The circadian syndrome predicts cardiovascular disease better than metabolic syndrome in Chinese adults.

BACKGROUND: To compare the predictive value of the circadian syndrome (CircS) and Metabolic syndrome (MetS) for cardiovascular disease. METHOD: We used the data of 9360 Chinese adults aged ≥40 years from the 2011 China Health and Retirement Longitudinal Study (CHARLS). Of the participants, 8253 people were followed in the 2015 survey. MetS was defined using the harmonized criteria. CircS was based on the components of the International Diabetes Federation (IDF) MetS plus short sleep and depression. The cut-off for CircS was set as ≥4. Multivariable logistic regression analysis was used to examine the associations. RESULTS: The prevalence of CircS and MetS was 39.0% and 44.7%. Both MetS and CircS were directly associated with prevalent CVD. The odds ratios for prevalent CVD comparing CircS with MetS, respectively, were 2.83 (95%CI 2.33-3.43) and 2.34 (1.93-2.83) in men, and 2.33 (1.98-2.73) and 1.79 (1.53-2.10) in women. Similar associations were found for incident CVD. The five-year incidence of CVD was 15.1% in CircS and 14.0% in MetS. The number of CircS components has a better predictive power for both prevalent and incident CVD than those of Mets components as indicated by the area under the ROC (AUC). AUC values for CVD in 2011 were higher for CircS than MetS in both men (0.659 (95%CI 0.634-0.684) vs 0.635 (95%CI 0.610-0.661)) and women (0.652 (95%CI 0.632-0.672) vs 0.619 (95%CI 0.599-0.640)). CONCLUSION: The circadian syndrome is a strong and better predictor for CVD than the metabolic syndrome in Chinese adults.

The Circadian Syndrome: is the Metabolic Syndrome and much more!

The Metabolic Syndrome is a cluster of cardio-metabolic risk factors and comorbidities conveying high risk of both cardiovascular disease and type 2 diabetes. It is responsible for huge socio-economic costs with its resulting morbidity and mortality in most countries. The underlying aetiology of this clustering has been the subject of much debate. More recently, significant interest has focussed on the involvement of the circadian system, a major regulator of almost every aspect of human health and metabolism. The Circadian Syndrome has now been implicated in several chronic diseases including type 2 diabetes and cardiovascular disease. There is now increasing evidence connecting disturbances in circadian rhythm with not only the key components of the Metabolic Syndrome but also its main comorbidities including sleep disturbances, depression, steatohepatitis and cognitive dysfunction. Based on this, we now propose that circadian disruption may be an important underlying aetiological factor for the Metabolic Syndrome and we suggest that it be renamed the 'Circadian Syndrome'. With the increased recognition of the 'Circadian Syndrome', circadian medicine, through the timing of exercise, light exposure, food consumption, dispensing of medications and sleep, is likely to play a much greater role in the maintenance of both individual and population health in the future.

Disrupted seasonal biology impacts health, food security and ecosystems.

The rhythm of life on earth is shaped by seasonal changes in the environment. Plants and animals show profound annual cycles in physiology, health, morphology, behaviour and demography in response to environmental cues. Seasonal biology impacts ecosystems and agriculture, with consequences for humans and biodiversity. Human populations show robust annual rhythms in health and well-being, and the birth month can have lasting effects that persist throughout life. This review emphasizes the need for a better understanding of seasonal biology against the backdrop of its rapidly progressing disruption through climate change, human lifestyles and other anthropogenic impact. Climate change is modifying annual rhythms to which numerous organisms have adapted, with potential consequences for industries relating to health, ecosystems and food security. Disconcertingly, human lifestyles under artificial conditions of eternal summer provide the most extreme example for disconnect from natural seasons, making humans vulnerable to increased morbidity and mortality. In this review, we introduce scenarios of seasonal disruption, highlight key aspects of seasonal biology and summarize from biomedical, anthropological, veterinary, agricultural and environmental perspectives the recent evidence for seasonal desynchronization between environmental factors and internal rhythms. Because annual rhythms are pervasive across biological systems, they provide a common framework for trans-disciplinary research.

Spontaneous caloric restriction associated with increased leptin levels in obesity-resistant αMUPA mice.

BACKGROUND: αMUPA mice carry as a transgene the cDNA encoding urokinase-type plasminogen activator, a member of the plasminogen/plasmin system that functions in fibrinolysis and extracellular proteolysis. These mice spontaneously consume less food when fed ad libitum and live longer compared with wild-type (WT) control mice. αMUPA mice are obesity resistant and they share many similarities with calorically restricted animals. However, extensive metabolic characterization of this unique transgenic model has never been performed. METHOD: Metabolism of αMUPA mice was analyzed by measuring hormone, lipid and glucose levels in the serum, as well as gene and protein expression levels in the liver, hypothalamus and brainstem. RESULTS: αMUPA mice were found to be leaner than WT mice mainly because of reduced fat depots. Serum analyses showed that αMUPA mice have high levels of the anorexigenic hormones insulin and leptin, and low levels of the orexigenic hormone ghrelin. Analyses of brain neuropeptides showed that the transcript of the anorexigenic neuropeptide Pomc is highly expressed in the brainstem, whereas the expression of the orexigenic neuropeptides Npy, Orexin and Mch is blunted in the hypothalamus of αMUPA mice. In addition, adenosine monophosphate (AMP)-activated protein kinase (AMPK) levels were higher in the liver and lower in the hypothalamus, thus promoting simultaneously central reduction in appetite and peripheral loss of fat. The levels of SIRT1 were low in the liver, but high in the hypothalamus, a feature that αMUPA mice share with calorically restricted animals. CONCLUSION: Taken together, αMUPA mice exhibit a unique metabolic phenotype of low-calorie intake and high leptin levels, and could serve as a model for both spontaneous calorie restriction and resistance to obesity.

Pro-inflammatory and oxidative effects of noncrystalline uric acid in human mesangial cells: contribution to hyperuricemic glomerular damage.

Hyperuricemia is associated with cardiovascular and renal diseases, as glomerulosclerosis. Noncrystalline uric acid induces deleterious effects on endothelial and vascular smooth muscle cells. In the present study, we analyzed the damage induced by UA on human mesangial cells (HMC), the potential mechanism involved in this injury, and its consequences during infection. HMC were exposed to noncrystalline UA (8 mg/dl) and/or lipopolysaccharide (LPS, 100 µg/ml) for 24 h. In the experiments of cellular viability, HMC were exposed to 8-50 mg/dl of UA. Necrosis was assessed by acridine orange and ethidium bromide. Reactive oxygen species (ROS) were analyzed by 2',7'-dichlorofluorescein. Prostaglandin E2 (PGE2) was evaluated by ELISA. Cyclooxygenase 2 (COX-2) expression was assessed by real-time PCR. UA induced necrosis only at supraphysiological concentrations. Nevertheless, it significantly increased ROS production at 8 mg/dl. LPS increased necrosis and ROS production. Interestingly, the association between UA and LPS decreased ROS and necrosis. UA associated or not with LPS induced COX-2 expression and PGE2 increases in HMC. Results suggest that UA has pro- and anti-oxidant effects in HMC. During infections, it acts like scavenger increasing cellular viability, but alone it can induce ROS production and cellular death in higher concentrations. Additionally, UA has direct pro-inflammatory effects inducing COX-2 expression and PGE2 synthesis. It is concluded that elevated concentrations of uric acid potentially contributes to glomerular damage.

Resistance exercise training ameliorates chronic kidney disease outcomes in a 5/6 nephrectomy model.

Chronic kidney disease (CKD) is defined by decreased glomerular filtration rate (GFR) or increased albumin excretion leading to renal injury. However, exercise training is an important non-pharmacological intervention that ameliorates and protects against Diabetes Mellitus, cardiovascular disease, and CKD. AIM: Our aim was to evaluate the capability of resistance exercise training (RET) to improve CKD outcomes and the contribution of the renal and muscular Akt/mTOR signaling pathway for RET beneficial effects on a CKD model. MAIN METHODS: Male Wistar rats were subjected to RET, followed for 10 weeks, and randomly divided into 5 groups: Sham: Sham-operated; sedentary and nephrectomy (5/6Nx) (SNS); exercising post-5/6Nx (SNE); exercising pre-5/6Nx (ENS); exercising pre- and post-5/6Nx (ENE). The systolic blood pressure (BP) was measured. Creatinine, proteinuria, and blood urea nitrogen (BUN) were evaluated. After euthanasia Renal and muscular Akt/mTOR signaling pathways were analyzed. KEY FINDING: Our study showed that the SNS presented renal injury, hypertension, weight and muscular mass loss and a higher mortality rate. SNS group also decreased renal IL-10 and increased TNF-alfa and TGF-Beta. Renal AKT, mTOR, and rpS6 pathway were increased, PTEN was decreased on SNS. And muscular Akt and mTOR were decreased on SNS. SIGNIFICANCE: The RET before and after the 5/6Nx ameliorates all these parameters mentioned above, suggesting that RET is a good non-pharmacological approach to diminish complications frequently found in CKD. We also suggest that the AKT-m-TOR pathway can play an important role in these beneficial outcomes of RET on the CKD animal model.

Drivers of Infectious Disease Seasonality: Potential Implications for COVID-19.

Not 1 year has passed since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). Since its emergence, great uncertainty has surrounded the potential for COVID-19 to establish as a seasonally recurrent disease. Many infectious diseases, including endemic human coronaviruses, vary across the year. They show a wide range of seasonal waveforms, timing (phase), and amplitudes, which differ depending on the geographical region. Drivers of such patterns are predominantly studied from an epidemiological perspective with a focus on weather and behavior, but complementary insights emerge from physiological studies of seasonality in animals, including humans. Thus, we take a multidisciplinary approach to integrate knowledge from usually distinct fields. First, we review epidemiological evidence of environmental and behavioral drivers of infectious disease seasonality. Subsequently, we take a chronobiological perspective and discuss within-host changes that may affect susceptibility, morbidity, and mortality from infectious diseases. Based on photoperiodic, circannual, and comparative human data, we not only identify promising future avenues but also highlight the need for further studies in animal models. Our preliminary assessment is that host immune seasonality warrants evaluation alongside weather and human behavior as factors that may contribute to COVID-19 seasonality, and that the relative importance of these drivers requires further investigation. A major challenge to predicting seasonality of infectious diseases are rapid, human-induced changes in the hitherto predictable seasonality of our planet, whose influence we review in a final outlook section. We conclude that a proactive multidisciplinary approach is warranted to predict, mitigate, and prevent seasonal infectious diseases in our complex, changing human-earth system.

The effects of lipopolysaccharide-induced reactive oxygen species were blunted by calcium oxalate in renal tubular epithelial cells.

BACKGROUND/AIM: Previously we demonstrated that calcium oxalate (CaOx) in LLC-PK1 cells and oxalate in MDCK cells induce tubular damage and greater glycosaminoglycan synthesis. We test the hypothesis that reactive oxygen species (ROS) and prostaglandins mediate these effects. METHODS: LLC-PK1 and MDCK cells were exposed to graded concentrations of CaOx, oxalate or both. Glycosaminoglycan synthesis was analyzed through metabolic labeling and gel electrophoresis. Cell permeability and lipid peroxidation were assessed by lactate dehydrogenase release and malondialdehyde levels. Hydrogen peroxide and superoxide anion were analyzed using 2',7'-dichlorofluorescein and luminol. Cyclooxygenase-2 expression and prostaglandin E2 production were assessed by RT-PCR and ELISA, respectively. RESULTS: In LLC-PK1 cells exposed to CaOx, we observed increased cell permeability, no induction of ROS or lipid peroxidation, inability to produce lipopolysaccharide-induced ROS and increases in prostaglandin E2. Indomethacin used alone increased glycosaminoglycan synthesis but did not potentiate CaOx-induced effects. In MDCK cells exposed to oxalate we observed increased cell permeability, ROS production only at higher concentrations and inability to produce lipopolysaccharide-induced ROS. Indomethacin alone had no effect but increased oxalate-induced glycosaminoglycan synthesis. CONCLUSIONS: Prostaglandins modulate endogenous production of glycosaminoglycans in LLC-PK1 cells, as well as regulate oxalate-induced glycosaminoglycan synthesis in MDCK cells. Rather than increasing, CaOx and oxalate blunted lipopolysaccharide-induced ROS production. We could speculate that patients with recurrent nephrolithiasis may lose antimicrobial protection induced by ROS during infections.

Targeted enhancement of the biological activity of the antineoplastic agent, neocarzinostatin. Studies in murine neuroblastoma cells.

The development of chemotherapeutic approaches to cancer has been hampered by the toxicity of proposed agents for normal rapidly dividing cells. By using neocarzinostatin, a "pro-drug" which is activated by reduction by thiol compounds, adjunctively with 6-mercaptodopamine, a thiol-containing dopamine analogue, we have been able to enhance neocarzinostatin toxicity for cells of the neural crest tumor neuroblastoma. Thiol compounds that are not neurotransmitter analogues do not act synergistically with neocarzinostatin in this system. Since most normal rapidly dividing cells do not have surface dopamine receptors, we propose this approach for the selective targeting of toxicity for neuroblastoma cells. We further introduce cell-selective activation of prodrugs as a new chemotherapeutic strategy which demands further development.

Role for intrarenal mechanisms in the impaired salt excretion of experimental nephrotic syndrome.

A unilateral model of puromycin aminonucleoside (PAN)-induced albuminuria was produced in Munich-Wistar rats to examine the mechanisms responsible for renal salt retention. 2 wk after selective perfusion of left kidneys with PAN (n = 8 rats) or isotonic saline (control, n = 7 rats), increases in albumin excretion and decreases in sodium excretion were demonstrated in PAN-perfused but not in nonperfused kidneys of PAN-treated rats although systemic plasma protein concentration remained at control level. Total kidney glomerular filtration rate (GFR) and superficial single nephron (SN) GFR were also reduced selectively in PAN-perfused kidneys, on average by approximately 30%, due primarily to a marked decline in the glomerular capillary ultrafiltration coefficient (Kf), which was also confined to PAN-perfused kidneys. Values for absolute proximal reabsorption (APR) were also selectively depressed in PAN-perfused kidneys, in keeping with a similarly selective decline in peritubular capillary oncotic pressure measured in these kidneys, the latter also a consequence of the fall in Kf. In a separate group of seven PAN-treated rats, however, no differences were detected between PAN-perfused and nonperfused kidneys in the absolute amount of sodium reaching the early (0.77 +/- 0.09 neq/min vs. 0.74 +/- 0.08, P greater than 0.40) and late portions of superficial distal tubules (0.31 +/- 0.02) neq/min vs. 0.32 +/- 0.05, P greater than 0.50), despite the lesser filtered load of sodium in PAN-perfused kidneys. Suppressed sodium reabsorption in both proximal convoluted tubules and short loops of Henle of PAN-perfused kidneys contributed to this equalization of sodium delivery rates to the late distal tubule, as did comparable reabsorption along distal convolutions. In two additional groups of PAN-treated rats, infusion of saralasin (0.3 mg/kg per h, i.v.) led to substantial increases in total kidney GFR and SNGFR in PAN-perfused but not in nonperfused kidneys. Despite these increases in total and SNGFR, urinary sodium excretion by PAN-perfused kidneys remained at a level far below that for nonperfused kidneys, again indicating that the antinatriuresis characterizing the PAN-perfused kidney is due to alterations in sodium handling by the tubules rather than changes in GFR. These results therefore indicate (a) that reductions in Kf and depressed sodium reabsorption by proximal tubules and Henle's loop segments in this model are brought about by intrarenal rather than circulating or systemic factors, and (b) assuming that superficial nephrons are representative of the entire nephron population, renal salt retention in this model is due primarily to intrarenal factor(s) acting beyond the distal convolution.

Anxiety and depression symptoms in recurrent painful renal lithiasis colic.

Several studies have reported that symptoms of anxiety and depression are significantly associated with diseases characterized by painful crises. However, there is little information about the psychological aspects of recurrent painful episodes of renal stone disease. Our objective was to evaluate the association of symptoms of anxiety, depression and recurrent painful renal colic in a case-control study involving 64 subjects (32 cases/32 controls) matched for age and sex. Cases were outpatients with a confirmed diagnosis of nephrolithiasis as per their case history, physical examination, image examination and other laboratory exams. Patients had a history of at least two episodes within a 3-year period, and were currently in an intercrisis interval. The control group consisted of subjects seen at the Ophthalmology Outpatient Clinic of this University Hospital with only eye refraction symptoms, and no other associated disease. Symptoms of anxiety were evaluated by the State-Trait Anxiety Inventory and symptoms of depression by the Beck Depression Inventory. Statistically significant differences were observed between patients with nephrolithiasis and controls for anxiety state (P = 0.001), anxiety trait (P = 0.005) and symptoms of depression (odds ratio = 3.74; 95%CI = 1.31-10.62). The Beck Depression Inventory showed 34.5% of respondents with moderate and 6% with severe levels of depression. There was a significant linear correlation between symptoms of anxiety (P = 0.002) and depression (P < 0.001) and the number of recurrent colic episodes (anxiety-state: P = 0.016 and anxiety-trait: P < 0.001). These data suggest an association between recurrent renal colic and symptoms of both anxiety and depression.

Endothelin-1 receptor antagonists protect the kidney against the nephrotoxicity induced by cyclosporine-A in normotensive and hypertensive rats.

Cyclosporin-A (CsA) is an immunosuppressant associated with acute kidney injury and chronic kidney disease. Nephrotoxicity associated with CsA involves the increase in afferent and efferent arteriole resistance, decreased renal blood flow (RBF) and glomerular filtration. The aim of this study was to evaluate the effect of Endothelin-1 (ET-1) receptor blockade with bosentan (BOS) and macitentan (MAC) antagonists on altered renal function induced by CsA in normotensive and hypertensive animals. Wistar and genetically hypertensive rats (SHR) were separated into control group, CsA group that received intraperitoneal injections of CsA (40 mg/kg) for 15 days, CsA+BOS and CsA+MAC that received CsA and BOS (5 mg/kg) or MAC (25 mg/kg) by gavage for 15 days. Plasma creatinine and urea, mean arterial pressure (MAP), RBF and renal vascular resistance (RVR), and immunohistochemistry for ET-1 in the kidney cortex were measured. CsA decreased renal function, as shown by increased creatinine and urea. There was a decrease in RBF and an increase in MAP and RVR in normotensive and hypertensive animals. These effects were partially reversed by ET-1 antagonists, especially in SHR where increased ET-1 production was observed in the kidney. Most MAC effects were similar to BOS, but BOS seemed to be better at reversing cyclosporine-induced changes in renal function in hypertensive animals. The results of this work suggested the direct participation of ET-1 in renal hemodynamics changes induced by cyclosporin in normotensive and hypertensive rats. The antagonists of ET-1 MAC and BOS reversed part of these effects.

The scientific production in health and biological sciences of the top 20 Brazilian universities.

Brazilian scientific output exhibited a 4-fold increase in the last two decades because of the stability of the investment in research and development activities and of changes in the policies of the main funding agencies. Most of this production is concentrated in public universities and research institutes located in the richest part of the country. Among all areas of knowledge, the most productive are Health and Biological Sciences. During the 1998-2002 period these areas presented heterogeneous growth ranging from 4.5% (Pharmacology) to 191% (Psychiatry), with a median growth rate of 47.2%. In order to identify and rank the 20 most prolific institutions in these areas, searches were made in three databases (DataCAPES, ISI and MEDLINE) which permitted the identification of 109,507 original articles produced by the 592 Graduate Programs in Health and Biological Sciences offered by 118 public universities and research institutes. The 20 most productive centers, ranked according to the total number of ISI-indexed articles published during the 1998-2003 period, produced 78.7% of the papers in these areas and are strongly concentrated in the Southern part of the country, mainly in São Paulo State.

Adjunctive use of ethiofos (WR-2721) with free radical-generating chemotherapeutic agents in mice: new caveats for therapy.

The neurotransmitter analogue 6-hydroxydopamine has been proposed as a selective chemotherapeutic agent for peripheral neural crest tumors. It exerts its action through the generation of cytotoxic oxygen free radicals. Unfortunately, it is toxic to normal peripheral neurons as well. Ethiofos (WR-2721) is a free radical scavenger which appears to be preferentially taken up by normal cells relative to some tumor cells. WR-2721 has been assayed as a protector of the normal autonomic nervous system in mice treated with 6-hydroxydopamine. Although WR-2721 has some activity in this regard, its therapeutic window is narrowed by its depletion of glutathione, a phenomenon which has not previously been noted with this drug. These findings raise issues regarding the safety of adjunctive use of WR-2721 with oxygen free radical-generating chemotherapeutic agents.

Potentiation of enediyne-induced apoptosis and differentiation by Bcl-2.

Bcl-2 overexpression has been shown to be protective against apoptosis induced by a variety of mechanistically diverse chemotherapeutic drugs. Recently, oxygen radical species have been implicated in the process of apoptosis, and Bcl-2 has been proposed to exert its protective effect by altering the redox state of the cell. Unlike most other chemotherapeutic agents, naturally occurring enediynes are rendered more cytotoxic in the presence of a higher reducing potential, because as prodrugs, they require reduction for activation. We demonstrate herein that induction of Bcl-2 expression in PC12 cells potentiates the induction of apoptosis and differentiation by the enediyne neocarzinostatin. In contradistinction, Bcl-2 abrogates the induction of apoptosis and differentiation by the autoactivating enediyne, enediyne-5, and the non-enediyne chemotherapeutic agent, cisplatin. We further demonstrate that enediyne potentiation by Bcl-2 is related to an increase in cellular glutathione. The present studies suggest that enediynes that require reductive activation might be critically useful agents in the therapy of tumors such as neuroblastomas and estrogen-responsive breast cancers, the resistance of which is related to up-regulation of Bcl-2.

Biochemical and quantitative histochemical study of reduced pyridine nucleotide dehydrogenation by human colonic carcinomas.

This study shows a marked increase in the activity of the soluble enzyme DT-diaphorase and of the histochemical activity of the reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate nitroblue tetrazolium menadione-mediated reductases in human colonic carcinomas when compared with the enzymatic activities of portions of the colon uninvolved by the carcinomatous process. The activity of the reductases in histological sections was quantitated with a microphotometer. It is believed that the increase in histochemical nitroblue-tetrazolium reductase activity in the histochemical reactions in colonic carcinomas is a real reflection of the activity of the DT-diaphorase, because the increase in the dehydrogenation of reduced nicotinamide adenine dinucleotide equals the dehydrogenation of reduced nicotinamide adenine dinucleotide phosphate when measured biochemically in the soluble fraction, or histochemically, by microspectophotometry in tissue sections; meanwhile, the biochemical dehydrogenation of NAD(P)H by the particulate fractions shows that the enzymatic activities are not altered by the neoplastic process. The biological significance of these changes is discussed in the text.

Adjunctive treatment of murine neuroblastoma with 6-hydroxydopamine and Tempol.

Currently available therapy for disseminated neuroblastoma affords only a 5-20% 5-year survival rate. We have attempted to design targeted chemotherapy for this disease by exploiting the dopamine uptake system on neuroblastoma cells. 6-Hydroxydopamine (6OHDA) is a neurotransmitter analogue, which generates cytolytic oxygen radicals in neuroblastoma cells that take it up. It is, however, predictably, systemically toxic, because of its spontaneous oxidation. Its toxicity is particularly severe in the sympathetic nervous system, because this tissue selectively concentrates dopamine and its analogues. Lowering the dose of 6OHDA below toxic levels prohibitively compromises its antitumor effect. To avoid both the systemic and sympathetic nervous system toxicity yet retain the antitumor efficacy of 6OHDA, we have used the antioxidant Tempol adjunctively with 6OHDA. Administration of Tempol (250 mg/kg, i.p.) 10 min prior to administration of toxic doses of 6OHDA (350 or 400 mg/kg, i.p.) resulted in a decrease in the mortality rate, sympathetic nervous system impairment, and activity impairment compared with those seen with 6OHDA alone. Tumor weights from mice administered saline or Tempol alone were 3.6 +/- 1.9 and 2.9 +/- 0.7 g, respectively. In contrast, mice administered Tempol followed by 6OHDA had an average tumor weight of 0.7 +/- 0.3 g. Tumor incidence was also reduced from 80-100% to 40%. Studies performed using electron spin resonance spectroscopy suggest that Tempol acts in this system by reacting directly with both the 6OHDA radical and, in the presence of iron, its oxidation product, the hydroxyl radical.

Apoptosis in the absence of caspase 3.

MCF-7 human breast cancer cells do not express caspase 3, thought by some to be a critical component of the apoptosis cascade. Nonetheless, both mock- and bcl-2-transfected MCF-7 cells undergo apoptosis after treatment with a variety of stimuli, including the DNA-cleaving antimitotic agent, neocarzinostatin (NCS). Transfection with bcl-2 shifts the concentration-response curve to NCS but does not change the phenomenology of apoptosis when it occurs. In both cases, NCS treatment results in condensation and fragmentation of MCF-7 cell nuclei and release of cytochrome c from the mitochondria to the cytosol. This apoptosis is accompanied by decreased levels of Bcl-2 and increased levels of Bax. Using a series of caspase inhibitors with overlapping specificities, enzyme-specific chromogenic substrates, and an antibody specific for activated caspase 7, we have determined that apoptosis in MCF-7 cells proceeds via sequential activation of caspases 9, 7 and 6. P21 is detected only after activation of caspase 7, and P53 is neither expressed at baseline nor up-regulated with apoptosis induction. This pathway bypasses the need for activated caspase 3 in these cells.

Cell line dependence of Bcl-2-induced alteration of glutathione handling.

Bcl-2 has been associated with both oxidative and antioxidative effects in vivo. Moreover, despite evidence that Bcl-2 is antiapoptotic by virtue of its effect on reactive oxygen species and their scavengers, Bcl-2 exerts its antiapoptotic effects even under anaerobic conditions. The reasons for the variable relationship between Bcl-2 and reactive oxygen species are not clear. The present studies demonstrate that the impact of Bcl-2 on glutathione (GSH) metabolism is cell line-dependent. Bcl-2 overproduction in PC12 cells is associated with increased functional thiol reserves, increased reductive activation of chemotherapeutic prodrugs, and GSH accumulation after treatment with N-acetylcysteine. In contrast, Bcl-2-overproducing MCF-7 breast cancer cells demonstrate neither altered GSH handling nor potentiation of chemotherapeutic prodrug reduction. These findings indicate that the effects of Bcl-2 on GSH handling are millieu-dependent. This could account for the variable effects of Bcl-2 in in vivo systems. Furthermore, since our previous studies have demonstrated that reduction-dependent prodrugs may be useful chemotherapeutic agents against tumors that demonstrate altered GSH handling, screening in vitro for alteration of GSH handling may predict responsiveness of such tumors to these reduction-dependent agents.

Proteoglycans and glycosaminoglycans synthesized in vitro by mesangial cells from normal and diabetic rats.

In the renal glomerulus, two extracellular matrices have been identified, the glomerular basement membrane and the mesangial matrix. Accumulation of glomerular extracellular matrix is a conspicuous feature of most forms of progressive glomerular disease, including diabetic nephropathy. Since proteoglycans are prominent components of the extracellular matrix, we examined the glycosaminoglycans and proteoglycans synthesized in vitro by mesangial cells from normal and diabetic rats. A mixture of dermatan sulfate and heparan sulfate was recovered. Dermatan sulfate was the predominant glycosaminoglycan synthesized and most of it was released to the culture medium, in contrast to heparan sulfate which was found to be cell associated to a higher degree. The dermatan sulfate chains are composed by D-glucuronic and L-iduronic acid-containing disaccharides and are highly sulfated. Mesangial cells from diabetic rats produce much more glycosaminoglycans than mesangial cells from normal rats, especially dermatan sulfate and this increase was proportional to the duration of diabetes. In contrast, exposure of mesangial cell from normal rats to elevated glucose did not lead to any changes in glycosaminoglycan synthesis, indicating that this short-term culture conditions may not adequately simulate diabetes mellitus. Other factors related to diabetes environment may be responsible for the observed alterations. The dermatan sulfate was secreted to the medium as proteoglycan. Two dermatan sulfate proteoglycans were identified, with molecular weights of 120 and 85 kDa respectively. The proteoglycan core protein M(r) was 45 kDa and the dermatan sulfate chains were 35 kDa. It is possible that the two proteoglycans represent two populations, one with two dermatan sulfate side chains (120 kDa) and the other with only one side chain (85 kDa), presumably fitting in the decorin/biglycan family of small proteoglycans.