Systemic arteriopathy in SIV-infected rhesus macaques (Macaca mulatta)

BACKGROUND: Severe disseminated vasculopathy was observed in two simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta). These animals developed clinical signs of AIDS, including lymphadenopathy, weight loss, diarrhea and collapse. RESULTS AND DISCUSSION: Grossly, both animals showed emaciation, lymphadenopathy, vegetations on the mitral valve, renal infarcts and a dilated intestine; one animal had multifocal hemorrhages in multiple organs. Histologically, both cases had disseminated arteriopathy characterized by intimal thickening and fibrosis with varying degrees of vasculitis. The lesion was prominent in the kidney, intestine, pancreas, liver, heart, lymph nodes, spleen and testis. Occasional venules had intimal thickening. Both cases had cytomegalovirus (CMV) infection with intranuclear inclusions, CMV antigen and nucleic acid; some inclusions were observed in endothelial cells within some of the vascular lesions in one of the two. These data suggest that CMV caused the unusual lesions.     

A Retraining Program for Inactive Physicians

During the past two years a pilot project was conducted in which 19 inactive physicians were retrained in preparation for resumption of active practice. The initial program consisted of a flexible training program of six months to one year patterned after conventional internship-residency concepts. During the second year the program was modified by providing an initial condensed indoctrination period of two months'' duration especially designed for this purpose, followed by a preceptorship type of training.The project was considered successful in permitting trainees to enter some form of active medical work, or to enroll in formal specialty training. The observations made by the faculty of the program and its accomplishments are discussed in the light of the effort expended and the cost of the project.     

Phosphorylation of mCRY2 at Ser557 in the Hypothalamic Suprachiasmatic Nucleus of the Mouse

Cryptochrome1 and 2 play a critical role in the molecular oscillations of the circadian clocks of central and peripheral tissues in mammals. Mouse Cryptochrome2 (mCRY2) is phosphorylated at Ser557 in the liver, in which the Ser557-phosphorylated form accumulates during the night in parallel with mCRY2 protein. Phosphorylation of mCRY2 at Ser557 allows subsequent phosphorylation at Ser553 by glycogen synthase kinase-3β (GSK-3β), resulting in efficient degradation of mCRY2 by a proteasome pathway. In the present study, we found that mCRY2 is phosphorylated at Ser557 also in the region of the mouse brain containing the suprachiasmatic nucleus (SCN), the central circadian clock tissue. Daily fluctuation of the Ser557-phosphorylation level in the SCN region suggests an important role of sequential phosphorylation of Ser557 and Ser553 in the rhythmic degradation of mCRY2 in both central and peripheral clocks of mice.     

Extraordinary proliferation of microorganisms in aposymbiotic pea aphids,Acyrthosiphon pisum

Aposymbiotic pea aphids, which were deprived of their intracellular symbiotic bacterium, Buchnera, exhibit growth retardation and no fecundity. High performance liquid chromatographic (HPLC) analysis revealed that these aposymbiotic aphids, when reared on broad bean plants, accumulated a large amount of histamine. To assess the possibility of extraordinary proliferation of microorganisms other than Buchnera, we enumerated eubacteria and fungi in aphids using the real-time quantitative PCR method that targets genes encoding small-subunit rRNAs. The result showed that these microorganisms were extremely abundant in the aposymbiotic aphids reared on plants. Microbial communities in aposymbiotic aphids were further profiled by phylogenetic analysis of small-subunit rDNAs. Of 172 nonchimeric sequences of fungal 18S rDNAs, 138 (80.2%) belonged to the phylum Ascomycota. Among them, 21 clustered within a monophyletic group consisting of insect-pathogenic fungi and yeast-like symbionts of homopteran insects. Thirty-one (18.0%), two (1.2%), and one (0.6%) clones were clustered within the Basidiomycota, Zygomycota, and Oomycota, respectively. Of 167 nonchimeric sequences of eubacterial 16S rDNAs, 84 (50.3%) belonged to the gamma-subdivision of Proteobacteria to which most primary endosymbionts of insects and prolific histamine producers belong. Forty (24.0%), 25 (15.0%), 10 (6.0%), and five (3.0%) clones were clustered within alpha-Proteobacteria, Cytophaga-Flavobacterium-Bacteroides (CFB) group, Actinobacteria, and beta-Proteobacteria, respectively. Three had no phylogenetic association with known taxonomic divisions. None of the sequences studied in this study coincided exactly with those deposited in GenBank.     

Prolonged effect of fluid flow stress on the proliferative activity of mesothelial cells after abrupt discontinuation of fluid streaming

Encapsulating peritoneal sclerosis (EPS) often develops after transfer to hemodialysis and transplantation. Both termination of peritoneal dialysis (PD) and transplantation-related factors are risks implicated in post-PD development of EPS, but the precise mechanism of this late-onset peritoneal fibrosis remains to be elucidated. We previously demonstrated that fluid flow stress induced mesothelial proliferation and epithelial-mesenchymal transition via mitogen-activated protein kinase (MAPK) signaling. Therefore, we speculated that the prolonged bioactive effect of fluid flow stress may affect mesothelial cell kinetics after cessation of fluid streaming. To investigate how long mesothelial cells stay under the bioactive effect brought on by fluid flow stress after removal of the stress, we initially cultured mesothelial cells under fluid flow stress and then cultured the cells under static conditions. Mesothelial cells exposed to fluid flow stress for a certain time showed significantly high proliferative activity compared with static conditions after stoppage of fluid streaming. The expression levels of protein phosphatase 2A, which dephosphorylates MAPK, in mesothelial cells changed with time and showed a biphasic pattern that was dependent on the duration of exposure to fluid flow stress. There were no differences in the fluid flow stress-related bioactive effects on mesothelial cells once a certain time had passed. The present findings show that fluid flow stress exerts a prolonged bioactive effect on mesothelial cells after termination of fluid streaming. These findings support the hypothesis that a history of PD for a certain period could serve as a trigger of EPS after stoppage of PD.     

Developmentally regulated expression of a cell surface protein,neuropilin, in the mouse nervous system

Neuropilin (previously A5) is a cell surface glycoprotein that was originally identified in Xenopus tadpole nervous tissues. In Xenopus, neuropilin is expressed on both the presynaptic and postsynaptic elements in the visual and general somatic sensory systems, suggesting a role in neuronal cell recognition. In this study, we identified a mouse homologue of neuropilin and examined its expression in developing mouse nervous tissues. cDNA cloning and sequencing revealed that the primary structure of the mouse neuropilin was highly similar to that of Xenopus and that the extracellular segment of the molecule possessed several motifs that were expected to be involved in cell-cell interaction. Immunohistochemistry and in situ hybridization analyses in mice indicated that the expression of neuropilin was restricted to particular neuron circuits. Neuropilin protein was localized on axons but not on the somata of neurons. The expression of neuropilin persisted through the time when axons were actively growing to form neuronal connections. These observations suggest that neuropilin is involved in growth, fasciculation, and targeting for a particular groups of axons. © 1996 John Wiley & Sons, Inc.     

Decreased apoptosis of beta 2- integrin-deficient bovine neutrophils

Stimulant-induced viability of neutrophils, nuclear-fragmentation, increase in intracellular calcium ([Ca2+]i), expression of annexin V on neutrophils and proteolysis of a fluorogenic peptide substrate Ac-DEVD-MCA (acetyl Asp-Glu-Val-Asp alpha-[4-methyl-coumaryl-7-amide]) by neutrophil lysates from five normal calves and three calves with leucocyte adhesion deficiency were determined to evaluate the apoptosis of normal and CD18-deficient neutrophils. Viability was markedly decreased in control neutrophils stimulated with opsonized zymosan (OPZ), compared to CD18-deficient neutrophils at 37 degrees C after incubation periods of 6 and 24 hours. The rate of apoptosis of control neutrophils stimulated with OPZ increased significantly depending on the incubation time, whereas no apparent increase in apoptosis was found in CD18-deficient neutrophils under the same conditions. Aggregated bovine (Agg) IgG-induced apoptosis of control neutrophils was not significantly different from that of CD18-deficient neutrophils. The expression of annexin V on OPZ-stimulated control neutrophils was greater than that of unstimulated ones 6 h after stimulation. No apparent increase in annexin V expression on CD18-deficient neutrophils was found with OPZ stimulation. A delay in apoptosis was demonstrated in CD18-deficient bovine neutrophils and this appeared to be closely associated with lowered signalling via [Ca2+]i, diminished annexin V expression on the cell surface, and decreased caspase 3 activity in lysates.     

Determination of nitrotyrosine and related compounds in biological specimens by competitive enzyme immunoassay.

A gas mediator, nitric oxide is converted to peroxynitrite in the presence of superoxide anion. Peroxynitrite is a potent oxidant, which injures various tissues and organs by nitration of the tyrosine residues of proteins, and it enhances the late response of inflammation. The determination of nitrated tyrosine, nitrotyrosine, which is a stable final metabolite of peroxynitrite, provides an important indicator of tissue disorders caused by peroxynitrite. This paper reports a competitive solid-phase immunoassay for measuring nitrotyrosine in various biological specimens. In this study, peroxidase-conjugated nitrotyrosine was prepared by reaction of nitrotyrosine with 1,4-benzoquinone treatment, and then it was allowed to compete with nitrotyrosine on an anti-nitrotyrosine antibody-coated 96-well multiplate. No amino acids or related compounds tested in the experiments interfered with the immune reaction of nitrotyrosine, except cysteine, which only slightly inhibited the immune reaction at the concentrations higher than 1000 times the concentration of nitrotyrosine. The limit of detection of free nitrotyrosine was approximately 500 pg/mL (2 nM) at a competition ratio (B/B(o)%) of 80%. The newly developed enzyme immunoassay (EIA) method was used for assay of nitrotyrosine in biological specimens, with the following results: (i) Lipopolysaccharide (LPS) activation of RAW264.7 cells induced a significant increase in nitrotyrosine production compared to that with nonactivated cells. N(omega)-nitro-L-arginine methyl ester decreased nitrotyrosine production with either LPS-activated or nonactivated RAW cells. There is a relationship between nitrotyrosine production and nitrite ion. (ii) The nitrotyrosine level detected in the plasma specimens from healthy volunteers was 35.21 +/- 4.87 ng/mL (135.4 +/- 18.7 nM). (iii) The concentration of nitrotyrosine in the nasal lavage fluid of allergic rhinitis patients was 41.40 +/- 20.96 ng/mL (159.02 +/- 80.6 nM). Thus, the EIA method combines sensitivity and specificity with the ability to process a large number of specimens to quantify nitrotyrosine produced with in vivo and in vitro sources.     

Isolation of Allo-isoleucic Acid (α-Hydroxy-β-methylvaleric Acid) and β-Hydroxy-β-methylvaleric Acid from Turkish Tobacco Leaves

A new method determining the activity of tannin acyl hydrolase (tannase) was made. This method was based on the change in optical density of substrate tannic acid at 310 mμ. In this method, the error of measurement was about 1~3%, and many samples could be tested at one time because of its simplicity.

The procedure was as follows; To four parts of substrate (0.350 w/v% of tannic acid dissolved in 0.05m citrate buffer, pH 5.5), one part of the enzyme solution was added.

After t minutes reaction at 30°C, 0.1 part of the mixture was added to ten parts of 90% ethanol.

The optical density of the ethanol solution at 310 mμ was measured. Tannase activity (unit/ml) was given by following equation. $u=114\times \frac{E_{t1}?E_{t2}}{t_2?t_1}$

Where E_t1 and E_t2 mean the optical density of the ethanol solution at 310 mμ prepared after t₁ and t₂ minutes reaction, and one unit of the enzyme means the amount of the enzyme which is able to hydrolyze one μ mole of the ester bond in tannic acid in one minute.

The substrate tannic acid used in this determining method was purified. It was composed of one mole of glucose and nine moles of gallic acid, and eight moles of which formed four moles of m-digallic acid.