Biomedical subjects

L J Greene

Publications and source records attributed to L J Greene.

At least 19 recordsLinked to original sources

PubMed2002

Epidemiological data of patients hospitalized with burns and other traumas in some cities in the southeast of Brazil from 1991 to 1997.

This retrospective analysis of burn patients and victims of other forms of trauma from Ribeirão Preto and nearby cities admitted to hospitals in the city of Ribeirão Preto, São Paulo, Brazil, was carried out to determine the frequency of injuries of all types in order to identify the extent of the problem of burns relative to other forms of trauma. Data concerning 921 patients with burns and 60,344 patients with other traumatic injuries hospitalized during the period from 1991 to 1997 are described. Burns corresponded to 1.5% of the total number of traumatic injuries. When data are reported as absolute numbers or as incidence rate of hospitalized burn patients, burns were two times more frequent among men in most age groups. The case fatality ratio due to burns was 8.4% (77 deaths among 921 patients), with a rate of 6.4% for men and 12.2% for women. The case fatality ratio was higher among women than men regardless of the city of residence. The case fatality ratio was 3.2 and 4.4 times greater for men and women burn victims from other towns than for burn victims from Ribeirão Preto, indicating the need for additional equipment and training of medical and paramedical personnel in the initial measures to be taken with burn patients.

Adolescent↗

PubMed2001

Refolding and purification of Bothropstoxin-I, a Lys49-phospholipase A2 homologue, expressed as inclusion bodies in Escherichia coli.

Hydrolysis of phospholipids by Group II phospholipase A2 enzymes involves a nucleophilic attack on the sn-2 ester bond by the His48 residue and stabilization of the reaction intermediate by a Ca2+ ion cofactor bound to the Asp49 residue in the protein active site region. Bothropstoxin-I (BthTX-I) is a PLA(2) variant present in the venom of the snake Bothrops jararacussu which shows a Asp49 to Lys substitution and which lacks hydrolytic activity yet damages artificial membranes by a noncatalytic Ca2+-independent mechanism. In order to better characterize this unusual mechanism of membrane damage, we have established an expression system for BthTX-I in Escherichia coli. The DNA-coding sequence for BthTX-I was subcloned into the vector pET11-d, and the BthTX-I was expressed as inclusion bodies in E. coli BL21(DE3). The native BthTX-I contains seven disulfide bonds, and a straightforward protocol has been developed to refold the recombinant protein at high protein concentration in the presence of surfactants using a size-exclusion chromatography matrix. After refolding, recovery yields of 2.5% (corresponding to 4-5 mg of refolded recombinant BthTX-I per liter of bacterial culture) were routinely obtained. After refolding, identical fluorescent and circular dichroism spectra were obtained for the recombinant BthTX-I compared to those of the native protein. Furthermore, the native and refolded recombinant protein demonstrated identical membrane-damaging properties as evaluated by measuring the release of an entrapped fluorescent marker from liposomes.

Amino Acid Substitution↗

PubMed2001

Toxoplasma gondii micronemal protein MIC1 is a lactose-binding lectin.

Host cell invasion by Toxoplasma gondii is a multistep process with one of the first steps being the apical release of micronemal proteins that interact with host receptors. We demonstrate here that micronemal protein 1 (MIC1) is a lactose-binding lectin. MIC1 and MIC4 were recovered in the lactose-eluted (Lac(+)) fraction on affinity chromatography on immobilized lactose of the soluble antigen fraction from tachyzoites of the virulent RH strain. MIC1 and MIC4 were both identified by N-terminal microsequencing. MIC4 was also identified by sequencing cDNA clones isolated from an expression library following screening with mouse polyclonal anti-60/70 kDa (Lac(+) proteins) serum. This antiserum localized the Lac(+) proteins on the apical region of T. gondii tachyzoites by confocal microscopy. The Lac(+) fraction induced hemagglutination (mainly type A human erythrocytes), which was inhibited by beta-galactosides (3 mM lactose and 12 mM galactose) but not by up to 100 mM melibiose (alpha-galactoside), fucose, mannose, or glucose or 0.2 mg/ml heparin. The lectin activity of the Lac(+) preparation was attributed to MIC1, because blotted MIC1, but not native MIC4, bound human erythrocyte type A and fetuin. The copurification of MIC1 and MIC4 may have been due to their association, as reported by others. These data suggest that MIC1 may act through its lectin activity during T. gondii infection.

Amino Acid Sequence↗

PubMed2000

Purification and characterization of the fimbria F18ac (2134P) isolated from enterotoxigenic Escherichia coli (ETEC).

The adhesin F18ac purified on Sepharose CL 4B column chromatography and SDS-PAGE stained with Coomassie Blue and Western blotting using specific anti-F18ac serum presented one band of approximately 17kDa. Gold immunolabeling revealed that the adhesin F18ac has a fimbrial structure on the bacterial surface. The first 27 amino acid residues of the N-terminal portion of the adhesin F18ac, showed 92.5% homology (25 amino acids) with the F107 (F18ab) fimbriae.

Adhesins, Escherichia coli↗

PubMed2000

The Rubino test for leprosy is a beta2-glycoprotein 1-dependent antiphospholipid reaction.

We describe the isolation and identification of three components required for the Rubino reaction (RR), which is the rapid sedimentation of formalinized sheep red-blood cells (SRBC) initiated by serum from leprosy patients with defective Mycobacterium leprae-specific cell immunity. The Rubino reaction factor (RRF) required for this phenomenon, previously identified as an immunoglobulin M (IgM), was purified from leprosy patient serum by adsorption to formalinized SRBC. Purified RRF IgM, when added to formalinized SRBC, did not produce a positive RR. However, when the contact was carried out in the presence of normal human serum (NHS), cells rapidly sedimented. The purified cofactor from NHS contained two components of 70 000 and 50 000 molecular weight (MW), as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The latter was recognized by the RRF IgM on immunoblot and its N-terminal sequence indicated that it was beta2-glycoprotein 1 (beta2-GP1), an anionic phospholipid-binding protein. Methanol-treated formalinized SRBC did not support the RR. Thin-layer chromatography of an extract of membranes indicated that the SRBC ligand was a cell-surface phospholipid. Cardiolipin inhibited the RR. These data demonstrate that the RR involves a trimolecular interaction in which IgM, beta2-GP1 and an SRBC phospholipid participate. By analogy with the antiphospholipid antibodies (anti-PL) that occur in autoimmune processes, serum samples from 29 systemic lupus erythematosus patients with high levels of anticardiolipin antibodies were submitted to the RR. A positive RR was obtained for 45% (13 of 29 patients). These results modify the paradigm of the absolute specificity of the RR for leprosy and demonstrate that RRF IgM is a beta2-GP1-dependent anti-PL.

Animals↗

PubMed1999

Characterization of a tissue kallikrein inhibitor isolated from Bauhinia bauhinioides seeds: inhibition of the hydrolysis of kininogen related substrates.

Trypsin inhibitors were purified from a saline extract of Bauhinia bauhinioides seeds by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q ion-exchange chromatography or, alternatively, by affinity chromatography on trypsin-Sepharose. Both B. bauhinioides isolated inhibitors, BbTI-I and BbTI-II, inhibit trypsin being the dissociation constant 0.6 and 0.36 nM, respectively. BbTI-II only inhibits porcine pancreatic kallikrein hydrolysis of H-Pro-Phe-Arg-AMC (Ki 2.0 nM); the bradykinin-containing sequence LGMISLMKRPPGFSPFRSSRI-NH2 and the two kininogen related flanking quenched substrates Abz-MISLMKRP-EDDnp (Ki 2.0 nM) and Abz-FRSSRQ-EDDnp (Ki 2.5 nM).

Amino Acid Sequence↗

PubMed1999

Effect of chronic angiotensin converting enzyme inhibition on angiotensin I and bradykinin metabolism in rats.

We determined the effect of chronic administration of the angiotensin converting enzyme (ACE) inhibitor, enalapril, on the in vivo pulmonary inactivation of bradykinin (BK) and conversion of angiotensin I (Ang I). In addition we assessed whether chronic ACE inhibition influenced the activity of prolylendopeptidase (PEP), which metabolizes Ang I to generate angiotensin-(1-7) (Ang-[1-7]) and inactivates BK. Male Wistar rats were treated orally with enalapril (10 mg/kg once a day) for 7 to 15 days (n = 20) and 21 to 30 days (n = 11). Vehicle-treated rats (7 to 30 days, n = 11) were used as controls. Pulmonary inactivation of BK and conversion of Ang I were determined in conscious enalapril- or vehicle-treated rats before and after intravenous administration of the ACE inhibitor enalaprilat (MK-422, 10 mg/kg). Pulmonary inactivation of BK (%) was determined by comparing equipotent doses of BK injected by the intravenous and intraaortic routes, and Ang I conversion (%) by comparing the pressor effect of Ang I and Ang II injected intravenously. PEP-like activity in plasma and lung homogenates was determined fluorometrically using the synthetic substrate Suc-Gly-Pro-MCA. In control rats, pulmonary BK inactivation averaged 97.6% +/-0.54%. Acute ACE inhibition with MK-422 reduced BK inactivation to 42.0% +/- 2.7%. However, in rats treated chronically with enalapril, BK inactivation was increased as compared with acute ACE inhibition, averaging 58.8% +/- 3.7% at 7 to 15 days and 58.8% +/- 4.5% at 21 to 30 days of treatment. Intravenous administration of MK-422 to the enalapril-treated rats did not return the increased BK inactivation to the level observed during acute ACE inhibition. In contrast, Ang I conversion was significantly reduced from 46.7% +/- 6.5% to 0.9% +/-0.2% by MK-422, and this inhibition remained essentially unchanged during chronic treatment. PEP-like activity in plasma and lung homogenates of control rats was 4.4 +/- 0.3 nmol MCA/min/mL and 11.4 +/- 0.9 nmol MCA/min/mg protein, respectively. After chronic treatment with enalapril there was a progressive increase of PEP-like activity in both plasma and lung, which after 21 to 30 days of treatment averaged 10.7 +/- 1.7 nmol MCA/min/mL and 29.2 +/- 2.8 nmol MCA/min/mg protein, respectively. These data indicate that chronic ACE blockade induces alternative BK-inactivating mechanisms and increases Ang-(1-7)-generating mechanisms.

Angiotensin I↗

PubMed1999

KM+, a mannose-binding lectin from Artocarpus integrifolia: amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the beta-prism fold.

The complete amino acid sequence of the lectin KM+ from Artocarpus integrifolia (jackfruit), which contains 149 residues/mol, is reported and compared to those of other members of the Moraceae family, particularly that of jacalin, also from jackfruit, with which it shares 52% sequence identity. KM+ presents an acetyl-blocked N-terminus and is not posttranslationally modified by proteolytic cleavage as is the case for jacalin. Rather, it possesses a short, glycine-rich linker that unites the regions homologous to the alpha- and beta-chains of jacalin. The results of homology modeling implicate the linker sequence in sterically impeding rotation of the side chain of Asp141 within the binding site pocket. As a consequence, the aspartic acid is locked into a conformation adequate only for the recognition of equatorial hydroxyl groups on the C4 epimeric center (alpha-D-mannose, alpha-D-glucose, and their derivatives). In contrast, the internal cleavage of the jacalin chain permits free rotation of the homologous aspartic acid, rendering it capable of accepting hydrogen bonds from both possible hydroxyl configurations on C4. We suggest that, together with direct recognition of epimeric hydroxyls and the steric exclusion of disfavored ligands, conformational restriction of the lectin should be considered to be a new mechanism by which selectivity may be built into carbohydrate binding sites. Jacalin and KM+ adopt the beta-prism fold already observed in two unrelated protein families. Despite presenting little or no sequence similarity, an analysis of the beta-prism reveals a canonical feature repeatedly present in all such structures, which is based on six largely hydrophobic residues within a beta-hairpin containing two classic-type beta-bulges. We suggest the term beta-prism motif to describe this feature.

Amino Acid Sequence↗

PubMed1999

New approaches to the treatment of phenylketonuria.

Phenylketonuria (PKU) is the most common of all aminoacidopathies and is caused by autosomal recessive deficiency of the hepatic phenylalanine hydroxylase system. The diagnosis of PKU should be multifactorial and based on a protein overload test that reveals increased plasma phenylalanine levels during the ingestion of a normal diet, a phenylalanine tolerance test, and in vitro and in vivo activity of the liver enzyme. An individualized diagnosis that characterizes the severity of the disease in each patient provides objective and effective criteria for the dietary treatment of each particular case.

Animals↗

PubMed1998

The analgesic activity of crotamine, a neurotoxin from Crotalus durissus terrificus (South American rattlesnake) venom: a biochemical and pharmacological study.

Crotamine, a 4.88 kDa neurotoxic protein, has been purified to apparent homogeneity from Crotalus durissus venom by gel filtration on Sephadex G-75. When injected (i.p. or s.c.) in adult male Swiss mice (20-25 g), it induced a time-dose dependent analgesic effect which was inhibited by naloxone, thus suggesting an opioid action mechanism. When compared with morphine (4 mg/kg), crotamine, even in extremely low doses (133.4 microg/kg, i.p., about 0.4% of a LD50 is approximately 30-fold more potent than morphine (w/w) as an analgesic. On a molar basis it is more than 500-fold more potent than morphine. It is also much more potent than the lower molecular weight crude fractions of the same venom. The antinociceptive effects of crotamine and morphine were assayed by the hot plate test and by the acetic acid-induced writhing method. Therefore, both central and peripheral mechanisms should be involved. Histopathological analysis of the brain, liver, skeletal muscles, stomach, lungs, spleen, heart, kidneys and small intestine of the crotamine injected mice did not show any visible lesion in any of these organs by light microscopy. Since crotamine accounted for 22% (w/w) of the desiccated venom, it was identified as its major antinociceptive low molecular weight peptide component.

Analgesics↗

PubMed1998

Epidemiological data and mortality rate of patients hospitalized with burns in Brazil.

This retrospective analysis of burn patients in a University Hospital in the state of São Paulo, Brazil, was carried out to characterize this population and to identify the factors that affect the mortality rate. All patients hospitalized from January 1990 to April 1995 (n = 229, 3.6 patients/month) and who terminated treatment were included. Of these, 80.8% (185 patients) were hospitalized within 24 h of the burn. Occupational and/or domestic accidents were responsible for most of the burns (78.6%), which were mainly caused by a direct flame (71.2%). with alcohol being the flammable fluid most frequently used. The average patient treated at the center was a male of 9 years of age or less with 20-40% burned body surface, who received care within 24 h after suffering an accidental alcohol burn and who was hospitalized for < or =30 days. The mortality rate was 18.8% for all patients and increased with burned body surface and age, and for suicide patients. Suicide attempts for all patients > or = 18 years were the cause of 46 .5% (20/43) of the burns involving women and of 8.9% (8/90) of the burns involving men. The mortality rate was significantly higher for self-inflicted burns (42.9%) than for accidental burns (20.2%).

Adolescent↗

PubMed1998

Pharmacological nutrition after burn injury.

Burn patients develop pathophysiological alterations, which include extensive nitrogen loss, malnutrition, markedly increased metabolic rate and immunologic deficiency. This predisposes burn patients to frequent infections, poor wound healing, increased length of hospitalization and increased mortality. The nutritional support requires high protein and high energy diets preferably administered enterally soon after injury. The effects of increased dietary components such as glutamine, arginine and (n-3) fatty acids and related compounds have been evaluated in burn victims. These components, when supplied in quantities two to seven times of those in normal diets of healthy persons, appear to have beneficial pharmacological effects on the pathophysiological alterations associated with burns. However, the efficacy of immune-enhancing diets remains to be convincingly shown.

Arginine↗

PubMed1996

Amino acid sequence of a new 2S albumin from Ricinus communis which is part of a 29-kDa precursor protein.

The isolation and sequence determination of a new 2S albumin storage protein from Ricinus communis seeds denoted 2S ASP-Ib are described. The fragment approach using selective enzymatic cleavage, Edman degradation, and mass spectrometry was used to demonstrate that the 11-kDa heterodimer protein linked by disulfide bridges has the following structure: short chain, GEREGSSSQQCRQEVQRKDLSSCERYLRQSSS; long chain, <QQQESQQLQQCCNQVKQVRDECQCEAIKYIAEDQIQQGQLHGEESERVAQRAGEIVSSCGVRCMR . The molecular weight of the intact protein, 11,140 +/- 2, determined by matrix-assisted laser desorption mass spectrometry was consistent with the assigned structure. The S- and L-chains are identical to residues 18-49 and 66-130 of the precursor protein predicted by S. D. Irwin, J. N. Keen, J. B. C. Findlay, and J. M. Lord [(1990) Mol. Gen. Genet. 222, 400-408], on the basis of the structure of a cDNA isolated using probes based on the sequence of another 2S albumin, described by F. S. Sharief and S. S. L. Li [(1982) J. Biol. Chem. 257, 14753-14759], which we denote 2S ASP-Ia. Three of the four termini could have been produced by posttranslational processing by endopeptidase(s) and carboxypeptidase(s) which utilized basic residues as the cleavage sites. Mass spectrometric evidence suggested that the protein presented microheterogeneity at its termini, i.e., truncated forms presumably due to processing heterogeneity. The present characterization of the 2S ASP-Ib protein, the second 2S albumin from Ricinus communis seeds, demonstrates that the 237-residue precursor protein codes for two different heterodimer proteins containing 97 and 99 residues each. This system should be useful for studying the posttranslational processing of plant storage proteins.

Amino Acid Sequence↗

PubMed1996

Effect of acute volume expansion associated with salt load on the profile of plasma angiotensins in rats.

We measured the changes produced in renin and the peptide components of the circulating renin-angiotensin system by acute volume expansion alone or associated with salt load in rats. With both maneuvers, there was a decrease of plasma renin activity (PRA) and plasma angiotensin (Ang) concentrations, but the changes were more pronounced in the salt-load group. In contrasting to Ang I and Ang II, plasma Ang-(1-7) concentration decreased significantly only within 60 min of 5.0% NaCl infusion (19.5 +/- 2.9 vs 30.5 +/- 1.9 pg/ml in the control group). As expected, plasma vasopressin increased markedly in the animals submitted to acute salt load (144.0 +/- 42.0 vs 5.0 +/- 0.2 pg/ml in the control group). The dissociation between the changes in plasma Ang-(1-7) and that of other plasma angiotensins is consistent with our previous studies using chronic salt load, and suggests that during acute volume expansion associated with salt load Ang I is preferentially converted to Ang-(1-7). In contrast to vasopressin, however, plasma Ang-(1-7) concentration increased only after chronic salt load, suggesting that this angiotensin may be more involved with long-term mechanisms of control of hydromineral balance.

Angiotensin I↗

PubMed1995

The cDNA encoding canine dihydrolipoamide dehydrogenase contains multiple termination signals.

A 2288-bp cDNA sequence encoding dihydrolipoamide dehydrogenase (DLDH; dihydrolipoamide: NAD+ oxido-reductase; EC 1.8.1.4) was obtained by isolating a 1762-bp cDNA clone from a canine skeletal muscle library in the vector, lambda UNIZAP, combined with PCR amplification of the 5' end of the mRNA. The DLDH cDNA sequence contains a 49-bp G+C-rich 5'-untranslated region (UTR), followed by 1527 bp of coding region, and 695 bp of 3'-UTR preceding a 17-bp poly(A) tail. The single open reading frame encodes a precursor DLDH of 509 amino acids (aa) that begins with a 35-aa leader sequence. The 3'-UTR includes six possible polyadenylation signals (three AATAAA, one TATAAA and two AATGAA) and one potential stem-loop region extending from bp 1969-1991. Alignment studies of the canine and human DLDH demonstrate homology within the coding region of 98% at the aa level and 94% at the nt level. Northern blot analysis using the cDNA clone as probe showed wide tissue distribution of the mRNA, with differences in the level of expression among tissues and possible utilization of different polyadenylation sites.

Amino Acid Sequence↗

PubMed1995

Neuroendocrine control of osmotic regulation in the freshwater shrimp Macrobrachium olfersii (Wiegmann) (Crustacea, decapoda): free amino acid concentrations in the hemolymph.

The participation of neuroendocrine factors present within the central nervous system in the regulation of hemolymph free amino acid (FAA) concentrations was examined in the freshwater shrimp Macrobrachium olfersii. Test shrimps were injected intramuscularly with homogenates prepared from the eyestalks (ES), ventral nerve cord (VNC), supraesophageal (SEG), or thoracic ganglia (TG) of donor shrimps previously exposed for 6 hr to a high-salinity medium (HSM, 21% salinity). After injection of the homogenate, the shrimps were maintained for up to 6 hr in either freshwater (FW) or HSM. Hemolymph was sampled by cardiac puncture and prepared for reverse phase HPLC, derivatizing the FAA with phenylisothiocyanate. An FAA profile was determined and the [FAA]:[Cl-] ratios for the four FAA present in highest concentration (Gly, Arg, Ala, and Pro for ES and VNC experiments; Glu, Leu, Ala, and Val for SEG and TG experiments) were obtained. Nonparametric analyses revealed specific, notable effects resulting from homogenate injection, e.g., ES homogenate increased [Pro]/[Cl-] ratios in FW-exposed shrimps; SEG homogenate increased [Glu]/[Cl-] and [Val]/[Cl-] ratios in HSM-exposed shrimps; and TG homogenate increased [FAA]/[Cl-] ratios for Glu, Leu, Ala, and Val in HSM-exposed shrimps. Total FAA concentrations decreased after exposure of the shrimps to HSM but were increased by the injection of ES homogenate in FW-exposed shrimps and by TG homogenate in HSM-exposed shrimps. The total [FAA]/[Cl-] ratio was also increased by TG homogenate in HSM-exposed animals. There were no clear effects on [Cl-] alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

PubMed1995

Casein, hydrolyzed casein, and amino acids that simulate casein produce the same extent of mucosal adaptation to massive bowel resection in adult rats.

We determined the effect of the extent of protein polymerization on the intestinal hyperplastic adaptation of adult male Wistar rats after 80% resection of the jejunal-ileal segment. Rats received one of four chemically defined solid diets prepared by using casein, two casein hydrolysates of different peptide size distributions, or free amino acids simulating casein and identical in all other components for 12 d, starting 3 d after surgery. Semipaired feeding was used to ensure that the same quantity of food was ingested by each group and as a consequence, nitrogen and energy intakes were reduced to 63% of that obtained with ad libitum feeding of the casein diet to intact rats. No significant differences were demonstrable in food ingestion, weight gain, nitrogen balance, or morphometric data for the remaining jejunal and ileal segments (number of cells/villus, number of cells/crypt, and crypt cell mitosis rate). These data demonstrate that the extent of polymerization of the protein nitrogen source did not affect the hyperplastic adaptative process of the rat. Additional studies in humans are necessary to determine whether intact protein diets can be used first as a nitrogen source in nutritional support of patients with a nonspecific hyperplastic response to surgical resection before the use of expensive hydrolysates and the more expensive amino acid mixtures.

Adaptation, Physiological↗

PubMed1995

[Biochemical basis of enteral nutrition].

Basic biochemical studies have demonstrated the products of protein intraluminal digestion are amino acids and peptides, and the those amino acids as well as small peptides are absorbed by independent mechanisms. The formers are absorbed by specific absorption systems mediated by carriers. The small peptides (di-and tripeptides) are absorbed intact from and may be intracelularlly hydrolised. Peptides with four or more residues are hydrolysed by peptidases located on the brush border of the intestinal villi and then absorbed as amino acids and/or small peptides. Such an absorption through a peptide mechanism is faster than the absorption of free amino acids. These are basic aspects that should direct the use of protein partial enzymatic hydrolisate in human nutrition.

Amino Acids↗