Biomedical subjects

G L Matters

Publications and source records attributed to G L Matters.

13 recordsLinked to original sources

PubMed2001

Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity.

Meprin A and B are highly regulated, secreted, and cell-surface metalloendopeptidases that are abundantly expressed in the kidney and intestine. Meprin oligomers consist of evolutionarily related alpha and/or beta subunits. The work herein was carried out to identify bioactive peptides and proteins that are susceptible to hydrolysis by mouse meprins and kinetically characterize the hydrolysis. Gastrin-releasing peptide fragment 14-27 and gastrin 17, regulatory molecules of the gastrointestinal tract, were found to be the best peptide substrates for meprin A and B, respectively. Peptide libraries and a variety of naturally occurring peptides revealed that the meprin beta subunit has a clear preference for acidic amino acids in the P1 and P1' sites of substrates. The meprin alpha subunit selected for small (e.g. serine, alanine) or hydrophobic (e.g. phenylalanine) residues in the P1 and P1' sites, and proline was the most preferred amino acid at the P2' position. Thus, although the meprin alpha and beta subunits share 55% amino acid identity within the protease domain and are normally localized at the same tissue cell surfaces, they have very different substrate and peptide bond specificities indicating different functions. Homology models of the mouse meprin alpha and beta protease domains, based on the astacin crystal structure, revealed active site differences that can account for the marked differences in substrate specificity of the two subunits.

Amino Acid Sequence↗

PubMed2000

Structure of the mouse metalloprotease meprin beta gene (Mep1b): alternative splicing in cancer cells.

The mouse meprin beta gene encodes an integral membrane protease that is expressed in a tissue-specific manner in embryonic and adult epithelial cells, and in carcinoma cells. The meprin beta mRNA in the embryo, kidney and intestinal cells is 2.5kb, whereas the isoform in carcinoma cells (beta' mRNA) is 2.7kb. The work herein was initiated to explore the molecular mechanism responsible for the different isoforms. Overlapping fragments containing the Mep1b gene were obtained from a yeast artificial chromosome clone using polymerase chain reactions. The gene spans approximately 40kb and consists of 18 exons and 17 introns. The first three exons are unique to the 5' end of beta' mRNA; the next two exons correspond to the 5' end of beta mRNA. The rest of the exons (13 total) encode the regions common to both beta and beta' messages. In conjunction with the cDNA sequences, the gene structure establishes that alternative splicing of 5' exons is responsible for the generation of the mRNA isoforms. The DNA regions between beta'- and beta-specific exons and upstream of the first beta' exon have been completely sequenced to identify potential regulatory elements for beta and beta' transcription. There is significant homology between the two regions, indicating that a duplication event occurred during evolution of the Mep1b gene. Potential promoter elements and transcription factor-binding sites were identified from comparisons to sequences in the databanks. This is the first gene structure that has been completed for meprin subunits from all species. The work elucidates molecular mechanisms that regulate differential expression of the Mep1b gene.

Alternative Splicing↗

PubMed1999

Meprin B: transcriptional and posttranscriptional regulation of the meprin beta metalloproteinase subunit in human and mouse cancer cells.

A novel mRNA isoform encoding the cell surface metalloproteinase meprin beta is expressed in mouse teratocarcinoma cells and in a variety of cultured human cancer cells. In both mouse and human cells, the cancer cell-specific mRNA isoform, referred to as beta', has an extended 5' UTR as compared to the meprin beta mRNA isoform expressed in normal kidney and intestinal epithelium. The work herein aimed to determine the molecular mechanisms for the expression of meprin beta and beta' in normal and cancer cells, respectively. Analysis of the 5' end of the mouse meprin beta gene revealed that the unique sequences in the beta and beta' mRNA isoforms are encoded by separate exons that are alternately spliced, and transcribed from independent promoters. By contrast, the human meprin beta and beta' mRNAs have identical sequences except for 87 additional bases in the 5' UTR sequence of beta', indicating that a single, mixed usage promoter directs expression of the isoforms. The region upstream of the human meprin beta' transcription start site contained elements with homology to the promoters of intestine-specific genes, interspersed with AP-1 and PEA3 elements; the latter were essential to meprin beta' promoter activity in cancer cells. Phorbol myristal acetate increased meprin beta' mRNA levels in cultured human colon cancer cells, providing further evidence that AP-1/PEA3 sites are actively involved in meprin beta' expression.

Animals↗

PubMed1999

Expression and regulation of the meprin beta gene in human cancer cells.

A novel mRNA isoform (meprin beta') of the cell-surface protease subunit meprin beta was previously identified in human colon cancer cells. The study reported here revealed that this mRNA isoform was identical within the protein coding region and at the 3' end to the beta isoform of normal intestine but that it contained an extended 5' untranslated region. Meprin beta' mRNA was expressed in the human breast cancer cell lines MCF-7 and SK-BR-3, in the human osteosarcoma cell line U2 Os, and in the human pancreatic cancer cell line BxPC-3. Meprin beta mRNA, but not beta' mRNA, was expressed in human fetal kidney cells. We cloned and sequenced genomic DNA encoding portions of the promoter region of the meprin beta gene. The unique sequences present in the beta' mRNA were present in the human genomic DNA immediately upstream of the transcription start site for the beta mRNA. The human meprin promoter sequence was searched for potential transcription-factor binding sites, and putative activator protein-1, polyoma enhancer activator 3 (PEA3), CCAAT enhancer-binding protein beta, and estrogen-receptor binding sites were identified along with binding sites for the intestine-specific cdx-2 transcription factor. The activity of meprin promoter/luciferase reporter gene constructs transfected into U2 Os cells was highest with constructs containing 83 and 639 bp of promoter DNA. These regions of the promoter each contain a putative PEA3 element. Treatment of the human colon adenocarcinoma cell line HT29-18C1 with 50 or 100 ng/mL phorbol myristal acetate for 8 h increased meprin beta' mRNA levels. Likewise, U2 Os cells transfected with the -639/luciferase or -1800/luciferase constructs showed a phorbol myristal acetate-inducible increase in reporter gene activity, indicating that the PEA3 element within the -639 construct or other elements further upstream respond to phorbol ester.

Animals↗

PubMed1996

The gene promoter for a bean abscission cellulase is ethylene-induced in transgenic tomato and shows high sequence conservation with a soybean abscission cellulase.

Bean leaf abscission (organ separation) correlates with the de novo accumulation of a pI9.5 cellulase and its mRNA. Overlapping genomic clones encoding the bean abscission cellulase (BAC) were isolated and partially sequenced. In addition, a genomic clone for a soybean abscission cellulase (SAC) was identified and the sequence compared to the BAC genomic sequence. Two 5'-upstream regions are particularly well conserved in the two sequences. Of special interest here is the region between -1 and -200 in the BAC promoter which is highly conserved in the SAC gene. Particle gun bombardment with a BAC promoter construct containing 210 bp of BAC sequence 5' to the transcription start site was sufficient to drive abscission-specific and ethylene and auxin-regulated transient expression in bean. In addition to the transient expression assay, expression was examined in stably transformed tomato. A similar -210 bp BAC promoter construct supported a low level of ethylene-inducible reporter gene expression in tomato leaf abscission zones and adjacent petioles but not in ethylene-treated stem tissue or fruit. Expression from the -210 promoter in tomato abscission zones was inhibited by silver thiosulfate, an ethylene action inhibitor, and was partially inhibited by treatment with auxin.

Base Sequence↗

PubMed1996

Calcium and calmodulin are involved in blue light induction of the gsa gene for an early chlorophyll biosynthetic step in Chlamydomonas.

The Chlamydomonas reinhardtii nuclear gene gsa, which encodes the early chlorophyll biosynthetic enzyme glutamate 1-semialdehyde aminotransferase (GSAT), is specifically induced by blue light in cells synchronized in a 12-hr-light and 12-hr-dark regime. Light induction required the presence of a nitrogen source in the incubation medium. Maximal induction also required acetate. However, in the absence of acetate, partial induction occurred when Ca2+ was present in the medium at concentrations of > or = 1 microM. The Ca2+ channel-blocking agents Nd3+ and nifedipine partially inhibited the external Ca(2+)-supported induction of GSAT mRNA but did not inhibit acetate-supported induction. The calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited both external Ca(2+)-supported and acetate-supported induction. The Ca2+ ionophore A23187 caused a transient induction in the dark. These results suggest that Ca2+ and calmodulin are involved in the signal transduction pathway linking blue light perception to the induction of GSAT mRNA. The electron transport uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited acetate-supported induction of GSAT mRNA but did not inhibit external Ca(2+)-supported induction. It is proposed that in the presence of acetate, an internal pool of Ca2+ can be mobilized as a second message, whereas in the absence of acetate, internal Ca2+ is not available but the requirement for Ca2+ can be partially met by an external Ca2+ source. The mobilization of internal Ca2+ may require energy derived from metabolism of acetate.

Acetates↗

PubMed1995

Structure and expression of the Chlamydomonas reinhardtii alad gene encoding the chlorophyll biosynthetic enzyme, delta-aminolevulinic acid dehydratase (porphobilinogen synthase).

cDNA clones for the alad gene encoding the chlorophyll biosynthetic enzyme ALA dehydratase (ALAD) from Chlamydomonas reinhardtii were isolated by complementation of an Escherichia coli ALAD mutant (hemB). The C. reinhardtii alad gene encodes a protein that has 50 to 60% sequence identity with higher plant ALADs, and includes a putative Mg(2+)-binding domain characteristic of plant ALADs. Multiple classes of ALAD cDNAs were identified which varied in the length of their 3'-untranslated region. Genomic Southern analysis, using an ALAD cDNA as a probe, indicates that it is a single-copy gene. This suggests that the differently sized ALAD cDNAS are not the products of separate genes, but that a primary ALAD transcript is polyadenylated at multiple sites. A time course determination of ALAD mRNA levels in 12-h light:12-h dark synchronized cultures shows a 7-fold increase in ALAD mRNA at 2 h into the light phase. The ALAD mRNA level gradually declines but continues to be detectable up to the beginning of the dark phase. ALAD enzyme activity increases 3-fold by 6 h into the light phase and remains high through 10 h. Thus, there is an increase in both ALAD mRNA level and ALAD enzyme activity during the light phase, corresponding to the previously observed increase in the rate of chlorophyll accumulation.

Amino Acid Sequence↗

PubMed1994

Structure and light-regulated expression of the gsa gene encoding the chlorophyll biosynthetic enzyme, glutamate 1-semialdehyde aminotransferase, in Chlamydomonas reinhardtii.

The gsa gene, which encodes glutamate 1-semialdehyde (GSA) aminotransferase (GSAT), an enzyme in the chlorophyll and heme biosynthetic pathway, has been cloned from Chlamydomonas reinhardtii by complementation of an Escherichia coli hemL mutant. The deduced C. reinhardtii GSAT amino acid sequence has a high degree of similarity to GSAT sequences from barley, tobacco, soybean and various prokaryotic sources. In vitro enzyme activity assays from E. coli transformed with the C. reinhardtii GSAT cDNA showed that higher levels of GSAT activity are associated with the expression of the cDNA insert. Analysis of changes in mRNA levels in light:dark synchronized C. reinhardtii cultures was done by northern blotting. The level of GSAT mRNA nearly doubled during the first 0.5 h in the light and increased over 26-fold after 2 h in the light. This increase is comparable to previously reported increases in GSAT activity in dark-grown cultures transferred to the light, and is the first report of induction by light of a gene encoding an ALA biosynthetic enzyme in plant or algal cells. The accumulation of GSAT mRNA follows the pattern of chlorophyll accumulation and the pattern of chlorophyll a/b-binding protein (cabII-1) mRNA accumulation in these cells, suggesting that the two genes may be regulated by light through a common mechanism. Additional evidence that the GSAT mRNA may be transcriptionally regulated by light is found in the genomic sequence of the gsa gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

A gene/pseudogene tandem duplication encodes a cysteine-rich protein expressed during zygote development in Chlamydomonas reinhardtii.

The Class V zygote-specific gene from Chlamydomonas reinhardtii has been cloned and sequenced. This gene encodes a polypeptide of 86 amino acids, which contains a signal peptide and 6 cysteine residues arranged in an inverted symmetrical repeat. The Class V gene product is postulated to be a component of the zygote cell wall. Southern analysis revealed two tandemly oriented and closely linked copies of the Class V gene, designated A and B. The A gene appears to be a pseudogene, based on analysis of Class V cDNAs, primer extension with gene-specific primers, and Northern analysis which failed to detect an A gene transcript. Genetic analysis using a related Chlamydomonas species that lacks the A gene, but which produces normal zygotic progeny, further indicates that the A gene is not required for zygote development.

Amino Acid Sequence↗

PubMed1986

Effect of the free radical-generating herbicide paraquat on the expression of the superoxide dismutase (Sod) genes in maize.

10-day-old maize leaves were treated with the oxygen free radical-generating herbicide paraquat for 12 h. Paraquat treatments (10(-5) M) resulted in a 40% increase in superoxide dismutase activity and a smaller increase in catalase activity. The increase in total superoxide dismutase (SOD) activity correlates with higher levels of specific isozymes. The chloroplast (SOD-1) and cytosolic (SOD-2 and SOD-4) forms were increased significantly; however, the mitochondrial form (SOD-3) was increased only slightly. Higher levels of SOD-4 and SOD-3 after paraquat exposure were the result of increased synthesis of these proteins, as determined by labeling in vivo with [35S]methionine. Isolation and in vitro translation of polysomes from 10(-5) M paraquat-treated leaves indicated that paraquat increased the amount of polysomal mRNA which codes for SOD-4 and SOD-3. Superoxide dismutase induction does not appear to be a response that is specific to paraquat, since another superoxide-generating compound, juglone, caused a similar increase in total superoxide dismutase activity. Therefore, the effect of these compounds on the expression of the maize Sod genes is exerted via their ability to generate superoxide.

Catalase↗

PubMed1986

Changes in plant gene expression during stress.

Changes in gene expression which occur during periods of environmentally induced stress provide models for the study of gene regulation. Several types of stress have been shown to elicit a specific and reproducible pattern of gene expression in various plant species. These stress factors include heat shock, anaerobiosis, plant pathogens, oxygen free radicals, heavy metals, water stress, and chilling. In some cases, changes in specific genes have been identified, such as increases in the expression of the gene encoding the phytoalexin-synthesizing enzyme in pathogen elicitor-treated cells. However, in most cases, the functional identity of stress-induced genes is unknown. The alterations in gene expression during stress usually are rapid and repeatable, making these genetic systems ideal for examination of factors and mechanisms involved in gene regulation.

Environment↗

PubMed1986

Effect of elevated temperature on catalase and superoxide dismutase during maize development.

Seeds of the inbred maize lines, W64A, R6-67, and D10, were germinated and grown at 25 degrees, 35 degrees, or 40 degrees C for up to 10 days. The catalase activity in scutella of W64A seedlings grown at 40 degrees C was slightly lower than that in seedlings grown at 25 degrees C. The total superoxide dismutase activity in scutella was lower in seedlings grown at 40 degrees C than in those grown at 25 degrees C during the first 3 days of germination, but thereafter was not significantly different at these temperatures. The high-catalase mutant lines, R6-67 and D10, grown at 40 degrees C exhibited a developmental pattern of catalase activity that was severalfold lower than that seen in seedlings grown at 25 degrees C. The decrease in catalase activity in R6-67 seedlings grown at 40 degrees C was correlated with lower amounts of CAT-2 protein, which is normally present at significantly high levels in this line. The application of a catalase synthesis inhibitor revealed that the low levels of CAT-2 in R6-67 grown at 40 degrees C were due to slightly higher degradation rates and a significant drop in the rate of catalase protein synthesis.

Catalase↗

PubMed1982

Soluble starch synthases and starch branching enzymes from cotyledons of smooth- and wrinkled-seeded lines of Pisum sativum L.

Soluble starch synthase and branching enzyme were purified from 18-day-old cotyledons of the smooth-seeded pea cultivar Alaska (RR) and wrinkled-seeded pea cultivar Progress #9 (rr) by DEAE-cellulose chromatography. Two coeluting peaks of primed and citrate-stimulated starch synthase activity and a major and minor peak of branching enzyme activity were observed in Alaska. However, in Progress #9, only one peak of synthase activity was found. When crude extracts of Progress #9 were centrifuged, over 70% of the starch synthase activity was recovered in the pelleted fraction, and additional washings of the pellet released no further activity. The addition of purified starch granules to Alaska crude extracts also resulted in the recovery of a greater proportion of synthase activity in pelleted fractions. The two peaks of branching enzyme activity in Alaska differed in their stimulation of phosphorylase, amylose branching activity, and activity in various buffers. The DEAE-cellulose profile of Progress #9 showed no distinct peak of branching enzyme and less than 10% of the total activity found in Alaska. The association of one form of soluble starch synthase with the pelleted fraction and the greatly reduced levels of branching enzyme provide a partial explanation for the appearance of high-amylose starch in Progress #9 cotyledons.

1,4-alpha-Glucan Branching Enzyme↗