Human papillomavirus infection and p53 protein expression in vulvar intraepithelial neoplasia and invasive squamous cell carcinoma

The etiopathogenesis of vulvar intraepithelial neoplasia (VIN III) and invasive squamous cell carcinoma are largely unknown. Since there are few studies on Brazilian patients, our purpose was to determine the frequency of human papillomavirus (HPV) infection and the expression of p53 in these lesions, and associate them with other factors such as age, morphological subtypes, multicentric and multifocal disease. Thirty-eight cases of VIN III, nine of superficially invasive carcinoma, and 55 of invasive vulvar carcinoma were retrospectively evaluated from 1983 to 1995 for the presence of HPV by immunohistochemistry and in situ hybridization, and for p53 protein expression by immunohistochemistry on paraffin sections. All cases for whom material (slides and paraffin blocks) and clinical data were available were included. HPV and p53 were detected in 57.9 and 21.1% of the VIN III lesions, 33.3 and 66.7% of superficially invasive carcinomas, and 7.3 and 58.2% of invasive squamous cell carcinomas, respectively. HPV infection was associated with younger age in the VIN III and invasive carcinoma groups. In the latter, HPV infection was associated with the basaloid variant. p53 expression rate was higher in superficially invasive and invasive lesions and was not related to HPV infection. Our findings are similar to others and support the hypothesis that there are two separate entities of the disease, one associated with HPV and the other unrelated, with p53 inactivation possibly being implicated in some of the cases.

Interruption of the blood-stage cycle of the malaria parasite, Plasmodium chabaudi, by protein tyrosine kinase inhibitors

Malaria is a devastating disease caused by a unicellular protozoan, Plasmodium, which affects 3.7 million people every year. Resistance of the parasite to classical treatments such as chloroquine requires the development of new drugs. To gain insight into the mechanisms that control Plasmodium cell cycle, we have examined the effects of kinase inhibitors on the blood-stage cycle of the rodent malaria parasite, Plasmodium chabaudi. In vitro incubation of red blood cells for 17 h at 37ºC with the inhibitors led to a decrease in the percent of infected cells, compared to control treatment, as follows: genistein (200 µM - 75%), staurosporine (1 µM - 58%), R03 (1 µM - 75%), and tyrphostins B44 (100 µM - 66%) and B46 (100 µM - 68%). All these treatments were shown to retard or prevent maturation of the intraerythrocytic parasites. The diverse concentration ranges at which these inhibitors exert their effects give a clue as to the types of signals that initiate the transitions between the different developmental stages of the parasite. The present data support our hypothesis that the maturation of the intraerythrocytic cycle of malaria parasites requires phosphorylation. In this respect, we have recently reported a high Ca2+ microenvironment surrounding the parasite within red blood cells. Several kinase activities are modulated by Ca2+. The molecular identification of the targets of these kinases could provide new strategies against malaria.

A role for histone-like protein H1 (H-NS) in the regulation of hemolysin expression by Serratia marcescens

The histone-like protein H1 (H-NS) is an abundant structural component of the bacterial nucleoid and influences many cellular processes including recombination, transcription and transposition. Mutations in the hns gene encoding H-NS are highly pleiotropic, affecting the expression of many unrelated genes. We have studied the role of H-NS on the regulation of hemolysin gene expression in Serratia marcescens. The Escherichia coli hns mutant carrying S. marcescens hemolysin genes on a plasmid constructed by ligation of the 3.2-kb HindIII-SacI fragment of pR02 into pBluescriptIIKS, showed a high level of expression of this hemolytic factor. To determine the osmoregulation of wild-type and hns defective mutants the cells were grown to mid-logarithmic phase in LB medium with 0.06 or 0.3 M NaCl containing ampicillin and kanamycin, whereas to analyze the effect of pH on hemolysin expression, the cells were grown to late-logarithmic phase in LB medium buffered with 0.1 M Tris-HCl, pH 4.5 to 8.0. To assay growth phase-related hemolysin production, bacterial cells were grown in LB medium supplemented with ampicillin and kanamycin. The expression of S. marcescens hemolysin genes in wild-type E. coli and in an hns-defective derivative at different pH and during different growth phases indicated that, in the absence of H-NS, the expression of hemolysin did not vary with pH changes or growth phases. Furthermore, the data suggest that H-NS may play an important role in the regulation of hemolysin expression in S. marcescens and its effect may be due to changes in DNA topology influencing transcription and thus the amount of hemolysin expression. Implications for the mechanism by which H-NS influences gene expression are discussed.

Effect of hepatitis C serology on C-reactive protein in a cohort of Brazilian hemodialysis patients

Hepatitis C (HCV) is not an uncommon feature in hemodialysis (HD) patients and may be a cause of systemic inflammation. Plasma cytokine interleukin-6 (IL-6) is mainly produced by circulating and peripheral cells and induces the hepatic synthesis of C-reactive protein (CRP), which is the main acute phase reactant. The aim of this study was to investigate the influence of HCV on two markers of systemic inflammation, serum CRP and IL-6, in HD patients. The study included 118 HD patients (47% males, age 47 ± 13 years, 9% diabetics) who had been treated by standard HD for at least 6 months. The patients were divided into two groups depending on the presence (HCV+) or absence (HCV-) of serum antibodies against HCV. Serum albumin (S-Alb), plasma high sensitivity CRP (hsCRP), IL-6, and alanine aminotransferase (ALT) were measured and the values were compared with those for 22 healthy controls. Median hsCRP and IL-6 values and hsCRP/IL-6 ratio were: 3.5 vs 2.1 mg/l, P < 0.05; 4.3 vs 0.9 pg/ml, P < 0.0001, and 0.8 vs 2.7, P < 0.0001, for patients and controls, respectively. Age, gender, S-Alb, IL-6 and hsCRP did not differ between the HCV+ and HCV- patients. However, HCV+ patients had higher ALT (29 ± 21 vs 21 ± 25 IU/l) and had been on HD for a longer time (6.1 ± 3.0 vs 4.0 ± 2.0 years, P < 0.0001). Moreover, HCV+ patients had a significantly lower median hsCRP/IL-6 ratio (0.7 vs 0.9, P < 0.05) compared to the HCV- group. The lower hsCRP/IL-6 ratio in HCV+ patients than in HCV- patients suggests that hsCRP may be a less useful marker of inflammation in HCV+ patients and that a different cut-off value for hsCRP for this population of patients on HD may be required to define inflammation.

Dialysis water treated by reverse osmosis decreases the levels of C-reactive protein in uremic patients

Atherosclerosis is a major complication of chronic renal failure. Microinflammation is involved in atherogenesis and is associated with uremia and dialysis. The role of dialysate water contamination in inducing inflammation has been debated. Our aim was to study inflammatory markers in patients on chronic dialysis, before and 3 to 6 months after switching the water purification system from deionization to reverse osmosis. Patients had demographic, clinical and nutritional information collected and blood drawn for determination of albumin, ferritin, C-reactive protein (CRP), interleukin-6, and tumor necrosis factor-alpha in both situations. Acceptable levels of water purity were less than 200 colony-forming units of bacteria and less than 1 ng/ml of endotoxin. Sixteen patients died. They had higher median CRP (26.6 vs 11.2 mg/dl, P = 0.007) and lower median albumin levels (3.1 vs 3.9 g/l, P < 0.05) compared to the 31 survivors. Eight patients were excluded because of obvious inflammatory conditions. From the 23 remaining patients (mean age ± SD: 51.3 ± 13.9 years), 18 had a decrease in CRP after the water treatment system was changed. Overall, median CRP was lower with reverse osmosis than with deionization (13.2 vs 4.5 mg/l, P = 0.022, N = 23). There was no difference in albumin, cytokines, subjective global evaluation, or clinical and biochemical parameters. In conclusion, uremic patients presented a clinically significant reduction in CRP levels when dialysate water purification system switched from deionization to reverse osmosis. It is possible that better water treatments induce less inflammation and eventually less atherosclerosis in hemodialysis patients.

The powerful high pressure tool for protein conformational studies

The pressure behavior of proteins may be summarized as a the pressure-induced disordering of their structures. This thermodynamic parameter has effects on proteins that are similar but not identical to those induced by temperature, the other thermodynamic parameter. Of particular importance are the intermolecular interactions that follow partial protein unfolding and that give rise to the formation of fibrils. Because some proteins do not form fibrils under pressure, these observations can be related to the shape of the stability diagram. Weak interactions which are differently affected by hydrostatic pressure or temperature play a determinant role in protein stability. Pressure acts on the 2º, 3º and 4º structures of proteins which are maintained by electrostatic and hydrophobic interactions and by hydrogen bonds. We present some typical examples of how pressure affects the tertiary structure of proteins (the case of prion proteins), induces unfolding (ataxin), is a convenient tool to study enzyme dissociation (enolase), and provides arguments to understand the role of the partial volume of an enzyme (butyrylcholinesterase). This approach may have important implications for the understanding of the basic mechanism of protein diseases and for the development of preventive and therapeutic measures.

Curcumin induces human HT-29 colon adenocarcinoma cell apoptosis by activating p53 and regulating apoptosis-related protein expression

Curcumin, a major yellow pigment and active component of turmeric, has multiple anti-cancer properties. However, its molecular targets and mechanisms of action on human colon adenocarcinoma cells are unknown. In the present study, we examined the effects of curcumin on the proliferation of human colon adenocarcinoma HT-29 cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method and confirmed the curcumin-induced apoptosis by morphology and DNA ladder formation. At the same time, p53, phospho-p53 (Ser15), and other apoptosis-related proteins such as Bax, Bcl-2, Bcl-xL, pro-caspase-3, and pro-caspase-9 were determined by Western blot analysis. The colon adenocarcinoma cells were treated with curcumin (0-75 µM) for 0-24 h. We observed that p53 was highly expressed in HT-29 cells and curcumin could up-regulate the serine phosphorylation of p53 in a time- and concentration-dependent manner. An increase in expression of the pro-apoptotic factor Bax and a decrease in expression of the anti-apoptotic factor Bcl-2 were also observed in a time-dependent manner after exposure of 50 µM curcumin, while the expression of the anti-apoptotic factor Bcl-xL was unchanged. Curcumin could also down-regulate the expression of pro-caspase-3 and pro-caspase-9 in a time-dependent manner. These data suggest a possible underlying molecular mechanism whereby curcumin could induce the apoptosis signaling pathway in human HT-29 colon adenocarcinoma cells by p53 activation and by the regulation of apoptosis-related proteins. This property of curcumin suggests that it could have a possible therapeutic potential in colon adenocarcinoma patients.

Effect of intraperitoneally administered hydrolyzed whey protein on blood pressure and renal sodium handling in awake spontaneously hypertensive rats

The present study evaluated the acute effect of the intraperitoneal (ip) administration of a whey protein hydrolysate (WPH) on systolic arterial blood pressure (SBP) and renal sodium handling by conscious spontaneously hypertensive rats (SHR). The ip administration of WPH in a volume of 1 ml dose-dependently lowered the SBP in SHR 2 h after administration at doses of 0.5 g/kg (0.15 M NaCl: 188.5 ± 9.3 mmHg vs WPH: 176.6 ± 4.9 mmHg, N = 8, P = 0.001) and 1.0 g/kg (0.15 M NaCl: 188.5 ± 9.3 mmHg vs WPH: 163.8 ± 5.9 mmHg, N = 8, P = 0.0018). Creatinine clearance decreased significantly (P = 0.0084) in the WPH-treated group (326 ± 67 µL min-1 100 g body weight-1) compared to 0.15 M NaCl-treated (890 ± 26 µL min-1 100 g body weight-1) and captopril-treated (903 ± 72 µL min-1 100 g body weight-1) rats. The ip administration of 1.0 g WPH/kg also decreased fractional sodium excretion to 0.021 ± 0.019% compared to 0.126 ± 0.041 and 0.66 ± 0.015% in 0.15 M NaCl and captopril-treated rats, respectively (P = 0.033). Similarly, the fractional potassium excretion in WPH-treated rats (0.25 ± 0.05%) was significantly lower (P = 0.0063) than in control (0.91 ± 0.15%) and captopril-treated rats (1.24 ± 0.30%), respectively. The present study shows a decreased SBP in SHR after the administration of WPH associated with a rise in tubule sodium reabsorption despite an angiotensin I-converting enzyme (ACE)-inhibiting in vitro activity (IC50 = 0.68 mg/mL). The present findings suggest a pathway involving ACE inhibition but measurements of plasma ACE activity and angiotensin II levels are needed to support this suggestion.

Determination of renal function in long-term heart transplant patients by measurement of urinary retinol-binding protein levels

Significant improvements have been noted in heart transplantation with the advent of cyclosporine. However, cyclosporine use is associated with significant side effects, such as chronic renal failure. We were interested in evaluating the incidence of long-term renal dysfunction in heart transplant recipients. Fifty-three heart transplant recipients were enrolled in the study. Forty-three patients completed the entire evaluation and follow-up. Glomerular (serum creatinine, creatinine clearance measured, and creatinine clearance calculated) and tubular functions (urinary retinol-binding protein, uRBP) were re-analyzed after 18 months. At the enrollment time, the prevalence of renal failure ranged from 37.7 to 54% according to criteria used to define it (serum creatinine > or = 1.5 mg/dL and creatinine clearance <60 mL/min). Mean serum creatinine was 1.61 ± 1.31 mg/dL (range 0.7 to 9.8 mg/dL) and calculated and measured creatinine clearances were 67.7 ± 25.9 and 61.18 ± 25.04 mL min-1 (1.73 m²)-1, respectively. Sixteen of the 43 patients who completed the follow-up (37.2%) had tubular dysfunction detected by increased levels of uRBP (median 1.06, 0.412-6.396 mg/dL). Eleven of the 16 patients (68.7%) with elevated uRBP had poorer renal function after 18 months of follow-up, compared with only eight of the 27 patients (29.6%) with normal uRBP (RR = 3.47, P = 0.0095). Interestingly, cyclosporine trough levels were not different between patients with or without tubular and glomerular dysfunction. Renal function impairment is common after heart transplantation. Tubular dysfunction, assessed by uRBP, correlates with a worsening of glomerular filtration and can be a useful tool for early detection of renal dysfunction.

In silico analysis identifies a C3HC4-RING finger domain of a putative E3 ubiquitin-protein ligase located at the C-terminus of a polyglutamine-containing protein

Almost identical polyglutamine-containing proteins with unknown structures have been found in human, mouse and rat genomes (GenBank AJ277365, AF525300, AY879229). We infer that an identical new gene (RING) finger domain of real interest is located in each C-terminal segment. A three-dimensional (3-D) model was generated by remote homology modeling and the functional implications are discussed. The model consists of 65 residues from terminal position 707 to 772 of the human protein with a total length of 796 residues. The 3-D model predicts a ubiquitin-protein ligase (E3) as a binding site for ubiquitin-conjugating enzyme (E2). Both enzymes are part of the ubiquitin pathway to label unwanted proteins for subsequent enzymatic degradation. The molecular contact specificities are suggested for both the substrate recognition and the residues at the possible E2-binding surface. The predicted structure, of a ubiquitin-protein ligase (E3, enzyme class number 6.3.2.19, CATH code 3.30.40.10.4) may contribute to explain the process of ubiquitination. The 3-D model supports the idea of a C3HC4-RING finger with a partially new pattern. The putative E2-binding site is formed by a shallow hydrophobic groove on the surface adjacent to the helix and one zinc finger (L722, C739, P740, P741, R744). Solvent-exposed hydrophobic amino acids lie around both zinc fingers (I717, L722, F738, or P765, L766, V767, V733, P734). The 3-D structure was deposited in the protein databank theoretical model repository (2B9G, RCSB Protein Data Bank, NJ).

Protection against cephalosporin-induced lipid peroxidation and nephrotoxicity by (+)-cyanidanol-3 and vitamin E

The ability of the clinically used cephalosporins: cephalothin, cefotaxime and cefotiam to induce lipid peroxidation (LPO) and renal damage was compared to that of nephrotoxic cephaloridine under in vivo conditions. Glutathione was measured in rat liver or in renal cortex as non-protein sulfhydryls. LPO was measured in plasma, renal cortex and liver by the generation of malondialdehyde or as the increase in renal cortical concentration of conjugated dienes. Impairment of renal function was measured as the decrease in renal cortical accumulation of the organic anion p-aminohippurate (PAH). Administration of cephalosporins to rats as a single dose (2000 mg/kg, ip) induced a significant glutathione-depletion in the renal cortex with cephaloridine, and in the liver with cephaloridine, cephalothin and cefotiam. Treatment of rats with cephaloridine, cephalothin and cefotiam (200, 500, or 1000 mg kg-1 day-1, ip) for 5 days resulted in a dose-dependent increase of LPO in the renal cortex. While cephaloridine induced the highest concentration of conjugated diene, cefotaxime had no effect. Measurements of PAH accumulation in renal cortical slices from cephalosporin-treated rats showed a dose-dependent decrease in the renal cortical accumulation of PAH. Pretreatment with the antioxidants vitamin E or cyanidanol (400 mg kg-1 day-1, ip) 1 h before treatment with cephaloridine, cephalothin or cefotiam (1000 mg kg-1 day-1, ip) for 3 days inhibited cephalosporin-induced LPO and significantly reduced the impairment of renal cortical accumulation of PAH. The potential of different cephalosporins for inducing LPO and reducing PAH accumulation was ranked as follows: cephaloridine > cephalothin > cefotiam > cefotaxime.

Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.

Roles of mitogen-activated protein kinases and angiotensin II in renal development

Experimental and clinical evidence suggests that angiotensin II (AII) participates in renal development. Renal AII content is several-fold higher in newborn rats and mice than in adult animals. AII receptors are also expressed in higher amounts in the kidneys of newborn rats. The kidneys of fetuses whose mother received a type 1 AII receptor (AT1) antagonist during gestation present several morphological alterations. Mutations in genes that encode components of the renin-angiotensin system are associated with autosomal recessive renal tubular dysgenesis. Morphological changes were detected in the kidneys of 3-week-old angiotensin-deficient mice. Mitogen-activated protein kinases (MAPKs) are important mediators that transduce extracellular stimuli to intracellular responses. The MAPK family comprises three major subgroups, namely extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinases (JNK), and p38 MAPK (p38). Important events in renal growth during nephrogenesis such as cellular proliferation and differentiation accompanied by apoptosis on a large scale can be mediated by MAPK pathways. A decrease in glomerulus number was observed in embryos cultured for 48 and 120 h with ERK or p38 inhibitors. Many effects of AII are mediated by MAPK pathways. Treatment with losartan during lactation provoked changes in renal function and structure associated with alterations in AT1 and type 2 AII (AT2) receptors and p-JNK and p-p38 expression in the kidney. Several studies have shown that AII and MAPKs play an important role in renal development. However, the relationship between the effects of AII and MAPK activation on renal development is still unclear.

Gentamicin-induced preconditioning of proximal tubular LLC-PK1 cells stimulates nitric oxide production but not the synthesis of heat shock protein

Nephrotoxicity is the main side effect of antibiotics such as gentamicin. Preconditioning has been reported to protect against injuries as ischemia/reperfusion. The objective of the present study was to determine the effect of preconditioning with gentamicin on LLC-PK1 cells. Preconditioning was induced in LLC-PK1 cells by 24-h exposure to 2.0 mM gentamicin (G/IU). After 4 or 15 days of preconditioning, cells were again exposed to gentamicin (2.0 mM) and compared to untreated control or G/IU cells. Necrosis and apoptosis were assessed by acridine orange and HOESCHT 33346. Nitric oxide (NO) and endothelin-1 were assessed by the Griess method and available kit. Heat shock proteins were analyzed by Western blotting. After 15 days of preconditioning, LLC-PK1 cells exhibited a significant decrease in necrosis (23.5 ± 4.3 to 6.5 ± 0.3%) and apoptosis (23.5 ± 4.3 to 6.5 ± 2.1%) and an increase in cell proliferation compared to G/IU. NO (0.177 ± 0.05 to 0.368 ± 0.073 µg/mg protein) and endothelin-1 (1.88 ± 0.47 to 2.75 ± 0.53 pg/mL) production significantly increased after 15 days of preconditioning compared to G/IU. No difference in inducible HSP 70, constitutive HSC 70 or HSP 90 synthesis in tubular cells was observed after preconditioning with gentamicin. The present data suggest that preconditioning with gentamicin has protective effects on proximal tubular cells, that involved NO synthesis but not reduction of endothelin-1 or production of HSP 70, HSC 70, or HSP 90. We conclude that preconditioning could be a useful tool to prevent the nephrotoxicity induced by gentamicin.

Highly sensitive C-reactive protein and male gender are independently related to the severity of coronary disease in patients with metabolic syndrome and an acute coronary event

Patients with metabolic syndrome are at high-risk for development of atherosclerosis and cardiovascular events. The objective of this study was to examine the major determinants of coronary disease severity, including those coronary risk factors associated with metabolic syndrome, during the early period after an acute coronary episode. We tested the hypothesis that inflammatory markers, especially highly sensitive C-reactive protein (hsCRP), are related to coronary atherosclerosis, in addition to traditional coronary risk factors. Subjects of both genders aged 30 to 75 years (N = 116) were prospectively included if they had suffered a recent acute coronary syndrome (acute myocardial infarction or unstable angina pectoris requiring hospitalization) and if they had metabolic syndrome diagnosed according to the National Cholesterol Education Program/Adult Treatment Panel III. Patients were submitted to a coronary angiography and the burden of atherosclerosis was estimated by the Gensini score. The severity of coronary disease was correlated (Spearman’s or Pearson’s coefficient) with gender (r = 0.291, P = 0.008), age (r = 0.218, P = 0.048), hsCRP (r = 0.256, P = 0.020), ApoB/ApoA ratio (r = 0.233, P = 0.041), and carotid intima-media thickness (r = 0.236, P = 0.041). After multiple linear regression, only male gender (P = 0.046) and hsCRP (P = 0.012) remained independently associated with the Gensini score. In this high-risk population, male gender and high levels of hsCRP, two variables that can be easily obtained, were associated with more extensive coronary disease, identifying patients with the highest potential of developing new coronary events.