Structural basis of RasGRP binding to high-affinity PKC ligands

The Ras guanyl releasing protein RasGRP belongs to the CDC25 class of guanyl nucleotide exchange factors that regulate Ras-related GTPases. These GTPases serve as switches for the propagation and divergence of signaling pathways. One interesting feature of RasGRP is the presence of a C-terminal C1 domain, which has high homology to the PKC C1 domain and binds to diacylglycerol (DAG) and phorbol esters. RasGRP thus represents a novel, non-kinase phorbol ester receptor. In this paper, we investigate the binding of indolactam(V) (ILV), 7-(n-octyl)-ILV, 8-(1-decynyl)benzolactam(V) (benzolactam), and 7-methoxy-8-(1-decynyl)benzolactam(V) (methoxylated benzolactam) to RasGRP through both experimental binding assays and molecular modeling studies. The binding affinities of these lactams to RasGRP are within the nanomolar range. Homology modeling was used to model the structure of the RasGRP C1 domain (C1-RasGRP), which was subsequently used to model the structures of C1-RasGRP in complex with these ligands and phorbol 13-acetate using a computational docking method. The structural model of C1-RasGRP exhibits a folding pattern that is nearly identical to that of C1b-PKCdelta and is comprised of three antiparallel-strand beta-sheets capped against a C-terminal alpha-helix. Two loops A and B comprising residues 8-12 and 21-27 form a binding pocket that has some positive charge character. The ligands phorbol 13-acetate, benzolactam, and ILV are recognized by C1-RasGRP through a number of hydrogen bonds with loops A and B. In the models of C1-RasGRP in complex with phorbol 13-acetate, benzolactam, and ILV, common hydrogen bonds are formed with two residues Thr12 and Leu21, whereas other hydrogen bond interactions are unique for each ligand. Furthermore, our modeling results suggest that the shallower insertion of ligands into the binding pocket of C1-RasGRP compared to C1b-PKCdelta may be due to the presence of Phe rather than Leu at position 20 in C1-RasGRP. Taken together, our experimental and modeling studies provide us with a better understanding of the structural basis of the binding of PKC ligands to the novel phorbol ester receptor RasGRP.     

Novel dual inhibitors of AChE and MAO derived from hydroxy aminoindan and phenethylamine as potential treatment for Alzheimer's disease

Carbamate derivatives of N-propargylaminoindans (Series I) and N-propargylphenethylamines (Series II) were synthesized via multistep procedures from the corresponding hydroxy precursors. The respective rasagiline- and selegiline-related series were designed to combine inhibitory activities of both acetylcholine esterase (AChE) and monoamine oxidase (MAO) by virtue of their carbamoyl and propargylamine pharmacophores. Each compound was tested for these activities in vitro in order to find molecules with similar potencies against each enzyme. Compounds with such dual AChE and MAO inhibitory activities are expected to have potential for the treatment of Alzheimer's disease. The observed SAR also offers insight into the requirements of the active sites on these enzymes. A carbamate moiety was found to be essential for AChE inhibition, which was absent in the corresponding hydroxy precursors. The propargyl group caused 2-70-fold decrease in AChE inhibitory activity (depending on the position of the carbamoyl group) of Series I, but had little or no effect in Series II. Thus, the 6- and 7-carbamyloxyphenyls in Series I were either equipotent to, or slightly (2- to 5-fold) less active as AChE inhibitors than, the corresponding compounds in Series II, while the 4-carbamyloxyphenyls were more potent. The presence of the carbamate moiety in 6- and 7-carbamyloxyphenyls of Series I, considerably decreased MAO-A and -B inhibitory activity, compared to that of the parent hydroxy analogues, while the opposite was true for Series II. Thus, the 6- and 7-carbamyloxyphenyls in Series I were 2-3 orders of magnitude weaker MAO inhibitors while the 4- carbamyloxyphenyls were equipotent with the corresponding compounds in Series II. In both series, N-methylation of the propargylamine enhanced the MAO (A and B equally) inhibitory activities and decreased the AChE inhibitory activity. Two candidates belonging to the indan and tetralin ring systems (24c, 27b) and one phenethylamine (53d) were identified as possible leads for further development based on the following criteria: (a) comparable AChE and MAO-B inhibitory activities, (b) good to moderate AChE inhibitory activity, and (c) lack of strong MAO-A selectivity. However, it is likely that these compounds will be metabolized to the corresponding phenols, with inhibitory activities against AChE and/or MAO-A or -B, different from those of the parent carbamates. Thus, the apparent enzyme inhibition will be a result of the combined inhibition of all of these individual metabolites. The results of our ongoing in vivo screening programs will be published elsewhere.     

The antiplasmodial activity of isolates from Ajuga remota

Ajuga remota is the most frequently used medicinal herb for malaria treatment in Kenya. Its two known isolates ajugarin-1 (1) and ergosterol-5,8-endoperoxide (3) and a new isolate 8-O-acetylharpagide (2) were evaluated for their in vitro antiplasmodial activity. Ajugarin-1 was moderately active, with an IC(50) of 23.0 +/- 3.0 microM, as compared to chloroquine (IC(50) = 0.041 +/- 0.003 microM) against the chloroquine-sensitive (FCA 20/GHA) strain of Plasmodium falciparum. Ergosterol-5,8-endoperoxide was about 3x as potent (IC(50) = 8.2 +/- 1.1 microM), while 8-O-acetylharpagide, whose structure was established by spectroscopic evidence, was inactive. Both ajugarin-1 and ergosterol-5,8-endoperoxide did not exhibit cytotoxicity against A431 (skin carcinoma) cell line, but 8-O-acetylharpagide was significantly cytotoxic. This iridoid glucoside, which has been formerly isolated from Ajuga decumbens, was identified in A. remota for the first time.     

Decreased expression of heat shock protein 72 in skeletal muscle of patients with type 2 diabetes correlates with insulin resistance

Oxidative stress has been ascribed a role in the pathogenesis of diabetes and its complications, and stress proteins have been shown to protect organisms in vitro and in vivo against oxidative stress. To study the putative role of one of the most abundant cytoprotective stress proteins, inducible cytoplasmic 72-kDa-mass heat shock protein (Hsp-72), in the pathogenesis of diabetes, we measured its mRNA concentration in muscle biopsies from six type 2 diabetic patients and six healthy control subjects (protocol 1) as well as in 12 twin pairs discordant for type 2 diabetes and 12 control subjects undergoing a euglycemic-hyperinsulinemic clamp in combination with indirect calorimetry (protocol 2). The amount of Hsp-72 mRNA in muscle was significantly lower in type 2 diabetic patients than in healthy control subjects (in protocol 1: 5.2 +/- 2.2 vs. 53 +/- 32 million copies of Hsp-72 mRNA/microg total RNA, n = 6, P = 0.0039; in protocol 2: 3.2 +/- 3.3 vs. 43 +/- 31 million copies of Hsp-72 mRNA/microg total RNA, n = 12, P = 0.0001). Hsp-72 mRNA levels were also markedly reduced in the nondiabetic co-twins compared with healthy control subjects (5.8 +/- 5.0 vs. 43 +/- 31, n = 12, P = 0.0001), but they were also statistically significantly different from their diabetic co-twins when the difference between the pairs was compared (P = 0.0280). Heat shock protein mRNA content in muscle of examined patients correlated with the rate of glucose uptake and other measures of insulin-stimulated carbohydrate and lipid metabolism. In conclusion, the finding of decreased levels of Hsp-72 mRNA in skeletal muscle of patients with type 2 diabetes and its relationship with insulin resistance raises the question of whether heat shock proteins are involved in the pathogenesis of skeletal muscle insulin resistance in type 2 diabetes.     

Grape seed proanthocyanidins improved cardiac recovery during reperfusion after ischemia in isolated rat hearts

BACKGROUND: Increasing evidence shows that red wine consumption has cardioprotective effects. These effects have been attributed to the polyphenolic compounds in grapes. OBJECTIVE: We studied the effects of red grape seed proanthocyanidins on the recovery of postischemic function in isolated rat hearts. DESIGN: Two groups of rats were fed different doses of proanthocyanidin-rich extract for 3 wk and another group was untreated and served as controls. The animals were then anesthetized and the hearts were isolated and subjected to 30 min of ischemia followed by 2 h of reperfusion. Coronary effluents were collected during the third minute of reperfusion for measurement of oxygen free radicals by using electron spin resonance spectroscopy. RESULTS: In rats treated with 50 and 100 mg grape seed proanthocyanidins/kg, the incidence of reperfusion-induced ventricular fibrillation was reduced from its control value of 92% to 42% and 25%, respectively (P < 0.05 for both). The incidence of ventricular tachycardia showed the same pattern. In rats treated with 100 mg proanthocyanidins/kg, the recovery of coronary flow, aortic flow, and developed pressure after 60 min of reperfusion was improved by 32% +/- 8%, 98% +/- 8%, and 37% +/- 3%, respectively (P < 0.05 for all) compared with untreated control rats. Electron spin resonance studies indicated that proanthocyanidins significantly inhibited the formation of oxygen free radicals. In rats treated with 100 mg proanthocyanidins/kg, free radical intensity was reduced by 75% +/- 7% (P < 0.05) compared with the control rats. CONCLUSION: Grape seed proanthocyanidins have cardioprotective effects against reperfusion-induced injury via their ability to reduce or remove, directly or indirectly, free radicals in myocardium that is reperfused after ischemia.     

Carcinoma oesophagus--overview and update of literature

Oesophageal cancer is one of the most challenging pathological conditions confronting the surgeon. With advances in imaging techniques, correct evaluation is possible pre-operatively, which helps in planning of treatment and thereby improving overall survival. Treatment now-a-days is multimodality, but still surgery remains the mainstay of the treatment. Usually very less number of patients are amenable to surgical resection and in remainder, palliative treatment remains the primary aim of treatment. Chemoradiation has got some role as in some seen recent trials. Novel drugs, which target at molecular level, are in research phase at present.     

MIF in der therapiekontrolle von herpes simplex recidivans: Behandlung mit levamisole,BCG, urushiol und herpes antigen vaccine

Zusammenfassung Dreißig Patienten mit rezidivierendem Herpes simplex und 6 Kontrollpatienten wurden in unserer Studie erfaßt. Vor der Behandlung, sowie während und nach der Therapie mit Levamisole, BCG, Urushiol und Herpes Antigen Vaccine wurden MIF-Bestimmungen durchgeführt.Dabei erwies sich der MIF als wertvoller Test in der Diagnose der Bereitschaft Herpesrezidive zu bekommen und in der Beurteilung des Therapieerfolges.Die Behandlung mit Levamisole, BCG und Herpes Antigen Vaccine war erfolgreich.Dabei korrelierten die MIF-Bestimmungen mit dem Ergebnis der Levamisole-und Herpes Antigen Vaccine-Behandlung, nicht hingegen bei den mit BCG behandelten Patienten.

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Therapy of recurrent herpes simplex and its surveillance by MIF determination: with levamisole, BCG, urushiol and herpes antigen vaccine

Summary Thirty patients with recurrent herpes simplex and 6 controls were included in our study. Before initiation of treatment, during therapy with Levamisole, BCG, poison ivy and herpes antigen vaccine and, thereafter, MIF-determinations were performed.The latter test proved to be a useful parameter in evaluating the sensitivity against herpes infection, (and subsequent recurrent manifestations), and effect of therapy.Treatment with Levamisole, BCG and herpes antigen vaccine was successful. MIF-inhibition values paralleled the therapeutic effect in the Levamisole- and herpes antigen vaccine treated group, not however, in the BCG-treated patients.

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Auszugsweise vorgetragen auf der IV. Jahrestagung der Arbeitsgemeinschaft für Dermatologische Forschung, 22.–24. Oktober 1976 in Berlin     

Radiation-enhanced endostatin gene expression and effects of combination treatment

Targeting cells that support tumor growth by administering potent angiogenesis inhibitors is currently an area of intense interest. In the present study, a unique plasmid vector for the mouse endostatin gene, pXLG-mEndo, was constructed and evaluated with and without radiation using the Lewis lung carcinoma (LLC) cell line. The physical properties of the expressed endostatin protein were validated by PCR, gel electrophoresis, and Western blot. Enzyme-linked immunosorbent and immunocytochemical analyses for the therapeutic gene demonstrated that transfected LLC cells secreted the protein into the medium. Exposure of the cells to 2 gray (Gy) gamma-rays reduced the time to reach the maximum expression level of the endostatin gene and also increased the amount of secreted endostatin protein (P<0.001). Biological activity of the endostatin was demonstrated by the inhibition of tube formation by human umbilical vein endothelial cells (HUVEC). Based on (3)H-thymidine incorporation, endostatin expression significantly depressed DNA synthesis in HUVEC and LLC cells compared to controls transfected with parental vector or no vector (P<0.005). In addition, radiation increased the efficiency of endostatin-mediated inhibition of both cell types over a 3-day period post-exposure (P<0.05 or less). Intratumoral injection of 100 small mu g pXLG-mEndo combined with 10 Gy radiation significantly delayed LLC tumor growth, especially when each modality was delivered twice (P<0.05 or less compared to all other groups). No toxicity was observed. These findings are very promising and suggest that endostatin therapy with a plasmid vector, such as pXLG-mEndo, may enhance the efficacy of radiotherapy for lung cancer.