High glucose-induced Ca overload and oxidative stress contribute to apoptosis of cardiac cells through mitochondrial dependent and independent pathways

Background

Cardiac cell apoptosis is the initiating factor of cardiac complications especially diabetic cardiomyopathy. Mitochondria are susceptible to the damaging effects of elevated glucose condition. Calcium overload and oxidative insult are the two mutually non-exclusive phenomena suggested to cause cardiac dysfunction. Here, we examined the effect of high-glucose induced calcium overload in calpain-1 mediated cardiac apoptosis in an in vitro setting.

Methods

H9c2, rat ventricular myoblast cell line was treated with elevated glucose condition and the cellular consequences were studied. Intracellular calcium trafficking, ROS generation, calpain-1 activation and caspase-12 and caspase-9 pathway were studied using flow cytometry, confocal microscopy and Western blot analysis.

Results

High-glucose treatment resulted in increased intracellular calcium ([Ca^2 +]i) which was mobilized to the mitochondria. Concomitant intra-mitochondrial calcium ([Ca^2 +]m) increase resulted in enhanced reactive oxygen and nitrogen species generation. These events led to mitochondrial dysfunction and apoptosis. Cardiomyocyte death exhibited several classical markers of apoptosis, including activation of caspases, appearance of annexin V on the outer plasma membrane, increased population of cells with sub-G₀/G₁ DNA content and nuclear condensation. Key findings include elucidation of cell signaling mechanism of high-glucose induced calcium-dependent cysteine protease calpain-1 activation, which triggers non-conventional caspases as alternate mode of cell death.

Conclusion

This information increases the understanding of cardiac cell death under hyperglycemic condition and can possibly be extended for designing new therapeutic strategies for diabetic cardiomyopathy.

General significance

The novel findings of the study reveal that high glucose induces apoptosis by both mitochondria-dependent and independent pathways via concomitant rise in intracellular calcium.     

Aliidiomarina haloalkalitolerans sp. nov., a marine bacterium isolated from coastal surface seawater

A novel Gram-negative, rod shaped, motile, non-sporing strictly aerobic bacterium, designated strain AK5^T, was isolated from a sea water sample collected near Visakhapatnam coast, Bay of Bengal, India. Colonies on marine agar were circular, 3–4 mm in diameter, creamish and rose with entire margin. Growth occurred at 10–40°C, 0.5–12% (w/v) NaCl and pH of 7–11. Strain AK5^T was oxidase and catalase positive. The fatty acids were dominated by iso-branched saturated and unsaturated fatty acids with a high abundance of iso-C_15:0, iso-C_17:0 and summed feature 9 (as defined by MIDI). Q8 was found to be the major respiratory quinone and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and four unidentified phospholipids as polar lipids. The DNA G+C content of strain AK5^T was 54.7 ± 0.2 mol%. Phylogenetic analysis revealed that strain AK5^T was a member of the genus Aliidiomarina and closely related to Aliidiomarina taiwanensis with a phylogenetic distance of 5.3% (94.7% 16S rRNA gene sequence similarity) and clustered with the same species. Results from the polyphasic taxonomy study support the conclusion that strain AK5^T represents a novel Aliidiomarina species, for which the name Aliidiomarina haloalkalitolerans sp. nov. is proposed. The type strain of A. haloalkalitolerans is AK5^T (= MTCC 11064^T = JCM 17359^T).     

Parasite burden and CD36-mediated sequestration are determinants of acute lung injury in an experimental malaria model

Although acute lung injury (ALI) is a common complication of severe malaria, little is known about the underlying molecular basis of lung dysfunction. Animal models have provided powerful insights into the pathogenesis of severe malaria syndromes such as cerebral malaria (CM); however, no model of malaria-induced lung injury has been definitively established. This study used bronchoalveolar lavage (BAL), histopathology and gene expression analysis to examine the development of ALI in mice infected with Plasmodium berghei ANKA (PbA). BAL fluid of PbA-infected C57BL/6 mice revealed a significant increase in IgM and total protein prior to the development of CM, indicating disruption of the alveolar-capillary membrane barrier-the physiological hallmark of ALI. In contrast to sepsis-induced ALI, BAL fluid cell counts remained constant with no infiltration of neutrophils. Histopathology showed septal inflammation without cellular transmigration into the alveolar spaces. Microarray analysis of lung tissue from PbA-infected mice identified a significant up-regulation of expressed genes associated with the gene ontology categories of defense and immune response. Severity of malaria-induced ALI varied in a panel of inbred mouse strains, and development of ALI correlated with peripheral parasite burden but not CM susceptibility. Cd36(-/-) mice, which have decreased parasite lung sequestration, were relatively protected from ALI. In summary, parasite burden and CD36-mediated sequestration in the lung are primary determinants of ALI in experimental murine malaria. Furthermore, differential susceptibility of mouse strains to malaria-induced ALI and CM suggests that distinct genetic determinants may regulate susceptibility to these two important causes of malaria-associated morbidity and mortality.     

Molecular mimicry in pauci-immune focal necrotizing glomerulonephritis

Pauci-immune focal necrotizing glomerulonephritis (FNGN) is a severe inflammatory disease associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA). Here we characterize autoantibodies to lysosomal membrane protein-2 (LAMP-2) and show that they are a new ANCA subtype present in almost all individuals with FNGN. Consequently, its prevalence is nearly twice that of the classical ANCAs that recognize myeloperoxidase or proteinase-3. Furthermore, antibodies to LAMP-2 cause pauci-immune FNGN when injected into rats, and a monoclonal antibody to human LAMP-2 (H4B4) induces apoptosis of human microvascular endothelium in vitro. The autoantibodies in individuals with pauci-immune FNGN commonly recognize a human LAMP-2 epitope (designated P(41-49)) with 100% homology to the bacterial adhesin FimH, with which they cross-react. Rats immunized with FimH develop pauci-immune FNGN and also develop antibodies to rat and human LAMP-2. Finally, we show that infections with fimbriated pathogens are common before the onset of FNGN. Thus, FimH-triggered autoimmunity to LAMP-2 provides a previously undescribed clinically relevant molecular mechanism for the development of pauci-immune FNGN.     

Inheritance of Resistance to Bacillus thuringiensis subsp. kurstaki in Trichoplusia ni

The genetic inheritance of resistance to a commercial formulation of Bacillus thuringiensis subsp. kurstaki was examined in a Trichoplusia ni colony initiated from a resistant population present in a commercial vegetable greenhouse in British Columbia, Canada. Progeny of F₁ reciprocal crosses and backcrosses between F₁ larvae and resistant (P_R) and susceptible (P_S) populations were assayed at different B. thuringiensis subsp. kurstaki concentrations. The responses of progeny of reciprocal F₁ crosses were identical, indicating that the resistant trait was autosomal. The 50% lethal concentration for the F₁ larvae was slightly higher than that for P_S, suggesting that resistance is partially recessive. The responses of both backcross progeny (F₁ × P_R, F₁ × P_S) did not correspond to predictions from a single-locus model. The inclusion of a nonhomozygous resistant parental line in the monogenic model significantly increased the correspondence between the expected and observed results for the F₁ × P_R backcross but decreased the correspondence with the F₁ × P_S backcross results. This finding suggests that resistance to B. thuringiensis subsp. kurstaki in this T. ni population is due to more than one gene.     

Thermoregulation during cold exposure: effects of prior exercise.

This study examined whether acute exercise would impair the body's capability to maintain thermal balance during a subsequent cold exposure. Ten men rested for 2 h during a standardized cold-air test (4.6 degrees C) after two treatments: 1) 60 min of cycle exercise (Ex) at 55% peak O(2) uptake and 2) passive heating (Heat). Ex was performed during a 35 degrees C water immersion (WI), and Heat was conducted during a 38.2 degrees C WI. The duration of Heat was individually adjusted (mean = 53 min) so that rectal temperature was similar at the end of WI in both Ex (38.2 degrees C) and Heat (38.1 degrees C). During the cold-air test after Ex, relative to Heat 1) rectal temperature was lower (P < 0.05) from minutes 40-120, 2) mean weighted heat flow was higher (P < 0.05), 3) insulation was lower (P < 0.05), and 4) metabolic heat production was not different. These results suggest that prior physical exercise may predispose a person to greater heat loss and to experience a larger decline in core temperature when subsequently exposed to cold air. The combination of exercise intensity and duration studied in these experiments did not fatigue the shivering response to cold exposure.     

BLUE LIGHT PHOTOREACTIVATION IN ULTRAVIOLET-IRRADIATED YOUNG SPOROPHYTES OF ALARIA ESCULENTA AND LAMINARIA SACCHARINA (PHAEOPHYTA)1

Exposure of ultraviolet (UV)-irradiated young sporophytes of Alaria esculenta (L.) Grev. and Laminaria saccharina (L.) Lamour. to visible light resulted in recovery from UV damage that would otherwise cause much higher mortality. For this photoreactivation, blue light was highly effective, whereas negligible reactivation was produced in green or red light. The blue quantum requirements for a 50% response were 1.9 mol·m^?2 in A. esculenta and 1.2 mol·m^?2 in L. saccharina, which were comparable to those reported for other blue light responses requiring high energy found in brown algae.     

Caspase inhibition prevents staurosporine-induced apoptosis in CHO-K1 cells

Apoptosis is a distinct form of programmed cell death that plays an important role in many biological processes.Although the phenotypes of apoptotic cells are well documented, little is known of the central mechanismleading to programmed cell death. Over the past few years, a number of ICE/CED-3 family proteases(also termed caspases) have been discovered and implicated as the common effectors of apoptosis. Inthis report, we demonstrate that induction of apoptosis in CHO-K1 cells by staurosporine, a broad spectruminhibitor of protein kinases, results in an increase in DEVD-dependent protease activity. These events werefollowed by nuclear DNA fragmentation and cell death. Inhibition of the DEVD-cleaving activity by a synthetictetrapeptide inhibitor DEVD-CHO, blocked staurosporine-induced downstream apoptotic phenotypes, suchas morphological characteristics and DNA fragmentation. These results suggest that staurosporine-inducedapoptosis in CHO-K1 cells is mediated through the CPP32/caspase-3-like cysteine proteases.     

Exertional fatigue, sleep loss, and negative energy balance increase susceptibility to hypothermia

The purpose ofthis study was to determine how chronic exertional fatigue and sleepdeprivation coupled with negative energy balance affectthermoregulation during cold exposure. Eight men wearing only shortsand socks sat quietly during 4-h cold air exposure (10°C)immediately after (<2 h, A) they completed 61 days of strenuousmilitary training (energy expenditure ~4,150 kcal/day, energy intake~3,300 kcal/day, sleep ~4 h/day) and again after short (48 h, SR)and long (109 days, LR) recovery. Body weight decreased 7.4 kg frombefore training to A, then increased 6.4 kg by SR, with an additional6.4 kg increase by LR. Body fat averaged 12% during A and SR andincreased to 21% during LR. Rectal temperature(T_re) was lower before andduring cold air exposure for A than for SR and LR.T_re declined during cold exposurein A and SR but not LR. Mean weighted skin temperature(_sk)during cold exposure was higher in A and SR than in LR. Metabolic rate increased during all cold exposures, but it was lower during A and LRthan SR. The mean body temperature (0.67 T_re + 0.33 _sk) threshold for increasing metabolism was lower during A than SR and LR.Thus chronic exertional fatigue and sleep loss, combined withunderfeeding, reduced tissue insulation and blunted metabolic heatproduction, which compromised maintenance of body temperature. A shortperiod of rest, sleep, and refeeding restored the thermogenic responseto cold, but thermal balance in the cold remained compromised untilafter several weeks of recovery when tissue insulation had beenrestored.