Development of a PCR-RFLP assay for the identification of Lactococcus lactis ssp. lactis and cremoris

The development of new starter culture of Lactococcus lactis for the manufacture of fermented dairy products with unique characteristics usually requires the isolation and identification of L. lactis up to subspecies level. Therefore, a rapid and specific PCR-RFLP assay has been developed. Forward and reverse primer sets were designed targeting the conserved house keeping gene htrA and yueF encoding a trypsin-like serine protease and a non-proteolytic protein from peptidase family M16, respectively, of L. lactis. Amplicons of 265 bp and 447 bp of htrA and yueF, respectively, were subjected to restriction fragment length polymorphism analysis. Restriction of the 265 bp amplicons with TaqI produced DNA bands of 90 bp and 175 bp with ssp. lactis, and 66 bp and 199 bp with ssp. cremoris. Similarly, restriction of PCR product of 447 bp size with AluI produced digested fragments of 125 bp and 322 bp with ssp. lactis, and 71 bp and 376 bp with ssp. cremoris. The designed primer sets were observed to be specific to L. lactis because other bacteria could not be amplified. The ssp. lactis and cremoris of L. lactis could be identified by restriction of PCR products of htrA and yueF with TaqI and AluI, respectively.     

De novo methylation of nucleosomal DNA by the mammalian Dnmt1 and Dnmt3A DNA methyltransferases

In the cell, DNA is wrapped on histone octamers, which reduces its accessibility for DNA interacting enzymes. We investigated de novo methylation of nucleosomal DNA in vitro and show that the Dnmt3a and Dnmt1 DNA methyltransferases efficiently methylate nucleosomal DNA without dissociation of the histone octamer from the DNA. In contrast, the prokaryotic SssI DNA methyltransferase and the catalytic domain of Dnmt3a are strongly inhibited by nucleosomes. We also found that full-length Dnmt1 and Dnmt3a bind to nucleosomes much stronger than their isolated catalytic domains, demonstrating that the N-terminal parts of the MTases are required for the interaction with nucleosomes. Variations of the DNA sequence or the histone tails did not significantly influence the methylation activity of Dnmt3a. The observation that mammalian methyltransferases directly modify nucleosomal DNA provides an insight into the mechanisms by which histone tail and DNA methylation patterns can influence each other because the DNA methylation pattern can be established while histones remain associated to the DNA.     

New nonsynthetic medium for Rhizobium culture production from wastes

A nonsynthetic medium was formulated for placement of mannitol fully by saccharified pea husk (Pisum sativum L.) and water hyacinth (Eichhoornia crassipes) with Trichoderma viride QM 9414 and molasses. Yeast extract was Partially replaced by proteolysed pea husk, water hyacinth, and mycelium of T. viride QM 9414 by boiling 4 hr with 5% (v/v) HCl. The rhizobial growth was equal in both standard yeast extract mannitol (YEM) and formulated nonsynthetic media. However, barring Rhizobium phaseoli (urid) E-6, the rhizobial counts in thenon-synthetic medium were higher then the counts in YEM medium. In the fermentor, rhizobial growth was also almost equal to YEM medium. These results indicated that costly ingredients like mannitol and yeast extract can be replaced by hydrolysates of pea husk, water hyacinth, mycelium of T. viride, and molasses.     

Water quality research in Loosdrecht lakes: the salient features

Ecosystem research by the working group Water Quality Research Loosdrecht lakes (WQL) was carried out from 1979 to 1990. A coordinated research programme, involving several research institutes and laboratories in The Netherlands, was initiated in 1983, i.e. a year before the reduction of external phosphorus loading by stripping, became effective. The paper summarizes the main results, with emphasis on insight they provide into the lake ecosystems.     

Histochemical localization of gamma glutamyl transpeptidase in the paranodal region of peripheral nerve

Gamma glutamyl transpeptidase (GGT), an amino acid transport enzyme, was investigated in normal and degenerated sciatic nerve of rat. The enzyme activity, which is considered to be a marker for cerebrovascular endothelium, was found to be absent in microvessels of normal and degenerated nerves. In the perineurium of normal nerve, GGT activity was faint, while in degenerated nerve, it increased. The most striking finding of this study was the observation of GGT activity at the paranode of each normal myelinated axon. It is interesting that after axotomy (8 weeks), no GGT activity was observed in the Schwann cells of degenerated nerve. Thus, Schwann cell plasmalemma contributed to GGT staining only when this cell was in contact with an axon mature enough to cause it to produce myelin. We conclude that, in peripheral nerve, transmembrane amino acid transport is apparently regional and associated with the paranodal region of myelinated nerve fibers.     

Restoration by biomanipulation in a small hypertrophic lake: first-year results

Biomanipulation was carried out in order to improve the water quality of the small hypertrophic Lake Zwemlust (1.5 ha; mean depth 1.5 m). In March 1987 the lake was drained to facilitate the elimination of fish. Fish populations were dominated by planktivorous and benthivorous species (total stock c. 1500 kg) and were collected by seine- and electro-fishing. The lake was subsequently re-stocked with 1500 northern pike fingerlings (Esox lucius L.) and a low density of adult rudd (Scardinius erythrophthalmus). The offspring of the rudd served as food for the predator pike. Stacks of Salix twigs, roots of Nuphar lutea and plantlets of Chara globularis were brought in as refuge and spawning grounds for the pike, as well as shelter for the zooplankton.The impact of this biomanipulation on the light penetration, phytoplankton density, macrophytes, zooplankton and fish communities and on nutrient concentrations was monitored from March 1987 onwards. This paper presents the results in the first year after biomanipulation.The abundance of phytoplankton in the first summer (1987) after this biomanipulation was very low, and consequently accompanied by increase of Secchi-disc transparency and drastic decline of chlorophyll a concentration.The submerged vegetation remained scarce, with only 5 % of the bottom covered by macrophytes at the end of the season.Zooplankters became more abundant and there was a shift from rotifers to cladocerans, comprised mainly of Daphnia and Bosmina species, the former including at least 3 species.The offspring of the stocked rudd was present in the lake from the end of August 1987. Only 19% of the stocked pike survived the first year.Bioassays and experiments with zooplankton community grazing showed that the grazing pressure imposed by the zooplankton community was able to keep chlorophyll a concentrations and algal abundance to low levels, even in the presence of very high concentrations of inorganic N and P. The total nutrient level increased after biomanipulation, probably due to increased release from the sediment by bioturbation, the biomass of chironomids being high.At the end of 1987 Lake Zwemlust was still in an unstable stage. A new fish population dominated by piscivores, intended to control the planktivorous and benthivorous fish, and the submerged macrophytes did not yet stabilize.     

Antioxidant and Antimicrobial Activity in Some Indian Herbal Plants: Protective Effect against Free Radical Mediated DNA Damage

Indian herbal plant species Lantana indica, Adhatoda vasica, Pandanus furcatus, Tylophora indica and Centella asiatica, traditionally used in ethno medicines to treat common infections and various disorders, have been studied for their antimicrobial and antioxidant activity. The methanolic extracts of the plant leaves exhibited significant and dose-dependent antioxidant activities in DPPH radical scavenging, ferric ion reducing and phosphomolybdate assays. These leaf extracts showed antimicrobial activity against selected Gram +ve and Gram ?ve bacterial strains. A. vasica and L. indica extracts possessed maximum antioxidant and antimicrobial activity, respectively. The activities could be correlated to phenolics and flavonoid content of the leaf extracts which ranged from 30.25 to 91.98 mg GAE g^?1 dw leaf extract and 2.67 to 96.45 mg RE g^?1 dw leaf extract respectively. The aqueous extracts of plant leaves significantly protected the DNA damage against the oxidative damage caused by hydroxyl radicals.     

Polynitroxyl hemoglobin: a pharmacokinetic study of covalently bound nitroxides to hemoglobin platforms

Adding antioxidant activities to hemoglobin-based oxygen carriers (HBOCs) represents a means of reducing cell-free hemoglobin-mediated oxidative cascades. We have covalently bound nitroxides, a class of antioxidant enzyme mimetics, to HBOCs. The objectives of this study were (1) to evaluate the pharmacokinetic (PK) effects of administering nitroxide covalently bound to HBOCs compared to those of free nitroxide coadministered with HBOCs and (2) to elucidate the effects of differing molecular weight HBOCs on the PK of bound nitroxide in a conscious guinea pig model of 25% blood exchange transfusion. Two HBOC platforms were used, intramolecular cross-linked hemoglobin (XLHb) and dextran polymerized/conjugated XLHb (PolyHb). Polynitroxylation was achieved by reacting 4-(2-bromoacetamido)-2,2,6,6,-tetramethylpiperidine-1-oxyl with XLHb or PolyHb to form polynitroxylated XLHb and polynitroxylated PolyHb, respectively, whereas a physical mixture of XLHb or PolyHb with 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl was prepared to reflect a molar equivalence to HBOC-bound nitroxide. Plasma concentrations of two redox states, nitroxide and hydroxylamine, were determined by electron paramagnetic resonance spectroscopy. Results are presented to illustrate the influence of covalent labeling and HBOC molecular weight on nitroxide PK. The therapeutic potential of polynitroxylation of HBOCs as it relates to observations from the current and previously reported studies is discussed.     

Use of blood outgrowth endothelial cells as virus-producing vectors for gene delivery to tumors

Cell-based delivery of therapeutic viruses has potential advantages over systemic viral administration, including attenuated neutralization and improved viral targeting. One of the exciting new areas of investigation is the potential ability of endothelial-lineage cells to deliver genes to the areas of neovascularization. In the present study, we compared two types of endothelial-lineage cells [outgrowth endothelial cells (OECs) and culture-modified mononuclear cells (CMMCs), also known as "endothelial progenitor cells"] for their ability to be infected with adenovirus and to home to the areas of neovascularization. Both cell types were isolated from peripheral blood of healthy human donors and expanded in culture. We demonstrate that OECs are more infectable and home better to tumors expressing VEGF on systemic administration. Furthermore, we used an adenoviral/retroviral chimeric system to convert OECs to retrovirus-producing cells. When injected systemically into tumor-bearing mice, OECs retain their ability to produce retrovirus and infect surrounding tumor cells. Our data demonstrate that OECs could be efficient carriers for viral delivery to areas of tumor neovascularization.