Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20–25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20–21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO₃)₂, K₂SO₄, KCl, and NH₄(SO₄)₂. The maximum shoot multiplication (29–30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2–3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.     

Enzymatic activity of atypical oriental types of aldehyde dehydrogenases

Catalytic activity of the atypical Oriental-type aldehyde dehydrogenase-2 (ALDH₂) was considered to be null or severely diminished. Recently it was suggested that the atypical ALDH ₂ ² retained about 30% of the specific activity of the usual ALDH ₂ ¹ . We reexamined the problem by two-dimensional crossed immunoelectrophoresis. The usual Caucasian livers exhibited two distinctive precipitin peaks, one corresponding to the cytosolic ALDH₁ and the other corresponding to the usual mitochondrial ALDH ₂ ¹ , in both protein stain and enzyme activity stain. In contrast, the atypical Oriental livers exhibited two precipitin peaks in protein stain, but only one peak, corresponding to ALDH₁, in enzyme activity stain. These results support the original notion that the atypical ALDH ₂ ² is enzymatically inactive or far less active than the usual enzyme, refuting the idea of the atypical ALDH ₂ ² with substantial enzyme activity.This work was supported by U.S. Public Health Service Grants HL-29515 and AA05763.     

Synthesis and evaluation of triazolones as checkpoint kinase 1 inhibitors

Checkpoint kinase 1 (Chk1, CHEK1) is a Ser/Thr protein kinase that plays a key role in mediating the cellular response to DNA-damage. Synthesis and evaluation of a previously described class of Chk1 inhibitors, triazoloquinolones/triazolones (TZs) is further described herein. Our investigation of structure-activity relationships led to the identification of potent inhibitors 14c, 14h and 16e. Key challenges included modulation of physicochemical properties and pharmacokinetic (PK) parameters to enable compound testing in a Chk1 specific hollow fiber pharmacodynamic model. In this model, 16e was shown to abrogate topotecan-induced cell cycle arrest in a dose dependent manner. The demonstrated activity of TZs in this model in combination with a chemotherapeutic agent as well as radiotherapy validates this series of Chk1 inhibitors. X-ray crystal structures (PDB code: 2YEX and 2YER) for an initial lead and an optimized analog are also presented.     

Inhibition of HIV-1 Integrase gene expression by 10-23 DNAzyme

HIV Integrase (IN) is an enzyme that is responsible for the integration of the proviral genome into the human genome, and this integration step is the first step of the virus hijacking the human cell machinery for its propagation and replication. 10-23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. We have designed three novel DNAzymes, DIN54, DIN116, and DIN152, against HIV-1 Integrase gene using Mfold software and evaluated them for target site cleavage activity on the in vitro transcribed mRNA. All DNAzymes were tested for its inhibition of expression of HIV Integrase protein in the transiently transfected cell lines. DIN116 and DIN152 inhibited IN-EGFP expression by 80 percent and 70 percent respectively.     

Synthesis and characterization of monosaccharide lipids as novel hydrogelators

Self-assembly of small molecules is a useful strategy for forming functional supramolecular structures. Three new series of methyl alpha-D-glucopyranoside derivatives, including esters and carbamates, have been synthesized and characterized. Several of these compounds are excellent hydrogelators and formed interesting self-assembled network structures, including birefringent fibers and tubules. The gelation properties depend on the acyl chain length and the headgroup structures. Small molecule sugar-based hydrogelators have potential applications in drug delivery and enzyme immobilization.     

Synthesis and antibacterial activity of substituted 1,2,3,4-tetrahydropyrazino [1,2-a] indoles

A series of substituted 1,2,3,4-tetrahydropyrazino [1,2-a] indole derivatives have been synthesized and tested against the Gram positive and Gram negative strains of bacteria namely Staphylococcus aureus (MTCCB 737), Salmonella typhi (MTCCB 733), Pseudomonas aeruginosa (MTCCB 741), Streptomyces thermonitrificans (MTCCB 1824) and Escherichia coli (MTCCB 1652). All synthesized compounds showed mild to moderate activity. However, compounds 4d-f were found to have potent activity against pathogenic bacteria used in the study. Their MIC ranged from 3.75 to 60 microg/disc. In vitro toxicity tests demonstrated that toxicity of 4d-f was not significantly different than that of gentamycin. However, at higher concentration (1000-4000 microg/ml) difference was highly significant.     

Increase in ovulation rate by active immunization against bovine inhibin-based synthetic peptides in a non-prolific sheep breed

The objective of this study was to explore the possibility of a recurrent increase in the ovulation rate of Malpura sheep, a non-prolific breed, by immunization against inhibin-based peptide immunogens over a period of 3 years. Adult ewes (4–7 years of age) and weighing between 28 and 38 kg were randomly allocated equally to three treatment groups. The immunization of the ewes was initiated during the autumn breeding season. Ewes were divided into three groups (n = 5 ewes/group) and actively immunized against the synthetic peptides from the α_C [bIα(1–29)-Tyr³⁰] (Group I) or α_N [bI-₄₃-Tyr¹⁵²(153-167)Cys¹⁶⁸] (Group II) area of the bovine inhibin α-subunit, conjugated to ovalbumin or against ovalbumin (control). Each ewe received a primary immunization of 400 μg immunogen and 3 booster injections, 200 μg immunogen each at 4-week intervals. Estrous was synchronized in all the ewes by administering two doses of PGF₂α at 10-day intervals for three consecutive years. Ovaries of ewes were examined each year between days 4 and 6 of the synchronized cycle, with the aid of the laparoscope to determine the ovulation rate. Active immunization significantly (p < 0.05) increased the ovulation rate. The overall ovulation rate, irrespective of the treatment period was 5.2 ± 0.44, 2.3 ± 0.38 and 0.9 ± 0.11 in Group I, Group II and the control, respectively. Although the beneficial effect of immunization on ovulation rate persisted for the entire period of the study, the interaction between immunization treatment and the time period was non-significant. The results clearly indicate that the active immunization against inhibin peptides can induce multiple ovulations in Malpura ewes and its effect on multiple ovulations is sustained for a prolonged period of time after the initial immunization.     

Microwave-assisted synthesis of antimicrobial dihydropyridines and tetrahydropyrimidin-2-ones: novel compounds against aspergillosis

Ten 4-aryl-1,4-dihydropyridine and three 4-aryl-1,2,3,4-tetrahydropyrimidin-2-one derivatives have been synthesized and examined for their activity against pathogenic strains of Aspergillus fumigatus and Candida albicans. Although none of the three compounds belonging to pyrimidin-2-one series showed any activity against two pathogens, two of the compounds of the dihydropyridine series, that is, diethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate and dimethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate, exhibited significant activity against A. fumigatus in disc diffusion, microbroth dilution and percent spore germination inhibition assays. The most active diethyl dihydropyridine derivative exhibited a MIC value of 2.92 microg/disc in disc diffusion and 15.62 microg/ml in microbroth dilution assays. The MIC(90) value of the most active compound by percent germination inhibition assay was found to be 15.62 microg/ml. The diethyl dicarboxylate derivative of dihydropyridine also exhibited appreciable activity against C. albicans. The in vitro toxicity of the most active diethyl dihydropyridine derivative was evaluated using haemolytic assay, in which the compound was found to be non-toxic to human erythrocytes even at a concentration of 625 microg/ml. The standard drug amphotericin B exhibited 100% lysis of erythrocytes at a concentration almost 16 times less than the safer concentration of the most active dihydropyridine derivative.     

Temporal and spatial analysis of lipid accumulation, oleosin expression and fatty acid partitioning during seed development in sunflower (Helianthus annuus L.)

Biochemical and fluorescence microscopic imaging approach has been adopted to investigate the accumulation of oil bodies at specific stages of seed development in Helianthus annuus L. cv. Morden. Seed filling in sunflower is marked with a rapid accumulation of proteins and lipids upto 30 DAA, after which protein accumulation declines whereas lipids continue to accumulate. Earliest signs of lipid accumulation are evident as early as during globular stage of embryo development. Spatially, a developing seed exhibits enhanced lipid deposition in peripheral cells. Oil body biogenesis is observed as early as 10 DAA, as is evident from the fluorescence microscopic detection of Nile red-positive entities in the protoplasts. To begin with, expression of one of the oleosin (the principal oil body membrane proteins) isoforms (16 kDa), is slower than the other two (17.5 and 20 kDa). Fatty acid composition of oil body lipids is quite similar to that of total seed lipids. An enhanced accumulation of linoleic acid is evident during later stages of seed filling. The proportion of major saturated fatty acids, palmitic (16:0) and stearic (18:0), however, do not alter much during the later phases of seed development. Present findings provide new information on oil body development, lipid accumulation and fatty acid composition, for a better understanding of the phasing of physiological and biochemical events associated with oilseed development.     

On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg

Background  

Enzymes have been extensively used in organic solvents to catalyze a variety of transformations of biological and industrial significance. It has been generally accepted that in dry aprotic organic solvents, enzymes are kinetically trapped in their conformation due to the high-energy barrier needed for them to unfold, suggesting that in such media they should remain catalytically active for long periods. However, recent studies on a variety of enzymes demonstrate that their initial high activity is severely reduced after exposure to organic solvents for several hours. It was speculated that this could be due to structural perturbations, changes of the enzyme's pH memory, enzyme aggregation, or dehydration due to water removal by the solvents. Herein, we systematically study the possible causes for this undesirable activity loss in 1,4-dioxane.