngeal endoderm was removed at the beginning of gastrulation, experimental embryos formed hearts, as did explants of prospective heart mesoderm from such embryos. These results indicate that the inductive interactions responsible for the establishment of heart mesoderm occur prior to the end of gastrulation and do not require the participation of the superficial pharyngeal endoderm.     

Demonstration of distinct agonist and antagonist conformations of the A1 adenosine receptor

A1 adenosine receptor-binding subunits can be visualized using high affinity antagonist and agonist photoaffinity radioligands. In the present study, we examined whether agonists and antagonists bind to the same receptor-binding subunit and if agonists and antagonists induce different conformational states of the receptor in intact membranes. It was demonstrated that several agonist and antagonist photoaffinity receptor-binding subunit. When the agonist and antagonist photoaffinity labeled peptides were denatured and subjected to partial peptide map analysis using a two-dimensional gel electrophoresis system similar peptide fragments were generated from each specifically labeled protein. This suggests that both classes of ligand label and incorporate into the same binding subunit. Proteolytic digestions of agonist- and antagonist-occupied receptors in native intact membranes revealed distinct and different peptide fragments depending on whether the ligand was an agonist or an antagonist. Manipulation of incubation conditions to perturb ligand-receptor interactions alter the pattern of peptide fragments generated with each specific protease. These data suggest that agonist and antagonist photoaffinity probes interact with an incorporate into the same binding subunit but that agonist binding is associated with a unique and detectable receptor conformation.     

Potent convulsant actions of the adenosine receptor antagonist, xanthine amine congener (XAC)

The convulsant properties of xanthine amine congener (XAC, 8-(4-(2-aminoethyl)-aminocarboxylmethyloxy)phenyl-1,3-dipropylxant hine) are compared to those of caffeine. Male Swiss albino mice were infused with convulsants through a lateral tail vein. Convulsion thresholds (i.e. the amount of convulsants required to elicit convulsions) of 39.8 +/- 2.0 mg/kg (n = 10) and 109.8 +/- 2.3 mg/kg (n = 10) were calculated for XAC and caffeine respectively. Pretreatment of animals with the adenosine receptor agonists 2-chloroadenosine, N6-cyclohexyladenosine or 5'-N-ethylcarboxamido-adenosine (1 mg/kg, i.p., 20 minutes prior to infusion) significantly decreased the seizure threshold of both XAC and caffeine. The adenosine uptake blockers, 6-nitrobenzylthioinosine or dipyridamole (0.25 mg/kg, i.p., 20 minutes prior to infusion) did not significantly affect the seizure threshold to either XAC or caffeine. The benzodiazepine agonist diazepam (5 mg/kg, i.p., 20 minutes prior to infusion) significantly increased the seizure threshold to both XAC (p less than 0.05) and caffeine (p less than 0.01), whereas the benzodiazepine antagonist Ro 15-1788 (10 mg/kg, i.p., 20 minutes prior to infusion) significantly increased the seizure threshold to caffeine (p less than 0.01), but not XAC. The results suggest that actions at benzodiazepine receptors may be a tenable hypothesis to explain the convulsant actions of caffeine, but not those of XAC.