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Biomedical subjects

J E Suarez

Publications and source records attributed to J E Suarez.

16 recordsLinked to original sources
PubMed2001

Plasmodium falciparum acid basic repeat antigen (ABRA) peptides: erythrocyte binding and biological activity.

Non overlapping 20-mer peptides, covering the complete sequence of acid basic repeat antigen (ABRA) of Plasmodium falciparum, were synthesised and tested in binding assays to erythrocytes. Five peptides localised in the N-terminal region coded 2148 (121LQSHKKLIKALKKNIESYQN(140)), 2149 (141KKHLIYKNKSYNPLLLSCVK(160)), 2150 (161KMNMLKENVDYIQKNQNLFK(180)), 2152 (201YKSQGHKKETSQNQNENNDN(220)) and 2153 (221QKYQEVNDEDDVNDEEDTND(240)) specifically bind to erythrocytes. These peptides bind independently of the peptide and erythrocyte charge, with high affinity (Kd between 70 and 180 nM) and the hydrophobic interaction is important for this binding ( approximately 30% hydrophobic critical residues). These results allow us define a specific erythrocyte binding region (residues 121-240), which may bound to at least three different binding sites on erythrocytes. Peptide 2153 shares the underlined sequence 221QKYQEVNDEDDVNDEEDTND(240) with an earlier 18-mer peptide recognised by human exposed sera. Peptides number 2148 and 2149 in vitro inhibit erythrocyte invasion by merozoites. We found that 2149 peptide and some of its glycine analogues show specific haemolytic and/or antimicrobial activity. We discuss a possible role of ABRA or its regions in the merozoite invasion of erythrocyte.

Amino Acid Sequence↗
PubMed2001

Plasmodium falciparum circumsporozoite (CS) protein peptides specifically bind to HepG2 cells.

Hepatocyte invasion by malaria parasites is mediated by specific molecular interactions. Several lines of evidence suggest the importance of the surface plasmodial circumsporozoite (CS) protein in the sporozoite invasion of hepatocytes. Identification of the sequences involved in binding to hepatocytes is an important step towards understanding the structural basis for the sporozoite-hepatocyte interaction. In this study, binding assays between Plasmodium falciparum CS peptides and HepG2 cells were performed. Fifteen overlapping residue 20 mer long peptides, spanning the entire CS sequence, were tested in HepG2 cell binding assays. Five High Binding Activity Peptides (HBAPs) to HepG2 cells were identified: 4593, (NANPNANPNANP); 4383, (NSRSLGENDDGNNEDNEKLR); 4388, (GNGQGHNMPNDPNRNVDENA); 4389, (HNMPNDPNRNVDENANANSA) and 4390, (DPNRNVDENANANSAVKNNN). The HBAP HepG2 interaction is independent of charge and amino-acid composition, but sequence dependent. Four HBAPs (4383, 4388, 4389 and 4390) are bound with similar affinity to a 50 kDa molecule. These HBAPs define three Hepatocyte Binding Sequences (HBSs): HBS-1, located between residues 68 and 87 (HBAP 4383); HBS-11, the repeat NANP region (HBAP 4593), for which anti repeat antibodies are able to specifically inhibit sporozoite invasion of hepatocytes have been reported; and HBS-111, between residues 286 and 315 (HBAPs 4388, 4388 and 4390), respectively. Interestingly, HBS 111 carries two earlier-reported B-epitopes (underlined) in peptides 4388, 4389 and 4390 (GNGQGHNMPNDPNRNVD ENANANSAVKNN) in its sequence. The HBSs reported here show lesser interspecie-variability than the entire protein in species invading the same kind of hepatic cells. This data supports these HBSs' important role in CS-protein function; they could be used as ligand by the sporozoite to invade hepatic cells.

Amino Acid Sequence↗
PubMed2000

Plasmodium falciparum AMA-1 erythrocyte binding peptides implicate AMA-1 as erythrocyte binding protein.

The role of AMA-1 during merozoite invasion has not yet been determined. However, reported experimental evidence suggests that this protein can be used, in particular as erythrocyte-binding protein, since, Fab fragments against this protein are able to block merozoite invasion. Using a previously described methodology, eight peptides with high binding activity to human erythrocyte, scattered along the different domains and having around 130 nM affinity constants, were identified in the Plasmodium falciparum AMA-1 protein. Their binding activity was sialic acid independent. Some of these peptides showed homology with the erythrocyte binding domains of one of the apical organelle protein family, MAEBL, identified in rodent malarial parasites. One of these peptides shares amino acid sequence with a previously reported B-cell epitope which induces antibodies to block parasite growth. The critical residues were identified for erythrocyte binding conserved peptides 4313 (DAEVAGTQYRLPSGKCPVFG), 4321 (VVDNWEKVCPRKNLQNAKFG), 4325 (MIKSAFLPTGAFKADRYKSH) and 4337 (WGEEKRASHTTPVLMEKPYY). All conserved peptides were able to block merozoite invasion of new RBC and development, suggesting that these peptides are involved in P. falciparum invasion.

Amino Acid Sequence↗
PubMed1996

Identification of Plasmodium falciparum MSP-1 peptides able to bind to human red blood cells.

To determine amino acid sequences of the Plasmodium falciparum MSP-1 protein that interact with red blood cell membranes in a specific receptor-ligand interaction, 78 sequential peptides, 20 amino acids long and spanning the entire length of the molecule, were synthesized and analysed with a specific binding assay developed for this purpose. Results show that peptides based on conserved and dimorphic regions of MSP-1, interact with human red blood cells (RBCs). This interaction occurs predominantly with peptides contained within the MSP-1 proteolytic fragments of 83 kDa, 38 kDa, 33 kDa and 19 kDa. Affinity constants of these peptides were between 140 and 250 nM. Peptide-RBC binding post enzyme treatment showed that the RBC receptors are not sialic acid dependent and appear to be proteic in nature. Some of these peptides inhibited merozoite invasion of RBCs yet did not inhibit intraerthrocytic development. These peptides, in conjunction with those from other merozoite surface proteins, may be used to rationally design a second generation of synthetic peptide-based malaria vaccines.

Amino Acid Sequence↗
PubMed1996

Bactericidal mode of action of plantaricin C.

Plantaricin C is a bacteriocin produced by Lactobacillus plantarum LL441 that kills sensitive cells by acting on the cytoplasmic membrane. In contrast to its lack of impact on immune cells, plantaricin C dissipates the proton motive force and inhibits amino acid transport in sensitive cells. In proteoliposomes, plantaricin C dissipates the transmembrane electrical potential, and in liposomes, it elicits efflux of entrapped carboxy-fluorescein. It is concluded that plantaricin C is a pore-forming bacteriocin that functions in a voltage-independent manner and does not require a specific protein receptor in the target membrane.

Journal Article↗
PubMed1985

Morphological characteristics of colony development in Micromonospora chalcea.

Colonies of Micromonospora chalcea display two types of mycelia. Vegetative mycelia are nonbranched, have a propensity for parallel disposition, and possess infrequent septa. The reproductive mycelia develop on their apical portion and are characterized by branching, frequent septation, and bearing of spores that are either sessile or at the end of small sporophores.

Micromonospora↗
PubMed1983

Pattern of protein degradation during germination of Streptomyces antibioticus spores.

The pattern of protein degradation during germination of Streptomyces antibioticus spores was studied by the pulse and chase technique. Two different protein fractions were found. First, a fraction of the proteins synthesized during the darkening process (20-30%) was quickly degraded in the 30 min following the labelling period. This rapid protein degradation was partially inhibited by protease inhibitors: p-chloromercuribenzoic acid, phenylmethylsulphonylfluoride, and o-phenanthroline. Second, the remaining 70-80% and the entire protein population formed during spore swelling and germ tube emergence were degraded with a lower and constant rate (3.3-6.0% /h). A stable mRNA fraction of the dormant spores was translated upon incubation of the spores in a minimal synthetic medium (MSM) or in distilled water. However, the degradation of these proteins did not occur unless the spores were then incubated in the MSM. A strong correlation between the degradation pattern of these proteins and that of those quickly degraded at the beginning of germination was observed. Protease activity in cell-free extracts of dormant spores was detected. Inhibition studies suggest the presence of serine, thiol, and metalloproteases. The protease activity, using casein as substrate, remained constant during the darkening process and started to increase progressively from the beginning of spore swelling.

Bacterial Proteins↗
PubMed1981

Restriction mapping of the DNA of the Streptomyces temperate phage phi C31 and its derivatives.

DNA of phi C31 propagated on Streptomyces lividans 66 contained no sites for the restriction enzymes BamHI, SalPI (=PstI) and XhoI; one for XbaI; three for HpaI; five for ClaI and KpnI; six for EcoRI; about 13 for HindIII; about 14 for BclI; and more than 15 for FspAI, HgiAI, SacI, SalGI and SmaI. A complete map of 20 sites (XbaI, HapI, ClaI, KpnI and EcoRI) was obtained using partial digestion and double digestion of DNA of the wild-type and deletion and insertion mutants. The total molecular size was estimated to be 41.2 kb.

Bacteriophages↗
PubMed1981

Dispensable sequences and packaging constraints of DNA from the Streptomyces temperate phage phi C31.

Deletion mutants of the temperate Streptomyces phage phi C31 were selected by two methods: resistance to the chelating agent sodium pyrophosphate, and plating of a phi C31::pBR322 hybrid phage on Streptomyces albus G to obtain large plaque mutants. The deletions defined a 7.7 kilobase (kb) segment of the phi C31 genome which is inessential for plaque formation, in addition to a shorter segment including the repressor gene. Analysis of deletions and insertions suggested that the minimum size of the phi C31 genome allowing plaque formation is 37.5 kb (91% of the wild-type length of 41.2 kb), and the maximum is at least 42.4 kb (103%). These results indicate that it should be possible to introduce up to 10 kb of foreign DNA into a suitably developed phi C31 vector.

Bacteriophages↗
PubMed1980

DNA cloning in Streptomyces: a bifunctional replicon comprising pBR322 inserted into a Streptomyces phage.

The Gram-positive, mycelial, differentiating streptomycetes are responsible for the production of many important antibiotics. The availability of gene cloning systems in this microbial group would have many industrial applications besides allowing more penetrating study of the genetics of Streptomyces coelicolor A3(2) (which, as the best understood streptomycete genetically, serves as a model for much other Streptomyces genetics). Recent successes (see previous paper) in introducing Streptomyces DNA into S. coelicolor and Streptomyces lividans on plasmid vectors would be nicely complemented by the availability of Streptomyces bacteriophage vectors (discussed in ref. 5): for example, many phages have wide and easily defined host ranges; heat-inducible prophages might be used to give high copy number of cloned DNA; efficient phage promoters might be used to increase gene expression; there may be differential stabilities for particular DNA sequences cloned in plasmids vis-à-vis phages; selective insertion of DNA, utilizing packaging constraints, may be possible with phages; and in situ hybridization of radioactive probes to DNA in plaques is likely to be simple. We describe here the use of the moderately wide host range temperate phage, phi C31, for this purpose.

Bacteriophages↗
PubMed1980

Germination of spores of Micromonospora chalcea: physiological and biochemical changes.

Germinating spores of Micromonospora chalcea pass through three morphological stages: darkening, swelling and germ tube emergence. The process of germination has pH and temperature optima of 8.0 and 40 degrees C, respectively, and is not affected by activation treatments. Darkening, accompanied by a loss of heat resistance and refractility and a decrease in absorbance of the dormant spores, needs only energy, which can be obtained from endogenous sources, and exogenous cations. Agents that inhibit ATP formation block darkening, but inhibitors of macromolecular synthesis do not affect it. Swelling requires exogenous carbon but not nitrogen sources and is characterized by a 30 to 40% increase in spore diameter. RNA synthesis is necessary for swelling and inhibitors of protein synthesis delay this process. During this stage, maximum respiratory, cytochrome oxidase and catalase activities are reached. DNA synthesis starts at the beginning of germ tube emergence. This final stage requires both exogenous carbon and nitrogen sources and the sequence of macromolecular synthesis is RNA, protein and, finally, DNA. Rifampicin, streptomycin and mitomycin C prevent protein and DNA synthesis regardless of when added during germination. Rifampicin inhibits [3H]uridine incorporation immediately but there is a delay of about 160 min in the case of streptomycin or mitomycin C.

Bacterial Proteins↗
PubMed1980

Polyethylene glycol-assisted transfection of Streptomyces protoplasts.

In the presence of polyethylene glycol (concentration optimum 20%), protoplasts of appropriate Streptomyces strains could be transfected by deoxyribonucleic acid (DNA) of five temperate phages (phi C31, VP5, R4, phi 448, and S14) belonging to four different immunity groups. Quantitation of transfection was made possible by plating the transfection mixture with excess uninfected protoplasts in soft agar overlays on protoplast regeneration medium so that plaques were easily detected. Optimum frequencies of transfection in the ranges of 10(-6)/DNA molecule and 10(-5)/viable protoplast were invariably obtained. It appeared that single DNA molecules initiated transfection events, and that the conformation of the DNA (i.e., circular or linear) was not important. Inhibition of transfection by ethylenediaminetetraacetic acid suggested that divalent cations were also observed. A minor subpopulation of protoplasts appeared to be particularly sensitive to transfection (i.e., "competent") in some DNA-host combinations. In such cases the size of this subpopulation was the major limiting factor in obtaining high transfection frequencies. The same protoplast

Bacteriophages↗
PubMedDate not supplied

The expression of Streptomyces and Escherichia coli drug-resistance determinants cloned into the Streptomyces phage phi C31.

Lysogens obtained by infecting Streptomyces albus G with a phi C31-pBR322 chimaeric prophage or its delta W12 deletion derivative had increased tetracycline resistance. The ability of the delta W12 derivative to transduce tetracycline resistance was inactivated by inserting a viomycin resistance determinant (vph) into the BamHI site of the pBR322 tet gene, and restored by excising the vph gene. Another deletion mutant (delta W17) of the chimaera, carrying an intact tet gene, was normally unable to transduce tetracycline resistance. This inability was correlated with the finding, by Southern hybridisation analysis, that the att site required for insertion of phi C31 prophage into the host chromosome was located within the delta W17 deletion. Use of phi C31 lysogenic recipient permitted the integration of the att-deleted phage, presumably by homologous recombination, giving tetracycline-resistant double lysogens. This technique was extended to S. coelicolor A3(2) in the detection of derivatives of the att-deleted phage into which a thiostrepton-resistance determinant (tsr) had been inserted in vitro. Phage released from double lysogens were mainly recombinants. One such recombinant is a PstI vector for DNA cloning, able to accommodate up to 6 kb of introduced DNA.

Bacteriophages↗
PubMedDate not supplied

A GBP 130 derived peptide from Plasmodium falciparum binds to human erythrocytes and inhibits merozoite invasion in vitro.

The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720) which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD) were found to be critical for peptide binding to erythrocytes.

Amino Acid Sequence↗