Attachment forces of the hemelytra-locking mechanisms in aquatic bugs (Heteroptera: Belostomatidae)

Combined hemelytra-locking system of Heteroptera, consisting of several locking mechanisms, aids the mechanical stabilisation of the body at rest, resists external loads, and keeps air stored with the option to easily unlock hemelytra prior to flight. The resistance to unlocking of the hemelytron was measured (in mN) with the aid of a load cell force transducer combined with a three-axial micromanipulator. It is shown that macro- and microstructural features of several submechanisms are responsible for their directionality. The highest resistance to unlocking was measured in lateral and dorsal directions. Summarised force of separately measured submechanisms was considerably lower than the force measured in the combined mechanism. Each submechanism is optimised for achieving high resistance to the hemelytron uncoupling in particular direction(s) and to be easily unlocked in another direction. It was demonstrated in the high-speed videorecordings that hemelytra uncoupling is promoted by their short anterior displacement.     

The characteristics of the electrodermal activity during changes in the level of human wakefulness

The wake-drowsiness transition during the performance of a monotonous psychomotor test was studied using polygraphic recording, with particular emphasis on changes in the electrodermal activity (EDA) and occurrence of electrodermal reactions (EDR). It was found that decrease in wakefulness was accompanied by a drop of the EDA. The EDR gradually disappeared for several minutes and did not reappear without activation; their dependence on sex and individual features of a subject significantly increased. It is suggested that discrepancies in the experimental results of psychophysiological studies with EDA may be explained by an insufficient control of the subject's alertness throughout the experiment. Examples of such discrepancies are given: disagreement of sex differences in the EDA, differentiation between EDA-labile and EDA-stable persons, on a possibility of recording the "emotional" EDA from different parts of human body etc. The ways of reproducibility of the results are discussed including the recording of several wake-drowsiness transitions and better recognition of the EDR.     

A new dipeptide,O-phosphoserylethanolamine isolated from Agkistroden blomhoffi (mamushi)

A new dipeptide was isolated from several tissues of Agkistroden blomhoffi (mamushi: a venomous snake in Japan), using ion-exchange resins and thin-layer chromatography. It was identified as O-phosphoserylethanolamine by mass spectrometry and comparison with synthetic compounds using several methods. This compound was contained in several mamushi tissues including the liver, heart, brain, bile, and muscle. The concentrations of O-phosphoserylethanolamine in the liver, brain, muscle, skin, heart, and bile were 7.17+/-3.11,16.98+/-4.25,37.37+/-7.88,37.56+/-8.97,23.93+/-6.11, and 22.21+/-5.76 micromol/g, respectively.     

Directly acting geno- and cytotoxic agents from a wild mushroom Dermocybe sanguinea.

The wild mushroom, Dermocybe sanguinea, contains several anthraquinone pigments, of which emodin (1,3,8-trihydroxy-6-methylanthraquinone) is quantitatively the most important. In our preliminary tests, Dermocybe sanguinea extracts were genotoxic without metabolic activation. The ethanol extract of Dermocybe sanguinea was fractionated by flash chromatography, and the emodin contents of the fractions were determined by HPLC. Their genotoxicities were assayed using a bacterial repair assay and sister-chromatid exchange analysis. The cytotoxicity of the fractions was assayed with mouse hepatoma cells using growth inhibition as the endpoint. The results of the biological tests were compared with those obtained with pure emodin. It was concluded that, in addition to emodin, Dermocybe sanguinea contains several other geno- and cytotoxic compounds.     

Synthesis of 9-N-cinchona alkaloid peptide hybrid derivatives: preparation and conformational study of 9-N-acylamino(9-deoxy)cinchona alkaloids

The synthesis and conformational analyses of several 9-N-acylamino(9-deoxy)cinchona alkaloids is presented. Peptides were connected to cinchona alkaloids via a 9-amino group. The synthesis of the new cinchona alkaloid derivatives was performed straightforwardly from 9-amino(9-deoxy)dihydroquinidine via coupling with carboxylic acid chlorides and several dipeptides. Both alkaloid derivatives with the configuration of the corresponding natural product as well as its unnatural epimer were studied. The conformations of the prepared derivatives in solution were determined by NMR spectroscopy. It is shown that the conformation is strongly influenced by the configuration at the 9-position.     

快速简便筛选cDNA文库的SSS法

建立了一种快速简便筛选cDNA文库的方法—SSS法（subsection screening）。该方法用cDNA噬菌体平板划块分组和根据目的基因设计的一对特异性引物,用PCR技术逐级筛选cDNA文库获得目的基因。与其它筛选cDNA文库的方法相比,该方法范围可控,目标明确,易于获得目的基因,而且快速、简捷、省时,一般可在一周内筛选出目的基因。本实验室利用该方法在半个月内筛选出了一个查耳酮异构酶(chalcone isomerase)(CHI)基因,一个黄酮类化合物3′-羟化酶(flavonoid 3′hydroxylase)(F3′H)基因,一个热激蛋白(heat shock protein)(HSP)基因和一个1 484 bp 热激蛋白基因片段。此方法用于同时筛选多个基因,可收到事半功倍的效果,对其它文库筛选也有借鉴意义。 Abstract:A quick and simple method subsection screening (SSS) method for screening cDNA library by PCR was established.With this method,cDNA phage plate was cut into several blocks and a couple of primers was designed according to target gene.And then the target genes were obtained by screening cDNA library.Comparing with other methods,this method has many advantages such as controlled range and clear target,and also quick and simple for obtaining the target genes.It is possible to get a target gene in one week in general by this method.Thus,the CHI gene,F3′H gene,HSP gene and one HSP partial fragment,were obtained respectively in half month in our lab.We can get twice the result with half the effort when we screen several genes in the same time.It is also suitable to screen other libraries.     

Mechanistic aspects of biosynthesis of silver nanoparticles by several <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strains

Extracellular production of metal nanoparticles by several strains of the fungus Fusarium oxysporum was carried out. It was found that aqueous silver ions when exposed to several Fusarium oxysporum strains are reduced in solution, thereby leading to the formation of silver hydrosol. The silver nanoparticles were in the range of 20–50 nm in dimensions. The reduction of the metal ions occurs by a nitrate-dependent reductase and a shuttle quinone extracellular process. The potentialities of this nanotechnological design based in fugal biosynthesis of nanoparticles for several technical applications are important, including their high potential as antibacterial material.     

Effect of anions on the photocycle of halorhodopsin. Substitution of chloride with formate anion

Halorhodopsin from Natronomonas pharaonis is a light-driven chloride pump which transports a chloride anion across the plasma membrane following light absorption by a retinal chromophore which initiates a photocycle. It was shown that the chloride anion bound in the vicinity of retinal PSB can be replaced by several inorganic anions, including azide which converts the chloride pump into a proton pump and induces formation of an M-like intermediate detected in the bR photocycle but not in native halorhodopsin. Here we have studied the possibility of replacing the chloride anion with organic anions and have followed the photocycle under several conditions. It is revealed that the chloride can be replaced with a formate anion but not with larger organic anions such as acetate. Flash photolysis experiments detected in the formate pigment an M-like intermediate characterized by a lifetime much longer than that of the O intermediate. The lifetime of the M-like intermediate depends on the pH, and its decay is significantly accelerated at low pH. The decay rate exhibited a titration-like curve, suggesting that the protonation of a protein residue controls the rate of M decay. Similar behavior was detected in N. pharaonis pigments in which the chloride anion was replaced with NO(2)(-) and OCN(-) anions. It is suggested that the formation of the M-like intermediate indicates branching pathways from the L intermediate or basic heterogeneity in the original pigment.     

The role of superoxide anion in peroxidase-catalyzed chemiluminescence of luminol

The chemiluminescence associated with peroxidation of luminol in buffered aqueous solution is a complex process involving several intermediates. It can be inhibited by removal of oxygen from the incubation medium. Superoxide radical is both an intermediate in this reaction and an essential component in light-producing steps. The importance of O₂^? in propagating this reaction was shown by the inhibition of luminescence by superoxide dismutase. A mechanism was proposed which is consistent with the data. It appears likely that the diverse biological effects of peroxidases are largely due to the reactivities of these intermediates and products.     

Immunological Characterization of Secretory Proteins of Chromaffin Granules: Chromogranins A, Chromogranins B, and Enkephalin-Containing Peptides

The soluble proteins of bovine chromaffin granules can be resolved into about 40 proteins by two-dimensional electrophoresis. Use of several antisera enabled us to characterize most of these proteins with the immune replica technique. An antiserum against dopamine beta-hydroxylase reacted with one protein of Mr 75,000. Met-enkephalin antisera labeled eight proteins of Mr 23,000-14,000. A new method was developed to obtain highly purified chromogranin A for immunization. The antiserum reacted with chromogranin A and several smaller proteins of similar pI. This specific antiserum did not react with a second family of hitherto undescribed proteins, which we propose to call chromogranins B. An antiserum against these proteins was raised. It labeled several proteins ranging in Mr from 100,000 to 24,000 and focusing at pH 5.2. Subcellular fractionation established that chromogranins B are specifically localized in chromaffin granules of several species. They are secreted from the adrenal medulla during cholinergic stimulation. We conclude that apart from dopamine beta-hydroxylase chromaffin granules contain three families of immunologically unrelated proteins.     

Origin and mediation of secretion induced by oral phytohaemagglutinin (PHA) in rats

After oral administration several gut-binding lectins induce accumulation of liquor and amylase in the proximal small intestine. Orally administered Phaseolus vulgaris phytohaemagglutinin (PHA) was used to study the mediation of these effects in rats. The regulation of amylase secretion clearly differed from that of the liquor. The amylase activity was of pancreatic origin, in agreement with the known cholecystokinin-releasing effect of PHA. It appears that CCK exerts its effect both directly and by facilitating neural stimulatory pathways. Intestinal secretion was identified as the source of the liquor, without a contribution by other secretions. It was mediated by a local cholinergic reflex with the involvement of both muscarinic and nicotinic acetylcholine receptors. It is speculated that the observed enteric reflex may enable the gut to transport secreted antibacterial peptides or secretory antibodies from the crypts to adherent bacteria on adjacent villi.     

The determination of methionine in proteins by gas-liquid chromatography.

Intact methionine residues in proteins were rapidly and precisely determined by measuring methyl thiocyanate released during the reaction with CNBr and separated by g.l.c. Conditions for the reaction and for chromatography on columns of Porapak P-S are described. The recovery of methyl thiocyanate from several methionine derivatives and analogues were examined. Carbamoylmethionine was adopted as a stable primary standard and ethyl thiocyanate as internal standard. The measured methionine content of several isolated proteins was close to the theoretical value indicated by previous work and the results for these and a range of food proteins agreed well with results obtained by ion-exchange chromatography after performic acid oxidation. Since CNBr does not react with methionine sulphoxide and a preliminary hydrolysis is not required, the method discriminates between methionine and any methionine sulphoxide that may be present. It could be useful in studies on the nutritional availability of methionine in processed foods.     

Application of irreversible thermodynamics to a functional description of the tumor cell membrane

The permeability of the membranes of several strains of Ehrlich mouse ascites tumor cells to a series of homologous glycols was analyzed with equations from irreversible thermodynamics. Experimental data were fitted to the appropriate equations of Kedem and Katchalsky by means of an analog computer and values for σ, L_p, and ωRT were calculated. These parameters would become part of a functional profile of the membrane of the tumor cell. The glycols cross the membrane with energies of activation ranging from 16 kcal/mole for ethylene glycol to 20 kcal/mole for triethylene glycol. Entropies of activation ranged from 17 to 27 entropy units. This analysis provided several additional dividends. It is one of the few examples with a mammalian cell where the kinetics of non-electrolyte transport have been described completely by the equations of irreversible thermodynamics. It has provided the means to distinguish how much of the transport occurred through porous paths in the membrane and how much involved solution within the matrix of the membrane. Finally, several tumor cell populations have been distinguished through these functional properties of their membranes.     

Stopped-flow studies of guanine binding by calf spleen purine nucleoside phosphorylase

The binding of guanine to calf spleen purine nucleoside phosphorylase at 20 degrees C, in 20 mM Hepes-NaOH buffer, pH 7.0, at several ionic strength between 5 and 150 mM was investigated using a stopped-flow spectrofluorimeter. The kinetic transients registered after mixing a protein solution with ligand solutions of different concentrations were simultaneously fitted by several association reaction models using nonlinear least-squares procedure based on numerical integration of the chemical kinetic equations appropriate for given model. It is concluded that binding of a guanine molecule by each of the binding sites is a two-step process and that symmetrical trimeric calf spleen purine nucleoside phosphorylase represents a system of (identical) interacting binding sites. The interaction is visible through relations between the rate constants and non-additivity of changes in "molar" fluorescence for different forms of PNP-guanine complexes. It is also probable that electrostatic effects in guanine binding are weak, which indicates that it is the neutral form of the ligand which is bound and dissociated by PNP molecule.     

Protein stability to the effect of denaturing agents and elevated temperature is directly proportional to the fraction of hydrophobic residues in its molecule

The dependence of protein stability against the action of urea, guanidine hydrochloride and elevated temperature on the fraction of hydrophobic residues in their molecules was studied. It was shown that proteins can be divided into several stability classes. The stability of proteins within each class is directly proportional to the fraction of hydrophobic residues in their molecules and this dependence is a linear one. As shown on several examples proteins can pass from one class to another and such discrete transitions are determined by the formation or disruption of bonds with cofactors. The effect of point mutations on the stability of proteins is discussed from the viewpoint of polar group dehydration.     

Difference-derivative absorbance spectrophotometry as a technique to measure state changes of phenylalanine residues in proteins.

The first derivatives of difference absorbance spectra of several proteins were measured to examine the applicability of this technique as a tool to investigate state changes of phenylalanine residues in proteins. It was found by this technique that phenylalanine residues in insulin and those in lysozyme are exposed to more aqueous environment by denaturation with guanidine hydrochloride. Heat denaturation of collagen caused similar changes of some of its phenylalanine residues. It was thus demonstrated that difference-derivative absorbance spectrophotometry gives the information about state changes of phenylalanine residues in native proteins, which are hardly detected by common difference spectrophotometry.     

Thrombophilic states in late gestosis

In 80 pregnant women with pre-eclampsia and in 38 healthy pregnant women malondialdehyde production by blood platelets, platelet survival and several haemostatic variables were determined. It was demonstrated that due to the enhanced platelet turnover pre-eclampsia was invariably accompanied by a thrombophilic state which in severe cases developed into chronic consumption coagulopathy.     

Processing of the electroencephalogram in cardiac surgery

The objective of this investigation was to find a method to automatically detect several types of abnormal EEG patterns which occurred during cardiac surgery. The EEGs were analyzed by means of several EEG processing and pattern recognition methods. It was found that a good classification into normal and abnormal EEG patterns was generally obtained if only two EEG features were used. The results of this investigation are of importance for implementing into a computer-based monitoring system for use during cardiac surgery.     

Properties of phosphoribulokinase from Thiobacillus neapolitanus

Partially purified preparations of ribulose-5-phosphate kinase (specific activity, 50 to 125 mumoles per min per mg of protein) were employed in a series of kinetic experiments in the presence of several concentrations of H(+), Mg(2+), adenosine triphosphate (ATP), and phosphoenolpyruvate (PEP). The pH optimum of the enzyme was found to be 7.9; at this pH and above, response of the enzyme to variations in ATP concentration was hyperbolic, exhibiting a K(m) of 7 x 10(-4)m ATP. At pH values below the optimum the response to ATP was sigmoidal, as it was throughout the entire pH range in the presence of PEP at a concentration greater than 5 x 10(-4)m. In the presence of PEP the pH optimum shifted to pH 8.4. In contrast, phosphoribulokinase from spinach exhibited hyperbolic responses throughout its pH range with no inhibition caused by PEP. Thiobacillus neapolitanus phosphoribulokinase was inhibited by PEP in a sigmoidal manner; however, in the presence of suboptimal concentrations of Mg(2+) the addition of PEP caused significant stimulation of activity. It is postulated that the enzyme consists of interacting subunits with several sites on the enzyme for binding ATP and with several separate sites binding PEP. It is suggested that PEP functions as a regulator of CO(2) fixation when the organism is under conditions of unlimited concentrations of substrate and CO(2).     

Antibiotic formation in plasmid and plasmid-free strains]

Antibiotic activity of three plasmid and three plasmid-free strains of Bacillus pumilus was studied. The antibiotic activity was found in one plasmid and two plasmid-free strains. It was supposed that both the chromosomic and extrachromosomic genes could participate in regulation of the antibiotic production in Bac. pumilus. It was shown that the antibiotics produced by Bac. pumilus had a very narrow spectrum and inhibited multiplication only of several grampositive bacteria.