Novel epoxy steroids from the indian ocean soft coral Sarcophyton crassocaule

A detailed further examination of the Indian Ocean soft coral Sarcophyton crassocaule resulted in the isolation of altogether 17 compounds of which two (1 and 2) are novel 17beta,20beta-epoxy steroids and one is a new dihydroxygorgost-5-en (3). The other compounds include the four hippurin steroids (4-7) reported earlier, and some known derivatives such as methyl arachidonate, batyl alcohol, a mixture of monohydroxy sterols, 3beta-hydroxypregn-5-en-20-one, two prostaglandin derivatives (PGB(2) acid and its methyl ester), and 9-oxo-9, 11-secogorgost-5-ene-3beta,11-diol (8). The structure of new dihydroxygorgostene derivative was established as gorgost-5-ene-3beta,11alpha-diol (3), while the structures of the novel epoxy steroids were established as 17beta,20beta-epoxy-23, 24-dimethylcholest-5-ene-3beta,22-diol (2) and its 3beta, 22-diacetate (1), respectively.     

Effect of reinstitution of good glycemic control on retinal oxidative stress and nitrative stress in diabetic rats

Clinical and experimental studies have shown that reinstitution of good glycemic control (GC) after a period of poor glycemic control (PC) does not produce immediate benefits on the progression of retinopathy, and hyperglycemia is sufficient to initiate the development of diabetic retinopathy. In this study, the effect of reinstitution of GC on hyperglycemia-induced increased oxidative stress and nitrative stress was evaluated in the retina of rats maintained in PC before initiation of GC. In diabetic rats, 2 or 6 months of PC (GHb >11.0%) was followed by 7 months of GC (GHb <5.5%). Reinstitution of GC after 2 months of PC inhibited elevations in retinal lipid peroxides and NO levels by approximately 50%, but failed to have any beneficial effects on nitrotyrosine formation. However, reversal of hyperglycemia after 6 months of PC had no significant effect on retinal oxidative stress and NO levels (P < 0.02 vs. normal). In the same rats, inducible nitric oxide synthase expression and nitrotyrosine levels remained elevated by >80% compared with normal rats or rats kept in GC for the duration. This suggests that oxidative and nitrative modifications in retina occur early in the course of development of retinopathy in diabetes. These abnormalities are not easily reversed by reinstitution of GC, and the duration of PC before initiation of GC influences the outcome of the reversal. Characterization of the abnormalities responsible for the resistance of retinopathy to arrest after reinstitution of GC will help identify potential future therapies to inhibit progression of diabetic retinopathy.     

Response of capillary cell death to aminoguanidine predicts the development of retinopathy: comparison of diabetes and galactosemia

PURPOSE: To examine the relationship between early retinal capillary cell apoptosis and late histologic lesions of diabetic retinopathy and to compare the effects of aminoguanidine (AMG) on the retinopathies caused by diabetes and galactose feeding. METHODS: Rats with alloxan-induced diabetes and rats fed a 30% galactose diet (known to induce diabetic-like retinopathy) were assigned randomly to receive diet with (2.5 g/kg diet) or without AMG. After 6 to 8 months of diabetes or galactosemia, retinal trypsin digests were prepared, and capillary cell apoptosis was quantitated using the Tdt-mediated dUTP nick-end labeling (TUNEL) reaction in association with morphologic evidence of nuclear fragmentation. At 18 months duration, pericyte ghosts and acellular capillaries were quantitated in the isolated vasculature. Several advanced glycation end products (AGEs) were measured at 4 months of study and at 18 months of study by established methods to assess biochemical effects of AMG. RESULTS: As expected, both diabetic and galactosemic rats showed increased frequency of TUNEL-positive capillary cells at 6 to 8 months and vascular lesions characteristic of retinopathy at 18 months. AMG inhibited both the early apoptosis and late histopathology in the diabetic rats, but neither of these abnormalities in the galactosemic rats. In contrast to its preventative effect on retinopathy in the diabetic rats, AMG showed no inhibitory effect on levels of hemoglobin AGE, or tail collagen pentosidine, fluorescence, and thermal breaking time. Diabetes of 4 months' duration did not cause a detectable increase in retinal levels of several AGEs. CONCLUSIONS: The frequency of early apoptosis in retinal microvascular cells predicted the development of the histologic lesions of retinopathy in diabetes as well as in galactosemia. The beneficial effect of AMG on retinal lesions in diabetes is exerted on pathways that are either not operative or are less important in galactosemia and that may not relate to the accumulation of AGEs.     

Primary choriocarcinoma of stomach

A 45 year old patient wad admitted with pain abdomen and a palpable mass in the epigastrium of 3 months duration. Endoscopy revealed growth in the stomach and biopsy showed poorly differentiated Carcinoma. Distal radical subtotal gastrectomy was done. Histopathology revealed choriacarcinoma with Syncytiotrophoblastic and cytotrophoblastic and foci of adenocarcinoma. Postoperatively urine and serum had very high levels of beta-human chorionic gonogotrophins (B-HCG). Immunochemistry showed positivity for B-HCG. Clinically and on scan both the testis were normal. Because of its rarity, we are presenting this case with brief review of literature.     

Diabetes-induced elevations in retinal oxidative stress, protein kinase C and nitric oxide are interrelated

Hyperglycemia results in various retinal metabolic abnormalities that can contribute to the development of retinopathy, but it has been difficult to recognize which abnormalities are critical. In this study, the possible inter-relationship between hyperglycemia-stimulated oxidative stress, protein kinase C (PKC), and nitric oxide (NO) was investigated by examining the effects of inhibitors of oxidative stress, PKC and NO on glucose-induced retinal oxidative stress, PKC activity and NO levels concentrations, both under in vitro conditions in retinal endothelial cells and isolated retina, and in vivo in the retina from diabetic rats. Bovine retinal endothelial cells were incubated in 5 or 30 mM glucose for 3 days in the presence or absence of inhibitors of oxidative stress (N-acetyl cysteine), PKC (LY333531), or NO (L-NAME). Incubation of retinal endothelial cells in 30 mM glucose resulted in an approximately 2-fold elevation in retinal TBARS, PKC and NO. Addition of N-acetyl cysteine, LY333531, or L-NAME significantly inhibited glucose-induced elevation in oxidative stress, NO and PKC. Similar results were obtained when intact retinas from normal rats were incubated with 30 mM glucose for 6 hours. In diabetic rats, elevations in retinal TBARS, PKC and NO were observed at 2 months of diabetes, and administration of N-acetyl cysteine, LY333531 or aminoguanidine prevented diabetes-induced elevation in retinal TBARS and NO levels, and PKC activity. Thus, these results suggest that diabetes-induced metabolic abnormalities, are apparently interrelated in retina; inhibiting a single retinal abnormality may have multiple beneficial effects to correct retinal dysmetabolism and to inhibit the development of retinopathy. Received: 14 March 2001 / Accepted in revised form: 12 November 2001     

Glucose activates the carboxyl methylation of gamma subunits of trimeric GTP-binding proteins in pancreatic beta cells. Modulation in vivo by calcium, GTP, and pertussis toxin.

The gamma subunits of trimeric G-proteins (gamma1, gamma2, gamma5, and gamma7 isoforms) were found to be methylated at their carboxyl termini in normal rat islets, human islets and pure beta [HIT-T15] cells. Of these, GTPgammaS significantly stimulated the carboxyl methylation selectively of gamma2 and gamma5 isoforms. Exposure of intact HIT cells to either of two receptor-independent agonists--a stimulatory concentration of glucose or a depolarizing concentration of K+--resulted in a rapid (within 30 s) and sustained (at least up to 60 min) stimulation of gamma subunit carboxyl methylation. Mastoparan, which directly activates G-proteins (and insulin secretion from beta cells), also stimulated the carboxyl methylation of gamma subunits in intact HIT cells. Stimulatory effects of glucose or K+ were not demonstrable after removal of extracellular Ca2+ or depletion of intracellular GTP, implying regulatory roles for calcium fluxes and GTP; however, the methyl transferase itself was not directly activated by either. The stimulatory effects of mastoparan were resistant to removal of extracellular Ca2+, implying a mechanism of action that is different from glucose or K+ but also suggesting that dissociation of the alphabetagamma trimer is conducive to gamma subunit carboxyl methylation. Indeed, pertussis toxin also markedly attenuated the stimulatory effects of glucose, K+ or mastoparan without altering the rise in intracellular calcium induced by glucose or K+. Glucose-induced carboxyl methylation of gamma2 and gamma5 isoforms was vitiated by coprovision of any of three structurally different cyclooxygenase inhibitors. Conversely, exogenous PGE2, which activates Gi and Go in HIT cells and which thereby would dissociate alpha from beta(gamma), stimulated the carboxyl methylation of gamma2 and gamma5 isoforms and reversed the inhibition of glucose-stimulated carboxyl methylation of gamma subunits elicited by cyclooxygenase inhibitors. These data indicate that gamma subunits of trimeric G-proteins undergo a glucose- and calcium-regulated methylation-demethylation cycle in insulin-secreting cells, findings that may imply an important role in beta cell function. Furthermore, this is the first example of the regulation of the posttranslational modification of G-protein gamma subunits via nonreceptor-mediated activation mechanisms, which are apparently dependent on calcium influx and the consequent activation of phospholipases releasing arachidonic acid.     

Lead-induced Hepatotoxicity and Evaluation of Certain Anti-stress Adaptogens in Poultry

A total of 225 day-old sexed male broiler chicks (Vencobb strain) were divided randomly into 15 groups consisting of 15 chicks in each group to study the toxicity of lead on hepatocytes. Group 1 was maintained on basal diet, group 2 on polyherbal formulation (PHF; stressroak), group 3 on shilajith, group 4 on amla and group 5 on vit E + Se. Group 6 was maintained on lead for 6 weeks and group 7 on lead for 4 weeks and subsequently on basal diet without lead for the remaining 2 weeks. Groups 8, 9, 10 and 11 were given lead along with PHF, shilajith, amla and vit E + Se, respectively, throughout 6 weeks. Groups 12, 13, 14 and 15 were given lead containing diet for the first 4 weeks and subsequently treated with PHF, shilajith, amla and vit E + Se, respectively, for the remaining 2 weeks. The activity of alanine transaminase (ALT) was significantly (P<0.05) increased in the toxic control groups at the end of 4(th) week as compared to group 1. However, following treatment, there was a significant (P<0.05) reversal in groups 12-15. The activity of Na(+)/K(+)-ATPase, Ca(2+)ATPase, Mg(2+)ATPase and CYP(450) was significantly (P<0.05) reduced in the liver of toxic control groups 6 and 7 as compared to groups1 through 5, which had the maximum activity of all the groups. Groups 8 through 15 revealed a significant (P<0.05) increase in the activity of these hepatocytic enzymes. The histological sections of the liver in lead toxic control (group 6) showed moderate focal lymphoid aggregates in liver, whereas the lesions were mild to moderate in treated groups and there were no observable lesions in plain control groups. The study revealed protective effect of PHF (stressroak), shilajith, amla and vit E + Se in lead-induced hepatocytic damage.     

A comparison of HWRF, ARW and NMM models in Hurricane Katrina (2005) simulation

The life cycle of Hurricane Katrina (2005) was simulated using three different modeling systems of Weather Research and Forecasting (WRF) mesoscale model. These are, HWRF (Hurricane WRF) designed specifically for hurricane studies and WRF model with two different dynamic cores as the Advanced Research WRF (ARW) model and the Non-hydrostatic Mesoscale Model (NMM). The WRF model was developed and sourced from National Center for Atmospheric Research (NCAR), incorporating the advances in atmospheric simulation system suitable for a broad range of applications. The HWRF modeling system was developed at the National Centers for Environmental Prediction (NCEP) based on the NMM dynamic core and the physical parameterization schemes specially designed for tropics. A case study of Hurricane Katrina was chosen as it is one of the intense hurricanes that caused severe destruction along the Gulf Coast from central Florida to Texas. ARW, NMM and HWRF models were designed to have two-way interactive nested domains with 27 and 9 km resolutions. The three different models used in this study were integrated for three days starting from 0000 UTC of 27 August 2005 to capture the landfall of hurricane Katrina on 29 August. The initial and time varying lateral boundary conditions were taken from NCEP global FNL (final analysis) data available at 1 degree resolution for ARW and NMM models and from NCEP GFS data at 0.5 degree resolution for HWRF model. The results show that the models simulated the intensification of Hurricane Katrina and the landfall on 29 August 2005 agreeing with the observations. Results from these experiments highlight the superior performance of HWRF model over ARW and NMM models in predicting the track and intensification of Hurricane Katrina.     

Abrogation of MMP-9 gene protects against the development of retinopathy in diabetic mice by preventing mitochondrial damage

OBJECTIVE

In the development of diabetic retinopathy, mitochondrial dysfunction is considered to play an important role in the apoptosis of retinal capillary cells. Diabetes activates matrix metalloproteinase-9 (MMP-9) in the retina and its capillary cells, and activated MMP-9 becomes proapoptotic. The objective of this study is to elucidate the plausible mechanism by which active MMP-9 contributes to the mitochondrial dysfunction in the retina.

RESEARCH DESIGN AND METHODS

Using MMP-9 gene knockout (MMP-KO) mice, we investigated the effect of MMP-9 regulation on diabetes-induced increased retinal capillary cell apoptosis, development of retinopathy, mitochondrial dysfunction and ultrastructure, and mitochondrial DNA (mtDNA) damage. To understand how diabetes increases mitochondrial accumulation of MMP-9, interactions between MMP-9 and chaperone proteins (heat shock protein [Hsp] 70 and Hsp60) were evaluated. The results were confirmed in the retinal mitochondria from human donors with diabetic retinopathy, and in isolated retinal endothelial cells transfected with MMP-9 small interfering RNA (siRNA).

RESULTS

Retinal microvasculature of MMP-KO mice, diabetic for ∼7 months, did not show increased apoptosis and pathology characteristic of retinopathy. In the same MMP-KO diabetic mice, activation of MMP-9 and dysfunction of the mitochondria were prevented, and electron microscopy of the retinal microvasculature region revealed normal mitochondrial matrix and packed lamellar cristae. Damage to mtDNA was protected, and the binding of MMP-9 with Hsp70 or Hsp60 was also normal. As in the retina from wild-type diabetic mice, activation of mitochondrial MMP-9 and alterations in the binding of MMP-9 with chaperone proteins were also observed in the retina from donors with diabetic retinopathy. In endothelial cells transfected with MMP-9 siRNA, high glucose–induced damage to the mitochondria and the chaperone machinery was ameliorated.

CONCLUSIONS

Regulation of activated MMP-9 prevents retinal capillary cells from undergoing apoptosis by protecting mitochondrial ultrastructure and function and preventing mtDNA damage. Thus, MMP-9 inhibitors could have potential therapeutic value in preventing the development of diabetic retinopathy by preventing the continuation of the vicious cycle of mitochondrial damage.Diabetic retinopathy is one of the major causes of acquired blindness in working adults, but despite extensive research in the field, the molecular mechanism of its development remains elusive. In the pathogenesis of this slow progressing disease, capillary cells and other retinal cells are lost by apoptosis before histopathology characteristic of diabetic retinopathy can be seen in the retina (1–4). However, how a diabetic environment accelerates retinal cell apoptosis is unclear.Matrix metalloproteinases (MMPs), a class of approximately 25 zinc-dependent proteinases, regulate a variety of cellular functions, including apoptosis, proliferation, differentiation, and angiogenesis. In diabetes, MMPs are elevated in the retina and other tissues, and MMP-9, the largest member of the MMP family (5), is associated with many diabetes complications, including nephropathy, cardiomyopathy, and retinopathy (6). Our previous work has shown that in a diabetic environment, the activation of MMP-9 in the retina and its capillary cells is mediated by the Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase pathway, and activated MMP-9 induces the apoptosis of retinal capillary cells (7,8). The mechanism by which MMP-9 induces apoptosis in the pathogenesis of diabetic retinopathy remains to be explored.Damage to the retinal mitochondria is considered to play a major role in the development of diabetic retinopathy. Retinal mitochondria become dysfunctional, superoxide levels are elevated, and mitochondrial permeability is significantly increased, which leads to leakage of cytochrome c into the cytosol and acceleration of the apoptosis of retinal capillary cells (9,10). Emerging work has shown that MMPs are not present in the matrix alone, they are also found in the mitochondria, and induction of MMP-9 in the myocyte mitochondria is considered to act as a negative regulator of mitochondrial function (11). Our recent work has shown that MMP-2, another important member of MMP family, is present in the retinal mitochondria, and in diabetes, MMP-2 damages retinal mitochondria by modulating connexin43 (12). As diabetes activates both MMP-2 and MMP-9 in the retina, accelerating apoptosis of retinal capillary cells (7,8,12), the role of MMP-9 in retinal mitochondrial dysfunction in diabetes, leading to retinal capillary cell loss, needs further investigation.The aim of this study is to elucidate the plausible mechanism by which active MMP-9 contributes to the mitochondrial dysfunction in the retina, accelerating the apoptosis of capillary cells and ultimately resulting in retinopathy. Using MMP-9 gene knockout (MMP-KO) mice, we have investigated the effect of MMP-9 regulation on diabetes-induced increased retinal capillary cell apoptosis and the development of retinopathy. To understand the mechanism, the effect of regulation of MMP-9 on retinal mitochondrial dysfunction, mitochondrial DNA (mtDNA) damage and ultrastructure was evaluated in MMP-KO mice. As mitochondrial function is largely controlled by their membrane structure, and translocases in the outer membrane (the TOM complex) and translocases in the mitochondrial inner membrane (the TIM complex) help facilitate import of proteins (13,14), we have also investigated the mechanism by which diabetes increases mitochondrial accumulation of MMP-9. The results are confirmed in the retina from human donors with diabetic retinopathy and also in isolated retinal endothelial cells exposed to high glucose with the MMP-9 gene silenced.     

Gefitinib resistance resulted from STAT3-mediated Akt activation in lung cancer cells

Hyperactivation of Epidermal Growth Factor Receptor (EGFR) tyrosine kinase is prevalent in human lung cancer and its inhibition by the tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, initially controls tumor growth. However, most patients ultimately relapse due to the development of drug resistance. In this study, we have discovered a STAT3-dependent Akt activation that impairs the efficacy of gefitinib. Mechanistically, gefitinib increased association of EGFR with STAT3, which de-repressed STAT3 from SOCS3, an upstream suppressor of STAT3. Such a de-repression of STAT3 in turn fostered Akt activation. Genetic or pharmacological inhibition of STAT3 abrogated Akt activation and combined gefitinib with STAT3 inhibition synergistically reduced the growth of the tumor cells. Taken together, this study suggests that activation of STAT3 is an intrinsic mechanism of drug resistance in response to EGFR TKIs. Combinational targeting on both EGFR and STAT3 may enhance the efficacy of gefitinib or other EGFR TKIs in lung cancer.