Some factors involved in the dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by digestive fluids of Trichoplusia ni larvae

Factors involved in the dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus (AcNPV) by digestive fluid collected from fifth stage Trichoplusia ni larvae were studied in vitro. Observations were made at timed intervals using phase contrast microscopy and transmission electron microscopy. When digestive fluid was heated at 50°C proteases retained activity. Exposure of polyhedra to digestive fluid previously heated to 50°C resulted in polyhedral matrix dissolution and envelope disruption in a manner similar to that of unheated digestive fluid, only delayed slightly. After exposure of polyhedra for 3 min, only enveloped virons were observed. Heating the digestive fluid to 60° or higher inactivated the proteases and altered the effect on polyhedra. Dissolution of the occlusion body matrix occurred but the polyhedral envelope remained and only a few weakened areas were observed in its structure. Within the polyhedral envelope, enveloped virons were not observed, only nucleocapsids and capsids. Exposure of polyhedra to 0.1 m sodium carbonate buffer at pHs of 9.5 or higher had effects similar to those of the digestive fluid with heat (60°C)-inactivated proteases. The addition of trypsin and chymotrypsin to the 0.1 m sodium carbonate buffer had no effect, while the addition of a bacterial protease (Streptomyces griseus) at pHs of 9.5 or higher resulted in dissolution of the matrix and disruption of the polyhedral envelope like the digestive fluid. Material infectious to TN-368 cells was obtained by exposure of AcNPV to T. ni digestive fluid. Maximum infectivity resulted from a 5-min exposure to unheated digestive fluid, with a dramatic decrease in infectivity with longer exposure. Exposure to digestive fluid with heat (60°C)-inactivated proteases resulted in a slower release of infectious material from the occlusion body, with a steady increase in the level of infectivity throughout the 30-min digestion period.     

A Cytokinetic Study of Small-Intestinal and Colonic Mucosa After Resection of 70% of the Small Intestine

Abstract. Pulse-labelling with tritiated thymidine and a fraction of labelled mitoses experiments have been performed in order to investigate the proliferative changes induced at various sites in the hyperplastic small-intestinal mucosa of rats previously subjected to resection of 70% of the small intestine. Proliferative activity in the colon was also studied.
In the distal ileum there is a significant reduction in cell cycle time (T_c) of cells at all levels within the crypt and the growth fraction falls. In the jejunum and proximal ileum the crypts contain an increased number of proliferating cells, but as the size of the maturation zone is also increased, there is no significant alteration in the relative number of proliferating cells per crypt. Nor does the distribution of proliferating cells in these crypts seem to alter. There is no general reduction in T_c at these sites, but there does appear to be a significant reduction in T_c on the part of the cells in the stem-cell zone at the crypt base.
In neither proximal nor distal colon was there any significant proliferative change apparent after small-intestinal resection.     

Basis for the potent inhibition of influenza virus infection by equine and guinea pig alpha 2-macroglobulin

The unique properties of equine and guinea pig sera which make them potent inhibitors of influenza virus adsorption and infection have been investigated. The inhibitory activities of both sera are found to reside entirely in their respective alpha 2-macroglobulins, high molecular weight glycoproteins which bind to viral hemagglutinins via sialic acids of their N-linked carbohydrate groups. Structure analysis has shown that both proteins contain 4-O-acetyl-N-acetylneuraminic acid (4-O-Ac-NeuAc) (Hanaoka, K., Pritchett, T. J., Takasaki, S., Kochibe, N., Sabesan, S., Paulson, J.C., and Kobata, A. (1989) J. Biol. Chem. 264, 9842-9849). These 4-O-acetylated sialic acids have been found in few species, making their coincidence with high inhibitory potency in equine and guinea pig alpha 2-macroglobulin striking. However, 4-O-Ac-NeuAc does not appear to increase the avidity of interaction with influenza virus since isolated oligosaccharides of equine alpha 2-macroglobulin are no more potent inhibitors of adsorption than isolated oligosaccharides of human alpha 2-macroglobulin, which is a relatively poor inhibitor and contains only NeuAc. Since 4-O-Ac-NeuAc is resistant to cleavage by viral sialidase it may serve to protect the inhibitor from inactivation. These and supporting results suggest that the key property of equine and guinea pig alpha 2-macroglobulin which make them high potency inhibitors is a spatial arrangement of sialic acid containing oligosaccharide groups which allows optimal interaction with multiple hemagglutinins. The implications of these results for the design of low molecular weight inhibitors of influenza virus infection are discussed.     

Structural and functional characterization of the gamma 1 subunit of GABAA/benzodiazepine receptors.

The GABAA receptor gamma 1 subunit of human, rat and bovine origin was molecularly cloned and compared with the gamma 2 subunit in structure and function. Both gamma subunit variants share 74% sequence similarity and are prominently synthesized in often distinct areas of the central nervous system as documented by in situ hybridization. When co-expressed with alpha and beta subunits in Xenopus oocytes and mammalian cells, the gamma variants mediate the potentiation of GABA evoked currents by benzodiazepines and help generate high-affinity binding sites for these drugs. However, these sites show disparate pharmacological properties which, for receptors assembled from alpha 1, beta 1 and gamma 1 subunits, are characterized by the conspicuous loss in affinity for neutral antagonists (e.g. flumazenil) and negative modulators (e.g. DMCM). These findings reveal a pronounced effect of gamma subunit variants on GABAA/benzodiazepine receptor pharmacology.     

Human colorectal tumours in short-term organ culture A stathmokinetic study

Abstract. Short-term organ culture, using a technique to preserve epithelial/stromal interaction and metabolism, is a useful technique for carrying out kinetic studies on human colorectal carcinoma and adjacent normal mucosa, providing initial perturbations of proliferative indices are allowed to settle. Tumours require 3.0 μg/ml vincristine for complete metaphase arrest compared with mucosa, which needs 0.5 μg/ml, a 6-fold difference. Using a stathmokinetic technique, the birth rate of tumour cells is 10.21 cells/1000 cells per hr, compared with 7.73 cells/1000 cells per hr for mucosa, a statistically significant difference ( P < 0.01).     

Dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by the digestive fluid of Trichoplusia ni (Lepidoptera:Noctuidae) larvae

The dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus by digestive fluid collected from 5th stage Trichoplusia ni larvae was studied in vitro. Observations were made at timed intervals using phase contrast microscopy, and scanning and transmission electron microscopy. Dissolution occurred rapidly and in a detectable sequence. Under phase contrast, most polyhedra lost their refringence by 0.5 min. The polyhedra became rounded in appearance with small protuberances on the surface and Brownian movement was observed within. After 1 min, the envelope of most polyhedra had ruptured, releasing the enclosed virions. The protuberances were also observed under the scanning electron microscope after digestion for 0.5 min. Many shell fragments devoid of internal contents were seen after more lengthy digestion. Internal structural changes were revealed by electron microscopy. After 1 min of exposure, polyhedra were observed in all stages of dissolution. By 3 min, only virions, scattered about in heterogeneous material, could be distinguished.     

DNA nucleotide sequence heterogeneity between the Towne and AD169 strains of cytomegalovirus.

The results of reciprocal DNA-DNA reassociation kinetics indicated that although the DNAs of human cytomegalovirus (CMV) strains Towne and AD169 shared approximately 90% of their nucleotide sequences, about 10% heterogeneity did exist. The implication was that, with respect to one another, the DNAs of CMV Towne and CMV AD169 contained unique nucleotide sequences. To obtain more direct evidence, 32P-labeled DNA of one virus strain was reassociated in the presence of excess unlabeled DNA of the heterologous virus strain. Those 32P-labeled DNA sequences remaining single stranded were separated from double-stranded DNA on hydroxyapatite columns and incubated with Southern blots containing XbaI restriction enzyme fragments of the homologous virus DNA. This approach not only enriched for nucleotide sequences unique to each strain of virus, but also provided for the identification of the restriction enzyme fragments in which the unique sequences were contained. The CMV Towne unique sequences were found in XbaI fragments A, C, G, L, N, and Q of CMV Towne DNA. The CMV AD169 unique sequences were found in XbaI fragments A, C, G, and J of CMV AD169 DNA. The possible significance of these data with respect to variation among other CMV isolates is discussed.     

Proteins of Epstein-Barr virus. I. Analysis of the polypeptides of purified enveloped Epstein-Barr virus.

Epstein-Barr virus (EBV) was purified from the extracellular fluid of HR-1 and B95-8 cell lines. The preparations of purified virus consisted of enveloped particles and had EBV-specific antigneic reactivity. Comparison of the amount of labeled protein in preparations of virus purified from cultures incubated in [35S]methionine with the amount of labeled protein in preparations obtained following a mixture of unlabeled virus with [35S]methionine-labeled cellular proteins indicated that less than 2% of the labeled protein in the purified virus preparation could be attributed to contamination with labeled cellular proteins. No extraneous membranous material was seen in thin sections of the purified virus preparations. Analysis of the polypeptides of purified enveloped EBV indicated the following. (i) Eighteen polypeptides could be resolved in Coomassie brilliant blue-stained electropherograms of extracellular virus purified from HR-1 and B95-8 cultures. (ii) Thirty-three polypeptides could be resolved in fluorograms of labeled EBV purified from B95-8 cultures and subjected to electrophoresis in acrylamide gels cross-linked with diallyltartardiamide. The molecular weight of the EBV polypeptides was estimated by co-electrophoresis with the polypeptides of purified herpes simplex virus and purified polypeptides of known molecular weight to range from 28 x 10(3) to approximately 290 x 10(3) (iii) The polypeptides of EBV could be grouped by their relative molar abundancy into three classes: VP6, 7, and 27 present in high abundance; VP1, 12, 20, 23, and 29 present in moderate abundance; and a third class of less abundant polypeptides, VP4, 5, 8, 9, 10, 11, 15, 16, 21, and 22. The remainder of the polypeptides could not be precisely quantitated. (iv) The polypeptides of purified EBV, although similar in number and in range of molecular weight to the polypeptides of purified herpes simplex virus, differ sufficiently from those of herpes simplex virus so as to preclude comparison of individual polypeptide components.     

Roux-en-Y Gastric Bypass Increases Intravenous Ethanol Self-Administration in Dietary Obese Rats

Roux-en-Y gastric bypass surgery (RYGB) is an effective treatment for severe obesity. Clinical studies however have reported susceptibility to increased alcohol use after RYGB, and preclinical studies have shown increased alcohol intake in obese rats after RYGB. This could reflect a direct enhancement of alcohol’s rewarding effects in the brain or an indirect effect due to increased alcohol absorption after RGYB. To rule out the contribution that changes in alcohol absorption have on its rewarding effects, here we assessed the effects of RYGB on intravenously (IV) administered ethanol (1%). For this purpose, high fat (60% kcal from fat) diet-induced obese male Sprague Dawley rats were tested ∼2 months after RYGB or sham surgery (SHAM) using both fixed and progressive ratio schedules of reinforcement to evaluate if RGYB modified the reinforcing effects of IV ethanol. Compared to SHAM, RYGB rats made significantly more active spout responses to earn IV ethanol during the fixed ratio schedule, and achieved higher breakpoints during the progressive ratio schedule. Although additional studies are needed, our results provide preliminary evidence that RYGB increases the rewarding effects of alcohol independent of its effects on alcohol absorption.     

Deletion of Metabotropic Glutamate Receptors 2 and 3 (mGlu2 & mGlu3) in Mice Disrupts Sleep and Wheel-Running Activity,and Increases the Sensitivity of the Circadian System to Light

Sleep and/or circadian rhythm disruption (SCRD) is seen in up to 80% of schizophrenia patients. The co-morbidity of schizophrenia and SCRD may in part stem from dysfunction in common brain mechanisms, which include the glutamate system, and in particular, the group II metabotropic glutamate receptors mGlu2 and mGlu3 (encoded by the genes Grm2 and Grm3). These receptors are relevant to the pathophysiology and potential treatment of schizophrenia, and have also been implicated in sleep and circadian function. In the present study, we characterised the sleep and circadian rhythms of Grm2/3 double knockout (Grm2/3^-/-) mice, to provide further evidence for the involvement of group II metabotropic glutamate receptors in the regulation of sleep and circadian rhythms. We report several novel findings. Firstly, Grm2/3^-/- mice demonstrated a decrease in immobility-determined sleep time and an increase in immobility-determined sleep fragmentation. Secondly, Grm2/3^-/- mice showed heightened sensitivity to the circadian effects of light, manifested as increased period lengthening in constant light, and greater phase delays in response to nocturnal light pulses. Greater light-induced phase delays were also exhibited by wildtype C57Bl/6J mice following administration of the mGlu2/3 negative allosteric modulator RO4432717. These results confirm the involvement of group II metabotropic glutamate receptors in photic entrainment and sleep regulation pathways. Finally, the diurnal wheel-running rhythms of Grm2/3^-/- mice were perturbed under a standard light/dark cycle, but their diurnal rest-activity rhythms were unaltered in cages lacking running wheels, as determined with passive infrared motion detectors. Hence, when assessing the diurnal rest-activity rhythms of mice, the choice of assay can have a major bearing on the results obtained.