bluuebunny/crossref_metadata_2025 huggingface dataset:
Stromal–epithelial interactions and heterogeneity of proliferative activity within the prostate
Authors: Cunha Gerald R. , Donjacour Annemarie A. , Sugimura Yoshiki
Published: December 2009

Growth and functional activity within the prostate gland is known to be regulated by androgens whose effects are thought to be mediated via androgen receptors. This concept has been derived in large part through analysis of whole organ homogenates, an approach which ignores potential heterogeneity of biological activity within the gland and the importance of cell–cell interactions. In this review recent findings are summarized which demonstrate that growth of the prostatic ductal network during prepubertal periods, as well as during prostatic regeneration in androgen-treated adult castrates, is nonuniform, with ductal growth being highest at the ductal tips and much lower in proximal ducts closer to the urethra. Androgen dependency for maintenance of ductal architecture following castration follows a similar pattern in that castration results in total destruction of distal ductal architecture, while proximal ducts are maintained albeit in an atrophic state. Thus, striking differences in biological properties are found in distal versus proximal prostatic ducts. Morphogenesis, growth, and secretory cytodifferentiation within the developing prostate is eleicited by androgens which act via mesenchymal–epithelial interactions. Through analysis of chimeric prostates constructed with androgen-receptor-positive wild-type mesenchyme and androgen-receptor-negative Tfm (testicular feminization) bladder epithelium, it is now evident that androgenic effects can be elicited in androgen-receptor-deficient (androgen-insensitive) Tfm prostatic epithelium, provided that the connective tissue component of the chimeric prostate is wild type. This observation has been made for both the developing and adult prostate. From this data it is evident that certain androgenic effects (ductal morphogenesis, epithelial growth, and secretory cytodifferentiation) do not require the presence of intraepithelial androgen receptors. In fact, recent evidence indicates that the epithelial androgen receptor is neither necessary or sufficient for induction of androgenic effects. Instead, many lines of evidence support the view that epithelial response to androgens is mediated via inductive trophic influences from mesenchymal (stromal) cells which are exceedingly important androgen targets.
Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase
Authors: Lim Megan S. , Walsh Michael P.
Published: December 2009

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[γ-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143 000, 60 000, 42 000, 33 000, and 11 000. The 143 000 MW substrate was identified as C-protein; the 42 000 MW substrate is probably actin; the 33 000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60 000 and 11 000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein was phosphorylated to the extent of ~0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5–6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of ~0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin). Phosphorylation of skeletal or cardiac C-proteins had no effect on their inhibition of this actomyosin Mg2+-ATPase activity. Furthermore, cardiac C-protein inhibited the Mg2+-ATPase activity of desensitized cardiac actomyosin; in this case, phosphorylation of cardiac C-protein enhanced its inhibitory effect on the actomyosin Mg2+-ATPase. These observations suggest that C-proteins of fast- and slow-twitch skeletal muscle fibers and cardiac muscle fibers are phosphorylated in response to catecholamines and other agents which induce cAMP formation and that, at least in the heart, this phosphorylation may affect actin–myosin interaction and the contractile state of the muscle.
Synthesis and use of galactolipid sulfotransferase substrate–analogue affinity probes: possible localization of the testicular enzyme
Authors: Lingwood Clifford , Taylor Trudy
Published: December 2009

Galactosyl ceramide (GC) is a substrate in vitro for galactolipid sulfotransferase preparations from brain, kidney, and testis. Biotinylated and light-sensitive azido derivatives of lysogalactosyl ceramide have been synthesized. These derivatives remain effective substrates for the testicular enzyme. Biotinylated GC has been used to localize specific GC-binding sites in frozen sections of rat testis. The binding pattern is consistent with the expected location of the testicular galactolipid sulfotransferase.
Monoclonal antibodies to cuticular proteins of Panagrellus silusiae: identification of a common collagenous epitope
Authors: Martin L. H. , Bols N. C. , Pasternak J. J.
Published: December 2009

The cuticle of Panagrellus silusiae contains at least 20 proteins that are linked to each other, to some extent, by disulfide bonds. These components range in apparent molecular mass from about 40 to 270 kilodaltons (kDa). After denaturation of cuticular fragments that had been extensively digested with pepsin, electrophoresis revealed five intact components (95, 117, 125, 144, and 197 kDa). Virtually all of the other native cuticular components are susceptible to degradation by both pepsin and collagenase, which indicates that these proteins contain both noncollagenous and collagenous sectors. Monoclonal antibodies were raised against reduced and solubilized cuticular preparation from Panagrellus. Each of the monoclonal antibodies reacted to a reduced and solubilized cuticular preparation from Ascaris lumbricoides and none cross-reacted with vertebrate collagens (including types I, II, in, IV, V, and VI), synthetic collagen-like polymers, or glycoproteins. Immunoblotting showed that each monoclonal antibody reacted with a group of, at least, seven cuticular components. Based on the susceptibility of these components to collagenase and pepsin, it was deduced that the common epitope is a shared collagenous domain.
Hydrophobic binding domains of rat intestinal maltase–glucoamylase
Authors: Lee L. , Forstner G.
Published: December 2009

Rat intestinal microvillus maltase–glucoamylase was isolated by detergent extraction and purification in the presence of protease inhibitors as previously described and incorporated into phospholipid vesicles. After purification of the vesicles on Scphadex G-50, maltase was labelled with 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ([125I]T1D) by photolysis using a water-jacketed mercury vapour lamp with a saturated CuSO4 filter. The labelled enzyme was extracted with acetone, resuspended in 1% Triton X-100, reincorporated into phospholipid vesicles, and digested with activated papain to release the hydrophilic polar head of the enzyme from the vesicle bilayer. Vesicle-bound and free enzyme components were separated on Scpharose 4B. Ninety percent of the enzymatic activity was free, while a similar percentage of radioactive label remained with the vesicles in keeping with the separation of an active polar headpiece from a labelled apolar peptide in the lipid bilayer. The vesicle fractions were subjected to chromatography on Sephadex LH-60 with cthanol – formic acid (7:3) as the eluant. A single radioactive peak (14 kilodaltons (kDa)) was separated from labelled lipid. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of the peak showed a radioactive doublet of 26–28 kDa, possibly representing a dimer. No other labelled peptides were found. These results suggest that detergent-solubilized maltase–glucoamylase is inserted into the phospholipid bilayer via an apolar peptide with a minimum molecular mass of 14 kDa. The peptide probably represents a terminal anchor segment of the 145-kDa subunit which is converted to 130 kDa when the membrane-bound enzyme is solubilized by papain.
Effect of acidic phospholipids on apolipoprotein binding by artificial lipid particles in vivo
Authors: Tong Ming-For , Kuksis Arnis
Published: December 2009

Soybean triacylglycerol particles stabilized with soybean phosphatidylinositol (PI), bovine brain phosphatidylserine (PS), egg yolk phosphatidylcholine (PC) or mixtures of these acidic and neutral phospholipids were prepared with diameters ranging from 250 to 520 nm. Binding of apoproteins to the lipid particles was studied using the strategy of Connelly and Kuksis. The recoveries of the injected particles, which had undergone minimal changes in lipid composition, ranged from 57% for the PC-stabilized emulsions to 21% for the emulsions stabilized with PS and 8% for the emulsions stabilized with PI. The apoprotein (apo) composition of the recovered particles showed characteristic qualitative and quantitative differences. The particles stabilized with PI and PS or PI-phosphatidylethanolamine contained an unknown protein of molecular weight 117 000 (43–48%) and albumin (9–13%) as major components. The apoC-II, apoC-III, apoA-I, apoE, and apoA-IV were present as minor components in ratios that were the reverse of those seen for the PC-stabilized particles, which contained these proteins as major components. The relative strength of the binding of the proteins, which was determined by washing the particles with saline under standard conditions, also showed variations among the different particles and different apoproteins. The lipid particles stabilized with the acidic phospholipids had less total apoprotein and held it less tightly than the particles stabilized with PC. It is concluded that the binding of apoproteins by lipid particles stabilized with acidic phospholipids involves hydrophobic and ionic interactions, both of which may be physiologically important.
The hysteretic effect of Gpp(NH)p on adenylate cyclase is not altered by Mg2+ in adipocyte membranes of ob/ob mice
Authors: Bégin-Heick Nicole
Published: December 2009

We have established previously that the regulation of adenylate cyclase is abnormal in adipose tissue membranes of ob/ob mice. To help establish the nature of the defect, we studied the time course of guanine nucleotide activation and inhibition of adenylate cyclase. The activation of adenylate cyclase by Gpp(NH)p in adipocyte membranes of normal (+/+) and ob/ob mice proceeds with a lag phase. In +/+ membranes, this lag could be shortented by increasing the concentration of Mg2+ in the incubation medium or by pretreatment of the membranes with cholera toxin, and it could be abolished by isoproterenol in combination with 4 mM MgCl2. In contrast, in the ob/ob membranes, only pretreatment with cholera toxin was effective in shortening the lag phase. These results indicate an impediment in the activation of adenylate cyclase in ob/ob membranes. In the +/+ membranes, Gpp(NH)p inhibited forskolin-stimulated adenylate cyclase, following a short lag phase, producing lower steady-state velocities than those seen with forskolin alone. The inhibitory effect of Gpp(NH)p on forskolin-stimulated activity was abolished by pertussis but not by cholera toxin treatment. In the ob/ob membranes, neither Gpp(NH)p nor pertussis treatment had any effect on the steady-state velocity of the forskolin-stimulated activity. These data have been interpreted as meaning that an anomaly in Ni rather than in Ns is likely to be responsible for the impairment of adenylate cyclase activity in the membranes of the ob/ob mouse.
Long-term distribution and metabolism of [1-14C]dolichol injected intravenously into rats
Authors: Rip Jack W. , Carroll Kenneth K.
Published: December 2009

We have previously shown that [1-14C]dolichol mixed in vitro with rat serum and injected intravenously is rapidly cleared from the circulation and appears primarily in the liver. One day after injection the liver accounted for 80% of the isotope in whole animals, whereas after 130 days it represented only 50%. During the 130 days the specific radioactivity (dpm/g liver) decreased by more than 20-fold. In contrast, the spleen retained at 130 days 85% of the radioactivity initially present and its specific radioactivity decreased by only a factor of two. At this time small amounts of isotope were also found in carcass (internal organs removed), gastrointestinal tract and contents, and lungs. Trace amounts of radioactivity were extractable from testes and kidneys, while the heart and brain were essentially free of radioactivity. At all times after injection nearly all the radioactivity present in all tissues was still associated with dolichol. Only trace amounts of [1-14C]dolichyl fatty acyl ester and no [1-14C]phosphorylated derivatives of dolichol were present in the liver and spleen removed 156 days postinjection. Fractionation of liver between 1 h and 93 days after injection suggested that [1-14C]dolichol becomes associated primarily with a lysosome-enriched fraction. The accumulation of [1-14C]dolichol in this and other subcellular compartments involved both an inward and outward flow of radioactivity, suggesting that deposition of dolichol in lysosomes is not a one-way terminal process.
Activation of phosphoenolpyruvate carboxykinase isolated from Veillonella parvula
Authors: Chau Helen S. , Ng Stephen K.
Published: December 2009

Phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) has been purified 940-fold from Veillonella parvula using protamine sulphate treatment, ammonium sulphate precipitation, and column chromatography. The purified enzyme was substantially free of contaminating enzymes or proteins. Maximum activity in the direction of oxaloacetate (OAA) decarboxylation was exhibited at pH 9.0. At this pH, the V. parvula enzyme catalysed phosphenolpyruvate formation in the presence of Mn2+ ions. In the presence of varying concentrations of OAA and ATP, the PEPCK from V. parvula exhibited hyperbolic kinetics with Kms of 0.16 and 0.46 mM, respectively. PEPCK from the anaerobe was not inhibited by NADH, succinate, glutamate, D-glucose 6-phosphate, acetyl phosphate, D-fructose 1,6-bisphosphate, pyruvate, ribose 5-phosphate, and aspartate. However, acetyl CoA, glyceraldehyde 3-phosphate, 3-phospho-D-glycerate, CTP, and GTP activated the enzyme. The activation of acetyl CoA was uncompetitive and noncooperative.
Purification and polyamine activation of a high-affinity 3′,5′-cAMP phosphodiesterase from rabbit muscle
Authors: Narindrasorasak Suree , Sanwal Bishnu D.
Published: December 2009

A high-affinity phosphodiesterase, termed PDE II, has been purified about 1400-fold from rabbit skeletal muscle. This enzyme is activated by treatment with proteases. It is also activated specifically by polyarginine and arginine-rich histones, but not by other polyanions. The activation is counteracted nonspecifically by polycations, such as heparin and chrondroitin sulphate. When the enzyme is fully activated by polyarginine it is no longer susceptible to activation by proteases. A conformational or structural change must thus occur in the enzyme by the binding of the polyanions.
The effects of pH, buffers, and fatty acid concentration on the incorporation of radioactive arachidonate into endogenous neuronal nuclear lipids
Authors: Baker R. Roy , Chang Huu-Yi
Published: December 2009

Using neuronal nuclei (N1) isolated from cerebral cortices of 15-day-old rabbits the incorporation of [3H]arachidonate into N1 lipids was followed in vitro. Arachidonate was principally incorporated into triacylglycerol and phosphatidylinositol. When low concentrations (32 mM) of Tris–HCl (pH 7.4) were used, rates of total arachidonate incorporation were small and phosphatidylinositol received the bulk (> 84%) of the arachidonate. When the concentration of Tris–HCl (pH 7.4) or, in certain cases, the concentration of arachidonate was increased, there was a rise in total arachidonate incorporation into N1, with an increasing proportion of radioactivity entering triacylglycerol until it was the predominantly labelled lipid. Using other buffers (phosphate, imidazole, HEPES, pH 7.4), the shift from phosphatidylinositol to triacylglycerol as principal labelled lipid, with buffer concentration, was not as marked as with Tris–HCl (pH 7.4). When the buffer concentration was maintained at 107 mM and the pH was lowered to 6.5, the three amine-containing buffers showed a sizeable decline in arachidonate incorporation into N1 lipids and a corresponding decrease in triacylglycerol labelling. The proportion of the total radioactivity in N1 phosphatidylinositol rose as the pH declined. Of the buffers used, Tris–HCl showed the greatest changes over the pH range. Based upon pK values for the amine buffers, it is suggested that an increased proportion of the protonated amine may be inhibitory to arachidonate incorporation in N1. Studies of acyl-CoA synthetase in N1 indicated this enzyme as the site of the inhibition.
Yeast enolase carboxyl modification using Woodward's reagent K
Authors: Sinha Uma , Brewer John M.
Published: December 2009

Yeast enolase is inactivated by Woodward's reagent K. Substantial protection is afforded by binding of 1 mol of "conformational" metal ion/subunit. Inactivation is correlated with modification of 13 carboxyl groups/subunit in the absence of conformational metal ion and 17 in its presence. Ten tryptic peptides labeled by Woodward's reagent K can be isolated, mostly from the C-terminal half of the protein. The changes in reactivity of these peptides produced by conformational metal ion suggest direct coordination to Glu-181 together with a contraction of the protein.
Diacylglycerol lipase and kinase activities in rabbit aorta and coronary microvessels
Authors: Severson David L. , Hee-Cheong Mariette
Published: December 2009

Diacylglycerol lipase and kinase activities were measured in particulate and soluble fractions from rabbit aorta (intima–media) and coronary microvessels. With rabbit aorta, the hydrolysis at the sn-1 position of 1-palmitoyl-2-oleoyl-sn-glycerol had a pH optimum of 5–6 and was greater than hydrolysis at the sn-2 position (pH optimum of 6.5). Only the 2-monoacylglycerol accumulated during incubations at pH 5 and 6.5. These results are consistent with an ordered two-step reaction sequence where the fatty acid at the sn-1 position is released first, followed by the hydrolysis of the fatty acid from the 2-monoacylglycerol by a monoacylglycerol lipase with a neutral pH optimum. Lipase activity (sn-2 hydrolysis) at pH 6.5 was greater than kinase activity at all substrate concentrations. The presence of arachidonate at the sn-2 position of the diacylglycerol increased kinase activity but had little effect on lipase activity. Kinase activity was mainly particulate, whereas 50–60% of diacylglycerol lipase and 50% of monoacylglycerol lipase activity were soluble. Diacylglycerol lipase and kinase were also present in coronary microvessel preparations. Diacylglycerol lipase (sn-2 hydrolysis) activity in coronary microvessels was not enhanced by preincubation of the enzyme preparation with cAMP-dependent protein kinase.
The quantitative comparison of the effects of denaturants on the stability of proteins
Authors: Thomson James A. , Bigelow Charles C.
Published: December 2009

A novel quantitative comparison of denaturants involving the complete reversible unfolding of proteins is presented. Ribonuclease A was denatured with guanidinium chloride in the presence of low fixed concentrations of various partial denaturants, with the unfolding process being monitored by circular dichroism and difference spectroscopy. The major advantage of this method is that it allows a direct quantitative comparison of the effects of denaturants on the stability of proteins. The effect on the stability of ribonuclease A was shown to be linearly dependent upon the concentration of denaturant. An investigation of the constitutive ions of salts revealed that their effects were additive only in the case of salts that have no specific binding capability. This method can also be useful in detecting the specific binding of salts.
Evidence for a defective thiol protease inhibitor in skeletal muscle of mice with hereditary muscular dystrophy
Authors: Gopalan P. , Dufresne M. J. , Warner A. H.
Published: December 2009

The thiol protease inhibitor (TPI-d) from hind-limb skeletal muscle of dystrophic 60-day-old male mice (strain 129/ReJ-dy) has been purified to apparent homogeneity and compared with the thiol protease inhibitor (TPI-n) from hind-limb skeletal muscle of normal 60-day-old male littermates. While both TPI-d and TPI-n displayed identical properties on sodium dodecyl sulfate – polyacrylamide gels (14 800 relative mass), analytical isoelectric focusing gels (pI 4.5), and high performance liquid chromatography columns, TPI-d was unable to inhibit papain and cathepsin B after purification by isoelectric focusing. However, a component in the purified TPI-d preparation with an isoelectric point of 4.9 initially masked the functional state of TPI-d, using papain when assayed with the test proteases papain and cathepsins H and L. This inhibitory component was absent from TPI-n preparations. Pure TPI-d was also unable to inhibit in vitro myosin hydrolysis by cathepsin B, whereas TPI-n completely blocked cathepsin B catalyzed myosin hydrolysis. Given the central role of the thiol proteases, especially cathepsin B, in intracellular protein metabolism and the possibility that uncontrolled thiol protease activity in muscle leads to muscle protein breakdown and dystrophy, our data suggest that a modified (defective) thiol protease inhibitor (TPI-d) may be (one of) the end product(s) of the dystrophy gene in mice with the hereditary form of the disease.
Nonlogical factors in research: chance and serendipity
Authors: Rothstein Aser
Published: December 2009

Although successful biological research appears to be based on logical inference, on systematic accumulation of information, and on evaluation and hypothesis testing, many nonlogical, unpredictable factors may play an important role or even open new avenues of research. These may include emotional decisions, laboratory accidents, the impact of unanticipated external events, unplanned interactions with other scientists, and relationships with graduate students and postdoctoral fellows. Based on personal experiences, the impact of such nonlogical factors, if exploited fully, can be considerable.
Experimental approaches to the study of the biogenesis of mammalian mitochondrial proteins
Authors: Freeman Karl B. , Yatscoff Randall W. , Ridley Robert G.
Published: December 2009

Mitochondrial proteins are synthesized in mitochondria and on cytosolic ribosomes. Several approaches used to establish the site of synthesis and the identity of mitochondrially synthesized proteins are described. These include the specific inhibition of mitochondrial translation by inhibitors or mutation and the specific elimination of cytosolic translation either by using isolated mitochondria or specific inhibitors. Experimental approaches to study the import of proteins into mitochondria are also discussed.
Antigenic analysis of testicular cytochromes c using monoclonal antibodies
Authors: Kim In C. , Nolla Hector
Published: December 2009

Testicular cytochrome c (cyt ct) was isolated from testes of sexually mature, rat, mouse, rabbit, and bull, among which rat testis is highly rich in cyt c1. By fusion of NS-1 myeloma cells and spleen cells of mice immunized with rat cyt ct, 11 stable mouse hybridoma cell lines were established. Using an enzyme-linked immunosorbent assay, it was determined that 4 of the 11 anti-rat cyt ct monoclonal antibodies (McAb) did not bind to somatic cyt c (cyt cs) of vertebrates nor to cyt ct of mouse, rabbit, and bull. Four other McAb showed no binding to cyt cs but showed different patterns of cross-reactivity with these four cyt ct. Therefore, these McAb appear to be very sensitive and useful probes for the discrimination or identification of extremely similar isocytochromes c. Although the primary amino acid sequences between cyt cs of rat and mouse are identical, the antigenic structure of cyt ct of rat and mouse are clearly distinct with regard to cross-reactivity with some anti-rat cyt ct McAb. Furthermore, these McAb also reveal that the primary amino acid sequences of cyt ct, which reflect differences in the surface conformation of the molecule, are probably species specific.
Inhibition of the cathepsin B like proteinase by a low molecular weight cysteine-proteinase inhibitor from ascitic fluid and plasma α2 macroglobulin
Authors: Pagano Maurice , Keppler Daniel , Fumeron-Dalet Veronique , Engler Robert
Published: December 2009

The cathepsin B like proteinase present in ascitic fluid of a patient with neoplasia has been purified and characterized after pepsin activation. From this fluid we have prepared the low molecular weight (LMW) cysteine-proteinase inhibitors. Three major inhibitor forms were found with isoelectric points of 7.4, 5.4, and 5.1, respectively. The interaction of the enzyme with the former inhibitor was studied because this inhibitor was the most abundant. The Ki value was found to be 4.3 × 10−8 M. Two molecules of this proteinase were bound by one molecule of plasma α2 macroglobulin (α2M). The LMW inhibitor was able to bind to the enzyme entrapped in α2M and reduced its endopeptidase activity on benzyloxycarbonyl-L-phenylalanyl-L-arginine-4-methyl-7-coumarylamide. These results may have a physiological significance in the regulation of the enzyme which, among other extracellular hydrolases, probably plays an important role in tumor invasion.
Activities of NAD- and NADP-specific isocitrate dehydrogenases in kidney mitochondria of rachitic rat
Authors: Treves Cristina , Vincenzini Maria Teresa , Vanni Paolo , Iantomasi Teresa , Stio Maria , Baccari Vincenzo
Published: December 2009

In the previous research we have demonstrated that rats fed on a rickets-inducing diet show increasing citrate levels in kidney and intestinal mucosa. The study of the enzymes related to citrate metabolism has shown that both NAD+- and NADP+-dependent isocitrate dehydrogenases decrease in kidney mitochondria of rachitic rat. The inhibitory effect of Ca2+ and citrate on the activity of the two dehydrogenases has been also investigated; these metabolites behave as competitive inhibitors against Mg2+ both in normal and in rachitic rats.
Synthetic peptides as substrates for processing enzymes in the bovine pituitary
Authors: Lee K. K. , Conway Aoife M. , Carne Thomas J.
Published: December 2009

Synthetic peptides, based on sequences of proopiomelanocortin (POMC) cleaved in both the bovine anterior and intermediate pituitaries (-Phe-Pro-Leu-Gly-Phe-Lys-Arg-Glu-Leu-Thr-Gly-) and only in the intermediate lobe (-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-), were used as substrates for the enzymes that process POMC to active hormones in the anterior and intermediate lobes of the pituitary. Cleavage of these peptides at the dibasic pair of residues, the expected cleavage site, was observed with a lysate from bovine pituitary secretory granules. Cleavage occurred optimally at a pH between 4 and 5 and was inhibited with sulfhydryl reagents, pepstatin, and leupeptin. Little specificity for the nature of the basic residues at the cleavage site was observed. An additonal cleavage, following glutamic acid residues, was also seen.
Carbodiimide-mediated conjugation of hippuric acid to concanavalin A: retention of ligand binding and hemagglutinating activities
Authors: Preston III James F. , Hencin Ronald S.
Published: December 2009

Conditions for conjugating small molecules with reactive carboxyl groups to concanavalin A (ConA), with retention of biological activity of the lectin, are described. Hippuric acid was conjugated to reactive amino groups on ConA with N-ethyl-N′-(dimethylaminopropyl)carbodiimide under conditions in which neither inter- nor intra-molecular cross-linking was detectable. These same conditions provided for the loading of variable amounts of hippurate as a function of reaction time; conjugates were synthesized with 9.6 mol hippurate∙mol ConA−1. Conjugation in the presence of 0.1 M phosphate provided a condition for limiting the extent of coupling; only amide linkages were formed. These conjugates retained both the ability to bind ligands and the hemagglutination titer of the native lectin.
Structural elucidation of the O-specific polysaccharide of the phenol-phase soluble lipopolysaccharide of Vibrio anguillarum
Authors: Banoub Joseph H. , Michon Francis , Hodder Howard J.
Published: December 2009

The structure of the O-specific polysaccharide of the phenol-soluble cellular lipopolysaccharide of Vibrio anguillarum has been investigated. The studies involved the use of methylation analysis, partial hydrolysis with 48% hydrogen fluoride, Smith degradation, oxidation with chromium trioxide, and comprehensive proton and carbon-13 nuclear magnetic resonance studies, in which one- and two-dimensional experiments were carried out. As a result of these studies it is proposed that the O-specific polysaccharide of Vibrio anguillarum is composed of a regular heteropolymer, i.e., a main chain of (1→4)-linked 3-acetamido-3,6-dideoxy-β-L-glucose residues alternately substituted through O-2 with side chain residues of 2-acetamido-2,6-dideoxy-α-D-glucose, which seem to be substituted through either O-3 or O-4 with propionyl groups (R), as in the following structure.[Formula: see text]
Activities of both ribonucleotide reductase subunits, M1 and M2, decrease upon serum starvation of baby hamster kidney 21/C13 cells
Authors: Cohen Eric A. , Filion Mario , Suh Martha , Langelier Yves
Published: December 2009

Ribonucleotide reductase from mammalian cells is composed of two nonidentical subunits M1 and M2 which are both required to form the catalytic site. The level of ribonucleotide reductase activity is cell cycle controlled and several reports suggest that this control is achieved mainly by the regulation of M2 subunit synthesis. In the present study, we have found that the activities of both subunits decreased markedly upon serum starvation in the Syrian baby hamster kidney 21/C13 cell line. These decreases did not seem to be correlated with the appearance of an inhibitory factor in serum-starved cells. Quantification of the amount of the M1 subunit protein (89 000 molecular weight) by [12P]dTTP photoaffinity labelling revealed that the decrease in M1 activity was not due to variation in M1 protein level. Therefore, a posttranslational mechanism probably exists which inactivates M1 subunit when cells stay in the quiescent (G0) state and this mechanism could play an important role in the control of ribonucleotide reductase activity.
Variability in the posttranslational processing of penicillin-binding protein 1b among different strains of Escherichia coli
Authors: Rojo Fernando , Berenguer José , Ayala Juan A. , De Pedro Miguel A.
Published: December 2009

Screening of a number of unrelated strains of Escherichia coli confirms the existence of at least two patterns of molecular forms for penicillin-binding protein 1b in E. coli cell envelopes. Our data support that the β-form of this protein is produced by posttranslational modification of the α-form and suggest that the absence of the β-form in some strains is due to a strain-dependent variability in the α-form processing mechanism.
Multicompartmental distribution of ribonuclease II in mouse liver
Authors: Trzaskos James M. , Meyer William L.
Published: December 2009

Thirty percent of RNase II (EC 3.1.27.5) is present in the cytosol of mouse liver where it exists in an inactive complex with a protein inhibitor. The remaining 70% of RNase II is active, soluble enzyme unassociated with inhibitor and is distributed in a ratio of 1.3 to 1 between the lumen of reticular elements and the interior of heavy particles. Although heavy particle RNase II resembles acid hydrolases in centrifugal behavior, in other tests including density shift experiments the resemblance is incomplete. In experiments employing lysis induced by L-amino acid methyl esters, RNase II activity is much more latent than the activity of the lysosomal marker, acid RNase. It is postulated that the heavy particle component of RNase II is contained in a secretory vesicle rather than in classic lysosomes.
Regulation of the number of Na+,K+-pump sites after mitogenic activation of lymphocytes
Authors: Severini Alberto , Prasad K. V. S. , Almeida Anthony F. , Kaplan J. Gordin
Published: December 2009

The early activation of Na+,K+-ATPase-mediated ion fluxes after concanavalin A (ConA) stimulation of pig lymphocytes is caused by an increase in intracellular Na+ concentration. A second mechanism of regulation of Na+,K+-ATPase activity becomes apparent between 3 and 5 h after mitogenic stimulation, but prior to onset of increase in cell volume; this consists of an increase (about 75%) in the number of ouabain-binding sites (from 35 × 103 ± 12 × 103/cell in resting to 60 × 103 ± 27 × 103/cell in activated lymphocytes). The increase in ouabain binding was attributed to an increase in the number of active Na+,K+-ATPase molecules, based on the following evidence: (i) there was an increase in the Vmax of ouabain binding, without variation in the Km; (ii) the increase in ouabain binding was accompanied by a proportional increase in K+ influx, when the assay was performed in the presence of the Na+ ionophore monesin, which was used to eliminate the difference in intracellular Na+ concentration between resting and activated cells; (iii) there was proportionality between ouabain-inhibitable ATPase activity in permeabilized cells and the number of ouabain-binding sites in resting and activated lymphocytes. The ConA-induced increase in ouabain-binding sites was influenced neither by amiloride nor by incubation in low Na+ medium, under conditions which prevented both increase in intracellular Na+ concentration and K+ influx. Increase in intracellular Na+ concentration was ineffective in altering the number of active pump molecules in resting cells. During incubation with ConA, the presence of ouabain did not affect the increase in ouabain-binding sites; thus, regulation of the number of pump sites is independent of the regulation of their activity. The ConA-induced increase in number of ouabain-binding sites did not require protein synthesis; indeed, cycloheximide, anisomycin, and puromycin, under conditions in which they inhibited protein synthesis by 95%, induced the increase to approximately the same extent as did ConA. This suggests the presence in resting lymphocytes of a rapidly turning over protein that either prevents the ATPase subunits from assembling or from integrating into the membrane.
Cannabinoids reduce fertility of sea urchin sperm
Authors: Schuel Herbert , Schuel Regina , Zimmerman Arthur M. , Zimmerman Selma
Published: December 2009

Cannabinoids are potent pharmacological substances derived from marihuana. The effects of Δ9-tetrahydrocannabinol (THC), cannabinol (CBN), and cannabidiol (CBD) on fertilization in the sea urchin Strongylocentrotus purpuratus were investigated. Insemination of THC-treated eggs (5–400 μM) with excess sperm did not result in polyspermic fertilization. At minimal sperm densities, THC (0.1–10 μM) inhibited fertilization in a dose-dependent manner. Pretreatment of eggs with THC did not reduce their receptivity to sperm. Pretreatment of sperm with THC reduced their fertilizing capacity. The concentration of THC required to reduce sperm fertility by 50% was 1.1 ± 1.1 μM. The fertilizing capacity of THC-treated sperm depended on concentration of sperm and duration of pretreatment. The fertility of sperm at minimal densities was reduced by 50% at 129.3 ± 43 s treatment with 10 μM THC. The adverse effect of THC on sperm fertility was reversible. CBN and CBD at comparable concentrations (0.1–10 μM) inhibited fertilization in a manner similar to THC. First division was not delayed in zygotes that were fertilized with sperm pretreated with 10 μM THC. These studies show that cannabinoids directly affect the process of fertilization in sea urchins by reducing the fertilizing capacity of sperm.
Saturation analysis of cellular retinoid binding proteins: application to retinoic acid resistant human neuroblastoma cells and to human tumors
Authors: Kelly Michael A. , Sidell Neil , Haussler Mark R.
Published: December 2009

A method for saturation analysis of cellular retinoic acid and retinol binding proteins, CRABP and CRBP, respectively, in cultured cells and human tumor samples, and its application to a retinoic acid resistant subline of the human neuroblastoma LA-N-5 cell line is described. Assessment of retinoid binding was accomplished by incubation of cytosols with increasing concentrations of [3H]retinoid (28–43 Ci/mmol; 1 Ci = 37 GBq) for 24 h. Bound retinoid was separated from free retinoid by adsorption with dextran-coated charcoal. Nonspecific binding was quantitated in parallel incubations which had been treated with p-chloromercuribenzene sulfonate (PCMBS), resulting in selective elimination of sulfhydryl-dependent ligand binding to both CRABP and CRBP. Quantitation was accomplished by Scatchard analysis of specific (PCMBS sensitive) binding. Employing this technique, specific retinoid binding was attributed to the presence of 2S macromolecules which displayed the known properties of CRABP and CRBP, namely ligand specificity, saturability, high ligand affinity, and PCMBS sensitivity. The apparent dissociation constants (Kd) for retinoic acid binding in cytosols prepared from murine 3T6 fibroblasts, rat testes, and a human ovarian tumor were 7, 11, and 35 nM, respectively. These preparations also bound retinol with high affinity, exhibiting Kds of 12, 26, and 48 nM, respectively. A retinoic acid resistant subline of LA-N-5 cells designated LA-N-5-R9 was established by long-term culture in the presence of 10−6 M retinoic acid. This subline is resistant to the effects of retinoic acid in that it requires a 10-fold higher concentration of retinoic acid for 50% inhibition of growth than the parent line and displays no retinoic acid induced morphologic differentiation. Saturation analysis of CRABP in the parent and resistant subline reveal no significant alteration in either CRABP content or affinity. These results indicate that resistance to retinoic acid induced differentiation in LA-N-5-R9 occurs distal to CRABP binding or that CRABP does not mediate this response to retinoic acid.
Isolation and characterization of a mitochondrial RNA polymerase from Drosophila melanogaster
Authors: Goldenthal Michael , Nishiura James T.
Published: December 2009

A DNA-dependent RNA polymerase was solubilized from sucrose gradient isolated, DNase-treated mitochrondria of Drosophila melanogaster. The isolated mitochondria were not detectably contaminated with nuclear DNA as shown by CsCl gradient centrifugation and polylysine Kieselguhr chromatography. The detergent-solubilized RNA polymerase was sensitive to rifampicin, resistant to α-amanitin, had an apparent molecular mass of about 60 kilodaltons, and displayed a tendency to aggregate, both in crude extracts or when purified. The mitochondrial RNA polymerase could be distinguished from nuclear RNA polymerases on the basis of size, salt optima, rifampicin sensitivity, and α-amanitin resistance.
Characterization of serum, liver, and intestinal sialyltransferases from rats treated with colchicine
Authors: Ratnam Shobhitha , Nagpurkar Arun , Mookerjea Sailen
Published: December 2009

A modified high pressure liquid chromatographic method using lactose (Galβ1 → 4G1c) as an exogenous acceptor has been used to characterize the sialyltransferases known to increase in the serum of colchicine-treated rats. The results show a 10-fold increase of Galβ1 → 4GlcNAc α2 → 6sialytransferase (α2 → 6 ST), whereas the Galβ1 → 3GlcNAc α2 → 3sialyltransferase showed only 1.6-fold increase in the serum after 17 h of colchicine treatment. The sialyltransferase activity in serum using exogenous desialylated, α1-acid glycoprotein as acceptor also showed an eightfold increase. In liver homogenate and Golgi membrane, the sialyltransferase activity when assayed with desialylated α1-acid glycoprotein as acceptor showed a slight decrease after 4 h, but returned to normal level after 17 h. A similar trend was seen when the two transferases were assayed with lactose as acceptor. The antiserum to rat α2 → 6 ST inhibited the sialyltransferase activity in serum, liver, and jejunal incubation medium. Jejunal sections from rats treated with colchicine for 4 h in presence of heated serum showed a decrease of sialyltransferase, with consequent increase of the α2 → 6 ST enzyme activity in the medium. This result suggests that intestinal tissue could be a source of increased serum enzyme activity in colchicine treatment.
Patterns of protein synthesis during the cell cycle of Chinese hamster ovary cells
Authors: Westwood J. Tim , Church Robert B. , Wagenaar Emile B.
Published: December 2009

The protein synthesis patterns at various stages of the cell cycle of Chinese hamster ovary cells were examined by labelling cells with [35S]methionine and then separating the proteins by isoelectric focussing and two-dimensional, nonequilibrium pH gradient gel electrophoresis. We have observed a number of proteins which display quantitative differences in synthesis at specific cell cycle stages and of these the α- and β-tubulins have been identified. A few proteins appear to be uniquely synthesized at specific times during the cell cycle. These include the histones and a modified version of them, which are synthesized only in S phase, and a pair of 21 kilodalton (kDa), pI 5.5 proteins, which appear only in late G2 and mitosis. We have also identified a 58-kDa, pI 7.5 protein which is present at all cell cycle stages except during late G2. This protein appears to have the same temporal properties as a 57-kDa protein called "cyclin" originally described in sea urchin embryos.
Purification and properties of the cytosolic and mitochondrial forms of aspartate aminotransferase and malate dehydrogenase from rat heart
Authors: Mavrides Charalampos , Nadeau Guylaine
Published: December 2009

The present work describes the purification from rat heart of the mitochondrial and cytosolic forms of the enzymes of the malate–aspartate shuttle, aspartate aminotransferase (EC 2.6.1.1) and malate dehydrogenase (EC 1.1.1.37), by a single procedure after the preparation of the original crude extract. In 10 purification steps, the four enzymes were obtained electrophoretically pure in yields ranging from 6 to 54% of their respective isoenzyme levels in the crude extract. Apoenzymes were formed from the aminotransferases by reacting them with cysteine sulfinate and dialyzing. Complete reconstitution was obtained after a brief incubation with pyridoxal phosphate. All four enzymes are dimers. The mitochondrial isoenzymes are of slightly lower molecular weight than their respective cytosolic forms. Michaelis constants and maximal velocities were derived by the use of primary and secondary plots. In general, the properties of the enzymes from rat heart are similar to the properties of the enzymes from other animal sources.
The interaction of lipolysis products of very low density lipoprotein with plasma high density lipoprotein (HDL): perfusate HDL with plasma HDL subfractions
Authors: Tam S. P. , Breckenridge W. C.
Published: December 2009

The nature of the interaction of high density lipoproteins (HDL), formed during lipolysis of human very low density lipoprotein (VLDL) by perfused rat heart, with subfractions of human plasma HDL was investigated. Perfusate HDL, containing apoliproproteins (apo) E, C-II, and C-III but no apo A-I or A-II, was incubated with a subfraction of HDL (HDL-A) containing apo A-I and A-II, but devoid of apo C-II, C-III, and E. The products of the incubation were resolved by heparin-Sepharose or hydroxylapatite chromatography under conditions which allowed the resolution of the initial HDL-A and perfusate HDL. The fractions were analyzed for apolipoprotein content and lipid composition and assessed for particle size by electron microscopy. Following the incubation, the apo-E-containing lipoproteins were distinct from perfusate HDL since they contained apo A-I as a major component and apo C-II and C-III in reduced proportions. However, the HDL-A fraction contained apo C-II and C-III as major constituents. Associated with these changes in apolipoprotein composition, the apo-E-rich lipoproteins acquired cholesteryl ester from the HDL-A fraction and lost phospholipid to the HDL-A fraction. The HDL-A fraction maintained a low unesterified cholesterol/phospholipid molar ratio (0.23), while the apo-E-containing lipoproteins possessed a high ratio (0.75) characteristic of the perfusate HDL. The particle size of apo-E-containing lipoproteins (138.9 ± 22.5 Å; 1 Å = 0.1 nm) was larger than the initial HDL-A (126.5 ± 17.6 Å) or the new HDL-A-like fraction (120.9 ± 17.4 Å) obtained following incubation with perfusate HDL. It is concluded that incubation of perfusate HDL containing apo E, C-II, and C-III with plasma HDL subfractions results in the acquisition of apo A-I and cholesteryl esters by the apo-E-containing perfusate HDL and the loss of apo C-II, C-III, and phospholipid to the plasma HDL-A fraction. The process does not appear to be due to fusion of the particles, since the apo-E-containing lipoproteins maintain a cholesterol/phospholipid ratio distinct from the HDL-A fraction. The data provide evidence for a potential mechanism for the formation of HDL-E, an apo-E-containing lipoprotein of HDL size and density, through lipolysis of VLDL.
Heat and sodium arsenite act synergistically on the induction of heat shock gene expression in Xenopus laevis A6 cells
Authors: Heikkila J. J. , Darasch S. P. , Mosser D. D. , Bols N. C.
Published: December 2009

Heat shock protein (HSP) synthesis was studied in the Xenopus epithelial cell line A6 in response to heat and sodium arsenite, either singly or together. Temperatures of 33–35 °C consistently brought about the synthesis of HSPs at 87,73,70,54,31, and 30 kilodaltons (kDa), whereas sodium arsenite at 25–100 μM induced the synthesis of HSPs at 73 and 70 kDa. In cultures exposed to 10 μM sodium arsenite at 30 °C, HSP synthesis in the 68- to 73-kDa and 29- to 31-kDa regions was much greater than the HSP synthesis in response to each treatment individually. RNA dot blot analysis using homologous genomic subclones revealed that heat shock induced the accumulation of HSP 70 and 30 mRN As. The sizes of the HSP 70 and 30 mRN As determined by Northern hybridization were 2.7 and 1.5 kilobases, respectively. Sodium arsenite (10–100 μM) also induced the accumulation of both HSP 70 and 30 mRNAs. Finally, a mild heat shock (30 °C) plus a low concentration of sodium arsenite (10 μM) acted synergistically on HSP 70 and 30 mRN A accumulation in A6 cells. Thus sodium arsenite and heat act synergistically at the level of both HSP synthesis and HSP mRNA accumulation.