Low-frequency dynamics and Raman scattering of crystals, of B-, A-, and Z-DNA, and fibers of C-DNA

Normal modes of vibration of DNA in the low-frequency region (10-300 cm-1 interval) have been identified from Raman spectra of crystals of B-DNA [d(CGCAAATTTGCG)], A-DNA [r(GCG)d(CGC) and d(CCCCGGGG)], and Z-DNA [d(CGCGCG) and d(CGCGTG)]. The lowest vibrational frequencies detected in the canonical DNA structures--at 18 +/- 2 cm-1 in the B-DNA crystal, near 24 +/- 2 cm-1 in A-DNA crystals, and near 30 +/- 2 cm-1 in Z-DNA crystals--are shown to correlate well with the degree of DNA hydration in the crystal structures, as well as with the level of hydration in calf thymus DNA fibers. These findings support the assignment [H. Urabe et al. (1985) J. Chem. Phys. 82, 531-535; C. Demarco et al. (1985) Biopolymers 24, 2035-2040] of the lowest frequency Raman band of each DNA to a helix mode, which is dependent primarily upon the degree of helix hydration, rather than upon the intrahelical conformation. The present results show also that B-, A-, C-, and Z-DNA structures can be distinguished from one another on the basis of their characteristic Raman intensity profiles in the region of 40-140 cm-1, even though all structures display two rather similar and complex bands centered within the intervals of 66-72 and 90-120 cm-1. The similarity of Raman frequencies for B-, A-, C-, and Z-DNA suggests that these modes originate from concerted motions of the bases (librations), which are not strongly dependent upon helix backbone geometry or handedness. Correlation of the Raman frequencies and intensities with the DNA base compositions suggests that the complex band near 90-120 cm-1 in all double-helix structures is due to in-plane librational motions of the bases, which involve stretching of the purine-pyrimidine hydrogen bonds. This would explain the centering of the band at higher frequencies in structures containing G.C pairs (greater than 100 cm-1) than in structures containing A.T pairs (less than 100 cm-1), consistent with the strengths of G.C and A.T hydrogen bonding.     

Synthesis, and cytotoxic and antiplatelet activities of oxime- and methyloxime-containing flavone, isoflavone, and xanthone derivatives

A series of oxime- and methyloxime-containing flavone, isoflavone, and xanthone derivatives (1-12) were synthesized (Scheme) and evaluated for their cytotoxic (Table 1) and antiplatelet activities (Table 2). The in vitro anticancer assay indicated that the cytotoxicity of structurally related compounds decreases in the order isoflavones (7a-7c) > flavones (8a-8c) > xanthones (9a-9c), electron-releasing substituents (R) on the Ph ring being favorable (mean GI50 values of 2.84, 12.3, and 20.9 microM for 7c, 8c, and 9c, resp.). The inhibition of platelet aggregation induced by arachidonic acid (AA) similarly decreased from the isoflavone 1 (IC50 = 2.97 microM) to the flavone 2 (7.70 microM) to the xanthone 3 (inactive). Thereby, compound 1 seems to be a promising lead, since it was not only the most-potent aggregation inhibitor (IC50 = 2.97 microM), but was also found to be noncytotoxic at a concentration of 100 microM.     

Metabolism of the endocannabinoids, 2-arachidonylglycerol and anandamide,into prostaglandin,thromboxane, and prostacyclin glycerol esters and ethanolamides

Cyclooxygenase-2 (COX-2) action on the endocannabinoids, 2-arachidonylglycerol (2-AG) and anandamide (AEA), generates prostaglandin glycerol esters (PG-G) and ethanolamides (PG-EA), respectively. The diversity of PG-Gs and PG-EAs that can be formed enzymatically following COX-2 oxygenation of endocannabinoids was examined in cellular and subcellular systems. In cellular systems, glycerol esters and ethanolamides of PGE(2), PGD(2), and PGF(2alpha) were major products of the endocannabinoid-derived COX-2 products, PGH(2)-G and PGH(2)-EA. The sequential action of purified COX-2 and thromboxane synthase on AEA and 2-AG provided thromboxane A(2) ethanolamide and glycerol ester, respectively. Similarly, bovine prostacyclin synthase catalyzed the isomerization of the intermediate endoperoxides, PGH(2)-G and PGH(2)-EA, to the corresponding prostacyclin derivatives. Quantification of the efficiency of prostaglandin and thromboxane synthase-directed endoperoxide isomerization demonstrated that PGE, PGD, and PGI synthases catalyze the isomerization of PGH(2)-G at rates approaching those observed with PGH(2). In contrast, thromboxane synthase was far more efficient at catalyzing PGH(2) isomerization than at catalyzing the isomerization of PGH(2)-G. These results define the in vitro diversity of endocannabinoid-derived prostanoids and will permit focused investigations into their production and potential biological actions in vivo.     

Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, beta-mercaptoethanol, DTT, SDS, Triton X-100, and urea

Effects of common electrophoretic reagents, reducing agents (beta-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pln) and human plasmin (h-pln) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two plns, whereas SDS (1%) and urea (1 M) denatured plns recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated plns did not restore their activities. Based on a fibrin zymogram gel, five (from b-pln) and four (from h-pln) active fragments were resolved. Two plns exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pln were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pln. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pln and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pln were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both plns was reversible.     

Carbonic anhydrase inhibitors. Inhibition of isozymes I, II, IV, V, and IX with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate

The inhibition of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes; the cytosolic hCA I and II, the membrane-bound hCA IV, the mitochondrial hCA V, and the tumor-associated, transmembrane hCA IX, with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate; such as chlorate, perchlorate, bromate, iodate, periodate, silicate, bismuthate, vanadate, molybdate, and wolframate is reported. Apparently, the geometry of the inhibitor (tetrahedral or trigonal) does not influence its binding to the Zn(II) ion of the enzyme active site, but the nature of the central element is the most important factor influencing potency. Isozymes hCA I and II are best inhibited by chlorate, perchlorate, and silicate, together with the anions structurally related to sulfate, sulfamate, and sulfamidate, but sulfate itself is a weak inhibitor (inhibition constant of 74 mM against hCA I and 183 mM against hCA II). Molybdate is a very weak hCA I inhibitor (K(I) of 914 mM) but it interacts with hCA II (K(I) of 27.5mM). Isozyme IV is well inhibited by sulfate (K(I) of 9 mM), sulfamate, and sulfamidate (in the low micromolar range), but not by perchlorate (K(I) of 767 mM). The mitochondrial isozyme V has the lowest affinity for sulfate (K(I) of 680 mM) and carbonate (K(I) of 95 mM) among all the investigated isozymes, suggesting on one hand its possible participation in metabolon(s) with sulfate anion exchanger(s), and on the other hand an evolutionary adaptation to working at higher pH values (around 8.5 in mitochondria) where rather high amounts of carbonate in equilibrium with bicarbonate may be present. Metasilicate, isosteric to carbonate, is also about a 10 times weaker inhibitor of this isozyme as compared to other CAs investigated here (K(I) of 28.2 mM). Surprisingly, the tumor-associated isozyme IX is resistant to sulfate inhibition (K(I) of 154 mM) but has affinity in the low micromolar range for carbonate, sulfamate, and sulfamidate (K(I) in the range of 8.6-9.6 microM). This constitutes another proof that this isozyme best works at acidic pH values present in tumors, being inhibited substantially at higher pH values when more carbonate may be present. Bromate and chlorate are quite weak CA IX inhibitors (K(I) s of 147-274 mM).     

Effects of Antimycin, Glucose Deprivation, and Serum on Cultures of Neurons, Astrocytes, and Neuroblastoma Cells

The resistance of cultured mouse neuroblastoma cells, primary cultures of rat cerebellar neurons, and rat brain astrocytes to a block of aerobic metabolism was studied. Parameters such as lactate production and ATP content were measured in the presence of antimycin A and under various conditions of glucose, oxygen, and serum supply. The following conclusions can be drawn: (1) All cell types studied were characterized by an active production of lactate; (2) Incubation of the various cell types in the absence of glucose at normal oxygen tension did not affect ATP levels; (3) Respiration blocked by antimycin led to a Pasteur effect; (4) Neuroblastoma cells, but not the other cell types, were fully resistant to inhibition of respiration provided that sufficient glucose was supplied; (5) In the absence of glucose no stores of energy or utilizable substrate were present in the cell types studied when respiration was blocked; (6) In the presence of fetal calf serum anoxic neurons showed irreversible signs of degeneration.     

Involvement of cis-Zeatin, Dihydrozeatin, and Aromatic Cytokinins in Germination and Seedling Establishment of Maize, Oats, and Lucerne

The aims of this study were to monitor endogenous cytokinin levels during germination and early seedling establishment in oats, maize, and lucerne to determine which cytokinin forms are involved in these processes; to quantify the transfer ribonucleic acid (tRNA)-bound cytokinins; and to measure cytokinin oxidase/dehydrogenase (CKX) activity. Cytokinins were identified using UPLC-MS/MS. The predominant free cytokinins present in the dry seeds were dihydrozeatin-type (DHZ) in lucerne and maize and cZ-type (cis-zeatin) in oats. Upon imbibition, there was a large increase in cZ-type cytokinins in lucerne although the cZ-type cytokinins remained at high levels in oats. In maize, the high concentrations of DHZ-type cytokinins decreased prior to radicle emergence. Four tRNA-bound cytokinins [cis-zeatin riboside (cZR)>N ⁶-(2-isopentenyl)adenosine (iPR), dihydrozeatin riboside (DHZR), trans-zeatin riboside (tZR)] were detected in low concentrations in all three species investigated. CKX activity was measured using an in vitro radioisotope assay. The order of substrate preference was N ⁶-(2-isopentenyl)adenine (iP)>trans-zeatin (tZ)>cZ in all three species, with activity fluctuating as germination proceeded. There was a negative correlation between CKX activity and iP concentrations and a positive correlation between CKX activity and O-glucoside levels. As O-glucosides are less resistant to CKX degradation, they may provide a readily available source of cytokinins that can be converted to physiologically active cytokinins required during germination. Aromatic cytokinins made a very small contribution to the total cytokinin pool and increased only slightly during seedling establishment, suggesting that they do not play a major role in germination.     

Cloning, overexpression, and characterization of cobrotoxin

Cobrotoxin (CBTX) is a highly toxic short neurotoxin, isolated from the Taiwan cobra (Naja naja atra) venom. In the present study for the first time we report the cloning and expression of CBTX in high yields (12mg/L) in Escherichia coli. CBTX fused to the IgG-binding domain of protein A (IgG-CBTX) was expressed in the soluble form. The misfolded CBTX portion (of the overexpressed fusion protein) was refolded under optimal redox conditions. The fusion protein (IgG-CBTX) was observed to undergo auto-catalytic cleavage to yield CBTX with additional 5 amino acids upstream of its N-terminal end. The far UV and near UV circular dichroism spectra of the recombinant CBTX were identical to those of the toxin isolated from the crude venom source. Recombinant CBTX was isotope labeled (15N and 13C) and all the resonances ('H, 13C, and 15N) in the protein have been unambiguously assigned. ' H '5N HSQC spectrum of recombinant CBTX revealed that the protein is in a biologically active conformation. 1H-15Nchemical shift perturbation data showed that recombinant CBTX binds to a peptide derived from the alpha7 subunit of the Torpedo acetylcholine receptor (AchR) with high affinity. The AchR peptide is found to bind to residues located at the tip of Loop-2 in CBTX. The results of the present study provide an avenue to understand the structural basis for the high toxicity exhibited by CBTX. In addition, complete resonance assignments in CBTX (reported in this study) are expected to trigger intensive research towards the design of new pharmacological agents against certain neural disorders.     

Active, water-insoluble derivatives of D-glucose oxidase and alginic acid, chitin, and Celite

Treatment of 2-benzimidazolemethanol (4) with methanesulfonyl chloride and pyridine in chloroform afforded 2-(chloromethyl)-1-(methylsulfonyl)benzimidazole (6), which was also prepared by methanesulfonylation of 2-(chloromethyl)benzimidazole. Methanesulfonylation of α-(2-benzimidazolyl)benzyl alcohol (8) in chloroform yielded 2-(α-chlorobenzyl)-1-(methylsulfonyl)benzimidazole. 1-(Methylsulfonyl)-2-benzimidazolemethanol was obtained on methanesulfonylation of 4 pyridine at 0°, and α-[1-(methylsulfonyl)-2-benzimidazolyl]benzyl alcohol (12) was prepared from 8 by using the same reaction conditions. The reaction of 1-acetyl-2-(chloromethyl)-benzimidazole with silver methanesulfonate in benzene gave 1-acetyl-O-(methylsulfonyl)-2-benzimidazolemethanol. Compound 6 has some antitumor activity in the KB cell-culture system, and some antibacterial activity in the Staphylococcus aureus test-system; it is also active in preventing anaphylactic shock in a mouse test-system.     

Biosynthesis,isolation, purification and seperation of Zearalenone,deoxynivalenol and 15-Acetyldeoxynivalenol

Fusarium graminearum KF-376 isolate was found to be able to form simultaneously three toxic metabolites: zearalenone (FF-2), deoxynivalenol (DON) and 15-acetyldeoxynivalenol (15-AcDON). Toxins were extracted with methanol — water 3:1 (v/v) and purified by liquid chromatography on charcoal — Kieselgel 60 column (preliminary) and Aluminiumoxid 90 column. Final separation of the metabolites was achived on Kieselgel 60 — Aluminiumoxid 90 column.     

Stability of His-Phe-Arg-Trp-Pro-Gly-Pro to Leucine Aminopeptidase,Carboxypeptidase Y,and Rat Nasal Mucus,Blood, and Plasma

The His-Phe-Arg-Trp-Pro-Gly-Pro [ACTH-(6–9)-PGP] peptide was synthesized. Proteolysis of ACTH-(6–9)-PGP and semax (Met-Glu-His-Phe-Pro-Gly-Pro) in the presence of leucine aminopeptidase and carboxypeptidase Y was studied. If the proteolysis of ACTH-(6–9)-PGP is mainly defined by aminopeptidases, the basic metabolite is Trp-Pro-Gly-Pro. Identification of major metabolites of ACTH-(6–9)PGP when incubated with the enzymes in vitro made it possible to evaluate proteolysis pathways of the peptide and prepare necessary amounts of its metabolites for using them as standards. Kinetics of ACTH-(6–9)PGP degradation in the presence of enzyme systems of nasal mucus, blood, and plasma were also explored. It was found that proteolysis of ACTH-(6–9)-PGP in rat blood and plasma occurs mainly under the effect of enzymes whose action is similar to leucine aminopeptidase.     

Twenty-four-Hour Rhythms of Serum Acth,Prolactin, Growth Hormone,and Thyroid-Stimulating Hormone,and of Median-Eminence Norepinephrine,Dopamine, and Serotonin,in Rats Injected with Freund's Adjuvant

The effect of Freund's adjuvant injection on 24-h variation of circulating ACTH, prolactin, growth hormone (GH), and thyroid-stimulating hormone (TSH) levels, and of norepinephrine (NE) content, and dopamine (DA) and serotonin (5HT) turnover in median eminence, was examined in adult rats kept under light between 0800 and 2000 h daily. Groups of 6–10 animals Freund's complete adjuvant or its vehicle at 1 lOOh 3 days before sacrifice and were killed by decapitation at six different time intervals throughout a 24-h cycle. In rats injected with adjuvant's vehicle, serum ACTH and prolactin exhibited peak values around the light-dark transition (p < 0.0001 and < 0.04, respectively), while the maximum in TSH was found in the late afternoon (p < 0.0001, one-way ANOVA). GH levels did not vary on a 24-h basis. In Freund's adjuvant-injected rats, 24-h variations of TSH levels became blunted, while 24-h variations of prolactin and ACTH persisted. Freund's adjuvant augmented serum ACTH and prolactin levels, and decreased GH and TSH levels (p < 0.0007, factorial ANOVA). Median-eminence NE content, and turnover of DA, assessed by measuring dihydroxyphenylacetic acid, DOPAC/DA ratio, and of 5HT, assessed by measuring 5-hydroxyindoleacetic acid, HIAA/5HT ratio, varied on a 24-h basis in rats receiving adjuvant's vehicle (p < 0.02). Median-eminence NE content attained its maximum at 1600–2000 h, while maxima in DOPA/DA and HIAA/5HT ratios occurred at 0400 h. Injection with Freund's adjuvant reduced the amplitude of the daily variation of NE content, shifted the maximum of DOPAC/DA ratio toward the light-dark transition, and blunted the daily variation in HIAA/5HT ratio in median eminence. The administration at 1200 of the immunosuppressant drug cyclosporine (5 mg/kg, 5 days) restored the augmented ACTH and prolactin levels (p < 0.0001, factorial ANOVA) and depressed GH and TSH levels (p < 0.02) found in Freund's adjuvant-injected rats. Cyclosporine was also effective in restoring 24-h rhythmicity of serum ACTH and TSH, but not of prolactin, levels. Cyclosporine did not modify the effect of Freund's adjuvant on time-of-day changes of median-eminence NE content, but it was effective in counteracting the changes of DA and 5HT turnover found after immunization. The results are compatible with a significant effect of immune-mediated inflammatory response at an early phase after Freund's adjuvant injection on ACTH, GH, prolactin, and TSH release, which is partially sensitive to immunosuppression by cyclosporine. (Chronobiology International, 14(3), 253–265, 1997)     

Prolamellar bodies of oat,wheat, and rye: Structure,lipid composition,and adsorption of saponins

Summary Soybean seedlings (Glycine max) were incubated in narrow temperature regimes to study the effects of heat shock on cell structures. The incubation temperatures used were as follows: 1. 28 °C (2h); 2. 40 °C (2h); 3. 45 °C (2h); 4. 40 °C (2h)45 °C (2h); 5. 47. 5 °C (10 min); 6. 40 °C (2h)47. 5 °C (10 min). Both optical and electron micrographs were taken of the different tissues of root meristems as they responded to heat shock. Cells of roots heated to 45 °C (2h) or 47.5 °C (10 min) with lethal treatment showed drastic heat injuries:e.g., membrane damage, coagulated plasmolysis, protoplasmic contraction, and leakage of cell content. Nucleolar segregation occurred in cells treated at both lethal and supraoptimal temperatures. Seedlings preincubated at 40 °C (2 h) became thermo-tolerant to lethal temperature treatment of 45 °C (2 h) or 47.5 °C (10 min), by protecting the plasmalemma, mitochondria, plastids and nuclei from heat damage. Without preincubation, however, these structures were destroyed.Abbreviations CC Central cylinder - CR Cortex - M Mitochondria - N Nuclei - Nu Nucleoli - P Plastids - RC Root cap - RE Region of elongation - RM Region of meristem     

Concentrations of copper, zinc, manganese, rubidium, and magnesium in thoracic empyemata and corresponding sera

In this study, a number of selected trace elements and clinically relevant parameters were compared between thoracic empyemata and the corresponding sera for a better understanding of the trace element distribution between these two compartments. Serum-empyema pairs were obtained from 13 patients and quantified for selected and essential trace elements, namely copper (Cu), zinc (Zn), manganese (Mn), rubidium (Rb), and magnesium (Mg), by inductively coupled plasma-mass spectrometry (ICP-MS). In addition, the concentrations of the following clinical laboratory parameters were analyzed by standard methods: total protein, leukocyte count, lactate dehydrogenase, glucose, pH, and the C-reactive protein. Individual concentrations of the elements determined in the empyemata were frequently higher than in pleural effusions of any other benign or malignant condition except for Cu. Serum Cu exceeded the normal range (600–1400 μg/kg) in 6 out of 13 patients (median 1410 μg/kg). In the empyemata, Zn concentrations (median 2000 μg/kg) were characteristically higher than in the sera (median 450 μg/kg) and exceeded the upper limit for serum (1200 μg/kg) in 8 of the 13 patients. Manganese concentrations in the empyemata (median 2.7 μg/kg) were also higher compared to corresponding sera, although they stayed within the limits considered normal for serum of healthy adults (upper limit 2.9 μg/kg). Rubidium was also moderately higher in most empyemata (median 290 μg/kg) and exceeded the upper limit for serum (560 μg/kg) in two patients. The median concentration of the essential element magnesium was higher in the empyemata (23 mg/kg) than in the sera (21 mg/kg). However, all serum Mg concentrations except three remained within the normal range (17–22 mg/kg). Removal of large amounts of empyematous fluid may deprive the body of trace elements and can cause suboptimal or deficient trace element status and homeostasis. Recuperation will be accelerated by compensatory supplementation of trace elements. Therefore, selective medication with adequate trace element compounds in patients with thoracic empyema can be generally recommended for zinc. The other elements need not necessarily be monitored or substituted, because of their stable concentrations in the serum. Rb may have a biological impact, but deficiency symptoms in man are not clearly defined. Deceased.     

Effects of fat, protein, and carbohydrate and protein load on appetite, plasma cholecystokinin, peptide YY, and ghrelin, and energy intake in lean and obese men

While protein is regarded as the most satiating macronutrient, many studies have employed test meals that had very high and unsustainable protein contents. Furthermore, the comparative responses between lean and obese subjects and the relationships between energy intake suppression and gut hormone release remain unclear. We evaluated the acute effects of meals with modest variations in 1) fat, protein, and carbohydrate content and 2) protein load on gastrointestinal hormones, appetite, and subsequent energy intake in lean and obese subjects. Sixteen lean and sixteen obese men were studied on four occasions. Following a standardized breakfast, they received for lunch: 1) high-fat (HF), 2) high-protein (HP), 3) high-carbohydrate/low-protein (HC/LP), or 4) adequate-protein (AP) isocaloric test meals. Hunger, fullness, and gut hormones were measured throughout, and at t = 180 min energy intake at a buffet meal was quantified. In lean subjects, hunger was less and fullness greater following HF, HP, and AP compared with HC/LP meals, and energy intake was less following HF and HP compared with HC meals (P < 0.05). In the obese subjects, hunger was less following HP compared with HF, HC/LP, and AP meals, and energy intake was less following HP and AP compared with HF and HC meals (P < 0.05). There were no major differences in hormone responses to the meals among subject groups, but the CCK and ghrelin responses to HP and AP were sustained in both groups. In conclusion, HP meals suppress energy intake in lean and obese subjects, an effect potentially mediated by CCK and ghrelin, while obese individuals appear to be less sensitive to the satiating effects of fat.     

Spirobipyridopyrans, spirobinaphthopyrans, indolinospiropyridopyrans, indolinospironaphthopyrans and indolinospironaphtho-1,4-oxazines: synthesis, study of X-ray crystal structure, antitumoral and antiviral evaluation

The novel racemic indolinospirobenzopyrans (5-7), indolinospironaphthopyrans (11-14) and indolinospironaphtho-1,4-oxazine (17) were synthesized by an aldol type of condensation of 1',3',3'-trimethyl-2 '-methyleneindoline and its 5-substituted derivatives with an appropriately substituted hydroxybenzaldehyde, hydroxynaphthaldehyde or nitrosonaphthol. An unequivocal proof of the stereostructures of 9 and 17 was obtained by the single-crystal X-ray diffraction method. A substituted indoline ring and the benzopyran ring in 9 and the naphtho-1,4-oxazine moiety in 17 are interconnected via the common chiral atom and positioned almost perpendicularly to each other. The five-membered 2,3-dihydropyrrolo moiety of the indoline ring adopts an envelope conformation in both structures. Of all the compounds of this series, spirobipyridopyran (1) inhibited specifically the growth of human melanoma (HBL) (IC(50): 0.9 microM) cells but not the growth of normal fibroblasts (WI38). Indolinospirobenzopyrans (8-10) showed significant cytostatic activities against all tumor cell lines. However, these compounds also exhibited a cytotoxic effect on normal human fibroblasts. The indolinospirobenzopyrans 4, 6-8, 10 and the indolinospironaphtho-1,4-oxazine 16 showed, albeit modest, selectivity as antiviral agents against varicella-zoster virus (VZV) and/or cytomegalovirus (CMV) (EC(50) within the concentration range of 1.0-12.6 microM).     

Design,synthesis, and evaluation of anti-inflammatory,analgesic, ulcerogenicity,and nitric oxide releasing studies of novel indomethacin analogs as non-ulcerogenic derivatives

Most non-steroidal anti-inflammatory drugs (NSAIDs) suffer from the deadlier gastrointestinal (GI) toxicities. The free -COOH group is responsible for the GI toxicity associated with all traditional NSAIDs. In the present research work, the main objective was to develop new chemical entities as potential anti-inflammatory agents with no GI toxicities. The results of synthesis and pharmacological screening of a series of hybrid molecules having general formula 2-(5-(5-(substituted phenyl)-2-oxo-ethylthio)-1,3,4-oxadiazole-2-yl)-2-phenyl-1H-indol-1-yl)-2-oxoethyl nitrate are described. These compounds were tested in vivo for their anti-inflammatory, analgesic, and ulcerogenic properties, and subjected to histopathological studies. Compound 7c, 2-(5-(5-(3-hydroxyphenyl)-2-oxo-ethylthio)-1,3,4-oxadiazole-2-yl)-2-phenyl-1H-indol-1-yl)-2-oxoethyl nitrate, was the most potent in this series. The compounds that showed significantly reduced GI ulcerogenicity also showed promising results in histopathological studies, and they were found to cause no mucosal injury. All the synthesized compounds were found to exhibit significant nitric oxide releasing activity in an in vitro method. In conclusion, the designed hybrid molecules were found to be significantly promising.     

Capture,Movement, Trade,and Consumption of Mammals in Madagascar

Wild meat trade constitutes a threat to many animal species. Understanding the commodity chain of wild animals (hunting, transportation, trade, consumption) can help target conservation initiatives. Wild meat commodity chain research has focused on the formal trade and less on informal enterprises, although informal enterprises contribute to a large portion of the wild meat trade in sub-Saharan Africa. We aimed to provide a more comprehensive understanding of the formal and informal components of these commodity chains by focusing on the mammalian wild meat trade in Madagascar. Our objectives were to: (1) identify hunting strategies used to capture different wild mammals; (2) analyze patterns of movement of wild meat from the capture location to the final consumer; (3) examine wild meat prices, volumes, and venues of sale; and (4) estimate the volume of wild meat consumption. Data were collected in May-August 2013 using semi-structured interviews with consumers (n = 1343 households, 21 towns), meat-sellers (n = 520 restaurants, open-air markets stalls, and supermarkets, 9 towns), and drivers of inter-city transit vehicles (n = 61, 5 towns). We found that: (1) a wide range of hunting methods were used, though prevalence of use differed by animal group; (2) wild meat was transported distances of up to 166 km to consumers, though some animal groups were hunted locally (<10 km) in rural areas; (3) most wild meat was procured from free sources (hunting, gifts), though urban respondents who consumed bats and wild pigs were more likely to purchase those meats; and (4) wild meat was consumed at lower rates than domestic meat, though urban respondents consumed wild meat twice as much per year compared to rural respondents. Apart from the hunting stage, the consumption and trade of wild meat in Madagascar is also likely more formalized than previously thought.     

Nuclear and nucleolar targeting sequences of c-erb-A, c-myb, N-myc, p53, HSP70, and HIV tat proteins

Protein import into the cell nucleus requires specific binding of nuclear proteins to the nuclear pore complex. Based on amino acid sequence "motifs" of known nuclear targeting signals, we identified peptides within a number of nuclear proteins with likely nuclear targeting potential and tested their function by transfecting into cells fusion genes that produce the cytoplasmic "reporter" protein, pyruvate kinase (PK), joined to the test sequence. Sequences within c-myb (PLLKKIKQ), N-myc (PPQKKIKS), p53 (PQPKKKP), and c-erb-A (SKRVAKRKL) oncoproteins that direct PK hybrids into the nucleus were identified. A peptide (GRKKRRQRRRAP) of the human immunodeficiency virus (HIV) tat protein (Tat), which contains two short basic regions, targets fusion proteins to the nucleolus. The COOH-terminal basic Tat region (QRRRAP) does not target PK hybrid proteins into the nucleus, but mutation of two basic amino acids in this region decreases but does not abolish nucleolar accumulation mediated by the entire Tat nucleolar targeting sequence. Moreover, the c-Myc nuclear targeting sequence fused to the COOH-terminal basic Tat region (PAAKRVKLDQRRRAP) effectively localizes PK hybrids to the nucleus and nucleolus. A similar sequence (FKRKHKKDISQNKRAVRR) in the human heat-shock protein HSP70 also localizes PK to the nucleus and nucleolus.     

The effects of cryopreservation on the acrosome structure,enzyme activity,motility, and fertility of bovine,ovine, and goat sperm

The study was designed to investigate the effects of cryopreservation on bovine, ovine, and goat sperm motility, acrosome structure, enzyme activity, and fertilization ability. Percentage of sperm with hyaluronidase enzyme (HYD) activity was detected by a modified sodium hyaluronate-gelatin membrane. The N-α-benzoyl-DL-arginine-p-nitroanilide (BNPNA) method was used to assess the sperm acrosome enzyme (ACE). The mean percentage of sperm acrosome integrity dropped significantly (P < 0.01) after cryopreservation. The ACE activity of bovine sperm (100.48) was higher (P < 0.01) than that of ovine (57.88) or goat sperm (50.30), while the percentage of sperm with HYD activity of bovine (71.10%) and ovine (67.60%) sperm was higher than that of goat sperm (58.52%) after cryopreservation (P < 0.01). Sperm motility was positively correlated with the activity of the two acrosome enzymes before and after cryopreservation (P < 0.01). Cryopreservation had a negative effect on acrosomal morphology, motility, and acrosomal enzyme activity in their sperm. The fertilization ability of ovine and goat sperm decreased significantly after cryopreservation, but that of frozen bovine sperm did not differ significantly when compared with fresh sperm. There was no significant difference between ovine and goat sperm indices, except for percentage of sperm with HYD activity.