Single-step,rapid separation of acidic and basic isoenzymes from commercial horseradish peroxidase

A simple and sensitive high-performance liquid chromatography method with electrochemical detector is described for the determination of free 3-methoxy-4-hydroxyphenylacetic acid (HVA) in human plasma. The method does not involve any extraction, is specific and reproducible, and has the potential to measure serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) simultaneously. The plasma concentration of free HVA in eight normal, healthy adult volunteers was 10.9 ± 4.6 (mean ± SD). In a preliminary study, in one schizophrenic patient the plasma HVA increased twofold after neuroleptic treatment.     

Bioactive polymers XXX. Immobilization of invertase on the diazonium salt of 4-aminobenzoylcellulose

Invertase was immobilized on diazotized 4-aminobenzoylcellulose. The optimum coupling conditions, namely enzyme concentration, time, and pH, were determined. The temperature and pH value for the maximum activity of the immobilized enzyme were determined and the apparent Michaelis constant was estimated.     

H－ras在颊粘膜正常、良性、癌前及恶性变上皮中的表达：免疫组织化学定量研究

本文通过建立图象分析方法对免疫组织化学反应结果进行定量,检测观察Ｈ－ｒａｓ在口腔颊粘膜上皮在正常（Ｎ）、慢性炎症（ＩＦ）、癌旁上皮（ＥＡＣ）和鳞癌（ＳＣＣ）的变化过程中的表达并进行分析。结果显示Ｈ－ｒａｓ在ＳＣＣ组中,以中等分化的ＳＣＣ无论是Ｈ－ｒａｓ表达的量还是细胞阳性率都较高。此外,组织学观察显示,Ｈ－ｒａｓ在处于分化末期但尚未角化的正常上皮细胞中有较高的表达。本文结果显示了Ｈ－ｒａｓ的过表达与上皮细胞的会化程度密切相关。本研究还显示,所采用的阳性区域透光值、平均总透光值及阳性反应区域与阴性反应区域比值可靠并有相关性。这进一步说明了用免疫组化定量方法检测Ｈ－ｒａｓ癌基因表达的精确和可靠性。     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.     

A peculiar lymph node anti-carcinoma reaction.

A case of a laryngeal extensive carcinoma determining a severe obstruction is reported, where the development of a monstrous adenopathy is followed by the regression of the initial tumour until complete laryngoscopical disappearance, as well as of obstructive and general symptoms. Cytological and histological examinations of the above-mentioned lymph node demonstrated (besides metastatic cells) a peculiar cellular reaction, similar to rhinoscleroma, which represented an original anti-carcinoma defensive mode. The medical importance of this very rare case is discussed and the probable immune origin of this reactive cellular complex is explained by analogy with "Hodgkinom", where granuloma are the immune response of the body against Sternberg-Reed cell.     

Isolation of Thiomonas thermosulfatus Strain 51, a Species Capable of Coupling Biogenic Pyritization with Chemiosmotic Energy Transduction

The precipitation of iron sulfides potentially offers enough energy and reducing power to sustain life but organisms harnessing this reaction have not to our knowledge been previously described. We isolated a bacterial strain, capable of forming the iron sulfide minerals troilite (FeS), greigite (Fe₃S₄), and pyrite (FeS₂), from subsurface, microbial mats in Mangalia, Romania. This strain, most closely related to strains of Thiomonas sp., forms pyrite only if the redox conditions remain negative (< ?60 mV), sulfides are provided continually (≈1 mM), and the concentration of iron remains low (≤ 0.08 mM) but constant. Pyrite formation by this microbial strain is proposed as an example of biologically controlled mineralization because it is controlled by uncouplers of oxidative phosphorylation, it is larger in living than in dead cells, it is additive (controlled less by the amount of cell surfaces and more by reagents), and it results in the formation of ATP. This study indicates that precipitation and crystal formation can represent an energy resource for life and provides support for the “iron-sulfide world hypothesis” regarding the early evolution of life on Earth.     

Occurrence of species of the genus Pityophthorus Eichhoff (Coleoptera,Curculionidae, Scolytinae) in?the?province of Quebec,Canada

Twig beetles in the genus Pityophthorus Eichhoff, 1864 include more than 300 species worldwide, with maximum diversity in tropical and subtropical regions. To date, approximately 50 species of Pityophthorus have been recorded in Canada, and these species are associated mainly with coniferous trees. Since 1981, no comprehensive study on this difficult taxonomic group has been conducted in Quebec, Canada, most likely due to their limited significance as forest pests. Based on data gathered from five years of field sampling in conifer seed orchards and compiled from various entomological collections, the distribution of Pityophthorus species in Quebec is presented. Approximately 291 new localities were recorded for the Pityophthorus species. Five species-group taxa, namely Pityophthorus puberulus (LeConte, 1868), Pityophthorus pulchellus pulchellus Eichhoff, 1869, Pityophthorus pulicarius (Zimmermann, 1868), Pityophthorus nitidus Swaine, 1917,and Pityophthorus cariniceps LeConte&Horn, 1876 were the most widespread. In contrast, Pityophthorus consimilis LeConte, 1878, Pityophthorus intextus Swaine, 1917, Pityophthorus dentifrons Blackman, 1922, Pityophthorus ramiperda Swaine, 1917, and Pityophthorus concavus Blackman, 1928 display a notably limited distribution. In addition, the first distribution records of Pityophthorus intextus and Pityophthorus biovalis Blackman, 1922 are furnished, and the subspecies Pityophthorus murrayanae murrayanae Blackman, 1922is reported from Quebec for the second time. Moreover, distribution maps are provided for all Pityophthorus species recorded in the province of Quebec.     

Interaction of piroxicam with bovine serum albumin investigated by spectroscopic,calorimetric and computational molecular methods

Abstract

The binding of drugs to serum proteins is governed by weak non-covalent forces. In this study, the nature and magnitude of the interactions between piroxicam (PRX) and bovine serum albumin (BSA) was assessed using spectroscopic, calorimetric and computational molecular methods. The fluorescence data revealed an atypical behavior during PRX and BSA interaction. The quenching process of tryptophan (Trp) by PRX is a dual one (approximately equal static and dynamic quenched components). The FRET results indicate that a non-radiative transfer of energy occurred. The association constant and the number of binding sites indicate moderate PRX and BSA binding. The competitive binding study indicates that PRX is bound to site I from the hydrophobic pocket of subdomain IIA of BSA. The synchronous spectra showed that the microenvironment around the BSA fluorophores and protein conformation do not change considerably. The Trp lifetimes revealed that PRX mainly quenches the fluorescence of Trp-213 situated in the hydrophobic domain. The CD and DSC investigation show that addition of PRX stabilizes the protein structure. ITC results revealed that BSA-PRX binding involves a combination of electrostatic, hydrophobic and hydrogen interactions. The analysis of the computational data is consistent with the experimental results. This thorough investigation of the PRX-BSA binding may provide support for other studies concerning moderate affinity drugs with serum protein.

Communicated by Ramaswamy H. Sarma