Ultrastructural Pathology of Murine Cytomegalovirus Infection in Cultured Mouse Nervous System Tissue

Mouse spinal cord-ganglia cultures were innoculated with murine cytomegalo-virus 14 days after explantation. Intranuclear virus was first observed 4 days after infection. The viruses, which occurred in four forms, were observed in increasing numbers during the ensuing 4 days. Differences were noted in the relative prevalence of certain of these forms in older as compared to younger cultures. This suggests that variations in virus form are related to virus maturation. Cytoplasmic viruses were occasionally observed, but their site of origin is not certain. A variety of cytoplasmic inclusions were seen, particularly in the older cultures. It seems likely that they represent specific cell responses to the presence of the virus. They were not observed in the control cultures, even though some of the latter did show severe degenerative changes.     

Poly(ethylene glycol)-containing Hydrogels for Oral Protein Delivery Applications

Novel pH-sensitive hydrogels were developed as suitable candidates for carriers in bioMEMS devices as well as for oral delivery of therapeutic peptides and proteins due to their ability to respond to environmental pH change. Macromonomers containing various PEG molecular weights were synthesized and used to prepare P(MAA-g-EG) hydrogels were by photopolymerization. P(MAA-g-EG) hydrogels showed a drastic change of the equilibrium swelling ratio between pH 2.2 and 7.0. At pH 7.0, hydrogels with PEGMA2000 exhibited higher swelling ratio than hydrogels with PEGMA1000. For both hydrogels with PEGMA1000 and PEGMA2000, the swelling mechanism became more relaxation-controled as the environmental pH changed from 2.2 to 7.0 due to the ionization of the functional groups in polymer networks at high pH. In vitro release studies of insulin were conducted. P(MAA-g-EG) hydrogels exhibited drastic increase of insulin release as the pH of the medium was changed from acidic to basic. Insulin release from P(MAA-g-EG) hydrogels with PEGMA2000 was slower than from hydrogels with PEGMA1000 at both low and high pH. These results were used to design and improve protein release behavior from these carriers.     

Comparison of Ashdown's medium, Burkholderia cepacia medium, and Burkholderia pseudomallei selective agar for clinical isolation of Burkholderia pseudomallei

Ashdown's medium, Burkholderia pseudomallei selective agar (BPSA), and a commercial Burkholderia cepacia medium were compared for their abilities to grow B. pseudomallei from 155 clinical specimens that proved positive for this organism. The sensitivity of each was equivalent; the selectivity of BPSA was lower than that of Ashdown's or B. cepacia medium.     

Mating ability in laboratory-adapted and field-derivedDrosophila melanogaster: The stress of domestication

Mating ability differences between flies of different alcohol dehydrogenase (Adh) genotypes have been assessed in the temperature range 15 to 29°C for laboratory-adapted and field-derivedDrosophila melanogaster. Significant differences amongAdh genotypes were detected principally for the laboratory-adapted strains due to departures from random mating associated with heterozygote superiority at the relatively extreme temperature of 29°C, although mating ability differences could not be attributed directly to theAdh locus. The difference between the laboratory and the field populations can be explained by the effects of genetic back-ground manifested in the form of fitness differences, being enhanced for the laboratory-adapted flies as a consequence of the stress of laboratory culture. In contrast with larval survival and development time, laboratory and field flies do not differe appreciably in their overall abilities to obtain mates, which indicates that mating ability is a direct fitness character not greatly affected by laboratory culture. It follows that direct fitness traits are the least amenable to change under domestication.     

UNILATERAL BRAIN DAMAGE AND BILATERAL SKIN CONDUCTANCE LEVELS IN HUMANS

Left and right, palmar and dorsal skin conductance levels (SCLs) were obtained from hospital controls, left hemisphere lesion Ss, right hemisphere lesion Ss, and diffuse or bilateral lesion Ss during several experimental conditions involving rest, passive auditory stimulation, motor reactions, and simple “perception”. The unilateral lesion groups generally displayed significantly higher palmar SCLs on the side contralateral to their lesion. Such “laterality” was not demonstrated in dorsal recordings or in the hospital controls or diffuse lesion group. These unilateral lesion groups had higher palmar SCLs during passive stimulation than during rest, motor, or perception phases. Results were discussed in terms of possible neural mechanisms underlying the phenomena.     

Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of immunogenicity following liver fluke infection

The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.     

Oligopeptidases of brush border membranes of rat small intestinal mucosal cells

1. The properties of peptidases located in the brush borders of rat small intestinal mucosal cells have been investigated using a new method for the assay of peptidase activity. In this method amino acids produced by hydrolysis of peptides are oxidized by ophidian L-amino acid oxidase and the hydrogen peroxide produced during reoxidation of the reduced enzyme is estimated spectrophotometrically.2. 10-20% of the total cellular peptidase activity and 50% of the leucylnaphthylamidase (LNAase) activity were found to be tightly bound to the brush borders and to possess different substrate specificity from the supernatant peptidase activity.3. Evidence from kinetic and competition studies indicates the presence of more than one peptidase in the brush borders. The peptidases exhibit pH optima of 8.0-8.5, are inhibited by EDTA, but are not usually activated by divalent metal ions. The brush border peptidases hydrolyse di- and tripeptides, some oligopeptides, leucinamide and leucyl-beta-naphthylamine. On the basis of the Michaelis constants, these substrates differ in their affinities for the enzymes.4. It is proposed that the brush border peptidases serve an analogous function in the terminal stages of protein digestion to that of the disaccharidases in the case of carbohydrate digestion and absorption.     

Characterization of the major secreted zinc metalloprotease- dependent glycerophospholipid:cholesterol acyltransferase, PlaC, of Legionella pneumophila

Legionella pneumophila, an intracellular pathogen causing a severe pneumonia, possesses distinct lipolytic activities which have not been completely assigned to specific enzymes so far. We cloned and characterized a gene, plaC, encoding a protein with high homology to PlaA, the major secreted lysophospholipase A of L. pneumophila and to other hydrolytic enzymes belonging to the GDSL family. Here we show that L. pneumophila plaC mutants possessed reduced phospholipase A and lysophospholipase A activities and lacked glycerophospholipid:cholesterol acyltransferase activity in their culture supernatants. The mutants' reduced phospholipase A and acyltransferase activities were complemented by reintroduction of an intact copy of plaC. Additionally, plaC conferred increased lysophospholipase A and glycerophospholipid:cholesterol acytransferase activities to recombinant Escherichia coli. Furthermore, PlaC was shown to be another candidate exported by the L. pneumophila type II secretion system and was activated by a factor present in the bacterial culture supernatant dependent on the zinc metalloprotease. Finally, the role of plaC in intracellular infection of Acanthamoeba castellanii and U937 macrophages with L. pneumophila was assessed, and plaC was found to be dispensable. Thus, L. pneumophila possesses another secreted lipolytic enzyme, a protein with acyltransferase, phospholipase A, and lysophospholipase A activities. This enzyme is distinguished from the previously characterized phospholipases A and lysophospholipases A by its capacity not only to cleave fatty acids from lipids but to transfer them to cholesterol. Cholesterol is an important compound of eukaryotic membranes, and an acyltransferase might be a tool for host cell modification to fit the needs of the bacterium.     

Multivariate Genetic Analysis of Chronic Pelvic Pain and Associated Phenotypes

Chronic pelvic pain (CPP) is a common condition in women that is difficult to diagnose. Although heritability estimates have been published for some conditions potentially underlying pelvic pain, the heritability of CPP itself has never been investigated. Using data from 623 MZ and 377 DZ female twin pairs aged 29–50 from an Australian twin cohort, we found an increased CPP concordance among MZs compared to DZs, with tetrachoric correlations of 0.43 (95% CI: 0.26–0.58) and 0.11 (95% CI: –0.16–0.38), respectively. This corresponded to a heritability of 0.41 (95% CI: 0.25–0.56). Lack of correlations with environmental indicators suggested that violation of the equal environments assumption was not responsible for this effect. Multivariate Cholesky decomposition models incorporating CPP and significantly correlated phenotypes showed that the entire CPP heritability could be explained by genetic variance underlying endometriosis (38%), dysmenorrhoea (23%), fibroids (24%), and somatic distress (15%), the latter a possible indicator of increased nociception. CPP itself is unlikely to be a useful independent phenotype to conduct genetic aetiological studies; contributing conditions such as endometriosis and variation in nociception are likely to provide more useful phenotypes.