Intramolecularly quenched fluorogenic peptide substrates for human renin.

Six intramolecularly quenched fluorogenic peptides related to the sequences Phe8 to His13, His6 to His13, and Tyr4 to His13 of the human angiotensinogen, containing o-aminobenzoyl (Abz) and ethylenediamine dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acids residues, were synthesized by classical solution methods. The Leu-Val is the only bond of all obtained peptides that was hydrolyzed by human renin with different degrees of purity and was resistant to hydrolysis by pig renin and cathepsin D. The hydrolysis of Abz-His-Pro-Phe-His-Leu-Val-Ile-His-EDDnp by human renin was inhibited by a highly specific transition-state analog of angiotensinogen (IC50 = 7.8 x 10(-9) M), described by K. Iizuka et al. (1990, J. Med. Chem. 33, 2707-2714). Therefore, specific and sensitive substrates for the continuous assay of human renin in which as little as 70 microGU of human renin could be detected by Abz-Phe-His-Leu-Val-Ile-His-EDDnp were described. The optimal pHs of hydrolysis of the substrates were in the range 4 to 6.     

Contribution of unusual Arginine-Arginine short-range interactions to stabilization and recognition in proteins

Although the majority of the ion pairs found in proteins consists of two charges of opposite sign, the observation of some unusual arrangements of two arginines led us to a search of such occurrences in the Brookhaven Protein Data Bank. We have found 41 Arginine-Arginine interactions with a C...C distance less than 5 å. Computer graphics analysis of these structures shows that most of the Arg-Arg pairs are found in the vicinity of the surface of the proteins, in an easily hydrated region. In order to determine which factors could stabilize such arrangements of species of similar charge, we have carried out AM1 semi-empirical calculations on a model of two guanidinium ions surrounded by several water molecules. The results show the existence of stable clusters with six or more water molecules, with distances between C atoms around 3 å. The bridging role of the water molecules is an important structural and energetic feature and we find bridges of two and three molecules between the guanidinium ions. These results are in good agreement with the structures found in our search of the experimental data. Enhancement of the electrostatic potential around these clusters, when compared to one of the guanidinium ions alone, is also demonstrated.     

Chromosomes of the Antarctic amphipod Waldeckia obesa Chevreux

Mitotic and meiotic chromosomes of the Antarctic lysianassoid amphipod Waldeckia obesa are described. The modal chromosome number is n = 25 and 2n = 50. The potential applications of cytogenetical studies in this group are discussed.     

Characterization of hemocytes from the marine amphipod Parhyale hawaiensis (Dana 1853): Setting the basis for immunotoxicological studies

Hemocytes are circulating blood cells that play a crucial function in amphipods and other crustacean immune systems. The hemocytes of the marine tropical amphipod Parhyale hawaiensis have been used for the evaluation of DNA damage and micronuclei, but they have not been characterized in the scientific literature. The aim of this study was to describe the hemolymph cells of P. hawaiensis and study their phagocytotic activity. Basic dyes were used to differentiate the cell types and the presence of lipids. The total hemocyte counts (THCs) and the proportion and sizes of the hemocyte types were determined. Hemolymph was exposed to Escherichia coli for verification of the presence of phagocytosis. Three cell types, all containing lipids, were identified in P. hawaiensis: granulocytes (oval shape, 13.4 × 7.6 μm), semi-granulocytes (oval shape, 14.1 × 7.2 μm), and hyalinocytes (round shape, 9.6 × 7.2 μm). Those three cell types were found in different percentages in males (64.8%, 31.1%, and 4.2%) and females (70.1%, 28.2%, and 1.7%). THCs for males were 9007 ± 3800 cells per individual and 4695 ± 1892 cells per individual for females. The cells of E. coli were phagocytized by the hemocytes. Our findings increased the knowledge of hemocytes in P. hawaiensis and is a step forward in using hemocyte-based immune responses as an endpoint in ecotoxicology.     

Phytochemical Characterization of Cannabis sativa L. Roots from Northeastern Brazil

This article describes the phytochemical study of Cannabis sativa roots from northeastern Brazil. The dried plant material was pulverized and subjected to exhaustive maceration with ethanol at room temperature, obtaining the crude ethanolic extract (Cs-EEBR). The volatile compounds were analyzed by gas chromatography coupled with mass spectrometry (GC/MS), which allowed to identify 22 compounds by comparing the linear retention index (LRI), the similarity index (SI) and the fragmentation pattern of the constituents with the literature. By this technique the major compounds identified were: friedelan-3-one and β-sitosterol. In addition, two fractions were obtained from Cs-EEBR by classical column chromatography and preparative thin layer chromatography. These fractions were analyzed by NMR and IR and together with the mass spectrometry data allowed to identify the compounds: epifriedelanol, friedelan-3-one, β-sitosterol and stigmasterol. The study contributed to the phytochemical knowledge of Cannabis sativa, specifically the roots, as there are few reports on the chemical constituents of this part of the plant.     

Computer-aided comparison of protein electrophoretic patterns for grouping and identification of heterotrophic bacteria from mineral water

The microflora of a natural mineral water was studied immediately after bottling (T0) and after 7 d storage (T7) during 6 months, and isolates were clustered by SDS-PAGE of wholecell protein profiles. Isolates from each cluster were further characterized by API 20NE, fatty acid composition and quinone profiles. The numerical analysis of the electrophoregrams of all bacteria isolated from the mineral water formed 15 clusters and five unclustered strains. Except for five minor clusters, all clusters were composed of strains isolated over several months. The numerical analysis of the electrophoregrams of bacteria isolated immediately after bottling formed 15 clusters while after 7 d storage only four of these populations could be isolated, indicating that populations present in the mineral water were stable and that changes occurring after bottling probably resulted from a selection process. Only one unclustered strain was identified simultaneously by all the systems, as Sphingomonas paucimobilis. The monitoring of the aquifer and the bottling system, and the construction of a large database with bacteria of the autochthonous flora allows the detection of alterations in the aquifer by changes in the microflora.     

Circadian and seasonal variation of the body temperature of sheep in a tropical environment

Nychthemeral and annual rhythms of the rectal temperature were determined for Corriedale sheep in a tropical climate. The minimum rectal temperature averaged 39.55° C at 0500 hours in summer, and 38.87° C at 0600 hours in winter. The maximum was 40.03° C in summer (1700 hours) and 39.33° C in winter (1830 hours). Annual cycle of the rectal temperature showed a minimum in July and maximum in December.     

Internally quenched fluorogenic protease substrates: Solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs

Summary A general procedure, using the commonly employed solid-phase peptide synthesis methodology for obtaining internally quenched fluorogenic peptides with ortho-aminobenzoyl/dinitrophenyl groups as donor-acceptor pairs, is presented. The essential feature of this procedure is the synthesis of an N -Boc or-Fmoc derivative of glutamic acid with the -carboxyl group bound to N-(2,4-dinitrophenyl)-ethylenediamine (EDDnp), which provides the quencher moiety attached to the C-terminus of the substrate. The fluorescent donor group, ortho-aminobenzoic acid (Abz), is incorporated into the resin-bound peptide in the last coupling cycle. Depending on the resin type used, Abz-peptidyl-Gln-EDDnp or Abz-peptidyl-Glu-EDDnp is obtained. Using the procedure described above, substrates for human renin and tissue kallikreins were synthesised. Spectrofluorimetric measurements of Abz bound to the -amino group of proline showed that strong quenching of Abz fluorescence occurs in the absence of any acceptor group.     

Studies on the adenosine 3′,5′-monophosphate-dependent protein kinase of Blastocladiella emersonii

Using a combination of Chromatographic and sucrose density gradient techniques under carefully controlled conditions of pH and protease inhibitors, we demonstrate that there is only one form of adenosine 3′,5′-monophosphate-dependent protein kinase in the cytosol fraction of the Blastocladiella emersonii zoospore. If any of these conditions are omitted during extract preparation, one obtains what are apparently multiple forms of the enzyme, which are in reality artifacts due to extensive endogenous proteolytic activity. This endogenous protease is stimulated by alkaline pH and inhibited by antipain. The zoospore protein kinase is similar to type II protein kinase from mammalian cells in several aspects including Chromatographic behavior on DEAE-cellulose column, conditions for subunit dissociation and reassociation, as well as the molecular weight value of the regulatory subunit.