Influence of mixed and single infection of grapevine leafroll-associated viruses and viral load on berry quality.

Grapevine leafroll disease is a viral disease that affects grapevines (Vitis vinifera L.) and has a severe economic impact on viticulture. In this study, the effect of grapevine leafroll-associated viruses (GLRaV) on berry quality was investigated in clones of cultivar cv.

Differentiation of mucinous from non-mucinous pancreatic cyst fluid using dual-stained, 1 dimensional polyacrylamide gel electrophoresis.

Background: Pancreatic cysts are being increasingly identified in patients. Mucinous cysts have malignant potential whereas non-mucinous cysts do not. Distinguishing potentially malignant cysts from harmless ones by the characterization of cyst fluid contents remains a difficult problem.

Analysis of protein expression patterns in Barrett's esophagus using MALDI mass spectrometry, in search of malignancy biomarkers.

In order to detect early changes of malignant degeneration in Barrett's esophagus (BE), and to reduce the cost of surveillance, molecular biomarkers of early malignancy have been sought, with limited success, using genomic and immunohistochemical tools. We postulate that direct analysis of epithelial proteins using mass spectrometry will provide protein profiles capable of identifying patients at high risk of developing malignancy. Our aim is to find transitional protein signals that show a cancer profile within histologically benign BE, which can be used as indicators of early malignant change.

A 9,000-year record of Chagas' disease.

Tissue specimens from 283 principally spontaneously (naturally) desiccated human mummies from coastal and low valley sites in northern Chile and southern Peru were tested with a DNA probe directed at a kinetoplast DNA segment of Trypanosoma cruzi. The time interval spanned by the eleven major cultural groups represented in the sample ranged from approximately 9,000 years B.P.

Hybridization screening of very short PCR products for paleoepidemiological studies of Chagas' disease.

Single strands of very short PCR products can be covalently immobilized to a slide and then easily detected by probe hybridization. In this work, the PCR product was a 70-nucleotide segment of ancient DNA, representing a portion of repeat mini-circle DNA from the kinetoplast of Trypanosoma cruzi, the infectious agent of Chagas' disease (American Trypanosomiasis). The target segment was initially established to be present in soft tissue samples taken from four "naturally" mummified Andean bodies using PCR followed by cloning and sequencing.

Induction of carbamoyl phosphate synthetase III and glutamine synthetase mRNA during confinement stress in gulf toadfish (Opsanus beta).

Gulf toadfish (Opsanus &bgr;) rapidly switch to excretion of urea as their main nitrogenous waste product under several laboratory conditions, including confinement to small volumes of water. Prior evidence suggested that the activities of two key enzymes of urea synthesis exhibited potentially different modes of upregulation during this switch, with carbamoyl phosphate synthethase III (CPSase III) activated allosterically by N-acetylglutamate, and glutamine synthetase (GSase) activated by increases in the concentration of protein. The present study was undertaken to examine additional aspects of the regulation of these enzymes.

Nitrogen excretion and expression of carbamoyl-phosphate synthetase III activity and mRNA in extrahepatic tissues of largemouth bass (Micropterus salmoides).

Low levels of all of the enzymes required for urea synthesis via the urea cycle, including mitochondrial glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase III (CPSase III) and cytosolic glutamine synthetase, are known to be present in liver of the teleost fish largemouth bass (Micropterus salmoides). The levels of these enzymes are higher than those in most other teleosts, but they are significantly lower than the levels present in liver of ureoosmotic elasmobranchs. The purpose of this study was to assess the physiological role of CPSase III in the context of urea synthesis in adult bass.

Expression of carbamoyl-phosphate synthetase III mRNA during the early stages of development and in muscle of adult rainbow trout (Oncorhynchus mykiss).

It has been reported that the activities of the urea cycle-related enzymes ornithine carbamoyltransferase and carbamoyl-phosphate synthetase III (CPSase III) are induced during early life stages of ammonotelic rainbow trout (Oncorhynchus mykiss), suggesting that the urea cycle may play a physiological role in early development in teleost fish (Wright, P. A., Felskie, A.

The promoter region of the carbamoyl-phosphate synthetase III gene of Squalus acanthias.

Carbamoyl-phosphate synthetase III (CPSase III) of Squalus acanthias (spiny dogfish) is a nuclear-encoded mitochondrial enzyme that catalyzes glutamine-dependent formation of carbamoyl phosphate for urea synthesis. In this paper we report the results of cloning a 10-kb segment of genomic DNA which includes the region flanking the 5' end of the spiny dogfish CPSase III gene. A total of 1,295 base pairs of sequence straddling the start codon was obtained.

Human enzyme polymorphism in the Canary Islands. VII. G6PD Seattle in Canarians and North African Berbers.

Sequence analysis of the glucose-6-phosphate dehydrogenase (G6PD) variant Gc, characterized by slower electrophoretic mobility than G6PD-B, in 14 Canarian and 2 Berber males, has revealed that all of them share the G-->C mutation at nucleotide 844 in exon 8 leading to an Asp-->His substitution at amino acid 282 known as G6PD Seattle. No additional differences have been detected among them nor with the common haplotype previously found for this variant.

Canine brain glucose transporter 3: gene sequence, phylogenetic comparisons and analysis of functional sites.

There has been a sparsity of various mammalian neuronal glucose transporter 3-encoding sequences(Glut3) available for the purposes of alignment studies. We report here a 2355-bp sequence of canine Glut3 that encodes a deduced protein of 496 amino acids (aa). The full-length canine aa sequence was compared to those of the human, mouse and rat glucose transporter 3 (Glut3), and found to be 88.

Pre-Columbian tuberculosis in northern Chile: molecular and skeletal evidence.

Analysis of 483 skeletons from Arica (Chile) and review of mummy dissection records demonstrates an overall 1% prevalence rate for tuberculosis between 2000 B.C. and A.

Nucleotide sequence and tissue-specific expression of the multifunctional protein carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) mRNA in Squalus acanthias.

Carbamoyl-phosphate synthetase II (CPSase II), aspartate transcarbamoylase (ATCase), and dihydroorotase (DHOase) catalyze the first three steps of de novo pyrimidine nucleotide biosynthesis, respectively. In mammalian species, these three enzyme activities exist in the cytosol in liver and other tissues as a multifunctional complex on a single polypeptide called carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase (CAD) in the order of NH2-CPSase II-DHOase-ATCase-COOH. Previous studies provided evidence that in Squalus acanthias (spiny dogfish) these enzymes are not expressed in liver and that they exist as separate entities in the cytosol of extra-hepatic tissues such as testes and spleen (Anderson, P.

Carbamyl phosphate synthetase III, an evolutionary intermediate in the transition between glutamine-dependent and ammonia-dependent carbamyl phosphate synthetases.

The amino acid sequence of carbamyl phosphate synthetase (CPS) III from liver of spiny dogfish shark Squalus acanthias was deduced from the nucleotide sequence of its cDNA. Alignment of the derived amino acid sequence of CPS III with sequences of rat and frog CPS I and hamster CPS II reveals a high degree of amino acid identity, indicating that CPS III shares the same common ancestral genes as CPSs I and II. All of the CPSs examined show a high conservation of sequences in the adenine nucleotide binding domains and in residues that have been implicated in catalysis.

Identification of Mycobacterium tuberculosis DNA in a pre-Columbian Peruvian mummy.

The existence of tuberculosis in the pre-Columbian Americas is controversial because the morphology of the lesion is not specific, the organism is culturally nonviable in ancient tissues, and nonpathogenic soil mycobacteria can contaminate buried bodies. We report the recovery of DNA unique to Mycobacterium tuberculosis from a lung lesion of a spontaneously mummified, 1000-year-old adult female body in southern Peru. This provides the most specific evidence possible for the pre-Columbian presence of human tuberculosis in the New World.

Application of novel PCR strategies to amplify and sequence glucose transporters in canine brain.

We employ here a modified oligo(dT) primer called a "lock-docking" primer which enables the production of a cDNA template that can subsequently be amplified in the 3'-RACE PCR procedure to produce discrete, first-round products for 3'-cDNA ends of any reverse-transcribed poly(A) RNAs. An upstream consensus primer targeted to a highly conserved region (amino acid region 448-455 in human Glut1) among all glucose transporter isoforms was used in the 3'-RACE PCR procedure with lock-docked cDNA template. Sources of lock-docked cDNA template were canine intestine, kidney, brain cortex, and brain microvessels.

A lock-docking oligo(dT) primer for 5' and 3' RACE PCR.

We describe a method that can be used to obtain and sequence 3' and 5' ends of cDNA transcripts directly from PCR products. The method employs a modified oligo(dT) primer that enables it to "lock-dock" at the junction of gene-specific cDNA sequence and a natural (3') or appended (5') poly(A) tail. As a result, discrete, first-round PCR products are obtained that are easily isolated and sequenced directly.

PCR libraries of ancient DNA using a generalized PCR method.

We describe a generalized PCR method that will amplify fragments of DNA without any knowledge of sequence using a single primer. Although we are presently using this method to amplify DNA fragments isolated from ancient preserved tissues, in effect, producing PCR libraries, it may prove to have other applications.

The preparation of poly(A)+mRNA from the hagfish slime gland.

The isolation of translatable poly(A)+mRNA from the slime glands of the Pacific hagfish, Eptatretus stouti, is not possible by the commonly used procedures because of the viscous slime that is formed when the contents of the glands are hydrated. This paper reports on a procedure developed to overcome this problem. Briefly, the tissue was powdered in liquid nitrogen, mixed with sodium lauroylsarcosine and proteinase K and lyophilized.

Hagfish slime gland thread cells. II. Isolation and characterization of intermediate filament components associated with the thread.

The slime glands of hagfish have two major cell types, gland thread cells (GTCs) and gland mucous cells (GMCs), both of which upon contact with water contribute to the formation of an abundant quantity of viscous mucus. In previous studies we reported a method for the isolation of GTCs and showed that each ellipsoidal thread cell normally contains a single tapered thread which is uniquely coiled into a space-saving conformation and occupies most of the cell volume. Subsequently, the developing thread was found to consist mainly of intermediate filaments (IFs) aligned in parallel not only to one another but also to a far fewer number of interspersed microtubules (see accompanying paper).

The hagfish slime gland thread cell. I. A unique cellular system for the study of intermediate filaments and intermediate filament-microtubule interactions.

Thread cell differentiation in the slime gland of the Pacific hagfish Eptatretus stouti has been studied using light microscopy and scanning and transmission electron microscopy. Thread cell differentiation is remarkable in that the life history of the cell is largely dedicated to the production of a single, tapered, cylindrical, highly coiled, and precisely packaged cytoplasmic thread that may attain lengths of 60 cm and diameters approaching 1.5 micron.

Fractionation of hagfish slime gland secretions: partial characterization of the mucous vesicle fraction.

This paper deals with the collection, fractionation and partial characterization of the slime gland secretion of the Pacific hagfish (Eptatretus stouti) with emphasis on the mucous fraction. Secretions were collected by electrical stimulation of the glands of anesthetized hagfish and, using three different methods, separated into three fractions: 1) the thread cells, 2) the mucous vesicles of the mucous cells, and 3) the soluble fraction. The methods take advantage of the stabilization of the thread cells and mucous vesicles by ammonium sulfate and sodium citrate.

The hagfish slime gland: a model system for studying the biology of mucus.

The hagfish slime gland may provide a model system for studying certain aspects of the biology of mucus. Mucus is obtained in nonhydrated form by electrically stimulating the anesthetized hagfish and the secretions are stirred into ammonium sulfate. Centrifugation and filtration are than used to isolate the two major secretory products, mucous vesicles and threads.

Threads in the hagfish slime gland thread cells: organization, biochemical features, and length.

Scanning electron microscopy in conjunction with cell isolation procedures revealed details of the packing of threads in hagfish slime gland thread cells. Biochemical studies indicate that the thread is largely composed of a protein subunit with a molecular weight of 63,500. Mathematical calculations suggest that the thread may attain lengths of 60 centimeters or more.

The incorporation of tritium from tritium-enriched water into UDP-N-acetylglucosamine and UDP-N-acetylmannosamine catalyzed by UDP-N-adetylglucosamine 2-epimerase from Escherichia coli.

Uridine diphosphate N-acetylglucosamine 2-epimerase from Escherichia coli 014 K7 H- catalyzes the reversible epimerization of uridine diphosphate N-acetylglucosamine to uridine diphosphate N-acetylmannosamine. During epimerization, tritium from tritium-enriched water is incorporated into both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylmannosamine. The position of incorporation is C-2 of the N-acetylhexosamine moieties.