Manipulation of the cytoplasmic and transmembrane domains alters cell surface levels of the coxsackie-adenovirus receptor and changes the efficiency of adenovirus infection

Expression of the coxsackie-adenovirus receptor (CAR) is a critical determinant in cellular susceptibility to infection with adenovirus-based gene transfer vectors. This study is focused on the hypothesis that manipulation of the cytoplasmic tail and transmembrane regions of CAR can be used to change cell surface levels of CAR and, consequently, to alter the efficiency of Ad-mediated gene transfer. To accomplish this, Flag-tagged ([F]) human CAR ([F]CAR), [F]tailless-CAR (lacking the cytoplasmic tail), and [F]GPI-CAR (containing a GPI lipid anchor instead of the transmembrane and cytoplasmic regions) were exogenously expressed in CHO cells. Analysis of (125)I-labeled anti-Flag antibody binding to transfected cells revealed that [F]tailless-CAR and [F]GPI-CAR were expressed on the cell surface in 1.8- to 2.5-fold higher amounts than [F]CAR, while the total expression levels were similar. Infection with replication-deficient adenovirus encoding beta-galactosidase (Ad-betagal) demonstrated 1.5- to 2-fold higher levels of transgene expression in CHO cells expressing [F]tailless-CAR or [F]GPI-CAR, respectively, compared with cells containing [F]CAR. The form of CAR expressed did not affect the transport of fluorescent Cy3-Ad particles from the cell surface to the nuclear region. These observations indicate that transduction of target cells by Ad vectors can be optimized by increasing cell surface levels of CAR through functional deletion of the tail and membrane protein domains.     

Allopregnanolone as a mediator of affective switching in reproductive mood disorders

Rationale

Reproductive mood disorders, including premenstrual dysphoria (PMD) and postpartum depression (PPD), are characterized by affective dysregulation that occurs during specific reproductive states. The occurrence of illness onset during changes in reproductive endocrine function has generated interest in the role of gonadal steroids in the pathophysiology of reproductive mood disorders, yet the mechanisms by which the changing hormone milieu triggers depression in susceptible women remain poorly understood.

Objectives

This review focuses on one of the neurosteroid metabolites of progesterone — allopregnanolone (ALLO) — that acutely regulates neuronal function and may mediate affective dysregulation that occurs concomitant with changes in reproductive endocrine function. We describe the role of the “neuroactive” steroids estradiol and progesterone in reproductive endocrine-related mood disorders to highlight the potential mechanisms by which ALLO might contribute to their pathophysiology. Finally, using existing data, we test the hypothesis that changes in ALLO levels may trigger affective dysregulation in susceptible women.

Results

Although there is no reliable evidence that basal ALLO levels distinguish those with PMD or PPD from those without, existing animal models suggest potential mechanisms by which specific reproductive states may unmask susceptibility to affective dysregulation. Consistent with these models, initially euthymic women with PMD and those with a history of PPD show a negative association between depressive symptoms and circulating ALLO levels following progesterone administration.

Conclusions

Existing animal models and our own preliminary data suggest that ALLO may play an important role in the pathophysiology of reproductive mood disorders by triggering affective dysregulation in susceptible women.     

When the At-Risk Do Not Develop Heart Failure: Understanding Positive Deviance Among Postmenopausal African American and Hispanic Women

BackgroundAfrican American and Hispanic postmenopausal women have the highest risk for heart failure compared with other races, but heart failure prevalence is lower than expected in some national cohorts. It is unknown whether psychosocial factors are associated with lower risk of incident heart failure hospitalization among high-risk postmenopausal minority women.Methods and ResultsUsing the Women's Health Initiative Study, African American and US Hispanic women were classified as high-risk for incident heart failure hospitalization with 1 or more traditional heart failure risk factors and the highest tertile heart failure genetic risk scores. Positive psychosocial factors (optimism, social support, religion) and negative psychosocial factors (living alone, social strain, depressive symptoms) were measured using validated survey instruments at baseline. Adjusted subdistribution hazard ratios of developing heart failure hospitalization were determined with death as a competing risk. Positive deviance indicated not developing incident heart failure hospitalization with 1 or more risk factors and the highest tertile for genetic risk. Among 7986 African American women (mean follow-up of 16 years), 27.0% demonstrated positive deviance. Among high-risk African American women, optimism was associated with modestly reduced risk of heart failure hospitalization (subdistribution hazard ratio 0.94, 95% confidence interval 0.91–0.99), and social strain was associated with modestly increased risk of heart failure hospitalization (subdistribution hazard ratio 1.07, 95% confidence interval 1.02–1.12) in the initial models; however, no psychosocial factors were associated with heart failure hospitalization in fully adjusted analyses. Among 3341 Hispanic women, 25.1% demonstrated positive deviance. Among high-risk Hispanic women, living alone was associated with increased risk of heart failure hospitalization (subdistribution hazard ratio 1.97, 95% confidence interval 1.06–3.63) in unadjusted analyses; however, no psychosocial factors were associated with heart failure hospitalization in fully adjusted analyses.ConclusionsAmong postmenopausal African American and Hispanic women, a significant proportion remained free from heart failure hospitalization despite having the highest genetic risk profile and 1 or more traditional risk factors. No observed psychosocial factors were associated with incident heart failure hospitalization in high-risk African Americans and Hispanics. Additional investigation is needed to understand protective factors among high-risk African American and Hispanic women.     

V̇O2 of resting muscle during arterial hypoxia: Role of reflex vasoconstriction

To examine the influence of chemoreflex vasoconstriction on the change in O₂ consumption in resting muscle during arterial hypoxia, responses in intact and denervated gracilis muscles were compared. Hypoxia was induced in anesthetized, paralyzed, and artificially ventilated dogs by inhalation of low O₂ gases. Electromagnetic blood flow, AVO₂ difference, and O₂ consumption were measured. Small vessel blood content served as relative index of open capillary density. Before denervation, muscle O₂ consumption was maintained during hypoxia, unless SaO₂ fell below 50%. Maintenance of muscle O₂ consumption during hypoxia was associated with an unchanging AVO₂ difference, a large increase in small vessel blood content, and a minimal increase in flow. Relative changes in parameters of O₂ supply and demand during hypoxia were not significantly different after denervation, except that flow increases were modestly higher. Results suggest that O₂ delivery to muscle during hypoxia is maintained primarily by an opening of capillaries rather than by increases in flow and that chemoreflex vasoconstriction does not interfere with this local vascular adjustment.     

Initial Dipeptide Formation in Hemoglobin Biosynthesis

Initiation factors M(1) + M(2) from reticulocyte ribosomes bind Met-tRNA(F) to rabbit reticulocyte ribosomes containing endogenous hemoglobin mRNA. The initial binding of Met-tRNA(F) appears to be to the small ribosomal subunit. The Met-tRNA(F) is able to participate in what is presumed to be the first peptide bond in the formation of hemoglobin, namely the synthesis of a methionyl-valine dipeptide. The formation of this methionyl-valine dipeptide requires Met-tRNA(F), initiation factors M(1), M(2), and M(3), as well as Val-tRNA and T(1). No synthesis of methionyl-valine dipeptide takes place if Met-tRNA(F) is replaced by Met-tRNA(M), or if initiation factor M(3) is omitted. Thus, Met-tRNA(F) appears to be the initiator tRNA for hemoglobin biosynthesis and M(3), although required for the synthesis of the first peptide bond of hemoglobin, does not appear to be necessary, under the experimental conditions studied, for Met-tRNA(F) binding.     

Lung Collagen Heterogeneity

The structural heterogeneity of rabbit lung collagen was examined by extracting labeled collagen from short-term cultures of lung minces with 1 M NaCl-50 mM Tris.HCl (pH 7.4), 0.5 M acetic acid, or 0.4 ionic strength phosphate buffer. The extracted collagens were purified by carboxymethyl-cellulose chromatography, and their cyanogen bromide peptides were mapped by ion exchange chromatography and acrylamide gels. Rabbit skin alpha1(I) and alpha2 chains and rabbit sternal cartilage alpha1(II) chains were used as markers.The peripheral lung, containing alveoli, small blood vessels, and small airways, synthesized alpha1(I) and alpha2 chains. The trachea and the bronchial tree (first through seventh order branches) both synthesized alpha1(II) chains. Lung alpha1(I), alpha2, and alpha1(II) chains all have a molecular weight of about 100,000 and are all sensitive to Clostridial collagenase.The extraction and purification methods used isolate only 50% of the collagen synthesized by these structures in vitro. Once all collagen types in lung can be described and quantitated, it should be possible to utilize lung collagen types as biochemical markers to study normal lung development and to define the lung fibrotic diseases.     

Outcome of myocardial infarction in patients treated with aspirin is enhanced by pre-hospital administration

OBJECTIVE: Reducing time to reperfusion therapy is one of the goals in the management of acute myocardial infarction (AMI). We assessed the association between timing of aspirin administration and outcome of patients with AMI. PATIENTS: We studied 922 consecutive AMI patients with ST-segment elevation in Killip class I-III on admission. Patients were divided into two groups based upon the timing of emergency aspirin administration: before (early aspirin users) or after (late aspirin users) hospital admission. RESULTS: Early aspirin users (n = 338; 37%) were younger, less likely to be women, and more likely to smoke (p < 0.006) than late users (n = 584; 63%). Other baseline and clinical characteristics were similar. Early aspirin users were more likely to be treated with thrombolysis or primary percutaneous transluminal coronary angioplasty. Compared with late users, early aspirin users had significantly lower in-hospital complications and lower mortality rates at 7 (2.4 vs. 7.3%, p = 0.002) and 30 days (4.9 vs. 11.1%, p = 0.001). By multivariate adjustment, pre-hospital aspirin was an independent determinant of survival at 7 (odds ratio 0.43; 95% confidence interval 0.18-0.92) and at 30 days (odds ratio, 0.60; 95% confidence interval 0.32-1.08). Survival benefit associated with aspirin persisted for subgroups treated or not with reperfusion therapy. CONCLUSIONS: Outcome of AMI patients treated with aspirin is improved by pre-hospital administration. Our findings suggest that emergency pre-hospital aspirin might facilitate early reperfusion.