The relative distribution of soluble and insoluble cholinesterases in rat excitable tissues

Three ratios were studied here: bound to free AChE (R1), bound to free BChE (R2), and the ratios between these two (R3). The first one proved relevant in that it contributed to the division of the cholinergic tissues into 3 classes: high values (nicotinic tissues: skeletal muscle), low values (muscarinic tissues: small intestine, uterus, heart), and middle values (mixed, nicotinic and muscarinic cholinergic innervation: brain). The third ratio (R3) showed different values in the muscarinic tissues studied; no significant differences could, however, be found between the ratios of brain and skeletal muscle. Further exploration of this ratio should indicate whether it is of some importance for the characterization of excitable tissues.     

Enthalphy changes associated with the denaturation of collagens of different imino acid content

The enthalpy changes associated with the denaturation of acid-soluble and insoluble collagens prepared from sheep, cod, halibut and pike skin were determined by differential scanning calorimetry. The enthalpy change associated with the soluble collagens decreased with decreasing imino acid content (from 1420 cal/mol for sheep to 736 cal/mol for cod) while the value for insoluble collagens was approximately constant at 1360 cal/mol. A possible explanation for these values in terms of the nautre of the bonds present in collagen is discussed.     

Effects of amount and type of dietary fibre (soluble and insoluble) on short-term control of appetite

OBJECTIVE: The calcium (Ca) pump of cardiac sarcoplasmic reticulum (SR) membranes is vulnerable to oxidation and hence likely to be damaged by chlorinated compounds, specifically hypochlorite (NaOCl) and monochloramine (NH2Cl), the most potent oxidants produced upon neutrophil activation. This could occur during prolonged ischemia or myocardial infarction when tissue levels of catecholamines are high. Phospholamban (PLN), the phosphorylatable regulator of the Ca pump, plays a central role in the effects of beta-adrenergic agonists on the heart. The purpose of this study was to investigate a possible role of PLN in determining the pump's sensitivity to NaOCl and NH2Cl. METHODS: Ca-uptake and Ca(2+)-ATPase activities in purified phosphorylated and control canine cardiac microsomes, incubated at increasing concentrations of NaOCl or NH2Cl, were related to the extent of PLN phosphorylation by protein kinase A, which was quantitated by PhosphorImager analysis. RESULTS AND CONCLUSIONS: Our data indicate that microsomal phosphorylation protects the Ca pump fully against 10 microM NaOCl or NH2Cl, which inhibit Ca-uptake by 21-41% when assayed at 25 or 37 degrees C and saturating Ca2+ in unphosphorylated microsomes, and protects partially at higher oxidant concentrations. The protective effect of protein kinase A on Ca-uptake is proportional to the amount of phosphorylated PLN. No comparable protection against similar oxidative damage of the Ca pump is observed when light fast skeletal muscle microsomes, which lack PLN, are incubated under conditions favorable for phosphorylation nor when PLN's inhibition of the cardiac Ca pump is relieved by proteolytic cleavage of its cytoplasmic domain. Our findings contribute toward an understanding of possible endogenous protective mechanisms that may promote calcium homeostasis in myocardial cells in inflammatory states associated with neutrophil activation and may suggest an approach toward development of protective strategies against oxidative damage in the heart.     

Outside-In signaling of soluble and solid-phase fibrinogen through integrin alphaIIbbeta3 is different and cooperative with each other in a megakaryoblastic leukemia cell line, CMK

The function and the outside-in signaling pathways of alphaIIbbeta3 were examined in relation to cell adhesion using a megakaryoblastic leukemia cell line, CMK. After 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, the cells adhered to the culture plate and underwent megakaryocytic differentiation with expression of alphaIIbbeta3. Binding of soluble fibrinogen to the cells via alphaIIbbeta3 was dependent on cell adhesion. Cell detaching reduced the affinity of this integrin for soluble fibrinogen, although its surface expression was almost unchanged. In contrast, detached cells became tightly adherent to the fibrinogen-coated plate (solid-phase fibrinogen). The same ligand, fibrinogen, present either in soluble or solid-phase form, triggered differential signaling pathways mediated by alphaIIbbeta3. By the stimulation with soluble fibrinogen, Syk was tyrosine-phosphorylated but FAK was dephosphorylated, whereas solid-phase fibrinogen promptly caused tyrosine phosphorylation of FAK followed by delayed phosphorylation of Syk. In addition, the binding of soluble fibrinogen to the cells adherent to fibrinogen-coated plate resulted in tyrosine phosphorylation of integrin beta3 and a complex formation of integrin beta3 with Syk. This implies the cooperation of both soluble and solid-phase fibrinogen-mediated signaling pathways.     

Both lobes of the soluble receptor of the periplasmic histidine permease, an ABC transporter (traffic ATPase), interact with the membrane-bound complex. Effect of different ligands and consequences for the mechanism of action

The histidine permease of Salmonella typhimurium is an ABC transporter (traffic ATPase). The liganded soluble receptor, the histidine-binding protein HisJ, interacts with the membrane-bound complex HisQMP2 and stimulates its ATPase activity, which results in histidine translocation. In this study, we utilized HisJ proteins with mutations in either of the two lobes and wild type HisJ liganded with different substrates to show that each lobe carries an interaction site and that both lobes are involved in inducing (stimulating) the ATPase activity. We suggest that the spatial relationship between the lobes is one of the factors recognized by the membrane-bound complex in dictating the efficiency of the induction signal and of translocation. Several of the key residues involved have been identified. In addition, using constitutive ATPase mutants, we show that the binding protein provides some additional essential function(s) in translocation that is independent of the stimulation of ATP hydrolysis, and one possible mechanism is proposed, which includes the notion that liganded HisJ has different optimal conformations for signaling and for translocation.     

Biochemical and ultra-structural reactions to parenteral nutrition with two different fat emulsions in rats

OBJECTIVE: To compare the effects on fat metabolism and Kupffer cell morphology by total parenteral nutrition (TPN) with two different fat emulsions. DESIGN: Thirty-two male Sprague-Dawley rats, divided into three groups, were investigated. Rats fed orally were used as a reference group, and a group of rats receiving TPN with fat emulsions containing pure long-chain triglycerides (LCT) was compared to a group of rats receiving fat emulsions containing both long-chain triglycerides and medium-chain triglycerides (MCT/LCT). The TPN regimens were equicaloric and administered continuously via a jugular catheter for 10 days. INTERVENTIONS: After suffocation, blood of the rats was collected for the determination of serum lipids. Epididymal fat and heart were collected for the analysis of lipoprotein lipase (LPL) activities, and liver specimens were saved for analyses of hepatic triglyceride concentration, as well as activities of hepatic lipase (HL) and lysosomal enzymes. Light and electron microscopy were used for examination of the Kupffer cell reaction. RESULTS: Directly after termination of parenteral feeding, the levels of serum triglycerides and high density lipoprotein (HDL) triglycerides were higher in the MCT/LCT group than in the LCT group, while no differences concerning cholesterol and phospholipid concentrations were found. No significant difference in liver steatosis was found between the two TPN groups. Comparison of the TPN groups showed that the MCT/ LCT group had significantly decreased LPL activity in adipose tissue, while the LCT group had significantly increased LPL activity in the heart. The activity of HL was low in both groups, but significantly lower in the LCT group. Lipid accumulation and an increased number of lysosomes were found in all Kupffer cell when TPN with LCTemulsions was used. Moreover, TPN induced a pronounced increase in various liver lysosomal enzyme activities, but there was no notable difference between LCT and MCT/LCT effects. CONCLUSIONS: Compared to treatment with pure LCTemulsions, treatment with MCT/LCT emulsions evoked weaker biochemical reactions in terms of lower activity of lipoprotein lipase in fat and heart together with higher serum and HDL triglyceride levels. Morphological signs of increased Kupffer cell activity such as the appearance of multiple lysosomes and fat vacuoles in the cytoplasm followed treatment with pure LCT emulsions. However, both TPN groups showed a marked increase in activities of liver lysosomal enzymes.     

The membrane-spanning proteoglycan NG2 binds to collagens V and VI through the central nonglobular domain of its core protein

NG2 is a membrane-spanning proteoglycan with a primary structure unique among cell surface or extracellular matrix proteins. To characterize the interaction between NG2 and extracellular matrix proteins, we have used a eukaryotic expression system to produce and purify several recombinant fragments covering not only the entire ectodomain of NG2 but also distinct subdomains of the molecule. Using a solid phase binding assay with various extracellular matrix proteins, we have identified two main ligands for NG2, namely, collagens V and VI. Consistent with previous models of glycosaminoglycan attachment, roughly 50% of the recombinant NG2 fragments containing the central domain have chondroitin sulfate chains attached to the protein core. These glycosaminoglycan chains are not directly involved in collagen binding, since chondroitinase-treated fragments exhibit an unimpaired ability to bind to both collagens. Using more restricted recombinant fragments of NG2, we mapped the binding site for both collagens to the central domain of NG2. Electron microscopy after rotary shadowing of native NG2 molecules indicates that this extended nonglobular domain provides a flexible connection joining the two N- and C-terminal globular regions of NG2. Rotary shadowing of mixtures of NG2 and collagen V or VI confirms a direct interaction between the molecules and indicates that the collagens align with the central region of NG2, giving the appearance of a rod between the N- and C-terminal globules.     

Mutations in ccf, a novel Drosophila gene encoding a chromosomal factor, affect progression through mitosis and interact with Pc-G mutations

We report herein the isolation of ccf, a new gene located in region 82E and essential for Drosophila development. This gene, expressed throughout development, encodes a novel product of 68 kDa which is found in the nucleus during interphase and labels, in a novel pattern, centrosomes and chromosome arms during mitosis. Mutations in ccf give rise to late larvae with small imaginal discs and to adults showing appendages of reduced size, consistent with CCF involvement in cell proliferation. Neuroblast squash analyses show that CCF is required for proper condensation of mitotic chromosomes and, therefore, for progression through mitosis. Furthermore, we observe that adult ccf mutants as well as animals overexpressing CCF during larval stages exhibit homeotic transformations. We also find that mutations in the Pc-G genes Polycomb, polyhomeotic and Enhancer of zeste are enhanced by ccf mutations. Finally, we show that the CCF protein binds to specific sites on polytene chromosomes, many of which are shared with the Posterior sex combs Pc-G protein. Together, these results suggest a role for the CCF protein in the maintenance of chromosome structure during mitosis and interphase.     

Hemorphins inhibit angiotensin IV binding and interact with aminopeptidase N

[125I]-Ang IV binding to rabbit collecting duct cell membranes was inhibited by hemorphins (H), a class of endogenous peptides obtained by hydrolysis of the beta chain of hemoglobin. The most potent competitors were those with a valine in their N-terminal part such as LVV-H7 and VV-H7 (IC50 = 1.3 nM) followed by VV-H8 and K6VV-H7 (5.1 nM). The same H, like Ang IV, interacted with aminopeptidase N (APN) as shown by their inhibitory effect (28-36%) on APN activity. HPLC analysis showed that only H with a N-terminal valine or leucine were hydrolyzed. Since H are detected in the body fluids, they are likely to act as endogenous competitors of Ang IV.     

Serum IgA1 and IgA2 subclass antibodies against collagens in patients with ankylosing spondylitis

Clear cell papulosis is a newly described skin disease characterized by multiple white papules. Histopathologically, diagnostic clear cells were seen among the basal cells of the epidermis. We report clear cell papulosis on the lumbar area and buttocks of a 1-year-old girl.     

The soluble granulocyte-macrophage colony-stimulating factor receptor's carboxyl-terminal domain mediates retention of the soluble receptor on the cell surface through interaction with the granulocyte-macrophage colony-stimulating factor receptor beta-subunit

The hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates its activity through binding to cell-surface receptors. The high-affinity GM-CSF receptor (GMR) consists of two transmembrane-anchored subunits: a ligand-specific, low-affinity subunit (GMRalpha); and a signal-transducing beta-subunit (GMRbeta). The human GMRalpha subunit also exists in a soluble isoform (SOLalpha) which antagonizes GM-CSF activity in vitro. Previous studies by us have shown that coexpression of SOLalpha and a mutated GMRbeta in BHK cells results in retention of SOLalpha on the cell surface and the formation of an intermediate affinity binding complex (Kd approximately 300 pM). This paper investigates the mechanism of the retention of SOLalpha on the cell surface. The data demonstrate that SOLalpha is anchored by a direct, ligand-independent interaction with GMRbeta which also occurs when SOLalpha is coexpressed with wild-type GMRbeta. However, SOLalpha and wild-type GMRbeta form a complex which binds GM-CSF with high affinity (Kd = 39 pM), indistinguishable from the binding characteristics of the TMalpha/GMRbeta complex. The experiments further reveal that the interaction between SOLalpha and GMRbeta is abrogated by removal of the unique 16 amino acid carboxyl-terminal domain of SOLalpha. Specific mutation of cysteine 323 in this carboxyl-domain to alanine also eliminates the cell-surface retention of SOLalpha identifying this residue as being necessary for the formation of the SOLalpha/GMRbeta complex.     

Identification and characterization of genes that interact with lin-12 in Caenorhabditis elegans

We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-17, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup-17 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup-17 and lag-2, suggest that both genes act at approximately the same time as lin-12 in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.     

Histones H1 and H5 interact preferentially with crossovers of double-helical DNA

The interaction of the linker histones H1 and H5 from chicken erythrocyte chromatin with pBR322 was studied as a function of the number of superhelical turns in circular plasmid molecules. Supercoiled plasmid DNA was relaxed with topoisomerase I so that a population with a narrow distribution of topoisomers, containing from zero to five superhelical turns, was obtained. None of the topoisomers contained alternative non-B-DNA structures. Histone-DNA complexes formed at either 25 or 100 mM NaCl final concentration and at histone-DNA molar ratios ranging from 10 to 150 were analyzed by agarose gel electrophoresis. The patterns of disappearance of individual topoisomer bands from the gel were interpreted as an indication of preference of the linker histones for crossovers of double-helical DNA. This preference was observed at both salt concentrations, being more pronounced under conditions of low ionic strength. Isolated H5 globular domain also caused selective disappearance of topoisomers from the gel, but it did so only at very high peptide-DNA molar ratios. The observed preference of the linker histones for crossovers of double-helical DNA is viewed as a part of the mechanism involved in the sealing of the two turns of DNA around the histone octamer.     

Signal peptide fragments of preprolactin and HIV-1 p-gp160 interact with calmodulin

Secretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide during transport into the lumen of the endoplasmic reticulum. Using site-specific photo-crosslinking we have followed the fate of the signal sequence of preprolactin in a cell-free system. This signal sequence has an unusually long hydrophilic n-region containing several positively charged amino acid residues. We found that after cleavage by signal peptidase the signal sequence is in contact with lipids and subunits of the signal peptidase complex. The cleaved signal sequence is processed further and an N-terminal fragment is released into the cytosol. This signal peptide fragment was found to interact efficiently with calmodulin. Similar to preprolactin, the signal sequence of the HIV-1 envelope protein p-gp160 has the characteristic feature for calmodulin binding in its n-region. We found that a signal peptide fragment of p-gp160 was released into the cytosol and interacts with calmodulin. Our results suggest that signal peptide fragments of some cellular and viral proteins can interact with cytosolic target molecules. The functional consequences of such interactions remain to be established. However, our data suggest that signal sequences may be functionally more versatile than anticipated up to now.     

Sexual standards and reactions to pornography: Enhancing behavioral consistency through self-focused attention.

Reports 3 studies which tested the hypothesis, derived from self-awareness theory, that behavior would be more consistent with personal attitudes or standards when attention was self-focused. In the 1st study, 52 male undergraduates' attitudes toward erotica were measured, and 1 mo later the Ss were asked to rate pictures of nude women, while either self-focused (in front of a mirror) or not. There was little relationship between pretested attitudes and reactions toward the pictures for the non-self-focused group; however, the same relationship was very strong for the group that rated pictures in front of a mirror. In the 2nd and 3rd studies, female Ss (51 and 48 undergraduates, respectively) were first pretested on the Mosher Sex-Guilt Scale. Two weeks later they read and rated pornographic passages, again, while either self-focused or not. The relationship between pretested standards (sex guilt) and reactions to sexual literature was weak in the non-self-aware condition, but considerably stronger for the self-focused Ss. Results suggest that focusing attention upon the self tends to inhibit behaviors that are inconsistent with personal attitudes or standards. (35 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Dietary flavonoids interact with trace metals and affect metallothionein level in human intestinal cells

Flavonoids are natural compounds found in food items of plant origin. The study examined systematically the interaction of structurally diverse dietary flavonoids with trace metal ions and the potential impact of dietary flavonoids on the function of intestinal cells. Spectrum analysis was first performed to determine flavonoid-metal interaction in the buffer. Among the flavonoids tested, genistein, biochanin-A, naringin, and naringenin did not interact with any metal ions tested. Members of the flavonol family, quercetin, rutin, kaempferol, flavanol, and catechin, were found to interact with Cu(II) and Fe(III). On prolonged exposure, quercetin also interacted with Mn(II). Quercetin at 1:1 ratio to Cu(II) completely blocked the Cu-dependent color formation from hematoxylin. When quercetin was added to the growth medium of cultured human intestinal cells, Caco-2, the level of metal binding antioxidant protein, metallothionein, decreased. The effect of quercetin on metallothionein was dose- and time-dependent. Genistein and biochanin A, on the contrary, increased the level of metallothionein. The interaction between dietary flavonoids and trace minerals and the effect of flavonoids on metallothionein level imply that flavonoids may affect metal homeostasis and cellular oxidative status in a structure-specific fashion.     

A comparison of the abilities of chlorpromazine and molindone to interact adversely with guanethidine

Chlorpromazine and molindone were tested for their abilities to impair conditioned avoidance behavior of rats. Chlorpromazine was effective within the dose range of 0.3 to 7.0 mg/kg (ID50approximately 2.0 mg/kg); molindone was effective within the range of 0.3 to 5.0 mg/kg (ID50 approximately 0.6 mg/kg). Behaviorally relevant doses of chlorpromazine and molindone were then tested for their effects on blood pressure and on adrenergic mechanisms. When given intravenously to anesthetized, hypertensive animals, both drugs (1.0 mg/kg) produced significant but transient vasodepression. When given intraperitoneally to anesthetized or to conscious hypertensive rats, the drugs did not produce significant effects on blood pressure. Both drugs (1.0 mg/kg) blocked responses to an alpha agonist (methoxamine), but chlorpromazine was significantly more potent than molindone. In addition, chlorpromazine produced a dose-dependent (1.0-10.0 mg/kg) inhibition of 3H-l-norepinephrine uptake into heart, but molindone at the same doses produced no inhibition of uptake. In related experiments, it was found that guanethidine (50 mg/kg) was an effective agent for lowering blood pressure of hypertensive rats. When chlorpromazine (3-10 mg/kg) was administered concomitantly with guanethidine, the blood pressure lowering properties of guanethidine were diminished or abolished. When molindone (1-10 mg/kg) was administered concomitantly with guanethidine, there was no loss of blood pressure control. It is concluded that molindone is an important drug, because it is an antipsychotic agent that does not interact adversely with guanethidine.     

Gabapentin does not interact with a contraceptive regimen of norethindrone acetate and ethinyl estradiol

Anticonvulsants that induce hepatic metabolism increase clearance of oral contraceptive hormones and thereby cause contraceptive failure. Gabapentin is not metabolized in humans and has little liability for causing metabolic-based drug-drug interactions. In healthy women receiving 2.5 mg norethindrone acetate and 50 microg ethinyl estradiol daily for three consecutive menstrual cycles, concurrent gabapentin administration did not alter the steady-state pharmacokinetics of either hormone. Thus, gabapentin is unlikely to cause contraceptive failure.