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Biomedical subjects

R W Hammond

Publications and source records attributed to R W Hammond.

52 records · Page 3Linked to original sources
PubMed1992

Analysis of the virulence modulating region of potato spindle tuber viroid (PSTVd) by site-directed mutagenesis.

A series of nucleotide substitutions (G46----C; C47----A; C315----U; U317----C) were introduced into the virulence modulating region of the intermediate strain of potato spindle tuber viroid (PSTVd) in order to examine their effect upon viroid infectivity and pathogenicity with the presence of all four mutations resulting in the sequence of a previously reported severe strain of PSTVd. Eight of the resulting mutant cDNAs were characterized for infectivity and symptom induction in tomato, and the secondary structure of their corresponding RNAs was examined. The combined results of infectivity, computer analysis, and chemical mapping data imply that a previously proposed correlation between thermodynamic stability and PSTVd pathogenicity does not hold true in all cases and suggest that conformation and/or sequence-specific interactions with host factors play a role in symptom development.

Base Sequence↗
PubMed1991

Indian bunchy top disease of tomato plants is caused by a distinct strain of citrus exocortis viroid.

A viroid has been isolated from tomato plants affected by Indian bunchy top disease of tomato (Lycopersicon esculentum Mill.). In dot blot hybridization assays with 32P-labelled cRNA probes specific for the detection of various viroids, the Indian viroid was shown to be most closely related to the citrus exocortis viroid (CEVd). Sequence determination showed that the viroid consists of 372 nucleotides and confirmed its close relationship with CEVd. The viroid, for which we propose the acronym CEVd-t, differs from the Australian CEVd strains A and B by 36 and 47 nucleotides, respectively, and from the Spanish grapevine isolate by 52 changes. A phylogenetic analysis confirmed the closet relationship with CEVd in all structural domains, except the pathogenicity and left-terminal domains, which are closely related to the corresponding domains of the potato spindle tuber and tomato apical stunt viroids, respectively.

Base Sequence↗
PubMed1989

Infectivity of chimeric viroid transcripts reveals the presence of alternative processing sites in potato spindle tuber viroid.

In an investigation of viroid replication and pathogenesis, we have assessed the effect of sequence duplication of the upper central conserved region (CCR) of the molecule on the infectivity of RNAs transcribed in vitro from partial dimers of wild-type and mutant viroid cDNAs. In one set of experiments, the relative infectivities of one monomeric potato spindle tuber viroid (PSTV) and five oligomeric SP6 transcripts [PSTV or PSTV-TASV (tomato apical stunt viroid) chimeras] were compared. With one exception, the extent of sequence duplication in the CCR, and thus the length of the so-called palindrome, does correlate with an increase in specific infectivity. In a second set of experiments, in vitro generated site-specific mutations in cloned PSTV were used as markers to determine if a cleavage/ligation at sites other than the palindrome could generate infectious molecules in vivo. The creation of a novel PSTV-TPMV (tomato planta macho viroid) chimera in these experiments provides evidence that multimeric RNAs can be processed at sites other than the CCR to yield monomeric progeny.

Base Sequence↗
PubMed1989

Molecular cloning, sequencing and expression in Escherichia coli of the bean yellow mosaic virus coat protein gene.

The sequence of 1015 nucleotides from the 3' poly(A) tract of the potyvirus bean yellow mosaic virus (BYMV) RNA has been determined from two cDNA clones. This sequence contained a single long open reading frame (ORF) starting upstream of the cloned region. The ORF was expressed as a fusion protein in Escherichia coli, and the product was detected by antibodies specific for the coat protein of BYMV. The predicted length of the coat protein gene was 822 nucleotides, corresponding to a 273 amino acid coat protein of Mr 30910. The deduced amino acid sequence of the BYMV coat protein was compared to the chemically determined amino acid composition of purified virion protein, and of protein prepared from trypsin-treated virions. The nucleotide and deduced amino acid sequences were compared to the sequences of the coat protein genes of other potyviruses. The BYMV coat protein gene was found to be 50 to 61% homologous to those of other potyviruses at both the nucleotide and amino acid levels; the greatest variation was between the 5'-proximal one-fifth of the genes. Amino acid sequences and hydrophilicity plots of the different potyvirus coat proteins showed similarities which indicated that the structure of the coat protein is highly conserved; a non-terminal region of variability was predicted to be exposed on the exterior of the virion. A putative cleavage site at a glutamine-serine dipeptide was identified by similarity in context to the cleavage sites of tobacco etch virus and tobacco vein mottling virus coat proteins from the viral polyproteins. The BYMV 3'-terminal non-coding region of 166 nucleotides is followed by a poly(A) tract.

Amino Acid Sequence↗
PubMed1988

Fibroblast growth factors from bovine pituitary and human placenta and their functions in the maturation of porcine granulosa cells in vitro.

Both acidic and basic fibroblast growth factors (FGFs) are present in pituitary, as demonstrated by Affigel-heparin affinity chromatography and immunoblot analysis. Unlike in the pituitary, only basic FGF appears to be present in human placenta. The purification of these growth factors from bovine pituitary and human placenta has been described. Immunoblot analysis using polyclonal antibodies specific for either basic FGF or both acidic and basic FGF have verified the results of purification. We have determined a novel function of acidic and basic FGF in the maturation of porcine granulosa cells in vitro. Basic FGF (1.0 ng/ml), purified from bovine pituitary functioned as a potent inhibitor of LH/hCG receptor induction (91 +/- 1.2%; P less than 0.0001), and progesterone secretion (45 +/- 2.9%; P less than 0.0001), two key steps in the maturation of granulosa cells. Human placental basic FGF demonstrated comparable inhibition characteristics. Bovine pituitary acidic FGF (50 ng/ml) appear to function as a relatively weak inhibitor (63.5 +/- 5.1% for LH/hCG receptor induction; 50 +/- 1.8% for progesterone secretion). Consequently, our analysis indicates that FGF may play an important role(s) in the maturation of ovarian granulosa cells.

Animals↗
PubMed1987

Mutational analysis of potato spindle tuber viroid reveals complex relationships between structure and infectivity.

Viroids are single-stranded, covalently closed circular RNA pathogens that can be isolated from certain higher plants afflicted with specific diseases. Their small size (246-375 nucleotides; M(r) 0.8-1.3 x 10(5)) and ability to replicate autonomously make viroids a unique model system in which to study the relationships between the structure of an RNA and its biological function. The demonstrated infectivity of certain cloned viroid cDNAs allows the use of site-specific mutagenesis techniques to probe structure-function relationships suggested by comparative sequence analysis. Several site-specific mutations that disrupt base pairing in either the native structure or secondary hairpin I destroyed the ability of potato spindle tuber viroid cDNA to initiate infection. Alterations in the terminal loops of the native structure also abolished cDNA infectivity. One pseudorevertant, a mutant cDNA containing compensating changes that restore base pairing in the native structure, was marginally infectious; a second pseudorevertant in which base pairing was restored within the stem of secondary hairpin I was not infectious. The behavior of these mutants dramatically demonstrates the effect of remarkably small structural changes on viroid infectivity and emphasizes the importance of the conserved rod-like native structure for viroid function.

Journal Article↗
PubMed1984

Molecular cloning and analysis of a gene coding for the Bowman-Birk protease inhibitor in soybean.

We have constructed cDNA clones from size-selected mRNA of developing soybean seeds to identify genes encoding the Bowman-Birk protease inhibitor. Recombinant clones containing sequences coding for the Bowman-Birk inhibitor were identified by mRNA hybrid selected translation and immunoprecipitation. The nucleotide sequence of the insert of plasmid pB38 corresponds to the mRNA for the Bowman-Birk inhibitor, although it is missing a portion of the coding sequence at the 5' end. Northern hybridization analysis of total RNA isolated from developing soybean seeds showed that the mRNA for the inhibitor is approximately 450 nucleotides long and that it accumulates early in embryogeny. Southern hybridization analysis of restriction enzyme-digested soybean nuclear DNA indicated that the gene encoding the Bowman-Birk inhibitor is not highly reiterated in the genome. We have isolated a gene encoding the Bowman-Birk inhibitor from a soybean genomic library constructed in Charon 4A. DNA sequence analysis of the genomic clone reveals that it is similar, although not identical, to the cDNA clone and that it contains no intervening sequences.

Amino Acid Sequence↗
PubMed1981

Stability of extemporaneous suspensions of carbamazepine.

The stability of carbamazepine in four suspending vehicles is reported. Suspensions of carbamazepine 200 mg/5 ml in sorbitol 70%, simple syrup, modified Hospital of the University of Pennsylvania Suspending Vehicle (HUP), and diluted HUP (HUP-A) were prepared. The first three suspensions were stored in amber glass bottles and oral syringes at 4, 25, and 37 degrees C, and the HUP-A suspension was stored at 4 degrees C. Physical stability was assessed by visual inspection of sedimentation, ease of pouring, and foaming upon shaking. Carbamazepine concentrations were determined periodically over 90 days by an enzyme-multiplied immunoassay. The assay was validated by acid-heat degradation of the drug, separation of breakdown products by thin-layer chromatography, and confirmation of non-reactivity of the breakdown products with the assay. The concentration of carbamazepine in sorbitol 70%, simple syrup, and HUP-A was at least 90% of the prepared concentration at all sampling times. Although separation occurred, the simple syrup suspensions could be redispersed. The suspension in HUP-A remained homogeneous, was easy to pour, and produced less foam than the HUP suspension. Extemporaneously compounded suspensions of carbamazepine in HUP-A or in simple syrup can be used for patients who require a liquid dosage form. Even though sorbitol 70% produced a pharmaceutically acceptable product, its use is not recommended because it has been reported to cause intractable diarrhea.

Carbamazepine↗