Transgenic cattle produced by reverse-transcribed gene transfer in oocytes

A critical requirement for integration of retroviruses, other than HIV and possibly related lentiviruses, is the breakdown of the nuclear envelope during mitosis. Nuclear envelope breakdown occurs during mitotic M-phase, the envelope reforming immediately after cell division, thereby permitting the translocation of the retroviral preintegration complex into the nucleus and enabling integration to proceed. In the oocyte, during metaphase II (MII) of the second meiosis, the nuclear envelope is also absent and the oocyte remains in MII arrest for a much longer period of time compared with M-phase in a somatic cell. Pseudotyped replication-defective retroviral vector was injected into the perivitelline space of bovine oocytes during MII. We show that reverse-transcribed gene transfer can take place in an oocyte in MII arrest of meiosis, leading to production of offspring, the majority of which are transgenic. We discuss the implications of this mechanism both as a means of production of transgenic livestock and as a model for naturally occurring recursive transgenesis.     

Circuit-specific alterations of N-methyl-D-aspartate receptor subunit 1 in the dentate gyrus of aged monkeys

Age-associated memory impairment occurs frequently in primates. Based on the established importance of both the perforant path and N-methyl-D-aspartate (NMDA) receptors in memory formation, we investigated the glutamate receptor distribution and immunofluorescence intensity within the dentate gyrus of juvenile, adult, and aged macaque monkeys with the combined use of subunit-specific antibodies and quantitative confocal laser scanning microscopy. Here we demonstrate that aged monkeys, compared to adult monkeys, exhibit a 30.6% decrease in the ratio of NMDA receptor subunit 1 (NMDAR1) immunofluorescence intensity within the distal dendrites of the dentate gyrus granule cells, which receive the perforant path input from the entorhinal cortex, relative to the proximal dendrites, which receive an intrinsic excitatory input from the dentate hilus. The intradendritic alteration in NMDAR1 immunofluorescence occurs without a similar alteration of non-NMDA receptor subunits. Further analyses using synaptophysin as a reflection of total synaptic density and microtubule-associated protein 2 as a dendritic structural marker demonstrated no significant difference in staining intensity or area across the molecular layer in aged animals compared to the younger animals. These findings suggest that, in aged monkeys, a circuit-specific alteration in the intradendritic concentration of NMDAR1 occurs without concomitant gross structural changes in dendritic morphology or a significant change in the total synaptic density across the molecular layer. This alteration in the NMDA receptor-mediated input to the hippocampus from the entorhinal cortex may represent a molecular/cellular substrate for age-associated memory impairments.     

Calcium-regulated protein tyrosine phosphorylation is required for endothelin-1 to induce prostaglandin endoperoxide synthase-2 mRNA expression and protein synthesis in mesangial cells

The role of endothelin (ET)-1-mediated cytosolic calcium ([Ca2+]i) elevation in regulating ET-1-induced prostaglandin endoperoxide synthase, prostaglandin G/H synthase (PGHS)-2 mRNA expression and protein synthesis was investigated in mesangial cells (MC). Ionomycin, a calcium ionophore, and thapsigargin, an inhibitor of calcium ATPase, mimicked the ET-1-stimulated PGHS-2 mRNA and protein induction. Inhibition of [Ca2+]i increases with (2-?C2-bis-(carboxymethyl)-amino-5 methylphenoxy]methyl?-6-methoxy-8-bis-(carboxymethyl)-aminoquinoline tetra-(acetoxymethyl)ester (Quin/AM), a calcium chelator, or with the combined presence of [8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, HCl] (TMB), an inhibitor of intracellular calcium stores release, and ethyleneglycol-bis-(beta-aminoethyl)- N,N,N',N'-tetra-acetic acid (EGTA) suppressed ET-1, as well as ionomycin and thapsigargin-mediated PGHS-2 mRNA and protein formation. Also, the ET-1-, ionomycin-, and thapsigargin-induced PGHS-2 mRNA expression and protein formation was inhibited in MC pretreated with inhibitors of calcium calmodulin kinase. In contrast, these conditions did not inhibit interleukin (IL)-1-induced PGHS-2 mRNA expression and protein synthesis. Pretreatment with tyrosine kinase inhibitors abolished the ET-1-, ionomycin-, thapsigargin-, and IL-1-mediated PGHS-2 mRNA and protein induction. ET-1-, ionomycin-, and thapsigargin- induced protein tyrosine phosphorylation, but not IL-1-induced protein tyrosine phosphorylation, was suppressed by inhibiting either [Ca2+]i elevation or calcium calmodulin kinase activation. It was concluded that elevation of [Ca2+]i and activation of calcium calmodulin kinases are upstream mediators of ET-1-induced PGHS-2 gene expression through activation of non-receptor-linked protein tyrosine kinase in MC.     

Plasma activity of contact coagulation factors in patients with IgA nephritis

A cellular electrical impedance device which can detect the activated state of eosinophils has been developed and tested. This impedance device consists of a small gold electrode (50 microns x 50 microns) and a large gold electrode (1.5 cm x 0.5 cm) on a glass substrate, and it was fabricated by standard photolithographic techniques. Eosinophils, which belong to the granulocytic class of white blood cells, exhibit different physical properties when they change from the nonactivated state to the activated state. Hypothetically, these changes should correspond to a change in the measured electrical impedance. In this paper, data from the measured electrical impedance of eosinophils is presented. The measurements show that the average impedance of the activated eosinophils is 26% lower than the average impedance of the nonactivated eosinophils. Statistical analysis of the measured data shows that there is a significant difference between the measured impedances of activated and nonactivated eosinophils.     

Description and evaluation of the Iowa Depression Awareness, Recognition, and Treatment Program

OBJECTIVE: The National Institute of Mental Health developed the Depression Awareness, Recognition, and Treatment Program to provide up-to-date information and training to health, mental health, and social service professionals regarding the identification and treatment of depression. This study was undertaken to evaluate a series of these programs for professionals who provide services to rural residents in the Midwest. METHOD: The 18 2-day training programs were attended by a total of 1,221 participants, physicians, psychologists, social workers, and nurses. Participants' knowledge regarding depression was assessed both before and after each program. Practice characteristics and perceived ability to assess and treat depression were assessed. Finally, 6-month follow-up evaluations of the usefulness of the training to the participants were undertaken. RESULTS: Following the programs, participants evidenced significant increases in levels of knowledge of depression and a high degree of satisfaction with most elements of the program. Six-month follow-up evaluations indicated a continued positive evaluation of the program. CONCLUSIONS: These outcomes suggest that the goals of the Depression Awareness, Recognition, and Treatment Program were met and provide support for the wider dissemination of these training programs.     

Diurnal rhythm of cytoplasmic estrogen receptors in the rat brain in the absence of circulating estrogens

The concentration of cytoplasmic estrogen receptors in the brain of ovariectomized female rats varies during the light-dark cycle. There is no variation in the affinity of the receptors for estradiol, and the rhythm is not due to estrogens from nonovarian sources. Pentobarbital reverses the reduction of receptors that occurs in the dark, and melatonin injection in the light partially mimics the action of darkness in reducing receptor levels. The factors that cause this rhythm is brain estrogen receptors may be one means by which light affects reproductive function.     

Use of aminoglycosides in surgical infections

Most infections on the surgical ward are due to one or more gram-negative rods, acting either as the sole pathogens or as principal components in a polymicrobial flora. To date, parenteral aminoglycosides have proven to be the most effective antibiotics for control or treatment of such sepsis. Unfortunately, however, serious complications as well as therapeutic failures do occur. During a 40-month period, 405 surgical patients receiving aminoglycosides (Gentamicin, Tobramycin, Sisomicin, or Amikacin) were prospectively studied with respect to: indications for antibiotic; patient population; serum concentrations of antibiotic according to route of administration, dose in mg/kg/day, and renal function; rapidity of antibiotic excretion in the urine; causative bacteria and their sensitivities to each aminoglycoside as determined by both disc and tube dilution methods; severity and frequency of drug complications; and clinical efficacy of each study antibiotic. Results supported the contention of a superior effectiveness from aminoglycosides for established abdominal and unspecified surgical infections, more rapid development of therapeutic blood levels by intravenous administration, need to alter drug dose according to frequent serum creatinine determinations, increased drug toxicity in dehydrated and shocked patients, preventability of complicating Candida sepsis, and the importance of early as well as adequate surgical debridement and drainage.     

Sterol synthesis. Syntheses of 15-oxygenated 5alpha, 14beta-cholest-7-en-3beta-ol derivatives

Treatment of 3beta-benzoyloxy-14alpha, 15alpha-epoxy-5alpha-cholest-7-ene with boron trifluoride-etherate gave, in 43% yield, 3alpha-benzoyloxy-5alpha, 14beta-cholest-7-en-15-one with the unnatural C-D ring juncture. Reduction of the latter compound with lithium aluminum hydride gave 15alpha, 14beta-cholest-7-en-3beta, 15alpha-diol and 5alpha, 14beta-cholest-7-en-3beta, 15beta-diol in 9% and 81% yields, respectively.