Virus protein changes and RNA termini alterations evolving during persistent infection

Cloned infectious vesicular stomatitis virus isolated following 5 years of persistent infection of BHK21 cells in vitro exhibits a number of peptide map changes in the G protein (spike glycoprotein), the M protein (membrane matrix protein) and the N protein (nucleocapsid structural protein). Only slight alterations have occurred in the peptide maps of the two VSV polymerase-associated proteins L and NS. Dideoxy sequencing of the 3′ ends of the cloned virus originally used to establish the persistent infection, and of the cloned virus recovered following 5 years of persistence, shows one base substitution in the three base junction between the 3′ leader sequence and the N protein-coding region. Repeated lytic passages of virus recovered from persistent infection led to no oligonucleotide map changes after 30 passages, but two map changes were present after 102 and remained after 133 lytic passages in BHK21 cells in vitro. Only one of these represented reversion to the original map position, and this “mutant” virus still exhibited a temperature-sensitive small plaque phenotype. Finally, the mutated virus recovered after more than $\text{5}\text{1}\text{2}$ years of persistent infection is now so slow-growing that it can establish persistent infection of BHK21 cells in the absence of DI particles (although DI particles are present constantly once the cells recover from the initial cytopathology).     

Adrenomedullary Secretion of DOPA, Catecholamines, Catechol Metabolites, and Neuropeptides

Abstract: Catecholamines and their metabolites have been proposed as markers of sympathetic nervous system stimulation. However, the adrenal medulla is a rich source of catecholamines and catecholamine metabolites and may play a significant role in plasma levels of these compounds. In addition to adrenal catecholamine metabolite efflux, the role of the catecholamine precursor 3,4-dihydroxyphenylalanine (DOPA) has not been fully evaluated. The simultaneous effluxes of catecholamines, metabolites, DOPA, and neuropeptides were measured in perfusates from isolated dog adrenals. The relative abundance of compounds detected consistently during unstimulated conditions was epinephrine ≫ norepinephrine > 3,4-dihydroxyphenylglycol > metanephrine > normetanephrine > dopamine > 3,4-dihydroxyphenylacetic acid > 3-methoxy-4-hydroxyphenylglycol ≥ DOPA ≫ [Met]enkephalin ≫ neuropeptide Y. Effluxes of analytes were not affected by cocaine and the ratios of catecholamines to metabolites increased dramatically with carbachol stimulation, consistent with negligible reuptake into adrenal cells. Thus, most of the 3,4-dihydroxyphenylglycol is expected to be derived from epinephrine and norepinephrine subsequent to translocation from chromaffin vesicles into the cytosol. The efflux of DOPA increased dramatically during stimulation with 30 µ M carbachol in a calcium-dependent manner. Efflux of DOPA during the initial stabilization period of the perfusion preparation declined exponentially, in parallel with the effluxes of the catecholamines and neuropeptides but not with metabolites. Evoked release of DOPA was Ca²⁺-dependent. These data suggest that DOPA can be stored and released exocytotically from chromaffin granules.     

Epidemiologic features of Lyme disease in New York

During 1982, surveillance identified 207 cases of Lyme disease in New York State. Cases were clustered in two geographic areas, eastern Long Island and northern Westchester counties. Symptoms and signs of Lyme disease in cases were consistent with previous reports, with erythema chronicum migrans (ECM) being the most frequently (77 percent) reported sign of disease. Facial palsy was reported in a surprisingly high 18 percent of cases. Of 160 cases whose sera were submitted for Lyme spirochete specific IgG antibody testing, 112 (70 percent) had titers greater than or equal to 64, while 88 (55 percent) had titers greater than or equal to 128. Positive titers were not associated with any single sign or symptom of disease, but were significantly associated with symptom onset or tick bite occurring during the three-month period of June, July, and August. We conclude that the incidence of Lyme disease in New York is much higher than previously recognized. In addition, our data suggest that a serologic test for Lyme-spirochete IgG antibody lacks sensitivity, but can be useful in confirming the diagnosis of Lyme disease when antibody titers are high.     

Characterisation of Age-Dependent Beta Cell Dynamics in the Male db/db Mice

Aim

To characterise changes in pancreatic beta cell mass during the development of diabetes in untreated male C57BLKS/J db/db mice.

Methods

Blood samples were collected from a total of 72 untreated male db/db mice aged 5, 6, 8, 10, 12, 14, 18, 24 and 34 weeks, for measurement of terminal blood glucose, HbA_1c, plasma insulin, and C-peptide. Pancreata were removed for quantification of beta cell mass, islet numbers as well as proliferation and apoptosis by immunohistochemistry and stereology.

Results

Total pancreatic beta cell mass increased significantly from 2.1 ± 0.3 mg in mice aged 5 weeks to a peak value of 4.84 ± 0.26 mg (P < 0.05) in 12-week-old mice, then gradually decreased to 3.27 ± 0.44 mg in mice aged 34 weeks. Analysis of islets in the 5-, 10-, and 24-week age groups showed increased beta cell proliferation in the 10-week-old animals whereas a low proliferation is seen in older animals. The expansion in beta cell mass was driven by an increase in mean islet mass as the total number of islets was unchanged in the three groups.

Conclusions/Interpretation

The age-dependent beta cell dynamics in male db/db mice has been described from 5-34 weeks of age and at the same time alterations in insulin/glucose homeostasis were assessed. High beta cell proliferation and increased beta cell mass occur in young animals followed by a gradual decline characterised by a low beta cell proliferation in older animals. The expansion of beta cell mass was caused by an increase in mean islet mass and not islet number.     

Primary Breast Cancer Tumours Contain High Amounts of IgA1 Immunoglobulin: An Immunohistochemical Analysis of a Possible Carrier of the Tumour-Associated Tn Antigen

The Tn antigen (GalNAc alpha-O-Ser/Thr) as defined by the binding of the lectin, helix pomatia agglutinin (HPA) or anti-Tn monoclonal antibodies, is known to be exposed in a majority of cancers, and it has also been shown to correlate positively with the metastatic capacity in breast carcinoma. The short O-glycan that forms the antigen is carried by a number of different proteins. One potential carrier of the Tn antigen is immunoglobulin A1 (IgA1), which we surprisingly found in tumour cells of the invasive parts of primary breast carcinoma. Conventional immunohistochemical analysis of paraffin-embedded sections from primary breast cancers showed IgA1 to be present in the cytoplasm and plasma membrane of 35 out of 36 individual primary tumours. The immunohistochemical staining of HPA and anti-Tn antibody (GOD3-2C4) did to some extent overlap with the presence of IgA1 in the tumours, but differences were seen in the percentage of stained cells and in the staining pattern in the different breast cancers analysed. Anti-Tn antibody and HPA were also shown to specifically bind to a number of possible constellations of the Tn antigen in the hinge region of IgA1. Both reagents could also detect the presence of Tn positive IgA in serum. On average 51% of the tumour cells in the individual breast cancer tumour sections showed staining for IgA1. The overall amount of staining in the invasive part of the tumour with the anti Tn antibody was 67%, and 93% with HPA. The intra-expression or uptake of IgA1 in breast cancer makes it a new potential carrier of the tumour associated and immunogenic Tn antigen.     

Expression of the RAI gene is conducive to apoptosis: studies of induction and interference

The RAI gene is also known as iASPP and PPP1R13L. Recent investigations have shown that the region encompassing RAI is important for the development of cancer in young and middle-aged persons. It has been speculated that the RAI product induces apoptosis by blocking NF-kappaB or inhibits apoptosis by blocking p53. Either way the gene could influence the survival of precancerous lesions. Here we report that the expression of RAI mRNA was increased in non-transformed lymphocytes and fibroblasts induced to undergo apoptosis by various means, such as treatment with etoposide, calcium ions, or interleukin-2 and/or serum deprivation. Treatment with etoposide increased the content of RAI protein, too, and caused it to translocate to the nucleus. Inhibition of RAI expression in lymphocytes and fibroblasts with siRNA reduced apoptosis, but treatment with the NF-kappaB-inhibiting substance sulfasalazine relieved this dependence. In the transformed cell line HEK-293 the association between RAI induction and apoptosis seemed broken. Thus, we hypothesize that RAI induction is necessary but not sufficient for apoptosis induction in non-transformed cells. Our results could be explained by a NF-kappaB mediated mechanism.     

Adipose tissue interleukin-18 mRNA and plasma interleukin-18: effect of obesity and exercise

Objectives: Obesity and a physically inactive lifestyle are associated with increased risk of developing insulin resistance. The hypothesis that obesity is associated with increased adipose tissue (AT) interleukin (IL)‐18 mRNA expression and that AT IL‐18 mRNA expression is related to insulin resistance was tested. Furthermore, we speculated that acute exercise and exercise training would regulate AT IL‐18 mRNA expression. Research Methods and Procedures: Non‐obese subjects with BMI < 30 kg/m² (women: n = 18; men; n = 11) and obese subjects with BMI >30 kg/m² (women: n = 6; men: n = 7) participated in the study. Blood samples and abdominal subcutaneous AT biopsies were obtained at rest, immediately after an acute exercise bout, and at 2 hours or 10 hours of recovery. After 8 weeks of exercise training of the obese group, sampling was repeated 48 hours after the last training session. Results: AT IL‐18 mRNA content and plasma IL‐18 concentration were higher (p < 0.05) in the obese group than in the non‐obese group. AT IL‐18 mRNA content and plasma IL‐18 concentration was positively correlated (p < 0.05) with insulin resistance. While acute exercise did not affect IL‐18 mRNA expression at the studied time‐points, exercise training reduced AT IL‐18 mRNA content by 20% in both sexes. Discussion: Because obesity and insulin resistance were associated with elevated AT IL‐18 mRNA and plasma IL‐18 levels, the training‐induced lowering of AT IL‐18 mRNA content may contribute to the beneficial effects of regular physical activity with improved insulin sensitivity.