Adenovirus type 2 late mRNA's: structural evidence for 3'-coterminal species.

Adenovirus type 2-infected HeLa cells were labeled with 32PO4 during the period 14 to 17 h postinfection. Viral mRNA's with polyadenylic acid were isolated by polyuridylic acid Sepharose chromatography and fractionated according to size by electrophoresis through an acrylamide-agarose slab gel. Messenger bands were eluted and partially degraded with alkali. RNA fragments from each band that contain polyadenylic acid were isolated by polyuridylic acid Sepharose chromatography and fingerprinted two-dimensionally after T1 RNase digestion. Three bands, with mobilities of approximately 26S, 21S, and 18S, shared two large characteristic T1 oligonucleotides in common in the fingerprints of their 3'-terminal sequences. These oligonucleotides were mapped with a Hpa II restriction fragment of adenovirus type 2 DNA with coordinates 49-50.2. We conclude that the three mRNA's are coterminal in sequence at their 3' ends and overlap at internal positions. Implications for the protein-coding potential of these mRNA's and the mechanisms of adenovirus tyep 2 late RNA processing are discussed.     

Autosomal dominant polycystic kidney disease: evidence for the existence of a third locus in a Portuguese family

Autosomal dominant polycystic kidney disease is characterized by clinical and genetic heterogeneity. Two loci implicated in the disease have previously been mapped (PKD1 on chromosome 16 and PKD2 on chromosome 4). By two point and multipoint linkage analysis, negative lod scores have been found for both chromosome 16 and chromosome 4 markers in a large Portuguese family, indicating that a third PKD locus is involved in the development of the disease.     

Structural evidence for a liver-specific glyceraldehyde-3-phosphate dehydrogenase.

The amino acid sequences near the amino termini of glyceraldehyde-3-phosphate dehydrogenase from bovine and porcine liver have been determined. Using classical peptide isolation techniques as well as automated Edman degradation, the NH₂-terminal 30 residues of the bovine liver enzyme were determined to be Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg-Leu-Val-Thr-Arg-Ala-Ala-Phe-Asn-Ser-Gly-Lys-Val-Asp-Ile-Val-Phe-Ile. Twenty-two residues from the NH₂-terminus of the porcine liver enzyme, determined using the automated Edman degradation, were identical to the corresponding sequence from bovine liver enzyme. Both liver enzymes have Asn at position 6. The corresponding residue 6 in the muscle and yeast glyceraldehyde-3-phosphate dehydrogenases is Asp. This evidence suggests that the Asn-6 residue is specific for the liver tissues. The exchange of Asn for Asp may significantly alter the allosteric properties of muscle and liver enzymes especially the activity of the liver enzymes in gluconeogenesis.     

5'-Nucleotide phosphodiesterase and alkaline phosphatase in tumor cells: evidence for existence of novel species in the cytosol

Characteristics of 5'-nucleotide phosphodiesterase (phosphodiesterase I, EC 3.1.4.1) and alkaline phosphatase (EC 3.1.3.1) activities in tumor cell lines of human and murine origin were examined. Of the 15 cell lines tested, 5'-nucleotide phosphodiesterase activity in 13 cell lines and alkaline phosphatase activity in 10 cell lines were inhibited by N-ethylmaleimide and activated by dithiothreitol (N-ethylmaleimide-sensitive), and suggested to be SH-enzymes. In contrast, the two phosphohydrolases from normal tissues were inactivated by dithiothreitol, but not by N-ethylmaleimide (dithiothreitol-sensitive). There was only one tumor cell line in which both activities were dithiothreitol-sensitive. Human hepatoma PLC/PRF/5 cells appear to possess both types of 5'-nucleotide phosphodiesterase and alkaline phosphatase, and the subcellular distribution of these enzymes in this cell line was investigated. Dithiothreitol-sensitive 5'-nucleotide phosphodiesterase and alkaline phosphatase of PLC/PRF/5 cells were localized in the plasma membrane as in normal tissues, but N-ethylmaleimide-sensitive phosphohydrolases were soluble cytosolic proteins. N-Ethylmaleimide-sensitive 5'-nucleotide phosphodiesterase and alkaline phosphatase activities from other cell lines were also recovered in the cytosol. Molecular masses of cytosolic N-ethylmaleimide-sensitive phosphohydrolases were apparently smaller than their membrane-bound dithiothreitol-sensitive counterparts, as judged from gel filtration. It was concluded that many tumor cell lines lack plasma membrane 5'-nucleotide phosphodiesterase and alkaline phosphatase, but express enzymes with similar activities in the cytosol, with properties clearly distinguishable from enzymes so far characterized.     

Mitogenome evidence for the existence of cryptic species in Coelomactra antiquata

Cryptic species which improve our understanding of species diversity and evolutionary histories within marine animals have been increasingly revealed in the marine realm. Coelomactra antiquate is an important commercial bivalve species, but has been suffering from severe population decline due to over-exploitation and deterioration of environmental conditions. To test the hypothesis that cryptic species might exist in C. antiquate presented in previous study, four complete mitogenomes of C. antiquate from northern and southern China were determined here. Comprehensive comparative analysis of the mitochondrial genomes of C. antiquate between northern and southern population reveals significant differences in genome composition, protein coding genes, tRNA genes, non-coding regions, genetic divergence and phylogenetic analysis. The results provide strong mitogenome evidence for the existence of cryptic species in C. antiquate. Besides, our results also support that comprehensive comparative analysis of mtDNA represents an accessible and powerful tool to identify cryptic species.     

The complete sequence of a full length cDNA for human liver glyceraldehyde-3-phosphate dehydrogenase: evidence for multiple mRNA species.

A recombinant M13 clone (O42) containing a 65 b.p. cDNA fragment from human fetal liver mRNA coding for glyceraldehyde-3-phosphate dehydrogenase has been identified and it has been used to isolate from a full-length human adult liver cDNA library a recombinant clone, pG1, which has been subcloned in M13 phage and completely sequenced with the chain terminator method. Besides the coding region of 1008 b.p., the cDNA sequence includes 60 nucleotides at the 5'-end and 204 nucleotides at the 3'-end up to the polyA tail. Hybridization of pG1 to human liver total RNA shows only one band about the size of pG1 cDNA. A much stronger hybridization signal was observed using RNA derived from human hepatocarcinoma and kidney carcinoma cell lines. Sequence homology between clone 042 and the homologous region of clone pG1 is 86%. On the other hand, homology among the translated sequences and the known human muscle protein sequence ranges between 77 and 90%; these data demonstrate the existence of more than one gene coding for G3PD. Southern blot of human DNA, digested with several restriction enzymes, also indicate that several homologous sequences are present in the human genome.     

Identification of a mitochondrial glycerol-3-phosphate dehydrogenase from Arabidopsis thaliana: evidence for a mitochondrial glycerol-3-phosphate shuttle in plants

We report molecular characterization of an Arabidopsis gene encoding a mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (FAD-GPDH) that oxidizes glycerol-3-phosphate (G-3-P) to dihydroxyacetone phosphate. We demonstrate through in vitro targeting assays that the encoded gene product can be imported into mitochondrial membrane systems. Enzyme activity of the protein was confirmed through heterologous expression in Escherichia coli. The Arabidopsis gene is expressed throughout plant development, but at the highest level during seed germination. We also show that expression of the Arabidopsis FAD-GPDH gene is coupled to oxygen consumption and affected by ABA and stress conditions. Together with an NAD(+)-dependent GPDH, this enzyme could form a G-3-P shuttle, as previously established in other eukaryotic organisms, and links cytosolic G-3-P metabolism to carbon source utilization and energy metabolism in plants.     

Myosin I heavy-chain genes of Acanthamoeba castellanii: cloning of a second gene and evidence for the existence of a third isoform

We have determined the complete sequence and structure of a second myosin I heavy-chain gene from Acanthamoeba castellanii. This gene, which we have named MIL, spans approx. 6kb, is split by 17 introns, encodes a 1147-aa polypeptide, and is transcribed in log-phase cells. The positions of six of the introns are conserved relative to a vertebrate muscle myosin gene. Similar to the previously characterized MIB heavy-chain gene, the deduced MIL heavy-chain aa sequence reveals a 125-kDa protein composed of a myosin globular head domain joined to a novel, approx. 50-kDa C-terminal domain that is rich in glycine, proline and alanine residues. There are differences, however, between MIL and MIB in the sequence organization of their unconventional C-terminal domains. We conclude from this and other data that Acanthamoeba express at least three myosin I heavy-chain isoforms: MIL, plus MIA and MIB, whose purifications have been published previously. Amoeba genomic DNA blots probed with a short, highly conserved sequence whose position is transposed between MIB and MIL indicate that the Acanthamoeba myosin I heavy-chain gene family may actually contain as many as six genes. Finally, we compared the myosin I sequences with those of two related proteins, Drosophila NinaC and the bovine myosin I-like protein, and found that a portion of the unconventional C-terminal domains of the amoeba myosins I and the bovine protein appear to be related.     

Kinetic evidence for interaction between aldolase and D-glyceraldehyde-3-phosphate dehydrogenase.

The possibility of interaction between purified rabbit muscle aldolase and D-glyceraldehyde-3-phosphate dehydrogenase was studied by rapid kinetic methods, by analyzing the kinetics of the consecutive reaction catalyzed by the coupled enzyme system. The Km of the intermediary product, glyceraldehyde 3-phosphate, produced by aldolase was determined in the coupled reaction for glyceraldehyde-3-phosphate dehydrogenase. Its value corresponds to that of the aldehyde (active) form of glyceraldehyde 3-phosphate, although in the given conditions the aldehyde leads to diol interconversion is faster than the enzymic reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase. We suggest that above a certain concentration of the enzymes the glyceraldehyde 3-phosphate produced by aldolase gets direct access to glyceraldehyde-3-phosphate dehydrogenase without participating in the aldehyde leads to diol interconversion which otherwise would occur if the substrate were to mix with the bulk medium.     

Pancreatic anionic trypsin: evidence for the existence of a 30 kDa form.

1. An anionic form of trypsin has been isolated from pancreas of various species (rat, pig, dog and cow). 2. The purification procedure included affinity chromatography on STI-Sepharose 4B and ion-exchange chromatography on DEAE-Sephadex A-50. 3. The preparation was homogenous as checked by SDS-polyacrylamide slab gel electrophoresis, resulting in an estimated molecular weight of 30 kilodaltons (kDa) for this anionic form. 4. Antibodies against the anionic form from rat pancreas cross-reacted towards the anionic enzyme from porcine pancreas but not with the dog or bovine enzyme, nor with all the studied cationic forms. 5. Limited proteolysis of tubulin, a cytoskeletal protein, with an anionic or cationic form of trypsin showed striking differences in the size of produced peptides.     

Spectral evidence for the existence of a second cytochrome o in whole cells of Vitreoscilla

Cytochrome o, a protoheme IX pigment, has been proposed as the terminal oxidase of the filamentous bacterium, Vitreoscilla. Aerobic and anaerobic photolysis of CO-liganded whole cells demonstrated the presence of a second CO-reactive pigment, cytochrome o'. At temperatures lower than -100 degrees C, anaerobic photolysis dissociated only about 25% of the total CO-liganded components to reveal the unliganded cytochrome o'. At these temperatures, the photolysis of cytochrome o could not be demonstrated. At warmer temperatures, recombination of CO with the reduced cytochrome o' occurred with an apparent energy of activation of 5.8 kcal/mol. Aerobic photolysis of whole cells demonstrated two oxygen-bound intermediates. At temperatures lower than -95 degrees C, a spectrally distinct compound with absorption maxima at 428, 534, and 564 nm appeared (form I'); the apparent second order rate constant (k+1) for the formation of this intermediate was found to be 9.1 M-1 s-1, the reverse rate (k-1) was 9.9 X 10(-5) s-1, and the equilibrium constant (Kd) was 1.1 X 10(-5) M. This oxygen intermediate of cytochrome o' is spectrally and kinetically similar to the oxygen intermediate of cytochrome o seen in Escherichia coli. At temperatures warmer than -90 degrees C, photolysis of aerobic samples resulted in the immediate formation of a second oxygen-bound intermediate (form I) with absorption maxima at 422, 534, and 564 nm. This second intermediate results from the binding of oxygen to the cytochrome o (oxygenated cytochrome o). These data support the proposal that whole cells of Vitreoscilla contain two alternative pathways of electron transport, one terminating with cytochrome o and the other with cytochrome o'.     

Control of enzyme synthesis in the oxalurate catabolic pathway of Streptococcus faecalis ATCC 11700: evidence for the existence of a third carbamate kinase

Streptococcus faecalis ATCC 11700 uses oxalurate as a sole energy source for growth. An oxamate carbamoyltransferase and a carbamate kinase, both induced by oxalurate, are involved in this process.The oxalurate-induced kinase is specific for the pathway. Its properties are different from those of the previously characterized agmatine and arginine-induced kinases.Glucose, but not arginine, nor agmatine, two other energy sources, represses the oxalurate pathway. In contrast, oxalurate was found to be at least as effective as glucose in repressing the arginine deiminase pathway in arginine grown cells or the agmatine deiminase pathway during growth on agmatine.     

Further evidence for the existence of B-lymphocyte subpopulations.

We have studied the homing properties of B lymphocytes by using ⁵¹Cr-labeled lymphoid cells obtained from athymic, nu/nu mice, and animals made T-lymphocyte deficient by thymectomy and lethal irradiation followed by reconstitution with syngeneic bone marrow. Comparison was made to the patterns of distribution observed when cell preparations containing normal numbers of T and B lymphocytes were migrated. A small but significant percentage of labeled lymphocytes from lymph nodes, spleen, Peyer's Patches, and bone marrow of T-cell-deficient animals was shown to be lymph node seeking. Secondary transfers of lymph node cells from primary recipients caused enrichment of this lymph node-seeking population. Treatment of T-lymphocyte-deficient lymphoid cell preparations with neuraminidase reduced the percentages of cells homing to the lymph nodes. The data showed that B lymphocytes exhibit unique homing properties when injected into normal recipients. In addition, direct comparison of the homing patterns of B lymphocytes prepared from spleen and lymph nodes of athymic mice revealed differences suggesting that these lymphoid organs contained unique mixtures of at least two different kinds of B cell. The evidence supports the notion that the B-lymphocyte populations contain at least two subpopulations, one of which possesses the ability to home to lymph nodes.     

Evidence for the existence of a novel enzyme system. myo-Inositol-1-phosphate dehydrogenase in Phaseolus aureus.

A novel enzyme system, myo-inositol-1-phosphate dehydrogenase, has been isolated from germinating mung bean seeds. The dehydrogenation and cleavage of myo-inositol 1-phosphate by this enzyme leads to the synthesis of a pentose phosphate which appears to be ribulose 5-phosphate. The pH optimum of the enzyme is 8.6; NAD+ is required as coenzyme and no other nucleotides can replace NAD+. Mono- or divalent cations are not essential for the enzyme activity. Stoichiometry of the reaction suggests that 2 mol of NAD+ are reduced per mol of ribulose-5-P generated.     

A role for phosphatidylinositol 3-phosphate in abscisic acid-induced reactive oxygen species generation in guard cells

Guard cells generate reactive oxygen species (ROS) in response to abscisic acid (ABA), which leads to stomatal closing. The upstream steps of the ABA-induced ROS generation pathway remain largely unknown. In animal cells, ROS generation in neutrophils is activated by phosphatidylinositol 3-phosphate (PI3P). Stomatal guard cells contain PI3P and PI 3-kinase activity. In this study, we tested whether PI3P has a role in ROS generation in guard cells exposed to ABA. We found that PI 3-kinase inhibitors wortmannin or LY294002 inhibited ABA-induced ROS generation and stomatal closing. Endosome-binding domain (of human EEA1), which specifically binds to PI3P, also inhibited ABA-induced ROS generation and stomatal closing when overexpressed in guard cells. Hydrogen peroxide partially reversed the effects of wortmannin or LY294002 on ABA-induced stomatal closing. These results support a role for PI3P in ABA-induced ROS generation and stomatal closing movement.     

Protein kinase C-mediated negative-feedback inhibition of unstimulated and bombesin-stimulated polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells.

Bombesin-related peptides stimulate a rapid increase in polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells. These peptides generate an increase in the efflux of 45Ca2+ from pre-labelled cells, a response consistent with an inositol trisphosphate-mediated mobilization of intracellular Ca2+. The bombesin-stimulated release of cellular 45Ca2+ is inhibited by tumour-promoting phorbol esters (e.g. 12-O-tetradecanoylphorbol 13-acetate, TPA). Although there are several possible sites of action at which this effect might occur, our results indicate that TPA induces an uncoupling of bombesin-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) without decreasing cellular binding of bombesin. In cultured cells, protein kinase C can be down-modulated by a prolonged incubation of the cells with phorbol esters. Such pretreatment greatly decreased the inhibitory effect of TPA on bombesin-stimulated PIP2 hydrolysis, suggesting that this action of the phorbol ester is mediated via protein kinase C. Since diacylglycerol is an endogenous activator of protein kinase C and a direct product of PIP2 hydrolysis, these results suggest that protein kinase C inhibition of polyphosphoinositide hydrolysis may function as a negative-feedback pathway. Cells in which protein kinase C has been down-modulated show elevated basal and bombesin-stimulated production of inositol phosphates, providing evidence that such a feedback loop limits polyphosphoinositide turnover in both unstimulated and mitogen-stimulated cells.