Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells

Abnormal regional increases in DNA methylation, which have potential for causing gene inactivation and chromosomal instability, are consistently found in immortalized and tumorigenic cells. Increased DNA methyltransferase activity, which is also a characteristic of such cells, is a candidate to mediate these abnormal DNA methylation patterns. We now show that, in NIH 3T3 mouse fibroblasts, constitutive overexpression of an exogenous mouse DNA methyltransferase gene results in a marked increase in overall DNA methylation which is accompanied by tumorigenic transformation. These transformation changes can also be elicited by dexamethasone-inducible expression of an exogenous DNA methyltransferase gene. Our findings provide strong evidence that the increase in DNA methyltransferase activity associated with tumor progression could be a key step in carcinogenesis and provide a model system that can be used to further study this possibility.     

Coronary revascularization after myocardial infarction in the very elderly: outcomes and long-term follow-up

OBJECTIVE: To determine the outcome of very elderly patients who had coronary revascularization during hospitalization for an acute myocardial infarction. DESIGN: Retrospective cohort study. SETTING: Community-based tertiary-care teaching hospital. PATIENTS: A total of 1215 consecutive patients 80 years and older were hospitalized with a myocardial infarction between 1985 and 1990. The study sample included all 93 patients (8%) who had cardiac catheterization before discharge and had not been excluded from study because of the following: severe valvular disease, absence of significant coronary disease, or death before a decision about revascularization could be made. MEASUREMENTS: Survival, quality of life, and functional status at least 1 year after discharge. RESULTS: After catheterization, 41 patients had angioplasty, 18 had coronary artery bypass surgery, and 34 did not have revascularization. Among the patients alive at discharge, those who had revascularization had a high likelihood of achieving a good or excellent quality of life (angioplasty, 86% [31 of 36]; surgery, 89% [16 of 18]; medical therapy, 44% [11 of 25]) and of being able to care for themselves (angioplasty, 89% [32 of 36], surgery, 89% [16 of 18], medical therapy, 52% [13 of 25]). Mortality rates at 1 year were 24% (95% CI, 15% to 47%) for the angioplasty group, 6% (CI, 0% to 27%) for the surgery group, and 44% (CI, 27% to 62%) for the medical therapy group. In a Cox proportional hazards model that adjusted for clinical, demographic, hemodynamic, and anatomic differences between the groups, the performance of coronary revascularization was associated with increased survival (hazard ratio, 0.42; CI, 0.18 to 0.98). CONCLUSIONS: A small percentage of very elderly patients with complicated acute myocardial infarctions, selected by their physicians for invasive cardiovascular procedures, can tolerate these procedures, avoid serious complications, return to independent living, and have excellent probability of survival. Although our results suggest that coronary revascularization may have benefited these patients, the study design did not permit definite conclusions, and future studies are needed to resolve this important question.     

Splenic lymphoid tissue of mice after irradiation with fast carbon ions

Reorganization of cellular content of splenic functional zones was studied morphometrically in mice after single irradiation with fast carbon ions. It was demonstrated that irradiation does not cause significant changes in microtopography of splenic structures but is accompanied with changes in cytoarchitecture and rearrangement of lymphoid cells throughout the experiment. Two arbitrary stages are determined in lymphoid tissue response. The first one (30 hrs-7 d) is characterized with destructive processes intensification and decrease of small lymphocytes number in all functional zones of the organ. Strong plasmocellular reaction was observed in the organ except for the lymphoid nodules mantle zone in this period as well. On the second stage (15-60 d) orientation of plasmocellular reaction changes along with the tendency of small lymphocytes to restore population (up to 57-79% from the control level on d 60 of the experiment. As a result plasma cell content decreases sharply in all the organ structures. Low level of lymphocytopoiesis was demonstrated in the course of the experiment resulting from cell mitotic activity decline following the irradiation. At the distant period (d 60 following the irradiation) another decrease of cell lymphoid population was noted, which indicates that irradiation with fast carbon ions is suppressive to the animal immune system.     

Recombinant measles viruses with mutations in the C, V, or F gene have altered growth phenotypes in vivo

An understanding of the determinants of measles virus (MV) virulence has been hampered by the lack of an experimental model of infection. We have previously demonstrated that virulence phenotypes in human infections are faithfully reproduced by infection of human thymus/liver (thy/liv) implants engrafted into SCID mice, where the virus grows primarily in stromal cells but induces thymocyte apoptosis (P. G. Auwaerter et al., J. Virol. 70:3734-3740, 1996). To begin to elucidate the roles of the C protein, V protein, and the 5' untranslated region of the F gene (F 5'UTR) in MV infection in vivo, the replication of strains bearing mutations of these genes was compared to that of the parent sequence-tagged Edmonston strain (EdTag). Growth curves show that mutants fall into two phenotypic classes. One class of mutants demonstrated kinetics of growth similar to that of EdTag, with decreased peak titers. The second class of mutants manifested peak titers similar to that of EdTag but had different replication kinetics. Abrogation of V expression led to delayed and markedly prolonged replication. Additionally, thymocyte survival was prolonged and implant architecture was preserved throughout the course of infection. In contrast, massive bystander thymocyte death occurred after infection with EdTag and all other mutants. A mutant which overexpressed V in Vero cells (V+) had the opposite phenotype of the A mutant not expressing V (V-). V+ grew more rapidly than EdTag with 100-fold-greater levels of virus production 3 days after infection. These results suggest that C, V, and the F 5'UTR are accessory factors required for efficient virus replication in vivo. In addition, thymocyte survival after V- infection suggests this protein may play multiple roles in pathogenesis of MV infection of thymus. Since these recombinant mutant viruses grew identically to the parent virus in Vero cells, the data show that thy/liv implants are an excellent model for investigating the determinants of MV virulence.     

A selective human beta3 adrenergic receptor agonist increases metabolic rate in rhesus monkeys

Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.     

Cross-linking of selected residues in the N- and C-terminal domains of Escherichia coli protein L7/L12 to other ribosomal proteins and the effect of elongation factor Tu

Five different variants of protein L7/L12, each with a single cysteine substitution at a selected site, were produced, modified with 125I-N-[4-(p-azidosalicylamido)-butyl]-3-(2'-pyridyldithio)propion amide, a radiolabeled, sulfhydryl-specific, heterobifunctional, cleavable photocross-linking reagent that transfers radiolabel to the target molecule upon reduction of the disulfide bond. The proteins were reconstituted with core particles depleted of wild type L7/L12 to yield 70 S ribosomes. Cross-linked molecules were identified and quantified by the radiolabel. No cross-linking of RNA was detected. Two sites in the dimeric N-terminal domain, Cys-12 and Cys-33, cross-linked strongly to L10 and in lower yield to L11 but to no other proteins. The three sites in the globular C-terminal domain all cross-linked strongly to L11 and, in lower yield, to L10. Weaker cross-linking to 50 S proteins L2 and L5 occurred from all three C-terminal domain locations. The 30 S ribosomal proteins S2, S3, S7, S14, S18 were also cross-linked from all three of these sites. Binding of the ternary complex [14C]Phe-tRNA-elongation factor Tu.guanyl-5'-yl imidodiphosphate) but not [14C]Phe-tRNA.elongation factor Tu.GDP.kirromycin increased labeling of L2, L5, and all of the 30 S proteins. These results imply the flexibility of L7/L12 and the transient proximity of three surfaces of the C-terminal domain with the base of the stalk, the peptidyl transferase domain, and the head of the 30 S subunit.     

A novel synaptobrevin/VAMP homologous protein (VAMP5) is increased during in vitro myogenesis and present in the plasma membrane

cDNA clones encoding a novel protein (VAMP5) homologous to synaptobrevins/VAMPs are detected during database searches. The predicted 102-amino acid VAMP5 harbors a 23-residue hydrophobic region near the carboxyl terminus and exhibits an overall amino acid identity of 33% with synaptobrevin/VAMP1 and 2 and cellubrevin. Northern blot analysis reveals that the mRNA for VAMP5 is preferentially expressed in the skeletal muscle and heart, whereas significantly lower levels are detected in several other tissues but not in the brain. During in vitro differentiation (myogenesis) of C2C12 myoblasts into myotubes, the mRNA level for VAMP5 is increased approximately 8- to 10-fold. Immunoblot analysis using antibodies specific for VAMP5 shows that the protein levels are also elevated approximately 6-fold during in vitro myogenesis of C2C12 cells. Indirect immunofluorescence microscopy and immunoelectron microscopy reveal that VAMP5 is associated with the plasma membrane as well as intracellular perinuclear and peripheral vesicular structures of myotubes. Epitope-tagged versions of VAMP5 are similarly targeted to the plasma membrane.     

Homocysteine and vascular disease

Teeth agenesis is an anomaly of frequent observation with a familiar heredity transmission pattern. In a frequency order the most common agenetic teeth are: third molars, upper lateral incisors and second lower bicuspids. Of exceptional rarity is the absence of second molars. Our observation confirm the heredity pattern of congenitally missing permanent teeth; in this case anomaly showed variable expressivity and different degree of severity.