Synthesis of 5-chloroformycin A, 5-chloro-2''-deoxyformycin A and certain related 5,7-disubstituted 3-beta-D-ribofuranosylpyrazolo[4,3-d] pyrimidines from formycin A.

A facile synthesis of 7-amino-5-chloro-3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine (5-chloroformycin A, 6), 7-amino-5-chloro-3-(2-deoxy-beta-D-erythro-pentofuranosyl) pyrazolo [4,3-d]-pyrimidine (5-chloro-2'-deoxyformycin A, 13) and certain related 5,7-disubstituted pyrazolo[4,3-d]pyrimidine ribonucleosides is described starting with formycin A. Thiation of tri-O-acetyloxoformycin B (4b) with phosphorus pentasulfide, followed 3-beta-D-ribofuranosyl-7-thioxopyrazolo[4,3-d] pyrimidin-5(1H,4H,6H)-one (3b) in excellent yield. Chlorination of 4b with either phosphorus oxychloride or phenyl phosphonicdichloride furnished the key intermediate 5,7-dichloro-3-(2,3, 5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine (5a), which on deacetylation afforded 5,7-dichloro-3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine (5b). Ammonolysis of 5a with liquid ammonia gave 6, whereas with MeOH/NH3, a mixture of 6 and 7-methoxy-5-chloro-3-beta-D-ribofuranosylpyrazolo[4,3-d]pyrimidine (7) was obtained. Reaction of 6 with lithium azide and subsequent hydrogenation afforded 5-aminoformycin A (10). Treatment of 5a with thiourea gave 5-chloro-3-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) pyrazolo[4,3-d]pyrimidine-7(1H,6H)-thione (8a), which on further reaction with sodium hydrosulfide furnished 3-beta-D-ribofuranosylpyrazolo [4,3-d]pyrimidine-5,7(1H,4H,6H)-dithione (11). The four-step deoxygenation procedure using phenoxythiocarbonylation of the 2'-hydroxy group of the 3', 5'-protected 6 gave 5-chloro-2'-deoxyformycin A (13).     

Isoenzyme Changes during Seed Germination of Saguaro Cactus (Carnegiea gigantea)

Esterases, acid phosphatases, leucine aminopeptidases, peroxidases and glutamic-oxalacetic transaminases from extracts made from seeds of Saguaro cactus at different stages of germination were studied using starch gelelectrophoretic techniques. In general, four of the five enzyme systems showed significant increase in the number of isoenzymes and their activity after 72 hours of germination. It is suggested that Saguaro cactus seeds reach their enzymatically most active state between 48 to 72 hours after being placed at optimum moisture conditions at 30°C in continuous light.     

Preparation and Antiviral Activity of Several Deoxygenated Ribavirin and Tiazofurin Derivatives

Abstract

Ribavirin and tiazofurin, two nucleosides of known antiviral activity, have been transformed by previously reported methods to yield several deoxy,epoxy, or dideoxy analogues. The deoxygenated derivatives were evaluated for antiviral activity against a host of DNA and RNA viruses; however, no significant in vitro activity was detected.     

Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry

Glycoproteins are involved in many important molecular recognition processes including invasion, adhesion, differentiation, and development. To identify the glycoproteins of Toxoplasma gondii, a proteomic analysis was undertaken. T. gondii proteins were prepared and fractioned using lectin affinity chromatography. The proteins in each fraction were then separated using SDS-PAGE and identified by tryptic in gel digestion followed by tandem mass spectrometry. Utilizing these methods 132 proteins were identified. Among the identified proteins were 17 surface proteins, 9 microneme proteins, 15 rhoptry proteins, 11 heat shock proteins (HSP), and 32 hypothetical proteins. Several proteins had 1–5 transmembrane domains (TMD) with some being as large as 608.3 kDa. Both lectin-fluorescence labeling and lectin blotting were employed to confirm the presence of carbohydrates on the surface or cytoplasm of T. gondii parasites. PCR demonstrated that selected hypothetical proteins were expressed in T. gondii tachyzoites. This data provides a large-scale analysis of the T. gondii glycoproteome. Studies of the function of glycosylation of these proteins may help elucidate mechanism(s) involved in invasion improving drug therapy as well as identify glycoproteins that may prove to be useful as vaccine candidates.     

Differential regulation of proteasome activity in the nucleus and the synaptic terminals

Proteasome is a multi-subunit proteolytic complex that degrades proteins covalently linked to multiple molecules of ubiquitin. Earlier studies showed a role for the ubiquitin-proteasome pathway in several models of long-term memory and other forms of synaptic plasticity. In Aplysia, the ubiquitin-proteasome pathway has been shown to contribute to the induction of long-term facilitation. In other model systems, ubiquitin-proteasome-mediated proteolysis has also been shown to play a role in synapse development. Previous studies of synaptic plasticity focused on changes in components or the substrates of the ubiquitin-proteasome pathway in whole neurons. Modification of specific synapses would require precise spatial and temporal regulation of the components of the ubiquitin-proteasome pathway within the subcellular compartments of neurons during learning. As a first step towards testing the idea of local regulation of the ubiquitin-proteasome pathway in neurons, we investigated proteasome activity in nuclear and synaptosomal fractions. Here we show that proteasome activity in the synaptic terminals is higher compared to the activity in the nucleus in the Aplysia nervous system as well as in the mouse brain. Furthermore, the proteasome activity in the two neuronal compartments is differentially modulated by protein kinases. Differential regulation of proteasome activity in neuronal compartments such as the synaptic terminals is likely to be a key mechanism underlying synapse-specific plasticity.     

Hesperidin Produces Cardioprotective Activity via PPAR-γ Pathway in Ischemic Heart Disease Model in Diabetic Rats

The present study investigated the effect of hesperidin, a natural flavonoid, in cardiac ischemia and reperfusion (I/R) injury in diabetic rats. Male Wistar rats with diabetes were divided into five groups and were orally administered saline once daily (IR-sham and IR-control), Hesperidin (100 mg/kg/day; IR-Hesperidin), GW9962 (PPAR-γ receptor antagonist), or combination of both for 14 days. On the 15^th day, in the IR-control and IR-treatment groups, rats were subjected to left anterior descending (LAD) coronary artery occlusion for 45 minutes followed by a one-hour reperfusion. Haemodynamic parameters were recorded and rats were sacrificed; hearts were isolated for biochemical, histopathological, ultrastructural and immunohistochemistry. In the IR-control group, significant ventricular dysfunctions were observed along with enhanced expression of pro-apoptotic protein Bax. A decline in cardiac injury markers lactate dehydrogenase activity, CK-MB and increased content of thiobarbituric acid reactive substances, a marker of lipid peroxidation, and TNF-α were observed. Hesperidin pretreatment significantly improved mean arterial pressure, reduced left ventricular end-diastolic pressure, and improved both inotropic and lusitropic function of the heart (+LVdP/dt and –LVdP/dt) as compared to IR-control. Furthermore, hesperidin treatment significantly decreased the level of thiobarbituric acid reactive substances and reversed the activity of lactate dehydrogenase towards normal value. Hesperidin showed anti-apoptotic effects by upregulating Bcl-2 protein and decreasing Bax protein expression. Additionally, histopathological and ultrastructural studies reconfirmed the protective action of hesperidin. On the other hand, GW9662, selective PPAR-γ receptor antagonist, produced opposite effects and attenuated the hesperidin induced improvements. The study for the first time evidence the involvement of PPAR-γ pathway in the cardioprotective activity of hesperidin in I/R model in rats.     

Expression profiling reveals differential gene induction underlying specific and non-specific memory for pheromones in mice

Memory for the mating male’s pheromones in female mice is thought to require synaptic changes in the accessory olfactory bulb (AOB). Induction of this memory depends on release of glutamate in response to pheromonal exposure coincident with release of norepinephrine (NE) in the AOB following mating. A similar memory for pheromones can also be induced artificially by local infusion of the GABA_A receptor antagonist bicuculline into the AOB. The natural memory formed by exposure to pheromones during mating is specific to the pheromones sensed by the female during mating. In contrast, the artificial memory induced by bicuculline is non-specific and results in the female mice recognizing all pheromones as if they were from the mating male. Although protein synthesis has been shown to be essential for development of pheromone memory, the gene expression cascades critical for memory formation are not known. We investigated changes in gene expression in the AOB using oligonucleotide microarrays during mating-induced pheromone memory (MIPM) as well as bicuculline-induced pheromone memory (BIPM). We found the set of genes induced during MIPM and BIPM are largely non-overlapping and Ingenuity Pathway Analysis revealed that the signaling pathways in MIPM and BIPM also differ. The products of genes induced during MIPM are associated with synaptic function, indicating the possibility of modification at specific synapses, while those induced during BIPM appear to possess neuron-wide functions, which would be consistent with global cellular changes. Thus, these results begin to provide a mechanistic explanation for specific and non-specific memories induced by pheromones and bicuculline infusion respectively.