Disrupting membrane raft domains by alkylphospholipids.

Using phase contrast and fluorescence microscopy we study the influence of the alkylphospholipid, ALP, 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate, ODPC, in giant unilamellar vesicles, GUVs, composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), brain sphingomyelin (SM) and cholesterol (Chol). The results show that adding 100µM ODPC (below CMC) to the outer solution of GUVs promotes DOPC membrane disruption over a period of 1h of continuous observation. On the other hand, the presence of SM and Chol in homogeneous fluid lipid bilayers protects the membrane from disruption. Interestingly, by adding 100µM ODPC to GUVs containing DOPC:SM:Chol (1:1:1), which display liquid ordered (Lo)-liquid disordered (Ld) phase coexistence, the domains rapidly disappear in less than 1min of ODPC contact with the membrane. The lipids are subsequently redistributed to liquid domains within a time course of 14-18min, reflecting that the homogenous phase was not thermodynamically stable, followed by rupture of the GUVs. A similar mechanism of action is also observed for perifosine, although to a larger extent. Therefore, the initial stage of lipid raft disruption by both ODPC and perifosine, and maybe other ALPS, by promoting lipid mixing, may be correlated with their toxicity upon neoplastic cells, since selective (dis)association of essential proteins within lipid raft microdomains must take place in the plasma membrane.

Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector.

Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1- kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (±931.7)- and 295,400 (±75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (±493,700)- and 308,000 (±139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/µg protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line.

Changes in hippocampal gene expression by 7-nitroindazole in rats submitted to forced swimming stress.

Nitric oxide (NO) is an atypical neurotransmitter that has been related to the pathophysiology of major depression disorder. Increased plasma NO levels have been reported in depressed and suicidal patients. Inhibition of neuronial nitric oxide synthase (nNOS), on the other hand, induces antidepressant effects in clinical and pre-clinical trials. The mechanisms responsible for the antidepressant-like effects of nNOS inhibitors, however, are not completely understood. In this study, genomic and proteomic analyses were used to investigate the effects of the preferential nNOS inhibitor 7-nitroindazole (7-NI) on changes in global gene and protein expression in the hippocampus of rats submitted to forced swimming test (FST). Chronic treatment (14 days, i.p.) with imipramine (15 mg/kg daily) or 7-NI (60 mg/kg daily) significantly reduced immobility in the FST. Saturation curves for Serial analysis of gene expression libraries showed that the hippocampus of animals submitted to FST presented a lower number of expressed genes compared to non-FST stressed groups. Imipramine, but not 7-NI, reverted this effect. GeneGo analyses revealed that genes related to oxidative phosphorylation, apoptosis and survival controlled by HTR1A signaling and cytoskeleton remodeling controlled by Rho GTPases were significantly changed by FST. 7-NI prevented this effect. In addition, 7-NI treatment changed the expression of genes related to transcription in the cAMP response element-binding pathway. Therefore, this study suggests that changes in oxidative stress and neuroplastic processes could be involved in the antidepressant-like effects induced by nNOS inhibition.

Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells.

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 microM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 microM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 microM, respectively. The critical micellar concentration (CMC) of ODPC was 200 microM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T(c)) of 41.5 degrees C and an enthalpy (H) variation of 7.3 kcal mol(-1). The presence of 25 microM ODPC decreased T(c) and H to 39.3 degrees C and 4.7 kcal mol(-1), respectively. ODPC at 250 microM destabilized the liposomes (36.3 degrees C, 0.46 kcal mol(-1)). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition.

Acute-phase protein alpha-1-acid glycoprotein mediates neutrophil migration failure in sepsis by a nitric oxide-dependent mechanism.

The reduction of circulating neutrophil migration to infection sites is associated with a poor outcome of severe sepsis. alpha-1-Acid glycoprotein (AGP) was isolated from the sera of severely septic patients by HPLC and acrylamide gel electrophoresis and identified by mass spectrometry. Both the isolated protein and commercial AGP inhibited carrageenin-induced neutrophil migration into the rat peritoneal cavity when administered i.v. at a dose of 4.0 microg per rat (95 pmol per rat). Analysis by intravital microscopy demonstrated that both proteins inhibited the rolling and adhesion of leukocytes in the mesenteric microcirculation. The inhibitory activity was blocked by 50 mg/kg aminoguanidine, s.c., and was not demonstrable in inducible nitric oxide synthase (iNOS) knockout mice. Incubation of AGP with neutrophils from healthy subjects induced the production of NO and inhibited the neutrophil chemotaxis by an iNOS/NO/cyclic guanosine 3,5-monophosphate-dependent pathway. In addition, AGP induced the l-selectin shedding by neutrophils. The administration of AGP to rats with mild cecal ligation puncture sepsis inhibited neutrophil migration and reduced 7-day survival from approximately 80% to 20%. These data demonstrate that AGP, an acute-phase protein, inhibits neutrophil migration by an NO-dependent process and suggest that AGP also participates in human sepsis.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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%).

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.

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,

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.