Characterization of Lactobacillus plantarum from wine must by PCR species-specific and RAPD-PCR

AIMS: Physiological and molecular analysis such as PCR species-specific and randomly amplified polymorphic PCR (RAPD-PCR) have been used for typing of Lactobacillus plantarum strains from typical wine must. METHODS AND RESULTS: Phenotypic tests such as API 50CH and evaluation of D-L-lactate production from glucose were used to perform a preliminary characterization of lactobacilli. Furthermore, 18 strains of lactobacilli were analyzed by PCR species-specific oligonucleotides based on short sequences of the recA gene. CONCLUSIONS: Four strains were identified as belonging to the L. plantarum species and were further analysed by RAPD-PCR. The RAPD-PCR profiles were similar in all strains that had positive results for species-specific PCR, suggesting that the four L. plantarum strains were closely related. SIGNIFICANCE AND IMPACT OF THE STUDY: Using PCR species-specific as a preliminary screening test and then RAPD-PCR can be as considered the most reliable method of performing a rapid and correct typing of L. plantarum from wine must.     

NADPH : protochlorophyllide oxidoreductases in white pine (Pines strobes) and loblolly pine (P. taeda)

Summary NADPH : protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) catalyzes the light-dependent reduction of protochlorophyllide in higher plants. Cloned cDNAs encoding two distinct pchlide reductases were isolated from a gt11 library constructed from poly(A)⁺ RNA prepared from the cotyledons of dark-grown white pine (Pines strobes) seedlings and a nuclear gene (lpcr) analogous to one of these cDNAs has been characterized from loblolly pine (P. taeda). The pine gene encodes an approximately 43 kDa precursor polypeptide consisting of a 334-amino acid mature protein and a 66-amino acid transit peptide. The deduced primary structures for the pine proteins are highly homologous to those reported from monocots and dicots. The coding portion of the pine lpcr gene is interrupted by four introns. The placement of these introns within the pine lpcr gene is identical to that observed in pea (Pisum sativum), suggesting conservation in gene organization between dicot and gymnosperm species. Western blot analysis using polyclonal antiserum against oat pchlide reductase detected in extracts of dark-grown pine cotyledons a single immunoreactive protein, which declined in abundance during a 48 h period of illumination with white light. Cotyledons of dark-grown seedlings were also found to accumulate high levels of pchlide reductase mRNA; however, little or no change in the steady-state levels of mRNA encoding pchlide reductase was observed in these tissues following illumination. Stem tissue of dark-grown seedlings did not contain significant levels of pchlide reductase mRNA, whereas stems of light-grown plants of the same age accumulated substantial amounts of the message. These results suggest that light and the developmental age of the tissue affect regulation of lpcr expression in pine.     

Postsynaptic density antigens: preparation and characterization of an antiserum against postsynaptic densities

Long-term immunization of rabbits with postsynaptic densities (PSD) from bovine brain produced an antiserum specific for PSD as judged by binding to subcellular fractions and immunohistochemical location at the light and electron microscope levels. (a) The major antigens of bovine PSD preparations were three polypeptides of molecular weight 95,000 (PSD-95), 82,000 (PSD-82), and 72,000 (PSD-72), respectively. Antigen PSD-95, also present in mouse and rat PSDs was virtually absent from cytoplasm, myelin, mitochondria, and microsomes from rodent or bovine brain. Antigens PSD-82 and PSD-72 were present in all subcellular fractions from bovine brain, especially in mitochondria, but were almost absent from rodent brain. The antiserum also contained low-affinity antibodies against tubulin. (b)Immunohistochemical studies were performed in mouse and rat brain, where antigen PSD-95 accounted for 90 percent of the antiserum binding after adsorption with purified brain tubulin. At the light microscope level, antibody binding was observed only in those regions of the brain where synapses are known to be present. No reaction was observed in myelinated tracts, in the neuronal cytoplasm, or in nonneuronal cells. Strong reactivity was observed in the molecular layer of the dentate gyrus, stratum oriens and stratum radiatum of the hippocampus, and the molecular layer of the cerebellum. Experimental lesions, such as ablation of the rat entorhinal cortex or intraventricular injection of kainic acid, which led to a major loss of PSD in well- defined areas of the hippocampal formation, caused a correlative decrease in immunoreactivity in these areas. Abnormal patterns of immunohistochemical staining correlated with abnormal synaptic patterns in the cerebella of reeler and staggerer mouse mutants. (c) At the electron microscopic level, immunoreactivity was detectable only in PSD. The antibody did not bind to myelin, mitochondria or plasma membranes. (d) The results indicate that antigen PSD-95 is located predominantly or exclusively in PSD and can be used as a marker during subcellular fractionation. Other potential uses include the study of synaptogenesis, and the detection of changes in synapse number after experimental perturbations of the nervous system.     

Phenological stages of natural species and their use as climate indicators

 The objectives of this paper are to: (1) present 10 years of phenological data for nine natural species growing in a Mediterranean-type climate, (2) present threshold temperatures that were derived for the computation of cumulative degree-days (CDD), and (3) evaluate the sensitivity of the nine natural species to weather variability. The study was conducted at the Phenological Research Garden of Oristano, Sardinia, Italy, during the period 1986–96. The observations were made on five typical Mediterranean species and four species that are typical of higher latitudes. The mean annual pattern of phenological events and the CDD from 1 January are given for each development stage. Temperature thresholds were evaluated by comparing the standard deviation about the mean number of days in the development period for each species. A good relationship between timing of phenophase occurrence and temperature was observed for the Mediterranean species, which were little affected by variations in rainfall. Phenological development of the non-native species was affected by springtime rainfall. Accepted: 28 October 1998     

Energy requirements and process design considerations in compression-milling pretreatment of cellulosic wastes for enzymatic hydrolysis

The effectiveness of compression-milling pretreatment of lignocellulosics for enzymatic hydrolysis has been demonstrated for a wide variety of substrate sources. Reductions in the degree of crystallinity and the degree of polymerization of cellulose and partial destruction of the structural integrity of lignocellulosics brought about by compression milling significantly increase the susceptibility of cellulose to enzymatic hydrolysis. The enzymatic hydrolysis yield was found to be directly related to the specific energy input to the cellulosic substrate (kWh/1b substrate) by compression milling, and the energy input can be controlled by the milling time. The enzymatic hydrolysis yeilds from cellulosic materials pretreated by compression milling also vary significantly depending on the source and kind, the composition milling also vary significantly depending on the source and kind, the composition (contents of lignin and other components), and the structure. The power requirements for compression milling which renders equivalent hydrolysis yields also depend on the source and kind of lignocellulosics to be pretreated. For newspaper, the specific energy input required for 55% sugar yield is estimated as 0.3 kWh/lb substrate including 15% power loss. The additional sugar yield gained from the enzymatic hydrolysis of compression-milled newspaper (over and above the sugar yield of untreated substrate) is determined as 453 g sugar/kWh energy input.     

Arginine metabolism in wineLactobacillus plantarum: in vitro activities of the enzymes arginine deiminase (ADI) and ornithine transcarbamilase (OTCase)

This work was carried out to determine the activity of enzymes involved in arginine metabolism inLactobacillus plantarum isolated from wine and previously characterised at molecular level. The activity of the enzymes arginine deiminase and ornithine transcarbamylase was determined and citrulline and ornithine formed were analysed by HPLC analysis. Although the enzymatic activity was detected in all the strains analysed, a strong variability was observed between strains.Lactobacillus plantrum strain Lp60 is the strain with more possibilities to accumulate citrulline, precursor of the carcinogenic ethyl-carbamate, as showed by its high arginine deiminase activity and low ornithine transcarbamylase activity.     

Isolation, characterization and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.)

Porphobilinogen deaminase catalyzes the condensation of four porphobilinogen monopyrrole units into hydroxymethylbilane, a linear tetrapyrrole necessary for the formation of chlorophyll and heme in higher plant cells. We report the purification to homogeneity of a chloroplast-localized form of the enzyme from pea (Pisum sativum L.) by a novel purification scheme involving dye-ligand affinity chromatography. The purified chloroplast porphobilinogen deaminase consists of a single polypeptide with a relative molecular mass of 36-45 kDa as determined by size-exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of the protein is acidic. The activity of the enzyme shows different levels of sensitivity to divalent cations and is most sensitive to FE2+. The amino terminus of pea enzyme has been obtained by microsequencing and determined to bear little similarity to the amino acid sequences of porphobilinogen deaminases purified from other organisms. Polyclonal antisera elicited against the purified protein has been used to examine the abundance and cellular distribution of the enzyme.     

Spawning seasons of Rasbora tawarensis (Pisces: Cyprinidae) in Lake Laut Tawar,Aceh Province,Indonesia

Background  

Rasbora tawarensis is an endemic freshwater fish in Lake Laut Tawar, Aceh Province, Indonesia. Unfortunately, its status is regarded as critical endangered with populations decreasing in recent years. To date no information on the spawning activities of the fish are available. Therefore, this study provides a contribution to the knowledge on reproductive biology of R. tawarensis especially on spawning seasons as well as basic information for conservation of the species.