Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.

Computerized invasive measurement of time-dependent intraocular pressure

Several methods have been described to measure intraocular pressure (IOP) in clinical and research situations. However, the measurement of time varying IOP with high accuracy, mainly in situations that alter corneal properties, has not been reported until now. The present report describes a computerized system capable of recording the transitory variability of IOP, which is sufficiently sensitive to reliably measure ocular pulse peak-to-peak values. We also describe its characteristics and discuss its applicability to research and clinical studies. The device consists of a pressure transducer, a signal conditioning unit and an analog-to-digital converter coupled to a video acquisition board. A modified Cairns trabeculectomy was performed in 9 Oryctolagus cuniculus rabbits to obtain changes in IOP decay parameters and to evaluate the utility and sensitivity of the recording system. The device was effective for the study of kinetic parameters of IOP, such as decay pattern and ocular pulse waves due to cardiac and respiratory cycle rhythm. In addition, there was a significant increase of IOP versus time curve derivative when pre- and post-trabeculectomy recordings were compared. The present procedure excludes corneal thickness and error related to individual operator ability. Clinical complications due to saline infusion and pressure overload were not observed during biomicroscopic evaluation. Among the disadvantages of the procedure are the requirement of anesthesia and the use in acute recordings rather than chronic protocols. Finally, the method described may provide a reliable alternative for the study of ocular pressure dynamic alterations in man and may facilitate the investigation of the pathogenesis of glaucoma.

TIMP-1 mediates the inhibitory effect of interleukin-6 on the proliferation of a hepatocarcinoma cell line in a STAT3-dependent manner

The tissue inhibitor of metalloproteinases (TIMP)-1 is a multifunctional protein which is not only an inhibitor of matrix metalloproteinases (MMPs) but also to have a possible "cytokine-like" action. Here, we first compared mRNA expression of TIMP-1 and MMP-9 in BEL-7402 (a hepatocellular carcinoma cell line), L-02 (a normal liver cell line) and QSG-7701 (a cell line derived from peripheral tissue of liver carcinoma) using real-time quantitative RT-PCR. By evaluating the variation of the MMP-9/TIMP-1 ratio as an index of reciprocal changes of the expression of the two genes, we observed that the MMP-9/TIMP-1 ratio was about 13- and 5-fold higher in BEL-7402 than in L-02 and QSG-7701, respectively. Significantly, overexpression of TIMP-1 decreased the MMP-9/TIMP-1 ratio in BEL-7402 and then inhibited the cell growth to 60% and reduced the migration to about 30%. Meanwhile, our data showed that interleukin-6 (IL-6) (100 ng/mL) could also inhibited the cell growth of BEL-7402. Further studies indicated that TIMP-1 mediated the inhibitory effect of IL-6 on BEL-7402 cell proliferation in a STAT3-dependent manner, which could further accelerate the expression of the cyclin-dependent kinase inhibitor p21. A dominant negative STAT3 mutant totally abolished IL-6-induced TIMP-1 expression and its biological functions. The present results demonstrate that TIMP-1 may be one of the mediators that regulate the inhibitory effect of IL-6 on BEL-7402 proliferation in which STAT3 signal transduction and p21 up-regulation also play important roles.

Growth inhibitory effect and Chk1-dependent signaling involved in G2/M arrest on human gastric cancer cells induced by diallyl disulfide

Diallyl disulfide (DADS) inhibits growth and induces cell cycle G2/M arrest in human gastric cancer MGC803 cells. In this study, 15 mg/L DADS exerted similar effects on growth and cell cycle arrest in human gastric cancer BGC823 cells. Due to the importance of cell cycle redistribution in DADS-mediated anti-carcinogenic effects, we investigated the role of checkpoint kinases (Chk1 and Chk2) during DADS-induced cell cycle arrest. We hypothesized that DADS could mediate G2/M phase arrest through either Chk1 or Chk2 signal transduction pathways. We demonstrated that DADS induced the accumulation of phosphorylated Chk1, but not of Chk2, and that DADS down-regulated Cdc25C and cyclin B1. The expression of mRNA and total protein for Chkl and Chk2 was unchanged. Chk1 is specifically phosphorylated by ATR (ATM-RAD3-related gene). Western blot analysis showed that phospho-ATR was activated by DADS. Taken together, these data suggest that cell cycle G2/M arrest, which was associated with accumulation of the phosphorylated forms of Chk1, but not of Chk2, was involved in the growth inhibition induced by DADS in the human gastric cancer cell line BGC823. Furthermore, the DADS-induced G2/M checkpoint response is mediated by Chk1 signaling through ATR/Chk1/Cdc25C/cyclin B1, and is independent of Chk2.

The role of Ca2+/calmodulin-dependent protein kinase II and calcineurin in TNF-α-induced myocardial hypertrophy

We investigated whether Ca2+/calmodulin-dependent kinase II (CaMKII) and calcineurin (CaN) are involved in myocardial hypertrophy induced by tumor necrosis factor α (TNF-α). The cardiomyocytes of neonatal Wistar rats (1-2 days old) were cultured and stimulated by TNF-α (100 μg/L), and Ca2+ signal transduction was blocked by several antagonists, including BAPTA (4 µM), KN-93 (0.2 µM) and cyclosporin A (CsA, 0.2 µM). Protein content, protein synthesis, cardiomyocyte volumes, [Ca2+]i transients, CaMKIIδB and CaN were evaluated by the Lowry method, [³H]-leucine incorporation, a computerized image analysis system, a Till imaging system, and Western blot analysis, respectively. TNF-α induced a significant increase in protein content in a dose-dependent manner from 10 µg/L (53.56 µg protein/well) to 100 μg/L (72.18 µg protein/well), and in a time-dependent manner from 12 h (37.42 µg protein/well) to 72 h (42.81 µg protein/well). TNF-α (100 μg/L) significantly increased the amplitude of spontaneous [Ca2+]i transients, the total protein content, cell size, and [³H]-leucine incorporation in cultured cardiomyocytes, which was abolished by 4 µM BAPTA, an intracellular Ca2+ chelator. The increases in protein content, cell size and [³H]-leucine incorporation were abolished by 0.2 µM KN-93 or 0.2 µM CsA. TNF-α increased the expression of CaMKIIδB by 35.21% and that of CaN by 22.22% compared to control. These effects were abolished by 4 µM BAPTA, which itself had no effect. These results suggest that TNF-α induces increases in [Ca2+]i, CaMKIIδB and CaN and promotes cardiac hypertrophy. Therefore, we hypothesize that the Ca2+/CaMKII- and CaN-dependent signaling pathways are involved in myocardial hypertrophy induced by TNF-α.

Malaria transmission model for different levels of acquired immunity and temperature-dependent parameters (vector)

OBJECTIVE: Describe the overall transmission of malaria through a compartmental model, considering the human host and mosquito vector. METHODS: A mathematical model was developed based on the following parameters: human host immunity, assuming the existence of acquired immunity and immunological memory, which boosts the protective response upon reinfection; mosquito vector, taking into account that the average period of development from egg to adult mosquito and the extrinsic incubation period of parasites (transformation of infected but non-infectious mosquitoes into infectious mosquitoes) are dependent on the ambient temperature. RESULTS: The steady state equilibrium values obtained with the model allowed the calculation of the basic reproduction ratio in terms of the model's parameters. CONCLUSIONS: The model allowed the calculation of the basic reproduction ratio, one of the most important epidemiological variables.

Effect of skin colour and selected physical characteristics on Schistosoma mansoni dependent morbidity

The effect of the colour group on the morbidity due to Schistosoma mansoni was examined in two endemic areas situated in the State of Minas Gerais, Brazil. Of the 2773 eligible inhabitants, 1971 (71.1%) participated in the study: 545 (27.6%) were classified as white, 719 (36.5%) as intermediate and 707 (35.9%) as black. For each colour group, signs and symptoms of individuals who eliminated S.mansoni eggs (cases) were compared to those who did not present eggs in the faeces (controls). The odds ratios were adjusted by age, gender, previous treatment for schistosomiasis, endemic area and quality of the household. There was no evidence of a modifier effect of colour on diarrhea, bloody faeces or abdominal pain. A modifier effect of colour on hepatomegaly was evident among those heaviest infected (> 400 epg): the adjusted odds ratios for palpable liver at the middle clavicular and the middle sternal lines were smaller among blacks (5.4 and 6.5, respectively) and higher among whites (10.6 and 12.9) and intermediates (10.4 and 10.1, respectively). These results point out the existence of some degree of protection against hepatomegaly among blacks heaviest infected in the studied areas.

T cell-dependent immunodepression in vivo in Schistosoma mansoni infected patients

T-cell function was evaluated in 29 patients with either hepatointestinal or hepatosplenic schistosomiasis by intradermal tests to recall antigens. Immunodepression was detected in 26% of the subjects with hepatointestinal schistosomiasis and in 50% of those with the hepatosplenic form. Cellular immunodepression was related to worm load and spleen size. This non specific T-cell immunodepression may represent a serious constraint to the elimination of intracellular pathogens both in hepatosplenic or hepatointestinal schistosomiasis.

Density-dependent morphological plasticity and trade-offs among vegetative traits in Eichhornia crassipes (Pontederiaceae)

Density-dependent responses are an important component of the organism life-history, and the resource allocation theory is a central concept to the life-history theory. When resource allocation varies due to environmental changes, a plant may change its morphology or physiology to cope with the new conditions, a process known as phenotypic plasticity. Our study aimed to evaluate how plant density affects Eichhornia crassipes allocation patterns. A total of 214 individuals in high and low density were collected. The density effect was observed in all plant traits examined including biomass accumulation. All traits of E. crassipes demonstrated higher values in high density conditions, except for biomass of leaves. Density exhibited a high influence on vegetative traits of E. crassipes, but did not influence allocation pattern, since a trade-off among the vegetative traits was not found. The morphological plasticity and the absence of trade-offs were discussed as strategies to overcome neighbor plants in competition situations. In high density conditions, there were clear changes in the morphology of the plants which probably allows for their survival in a highly competitive environment.

Alcohol-dependent patients receiving specialised outpatient care: an overview of gender issues

OBJECTIVE: The objective of the present study, with focus on gender, was aimed at evaluating alcohol-dependent individual in terms of socio-demographic variables related to alcohol consumption and therapeutic interventions. METHODS: This is a retrospective cross-sectional study of 1,051 patients (833 men and 218 women), with diagnosis of alcohol dependence syndrome, according to ICD-10 criteria, who had sought treatment for the first time at a specialised health centre between 2000 and 2006. RESULTS: The results showed that women, compared to men, are more likely to be unemployed and without partner, in addition to having higher educational level, latter age of alcohol initiation, needing less outpatient alcohol detoxification program, consuming more fermented beverage, presenting less psychiatric comorbidities, and using less coadjutant medications during treatment. CONCLUSION: We can state that some peculiarities exist permeating both gender and alcohol consumption. A further focus on the characteristics of each population is needed to facilitate the adequate use of therapeutic interventions according to gender specificities.

Comorbidities of mental and behavioral disorders in chemically dependent patients in different periods of abstinence

OBJECTIVE: To assess the frequency of comorbidities of mental and behavioral disorders (CMBD) in psychoactive substance (PAS)-dependent patients with different periods of abstinence cared for at Alcohol and Other Drug Psychosocial Care Centers (CAPS-ad). METHOD: All patients under treatment in the two CAPS-ad of the city of Uberlândia-MG, between April and September 2010, were consecutively assessed. The ICD-10 symptom checklist was used to diagnose CMBD; additional information was obtained from interviews and medical records. The patients were divided according to duration of abstinence: < 1 week (Group 1); 1-4 weeks (Group 2); and > 4 weeks (Group 3). RESULTS: Of all patients assessed, 62.8% were diagnosed with CMBD, which were more frequent (p < 0.05) in Group 1 (72%) than Group 3 (54.2%), and both groups were similar to Group 2 (61%). Depressive and anxiety disorders were more frequent among patients of Group 1. Mood disorders were more frequent (p < 0.05) in women [22/34 (65%) vs. 54/154 (35.1%)], whereas psychotic disorders were more frequent (p = 0.05) in men [16/154 (10.4%) vs. 0]. The presence of CMBD was associated with more severe clinical conditions. CONCLUSIONS: The higher frequency of diagnosis of CMBD in patients of Group 1 may have resulted from the difficulties in distinguishing mental disorders that are due to PAS intoxication or withdrawal from those that are not. However, to make the diagnosis of CMBD, even during detoxification, can increase the likelihood of better response to treatment.

A possible contribution to improving the therapeutic potentials of Babor's Typology of Alcohol Dependent Patients

ABSTRACT Objective The objective of this study was to replicate Babor's Typology and to explore clinical features related to personality traits that may underlie this classification, in order to improve its therapeutic possibilities. Methods Observational prospective study on a group of 273 male alcoholics. After a replication of Babor's variables, Cluster Analysis, Chi-Square – applied on clinical variables related to a Lappda Tipology – and Kappa tests were performed. Results The study identified two distinct clusters that held similar features to those described for the Type A/Type B classification. Besides presenting a lower socio-economic situation, Cluster 2 patients were associated with higher vulnerability and severe clinical features and also differed from Cluster 1 in their response to treatment. These replicated clusters retained connections and also differences in relation to the variables derived from the Lappda Typology. Conclusion Considering that each of the two replicated clusters seem to be associated to different personality traits – according to their correlations to the affective, cognitive and behavioral dimensions brought forward by the Lappda Typology – it is acceptable that this study may contribute to the development of more comprehensive and effective therapeutic strategies specifically tailored to target more specific personality traits of these subgroups of alcoholic patients.

Coronary artery disease, microalbuminuria and lipid profile in patients with non-insulin dependent diabetes mellitus

PURPOSE: To determine the frequency of coronary artery disease, microalbuminuria and the relation to lipid profile disorders, blood pressure and clinical and metabolic features. METHODS: Fifty-five type 2 diabetic patients (32 females, 23 males), aged 59.9±9 years and with known diabetes duration of 11±7.3 years were studied. Coronary artery disease (CAD) was defined as a positive history of myocardial infarction, typical angina, myocardial revascularization or a positive stress testing. Microalbuminuria was defined when two out of three overnight urine samples had a urinary albumin excretion ranging 20 - 200µg/min. RESULTS: CAD was present in 24 patients (43,6%). High blood pressure (HBP) present in 32 patients (58.2%) and was more frequent in CAD group (p=0.05) HBP. Increased the risk of CAD 3.7 times (CI[1.14-12]). Microalbuminuria was present in 25 patients (45.5%) and tended to associate with higher systolic blood pressure (SBP) (p = 0.06), presence of hypertension (p = 0.06) and know diabetes duration (p = 0.08). In the stepwise multiple logistic regression the systolic blood pressure was the only variable that influenced UAE (r = 0.39, r² = 0.14, p = 0.01). The h ypertensive patients had higher cholesterol levels (p = 0.04). CONCLUSION: In our sample the frequency of microalbuminuria, hypertension, hypercholesterolemia and CHD was high. Since diabetes is an independent risk factor for cardiovascular disease, the association of others risk factors suggest the need for an intensive therapeutic intervention in primary and in secundary prevention.

Ambulatory blood pressure monitoring and microalbuminuria in normotensive subjects with insulin-dependent diabetes mellitus

OBJECTIVE: To assess the association between microalbuminuria with ambulatory blood pressure monitoring in normotensive individuals with insulin-dependent diabetes mellitus. METHODS: Thirty-seven patients underwent determination of the rate of urinary excretion of albumin through radioimmunoassay and ambulatory blood pressure monitoring. Their mean age was 26.5±6.7 years, and the mean duration of their disease was 8 (1-34) years. Microalbuminuria was defined as urinary excretion of albumin > or = 20 and <200µg/min in at least 2 out of 3 urine samples. RESULTS: Nine (24.3%) patients were microalbuminuric. Ambulatory blood pressure monitoring in the microalbuminuric patients had higher mean pressure values, mainly the systolic pressure, during sleep as compared with that in the normoalbuminuric patients (120.1±8.3 vs 110.8±7.1 mmHg; p=0.007). The pressure load was higher in the microalbuminuric individuals, mainly the systolic pressure load during wakefulness [6.3 (2.9-45.9) vs 1.6 (0-16%); p=0.001]. This was the variable that better correlated with the urinary excretion of albumin (rS=0.61; p<0.001). Systolic pressure load >50% and diastolic pressure load > 30% during sleep was associated with microalbuminuria (p=0.008). The pressure drop during sleep did not differ between the groups. CONCLUSION: Microalbuminuric normotensive insulin-dependent diabetic patients show greater mean pressure value and pressure load during ambulatory blood pressure monitoring, and these variables correlate with urinary excretion of albumin.