Comparison of six in-house tests for the rapid diagnosis of feline immunodeficiency and feline leukaemia virus infections.

Six rapid tests for the diagnosis of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) infections which have recently been introduced in Europe for use in small animal practice were compared. Eight hundred serum samples were tested and those reacting FIV-positive in at least one of the tests were confirmed by Western blot, and those reacting FeLV-positive were confirmed by virus isolation. The specificity and sensitivity of each test and the quality of the results produced were compared.

Food-anticipatory activity persists after olfactory bulb ablation in the rat.

Restricted daily food access acts as an entraining stimulus (zeitgeber) for a circadian clock, the feeding-entrainable oscillator (FEO). There are many properties of a daily meal that could potentially convey timing information to the FEO. Olfactory cues associated with feeding are one such property. In order to rule out olfaction as a necessary entraining stimulus, olfactory bulbectomized and sham-operated male Sprague--Dawley rats had access to food for 2 h each day. Food bin approach behavior was monitored as an index of food-anticipatory activity (FAA). Both groups entrained to the daily meal with an increase in feeder approach time several hours before meal onset. There were no significant differences in the timing or the amount of FAA between groups. Furthermore, FAA was maintained during 3 days of food deprivation in both groups. In accordance with previous studies, the results show that olfactory cues are not necessary for the entrainment of FAA.

Kinetic isotope effect studies of the reaction catalyzed by uracil DNA glycosylase: evidence for an oxocarbenium ion-uracil anion intermediate.

The DNA repair enzyme uracil DNA glycosylase catalyzes the first step in the uracil base excision repair pathway, the hydrolytic cleavage of the N-glycosidic bond of deoxyuridine in DNA. Here we report kinetic isotope effect (KIE) measurements that have allowed the determination of the transition-state structure for this important reaction. The small primary (13)C KIE (=1.010 +/- 0.009) and the large secondary alpha-deuterium KIE (=1.201 +/- 0.021) indicate that (i) the glycosidic bond is essentially completely broken in the transition state and (ii) there is significant sp(2) character at the anomeric carbon. Large secondary beta-deuterium KIEs were observed when [2'R-(2)H] = 1.102 +/- 0.011 and [2'S-(2)H] = 1.106 +/- 0.010. The nearly equal and large magnitudes of the two stereospecific beta-deuterium KIEs indicate strong hyperconjugation between the elongated glycosidic bond and both of the C2'-H2' bonds. Geometric interpretation of these beta-deuterium KIEs indicates that the furanose ring adopts a mild 3'-exo sugar pucker in the transition state, as would be expected for maximal stabilization of an oxocarbenium ion. Taken together, these results strongly indicate that the reaction proceeds through a dissociative transition state, with complete dissociation of the uracil anion followed by addition of water. To our knowledge, this is the first transition-state structure determined for enzymatic cleavage of the glycosidic linkage in a pyrimidine deoxyribonucleotide.

Stressing-out DNA? The contribution of serine-phosphodiester interactions in catalysis by uracil DNA glycosylase.

The DNA repair enzyme uracil DNA glycosylase (UDG) pinches the phosphodiester backbone of damaged DNA using the hydroxyl side chains of a conserved trio of serine residues, resulting in flipping of the deoxyuridine from the DNA helix into the enzyme active site. We have investigated the energetic role of these serine-phosphodiester interactions using the complementary approaches of crystallography, directed mutagenesis, and stereospecific phosphorothioate substitutions. A new crystal structure of UDG bound to 5'-HO-dUAAp-3' (which lacks the 5' phosphodiester group that interacts with the Ser88 pinching finger) shows that the glycosidic bond of dU has been cleaved, and that the enzyme has undergone the same specific clamping motion that brings key active site groups into position as previously observed in the structures of human UDG bound to large duplex DNA substrates. From this structure, it may be concluded that glycosidic bond cleavage and the induced fit conformational change in UDG can occur without the 5' pinching interaction. The S88A, S189A, and S192G "pinching" mutations exhibit 360-, 80-, and 21-fold damaging effects on k(cat)/K(m), respectively, while the S88A/S189A double mutant exhibits an 8200-fold damaging effect. A free energy analysis of the combined effects of nonbridging phosphorothioate substitution and mutation at these positions reveals the presence of a modest amount of strain energy between the compressed 5' and 3' phosphodiester groups flanking the bound uridine. Overall, these results indicate a role for these serine-phosphodiester interactions in uracil flipping and preorganization of the sugar ring into a reactive conformation. However, in contrast to a recent proposal [Parikh, S. S., et al. (2000) Proc Natl. Acad. Sci. 94, 5083], there is no evidence that conformational strain of the glycosidic bond induced by serine pinching plays a major role in the 10(12)-fold rate enhancement brought about by UDG.

LDL-cholesterol: a risk factor for coronary artery disease--from epidemiology to clinical trials.

Discussion of potential strategies to modify lipids and lipoproteins other than low density lipoproteins (LDLs) should first recognize the convincing evidence in favour of the identification and aggressive treatment of elevated LDL cholesterol (LDL-C) levels in patients with established cardiovascular disease. Elevated LDL-C level is one of the few risk factors for which there is evidence of involvement in every pathophysiological step of the development of cardiovascular disease. Longitudinal studies have established the role of LDL-C as a risk factor for cardiovascular disease incidence, recurrence and fatal outcome. Clinical trials and economic analyses have proven that aggressive treatment of elevated LDL-C in patients at high risk can prevent cardiac events with excellent cost effectiveness.

Characterization of Rickettsia tsutsugamushi strains in two species of naturally infected, laboratory-reared chiggers.

The strains of Rickettsia tsutsugamushi found in naturally infected, laboratory-reared Leptotrombidium (Leptotrombidium) arenicola and L. (L.) fletcheri chiggers were characterized by direct immunofluorescence (FA) and by mouse and monkey virulence tests. The strains existing in the L. (L.) arenicola chiggers consisted of different combinations of TA716, TA763, TA686, Karp, and Kato. In addition to these five strains, Gilliam was found in the L. (L.) fletcheri chiggers. Results indicate that individual chiggers can be simultaneously infected with several antigenic strains of R. tsutsugamushi. Although these antigens appear to remain stable within familial lines when several generations were viewed, the antigenic patterns observed in two succeeding generations did not always correlate. This variable expression of antigens was considered to be due to a quantitative fluctuation from one generation to the next in the strains of rickettsiae combined with a lack of sensitivity of the direct FA test in detecting small numbers of antigenically different rickettsiae. Phenotypic variation was considered to be a less probable explanation. Morbidity and mortality were minimal in ICR mice fed upon by individual chiggers of either species, but infection rates were 85-99%. Tissue suspensions prepared from mice infected by L. (L.) arenicola produced higher mortality and longer duration of illness in mice than those prepared from L. (L.) fletcheri-infected mice. Silvered leaf and cynomolgus monkeys were fed upon by the two species of chiggers or inoculated with the mouse tissue suspensions. In both cases, minimal clinical responses were observed.

Clinical manifestations of mousepox in an experimental animal holding room.

A study of the clinical aspects of mousepox was conducted during the 1979-80 outbreak at the National Institutes of Health. The disease was detected serologically in a room located adjacent to the index room. The index room received animals prior to this outbreak from a noncommercial colony which later was found to be infected with mousepox. The infection was present in the room for at least 6 weeks prior to the completion of the study. The paucity of clinical signs and low mortality were striking when compared to previous descriptions of mousepox in the United States. Only 27 of the 939 mice in the room were infected, and only one of these had typical skin lesions. A few of the mice had non-specific signs such as ruffled hair coat and hunched appearance. Minimal spread of the disease was evidenced by clustering of infected cages on one of five animal racks in the room.

Pathology and diagnosis of mousepox.

The pathologic changes of mousepox were studied during an outbreak at the National Institutes of Health in 1979. The most consistent lesions were necrosis of lymphatic tissues, especially the spleen, lymph nodes, and Peyer's patches. Hepatic necrosis and jejunal hemorrhage also were found. In two transmission studies, the disease was experimentally induced in BALB/cAnN and C3H/HeN-nu mice. Athymic mice were found to be highly susceptible, and they developed fulminant disease. The diagnosis was confirmed by demonstration of pox virions in infected tissues by electron microscopy, staining of viral antigen by immunoperoxidase methods, and by isolation of the virus in chorioallantoic membranes of hen's eggs and in cultures of chick embryonic cells.

Establishment of a cynomolgus monkey (Macaca fascicularis) breeding colony in Malaysia: a feasibility study.

A breeding colony utilizing a harem mating system was established to study the feasibility of breeding cynomoglus monkeys, Macaca fascicularis, in Malaysia. Two groups consisting of 10 females and one male each were evaluated over a 3 period. Forty births were recorded; one was stillborn, 11 died while nursing, and 28 were weaned. The average time to wean offspring was 230 days with an average weight at weaning of 0.858 kg. The average time for conception to take place after weaning was 50 days. Of the 20 breeder females, six produced three offspring each, nine produced two offspring each, four produced one offspring each and one remained barren throughout the project. Three different weaning systems were evaluated. The best method was caging the mother-infant pair within or adjacent to the breeding room followed by a two-part cage system which allowed the infant to continue nursing and also obtain solid food inaccessible to the mother.

Comparison of experimental Rickettsia tsutsugamushi infections in silvered leaf (Presbytis cristatus) and cynomolgus (Macaca fascicularis) monkeys.

Both silvered leaf and cynomolgus monkeys were infected with the Gilliam, Karp and Kato strains of Rickettsia tsutsugamushi. The two species developed similar clinical syndromes, but the antibody responses were greater in cynomolgus monkeys. In both species of monkeys, the Gilliam strain induced more severe clinical manifestations. At 10 months post-infection, silvered leaf monkeys were immune to homologous intradermal (id) challenge. Cynomolgus monkeys, at 15 months post-infection, were relatively resistant to homologous intravenous challenge, but not to a homologous or heterologous id challenge.

Clinical responses of silvered leaf monkeys to infection with selected strains of Rickettsia tsutsugamushi.

Minimal clinical and hematologic signs were observed in silvered leaf monkeys inoculated intradermally with four strains of Rickettsia tsutsugamushi, both virulent and avirulent for laboratory mice. The clinical response of the monkeys to the infection was related to neither the virulence of the strains in mice nor the antigenic characteristics of the strains.

Experimental Rickettsia tsutsugamushi infections in dogs.

Dogs were infected intravenously and intradermally with the Gilliam and Karp strains of R. tsutsugamushi. Although the development of clinical signs was related to the dose of the organism, Gilliam-infected dogs developed severer infections than those infected with Karp. Specific antibodies were demonstrated in sera of experimentally infected dogs.

Decrease in the lethal effect of snake venom by serum of the opossum, Didelphis marsupialis.

Decrease in the lethal effect of several crotalid snake venoms were compared when diluted or mixed with opossum serum, normal horse serum, or normal dog serum and injected into mice. Normal dog and horse serum showed no protective qualities while opossum serum and diluted antivenin were approximately equivalent in their protective effect.