Both lumenal and cytosolic gating of the aqueous ER translocon pore are regulated from inside the ribosome during membrane protein integration.

Portions of the nascent chain are exposed to the lumen, the cytosol, or neither at different stages during the cotranslational integration of a protein into the ER membrane, as shown by compartment-specific collisional quenching of fluorophores incorporated into the polypeptide. The opening or closing of each end of the aqueous translocon pore is tightly controlled and occurs in a sequence that does not compromise the membrane's permeability barrier. Surprisingly, these structural changes at the membrane are effected by the transmembrane segment in the nascent protein from inside the ribosome.

Actions of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate in vascular smooth muscle cell cultures.

Aim: To study the effects of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) on vascular smooth muscle (VSM) cells A7r5.

Methods: The effects of TMB-8 were investigated in A7r5 cell cultures with 45CaCl2.

Results: TMB-8 reduced the intracellular free Ca2+ concentration, [Ca2+]i in a Ca(2+)-free medium and blocked Ca2+ entry from the extracellular site in a regular Ca2+ medium.

Immune responsiveness of splenocytes after chronic daily melatonin administration in male Syrian hamsters.

The interrelationships between the immune system and the pineal hormone, melatonin, have been explored recently. The present studies investigated the effects of daily melatonin injections on reproductive and spleen function in male Syrian hamsters. Testes weights and serum testosterone levels were depressed after 8-10 weeks of daily melatonin injections.

Designed protein pores as components for biosensors.

Background: There is a pressing need for new sensors that can detect a variety of analytes, ranging from simple ions to complex compounds and even microorganisms. The devices should offer sensitivity, speed, reversibility and selectivity. Given these criteria, protein pores, remodeled so that their transmembrane conductances are modulated by the association of specific analytes, are excellent prospects as components of biosensors.

Calcium and mechanotransduction of the myogenic response.

The purpose of this study was to measure vascular smooth muscle (VSM) cytosolic calcium ([Ca2+]i) during the myogenic response. We examined the temporal and steady-state relationships between lumen diameter and VSM [Ca2+]i in isolated arterioles exposed to step changes in intravascular pressure. We also studied the relationship between step sizes in intravascular pressure and changes in [Ca2+]i.

Endothelial ATP-sensitive potassium channels mediate coronary microvascular dilation to hyperosmolarity.

Coronary arterial occlusion has been shown to increase osmolarity in the myocardial interstitium. Intracoronary injection of hyperosmolar solutions reduces coronary vascular resistance. However, the response of coronary microvessels to an abluminal increase in osmolarity is unclear, and the underlying mechanism for its vasomotor regulation has not been elucidated.

Cocaethylene exposure during the brain growth spurt period: brain growth restrictions and neurochemistry studies.

The concurrent use of alcohol and cocaine has recently attracted attention in the medical research field due to the prevalence of this drug abuse pattern and the exclusive formation of a pharmacologically active substance, cocaethylene (CE). This is the first study to examine the neuroteratogenic effects of cocaethylene exposure during the brain growth spurt (part of the third trimester equivalent) on brain growth restrictions and neurochemical profiles. For the brain growth restrictions study, three groups of artificially reared rat pups were given daily injections of 0, 10 or 20 mg/kg cocaethylene (s.

Synergistic effect of mutations in invA and lpfC on the ability of Salmonella typhimurium to cause murine typhoid.

Penetration of the intestinal mucosa at areas of Peyer's patches is an important first step for Salmonella typhimurium to produce lethal systemic disease in mice. However, mutations in genes that are important for intestinal invasion result in only moderately decreased virulence of S. typhimurium for mice.

A specific host cellular protein binding element near the 3' end of mouse hepatitis virus genomic RNA.

A distinct host cellular protein binding element was mapped within a 38-nucleotide (nt) sequence 166-129 nucleotides upstream of the 3' end of the MHV-JHM genome using a RNase T1 protection/gel mobility shift electrophoresis assay. The resultant RNA-protein complex contains six host cellular proteins, one protein of 120-kDa molecular mass, two poorly resolved species approximately 55 kDa in size, a second pair of poorly resolved 40-kDa proteins, and a minor component of 25 kDa. A series of RNA probes containing deletions or clustered transversion mutations were tested for their ability to form complexes with mock- and MHV-JHM-infected cytoplasmic extracts.

The aqueous pore through the translocon has a diameter of 40-60 A during cotranslational protein translocation at the ER membrane.

Eukaryotic secretory proteins are cotranslationally translocated through the endoplasmic reticulum (ER) membrane via aqueous pores that span the lipid bilayer. Fluorescent probes were incorporated into nascent secretory proteins using modified Lys-tRNAs, and the resulting nascent chains were sealed off from the cytosol in fully assembled translocation intermediates. Fluorescence quenching agents of different sizes were then introduced into the ER lumen in order to determine which were small enough to enter the pore and to quench the fluorescence of probes inside the ribosome and/or the pore.

Calcium-release-activated calcium influx in endothelium.

Signaling pathways activated by the tachykinin substance P (SP) were investigated in pig coronary artery endothelial cells (PCAECs). Single cells were obtained after enzymatic digestion of coronary arteries. Intracellular Ca2+ ([Ca2+]i) was measured from fura-2 fluorescence while membrane potential or ionic current was measured using patch-clamp techniques.

Alcohol, osteoporosis, and bone regulating hormones.

The mechanism of the production of ethanol-associated osteopenia seems to be a direct effect of alcohol on bone cells and an indirect or modulating effect through mineral regulating hormones such as vitamin D metabolites, parathyroid hormone, and calcitonin. The modulating effects of these hormones on bone and mineral metabolism in acute and chronic alcohol consumption is discussed herein.

Nitric oxide and renal effects of volume expansion in conscious monkeys.

Experiments were performed to determine the effects of nitric oxide (NO) synthase inhibition on the renal responses to volume expansion in conscious cynomolgus monkeys. All animals were volume expanded with 3% dextran in normal saline under three conditions: 1) during a control state, 2) during constant infusion of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 30 microg x kg(-1) x min(-1)), and 3) during simultaneous infusion of L-NAME and excess NO substrate L-arginine (0.6 mg x kg(-1) x min(-1)).

Integrin-mediated reduction in vascular smooth muscle [Ca2+]i induced by RGD-containing peptide.

It has previously been shown that synthetic peptides containing the sequence arginine-glycine-aspartic acid (RGD) cause vasodilation by activation of alpha(v)beta3-integrin present on vascular smooth muscle (VSM) cells. The purpose of this study was to determine whether this dilatory effect is mediated by a reduction in VSM cytosolic Ca2+ concentration ([Ca2+]i). First-order arterioles from the rat cremaster were isolated, cannulated, and pressurized.

Isolation and biological properties of osteopontin from bovine milk.

A procedure for the isolation of osteopontin (OPN) from bovine milk using ion-exchange and hydrophobic chromatography is described. A DEAE-Sephacel column followed by dual phenyl-Sepharose columns yielded approximately 8 mg of purified protein per liter of milk. SDS-PAGE analysis revealed that the protein migrated at M(r) 60,000.

Alcohol consumption by young actively growing rats: a histomorphometric study of cancellous bone.

Alcohol consumption by young actively growing rats has been previously demonstrated to decrease bone density. This study addresses the mechanism of alcohol action on the early phases of bone growth and development using histomorphometric techniques. Four-week-old, female Sprague-Dawley rats were divided into three groups.

L-glutamine inhibits nitric oxide synthesis in bovine venular endothelial cells.

This study was conducted to test the hypothesis that L-glutamine has differential effects on nitric oxide (NO) synthesis from L-arginine in bovine venular endothelial cells (EC) stimulated by A23187 (a Ca++ ionophore) and receptor-mediated vasodilators (bradykinin and substance P). EC were cultured at 37 degrees C for 24 h in the presence of 0.4 mM L-arginine and 0.

A direct comparison of helix propensity in proteins and peptides.

alpha-Helical secondary structure occurs widely in globular proteins and its formation is a key step in their folding. As a consequence, understanding the energetics of helix formation is crucial to understanding protein folding and stability. We have measured the helix propensities of the nonpolar amino acids for an alpha-helix in an intact protein, ribonuclease T1, and for a 17-residue peptide with a sequence identical to that of the alpha-helix in the protein.

8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate actions on calcium dynamics in cultured vascular smooth muscle cells.

Aim: To study 8-(N,N-Diethylamino)n-octyl-3,4,5-trimethoxybenzoate (TMB), a potent Ca(2+)-antagonist, actions on cellular calcium dynamics in vascular smooth muscle cell (VSMC) cultures.

Methods: A7r5 VSMC were cultured with Fura-2 measurements of intracellular Ca2+ concentration, [Ca2+]i.

Results: TMB reduced [Ca2+]i from control levels and blocked [Ca2+]i increase caused by norepinephrine (NE) and 2,5-di (t-butyl)-1,4-benzohydroquinone (BHQ).

Cellular and humoral immune responses to mycobacterial stress proteins in experimental pulmonary tuberculosis.

Objective: Immunity to mycobacterial stress protein antigens was studied in response to vaccination and/or virulent infection.

Design: Guinea pigs, either vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG), infected by the pulmonary route with virulent M. tuberculosis, or vaccinated then infected, were studied for the development of cellular and humoral immunity to two recombinant mycobacterial stress proteins, hsp 65 and hsp 70.

Ethanol inhibition of reduced frequency-dependent rundown of calcium currents in acutely dissociated MS/nDB neurons from chronic in vivo lead-exposed adult rats.

Although it is well known that lead (Pb2+0 acutely blocks voltage-gated calcium currents (VGCCs) in mammalian neurons, little is known about the long-term effects of continuous exposure to this metal on VGCCs. In the present study, the effects of chronic lead exposure on VGCCs (with barium ions as the charge carrier) were studied using whole-cell patch-clamp electrophysiological techniques in acutely dissociated medial septum (MS)/nucleus diagonal band (nDB) neurons. Neither peak, end current amplitudes, nor the current-voltage relationship were affected by chronic lead exposure.

Prenatal alcohol treatment attenuated postnatal cocaine-induced elevation of dopamine concentration in nucleus accumbens: a preliminary study.

Prenatal alcohol exposure has been shown to damage the developing central nervous system (CNS) in a variety of ways, including neuroanatomical anomalies, neurochemical imbalance, and neuropharmacological dysfunction. The present study investigated one of the functional aspects of dopaminergic system in neonatal rats exposed prenatally to a binge-like alcohol paradigm by measuring dopamine concentrations following a single postnatal cocaine challenge. Pregnant Sprague-Dawley rats were given daily intragastric intubations of 5.

Casein kinase II phosphorylates bovine papillomavirus type 1 E1 in vitro at a conserved motif.

The E1 protein of bovine papillomavirus type 1 (BPV-1) is a phosphoprotein which specifically binds and unwinds the virus replication origin by ATP-dependent helicase activity. The El protein has been shown to be multiply phosphorylated in vivo, although the sites of modification are incompletely mapped. Examination of the predicted amino acid sequence of all available E1 proteins revealed strong conservation between amino acids 25 and 60 of a motif consisting of a serine residue followed by a stretch of acidic residues.

Isolation of an amino-terminal region of bovine papillomavirus type 1 E1 protein that retains origin binding and E2 interaction capacity.

In vitro DNA binding results from a series of E1 proteins containing amino-terminal or carboxy-terminal truncations indicated that sequences between amino acids 121 and 284 were critical for origin binding. Additional binding experiments with E1 proteins containing internal, in-frame insertions or deletions confirmed the importance of the region defined by truncated E1 proteins and also demonstrated that downstream sequences were not required for binding activity in the context of the full-length E1 protein. On the basis of mapping results from the E1 mutants, a clone (pE1(121-311)) was constructed that expressed E1 amino acids within the approximate boundaries of the critical sequences for DNA binding.

Clarifying the relationship of veterinary medical education and moral development.

Objective: To clarify the relationship between veterinary medical education and moral development in response to 2 previous studies that presented conflicting evidence that the experience of veterinary medical education may inhibit moral development.

Design: The Defining Issues Test (DIT) was used to survey the moral reasoning of veterinary medical students at the beginning and end of their education.

Sample Population: First and fourth-year veterinary medical students.