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Biomedical subjects

H O Smith

Publications and source records attributed to H O Smith.

At least 19 recordsLinked to original sources
PubMed1992

Phase II trial of amonafide in previously treated patients with advanced ovarian cancer: a Southwest Oncology Group study.

Twenty-three patients with metastatic or recurrent Stage III or IV epithelial ovarian cancer who were refractory to or relapsed following previous chemotherapy with cisplatin or a cisplatin analog were entered into a phase II study of amonafide. The starting dose of amonafide was 300 mg/m2 delivered daily over 1 hr by intravenous infusion. In the absence of myelosuppression, the dose of amonafide was escalated by increments of 75 mg/m2 to a maximum of 450 mg/m2. There were 19 eligible and 17 fully evaluable patients. Grade 3 or 4 leukopenia occurred in 14 (74%) patients and grade 3 or 4 thrombocytopenia in 6 (32%) patients. No objective complete or partial responses were observed. Four patients had stable disease for 3, 4, 4.5, and 6 months, respectively. Therefore, amonafide in the doses used in the present trial does not have significant activity in previously treated patients with ovarian cancer.

Adenine↗
PubMed1991

Two-step affinity purification of U7 small nuclear ribonucleoprotein particles using complementary biotinylated 2'-O-methyl oligoribonucleotides.

U7 snRNP is a low-abundance small nuclear ribonucleoprotein particle essential for 3' processing of replication-dependent histone pre-mRNA. We have developed a two-step purification of the particle from TB21 mouse mastocytoma cell nuclear extracts, with about a 20% overall yield, using affinity binding to 2'-O-methyl oligoribonucleotides. The purified particle is homogeneous with respect to RNA content. SDS/PAGE of the U7 snRNP proteins revealed a full complement of the standard core proteins (B, DD', E, F, and G) found in the majority of snRNPs. In addition, two U7-specific polypeptides of 14 kDa and 50 kDa were identified. Summation of the molecular masses of the identified components of the U7 particle yields a particle mass of 249 kDa, in approximate agreement with estimates from sucrose gradient sedimentation (261 kDa) and nondenaturing gradient PAGE (217 kDa).

Animals↗
PubMed1991

Nucleotide sequence of a cluster of genes involved in the transformation of Haemophilus influenzae Rd.

A genetic locus implicated in the development of competence in Haemophilus influenzae Rd has been previously mapped to a 12.8-kb PstI region of the chromosome [Tomb et al., J. Bacteriol. 171 (1989) 3796-3802]. To define the boundaries of this locus and to identify the gene(s) involved in transformation, additional mini-Tn10kan mutagenesis was performed and the region containing all mutagenic insertions was sequenced. Three new transformation-deficient (Tfo-) mutants were found, bringing the number of distinct mutations mapped to this region up to eight. The transformation frequency of strains carrying the new insertions was 25- to 10(5)-fold less than wild type. The ends of the mini-Tn10kan element were used as starting points to sequence a 9.1-kb region. The position of the eight mutagenic insertions was determined and ten putative open reading frames (ORFs) were found. One of the mini-Tn10kan elements had inserted in an intergenic region while the rest had inserted in six of the ORFs. Based on the phenotypes of the mutant strains and the position of the insertions, we concluded that at least three of the genes should be involved in transformation. In addition, fourteen 9-11-bp uptake signal sequences (USS) were found, four of which were part of stem-loop structures and could function as attenuators of terminators of transcription.

Amino Acid Sequence↗
PubMed1991

Aggressive angiomyxoma of the female pelvis and perineum: review of the literature.

Aggressive angiomyxoma is an uncommon neoplasm which predominantly involves the pelvis and perineum of young White females. Misdiagnosis is common. Treatment typically involves surgery, and in spite of apparently complete resection, recurrences are common. Local spread into the adjacent fascia and musculature is frequently reported, and rarely, extension into intestine and bladder. The first reported case of pubic bone involvement, including its histology, radiologic features, and operative management, is discussed. Including this patient, 26 women with this tumor have been reported in the literature and are reviewed, along with 2 previously reported cases from the University of New Mexico Tumor Registry.

Adult↗
PubMed1990

Construction of an efficient overproducer clone of HinfI restriction endonuclease using the polymerase chain reaction.

We describe the use of the polymerase chain reaction (PCR) technique to alter transcriptional and translational signals surrounding a gene so as to achieve overexpression in Escherichia coli. By changing the ribosome-binding site sequence preceding the hinfIR gene to match the consensus E. coli signal and by adding a transcription terminator sequence immediately following the gene, the yield of HinfI was increased about tenfold over that obtained from the natural Haemophilus influenzae signals. The addition of the positive retroregulator stem-loop sequence derived from the crystal protein-encoding gene of Bacillus thuringiensis downstream from the hinfIR gene further increased yields by twofold to a level of 13% of the total cellular protein.

Base Sequence↗
PubMed1990

Finding sequence motifs in groups of functionally related proteins.

We have developed a method for rapidly finding patterns of conserved amino acid residues (motifs) in groups of functionally related proteins. All 3-amino acid patterns in a group of proteins of the type aa1 d1 aa2 d2 aa3, where d1 and d2 are distances that can be varied in a range up to 24 residues, are accumulated into an array. Segments of the proteins containing those patterns that occur most frequently are aligned on each other by a scoring method that obtains an average relatedness value for all the amino acids in each column of the aligned sequence block based on the Dayhoff relatedness odds matrix. The automated method successfully finds and displays nearly all of the sequence motifs that have been previously reported to occur in 33 reverse transcriptases, 18 DNA integrases, and 30 DNA methyltransferases.

Amino Acid Sequence↗
PubMed1989

Use of synthetic ribosome binding site for overproduction of the 5B protein of insertion sequence IS5.

Insertion sequence IS5 is a bacterial transposable element which contains three open reading frames designated 5A, 5B and 5C. Although there was no detectable expression from the 5B open reading frame when it was preceded by the native promoter and ribosome binding site or by a tac promoter and the native ribosome binding site, we have overproduced a 5B protein both in vitro and in Escherichia coli cells by using a tac promoter and a specially-designed synthetic ribosome binding site. beta-galactosidase fusion studies suggested that the synthetic binding site is at least 150-fold more efficient than the native binding site. The 5B protein amounted to 80-85% of the total protein made in vitro and 20-25% of the total protein pulse-labelled in whole cells. It is stable in vitro but rapidly degraded in vivo. Thus expression of the 5B gene appears to be limited by both poor translation initiation and protein degradation.

Bacterial Proteins↗
PubMed1989

Two Haemophilus influenzae Rd genes that complement the recA-like mutation rec-1.

Two Haemophilus influenzae Rd genes each complemented the pleiotropic defects of the recA-like mutation rec-1. One gene, fec, was isolated on a 3.6-kilobase-pair EcoRI restriction fragment by complementation of the Fec- phenotype of bacteriophage lambda. The other gene, rec, was identified on a 3.1-kilobase-pair EcoRI fragment by Southern hybridization by using recA-like gene probes from Erwinia carotovora and Pseudomonas aeruginosa PAO. In a rec-1 strain of H. influenzae, the cloned genes restored resistance to UV irradiation, transformation by chromosomal DNA, and spontaneous release of HP1 prophage to wild-type levels. The fec and rec genes were located on the cloned segments by insertion and deletion mutagenesis and subcloning. The restriction endonuclease cleavage maps of the two DNAs were similar but not identical. Southern hybridization demonstrated that the two EcoRI restriction fragments contained homologous DNA sequences, but a fec gene-specific probe was prepared. Each gene encoded a 38,000-dalton polypeptide.

Bacterial Proteins↗
PubMed1989

Organization of the Haemophilus influenzae Rd genome.

We present the first complete map of the Haemophilus influenzae genome, consisting of a detailed restriction map with a number of genetic loci. All of the ApaI, SmaI, and RsrII restriction sites (total of 45 sites) were mapped by Southern blot hybridization analysis of fragments separated by pulsed-field gel electrophoresis. Cloned genes were placed on the restriction map by Southern hybridization, and antibiotic resistance loci were also located by transformation with purified restriction fragments. The attachment site of the HP1 prophage was mapped. In addition, the number, locations, and orientations of the six rRNA operons in the H. influenzae chromosome were determined. The positions of conserved restriction sites in these rrn operons confirm that the direction of transcription is 16S to 23S, as in most other bacteria. The widely used strain BC200 appears to contain an unexpected 45-kilobase duplication.

Blotting, Southern↗
PubMed1989

Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd.

A plasmid library of PstI fragments of Haemophilus influenzae Rd genomic DNA was mutagenized in Escherichia coli with mini-Tn10kan. The mutagenized PstI fragments were introduced by transformation into the H. influenzae chromosome, and kanamycin-resistant transformants were screened for the transformation-deficient phenotype by a cyclic AMP-DNA plate method. Fifty-four mutant strains containing 24 unique insertions that mapped to 10 different PstI fragments were isolated. Strains carrying unique insertions were tested individually for DNA uptake, transformation efficiency, UV sensitivity, and growth rate. The transformation frequencies of these mutants were decreased by factors of 10(-2) to 10(-6). Five of the mutants had normal competence-induced DNA uptake, and the rest were variably deficient in competence development. Three strains were moderately UV sensitive. All strains but one had doubling times within 50% of that of the wild type. Mutated genes were cloned into an H. influenzae-E. coli shuttle vector, and wild-type loci were recovered by in vivo recombinational exchange. Hybridization of these clones to SmaI genomic fragments separated in pulsed-field gels showed that these insertions were not clustered in a particular region of the chromosome.

Alleles↗
PubMed1988

Overproduction and purification of the M.HhaII methyltransferase from Haemophilus haemolyticus.

The HhaII methyltransferase gene from Haemophilus haemolyticus was subcloned in an expression vector under control of the hybrid trp-lac promoter. Induction with isopropyl-beta-D-thiogalactopyranoside results in overproduction of the methyltransferase to about 3% of total cellular protein. The methyltransferase was purified to near electrophoretic homogeneity by phosphocellulose, DEAE, and gel chromatography. Its monomer Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25 kDa, in good agreement with that predicted from the nucleotide sequence. Crystals of the methyltransferase were obtained in the presence of a two-fold molar excess of the duplex oligodeoxynucleotide substrate 5'd-GGACTCC.CCTGAGG.

Amino Acid Sequence↗
PubMed1988

Cloning and sequencing the HinfI restriction and modification genes.

The HinfI restriction and modification genes were cloned on a 3.9-kb PstI fragment inserted into the PstI site of plasmid pBR322. Both genes are confined to an internal 2.3-kb BclI-AvaI subfragment. This subfragment was sequenced. Two large open reading frames (ORF's) are present. ORF1 codes for the methylase [predicted 359 amino acids (aa)] and ORF2 codes for the endonuclease (predicted 262 or 272 aa).

Amino Acid Sequence↗
PubMed1988

EcoRII can be activated to cleave refractory DNA recognition sites.

EcoRII restriction sites [5'-CC(A/T)GG] in phage T3 and T7 DNA are refractory to cleavage by EcoRII, but become sensitive to cleavage in the presence of DNAs which contain an abundance of EcoRII sensitive sites (e.g. pBR322 or lambda DNA). Studies using fragments of pBR322 containing different numbers of EcoRII sites show that the susceptibility to EcoRII cleavage is proportional to the number of sites in the individual fragment. We postulate that EcoRII is the prototype of restriction endonucleases which require at least 2 simultaneously bound substrate sites for their activation. EcoRII sites are refractory when they occur at relatively low frequency in the DNA. The restriction enzyme can be activated by DNA with a higher frequency of sites.

DNA Restriction Enzymes↗
PubMed1988

Synthesis and overproduction of the 5A protein of insertion sequence IS5.

We have demonstrated both the synthesis and overproduction of the 5A protein encoded by the longest open reading frame of the bacterial insertion sequence IS5. Expression was obtained in vitro and in Escherichia coli maxicells from plasmids containing IS5 in either orientation, as well as in vitro from a restriction fragment containing exclusively IS5 DNA. When IS5 was cloned in the appropriate orientation downstream of a strong tac promoter, production of the 5A protein was increased to 10 to 20% of the total protein synthesized in vitro.

Carrier Proteins↗
PubMed1988

Sizing of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis.

The four restriction enzymes ApaI (5'-GGGCCC), EagI (5'-CGGCCG), NaeI (5'-GCCGGC), and SmaI (5'-CCCGGG) were found to produce distributions of DNA fragment sizes useful for mapping of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis. ApaI produced 21 fragments (range, 1.6 to 305 kilobases [kb]), EagI yielded 30 fragments (0.6 to 339 kb), NaeI produced 32 fragments (2.3 to 290 kb), and SmaI yielded 16 fragments (6.0 to 377 kb). Summation of the fragment lengths in each digest yielded estimates for the size of the H. influenzae chromosome ranging from 1,834 kb.

Chromosome Mapping↗
PubMed1987

Conservative surgical management of isthmic ectopic pregnancies.

During the 12-month study interval ending March 30, 1986, there were 203 ectopic pregnancies at Grady Memorial Hospital, a ratio of one ectopic gestation per 34 deliveries. Twenty patients with isthmic ectopic pregnancies were selected for treatment by one of three operative modalities. Seven patients with ruptured isthmic ectopic pregnancies underwent segmental tubal resection without reanastomosis. All four patients who underwent segmental tubal resection with primary microsurgical reanastomosis had postoperative hysterosalpingograms demonstrating bilateral tubal patency. One pregnancy has occurred in this group. Nine patients underwent linear salpingostomy. In five of the six patients who had postoperative hysterosalpingography, patency of the involved fallopian tube was demonstrated. Four of these nine patients, including one patient with contralateral tubal occlusion, have conceived. We conclude that linear salpingostomy for isthmic ectopic pregnancies is as effective as segmental tubal resection with primary microsurgical reanastomosis in achieving tubal patency.

Adult↗