Identification of an interaction between the m-band protein skelemin and beta-integrin subunits. Colocalization of a skelemin-like protein with beta1- and beta3-integrins in non-muscle cells

Signaling across integrins is regulated by interaction of these receptors with cytoskeletal proteins and signaling molecules. To identify molecules interacting with the cytoplasmic domain of the beta3-integrin subunit (glycoprotein IIIa), a placental cDNA library was screened in the yeast two-hybrid system. Two identical clones coding for a 96-amino acid sequence were identified. This sequence was 100% identical to a sequence in skelemin, a protein identified previously in skeletal muscle. Skelemin is a member of a superfamily of cytoskeletal proteins that contain fibronectin-type III-like motifs and immunoglobulin C2-like motifs and that regulate the organization of myosin filaments in muscle. The amino acid residues in the isolated clones encompassed C2 motifs 4 and 5 of skelemin. A recombinant skelemin protein consisting of C2 motifs 3-7 interacted with beta1- and beta3-integrin cytoplasmic domains expressed as glutathione S-transferase (GST) fusion proteins, but not with GST-beta2-integrin cytoplasmic tail or GST alone. The skelemin-binding region was in the membrane proximal cytoplasmic domains of the integrins. Full-length skelemin interacted with integrin in intact cells as demonstrated by the colocalization of hemagglutinin-tagged skelemin in Chinese hamster ovary (CHO) cells containing alphaIIbbeta3-integrin and by the finding that microinjection of C2 motif 4 of skelemin into C2C12 mouse myoblast cells caused spread cells to round up. A skelemin-like protein was detected in CHO cells, endothelial cells, and platelets, and this protein colocalized with beta1- and beta3-integrins in CHO cells. This study suggests the presence of a skelemin-like protein in non-muscle cells and provides evidence that it may be involved in linking integrins to the cytoskeleton.     

Breast abscess with lethal septicemia due to Pseudomonas aeruginosa in a patient with AIDS

OBJECTIVE: The incidence of Pseudomonas aeruginosa in HIV-infected patients has increased over the last years. We describe a case of pseudomonal breast abscess complicated with fatal septicemia in an AIDS patient. CASE REPORT: A 21-year-old woman was admitted for fever, chills, nausea, vomiting and pain in the breast. She had a swelling in the right breast of 3 days duration. HIV infection had been confirmed 6 years earlier. CD4 count was 2/mm3. Surgical drainage produced a blue-green purulent discharge which grew Pseudomonas aeruginosa on culture. Despite cloxacilin, then ceftazidime and amikacin, initial improvement was followed 2 weeks later by nodular pulmonary infiltration with cavitation. P. aeruginosa was recovered from sputum and blood cultures, but stepwise resistance developed and the patient died 3 months after admission. DISCUSSION: Breast abscesses are infrequent in nonlactating women. P. aeruginosa is rarely involved, even in HIV patients. Due to the risk of resistance, prompt administration of appropriate antibiotics is required.     

The effect of a two-week anti-orthostatic hypokinesia on the cardiac function and on the latter''s regulation in monkeys

Medical practice guidelines are being developed rapidly and advocated as a crucial component in transforming and improving the delivery of health care services. However, little is known about the ability of hospitals to institute guidelines outside of experimental settings. This article approaches guideline implementation from the viewpoint of process innovation assimilation and presents specific recommendations for organizational tactics leading to successful implementation of practice guidelines.     

Gastroparesis after celiac plexus block

Pectin constituents, which were about 70 w/w% of extracellular polysaccharides (ECP) from a cell-suspension culture of Mentha, were purified by gel filtration chromatography, and their sugar composition and linkage were investigated. Two major constituents identified were (1-->3)-linked galactan carrying arabinosyl residues on C-6 and (1-->4)-alpha-linked galacturonan partially interspersed with (1-->2)-linked rhamnosyl resides. Acetylated or methylated pectins were not identified on 1H-NMR analysis.     

Complete relaxation and conformational exchange matrix (CORCEMA) analysis of NOESY spectra of interacting systems; two-dimensional transferred NOESY

A very general procedure entitled complete relaxation and conformational exchange matrix (CORCEMA) analysis has been developed to analyze the 2D-NOESY spectra of interacting systems undergoing multistate conformational exchange. This is an extension of earlier work from this laboratory on the methodological treatment of multistate conformational exchange [Krishna et al., Biopolymers 19, 2003 (1980)] and the theory of transferred NOESY for finite exchange off-rates [Lee and Krishna, J. Magn. Reson. 98, 36 (1992)]. The current theory is based on generalized rate matrices for relaxation and conformational exchange. The CORCEMA algorithm explicitly incorporates intermolecular dipolar cross relaxation between the molecules when they are complexed. It permits an analysis of NOESY intensities for the intra- as well as intermolecular contacts between the interacting molecules under a variety of binding conditions. Its application is illustrated on two examples of transferred NOESY simulations: (1) a two-state system involving a ligand and an enzyme forming a ligand-enzyme complex, and (2) a three-state system in which the ligand-enzyme complex can undergo a conformational transition from an "open state" to a "closed state," and can include conformational changes in both the complexed ligand and the complexed enzyme, such as hinge-bending motions. Simplifying expressions for generalized matrix analyses are derived for three limiting cases of the three-state system. This three-state example is illustrated using a hypothetical model of the hinge-bending motion in a thermolysin-inhibitor complex. It is shown that: (1) The neglect of cross relaxation between the interacting species in their complexed forms can lead to misleading conclusions on the "bound" conformation of the ligand. (2) If protein-mediated spin diffusion is dominant, caution is needed in analyses based on initial slopes alone due to one's inability to identify the exact range of the initial growth curve under poor signal/noise situations. (3) The neglect of conformational changes upon complexation, e.g., hinge-bending motions of the ligand-enzyme complex, can lead to erroneous results on the nature of "bound" conformations of the ligand. In this case, attempts to analyze the transferred NOESY data with a two-state model will result in a "virtual" conformation for the bound ligand. (4) When the hinge-bending rate is slower than the cross relaxation and enzyme off-rates, the bound conformation of a ligand deduced from the transferred NOESY experiment is more likely to represent nonspecific or weak binding in an open state of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dynamic course of neurological syndromes in very young children with the history of intrauterine growth retardation

90 infants with intrauterine growth retardation (IGR) and 100 normal infants (control group) were followed up from 5 days till 3 years of life. In IGR infants there was a more frequent combination of several neurologic syndromes, an early manifestation of motor disorders (from the very moment of birth), a delay of neuro-psychic development (during the first year of life), a tendency to development of moderate hydrocephalus by the age of 6 months. Autonomic-visceral disorders in them were mostly characterized by the symptoms of abaissement, but not of irritation.     

Toxicity of the styrene metabolite, phenylglyoxylic acid, in rats after three months' oral dosing

Male Wistar rats were dosed with 0, 1250, 3750 or 5000 mg/l of phenylglyoxylic acid (PGA) (CAS no. 611-73-4) in the drinking water ad libitum for 3 months. During the entire treatment period, there were no gross signs of toxicity related to PGA. No changes in neurobehavior were found after using a functional observational battery or radial arm maze. An increased relative kidney weight was seen in the highest dose-group (Controls: 0.504 +/- 0.031 g/100 g b.wt.; 5000 mg PGA/l: 0.579 +/- 0.033 g/100 g b.wt.; p<0.01). No other organ weights were affected. Histopathology revealed no change in kidney structure. No changes in clinical biochemistry. In the highest dose-group three animals out of ten showed reduction in peripheral nerve myelin sheath thickness. No such changes were seen in the control group. The study revealed no changes in auditory brain stem response but minor changes in electroretinography. The noradrenaline (NA) concentration decreased in pons and thalamus whereas it increased in medulla oblongata and whole brain. The dopamine (DA) concentration increased in cerebellum, hippocampus, pons, and whole brain. The most marked DA increase was seen in hippocampus (Controls: 0.56 +/- 0.10 nmol/g tissue; 5000 mg/l: 1.04 +/- 0.11 nmol/g tissue; p<0.001). The 5-hydroxytryptamine (5-HT) concentration decreased in cerebellum, cerebral cortex, hippocampus, and medulla oblongata, whereas it increased in thalamus. The yield of synaptosomal protein, synaptosomal NA, DA, and 5-HT concentrations, and DA uptake rate were not affected. When dosed males were mated with naive females, there were no differences between groups in the pregnancy rate, number of corpora luteae, implantations, live or dead fetuses, resorptions, preimplantation loss, or postimplantation loss. It is concluded that a part of the effects on kidney, peripheral nerves, and vision, which have previously been reported after exposure to styrene, might be induced by the styrene metabolite, PGA. If PGA has ototoxic effects in rats, the dosing in the present study is not sufficient to induce the necessary ototoxic concentration in blood. Alternatively, the ototoxicity of styrene, like toluene, may be caused the parent compound itself and not by a metabolite like PGA.     

Development of orally active oxytocin antagonists: studies on 1-(1-[4-[1-(2-methyl-1-oxidopyridin-3-ylmethyl)piperidin-4-yloxy]-2- methoxybenzoyl]piperidin-4-yl)-1,4-dihydrobenz[d][1,3]oxazin-2-one (L-372,662) and related pyridines

The previously reported oxytocin antagonist L-371,257 (2) has been modified at its acetylpiperidine terminus to incorporate various pyridine N-oxide groups. This modification has led to the identification of compounds with improved pharmacokinetics and excellent oral bioavailability. The pyridine N-oxide series is exemplified by L-372,662 (30), which possessed good potency in vitro (Ki = 4.1 nM, cloned human oxytocin receptor) and in vivo (intravenous AD50 = 0.71 mg/kg in the rat), excellent oral bioavailability (90% in the rat, 96% in the dog), good aqueous solubility (>8.5 mg/mL at pH 5.2) which should facilitate formulation for iv administration, and excellent selectivity against the human arginine vasopressin receptors. Incorporation of a 5-fluoro substituent on the central benzoyl ring of this class of oxytocin antagonists enhanced in vitro and in vivo potency but was detrimental to the pharmacokinetic profiles of these compounds. Although lipophilic substitution around the pyridine ring of compound 30 gave higher affinity in vitro, such substituents were a metabolic liability and caused shortfalls in vivo. Two approaches to prevent this metabolism, addition of a cyclic constraint and incorporation of trifluoromethyl groups, were examined. The former approach was ineffective because of metabolic hydroxylation on the constrained ring system, whereas the latter showed improvement in plasma pharmacokinetics in some cases.