bluuebunny/crossref_metadata_2025 huggingface dataset:
High salt and solvent induced Z‐conformation in native calf thymus DNA
Authors: Ali Naushad , Ali Rashid
Published: July 2007

The Z‐DNA forming potentiality of native calf thymus DNA using spectrophotometric measurements and diethyl pyrocarbonate reactivity have been investigated. The [image omitted] value was evaluated from absorbance ratio for B‐ and Z‐DNA and compared with the values obtained for native DNA. The results suggest that native DNA undergoes B‐ to Z‐/Z‐analogous transition to an extent of 20 per cent under constraint conditions. Possible Z‐DNA formation was also characterized by hyper‐reactivity of native DNA towards diethyl pyrocarbonate in aqueous solvent. The thermal melting profile of native and modified DNA and nuclease S1 digestibility data, in addition, reflects B‐ to Z‐/Z‐analogous transition in native DNA.
Nanomolar concentrations of gangliosides stimulate primary humoral response
Authors: Dozmorov I. M. , Prokhazova A. L. , Svirtshevskaya E. V. , Lutsan N. I. , Kozin I. I. , Lemenovskaya A. F. , Dyatlovitskaya E. V. , Bergelson L. D.
Published: July 2007

Exogenous gangliosides act as immunosuppressors when applied at micromolar concentrations corresponding to their average level in human plasma. Here we show that at nanomolar concentrations the gangliosides GD3, GD1a and GM1 can act as immunostimulators markedly enhancing the number of plaque‐forming cells in mouse splenocyte culture responding to sheep erythrocytes. At such low concentration these gangliosides as well as GM3 were not able to influence significantly proliferative responses of splenic B and T lymphocytes or of cytotoxic T‐cells. Neither did they change significantly the production of IL‐1 by antigen‐ representing cells, or of IL‐2 by Con A‐induced blasts in the splenocyte culture. It is suggested that the stimulatory effect of low ganglioside concentrations on humoral response is due to their influence on cooperative cell‐cell interactions required for the differentiation of B‐cells into Ig‐secreting cells.
Acetylcholinesterase activity of normal and diabetic human erythrocyte membranes: The effect of oxidative agents
Authors: Krajewska Emilia , Zavodnik Ilya , Kluska Beata , Szosland Konrad , Bryszewska Maria
Published: July 2007

The activity characteristics of membrane acetylcholinesterase from red blood cells of diabetic patients are very different from those of healthy donors: the limiting enzyme reaction rate is 17.2±0.8 μmol acetylthiocholine per ml packed cells per min compared with 13.1±0.8 μnol for control cells. The Michaelis constants for substrate are the same: 0.061±0.007 mM for diabetic and 0.061±0.004 mM for control cells. Cell exposure to oxidative agent (t‐butyl hydroperoxide) significantly changes the enzyme activity parameters. The limiting enzyme reaction rate increases but the affinity for the substrate decreases at lower oxidant concentrations (up to 0.1 mM for the “diabetic” erythrocytes and up to 0.4 mM for the control ones). At higher oxidant concentrations both the limiting reaction rate and the Michaelis constant decrease. The susceptibility of erythrocyte membranes of diabetic patients to oxidative stress is much higher in comparison with control erythrocyte membranes.
Effect of thiols on fructosamine assay
Authors: Liu Wei , He Rongqiao
Published: July 2007

Fresh human serum, gGAPDH and β‐mercaptoethanol were used to examine the effect of thiols on fructosamine assay. The kinetics of the reaction with both fresh human serum and gGAPDH displayed biphasic behaviour (fast and slow). When the thiols were modified with IAA, the kinetics only demonstrated the slow phase. Since the absorbance increase in the interval from 9 to 10 min was used in the fructosamine assay of glycated human serum we studied the effect of thiols on that measurement. In the case of gGAPDH, the value was approximately one‐half of the original after thiol modification, suggesting thiol interference. Nevertheless, gGAPDH may contain a fructosamine structure, β‐Mercaptoethanol itself gave a strong positive result in the fructosamine assay. Hence, thiol groups on glycated proteins should be modified before doing a fructosamine assay because of their substantial interference.
Activity and carboxylation specificity factor of mutant ribulose 1,5‐bisphosphate carboxylase/oxygenase from Anacystis Nidulans
Authors: Romanova Alla K. , Zhen‐qi‐Cheng  , McFadden Bruce A.
Published: July 2007

The values of molecular carboxylase activity kcat and carboxylation specificity factor τ for mutant ribulose 1,5‐bisphosphate carboxylase (rubisco) from Anacystis nidulans decreased as compared to those of the wild type recombinant rubisco. The substitution of five amino acid residues in rubisco large subunit Lys,AIa,Ser, Thr, Leu(339‐343)Phe,Leu,Met,Ile,Lys had kcat decreased by 90% and τ by 36.3%. The same parameters for mutants with the single replacements decreased: for Thr342Ile kcat by 40.5% and τ by16.7%, and for mutant Leu343Lys kcat by 48.1% and τ by 18.5%. Mutant rubisco with three amino acid resudues changed Val,Asp,Leu(346‐348)Tyr,His,Thr was inactive. The substitution Leu326Ile decreased kcat by 54.4% and τ by 34.2%; and change Ser328Ala decreased kcat only by 5.6% but τ by 41.5%. Replacement Asn123His decreased kcat by 16.5%. Significance of the non conservative amino acid residues for carboxylase activity and ribulose‐l,5‐bisphosphate partition is discussed.
Effect of 5‐fluorouracil on interleukin‐1 and interleukin‐2 receptor expression
Authors: Balaji K. N. , Nayak R.
Published: July 2007

We have studied the effect of anticancer drug 5‐Fluorouracil on the expression of human Interleukin‐1 and Interleukin‐2 receptor. In this report, we show that 5‐Fluorouracil increases the Interleukin‐1 expression upto 2.66 folds without significantly affecting the levels of surface expression of p55 IL‐2 receptor on human Peripheral blood mononuclear cells, CD4 and CD8 T cells. On contrary, the drug decreases the levels of Interferon‐γ secretion by upto 42%. In earlier studies we have shown that 5‐Fluorouracil increases the IL‐2 expression both at mRNA and protein levels. Taken together, 5‐Fluorouracil differentially affects the expression of Interleukin‐1, Interferon‐γ and Interleukin‐2 receptor.
Expression of the trefoil polypeptide ITF in PC12 cells
Authors: Probst Joseph Christopher , Landgraf Rainer , Behl Christian
Published: July 2007

Trefoil polypeptides are expressed mainly in the amphibian skin and the gastrointestinal tract of mammals, usually coexpressed with mucin‐glycoproteins. Recently, the trefoil polypeptides were shown to be expressed also in different areas of the human and routine brain. To investigate the expression and possible functions of ITF in rat pheochromocytoma (PC12) ceils were employed. PC12 cells show a low basal expression of this polypeptide as determined by reverse transcriptase‐polymerase chain reaction. After treatment with the synthetic glucocorticoid dexamethasone, the second messenger cyclic adenosine monophosphate and the neurotrophic factor nerve growth factor the expression of the trefoil polypeptide ITF was increased as shown by reverse transcriptase‐polymerase chain reaction and by immunocytochemistry. Since these various stimuli can directly alter the expression level of this peptide we conclude that the presented results may form the basis for further investigations of possible functions of this novel gut‐brain polypeptide in neurons using PC12 cells.
Human placenta cytidine deaminase: proton‐linked enzyme activity and substrate binding
Authors: Vincenzetti Silvia , Angeletti Mauro , Lupidi Giulio , Cambi Alessandra , Natalini Paolo , Vita Alberto
Published: July 2007

In this work we focused our attention on some catalytic site requirements for human placenta cytidine deaminase (CDA). The effect of pH on substrate binding and catalysis was studied between pH 3.0 and pH 11.0. The results could be discussed postulating the presence of two classes of ionizable groups in the active site of CDA. The kinetic parameters pH‐dependence has been discussed considering the presence of four zinc atoms per each enzyme tetramer. Furthermore fluorescence studies on the enzyme and on enzyme‐inhibitor complexes, examined by using polar and non polar quenchers, allowed to define the substrate(inhibitor)‐dependent accessibility of the tryptophan‐containing pocket on each CDA monomer.
Heat treated myosin does not bind ATPase ‐ inhibiting antibodies
Authors: Gröschel‐Stewart Ute , Christian Anna‐Luise , Mestan Jürgen
Published: July 2007

Polyclonal antibodies to native chicken pectoral fast‐twitch myosin are directed to all subfragments of the molecule (S1, S2 and LMM), as seen in the ELISA and Western blotting techniques. The antibodies inhibit the Ca2+‐activated myosin ATPase. Absorption of the antibodies with native myosin abolishes these reactions. Heat treatment of myosin for 2h at 40°C will inactivate myosin ATPase and alter its antibody binding pattern: the binding of antibodies to the rod fractions is reduced, that to the globular head (S1) completely abolished. Thus, these antibodies are useful as sensitive probes for the structural integrity of the myosin head.
Heterogeneous nuclear RNP protein A1‐arginine methylation during HCT‐48 cell cycle
Authors: Park Seung Hee , Park Gil Hong , Gu Hyunmin , Hwang Woo‐Ik , Lim In Kyoung , Paik Woon Ki , Kim Sangduk
Published: July 2007

Protein methylase I (protein‐arginine N‐methyltransferase) was examined in HCT‐48 cells, synchronized by serum deprivation and hydroxyurea treatment. The enzyme activity to methylate the added hnRNP protein A1 increased about 2‐fold from G0 to S phase, and then decreased during G2/M phase. The enzymatically [methyl‐3H]‐labeled hnRNP protein A1 was identified by SDS‐PAGE/fluorography, and the products were identified as NG‐monomethylarginine and NG,NG‐dimethyl‐(asymmetric)arginines by HPLC. Among endogenous proteins, the 20‐kDa species in the extract was most intensely [methyl‐3H]‐labeled. This 20‐kDa methylation was markedly inhibited by the addition of exogenous hnRNP protein A1, indicating that these two substrates compete for the same protein methylase. The possible role of this post‐translational modification has been discussed.
The activity of branched‐chain α‐keto acid dehydrogenase complex in rat digestive tracts: Effects of dietary protein and exercise
Authors: Tsukamura Midoriko , Goto Hidemi , Arisawa Tomiyasu , Hayakawa Tetsuo , Kobayashi Rumi , Nakai Naoya , Murakami Taro , Sugiyama Satoru , Shimomura Yoshiharu
Published: July 2007

Branched‐chain α‐keto acid dehydrogenase complex is the rate limiting enzyme in catabolism of branched‐chain amino acids. In this study, we examined effects of dietary protein and exercise on the complex activity in digestive tracts (stomach, small intestine and colon) of rats. Rats were fed a high (30%) or low (8%) protein diet for 3 weeks and a half of rats in each diet group was exercised by 85 min running just prior to sacrifice on the final day of the experiment. Total and actual (active form) activities of the complex were markedly high in stomach compared to other two tissues and the actual activity in stomach was significantly elevated by exercise only in rats fed the high protein diet. Both total and actual activities in colon were only a few percentage of those in stomach, and those in small intestine was further less. These results suggest that rat stomach is the tissue active in catabolism of branched‐chain amino acids, which is promoted by combination of high protein diet and exercise.
The structural analysis of the O‐glycans of the jacalin‐bound rabbit immunoglobulin G
Authors: Kabir S. , Gerwig G. J.
Published: July 2007

Rabbit immunoglobulin G (IgG) is a sialoglycoprotein (2.4% carbohydrate), containing both N‐glycolyl (Neu5Gc) and N‐acetyl (Neu5Ac) neuraminic acids in a ratio of 87:13. A small fraction of rabbit IgG (about 25% of total IgG) bound to jacalin as demonstrated by affinity chromatography. The jacalin‐bound rabbit IgG contained O‐linked glycans which were liberated from the protein by β‐elimination and isolated. Two O‐glycan structures were determined by 1H‐NMR spectroscopy, as being Neu5Gc(α2‐3)‐Gal (β31‐3)‐GalNAc‐ol and Neu5Ac(α2‐3)‐Gal(β1‐3)‐GalNAc‐ol in a ratio of 83…n17.
The priming effects of some human interleukins on the production of leukotrienes by calcium ionophore stimulated leukocytes
Authors: Mansour Mahmoud , EL‐Ahmady Omar , Motawi Tarek
Published: July 2007

This study demonstrates the changing levels of leukotrieneB4 (LTB4) and leukotrieneC4 (LTC4) generated by human white blood cells primed with different human interleukins IL‐3, IL‐5, IL‐6, IL‐8 and with human hematopoietic growth factor granulocyte‐macrophage colony stimulating factor (GM‐CSF). Preincubation of white blood cells at 37°C for 30 min with different human interleukins significantly increased leukotriene production in white blood cells preincubated with diluent control while IL‐3 was without priming effects. Addition of exogenous arachidonic acid to non primed white blood cells increased the production of leukotrienes in response to the calcium ionophore (A23187). The enhancement of the leukotrienes production by white blood cells stimulated by A23187 was only observed when the cells stimulated for periods of 2 min or less. The results reveal that GM‐CSF, IL‐4, IL‐6 and IL‐8 belong to particular set of cytokines with potent modulating effect on inducing inflammatory mediators released by white blood cells, while IL‐3 was ineffctive.
Nucleotide sequence of cDNA fragments coding for the FAD‐, glycerophosphate‐ and calcium‐binding domains of human islet mitochondrial glycerophosphate dehydrogenase
Authors: Novials Anna , Franco Carmen , Malaisse Willy J. , Gomis Ramon
Published: July 2007

The cDNA fragments coding for the FAD‐, glycerophosphate‐ and calcium‐ binding domains of mitochondrial glycerophosphate dehydrogenase (mGDH were synthetized using RNA extracted from freshly isolated pancreatic islets of a normal subject and two‐non‐insulin‐dependent diabetic patients. Single strand conformation polymorphism analysis of the PCR products yielded the same mobility as control cDNA probes. Likewise, the nucleotide sequence and corresponding amino acid sequence were identical to the normal gene bank sequence. These findings argue against the presence, in pancreatic islets, of an mGDH isoform distinct from that previously characterized in extrapancreatic organs.
Diminished response to food deprivation of the rat brown adipose tissue mitochondrial uncoupling system with age
Authors: Garcia‐Palmer Francisco J. , Pericas Jordi , Matamala Juan C. , Puigserver Pere , Bonet María L. , Palou Andreu , Gianotti Magdalena
Published: July 2007

The aim of the present work is to investigate the effect of starvation on brown adipose tissue thermogenic activity with aging. Interscapular brown adipose tissue from female Wistar rats of different ages was used; half of them were fed and the other half were starved for 24 hours. Mitochondria were isolated and mitochondrial protein content, GDP‐binding, Cytochrome‐c Oxidase activity and uncoupling protein levels were measured. Results show a decrease of all studied parameters, indicating a diminished thermogenic activity with age. The response to starvation is almost the same in all the parameters studied: a general reduction with starvation and a progressive disappearance of this response to starvation with aging. On the whole, these results would indicate a deficient regulation of brown adipose tissue thermogenic activity in old animals, as it happens in other animal models with an alterated thermogenesis.
Catalytic and physico‐chemical characteristics of goat spleen cathepsin B
Authors: Agarwal Sudhir K. , Choudhury Santanu D. , Lamsal Madhav , Khan M. Yahiya
Published: July 2007

To improve the level of purity of cathepsin B, we have modified the published procedure [Agarwal, S.K. and Khan, M.Y. (1987) Biochem. Int. 15, 785‐792] by incorporating CM‐Sephadex ion exchange chromatography and chromatofocusing. The enzyme thus isolated could be resolved into one 26 kDa major and a minor 27 kDa protein bands on SDS‐PAGE. The two components, however, could not be separated by gel filtration and they eluted, in a single peak corresponding to a molecular mass of 28.1 kDa . Among the various substrates tested, Z‐Phe‐Arg‐MCA with a Km of 0.058 mM and hemoglobin with a Km of 1.449 μM were the most preferred synthetic and protein substrates respectively. It was found to be a glycoprotein with an acidic pI of 4.8. The enzyme was activated by various thiol‐reducing reagents and inhibited by cysteine proteinase inhibitors, divalent cations, lysyl group modifiers, anti‐inflammatory drug and denaturing agents. The hydrodynamic behaviour of cathepsin B suggested a compact and globular conformation. Immunodiffusion studies with anti‐goat cathepsin B indicated a tissue/species dependence.
Modulation of the electrophoretic ATP‐induced K ‐transport in yeast mitochondria by ΔpH
Authors: Roucou Xavier , Manon Stéphen , Guérin Martine
Published: July 2007

The modulation of the ATP‐induced K+‐transport pathway of the yeast inner mitochondrial membrane by ΔpH was investigated in two ways. First, the inhibitory effect of phosphate was compared to the effect of other permeant acids, demonstrating that a part of the effect of phosphate was linked to its electroneutral transport down ΔpH. However, an additional effect specific for phosphate also occured inside the matrix. Second, the stimulation of the respiration by ATP in the presence of K+ was compared to the effects of the protonophore C1CCP2 and of the K+‐ionophore valinomycin. Quite unexpectedly, the effect of ATP looked more like the effect of CICCP than the effect of valinomycin. This and previous results (Manon et al., Biochimica et Biophysica Acta 1231, 282‐288 (1995)) show that the rate of electrophoretic K+‐entry is limited by the rate of electroneutral K+‐exit via the K+/H+ exchange and is therefore ΔpH‐dependent.
Determination of 8‐hydroxy‐deoxyguanosine formation in rat organs: Assessment of paraquat‐evoked oxidative DNA damage
Authors: Tokunaga Itsuo , Kubo Shin‐ichi , Mikasa Hiroaki , Suzuki Yasuo , Morita Kyoji
Published: July 2007

Paraquat has previously been shown to cause the oxidative damage to DNA in variety of cells and tissues. However, although paraquat‐evoked strand breaks has been extensively studied to assess the DNA damage, the effect of paraquat on base modifications, another marker for the oxidative damage, has not yet been investigated. To further characterize paraquat‐evoked DNA damage, the effect of paraquat on 8‐hydroxy‐deoxyguanosine (8‐OH‐dG) formation in various rat organs was examined. Paraquat markedly increased 8‐OH‐dG contents in various organs, particularly in brain, lung and heart, and this increase reached the maximum levels at 5 days after the drug administration. In contrast, the formation of 8‐hydroxy‐guanosine (8‐OH‐G), a marker for the oxidative damage to RNA, was not significantly affected by paraquat. These results indicate that paraquat causes base modifications as well as strand breaks as a consequence of the oxidative damage to DNA.
A study of the Na /H antiport in the archaeon Methanobacterium thermoautotrophicum strain ΔH
Authors: Majernik Alan , Smigán Peter , Greksák Miloslav
Published: July 2007

The ability of the cells of Mb. thermoautotrophicum strain ΔH to generate a proton gradient (driven by a concentration gradient of sodium ions) at pH 6.8 as well as at pH 8 was demonstrated. The electrogenic Na+/H+ antiport responsible for this process was shown to be inhibited by EIPA and also by DCCD. Artificially increasing of intracellular concentration of Ca2+ in these cells enhanced the Na+/H+ antiport activity, while the lowering of external Ca2+ by EGTA significantly decreased this activity. The apparent Km values for Na+ about 14 and 3 mM at pH 6.8 and 8, respectively, and Vmax about 214 (pH 6.8) and 155 (pH 8) ΔQ/min per mg of cell proteins, respectively, were calculated. It is concluded that the described processes are mediated by the Na+/H+ antiporter which might be a clue to the adaptive bioenergetic behaviour of the cells of Mb. thermoautotrophicum strain ΔH under the different physiological conditions.
Potentiation by its esterification of the inhibitory action of 2‐deoxy‐D‐glucose on D‐glucose metabolism and insulinotropic action
Authors: Vanhoutte C. , Kadiata M. M. , Sener A. , Malaisse W. J.
Published: July 2007

It was recently reported that selected esters of D‐glucose are more efficiently metabolized and display a greater insulinotropic action in pancreatic islets than the unesterified hexose. Likewise, in the present study, the inhibitory action of 2‐deoxy‐D‐glucose tetraacetate upon D‐glucose metabolism in rat erythrocytes and pancreatic islets was found to be more pronounced than that of unesterified 2‐deoxy‐D‐glucose. At a concentration of 10 mM, the ester also inhibited more severely than unesterified 2‐deoxy‐D‐glucose the release of insulin evoked by D‐glucose in isolated islets. It is proposed that 2‐deoxy‐D‐glucose tetraacetate represents a useful tool to facilitate the intracellular delivery of the glucose analog and to increase its efficiency as a potential therapeutic agent.
Sphingomyelinase increases 2‐deoxyglucose uptake and glucose metabolism of human platelets
Authors: Vasta Valeria , Meacci Elisabetta , Romiti Elena , Farnararo Marta , Bruni Paola
Published: July 2007

Recent studies have shown that the sphingomyelinase (SMase)‐catalyzed hydrolysis of sphingomyelin (SM) represents an important cell signalling pathway. Control of SMase activity appears to be crucial for the regulation of multiple biological events in different cell systems; in particular, SMase activity appears to be involved in the control of vascular functions and in atherogenic events. Here we report that SMase treatment of human platelets significantly increases 2‐deoxyglucose uptake by decreasing Km value of sugar transport and increasing sugar diffusion. In addition SMase treatment enhances basal glycolytic flux of platelets as well as the stimulation of the flux induced by suboptimal concentration of thrombin. The present study demonstrates that exposure of platelets to SMase, which may take place in vivo in physiological and/or in pathological conditions, modifies biochemical parameters of resting and stimulated platelets which are essential for cell physiological role.
High affinity bradykinin binding to human inflammatory cells
Authors: Rajasekariah Poornima , Warlow Robert S. , Walls Ronald S.
Published: July 2007

Specific direct bradykinin (BK) binding and competitive inhibition was detected in human neutrophil and peripheral blood mononuclear cell (PBMC) detergent solubilized extracts and purified plasma membranes using in vitro radioreceptor ligand binding. Scatchard analyses of [125I]‐BK binding revealed an equilibrium dissociation constant (Kd) of 2.9×10‐11 M for neutrophils and 5.6×10‐11 M for PBMC using [des‐arg9]‐BK a B1 agonist; 2.6×10‐11 M for neutrophils, 6.2×10‐11 M for PBMC with BK a B2 agonist; 5.4×10‐11 M for PBMC using Lys‐BK a B2 agonist. The number of binding sites (Bmax) was calculated to be 0.113 fM/μg protein (720 receptors per cell) for neutrophils and 0.200 fM/μg protein (1289 receptors per cell) for PBMC with the B1 agonist while with the B2 agonists the values were 0.128 fM/μg protein (818 receptors per cell) for neutrophils and 0.157 fM/μg protein (1005 receptors per cell) for PBMC with BK, and 0.293 fM/μg protein (1870 receptors per cell) with Lys‐BK for PBMC. In a competitive binding inhibition assay using neutrophil and PBMC glycerol purified plasma membranes, high affinity binding in the nanomolar range was detected to Lys‐BK and BK but with [des‐arg9]‐BK a 10‐100 fold lower order affinity was observed this being indicative of pharmacologically defined B2 characteristics.
Inhibition of platelet aggregation by L‐arginine and polyamines in alloxan treated rats
Authors: Méndez J. D. , Zarzoza E.
Published: July 2007

The antiaggregating effect of L‐arginine and polyamines (putrescine, spermidine and spermine) was studied in platelets of normal and diabetic rats (120 mg/Kg alloxan, i.p.). This effect was compared with insulin. The assays of platelet aggregation were carried out using platelet‐rich plasma (PRP) obtained from both groups of animals. The platelet aggregation test were first standardized by using PRP obtained from human health donors. 2.5 μmoles/ml ADP, 250 μmoles/ml epinephrine and 0.4 U/ml of thrombin were used as inductors of platelet aggregation. 60% of inhibition was observed with 10 μM of L‐arginine or polyamines in PRP of normal rats, and 50% with PRP of diabetic rats when thrombin was used as an agonist. These results show that L‐arginine and the polyamines putrescine, spermidine and spermine have an antagonist action in platelet aggregation. In addition, we demonstrated the platelet arginase activity not only in rat platelets but also in human, was less under hyperglycaemia. The activity of this enzyme has been associated with polyamine synthesis, required to regulate platelet function.
Sucrose‐phosphate synthase in tree species: light/dark regulation involves a component of protein turnover in Prosopis juliflora (SW DC)
Authors: Sinha Alok K. , Shirke P. A. , Pathre Uday , Sane P. V.
Published: July 2007

Light dependent modulation of sucrose‐phosphate synthase activity (SPS; EC 2.4.1.14) was studied in a tree species, namely Prosopis juliflora. In this paper we demonstrate that cycloheximide, an inhibitor of cytoplasmic protein synthesis, when fed to detached leaves of P. juliflora through transpiration stream in the dark or in light completely prevents in vivo light activation of Vlim and Vmax activities of SPS. In case of spinach, however, cycloheximide feeding affects only Vlim activity while Vmax activity remained unchanged. In contrast, chloramphenicol, an inhibitor of protein synthesis in chloroplast has no effect on the light activation of SPS in Prosopis. The treatment with cycloheximide showed slight reduction in the rate of O2 evolution indicating that cycloheximide had very little effect on overall photosynthesis. These results indicate that short term protein turnover of the SPS protein and some other essential component(s) (e.g., a putative protein that modifies SPS activity) is one of the primary steps in a complex and unique regulatory cascade effecting the reversible light activation of SPS.
Metabolic inhibitors as tools to delineate participation of distinct intracellular pathways in enhancement of lactose‐induced dissociation of neutrophil and thymocyte aggregates formed by mediation of a plant lectin
Authors: Timoshenko A. V. , Gorudko I. V. , Kaltner H. , Cherenkevich S. N. , Gabius H. ‐J.
Published: July 2007

Signaling processes in the course of the formation of the lectin‐mediated aggregates may partake in conveying enhanced stability to the cell clusters. To prove the validity of this reasoning in a model, we have studied the impact of addition of three metabolic inhibitors (N‐ethylmaleimide, nordihydroguaiaretic acid, and trifluoperazine on lactose‐dependent dissociation of cell aggregates, formed in the presence of the galactoside‐binding mistletoe lectin. Using both human neutrophils and rat thymocytes to avoid measurement of responses restricted to a single cell type, an enhanced dissociation of lectin‐formed cell aggregates was observed, when lactose and an inhibitor were present. Among the tested inhibitors, nordihydroguaiaretic acid and N‐ethylmateimide were more potent enhancers of cell dissociation than trifluoperazine. These results suggest that biosignaling pathways connected with lipoxygenase activity as well as the level of intracellular sulfhydryl groups confer further stability to lectin‐dependent cell aggregates. The systematic evaluation of inhibitors for defined activities is thus suggested as a tool to disclose the nature and the contribution of individual signaling mechanisms to post‐binding effects following lectin‐initiated cell contact formation.
Isolation and characterization of anti morphine analgesic peptide from canine brain
Authors: Chert ShuJuan , Chen YuZhen , Wang JingYan
Published: July 2007

The anti‐morphine peptide from canine brain which is homologous to α subunit of hemoglobin of canine, has been isolated and characterized. The canine brain was homogenized and extracted in 0.1 M acetic acid. The supernatant was collected, and purified by chromatography of Sephadex G‐50 and ion‐exchange and RP‐HPLC. A polypeptide was then obtained, which is termed as AMP5. It shows prominent anti‐morphine analgesic activity. AMP5 has an apparent molecular mass of 10.23kD. The isoelectric point was estimated to be around pH 6.7 by IEF. The amino acid composition of AMP5 and sequence of the N‐terminal 50 amino acid residues was determined.
Unusual AMP‐deaminase solubilization from teleost fish white muscle
Authors: Lushchak Voiodymyr I. , Smirnova Yulia D. , Storey Kenneth B.
Published: July 2007

A previous study described an unusual influence of neutral salts on the behavior of trout muscle AMP‐deaminase (AMPD) in its interactions with subcellular particulate matter (Lushchak and Storey 1994, Fish Physiol. Biochem. 13: 356‐368). The present study shows that this behavior is also shared by the muscle enzyme of two other fish species, sea scorpion (Scorpaena porcus) and corb (Sciena umbra), indicating that this describes a principle for AMPD interaction with cellular particulate material. AMPD binding to particulate matter increased with increasing KC1 concentration through the physiological range (100‐200 mM), but at higher salt concentrations the amount of bound enzyme was reduced. The pattern of binding was not influenced by hydrophobic interactions since addition of the nonionic detergents, Triton X‐100 or Tween‐80, did not alter the distribution of bound versus free enzyme although both detergents, at low concentrations, enhanced enzyme maximal activity. AMPD binding to particulate matter was also influenced by pH, the amount of free enzyme rising by nearly 3‐fold as pH fell within the physiological range from 7.5 to 6.5. It is concluded that neither electrostatic nor hydrophobic forrces alone can account for the unusual solubilization of AMPD from fish muscle and it is possible that the effect is also related to ion‐induced conforlnational changes in the structure of AMPD and/or of the myosin to which the enzyme binds.
Characterization of a helicase, DHEL III, from Drosophila melanogaster embryonic nuclei
Authors: Ristic Dejan , Todorovic Vesna , Kojic Snezana , Stefanovic Dragana
Published: July 2007

Nuclear extracts prepared from Drosophila preblastoderm embryos contain a nucleoside 5′‐triphosphate‐dependent helicase that unwinds circular or linear partial duplex DNA substrates and moves along the DNA in 3′ to 5′ direction. The helicase reaction is supported with both nucleoside and deoxynucleoside 5′‐triphosphates and requires divalent cations. Optimum activity in vitro is achieved with dATP or ATP and with MgCl2. In glycerol density gradients, embryonic enzyme migrates as a single peak of around 90kDa and it is the most prominent DNA‐unwinding activity detectable in nuclear extracts prepared from embryos 0‐2 hours after egg laying. In embryos collected 2‐4 hours after egg laying this DNA unwinding activity increases and then gradually decreases in embryos collected 4‐8 and 8‐16 hours after egg laying.
Identification of chlorella T‐1 ingredient which enhances DMPO‐OH adduct formation in fenton reaction
Authors: Ali M. Aktar , Oykawa Kikuo , Ohwada Minoru , Konishi Tetsuya
Published: July 2007

Chlorella T‐1 is a micro algae which carries out highly active photosynthetic carbon fixation. For searching the ·OH scavenging molecules in this strain, the methanol extract of T‐1 was fractionated by RP‐HPLC and the fractions were analyzed for DMPO‐OH formation by ESR after Fenton reaction. In the course of study, a single chlorella T‐1 ingredient was found to enhance the formation of DMPO‐OH rather than scavenge in the Fenton system. The ·OH enhancing chlorella component was further purified by chromatography and finally identified as lactic acid by NMR and LC‐MS. From the analysis of the reaction conditions for DMPO‐OH, it was found that lactic acid did not serve as direct source of ·OH but accelerated Fenton reaction.
Involvement of the GLUT 3 transporter in myogenic regulation
Authors: Broydell M. , Mazzuca D. M. , Abidi F. , Kudo P. A. , Lo T. C. Y.
Published: July 2007

We have recently demonstrated a close relationship between the GLUT 3 transporter and the myogenic ability of rat skeletal L6 myoblasts [1]. This investigation examined the effects of over‐and under‐expression of the GLUT 3 transporter on both biochemical and morphological differentiation. L6 transfectants expressing two to five times the normal L6 GLUT 3 transcript level were impaired in the expression of myogenesis‐associated genes, such as myogenin, MLC, MHC and TnT, and in myotube formation. Similar defects were also observed in myoblast mutants expressing less than 20% of the normal GLUT 3 level. Forced expression of an exogenous GLUT 3 cDNA could partially rescue the myogenic defect of these GLUT 3‐ mutants. However, such myogenic defects were not observed in L6 GLUT 3 antisense transfectants expressing 39% of the normal L6 GLUT 3 level. These data suggest that myogenic differentiation will proceed only within a critical level of the GLUT 3 transporter. The optimal GLUT 3 content for myogenesis ranges from around 2 × 105 to 5 × 105 molecules per cell in day 2 cultures; GLUT 3 levels outside this range have a negative effect on myogenesis. Our data suggest that GLUT 3 may regulate myogenesis by modulating the levels of signal transducers required for expression of myogenin and muscle‐specific contractile protein genes.
Occurrence of two missense mutations in Cu‐ATPase of the macular mouse, a menkes disease model
Authors: Ohta Yuriko , Shiraishi Noriyuki , Nishikimi Morimitsu
Published: July 2007

We have investigated the genetic defect of the Cu‐ATPase gene (Atp7a) in the macular mouse, a genetic model of classical Menkes disease. Northern blot analysis showed that its placenta and kidney possess a normal amount of the Cu‐ATPase mRNA of the normal size; sequencing analysis revealed two missense mutations, His674Arg and Ser1381Pro, in a PCR‐amplified cDNA for mutant Cu‐ATPase. The latter mutation was suspected to affect the function of the ATPase, because it lies in the transmembrane segment that is thought to form a channel for the transportation of copper ions.
Intracellular proteases in sporulated Bacillus thuringiensis subsp. tenebrionis: detection and analysis by gelatin zymography
Authors: Reddy Sreelatha T. , Venkateswerlu G.
Published: July 2007

Three intracellular proteases were identified from sporulated cultures of Bacillus thuringiensis subsp. tenebrionis by fractionation with ammonium sulfate. In this study, we detected protease activities at Mr 92 kDa, 81 kDa and 69 kDa employing gelatin zymography. The major proteolytic activity was due to the 81 kDa protease, which was identified as a metalloprotease being inhibited by both 1,10‐phenanthroline and ethylenediamine tetraacetic acid. The proteases showed maximal azocasein hydrolytic activity at 60°C and were heat‐activated from 40°C to 60°C. The 69 kDa and 81 kDa proteases were thermo‐inactivated at 70°C and 80°C respectively, while the 92 kDa protease was still active at 80°C.
Effect of pyrrolidine dithiocarbamate on photo‐induced proton transport through chloroplast membranes
Authors: Po Edwina Sau‐Man , Ho John W.
Published: July 2007

pH changes produced by photo‐induced proton transport through chloroplast membranes in spinach were measured by a glass microelectrode. Effect of pyrrolidine dithiocarbamate on proton translocation through chloroplast membranes has been studied. Kinetic analysis of proton translocation shows that the rate is reduced as the carbamate concentration increases. The rate of proton uptake follows firstorder kinetics and diminishes with increasing carbamate concentrations. The outward leakage of accumulated protons through thytakoid membranes in the dark also decreases likewise. However, the leakage of protons takes a much longer time. Pyrrolidine dithiocarbamate is an effective inhibitor of proton transport through chloroplast membranes. The results suggest that the photo‐induced proton translocation is regulated by conformation change in the membrane. Higher concentration of carbamate disrupts the tertiary conformation of the membrane. The inhibition of proton transport would affect ATPase function; thus, an excess use or accumulation of pyrrolidine thiocarbamate may compromise ATP production.
A new family of plasma membrane polypeptides differentially regulated during plant development
Authors: Logan David C. , Domergue Odile , de la Serve Bernard Teyssendier , Rossignol Michel
Published: July 2007

Two cDNAs encoding polypeptides identified in a tobacco leaf plasma membrane fraction prepared by phase partitioning were cloned. The deduced polypeptides, P16 and P17, exhibit a striking primary structure, similar to that of P19, a previously cloned plasma membrane polypeptide. Antibodies raised to the recombinant proteins were used to probe the cellular location of P16, P17 and P19 by means of western blotting of sucrose density‐gradient fractions; all three polypeptides were found to be located solely at the plasma membrane. Furthermore, P19 antigen accumulated transiently at the time of floral induction while P16 and P17 antigens accumulated towards the end of the life‐cycle. These results together with sequence database searches and multiple‐sequence alignments suggest that we have identified a new family of plasma membrane polypeptides that (i) are putatively plant specific and (ii) are differentially regulated during plant development. These polypeptides are termed DREPPs for developmentally regulated plasma membrane polypeptides.
Inhibition of proteases, myotoxins and phospholipases A2 from Bothrops venoms by the heteromeric protein complex of Didelphis albiventris opossum serum
Authors: Soares A. M. , Rodrigues V. M. , Borges M. H. , Andrião‐Escarso S. H. , Cunha O. A. B. , Homsi‐Brandeburgo M. I. , Giglio J. R.
Published: July 2007

The antibothropic complex (ABC) from opossum (species Didelphis albiventris) serum was purified by chromatography on DEAE‐Sephacel. It showed an acidic character and two polypeptide chains of ca. 45 kDa and 48 kDa, respectively. Lyophilized opossum serum or the ABC (1001μg), as well as ethylenediamine tetraacetate (0.25μmotes) were able to completely neutralise the hemorrhagic effect of 50μg of the desiccated venoms of Bothrops moojeni, Bothrops pirajai and Bothrops jararacussu. The myotoxic (100μg venom in mice) and edematogenic (90μg venom in rats) activities of Bothrops moojeni and Bothrops jararacussu venoms, as well as of the major myonecrotic protein (myotoxin‐I) isolated from Bothrops moojeni venom, were also totally inhibited by the ABC (200μg and 270μg, respectively). The lyophilized opossum serum (30μg) and the ABC (30μg) reduced to 50% the phospholipase A2 activity of Bothrops moojeni venom (10μg). The clotting activity of Bothrops alternatus and Bothrops moojeni (20μg) on bovine plasma was also significantly inhibited by the ABC (60μg).
The differential transforming activities of TAL1 oncoproteins
Authors: Hsu Shih‐Fang , Cheng Jiin‐Tsuey
Published: July 2007

TAL1 (Or SCL) oncogene produces both full‐length pp42 and truncated pp22 phosphoproteins in T‐cell acute lymphoblastic leukemia cells. To investigate the transforming properties of these two oncoproteins, permanent transfected NIH3T3 cell lines were generated that constitutively express either pp42 or pp22. Both cell lines exhibited similar morphology as parental NIH3T3 cells. However, they showed differential anchorage independent growth in soft‐agar. The clonogenicity of pp22 expressing cells was ten folds higher than that of pp42 expressing cells. This result might implicate the differential transformation potential of the two Tal1 oncoproteins.
Single microinjection of L‐glutamate induces oxidative stress in discrete regions of rat brain
Authors: Babu G. Nagesh , Bawari Meenakshi
Published: July 2007

Glutamate (Glu) is a major excitatory aminoacid neurotransmitter in the mammalian brain. Under Certain Circumstances Glu can also exert toxic effects on neuronal Cells. To unravel the biochemical mechanisms of Glu‐induced acute neuronal injury, Glu 1μmol/1μl was microinjected into cerebral Cortex, striatum and hippocampus of adult rats and oxidative stress and antioxidant parameters were evaluated. The results show that the rate of lipid peroxidation was significantly increased in the above brain regions following Glu administration suggesting neuronal membrane damage and also the total and free sulfhydryl groups were significantly depleted, indicating altered red‐ox status of the cells. There was also alteration in the activity of antioxidant enzyme catalase in cerebral cortex. Some of the above Glu‐induced effects were reversed or modified by NMDA receptor antagonist MK‐801.
RAPD analysis of Candida albicans strains recovered from different immunocompromised patients (ICP) reveals an apparently non‐random infectivity of the strains
Authors: Gyanchandani Anita , Khan Zafar K. , Farooqui Nuzhat , Goswami Mamta , Ranade Shirish A.
Published: July 2007

The opportunistic imperfect fungus Candida albicans causing life‐threatening infections in immunocompromised patients (ICP), especially in HIV‐positive cases, is recognized to be one of the most important nosocomial pathogens in the recent decades. The extent of strain‐to‐strain variation within a species and its relationship to the ability of the organism to colonize or invade a specific group of patients or even a body site is, however, not well known. We have analysed 19 strains of C.albicans recovered from ICP at different locales and times, employing the RAPD technique. No two strains generated identical RAPD profiles with any of the 21 primers tested. Further, the UPGMA clustering of the strains seemingly reflected a certain relationship or non‐randomness in the infection of the patients with the strain of C.albicans vis‐a‐vis the immunocompromised status due to underlying disease such as diabetes, cancer, asthma and meningitis. These results may have a profound impact on the management of candidiasis, especially in the ICP.
Conformation of polygalacturonase‐II from Aspergillus carbonarius ‐ A spectroscopic study
Authors: Devi Nemkal Anjana , Rao A.G. Appu
Published: July 2007

Solution conformation of polygalacturonase from Aspergillus carbonarius was determined by spectroscopy. UV absorption, second derivative, near‐UV CD, fluorescence emission spectra and fluorescence quenching measurements suggest that the tryptophan fluorophores are in a hydrophobic environment. Of the nine tryptophan residues, only one is exposed to the solvent. In the near UV region the enzyme exhibits very weak CD bands, the far UV CD spectrum has a minimum at 218 nm; the enzyme is rich in parallel β structure. Modification of solvent exposed tryptophan by N‐bromosuccinimide resulted in the complete loss of enzyme activity. The enzyme is very sensitive towards urea induced unfolding, with complete loss of activity at 3 M urea concentration.
Is the glutathione S‐conjugate pump a flippase?
Authors: Sokal Adam , Pułaski Łukasz , Rychlik Błazej , Fortuniak Anna , Bartosz Grzegorz
Published: July 2007

A hypothesis of the flippase nature of the glutathione S‐conjugate transport is presented. Experimental premises for this hypothesis include interaction of glutathione S‐conjugates with the membrane, as demonstrated by their effects on membrane fluidity, quenching of 1‐(4‐trimethylammoniumphenyl)‐6‐phenyl‐1,3,5‐hexatriene fluorescence and induction of echinocytosis by 2,4‐dinitrophenyl‐S‐glutathione (DNP‐SG). This hypothesis can rationalize, i. a., observations of the enhancement of DNP‐SG transport by butanol and stimulation of erythrocyte membrane Mg2+‐ATPase activity by albumin‐coupled DNP‐SG.
Fru 2,6‐P2 and citrate: Intracellular distribution in citrus tissues and effect on grapefruit leaf PFP.
Authors: Van Praag Esther , Gonzalez Pedro , Brune Andreas , Goren Raphael , Zehavi Uri , Echeverria Ed
Published: July 2007

Grapefruit leaf PFP was studied for its activation by fructose 2,6‐bisphosphate (Fru 2,6‐P2) in the forward and reverse reactions. In the glycolytic reaction, a Ka of 4.0±0.12 nM was obtained. This constant is affected by the presence of increasing concentrations of citrate (1, 5 and 20 mM) with a Ka(citrate) of 4.5±0.09, 6.9±0.05 and 38.2±1.4 respectively. The inhibition mode of citrate is competitive with Fru 2,6‐P2, but non‐linear in relation of increasing concentrations of the inhibitor. The intracellular distribution and concentration of the key regulatory metabolite Fru 2,6‐P2 was further investigated in citrus leaves and juice cells. Fru 2,6‐P2 was only found in the cytosol of juice cells. Fru 2,6‐P2 was detected under both conditions with higher concentrations found under aerobiosis.
Non‐synergistic interactions between strong allosteric effectors and human embryonic and adult haemoglobins
Authors: McLennan Amy. E. M. , Brittain Thomas
Published: July 2007

The binding of two strong allosteric effectors (2,3 Diphosphoglycerate D.P.G., and Bezafibrate, Bzf) to both adult and the three human embryonic haemoglobins, either individually or in combination, have been studied in detail. The adult protein exhibits one binding site for D.P.G and two for Bzf. When both effectors are present simultaneously their effects are simply additive. The same qualitative pattern of binding is observed in the case of the three human embryonic haemoglobins, although with different binding constants. The lack of synergism between these effectors and the different binding affinity expressed by these proteins are discussed in terms of the known amino acid sequence differences.
Carnitine palmitoyltransferase II activity is decreased in liver mitochondria of cachectic rats bearing the Walker 256 carcinosarcoma: Effect of indomethacin treatment
Authors: Seelaender Maríla C. L. , Curi Rui , Colquhoun Alison , Williams John F. , Zammitt Victor A.
Published: July 2007

The syndrome of cancer cachexia is accompanied by several alterations of lipid metabolism, especially that in the liver. In this study we have investigated a possible mechanism whereby the presence of the Walker 256 carcinosarcoma affects hepatic fatty acid oxidative capacity in tumour‐bearing rats. Hepatic mitochondrial outer membrane carnitine palmitoyltransferase I (CPT I), generally accepted as the main site of regulation of fatty acid oxidation, was unaffected by the presence of the extra‐hepatic tumour. However, mitochondrial inner‐membrane carnitine palmitoyltransferase II (CPT II) activity was markedly decreased in mitochondria isolated from the liver of tumour‐bearing rats. Immuno‐detection by Western blotting using a CPT II‐specific antibody identified two bands (corresponding to Mr 69,000 and 54,000) in tumour‐bearing rats whereas only the normal‐sized CPT II was present (at the expected Mr 69,000) in mitochondria from control rats. It is suggested that the emergence of the second, smaller protein may represent a catalytically less active protein that arises in vivo, since its appearance was not affected by the inclusion of proteolysis inhibitors in the mitochondrial preparation buffers. Treatment of the tumour‐bearing rats with indomethacin, a prostaglandin (including PGE2) synthesis inhibitor, increased CPT II activity to levels even higher than those found in the control animals. It is suggested that PGE2 may play a role in the control of CPT II expression in the liver of tumour‐bearing rats. Indomethacin treatment did not affect either of the two CPT activities of the mitochondria isolated from tumour tissue.