Pilot scale lactic acid fermentation of shrimp wastes for chitin recovery

Lactic fermentation of shrimp waste on solid substrates was studied as a means of preservation for chitin recovery. Shrimp wastes were fermented in 100-g flasks with varying levels of inoculation with lactobacilli as well as different types and levels of carbohydrate. Sucrose was selected as the carbohydrate source in further experimental work due to its better acid production potential as compared to lactose and milk whey powder. Lactic acid fermentation was scaled-up from 2 to 30 kg in column reactors using geometric similarity as the scale-up criterion. The pH rapidly decreased to less than 5.0, allowing preservation of wastes for at least 3 months. During ensilation, deproteinisation and demineralisation were observed. Chitin obtained from the silage was treated with acid and alkali for mineral and protein removal.     

An invasive cleavage assay for direct quantitation of specific RNAs.

RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in > or =20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.     

Mapping a forest mosaic – A comparison of vegetation and bird distributions using geographic boundary analysis

Many areas of ecological inquiry require the ability to detect and characterize change in ecological variables across both space and time. The purpose of this study was to investigate ways in which geographic boundary analysis techniques could be used to characterize the pattern of change over space in plant distributions in a forested wetland mosaic. With vegetation maps created using spatially constrained clustering and difference boundary delineation, we examined similarities between the identified boundaries in plant distributions and the occurrence of six species of songbirds. We found that vegetation boundaries were significantly cohesive, suggesting one or more crisp vegetation transition zones exist in the study site. Smaller, less cohesive boundary areas also provided important information about patterns of treefall gaps and dense patches of understory within the study area. Boundaries for songbird abundance were not cohesive, and bird and vegetation difference boundaries did not show significant overlap. However, bird boundaries did overlap significantly with vegetation cluster boundaries. Vegetation clusters delineated using constrained clustering techniques have the potential to be very useful for stratifying bird abundance data collected in different sections of the study site, which could be used to improve the efficiency of monitoring efforts for rare bird species.     

Linkage disequilibrium at the Angelman syndrome gene UBE3A in autism families.

Autistic disorder is a neurodevelopmental disorder with a complex genetic etiology. Observations of maternal duplications affecting chromosome 15q11-q13 in patients with autism and evidence for linkage and linkage disequilibrium to markers in this region in chromosomally normal autism families indicate the existence of a susceptibility locus. We have screened the families of the Collaborative Linkage Study of Autism for several markers spanning a candidate region covering approximately 2 Mb and including the Angelman syndrome gene (UBE3A) and a cluster of gamma-aminobutyric acid (GABA(A)) receptor subunit genes (GABRB3, GABRA5, and GABRG3). We found significant evidence for linkage disequilibrium at marker D15S122, located at the 5' end of UBE3A. This is the first report, to our knowledge, of linkage disequilibrium at UBE3A in autism families. Characterization of null alleles detected at D15S822 in the course of genetic studies of this region showed a small (approximately 5-kb) genomic deletion, which was present at somewhat higher frequencies in autism families than in controls.     

Arabidopsis UVH3 gene is a homolog of the Saccharomyces cerevisiae RAD2 and human XPG DNA repair genes

To identify mechanisms of DNA repair in Arabidopsis thaliana, we have analyzed a mutant (uvh3) which exhibits increased sensitivity to ultraviolet (UV) light, H2O2 and ionizing radiation and displays a premature senescence phenotype. The uvh3 locus was mapped within chromosome III to the GL1 locus. A cosmid contig of the GL1 region was constructed, and individual cosmids were used to transform uvh3 mutant plants. Cosmid N9 was found to confer UV-resistance, H2O2-resistance and a normal senescence phenotype following transformation, indicating that the UVH3 gene is located on this cosmid and that all three phenotypes are due to the same mutation. Analysis of cosmid N9 sequences identified a gene showing strong similarity to two homologous repair genes, RAD2 (Saccharomyces cerevisiae) and XPG (human), which encode an endonuclease required for nucleotide excision repair of UV-damage. The uvh3 mutant was shown to carry a nonsense mutation in the coding region of the AtRAD2/XPG gene, thus revealing that the UVH3 gene encodes the AtRAD2/XPG gene product. In humans, the homologous XPG protein is also involved in removal of oxygen-damaged nucleotides by base excision repair. We discuss the possibility that the increased sensitivity of the uvh3 mutant to H2O2 and the premature senescence phenotype might result from failure to repair oxygen damage in plant tissues. Finally, we show that the AtRAD2/XPG gene is expressed at moderate levels in all plant tissues.     

The cytoplasmic tail of CD4 targets chimeric molecules to a degradative pathway.

Many different cell surface receptors undergo endocytosis via coated pits. Once having entered the cell, the receptors are sorted into diverse pathways. Which path a given receptor will follow is determined by signals inherent in the receptor's structure. The nature of these structural features is not yet known. In this study, we have taken the approach of constructing chimeric molecules to localize the domain of the T-cell surface molecule CD4 which is responsible for targeting it for degradation. Chimeric molecules bearing the cytoplasmic domain of CD4 and the extracellular domain of either the low-density lipoprotein receptor or a major histocompatibility complex (MHC) class I molecule were both internalized in response to phorbol 12-myristate 13-acetate and were subsequently degraded, indicating that the cytoplasmic tail of CD4 contains all the information required for both processes. The ability to modulate the level of MHC class I molecules on the cell surface offers an approach to investigating quantitative aspects of antigen presentation, the initial possibilities of which are explored herein.     

Host specificity of nisin production by Lactococcus lactis

Kinetics of nisin production have been investigated in terms of endogenous features of the producer organism, Lactococcus lactis. Nisin-producing transposons (Tn Nip) were transferred to different hosts by conjugation. Constructs were cultivated in batch cultures and nisin produced was measured. The proteinase function of C2Prt (Tn Nip)-1 was eliminated by plasmid curing, resulting in the construct C2Prt - (Tn Nip)-1. C2Prt - (Tn Nip)-1 produced nisin to a higher concentration compared to C2Prt (Tn Nip)-1 and was able to maintain the maximum concentration till the end of cultivation. The final concentration of nisin produced was host-specific, because when different constructs carrying the same Tn Nip were cultivated they produced nisin to different concentrations. However, when the same host carried Tn Nip transposons derived from different donors the concentration of nisin produced was similar, suggesting that the two Tn Nip transposons may be similar.