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Biomedical subjects

C Bloch

Publications and source records attributed to C Bloch.

At least 19 recordsLinked to original sources
PubMed2006

Bradykinin-related peptides from Phyllomedusa hypochondrialis.

Bradykinin related peptides (BRPs) present in the water-soluble secretion and freshly dissected skin fragments of Phyllomedusa hypochondrialis were investigated by mass spectrometry techniques. Eighteen BRPs, along with their post-translational modifications, were characterized in the secretion by de novo MS/MS sequencing and direct MALDI imaging experiments of the frog skin. These molecules revealed strong sequence similarities to the main plasma kinin of some mammals and reptiles. Such a diversity of molecules, within the same peptide family, belonging to a single amphibian species may be related to functional specializations of these peptides and a variety of corresponding receptors that might be present in a number of different predators. Also, a novel analog, [Val]1,[Thr]6-bradykinyl-Gln,Ser had its biological activity positively detected in cell culture expressing the human bradykinin B2 receptor and in guinea pig ileum preparations.

Amino Acid Sequence↗
PubMed2005

Comparison of the partial proteomes of the venoms of Brazilian spiders of the genus Phoneutria.

The proteomes of the venoms of the Brazilian wandering "armed" spiders Phoneutria nigriventer, Phoneutria reidyi, and Phoneutria keyserlingi, were compared using two-dimensional gel electrophoresis. The venom components were also fractionated using a combination of preparative reverse phase HPLC on Vydac C4, analytical RP-HPLC on Vydac C8 and C18 and cation exchange FPLC on Resource S at pH 6.1 and 4.7, or anion exchange HPLC on Synchropak AX-300 at pH 8.6. The amino acid sequences of the native and S-pyridyl-ethylated proteins and peptides derived from them by enzymatic digestion and chemical cleavages were determined using a Shimadzu PPSQ-21(A) automated protein sequencer, and by MS/MS collision induced dissociations. To date nearly 400 peptides and proteins (1.2-27 kDa) have been isolated in a pure state and, of these, more than 100 have had their complete or partial amino acid sequences determined. These sequences demonstrate, as might be expected, that the venoms of P. reidyi and P. keyserlingi (Family: Ctenidae) both contain a similar range of isoforms of the neurotoxins as those previously isolated from P. nigriventer which are active on neuronal ion (Ca(2+), Na(+) and K(+)) channels and NMDA-type glutamate receptors. In addition two new families of small (3-4 kDa) toxins, some larger protein (>10 kDa) components, and two serine proteinases of the venom of P. nigriventer are described. These enzymes may be responsible for some of the post-translational modification observed in some of the venom components.

Amino Acid Sequence↗
PubMed2005

Occurrence of bufotenin in the Osteocephalus genus (Anura: Hylidae).

Bufotenin (5-hydroxy-N,N-dimetyltryptamine) is a tryptamine alkaloid widely spread among anuran families as a component of their chemical defense system, acting as a potent hallucinogenic factor, showing similar activity to LSD upon interaction with the 5HT2 human receptor. This work demonstrates the presence of bufotenin in the skin secretion of three arboreal amphibian species of the Osteocephalus genus (Osteocephalus taurinus, Osteocephalus oophagus and Osteocephalus langsdorffii) from the Amazon and the Atlantic rain forests using RP-HPLC, ESI-MS/MS, UV, IR and multidimensional NMR techniques. To our knowledge, this is the first description of bufotenin in the Osteocephalus genus, so far.

Animals↗
PubMed2005

Purification and characterization of a novel peptide with antifungal activity from Bothrops jararaca venom.

Different peptides have been isolated from a wide range of animal species. It is has become increasingly clear that due to the development of antibiotic-resistant microbes, antibacterial and antifungal peptides have attracted the attention in recent years, in order to find new therapeutic agents. In this work, a novel peptide with high inhibitory activity against fungi growth have been isolated from the venom of the Brazilian snake Bothrops jararaca. A Sephacryl S-100 gel filtration column was employed for further separation of proteins. The FV fraction with high antifungal activity was named Pep5Bj, pooled and submitted to reverse-phase chromatography in HPLC. The fraction containing the isolated peptide inhibited the growth of different phytopathogenic fungi (Fusarium oxysporum and Colletotrichum lindemuthianum) and yeast (Candida albicans and Saccharomyces cerevisiae). The peptide minimal inhibitory concentration is comparable to other known antifungal peptides, like insect defensins and cecropins, found in the last years in a large diversity of animals. We investigate F. oxysporum cells membrane permeabilization using SYTOX Green uptake, an organic compound that fluoresces upon interaction with nucleic acids after penetration in cell with compromised plasma membranes. When viewed under fluorescence optical microscopy, F. oxysporum cells exposed to Pep5Bj display strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. The SYTOX Green data suggested that this effect is related to membrane permeabilization. The molecular masses of this peptide was obtained by MALDI-TOF spectrometry and corresponded to 1370Da.

Animals↗
PubMed2005

Purification of a 6.5 kDa protease inhibitor from Amazon Inga umbratica seeds effective against serine proteases of the boll weevil Anthonomus grandis.

A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.

Chromatography, Ion Exchange↗
PubMed2005

Screening and purification of a novel trypsin inhibitor from Prosopis juliflora seeds with activity toward pest digestive enzymes.

Several pests are capable of decreasing crop production causing severe economical and social losses. Aiming to find novel molecules that could impede the digestion process of different pests, a screening of alpha-amylase and trypsin-like proteinase inhibitors was carried out in Prosopis juliflora, showing the presence of both in dry seeds. Furthermore, a novel trypsin inhibitor, with molecular mass of 13,292 Da, was purified showing remarkable in vitro activity against T. castaneum and C. maculatus.

Chromatography, Affinity↗
PubMed2002

Isolation and partial characterisation of a protein from buck seminal plasma (Capra hircus), homologous to spermadhesins.

Spermadhesins are a family of secretory proteins expressed in the male genital tract of pig, horse and bull. Their function and structure have been widely studied, especially those isolated from boar. However, there are no data concerning spermadhesins isolated from buck. Buck seminal plasma was collected and subjected to ion exchange chromatography on DEAE-Sephacel column followed by chromatography in a C18 column coupled to a HPLC system. The purification of the protein was determined by SDS-PAGE and MALDI-TOF analysis exhibiting a molecular mass of 12.5 KDa and showed to be structurally homologous to spermadhesins from boar and stallion.

Amino Acid Sequence↗
PubMed2002

Purification and characterization of a new lectin from the red marine alga Hypnea musciformis.

A lectin from the red marine alga Hypnea musciformis (HML) was purified by extraction with 20 mM PBS, precipitation with 70% saturated ammonium sulphate, ion-exchange DEAE-Cellulose chromatography and RP-HPLC. The 9.3 kDa polypeptide agglutinates erythrocytes from various sources and shows oligomerization tendencies under certain MALDI-TOF/MS conditions. Preliminary N-terminal sequencing and biological assays strongly suggest that the HML may belong to a new class of algae lectins.

Animals↗
PubMed2001

A novel heterodimeric antimicrobial peptide from the tree-frog Phyllomedusa distincta.

We present here the purification and the analysis of the structural and functional properties of distinctin, a 5.4 kDa heterodimeric peptide with antimicrobial activity from the tree-frog Phyllomedusa distincta. This peptide was isolated from the crude extract of skin granular glands by different chromatographic steps. Its minimal inhibitory concentration was determined against pathogenic Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa strains. Amino acid sequencing and mass spectrometric investigations demonstrated that distinctin is constituted of two different polypeptide chains connected by an intermolecular disulphide bridge. Circular dichroism and Fourier-transformed infrared spectroscopy studies showed that this molecule adopts, in water, a structure containing a significant percentage of anti-parallel beta-sheet. A conformational variation was observed under experimental conditions mimicking a membrane-like environment. Database searches did not show sequence similarities with any known antimicrobial peptides. In the light of these results, we can consider distinctin as the first example of a new class of antimicrobial heterodimeric peptides from frog skin.

Amino Acid Sequence↗
PubMed2000

Fast K(+) currents from cerebellum granular cells are completely blocked by a peptide purified from Androctonus australis Garzoni scorpion venom.

A novel peptide was purified from the venom of the scorpion Androctonus australis Garzoni (abbreviated Aa1, corresponding to the systematic number alpha KTX4.4). It contains 37 amino acid residues, has a molecular mass of 3850 Da, is closely packed by three disulfide bridges and a blocked N-terminal amino acid. This peptide selectively affects the K(+) currents recorded from cerebellum granular cells. Only the fast activating and inactivating current, with a kinetics similar to I(A)-type current, is completely blocked by the addition of low micromolar concentrations (K(i) value of 150 nM) of peptide Aa1 to the external side of the cell preparation. The blockade is partially reversible in our experimental conditions. Aa1 blocks the channels in both the open and the closed states. The blockage is test potential independent and is not affected by changes in the holding potential. The kinetics of the current are not affected by the addition of Aa1 to the preparation; it means that the block is a simple 'plugging mechanism', in which a single toxin molecule finds a specific receptor site in the external vestibule of the K(+) channel and thereby occludes the outer entry to the K(+) conducting pore.

Amino Acid Sequence↗
PubMed2000

Molecular characterization of a new arcelin-5 gene.

Arcelins are insecticidal proteins found in some wild accessions of the common bean, Phaseolus vulgaris. They are grouped in six allelic variants and arcelin-5 is the variant with the highest inhibitory effect on the development of Zabrotes subfasciatus larvae. Characterization of the protein and its genes resulted in the identification of three polypeptides and the isolation of two genes that encode the Arc5a and Arc5b polypeptides. Here we describe a new gene, Arc5-III. The protein it encodes has 81% amino acid identity with the derived amino acid sequences of Arc5-I and Arc5-II. The Arc5-III gene is highly expressed in developing seeds and at a much lower level in roots. Data obtained by a combination of two-dimensional gel electrophoresis, protein sequencing and MALDI-TOF mass spectrometry analysis support the conclusion that Arc5-III encodes a polypeptide present in Arc5c band. Using ion-exchange chromatography, three fractions containing arcelin-5 polypeptides were eluted by increasing the salt concentration. The three fractions contain various amounts of the three arc-5 polypeptides and inhibit the growth of Zabrotes subfasciatus larvae differentially, suggesting differences in insecticidal activity among the arcelin-5 isoforms.

Amino Acid Sequence↗
PubMed2000

The amino acid sequence of the agglutinin isolated from the red marine alga Bryothamnion triquetrum defines a novel lectin structure.

The primary structure of a lectin isolated from the red alga Bryothamnion triquetrum was established by combination of Edman degradation of sets of overlapping peptides and mass spectrometry. It contains 91 amino acids and two disulphide bonds. The primary structure of the B. triquetrum lectin does not show amino acid sequence similarity with known plant and animal lectin structures. Hence, this protein may be the paradigm of a novel lectin family.

Algal Proteins↗
PubMed2000

Activity of wheat alpha-amylase inhibitors towards bruchid alpha-amylases and structural explanation of observed specificities.

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Five alpha-amylase inhibitors of the structural 0.19 family were isolated from wheat kernels, and assayed against three insect alpha-amylases and porcine pancreatic alpha-amylase, revealing several intriguing differences in inhibition profiles, even between proteins sharing sequence identity of up to 98%. Inhibition of the enzyme from a commercially important pest, the bean weevil Acanthoscelides obtectus, is observed for the first time. Using the crystal structure of an insect alpha-amylase in complex with a structurally related inhibitor, models were constructed and refined of insect and human alpha-amylases bound to 0.19 inhibitor. Four key questions posed by the differences in biochemical behaviour between the five inhibitors were successfully explained using these models. Residue size and charge, loop lengths, and the conformational effects of a Cys to Pro mutation, were among the factors responsible for observed differences in specificity. The improved structural understanding of the bases for the 0.19 structural family inhibitor specificity reported here may prove useful in the future for the rational design of inhibitors possessing altered inhibition characteristics.

Amino Acid Sequence↗
PubMed2000

Depth ionization curves for an unmodulated proton beam measured with different ionization chambers.

Differences in depth dose curves for a 78 MeV unmodulated proton beam were measured with four commercially available ionization chambers. Measurements were performed both in water and in a commercially available solid water phantom. A depth scaling factor (Cpl) was determined from the ratio of depths distal to the Bragg peak where the dose is reduced to 80% of the maximum dose in water and in the solid water phantom. This scaling factor provides good agreement between the ionization curves at all depths in water and in this solid water phantom. There is no significant difference in the value of the depth scaling factor between the ratios (R80wat/R80med) and (R50wat/R50med), or (R100wat/R100med) for 78 MeV unmodulated proton beams. The depth scaling factor for this commercially available solid water phantom is 1.023. An effective point of measurement for a cylindrical ionization chamber was found to be slightly greater than the 50% of the cavity radius proposed by the AAPM-TG25 dosimetry protocol for electron beams and amounts to 62.5% of the cavity radius of cylindrical ionization chambers. The ion collection efficiency, Pion, and the polarity correction factor, Ppol, for all the ionization chambers studied are within 1% and 0.4% of unity, respectively. Absolute doses measured with a parallel plate ionization chamber in water and in the solid water phantom show that the doses measured in the solid water phantom are 1.4% +/- 0.5% lower than in water. The dose rate dependent response of the beam line monitor chamber was also investigated. Agreement between all the chambers was within 1.5% at the dose rates studied but the results showed that all four ionization chambers are less dose rate dependent than the monitor chamber.

Algorithms↗
PubMed2000

Characterization of a methionine-rich protein from the seeds of Cereus jamacaru mill. (Cactaceae).

We describe here the isolation and characterization of a major albumin from the seeds of Cereus jamacaru (Cactaceae), to which we gave the trivial name of cactin. This protein has a molecular mass of 11.3 kDa and is formed by a light chain (3.67 kDa) and a heavy chain (7.63 kDa). This protein was isolated using a combination of gel filtration chromatography and reverse-phase HPLC. The amino acid composition of cactin was determined and found to resemble that of the 2S seed reserve protein from the Brazil nut, a protein remarkable for its high methionine content. The usefulness of cactin as a molecular marker in the taxonomy of the Cactaceae is discussed.

Albumins↗
PubMed1999

Proton dosimetry intercomparison based on the ICRU report 59 protocol.

BACKGROUND AND PURPOSE: A new protocol for calibration of proton beams was established by the ICRU in report 59 on proton dosimetry. In this paper we report the results of an international proton dosimetry intercomparison, which was held at Loma Linda University Medical Center. The goals of the intercomparison were, first, to estimate the level of consistency in absorbed dose delivered to patients if proton beams at various clinics were calibrated with the new ICRU protocol, and second, to evaluate the differences in absorbed dose determination due to differences in 60Co-based ionization chamber calibration factors. MATERIALS AND METHODS: Eleven institutions participated in the intercomparison. Measurements were performed in a polystyrene phantom at a depth of 10.27 cm water equivalent thickness in a 6-cm modulated proton beam with an accelerator energy of 155 MeV and an incident energy of approximately 135 MeV. Most participants used ionization chambers calibrated in terms of exposure or air kerma. Four ionization chambers had 60Co-based calibration in terms of absorbed dose-to-water. Two chambers were calibrated in a 60Co beam at the NIST both in terms of air kerma and absorbed dose-to-water to provide a comparison of ionization chambers with different calibrations. RESULTS: The intercomparison showed that use of the ICRU report 59 protocol would result in absorbed doses being delivered to patients at their participating institutions to within +/-0.9% (one standard deviation). The maximum difference between doses determined by the participants was found to be 2.9%. Differences between proton doses derived from the measurements with ionization chambers with N(K)-, or N(W) - calibration type depended on chamber type. CONCLUSIONS: Using ionization chambers with 60Co calibration factors traceable to standard laboratories and the ICRU report 59 protocol, a distribution of stated proton absorbed dose is achieved with a difference less than 3%. The ICRU protocol should be adopted for clinical proton beam calibration. A comparison of proton doses derived from measurements with different chambers indicates that the difference in results cannot be explained only by differences in 60Co calibration factors.

Algorithms↗
PubMed1999

Antimicrobial peptides from the Brazilian frog Phyllomedusa distincta.

Different peptides were purified by chromatographic procedures from the skin-secretory glands of the frog Phyllomedusa distincta. These are the first peptides reported from this frog species. Their primary structure was determined by a combination of automated Edman degradation and mass spectrometry. Peptide Q2 contains 25 amino acid residues, peptide Q1 and L have 28 each, peptide M contains 31, and peptide K has 33 amino acid residues. They all showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria, presenting minimal inhibitory concentrations from 0.6 to 40 microM, when tested against Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Peptides K, L, and Q1 were chemically synthesized and shown to be active.

Amino Acid Sequence↗