bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of a Double Layer Agar Plate For Direct Enumeration of Vibrio parahaemolyticus
Authors: Duan Jingyun , Liu Chengchu , Su Yi‐Cheng
Published: August 2010

A double layer agar plate (DLAP) was developed according to the thin agar layer (TAL) method as a 1‐step procedure for direct enumeration of injured Vibrio parahaemolyticus cells based on the formation of unique purple colonies by V. parahaemolyticus. The DLAP was prepared by overlaying an equal volume (10 mL) of a nonselective medium (tryptic soy agar supplemented with 1.5% NaCl) onto a selective medium (Bio‐Chrome Vibrio medium). The DLAP was capable of detecting V. parahaemolyticus in mixed cultures containing non‐Vibrio bacteria. Production of purple colonies by V. parahaemolyticus on DLAP was not affected by the growth of other bacteria, even when V. parahaemolyticus was only a small fraction (5%) of the entire bacterial population. Direct plating on DLAP was found as effective as the most probable number (MPN) method for recovering heat‐and cold‐injured V. parahaemolyticus cells, which could not be detected by direct plating on Bio‐Chrome Vibrio medium or thiosulfate‐citrate‐bile salts‐sucrose agar. The DLAP offers an alternative to the MPN method for detecting injured V. parahaemolyticus cells and can be used as a simple 1‐step procedure for quick screening of V. parahaemolyticus in foods.
Characterization of the Fermented Milk “Laban” with Sensory Analysis and Instrumental Measurements
Authors: Chammas I. Gisele , Saliba Rachad , Béal Catherine
Published: August 2010

Sensory, chemical, and rheological properties of commercial and traditional laban samples were investigated to characterize this fermented milk. One commercial sample and 14 traditional samples, collected from various geographical locations in Lebanon, were evaluated by a descriptive panel in terms of appearance, color, texture, odor, taste, and after‐taste. Principal component analysis of the sensory data revealed high differences between laban samples. They were separated into 5 distinct groups that were identified by the following sensory characteristics: firm and sour, slimy and sweet, high butter odor, high yogurt odor, and moderate levels for all the descriptors. Six samples, showing different features, were selected from these 15 samples. Physicochemical analyses of acidity, lactose and fat contents, firmness, and apparent viscosity were assessed on these 6 samples. Laban, independently of its origin, displayed higher acidity than yogurt. Commercial laban showed higher acidity and viscosity than traditional samples. Statistical relationships between sensory and instrumental data showed significant correlation between apparent viscosity and smoothness, fat content and butter odor, titratable acidity and sourness, and penetrometry readings and sliminess. Finally, principal component analysis of the instrumental and sensory parameters revealed that both analyses characterized the samples in the same way.
Influence of Texture on Taste: Insights Gained During Studies of Hardness, Juiciness, and Sweetness of Apple Fruit
Authors: Harker F. Roger , Amos Rachel L. , Echeverríaa Gemma , Gunson F. Anne
Published: August 2010

Apple flesh can be considered as a living structure within which juice and tastants are encapsulated in individual cells. The breakdown of this structure and release of juice varies between hard and soft apples. We hypothesized that the texture of the fruit and the way it releases juice during chewing would therefore influence the perception of sweetness. Time‐intensity (TI) techniques were used by a trained panel to study the temporal changes in the intensity of “juiciness” and “sweetness” during consumption of apples that varied in mechanical hardness (measured by puncture test). All apple samples had a similar water content, but the pattern of juice release was markedly different. Juice was released into the mouth in a pattern of increasing and then deceasing intensity. The maximum intensity (Imax), duration of the stimulus (DUR), and area under the TI curve (AUC) were significantly higher for hard compared with soft apples. The impact of juice release on perception of sweetness was examined using apples in which the Imax and AUC for juiciness of soft apples were half the value of those for firm apples. There was no consistent difference in Imax, DUR, or AUC for sweet taste in these 2 sets of apples. We concluded that sufficient juice was released from soft and less juicy apples to ensure that sweetness perception was maximized. These results are discussed in relation to earlier studies that demonstrated that texture (thickening agents) suppresses flavor perception in solutions and juices.
Quality Characterization of Farmed Atlantic Halibut During Ice Storage
Authors: Guillerm‐Regost Christelle , Haugen Trine , Nortvedt Ragnar , Carlehöug Mats , Lunestad Bjørn Tore , Kiessling Anders , Rørár Anna Maria B.
Published: August 2010

A quality index method (QIM) was developed for farmed Atlantic halibut and together with instrumental, chemical, sensory, and bacteriological analysis, quality changes of halibut stored on ice for 26 d was evaluated. Two groups of fish were fed diets that differed only in the source of lipid, where 1 diet contained only marine oil sources and the other a 50/50 mixture of marine and soybean oil. Fish were slaughtered after 1 y and then stored on ice for 26 d. The fish were sampled on day 1, day 2, and every 2nd day after that. Dietary lipid sources had no effect on freshness, (ATP) degradation (K‐value), texture, color, or liquid‐holding capacity. The QIM scores increased with storage time, in particular the appearance and eyes parameters. The QIM is a good freshness indicator for halibut. The K‐value was strongly correlated with storage time (r= 0.99), while total bacterial counts increased after 7 to 8 d of ice storage. The texture, liquid‐holding capacity, and color were significantly affected by storage time during the early period of storage, probably due to rigor stiffness and rigor resolution. The texture, liquid‐holding capacity, and color did not change significantly from approximately day 8 of storage until the end of the experiment at day 26.
Distribution and Regulation by Oestrogen of Fully Processed and Variant Transcripts of Gonadotropin Releasing Hormone I and Gonadotropin Releasing Hormone Receptor mRNAs in the Male Chicken
Authors: Sun Y.‐M. , Dunn I. C. , Baines E. , Talbot R. T. , Illing N. , Millar R. P. , Sharp P. J.
Published: August 2010

The aim of this study was to increase understanding of the occurrence and regulation of chicken gonadotropin releasing hormone I (cGnRH I) and chicken gonadotropin releasing hormone receptor (cGnRH‐R) mRNA variants in the hypothalamic‐pituitary‐testicular axis (HPTA). The study was carried out in the cockerel. Fully processed cGnRH I mRNA (cGnRH Ia) and a variant transcript (cGnRH Ib) with a retained intron 1 were observed in the preoptic/anterior hypothalamus (POA), the basal hypothalamus, anterior pituitary gland, and testes. Fully processed cGnRH‐R mRNA (cGnRH‐Ra) and a variant transcript (cGnRH‐Rb) with a deletion were detected in the same tissues. In juvenile cockerels, concentrations of cGnRH Ia and b in the POA increased after castration, and this was prevented by oestrogen treatment. In the anterior pituitary gland, the concentration of cGnRH‐Ra increased after castration and this was reversed by oestrogen treatment. In intact adult cockerels, oestrogen treatment depressed plasma luteinizing hormone but did not affect concentrations of cGnRH I and cGnRH‐R mRNAs in the POA, basal hypothalamus, and anterior pituitary gland, suggesting that locally produced oestrogen, by aromatization, may exert maximal suppression on cGnRH I and GnRH‐R mRNAs. In intact adult cockerels, the concentrations of cGnRH Ia and b in the testis, but not cGnRH‐Ra and b, were depressed by oestrogen treatment. It was concluded that fully processed and variant cGnRH I and cGnRH‐R mRNAs occur in all components of the HPTA. Oestrogen appears to play a role in the regulation of cGnRH Ia and b in the POA and testes, and of cGnRH‐Ra in the POA and anterior pituitary gland.
Gene Expression of the Insulin‐Like Growth Factor System During Postnatal Development of the Rat Pituitary Gland
Authors: González‐Parra S. , Argente J. , Chowen J. A. , Van Kleffens M. , Van Neck J. W. , Lindenbeigh‐Kortleve D. J. , Drop S. L. S.
Published: August 2010

Insulin‐like growth factors I and II (IGFI and II) are synthesized by anterior pituitary cells and participate in cellular growth and differentiation, as well as the control of pituitary hormone secretion. Type 1 and 2 IGF receptors (IGFR1 and IGFR2) and the six IGF binding proteins (IGFBPs), which modulate IGF effects, are expressed in the anterior pituitary gland. We usedin situhybridization to analyse the temporal expression pattern of IGFI and II, IGFR1 and 2 and IGFBP1–6 in the anterior pituitary gland during postnatal development in both male and female rats (10, 20, 30, 40 and 60 days of age). We found all of the components of the IGF system to be expressed in the anterior pituitary gland, with each having a specific temporal pattern of expression. In addition, there exist differences between the sexes in the expression of some components of the IGF system. These data emphasize that in the anterior pituitary gland the IGF system is under tight regulation during postnatal life when this gland continues to develop. The distinct temporal expression of each member of the IGF system may indicate specific roles in the development and physiology of the anterior pituitary gland.
Characterization of an Antibody to the Human Melatonin mt1 Receptor
Authors: Williams L. M. , Drew J. E. , Bunnett N. W. , Grady E. , Barrett P. , Abramovich D. R. , Morris A. , Slater D.
Published: August 2010

Melatonin acts via high affinity, G‐protein coupled, seven transmembrane domain receptors. To precisely localize these receptors, antibodies were raised in chickens against a 15 amino acid fragment at the intracellular C‐terminal region of the human melatonin receptor subtype mt1 (DSSNDVADRVKWKPS, mt1338−352). A chimeric form of the receptor with a hydrophilic Flag peptide (DYKDDDDK) in sequence with the extracellular N‐terminus (Flag‐mt1) was generated by polymerase chain reaction and expressed in mammalian cell lines. An IgY antibody (Y31), which gave high antibody titres by enzyme‐linked immunosorbent assay, was used to localize Flag‐mt1 in stably transfected cells by immunofluoresence. Flag‐mt1 localization with Y31 was identical to that obtained with the M5 antibody directed against the Flag epitope and was mainly localized to the Golgi apparatus with some staining at the cell surface. No staining was seen in untransfected cells with either antibody. Y31 staining was abolished using antibody preabsorbed with peptide antigen. Y31 immunofluorescence in fetal human kidney sections was restricted to nephrogenic regions and matched that of 2‐(125I)iodomelatonin binding and mt1 gene expression by in situ hybridization. Y31 was used to immunoprecipitate biotinylated membrane proteins from Flag‐mt1 stably transfected and untransfected CHO cells. Western blotting of immunoprecipitated proteins revealed two major bands specific to stably transfected cells, one at 63 kDa and one at 86 kDa. The first band almost certainly corresponds to the glycosylated form of Flag‐mt1 and the second band to receptor dimers. Thus, Y31 antibody is suitable for use in detecting the human mt1 receptor subtype in tissues and in transfected cells.
Photoperiodic Regulation of Prolactin Gene Expression in the Syrian Hamster by a Pars Tuberalis‐Derived Factor
Authors: Stirland J. A. , Johnston J. D. , Cagampang F. R. A. , Morgan P. J. , Castro M. G. , White M. R. H. , Davis J. R. E. , Loudon A. S. I.
Published: August 2010

Syrian hamsters exhibit a marked seasonal variation in prolactin secretion. The aim of this study was to analyse the nature of the photoperiodic regulation of prolactin gene expression, and to define the role of melatonin and the pars tuberalis of the anterior pituitary in this process. Pituitary prolactin gene expression, restricted to the pars distalis, was increased in hamsters maintained in long daylengths (16 h : 8 h, light : dark) compared to hamsters exposed to short daylengths (8 h : 16 h, light : dark) for 8–12 weeks. Analysis of single cells by in situ hybridization showed that photoperiod had no effect on the percentage of pars distalis cells expressing prolactin mRNA, but shifted the frequency distribution of prolactin mRNA expression per cell, such that in long photoperiods a greater proportion of cells were recruited to a higher expressing population. In vitro coculture of hamster pars tuberalis fragments increased prolactin promoter‐driven luciferase activity in stably transfected GH3 cells in a dose‐ and duration‐dependent manner. Conditioned medium from hamster and ovine pars tuberalis also activated the prolactin promoter. Furthermore, basal and forskolin‐stimulated conditioned medium from hamster pars tuberalis increased prolactin mRNA expression in primary cultures of pars distalis cells. Melatonin attenuated the activity of pars tuberalis‐conditioned medium but had no direct effect on either prolactin mRNA expression or secretion in pars distalis cell cultures. Finally, pars tuberalis fragments from long photoperiod hamsters stimulated prolactin gene promoter activity to a greater extent than those from short photoperiod hamsters. In conclusion, this study provides the first evidence in a seasonal mammal that the synthesis of prolactin depends on photoperiodic modulation of a pars tuberalis‐derived factor. Our data support further the hypothesis that seasonal modulation of prolactin gene expression depends upon a melatonin‐dependent paracrine action of the pars tuberalis on pars distalis lactotrophic cells.
Actions of Leptin on Growth Hormone Secretagogue‐Responsive Neurones in the Rat Hypothalamic Arcuate Nucleus RecordedIn Vitro
Authors: Tung Y. C. L. , Hewson A. K. , Dickson S. L.
Published: August 2010

In the arcuate nucleus, the growth hormone (GH) secretagogue (GHS)‐responsive cells include a subpopulation of the neuropeptide Y (NPY) neurones. It is not known whether these include the orexigenic NPY population that are inhibited by the satiety hormone, leptin. Thus we investigated whether (i) the arcuate nucleus cells electrically excited by GHS are inhibited by leptin and (ii) chronic central leptin infusion alters GHS‐induced Fos expression. Of 36 cells recorded from a trimmed hypothalamic slice containing arcuate nucleus, 13 cells were excited by the nonpeptide GHS, CP‐459,599. The predominant response of these cells to leptin was inhibitory: six inhibited, three excited and four unresponsive. Similar responses were observed in a population of arcuate cells recorded from a preparation in which synaptic transmission was blocked, suggesting that leptin acts directly on a subpopulation of GHS‐responsive neurones. Intracerebroventricular infusion of leptin for 1 week did not alter the number of cells expressing Fos following GHS administration. Thus, while leptin does not appear to influence the central actions of GHS to induce immediate early gene expression, it does act directly on a subpopulation of cells excited by GHS, eliciting mostly inhibitory but also some excitatory responses. It will be interesting to discover the consequences of leptin's inhibitory effects on the hypothalamic circuits excited by GHS, particularly since leptin paradoxically has a stimulatory effect on GH secretion, presumed to reflect a suppression of central NPY pathways.
Diversity in Fibroblast Growth Factor Receptor 1 Regulation: Learning from the Investigation of Kallmann Syndrome
Authors: Kim S.‐H. , Hu Y. , Cadman S. , Bouloux P.
Published: August 2010

The unravelling of the genetic basis of the hypogonadotrophic hypogonadal disorders, including Kallmann syndrome (KS), has led to renewed interest into the developmental biology of gonadotrophin‐releasing hormone (GnRH) neurones and, more generally, into the molecular mechanisms of reproduction. KS is characterised by the association of GnRH deficiency with diminished olfaction. Until recently, only two KS‐associated genes were known: KAL1 and KAL2. KAL1 encodes the cell membrane and extracellular matrix‐associated secreted protein anosmin‐1 which is implicated in the X‐linked form of KS. Anosmin‐1 shows high affinity binding to heparan sulphate (HS) and its function remains the focus of ongoing investigation, although a role in axonal guidance and neuronal migration, which are processes essential for normal GnRH ontogeny and olfactory bulb histogenesis, has been suggested. KAL2, identified as the fibroblast growth factor receptor 1 (FGFR1) gene, has now been recognised to be the underlying genetic defect for an autosomal dominant form of KS. The diverse signalling pathways initiated upon FGFR activation can elicit pleiotropic cellular responses depending on the cellular context. Signalling through FGFR requires HS for receptor dimerisation and ligand binding. Current evidence supports a HS‐dependent interaction between anosmin‐1 and FGFR1, where anosmin‐1 serves as a co‐ligand activator enhancing the signal acitivity, the finer details of whose mechanism remain the subject of intense investigation. Recently, mutations in the genes encoding prokineticin 2 (PK2) and prokineticin receptor 2 (PKR2) were reported in a cohort of KS patients, further reinforcing the view of KS as a multigenic trait involving divergent pathways. Here, we review the historical and current understandings of KS and discuss the latest findings from the molecular and cellular studies of the KS‐associated proteins, and describe the evidence that suggests convergence of several of these pathways during normal GnRH and olfactory neuronal ontogeny.
Voltage‐Dependent Calcium Channels in Ventromedial Hypothalamic Neurones of Postnatal Rats: Modulation by Oestradiol and Phenylephrine
Authors: Lee A. W. , Kyrozis A. , Chevaleyre V. , Kow L.‐M. , Zhou J. , Devidze N. , Zhang Q. , Etgen A. M. , Pfaff D. W.
Published: August 2010

Oestradiol actions in the hypothalamus play an important role in reproductive behaviour. Oestradiol treatmentin vivoinduces α1b‐adrenoceptor mRNA and increases the density of α1B‐adrenoceptor binding in the hypothalamus. Oestradiol is also known to modulate neuronal excitability, in some cases by modulating calcium channels. We assessed the effects of phenylephrine, an α1‐adrenergic agonist, on low‐voltage‐activated (LVA) and high‐voltage‐activated (HVA) calcium channels in ventromedial hypothalamic (VMN) neurones from vehicle‐ and oestradiol‐treated female rats. Whole‐cell and gramicidin perforated‐patch recordings were obtained, with barium as the charge carrier. In the absence of phenylephrine, oestradiol treatment increased the magnitude of LVA currents compared to controls, but had no effect on HVA currents. Phenylephrine enhanced HVA currents in a significantly greater proportion of neurones from oestradiol‐treated rats (76%) than from vehicle‐treated (41%) rats. The L‐channel blocker nifedipine abolished this oestradiol effect on phenylephrine‐enhanced HVA currents. Preincubating slices with the N‐type channel blocker omega‐conotoxin GVIA completely blocked the phenylephrine response, suggesting that the N‐type channel is essential. Phenylephrine also stimulated LVA currents in approximately two‐thirds of neurones in slices from both vehicle‐ and oestradiol‐treated rats. Our data show that oestradiol increases LVA currents in the VMN. Oestradiol also amplifies α1‐adrenergic signalling by increasing the proportion of neurones showing phenylephrine‐stimulated HVA currents mediated by N‐ and L‐type calcium channels. In this way, oestradiol may increase excitatory responses to arousing adrenergic inputs to VMN neurones governing oestradiol‐dependent reproductive behaviour.
Nesfatin‐1 Influences the Excitability of Paraventricular Nucleus Neurones
Authors: Price C. J. , Hoyda T. D. , Samson W. K. , Ferguson A. V.
Published: August 2010

Nesfatin‐1 is a newly‐discovered satiety peptide found in several nuclei of the hypothalamus, including the paraventricular nucleus. To begin to understand the physiological mechanisms underlying these satiety‐inducing actions, we examined the effects of nesfatin‐1 on the excitability of neurones in the paraventricular nucleus. Whole‐cell current‐clamp recordings from rat paraventricular nucleus neurones showed nesfatin‐1 to have either hyperpolarising or depolarising effects on the majority of neurones tested. Both types of response were observed in neurones irrespective of classification based on electrophysiological fingerprint (magnocellular, neuroendocrine or pre‐autonomic) or molecular phenotype (vasopressin, oxytocin, corticotrophin‐releasing hormone, thyrotrophin‐releasing hormone or vesicular glutamate transporter), determined using single cell reverse transcription‐poylmerase chain reaction. Consequently, we provide the first evidence that this peptide, which is produced in the paraventricular nucleus, has effects on the membrane potential of a large proportion of different subpopulations of neurones located in this nucleus, and therefore identify nesfatin‐1 as a potentially important regulator of paraventricular nucleus output.
SOLUBLE FRAGMENTS OF ELASTIN. CIRCULAR DICHROISM STUDIES
Authors: Abatangelo G. , Daga‐Gordini D. , Tamburro A. M.
Published: August 2010

Using alcoholic KOH, elastin has been hydrolyzed and two homogeneous fragments have been isolated by Amberlite IR‐120 chromatography. The peptides have been studied by circular dichroism in different solvents and in presence of Ca++ ions. The results are discussed from the standpoint of the free conformation in solution and the Ca++ ions complexation.
STUDIES ON RIBONUCLEASE S: THE ROLE OF LYSINE‐7 FOR ACTIVATION OF S‐PROTEIN*
Authors: Marchiori Fernando , Borin Gianfranco , Moroder Luis
Published: August 2010

In order to describe further the role of the invariant amino acid residue lysine‐7 in the RNase S' system, potential catalytic activities of des‐Lys7‐[Orn10]‐, [Ala7, Orn10]‐, [Lys4, Ala7, Orn1.0]‐S‐peptide and corresponding guanidinated derivatives were studied. New information was obtained about S‐protein binding capacities of the analogs relative to that of unmodified S‐peptide, and about conformation of the corresponding RNase S' analogs by studies on competitive inhibition, difference spectroscopy, difference circular dichroism and thermal transition. Peptides modified in position 7 were no weaker in strength in peptide‐protein noncovalent interaction in the presence of substrate. In the absence of substrate the variously modified S‐peptide analogs were differentiated in their S‐protein affinity. Conformational changes in the geometry of the active site of the reconstituted enzymes, if present, must be smaller than the detection limits of the techniques used. There is strong implication that the change in catalytic efficiency has to be attributed to an alteration or to the position of the charge and that lysine‐7 in the natural enzyme is involved in stabilizing the transition state intermediates during decomposition of the substrate. Further substantiation was provided by substrate inhibition studies as well as 3‐CMP binding measurements, since 3‐CMP binding affinity of RNase S' modified in position 7 was found to be significantly lower than that of RNase S' itself.
TERTIARY STRUCTURES OF GASTRIN‐LIKE TETRAPEPTIDES
Authors: Yamada Tsutoo , Wako Hiroshi , Saitô Nobuhiko , Isogai Yoshinori , Watari Hiroshi
Published: August 2010

Tertiary structures of gastrin‐like tetrapeptide Trp‐Met‐Asp‐Phe‐NH2 and those substituted by Leu, Val or Gly for Met are studied. The lowest energy conformations of the side chains when the back bone is fixed in α‐helix are obtained by modified minimization algorithm. It is suggested that protein folding proceeds in the accessible conformation space as a self‐organization process leading to minimum energy conformation in this space.
CATALYTIC HYDROGENOLYSIS IN LIQUID AMMONIA.* CLEAVAGE OF Nα‐BENZYLOXYCARBONYL GROUPS FROM CYSTEINE‐CONTAINING PEPTIDES WITH tert‐BUTYL SIDE CHAIN PROTECTION
Authors: Felix Arthur M. , Jimenez Manuel H. , Mowles Thomas , Meienhofer Johannes
Published: August 2010

To exemplify the extension to synthesis of sulfur‐containing peptides of the Nα‐benzyloxycarbonyl and side chain tert‐butyl protective group combination, somatostatin has been synthesized via incremental chain elongation starting from the COOH‐terminal cysteine. Cleavage of the Nα‐benzyloxycarbonyl groups was achieved in high yield, at each stage, by palladium‐catalyzed hydrogenation in liquid ammonia. All side chain functionalities including the cysteine thiol groups were blocked by tert‐butyl‐derived groups. Each gave rise to individual n.m.r. signals which permitted sensitive characterization of all intermediate protected peptides. Mild conditions were used for the removal of all protecting groups from the completed somatostatin tetradecapeptide. The tert‐butyl thiol protective groups were readily and completely cleaved by mercuric acetate at pH 4. Oxidation of dihydrosomatostatin with potassium ferricyanide provided somatostatin in good yield. The results indicate that the absolute selectivity between α‐amine and side chain protective group cleavage afforded by Nα‐benzyloxycarbonyl hydrogenolysis in the presence of ‐tert‐butyl groups may now be extended to synthesis of cysteine‐ and methionine‐containing peptides.
TIGHT PACKING OF PROTEIN CORES AND INTERFACES
Authors: Bello Jake
Published: August 2010

The tightly packed protein interiors and interfaces are taken to be essentially solids. The tight packing, and the r‐6 dependence of the energy of van der Waals' interactions may account for the highly conservative core regions of homologous proteins. The hydrophobic free energies used to calculate conformational stability and the association constants for protein‐protein interactions are not adequate, since the free energies are obtained from liquid‐liquid transfer of model compounds. An additional term is required, the enthalpy of fusion. This provides an additional 7 kcal mol‐1 for the stabilization of the trypsin‐trypsin inhibitor complex.
SORGHUM PROTEINASE INHIBITORS:
Authors: Kumar P. M. Harish , Virupaksha Tumkur K. , Vithayathil Paul J.
Published: August 2010

An investigation has been carried out on the proteinase inhibitors of grain sorghum (Sorghum bicolor (L.) Moench). One of the inhibitors has been isolated in a pure form and characterized. The proteinase inhibitor was extracted from the acetone‐defatted sorghum meal and purified by selective thermal denaturation, ammonium sulfate fractionation, Sephadex gel filtration and DEAE‐cellulose chromatography (DEAE‐preparation II). This preparation was demonstrated to be a mixture of three inhibitor components by polyacrylamide disc gel electrophoresis. Further resolution of this mixture into Inhibitors I to III was achieved by QAE‐Sephadex chromatography. Sorghum Inhibitor III was homogeneous by the criteria of disc gel electrophoresis and has been more fully characterized. A molecular weight of 25,000 was obtained for Inhibitor III by gel filtration and was in agreement with the value calculated from the amino acid composition of the inhibitor. The N‐terminal amino acid residue of Inhibitor III, a single chain protein, was isoleucine. Sorghum proteinase inhibitors inhibit specifically the serine proteinases and are inactive towards the other classes of proteinases. Inhibitor III is primarily a chymotrypsin inhibitor, whereas Inhibitors I and II inhibit both trypsin and chymotrypsin.
VOLUME AND POLARITY CHANGES ACCOMPANIED BY AMINO ACID SUBSTITUTIONS IN PROTEIN EVOLUTION
Authors: Go Mitiko , Miyazawa Sanzo
Published: August 2010

We evaluated the volume and polarity changes accompanied by amino acid substitutions along branches of the phylogenetic trees of cytochrome c, myoglobin and hemoglobin α andβ chains. In most cases the volume changes accompanied by the substitutions were found to be much larger than the volume of cavities existing in the interior of X‐ray‐analysed proteins. This implies that the interior of the proteins is very flexible and the necessary space for a larger amino acid residue substitution can be provided by adjusting nearby structures. Also, the volume and polarity changes are not particularly dependent on whether the substituted site is located in the exterior or interior of the proteins. This result supports the concept of the covarions by Fitch and Markowitz, when combined with the known fact that the exterior sites are more variable than the interior ones during protein evolution.
I.R.‐STIMULATED CATALYSIS (A POTENTIAL SPECTRAL TOOL)
Authors: SMITH T.F. , ROSS M.
Published: August 2010

Catalytic rate is proposed as an infrared spectrographic index. Initial investigations of catalytic activity, on β‐galactosidase show a 1–2‐fold increase in rate for a mutant enzyme when exposed to 10.6 μm irradiation at 2°. No increase was obtained for the wild‐type enzyme.
HEXAHYDRO DERIVATIVE OF (D‐MET2, PRO‐NH25]‐ENKEPHALIN GIVES RISE TO PHYSICAL DEPENDENCE
Authors: MORLEY JOHN S. , WEI E.T.
Published: August 2010

Filippi et al. (1979) have claimed that substitution of cyclohexylalanine for phenylalanine in leucine‐enkephalin and its L‐Ser3derivative resulted in synthetic analogs which were non‐addicting. We have examined the hexahydro derivative of [D‐Mer2, Pro‐NH52]‐enkephalin, a potent analgesic agent, to determine if dependence liability can be separated from short‐term opiate actions after hydrogenation of the phenylalanine residue. In contrast to Filippi et al.'s observations, we found that the hexahydro derivative of [D‐Met2, Pro‐NH52]‐enkephalin gives rise to physical dependence and that its short‐term potencies generally paralleled its long‐term ability to produce physical dependence.
CYCLIC PEPTIDES
Authors: KANMERA TATSUHIKO , LEE SANNAMU , AOYAGI HARUHIKO , IZUMIYA NOBUO
Published: August 2010

Å number of cyclo (Δaminoacyl‐L‐aminoacyl) (Δaminoacyl =ΔAba, ΔVal, ΔLeu, ΔApp, ΔPhe and ΔTrp; L‐aminoacyl=L‐Ala, L‐Val, L‐Leu and L‐Lys (e‐Ac)) were synthesized by the condensation of cyclo (N‐acetyl‐Gly‐N‐acetyl‐L‐aminoacyl) with corresponding aldehyde or acetone and subsequent treatment with hydrazine. Each cyclo (Δaminoacyl‐L‐aminoacyl) except ones containing ΔPhe and ΔTrp was hydrogenated using Pd black in methanol to give cyclo (L‐aminoacyl‐L‐aminoacyl) with high chiral induction. In the case of dehydrocyclodipeptides containing ΔPhe or ΔTrp, slightly lower chiral induction was observed. Then, a mechanism of asymmetric hydrogenation was proposed. The influence of variation of solvent, temperature and catalyst on asymmetric hydrogenation was examined. Optically pure L‐2‐aminobutanoic acid, L‐2‐amino‐5‐phenylpentanoic acid and L‐phenylalanine were synthesized on a preparative scale by this method and subsequent acid‐hydrolysis.
FLUORESCENCE AND N.M.R. STUDIES OF THE BINDING OF CHOLINERGIC FLUORESCENT PROBES TO HORSE SERUM CHOLINESTERASE
Authors: NARAYANAN REVATHI , BALARAM P.
Published: August 2010

The interaction of the cholinergic fluorescent probes, 1‐(5‐dimethyl‐aminoaphthalene‐1‐sulfonamido) ethane‐2‐trimethylammonium perchlorate, 1‐(5‐dimethylaminonaphthalene‐1‐sulfonamido) pentane‐5‐trimethylammonium tartarate and 1‐(5‐dimethylaminonaphthalene‐1‐sulfonamido) decane‐10‐ trimethylammonium tartarate with horse serum cholinesterase has been examined by fluorescence and n.m.r. methods. Fluorescence titrations show binding of the decane derivative to two sites on the protein whereas the lower homologs bind largely to one site. Active site inhibitors like curbamylcholine and decamethonium abolish binding of the decane derivative to the high affinity site. The inhibitors are largely without effect on the binding of the lower homologs. N.m.r. studies clearly establish immobilization of both ends of the molecule on binding in the case of the decane derivative, whereas in the lower homologs the dimethylamino group on the naphthalene ring is significantly more affected in the presence of enzyme. The probes are effective inhibitors of the enzyme with the decane derivative being two orders of magnitude more effective than its lower homologs. Based on the n.m.r., fluorescence and inhibition studies, a model for probe binding to the enzyme is advanced. It appears that the decane derivative binds with high affinity to the catalytic anionic site while the lower affinity site is assigned to a peripheral anionic site. The lower homologs probe only the peripheral site. A comparison of fluorescence, n.m.r. and inhibition studies with acetylcholinesterases from electric eel and bovine erythrocytes is presented.
ELECTRON MICROSCOPY OF NETWORK STRUCTURES IN THERMALLY‐INDUCED GLOBULAR PROTEIN GELS
Authors: CLARK A.H. , JUDGE F.J. , RICHARDS J.B. , STUBBS J.M. , SUGGETT A.
Published: August 2010

Thin sections of heat‐set protein gels formed from bovine serum albumin, insulin, lysozyme, ribonuclease, and alpha‐chymotrypsin, have been studied by transmission electron microscopy. Micrographs have been interpreted as showing protein networks with strands between one and two times as thick as the native protein diameters. Considerable differences in the persistence characteristics, and frequencies of cross‐linking, of the strands are observed, and there are variations in network homogeneity over long distances which correlate well with changes in gel opacity caused by alterations in pH and ionic strength. Evidence that arte facts are unlikely to have influenced these interpretations has been obtained in the BSA case in particular, by studying the aggregation process in solution, using alternative microscope approaches such as heavy‐metal shadowing and negative staining. Assuming that artefacts are absent, gel section micrographs have been simulated by a computer procedure, and the results suggest that, in most cases, the simplest interpretation of the microscope data is in terms of a “string of beads” model for the aggregation process, involving only moderately unfolded, and still globular, protein molecules. Other structural interpretations cannot be ruled out, however, as the degree of protein unfolding, and the exact mode of incorporation of the monomers into the network filaments, cannot be established by the microscope technique alone.
POLYMERIC 2‐MERCAPTOPYRIDINE AND 2‐MERCAPTO‐NITROBENZENE DERIVATIVES: New Reagents for Peptide Synthesis
Authors: STERN MEIR , WARSHAWKSY ABRAHAM , FRIDKIN MATI
Published: August 2010

Insoluble 2‐mercaptopyridine and 2‐mercapto‐nitrobenzene derivatives were prepared by modification of commercially available polystyrene. Applicability of these polymers as reagents for the thiolytic removal of the 2‐nitrophenyl‐sulphenyl amino‐protecting group and as supports for preparation of polymeric active esters was evaluated. Polymeric 2‐mercaptopyridine (PMP) was found efficient for both purposes. It was used in the stepwise synthesis of Leu‐enkephalin via the polymeric reagent approach, serving as an Nps‐cleaver. Polymeric esters derived from PMP and Boc‐amino acids proved to be excellent acylators. Their usefulness is exemplified in the preparation of two dipeptides, which were produced rapidly and in high yields and purity.
AMINO ACID COMPOSITION AND SEQUENCE ANALYSIS OF SAUVAGINE, A NEW ACTIVE PEPTIDE FROM THE SKIN OF PHYLLOMEDUSA SAWAGEI
Authors: MONTECUCCHI PIER CARLO , HENSCHEN AGNES
Published: August 2010

The complete amino acid sequence of sauvagine, a new active polypeptide from the skin of Phyllomedusa sauvagei, a frog of Central and South America, has been determined by automated liquid‐phase procedure after specific removal of the N‐terminal pyrrolidonecarboxylic acid, and specific cleavages at the single methionine and at the two arginine residues. The proposed sequence is: Pyr‐Gly‐Pro‐Pro‐Ile‐Ser‐Ile‐Asp‐Leu‐Ser‐Leu‐Glu‐Leu‐Leu‐Arg‐Lys‐Met‐Ile‐GIu‐Ile‐Glu‐Lys‐Gln‐Glu‐Lys‐Glu‐Lys‐Gln‐Gln‐Ala‐Ala‐Asn‐Asn‐Arg‐Leu‐Leu‐Leu‐Asp‐Thr‐Ile‐NH2.

Sauvagine possesses a number of pharmacological actions on diuresis, the cardiovascular system and endocrine glands; it can be considered the prototype of a new family of amphibian peptides, in addition to the tachykinins, bradykinins, dermorphins, caerulein‐like and bombesin‐like peptides.
Mechanistic implications of cyanide binding to carboxypeptidase B
Authors: ZISAPEL NAVA , SOKOLOVSKY MORDECHAI
Published: August 2010

The putative metal coordinating ligand cyanide was used to study the effects of modifications of the metal coordination sphere on the spectral properties and catalytic activity of cobalt and zinc carboxypeptidases. The absorption spectra of Co2+‐carboxypeptidase B in the presence of cyanide pointed to a direct interaction of the ligands with the metal. Gel‐filtration experiments showed that the binding of one mole of ligand per mole of enzyme metal ion resulted in maximal spectral effects. Binding of cyanide to the metal ion as measured by absorption spectroscopy was inhibited by acetyl‐ L‐arginine, a peptide pseudosubstrate, and by acetyl‐D‐arginine, a competitive peptide inhibitor. Addition of acetyl arginine to the enzyme‐cyanide complex caused displacement of the ligand, as evidenced by the spectral parameters. Cyanide inhibited peptide hydrolysis in a partially noncompetitive manner, i.e. it did not prevent binding of the substrate to the enzyme but the enzyme‐substrate‐cyanide complex was hydrolyzed at a slower rate than the enzyme‐substrate complex. The dissociation constant evaluated from kinetic studies for the binding of cyanide to Co2+‐carboxy‐peptidase B was in good agreement with that obtained from spectral measurements. Hydrolysis of the ester analog of the basic peptide substrate was not affected by cyanide. Based on these data a model is proposed in which the peptide carbonyl group displaces the water molecule from the metal coordination sphere during catalysis without increasing the coordination number.
Reversed phase thin‐layer chromatography for the separation of β‐endorphin, β‐lipotropin and enkephalins
Authors: ZIRING B. , SHEPPERD S. , KREEK M.J.
Published: August 2010

A novel, simple technique using reversed phase thin‐layer chromatography has been developed to separate β‐endorphin, β‐lipotropin and Met‐and Leuenkephalins. This method is useful for the rapid determination of the purity of these opioid peptides. The effectiveness of several solvent systems has been assessed. The resolution between β‐endorphin and β‐lipotropin achieved by this method exceeds that reported using high performance liquid chromatography or a variety of column techniques. This system also eliminates the problem of unrecognized loss of peptide due to binding on HPLC or other types of columns since the entire stationary phase is visualized using fluorescamine detection. Efficient, inexpensive and reliable simultaneous separation with visualization of several opioid peptides, their peptide precursors and their degradation products is now possible using the new technique. Such separations are required for many purposes including preparation of samples for radioimmunoassay of opioid peptides.
Circular dichroism studies of helical oligopeptides: Can 310 and α‐helical conformations be chiroptically distinguished?
Authors: SUDHA T.S. , VIJAYAKUMAR E.K.S. , BALARAM P.
Published: August 2010

Circular dichroism studies of seven helical oligopeptides containing α‐aminoisobutyric acid (Aib) in methanol and trifluoroethanol (TFE) solutions are reported. Peptides ranging from 10 to 21 residues in length have been examined. In all cases distinct negative CD bands characteristic of helical peptides are obtained at 220 and 205 nm corresponding to the n‐π* π‐π* transitions, respectively. The ratio R = [θ]n‐π*/[θ]π‐π* is < 1.0 for all peptides studied. Using crystal structure and n.m.r. results for a 10 residue 310 helical peptide and literature values for an α‐helical 11‐residue peptide, it is shown that both helical conformations yield R values of ˜ 0.8 in alcoholic solvents. The CD data are considered in the light of 1H n.m.r. studies on these oligopeptides. The results suggest that 310 and α‐helical conformations cannot be distinguished by CD methods.
Conformation and structure of acidic dipeptides
Authors: EGGLESTON DRAKE S. , HODGSON DEREK J.
Published: August 2010

The crystal structures of the dipeptides L‐alanyl‐L‐aspartic acid, C7H12N2O5, and α‐L‐glutamyl‐L‐aspartic acid, C9H14N2O7, have been determined from three‐dimensional X‐ray diffractometer data. Alanylaspartic acid crystallizes in the orthorhombic space group P212121 with four formula units in a cell of dimensions a = 13.389(5), b = 14.467(3), c = 4.782(1)Å. Glutamylaspartic acid also crystallizes in space group P212121 with four formula units in a cell of dimensions a = 13.709(5), b = 16.126(7), c = 4.939(5)Å. Both structures were solved by direct methods and refined by full‐matrix least squares methods; the final value of the weighted R‐factors (on F) were 0.040 based on 790 independent intesities with I‐2s̀(I) for Ala‐Asp and 0.033 based on 1105 intensities with I‐2s̀(I) for Glu‐Asp. Each dipeptide occurs as a zwitterion with the amino terminus protonated and the main chain carboxyl group deprotonated. The conformation of the peptide linkage is trans in both molecules, the ω torsion angle being ‐175.9° in Ala‐Asp and 174.3° in Glu‐Asp. There is considerable intermolecular, but not intramolecular, hydrogen bonding in the crystals. The conformations and structures of Ala‐Asp and Glu‐Asp are compared to those of other structurally characterized acidic dipeptides.
Differential equation model to study dynamic behaviour of globular proteins
Authors: PONNUSWAMY P.K. , BHASKARAN R.
Published: August 2010

By regarding the globular proteins as spheroidal shaped bodies of uniform density, a differential equation model to study their low amplitude fluctuations was developed. It was then applied to the crystal structures of pancreatic trypsin inhibitor and ferrocytochrome c. The results were tested by comparing them with those of the dynamic simulation and temperature factor studies on the same proteins.
Lysolecithin:lysolecithin acyltransferase from rabbit lung.
Authors: Arche .R. , Acebal C. , Casals C. , Estrada P. , Herrera L.
Published: August 2010

The enzyme lysolecithin:lysolecithin acyltransferase from rabbit lung has been found to have a relatively disordered conformation in solutions of high ionic strength. The protein exhibited an ordering of structure when salt was suppressed. This conformational change was concomitant with the loss of transacylase activity, the hydrolytic reaction remaining unchanged. Addition of NaCl caused a progressive disordering of structure with a parallel increase of transacylase activity.

The acid denaturation of the protein, at low and high ionic strengths, showed that the ionization of groups with pK in the range 5.9–6.4 was essential for denaturation. The structure was stable at basic pH.

The addition of lipids resulted in a non‐specific stabilization of the disordered conformation, in the same manner as the addition of NaCl.

From these results, it is suggested that there are two conformations for this protein which differ in their ability to bind lysolecithin molecules in the enzyme deacylation step of the reaction. This hypothesis agrees with previously published properties of the enzyme, concerning aggregation with other proteins and kinetic data. From the amino acid composition and conformational properties, the authors suggest that this enzyme could be a peripheral membrane protein.
Binding affinities to rat brain synaptosomes – synthesis of biotinylated analogues of Substance P
Authors: LAVIELLE S. , CHASSAING G. , BEAUJOUAN J.C. , TORRENS Y. , MARQUET A.
Published: August 2010

The synthesis of four biotinylated analogues of Substance P is described. The affinities of these analogues and of their complexes with avidin for the 125I‐Bolton Hunter Substance P binding sites on rat brain synaptosomes were determined. While these biotinylated peptides complexed to avidin retain a good biological activity on the guinea‐pig ileum bioassay, we observe a net decrease in their binding affinities in the central nervous system. The present study confirms that in the central nervous system the higher affinity is related to the N‐terminal tetrapeptide and establishes that the free amino group (N‐α‐Arg and N‐α‐Lys) are not essential in the binding.
Theoretical π‐π* absorption and circular dichroic spectra of cyclic dipeptides
Authors: SATHYANARAYANA B.K. , APPLEQUIST JON
Published: August 2010

The dipole interaction model, treated by the partially dispersive normal mode method, is used to calculate circular dichroic spectra of cyclo(Gly‐Gly), cyclo (Ala‐Gly), cyclo(Ala‐Ala), cyclo(Pro‐Gly), cyclo(Pro‐Ala), cyclo(Pro‐Val), cyclo (Pro‐D‐Val), and cyclo(Pro‐Pro) in the amide π‐π* absorption band near 190 nm. Assuming a standard backbone geometry, spectra which are in fair to good agreement wth experiment are obtained for these molecules. The spectra are predicted to be sensitive to conformations of Pro and Val side chains. The effects of dipeptide ring folding on calculated CD spectra are mostly consistent with those found by other workers, except that it is found that a planar ring conformation of cyclo (Ala‐Ala) and cyclo (Ala‐Gly) gives predicted spectra comparable to experiment. The same model gives theoretical absorption spectra consistent with available experimental data.
Coupling in the absence of tertiary amines
Authors: BODANSZKY MIKLOS , BODANSZKY AGNES , CASARETTO MONIKA , ZAHN HELMUT
Published: August 2010

Several weak acids were tested as acidolytic reagents for the removal of acid sensitive amine‐blocking groups. Cleavage with tetrazole in trifluoroethanol yielded amine salts which could be acylated with active esters without the addition of a tertiary amine. Concurrent deprotection‐acylation was also possible. Scope and limitations of the tetrazole approach are discussed.
Selective ion transport and ion extraction by synthetic cyclic octapeptide
Authors: SHIMIZU TOSHIMI , TANAKA YOSHIO , TSUDA KEISHIRO
Published: August 2010

Cyclic octapeptide, cyclo[Gly‐L‐Lys(Z)‐Sar‐L‐Pro]2 (CGLSP2) was synthesized as an ionophore model. Its ion‐transport ability through a liquid membrane was investigated in connection with ion extractability and conformational properties. CGLSP2 transported the picrate salts of Ba2+ and Ca2+ efficiently through chloroform membrane in a U‐tube (Ba2+> Ca2+ > K+ > Rb+ > Mg2+> Na+). Cyclo[Gly‐L‐Lys(Z)‐Sar‐L‐Pro] or Boc‐[Gly‐L‐Lys(Z)‐Sar‐L‐Pro]n‐OR (n = 1 or 2; R = H or CH3) showed a significant decrease in ion‐transport ability. Extraction equilibrium constants (Kex) of CGLSP2 with metal picrates were determined in chloroform‐water system. In this case, CGLSP2/cation complexes were found to have a stoichiometry of 1:1. The Kex sequences were Ba2+ > Ca2+ > Mg2+ and K+ > Rb+ > Na+, showing good agreement with the selectivity in ion transport. Conformation of CGLSP2 in chloroform (CDC13) was investigated by 1H and 13C‐n.m.r. spectroscopy. Predominant conformation takes a C2 ‐symmetric structure containing two cis Lys‐Sar peptide bonds. Upon complexation with Ca2+ in CDC13/CD3CN mixture (5/3, v/v), the conformation of CGLSP2 converged into a single C2 ‐symmetric one that was predominant in a free state in CDC13.
Synthesis of an arginine rich fragment of a viral coat protein using guanidinium functions exclusively
Authors: TEN KORTENAAR PAUL B.W. , KRÜSE JAAP , HEMMINGA MARCUS A. , TESSER GODEFRIDUS I.
Published: August 2010

The N‐terminal pentacosapeptide of Cowpea Chlorotic Mottle Virus (CCMV) was synthesized as the Nα25‐acetyl, Cα25‐methylamide. The compound contains six arginyl residues, which were incorporated in the peptide chain protected by protonation. The compound was obtained from an azide and a carbodiimide mediated condensation, unifying three fragments. The identity of the compound was proved by amino acid analysis, 13C‐n.m.r. and 1H‐n.m.r. spectroscopy. It behaved like the native protein in its tendency to interact with viral RNA (fragments).