Antimicrobial alkaloids from Zanthoxylum tetraspermum and caudatum

Two benzophenanthrene alkaloids, 8-acetonyldihydronitidine and 8-acetonyldihydroavicine were isolated from Zanthoxylum tetraspermum stem bark along with liriodenine, sesamin, lichexanthone and (+)-piperitol-gamma,gamma-dimethylallylether. The species endemic to Sri Lanka, Z. caudatum, contained sesamin, savinin, liriodenine, decarine and 8-O-desmethyl-N-nornitidine. 8-Acetonyldihydronitidine and 8-acetonyldihydroavicine showed significant antibacterial activity while the former along with liriodenine was strongly antifungal. Savinin exhibited potent spermicidal activity. Both savinin and sesamin exhibited significant insecticidal activity.     

Rapid isolation of plant Ty1-copia group retrotransposon LTR sequences for molecular marker studies

The terminal sequences of long-terminal repeat (LTR) retrotransposons are a source of powerful molecular markers for linkage mapping and biodiversity studies. The major factor limiting the widespread application of LTR retrotransposon-based molecular markers is the availability of new retrotransposon terminal sequences. We describe a PCR-based method for the rapid isolation of LTR sequences of Ty1-copia group retrotransposons from the genomic DNA of potentially any higher plant species. To demonstrate the utility of this technique, we have identified a variety of new retrotransposon LTR sequences from pea, broad bean and Norway spruce. Primers specific for three pea LTRs have been used to reveal polymorphisms associated with the corresponding retrotransposons within the Pisum genus.     

Purification of muscle enzymes by pseudoaffinity chromatography

A method based on pseudoaffinity chromatography has been developed for the separation of lactate dehydrogenase (LDH), pyruvate kinase (PK) and aldolase from rabbit muscle extract using cross-linked guar (CLG) and cross-linked pectin (CLP) as the matrices, and dyes as the ligands. Screening of several dyes revealed that dyes No. 1014 and No. 1015, immobilized on CLG and CLP displayed a higher affinity for LDH and PK. Aldolase was not retained on any of the dye columns. It was observed that 1014-CLP and 1014-CLG columns retained 90% and 55% LDH activities, respectively, whereas 1015-CLP and 1015-CLG retained 83% LDH and 72% PK. A coupled-column system comprising 1014-CLP and 1015-CLP or 1014-CLG and 1015-CLG could separate LDH, PK, and aldolase from a mixture of these enzymes, as well as from rabbit muscle extract. Enzymes were found to be homogeneous on polyacrylamide gel electrophoresis. The method has been found to be simple and economical.     

Purification of Lac repressor protein using polymer displacement and immobilization of the protein

Lac repressor protein was purified from E. coli BMH8117 harboring plasmid pWB1000 and E. coli K₁₂BMH 71-18 strains. Displacement of the protein with poly(ethyleneimine) (PEI) from phosphocellulose cation exchange column was shown to be an effective elution strategy. It resulted in better recoveries and sharper elution profiles than traditional salt elution without effecting the purity of the protein. The elution is assumed to proceed via displacement of bound protein by PEI when the polymer binds to the ion exchanger. The minor impurities in the protein solution were finally removed by chromatography on immobilized metal affinity column. The repressor protein undergoes distinct conformational changes upon addition of specific inducer isopropyl--D-thiogalactoside (IPTG), which is evidenced by changes in ultraviolet absorption spectrum. The protein was immobilized covalently to the Sepharose matrix. The intact biological activity of the protein after immobilization was shown by binding of genomic DNA and lac operator plasmid DNA from E. coli to the immobilized lac repressor.     

Non-iron metalloporphyrins: potent antibacterial compounds that exploit haem/Hb uptake systems of pathogenic bacteria

A new group of potent antibacterial compounds, non-iron metalloporphyrins (MPs), is described. MPs possess a strong antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and mycobacteria. Anaerobically grown bacteria and microorganisms that do not respire and/or express haem uptake systems were resistant to MPs. Antibacterial activity of MPs was not affected by known antibiotic resistance mechanisms operating in bacteria. The most potent MP against Y. enterocolitica, methicillin-resistant S. aureus and M. smegmatis was gallium protoporphyrin IX (Ga-PPIX). When tested alone, Ga ions and metal-free porphyrins had approximately 100-fold higher minimum inhibitory concentration (MIC) values for these organisms. Ga-PPIX was not degraded by MP-sensitive bacteria, indicating that the whole molecule is responsible for antibacterial activity. MPs are antibacterial 'Trojan horses', as they exploit haem transport systems of Gram-negative bacteria as portals of entry into the cell. Bacterial mutants in superoxide dismutases, catalases and stationary-phase sigma factors were hypersensitive to Ga-PPIX. The extreme sensitivity of sod mutants to MPs and the requirement for active respiration for MP activity suggests that these compounds stimulate the production of reactive oxygen radicals in bacteria. Ga-PPIX was not toxic to primary human fibroblasts, several established cell lines and experimental animals at concentrations > 100-fold higher than the MIC for sensitive bacteria.     

The anhydrous milk fat, ghee, lowers serum prostaglandins and secretion of leukotrienes by rat peritoneal macrophages

Ghee, the anhydrous milk fat, is one of the most important sources of dietary fat in India. Male Wistar rats were fed diets containing 2.5, 5.0 and 10 wt% ghee for a period of 8 weeks. The diets were made isocaloric with groundnut oil. The results showed that serum thromboxane levels decreased by 27-35%, and 6-keto-prostaglandin F1alpha by 23-37% when ghee was incorporated at level of 10% in the diet. Prostaglandin E2 levels in serum and secretion of leukotrienes B4, C4 and D4 by peritoneal macrophages activated with calcium ionophore decreased when increased amounts of ghee from 2.5 to 10% were included in the diet. Arachidonic acid levels in macrophage phospholipids decreased when incremental amounts of ghee were fed to rats. These studies indicate that ghee in the diet not only lowers the prostaglandin levels in serum but also decreases the secretion of leukotrienes by macrophages.     

Purification and characterization of an extracellular polygalacturonase from the thermophilic fungus,Thermomyces lanuginosus

A polygalacturonase was purified from the thermophilic fungus, Thermomyces lanuginosus to apparent homogeneity by ultrafiltration, acetone precipitation and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60 °C. The apparent K_M with potassium pectate was 0.67 mg/ml and the V_max was 7.2 × 10⁵ mol/min/mg protein. The apparent molecular weight of the enzyme was 59 kDa and it contained approximately 10% carbohydrate. The enzyme was completely stable at room temperature (32 ± 3 °C) and retained about 50% activity at 50 °C for 6 h. The zymogram of the purified enzyme revealed two activity bands, one of which was a major one. Polyclonal antibodies raised against the enzyme did not show any immunological relatedness with other mesophilic polygalacturonases.     

Light sensitivity of the photoperiodic response system in higher vertebrates: wavelength and intensity effects

Most species use daily light in one way or the other in regulation of their short and/or long term activities. Light is perceived by pigment(s) present in the retinal (RP) and/or extra-retinal photoreceptors (ERPs). ERPs may be located at various sites in the body but in non-mammalian vertebrates they are found predominantly in the pineal body and hypothalamic region of the brain, Light radiations directly penetrate brain tissues to reach and stimulate the hypothalamic (deep-brain) photoreceptors. How does light information finally reach to the clock is not fully understood in many vertebrate groups? In mammals, however, the light information from the retina to the clock (the hypothalamic suprachiasmatic nuclei, SCN) is relayed through the retino-hypothalamic tract (RHT) which originates from the retinal ganglion cells, and through the geniculo-hypothalamic tract (GHT) which originates from the photically responsive cells of a portion of the lateral geniculate nucleus (LGN), called the intergeniculate leaflet (IGL). A response to light (the photoperiodic response) is the result of the interpretation of light information by the photoperiodic system. Apart from the duration, the animals use the gradual shifts in the intensity and wavelength of daily light to regulate their photoperiodic clock system. The wavelengths to which photoreceptors are maximally sensitive or the wavelengths which have greater access to the photoreceptors can induce a maximal response. There can also be differential effects of wavelength and intensity of light on circadian process(es) involved in the entrainment and induction of the photoperiodic clock. This may have some adaptive implications. Entrainment to daily light-dark (LD) cycle may be achieved at dawn or dusk, depending whether the animal is day- or night-active, when there is relatively low intensity of light. By contrast, photoperiodic induction in many species occurs during long days of spring and summer when plenty of daylight at higher intensity is available later in the day.