Biomedical subjects

H M Goodman

Publications and source records attributed to H M Goodman.

At least 37 records · Page 2Linked to original sources

PubMed1998

Tissue distribution, turnover, and glycosylation of the long and short growth hormone receptor isoforms in rat tissues.

Two isoforms of the GH receptor, the full-length receptor (GHRL) and a short isoform (GHRS) that lacks the transmembrane and intracellular domains of GHRL, have been analyzed in rat tissue extracts by Western blotting and immunoprecipitation. Although quantitative estimates of GHRS and GHRL based on coprecipitation of [125I]GH indicated similar amounts of both isoforms in tissue extracts, the 110 kDa band corresponding to GHRL was generally not detected on Western blots without enrichment by immunoprecipitation. Two bands with electrophoretic mobilities corresponding to 38 and 42 kDa were present in extracts prepared from liver, muscle, and adipocytes. Western blots of the GH binding protein in rat serum also revealed two bands, but these had electrophoretic mobilities corresponding to 44 and 52 kDa. After digestion by endoglycosidase F, a single band with an electrophoretic mobility corresponding to 31 kDa was detected in samples from adipocytes, liver or serum, indicating that GHRS retained in tissues is glycosylated less extensively than that in rat serum. Digestion with neuraminidase indicated that the smaller glycoproteins in tissue extracts lack sialic acid residues that are present in serum samples. Furthermore, endoglycosidase H degraded GHRS in liver extracts to a 31 kDa band but did not degrade serum samples, suggesting that tissues retain a high mannose form of GHRS. The abundance of GHRS or GHRL in tissues from male, virgin female, and pregnant rats was estimated from the amount of 125I-GH that was bound to each isoform after immunoprecipitation. Liver contained more than 10 times as much GHRS per gram of tissue as fat or muscle. In liver, muscle, and fat, the amount of GHRS exceeded that of GHRL, sometimes by as much as 6-fold. GHBP levels in serum of females exceeded those in males, and rose even higher in pregnant females. The abundance of GHRS in all tissue extracts paralleled serum levels. In muscle and fat, the levels of GHRL did not differ in male, female and pregnant rats, whereas in liver, the pattern was similar to the GHRS pattern. In all tissues, pools of GHRS exceeded those of GHRL by a factor that grew larger as tissue and serum levels increased. The half life of GHBP in serum was estimated to be 2.4 h in rats treated with cycloheximide, whereas that of GHRS was 20 min in liver and 8.5 h in fat. These results suggest that GHRS is synthesized in liver 8 times faster than it is released into serum, whereas synthesis in fat is less than 30% of the rate at which it is released into serum by all tissues. Therefore, liver appears to be the major source of GHBP in serum. Although secretion into the circulatory system accounts for little or perhaps none of its turnover in some tissues, GHRS pools in tissues do appear to be regulated, suggesting that GHRS may function primarily in the cells in which it is synthesized.

Adipocytes↗

PubMed1998

Application of fiber-FISH in physical mapping of Arabidopsis thaliana.

Arabidopsis thaliana has become a model plant species for genetic studies because of its small genome and short juvenility period. However, the small chromosomes of this species are not suitable for classical cytogenetic studies. Here we demonstrate that the fluorescence in situ hybridization (FISH) technique using extended DNA fibers can be a powerful tool in the physical mapping of the A. thaliana genome. Using a refined fiber-FISH technique we were able to measure DNA clusters as long as 1.71 Mb, more than 1% of the A. thaliana genome. Several small DNA loci, including the telomeres and a dispersed repetititve DNA sequence, mi167, were also analyzed with this technique. The results show that without known adjacent DNA markers such small DNA loci cannot be mapped precisely using fiber-FISH. One of the most difficult obstacles in physical mapping by contig assembly is closing the gaps that are present between adjacent contigs. Currently available molecular techniques are not sufficient to accurately estimate the physical sizes of these gaps. We isolated bacterial artificial chromosome (BAC) clones bordering gaps 2 and 3 on the physical contig map of A. thaliana chromosome II. The BAC clones were used in fiber-FISH analysis and the physical sizes of the two gaps were estimated as 31 kb and more than 500 kb, respectively. Thus, we have demonstrated that fiber-FISH is an efficient technique for determining the physical size of gaps on molecular contig maps.

Arabidopsis↗

PubMed1997

An Arabidopsis gene encoding a putative 14-3-3-interacting protein, caffeic acid/5-hydroxyferulic acid O-methyltransferase.

AFT1, a 14-3-3 protein from Arabidopsis thaliana, was used as a 'bait' in the two-hybrid system to identify its interacting proteins. A caffeic acid/5-hydroxyferulic acid O-methyltransferase, OMT1, was identified as one of the several proteins that specifically interacts with AFT1 in yeast cells. The physical interaction between AFT1 and a partial OMT1 polypeptide can be demonstrated in vitro by using bacterially expressed proteins. A single copy gene was found to encode OMT1 in Arabidopsis, and its expression is both spatially and temporally regulated.

14-3-3 Proteins↗

PubMed1997

Cloning and expression of an Arabidopsis gene encoding a putative peroxisomal ascorbate peroxidase.

An Arabidopsis 14-3-3 protein, AFT1, was used as a 'bait' in the two-hybrid system to identify its interacting proteins. One of the candidate proteins, APX3, was identified as a putative peroxisomal membrane-bound ascorbate peroxidase. Ascorbate peroxidases are important defense enzymes that protect plant cells from oxidative stress damage. DNA blot analysis indicates that APX3 is encoded by a single-copy gene in the Arabidopsis genome. RNA blot analyses show that APX3 transcript levels increase slightly in response to cold, UV light, and treatments with hydrogen peroxide and paraquat. The activity of APX3 in Arabidopsis may be controlled in two ways: its enzymatic activity through protein-protein interactions and its transcription by transcriptional or posttranscriptional regulation.

14-3-3 Proteins↗

PubMed1997

Construction of an approximately 2 Mb contig in the region around 80 cM of Arabidopsis thaliana chromosome 2.

A method for construction of bacterial artificial chromosome (BAC) contigs from a yeast artificial chromosome (YAC) physical map is described. An approximately 2 Mb contig, consisting of two large BAC contigs linked by a small YAC, has been assembled in the region around 80 cM of Arabidopsis thaliana chromosome 2. Clones from this contig will facilitate gene isolation in the region and can be used directly as substrates for DNA sequencing.

Arabidopsis↗

PubMed1997

Antisense inhibition of the photosystem I antenna protein Lhca4 in Arabidopsis thaliana.

The function of Lhca4, a gene encoding the photosystem 1 type IV chlorophyll a/b-binding protein complex in Arabidopsis, was investigated using antisense technology. Lhca4 protein was reduced in a number of mutant lines and abolished in one. The inhibition of protein was not correlated with the inhibition of mRNA. No depletion of Lhca1 was observed, but the low-temperature fluorescence emission spectrum was drastically altered in the mutants. The emission maximum was blue-shifted by 6 nm, showing that chlorophyll molecules bound to Lhca4 are responsible for most of the long-wavelength fluorescence emission. Some mutants also showed an unexplainable delay in flowering time and an increase in seed weight.

Arabidopsis↗

PubMed1996

Expression analysis of an Arabidopsis C2H2 zinc finger protein gene.

C2H2 zinc finger protein genes encode nucleic acid-binding proteins involved in the regulation of gene activity. AtZFP1 (Arabidopsis thaliana zinc finger protein 1) is one member of a small family of C2H2 zinc finger-encoding sequences previously characterized from Arabidopsis. The genomic sequence corresponding to the AtZFP1 cDNA has been determined. Molecular analysis demonstrates that AtZFP1 is a unique, intronless gene which encodes a 1100 nucleotides mRNA highly expressed in roots and stems. A construct in which 2.5 kb of AtZFP1 upstream sequences is linked to the beta-glucuronidase gene was introduced into Arabidopsis by Agrobacterium-mediated transformation of roots. Histochemical analysis of transgenic Arabidopsis carrying the AtZFP1 promoter: beta-glucuronidase fusion shows good correlation with RNA blot hybridization analysis. This transgenic line will be a useful tool for analyzing the regulation of AtZFP1 to further our understanding of its function.

Amino Acid Sequence↗

PubMed1996

Multiple non-LTR retrotransposons in the genome of Arabidopsis thaliana.

DNA sequence analysis near the Arabidopsis thaliana ABI3 gene revealed the presence of a non-LTR retrotransposon insertion that we have designated Ta11-1. This insertion is 6.2 kb in length and encodes two overlapping reading frames with similarity to non-LTR retrotransposon proteins, including reverse transcriptase. A polymerase chain reaction assay was developed based on conserved amino acid sequences shared between the Ta11-1 reverse transcriptase and those of non-LTR retrotransposons from other species. Seventeen additional A. thaliana reverse transcriptases were identified that range in nucleotide similarity from 48-88% (Ta12-Ta28). Phylogenetic analyses indicated that the A. thaliana sequences are more closely related to each other than to elements from other organisms, consistent with the vertical evolution of these sequences over most of their evolutionary history. One sequence, Ta17, is located in the mitochondrial genome. The remaining are nuclear and of low copy number among 17 diverse A. thaliana ecotypes tested, suggesting that they are not highly active in transposition. The paucity of retrotransposons and the small genome size of A. thaliana support the hypothesis that most repetitive sequences have been lost from the genome and that mechanisms may exist to prevent amplification of extant element families.

Amino Acid Sequence↗

PubMed1996

A physical map of chromosome 2 of Arabidopsis thaliana.

A yeast artificial chromosome (YAC) physical map of chromosome 2 of Arabidopsis thaliana has been constructed by hybridization of 69 DNA markers and 61 YAC end probes to gridded arrays of YAC clones. Thirty-four YACs in four contigs define the chromosome. Complete closure of the map was not attained because some regions of the chromosome were repetitive or were not represented in the YAC library. Based on the sizes of the YACs and their coverage of the chromosome, the length of chromosome 2 is estimated to be at least 18 Mb. These data provide the means for immediately identifying the YACs containing a genetic locus mapped on Arabidopsis chromosome 2.

Arabidopsis↗

PubMed1996

Growth hormone regulates cytosolic free calcium in rat fat cells by maintaining L-type calcium channels.

In freshly isolated individual rat adipocytes, cytosolic free Ca2+ concentration ([Ca2+]i) as measured with fura 2 slowly declined during incubation but was sustained, or even somewhat increased, by brief treatment with growth hormone (GH) at the beginning of a 3-h incubation period. GH-treated adipocytes were more permeable to Ca2+ than GH-deprived cells as indicated, using Mn2- as a surrogate and monitoring influx by the rate of quenching of fura 2 fluorescence. Blockage of Ca2- channels with 100 nM nimodipine lowered [Ca2+]i in GH-treated cells to the level seen in GH-deprived cells. Increases in [Ca2+]i or the rate of Mn2+ entry were twofold greater in GH-treated than in GH-deprived cells when extracellular K+ was increased to 30 mM. Similarly, the Ca2+ channel agonist BAY K 5552 or the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol increased [Ca2+]i more in GH-treated than in GH-deprived adipocytes. Ca(2+)-ATPase activity was two times higher in plasma membranes isolated from GH-treated than from GH-deprived cells. Continued synthesis of Ca(2+)-ATPase may depend on [Ca2+]i, since the effects of GH on [Ca2+]i and Ca(2+)-ATPase were blocked by a cycloheximide or verapamil. We suggest that voltage-sensitive L-type Ca2+ channels regulate steady-state [Ca2+]i in rat adipocytes and that GH maintains the number or functional integrity of these channels.

Adipocytes↗

PubMed1996

Growth hormone increases calcium uptake in rat fat cells by a mechanism dependent on protein kinase C.

Growth hormone (GH; 500 ng/ml) rapidly doubled cytosolic free Ca2+ concentration ([Ca2+]i) in rat adipocytes as determined with the Ca2+ indicator fura 2. No response was seen in Ca(2+)-free medium, suggesting that the increase in [Ca2+]i was due to Ca2+ influx. GH also doubled the influx of Mn2- as inferred from the rate of fluorescence quenching. Depolarization with 30 mMK+ also increased [Ca2+]i, and the increase in [Ca2+]i due to either GH or 30 mMK+ was blocked by 100 nM nimodipine, suggesting that GH increases [Ca2+]i by activating voltage-sensitive L-type Ca2+ channels. GH increased [Ca2+]i even when K+ channels were blocked, suggesting that activation of Ca2+ uptake was not secondary to closure of K+ channels and consequent depolarization. A diacylglycerol (PAG) analogue, 1,2-dioctanoyl-sn-glycerol (50 microM), duplicated, and the protein kinase C(PKC) inhibitors calphostin C (100 nM), chelerythrine (1 microM), and bis-indolylmaleimide (250 nM) inhibited the effects of GH on [Ca2+]i. Xanthogenate tricyclodecan-9-yl (D609), a specific inhibitor of phospholipase C(PLC), abolished the increase in [Ca2+]i due to GH but not to DAG. The results suggest that GH increases [Ca2+]i by activation of PLC, release of DAG, and activation of a Ca(2+)-independent isoform of PKC. PKC-catalyzed phosphorylation of either the Ca2+ channels or a protein that regulates them may account for the influx of Ca2+ produced by GH.

Adipocytes↗

PubMed1996

Use of a second-generation CA125 assay in gynecologic patients.

OBJECTIVE: To determine the correlation between the widely used CA125 assay and a second-generation assay, CA125 II, in a large population of women to determine the utility of measuring CA125 levels in the diagnosis of benign gynecologic disease. METHODS: A total of 822 patients who presented to a gynecology clinic completed a medical questionnaire and underwent a physical examination. Blood samples were obtained to determine serum CA125 levels. Hospital charts were reviewed, and diagnoses were correlated with serum CA125 levels as measured by the CA125 and CA125 II assays. RESULTS: CA125 and CA125 II assay values were highly correlated within four subject categories: age, ethnicity, smoking history, and menstrual category. Mean values decreased with increasing age but were unchanged across ethnic groups and cigarette-smoking status. When means were compared for results of the second-generation and current assays, little difference was found in the age, ethnicity, and smoking categories. Mean serum CA125 values on the CA125 II assay exceeded those on the CA125 assay when obtained during the menstrual and follicular/luteal phases of the menstrual cycle, but they did not differ in anovulatory, pregnant, and menopausal women or in those patients taking hormonal medications. CA125 levels were elevated on both assays in women with fibroids and were higher on the CA125 II assay than on the CA125 assay in women with benign ovarian cysts. CONCLUSIONS: Serum CA125 levels obtained using the CA125 II assay are closely correlated with those obtained using the more familiar CA125 assay. In the diagnosis of benign gynecologic disease, the usefulness of a single serum level measured with the newer CA125 II assay was the same as that of a single CA125 assay level.

Biomarkers↗

PubMed1995

The genome of Arabidopsis thaliana.

Arabidopsis thaliana is a small flowering plant that is a member of the family cruciferae. It has many characteristics--diploid genetics, rapid growth cycle, relatively low repetitive DNA content, and small genome size--that recommend it as the model for a plant genome project. The current status of the genetic and physical maps, as well as efforts to sequence the genome, are presented. Examples are given of genes isolated by using map-based cloning. The importance of the Arabidopsis project for plant biology in general is discussed.

Arabidopsis↗

PubMed1995

Identification of a plant serine-arginine-rich protein similar to the mammalian splicing factor SF2/ASF.

We show that the higher plant Arabidopsis thaliana has a serine-arginine-rich (SR) protein family whose members contain a phosphoepitope shared by the animal SR family of splicing factors. In addition, we report the cloning and characterization of a cDNA encoding a higher-plant SR protein from Arabidopsis, SR1, which has striking sequence and structural homology to the human splicing factor SF2/ASF. Similar to SF2/ASF, the plant SR1 protein promotes splice site switching in mammalian nuclear extracts. A novel feature of the Arabidopsis SR protein is a C-terminal domain containing a high concentration of proline, serine, and lysine residues (PSK domain), a composition reminiscent of histones. This domain includes a putative phosphorylation site for the mitotic kinase cyclin/p34cdc2.

Amino Acid Sequence↗

PubMed1995

Isolation and expression of an Arabidopsis 14-3-3-like protein gene.

We have isolated an Arabidopsis gene, AFT1, which encodes a 14-3-3-like protein (AFT1). The wide distribution of the 14-3-3 protein family in eukaryotes and the high steady-state mRNA levels of the AFT1 gene in most tissues throughout Arabidopsis growth and development suggest that AFT1 plays an important role(s) in Arabidopsis. DNA blot analysis indicates that AFT1 is a single copy gene, therefore AFT1 should be a good target for studies involving antisense or sense RNA technologies as a means to determine AFT1's function in vivo.

Amino Acid Sequence↗

PubMed1995

A single arginine residue determines species specificity of the human growth hormone receptor.

Although growth hormone (GH) receptors (GHRs) in many species bind human (h) GH as well as their own GH, the hGHR only binds primate GH. Arg43 in hGHR interacts with Asp171 of hGH. Nonprimates have a His in the position equivalent to residue 171 of primate GH and a Leu in position 43 of primate GHR. To determine whether Arg43 accounts for the species specificity of the hGHR, point mutations that changed Leu43 to Arg were introduced into the cDNAs encoding the bovine (b) GHR or the rat GH binding protein (GHBP) and these mutants or their wild-type (WT) counterparts were expressed in mouse L cells. Binding of hGH or bGH to transfected cells or to GHBP secreted into the incubation medium was assessed by displacement of 125I-labeled hGH. WT and mutant bGHR bound hGH with similar affinity, but the affinity of the mutant receptors for bGH was reduced 200-fold. Likewise, WT and mutant GHBP bound hGH with equal affinity, but only WT GHBP bound bGH. Cross-linking of 125I-labeled hGH to WT or mutant GHR produced a 141-kDa labeled complex whose appearance was blocked by unlabeled hGH, but bGH blocked cross-linking only to WT receptors. Both hGH and bGH stimulated tyrosine phosphorylation of a 95-kDa protein in cells transfected with WT GHR, but bGH was less effective in cells expressing mutant GHR. We conclude that incompatibility of Arg43 in the hGHR with His171 in nonprimate GH is the major determinant of species specificity.

Amino Acid Sequence↗

PubMed1995

Evidence for a multifocal origin of papillary serous carcinoma of the peritoneum.

Histopathological evidence suggests that papillary serous carcinoma of the peritoneum (PSCP) may be multifocal in origin. Utilizing a PCR based method to detect tandem repeat polymorphisms in formalin fixed tissue, loss of heterozygosity at eight loci on chromosomes 1, 3, 4, and 17 was studied in six cases of PSCP. Loss of heterozygosity was assessed at between 5 and 11 tumor sites/patient. Allelic losses at 4 loci (1q32-qter, 3p14.3-21.1, 17q12, 17q21.3-23) were noted. Three cases demonstrated a different pattern of allelic loss at various anatomic sites within the same patient. In an additional case, a mutation of the p53 gene, detected by quantitative PCR followed by single-strand conformation polymorphism analysis, was detected in only 2 of 5 tumor sites. The pattern of allelic loss and the mutational pattern of the p53 gene varied at tumor sites within the same patient in 4 of 6 cases of PSCP. These findings are consistent with histopathological evidence that PSCP is multifocal in origin.

Base Sequence↗

PubMed1995

Characterization of a family of Arabidopsis zinc finger protein cDNAs.

Work from animal systems indicates that C2H2 zinc finger proteins play an important role in development, and this is likely true for plant systems as well. To address this question we have used the sequence information from a previously isolated Arabidopsis thaliana C2H2 zinc finger protein gene to isolate additional cDNAs belonging to this gene family. While zinc finger genes isolated from other organisms encode multiple copies of this domain, the eight cDNAs isolated from Arabidopsis each contain only a single zinc finger. Outside of the finger region there is little sequence identity or similarity, although features characteristic of transcription factors are evident. While these genes are related, hybridization analysis indicates that each of them is a unique gene in the Arabidopsis genome. Analysis of mRNA demonstrates that the genes are expressed in different but overlapping sets of organs in the plant. These results are discussed in the light of recent analysis of zinc finger genes from other plant systems.

Amino Acid Sequence↗