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Biomedical subjects

C Richardson

Publications and source records attributed to C Richardson.

At least 145 records · Page 8Linked to original sources
PubMed1990

Identification and sequencing of the spheroidin gene of Choristoneura biennis entomopoxvirus.

Entomopoxviruses are a class of insect viruses whose virions are embedded in cytoplasmic occlusion bodies. The major component of these protective complexes is a protein called spheroidin. An open reading frame encoding the spheroidin gene of Choristoneura biennis entomopoxvirus has been identified and sequenced in our laboratory. This protein coding region is 1023 nucleotides long and specifies a polypeptide of 38,500 Da. Spheroidin was purified by SDS polyacrylamide gel electrophoresis, electroeluted, and its amino terminus sequence was determined on a gas phase sequencer. We observed that the first 20 N-terminal amino acids were absent in the mature processed form of the spheroidin molecule. Examination of these 20 residues revealed their hydrophobic nature and close resemblance to the consensus signal peptide sequence which is commonly found on membrane proteins. The DNA sequence of the spheroidin gene predicted a processed polypeptide with a molecular weight of 36 kDa. However, spheroidin was observed to aggregate in complexes composed of 50-kDa monomers. Intermolecular disulfide bonds were shown to play major roles in the formation and structure of these viral occlusion bodies. The difference in molecular weight between the predicted protein and its counterpart in infected cells is likely due to post-translational modifications. Indeed, two potential asparagine-linked glycosylation sites are present on the spheroidin molecule. The 5' flanking regions of the spheroidin gene and the vaccinia major core protein precursor gene P4b were shown to share substantial homology.

Amino Acid Sequence↗
PubMed1990

Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene.

An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining. Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells. On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays. Levels of synthesis were in the order of 50 to 150 mg of protein per 10(8) cells.

Animals↗
PubMed1990

Expression of mature pulmonary surfactant-associated protein B (SP-B) in Escherichia coli using truncated human SP-B cDNAs.

The present communication documents attempts to produce the mature form of human surfactant-associated protein B (SP-B) by modification of the 5' and 3' regions of the cDNA and expression of the truncated cDNAs after insertion into the vector pKK223-3. The 5' end of a cDNA for human SP-B (1407 base pairs) was reconstructed through the ligation of synthetic oligonucleotides to an internal PstI site in the 5' region. This construction coded for the initiation of protein synthesis at a Met codon adjacent to a codon for the N-terminal Phe of the mature polypeptide. Variable amounts of the 3' end of the human SP-B cDNA were deleted with mung bean nuclease and exonuclease III. The resulting blunt-ended 3' fragments were then ligated to a synthetic oligonucleotide linker designed to create a stop codon. The modified 5' and 3' ends were ligated to a short PstI-BamHI fragment isolated from the SP-B cDNA and inserted into the expression vector pKK223-3. In vitro translation of sense mRNAs derived from the truncated SP-B cDNAs yielded oligopeptides of appropriate molecular weights, as indicated by urea - sodium dodecyl sulphate - polyacrylamide gel electrophoresis of either intact or immunoprecipitated reaction mixtures. Expression of SP-B in Escherichia coli was confirmed by Northern blot analysis for the mRNAs corresponding to the truncated cDNAs in appropriately transformed bacteria induced with the galactose analog isopropyl-beta-thiogalactoside. Western blot analysis using rabbit antisera prepared against bovine SP-B confirmed the presence of mature SP-B in lipid extracts of transformed E. coli, but the amounts were very small.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

High-frequency ventilation and extracorporeal membrane oxygenation.

Dramatic improvement in morbidity and mortality associated with neonatal respiratory failure has evolved over the last 30 years. Favorable survival statistics can be directly related to the institution and refinement of assisted ventilation techniques. Short- and long-term pulmonary complications continue to be of major concern. New ways to support the neonate in respiratory failure are being investigated. Concentrated efforts are being undertaken to find ways to safely and effectively treat these infants while decreasing the morbidity associated with therapy. Two such therapies, both experimental and controversial, which are gaining widespread recognition, are high-frequency ventilation (HFV) and extracorporeal membrane oxygenation (ECMO).

Extracorporeal Membrane Oxygenation↗
PubMed1989

Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit.

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.

Amino Acid Sequence↗
PubMed1989

Mineral phases of calcium phosphate.

Many studies of calcium phosphate precipitation have been made using relaxation techniques in which the concentrations of the lattice ions are allowed to decrease as equilibrium is approached. Since the nature of the phases that form depend markedly on the solution composition, this decrease can lead to concomitant phase transformations during the crystallization experiments. The results of the present constant composition (CC) studies show that defect apatites may be formed under conditions of sustained supersaturation with a non-stoichiometric coefficient dependent on the pH of the growth medium. An important factor in analyzing these experiments is the initial surface modification and ion-exchange processes involving H+ and Ca2+ ions after inoculation of the supersaturated solutions. Thereafter, active growth sites may be eliminated as the crystals undergo lattice perfection. Transformation of dicalcium phosphate dihydrate to octacalcium phosphate, involving dissolution and subsequent nucleation and growth of the new phase, is also influenced by surface roughening of the initial phase. Typical inhibitors that reduce the rate of growth of seed crystals in supersaturated solutions may actually induce the nucleation of calcium phosphate phases when immobilized on inert surfaces. This may be a factor in the modulation of crystal growth in many biological systems.

Calcium Phosphates↗
PubMed1989

Intravesical bacillus Calmette-Guérin and second primary malignancies.

In an effort to answer the question, "Does intravesical bacillus Calmette-Guérin (BCG) therapy increase the incidence of second primary malignancies," the records of 153 patients treated with BCG for carcinoma in situ of the urinary bladder were reviewed. The weight of the available evidence suggests that intravesical BCG does not increase the incidence of second primary malignancies. While this question should be investigated, at the present time it appears that the advantage of intravesical BCG for high-risk patients with superficial bladder cancer outweighs the known disadvantages.

BCG Vaccine↗
PubMed1989

Primary structure of an oligomeric antigen of Treponema pallidum.

The properties and sequence of an oligomeric antigen of Treponema pallidum are presented. Antigen C1-5 assembles into oligomers of 140,000 and greater. The nucleotide sequence predicts an open reading frame for a protein monomer of 19,400, confirmed by amino-terminal sequencing of the recombinant antigen.

Amino Acid Sequence↗
PubMed1989

BCG in the management of superficial bladder cancer.

An overall comparison of different types of therapy for any disease is difficult. A definition of the disease, which included carcinoma in situ was not agreed upon in 1961 when thiotepa was first described. Most importantly, many series are not prospective and randomized. Response criteria have varied and have not always included cytologies. With these shortcomings in mind we have attempted to compare the various therapeutic agents. The response rate for BCG appears superior to chemotherapy for the treatment of superficial bladder cancer (59% vs 45%), for prophylaxis (77% vs 53%) and the management of carcinoma in situ (74% vs 52%). Moreover BCG also appears superior to chemotherapy for the management of prior treatment failures, particularly carcinoma in situ. In summary, this review of the literature suggests that BCG is superior, in terms of response rate, to available chemotherapy in the management of superficial bladder cancer.

Administration, Intravesical↗
PubMed1988

Peripheral regulation of stiffness during arm movements by coactivation of the antagonist muscles.

Two experiments investigated whether unexpected and differential loading of a rapid, unsighted arm movement resulted in the central nervous system (CNS) regulating limb stiffness by modifying the associated neuromuscular activity. In Experiment 1, subjects completed multiple, spring-loaded training trials until a prespecified criterion of learning was attained. On selected trials, the spring load was unexpectedly replaced by an inertial load. Results indicated that to maintain positional accuracy during this inertial load trial, limb stiffness was increased by coactivating the antagonist muscles, i.e. by changing the associated neuromuscular activity from a predominantly triphasic pattern to one of coactivation. In Experiment 2, the sequence of loading was reversed producing a change in the required limb stiffness from a relatively high to low level. This change was observed as a pattern of coactivation being replaced by a triphasic activity pattern. These results support the notion that limb stiffness is regulated primarily through modification of the neuromuscular activity pattern prior to movement termination. It was also demonstrated that the size of the unexpected load did not affect the basic activation pattern selected by the CNS. It is proposed that the signal which triggers the CNS to regulate limb stiffness is based on peripheral information generated as a result of agonist activity occurring during the first part of the movement.

Adult↗
PubMed1988

Cotside measurement of cerebral blood flow in ill newborn infants by near infrared spectroscopy.

A new method of quantifying cerebral blood flow which allows repeated cotside measurements is described. 31 observations were made on nine ill, mostly very preterm, infants. Cerebral blood flow was usually about 18 ml.100 g-1.min-1, but ranged from 7 ml.100g-1.min-1 (after the administration of indomethacin to a very preterm infant) to 33 ml.100 g-1.min-1 in a birth-asphyxiated post-term infant.

Cerebrovascular Circulation↗
PubMed1988

Some effects of commissurotomy on the reinstatement and potentiation of lesion deficits.

Two experiments investigated in rats the effects of cutting the corpus callosum after recovery from unilateral cortical lesions that produce transient symptoms of neglect and circling. Side of lesion was also examined. In Expt. I, 60 rats received left or right lesions of parietal, medial frontal, or motor cortex. After one month of testing for visual, auditory and somatosensory responsiveness and for circling, the callosum was cut, and the sequence of measures was repeated. Callosotomy reinstated neglect after recovery from the lesions in the parietal and medial frontal groups, more severely and consistently in the frontal group. Side of lesion made no difference. Circling was predominantly ipsiversive after the cortical lesions, due entirely to the frontal group. Callosum section markedly potentiated contraversive circling in the left parietal group; right parietal animals showed no preference. This was the only hemisphere difference found. Circling remained ipsiversive in medial frontal animals after callosotomy. These circling biases did not diminish in the postcallosotomy period. Expt. II replicated the circling procedures with 58 animals that were given the same unilateral cortical lesions or were unoperated controls. Callosotomy was performed one month postlesion. Again, left parietal animals circled contraversively, and there was no bias in the right parietal group. A left-right difference was also evident in the motor cortex group, left lesions producing contraversive turning. We confirm the reinstatement of neglect from frontal lesions by callosum section previously found in the monkey and show that it also occurs with parietal lesions. While neglect symptoms do not differ after left or right lesions, circling does: left parietal lesions plus callosotomy produce a marked contraversive tendency that may reflect an elemental spatial lateralization.

Animals↗
PubMed1987

Antitumor activity of intraperitoneal immunotoxins in a nude mouse model of human malignant mesothelioma.

Immunotoxins directed against human transferrin receptor have been evaluated in a nude mouse model of human malignant mesothelioma. Immunotoxins were constructed by linking ricin A chain to murine monoclonal antibodies reactive with the human transferrin receptor. A chain was obtained either by isolation from the parent toxin or by recombinant DNA techniques. These immunotoxins acted as potent in vitro cytotoxins against human malignant mesothelioma cells (H-MESO-1) (ID50, 2 X 10(-9) M). Cytotoxic potency and kinetics of cell kill were potentiated in vitro by the carboxylic ionophore monensin. For in vivo trials, nude mice were injected i.p. with 6-9 X 10(6) human malignant mesothelioma cells 24 h prior to the start of i.p. immunotoxin treatments. The survival of tumor-bearing mice was extended by 149-404%, representing a probable cell kill of 2-4 logs. Specificity of this antitransferrin receptor immunotoxin response was confirmed by the ineffectiveness of irrelevant control immunotoxins and blockade of specific immunotoxin action by excess free antibody. Monensin showed limited in vivo potentiation of immunotoxin effect, but a derivative formed by esterification of monensin with linoleic acid gave improved survival times over treatment with immunotoxin alone. Immunotoxins constructed with ricin A chain have significant tumoricidal activity in this model of regional antitumor therapy. These results may have direct relevance for treatment of i.p. malignancy in clinical settings.

Animals↗
PubMed1987

Partial splenic embolisation for hypersplenism of thalassaemia major: five year follow up.

Six patients with thalassaemia major were treated by partial splenic embolisation as an alternative to splenectomy and followed up for five years. Results were compared with those in a matched control group of seven patients treated by splenectomy. All patients treated by partial splenic embolisation showed a reduction in blood transfusion requirements comparable with those in the controls and which remained unchanged over the five years. Serious infections that commonly occur in patients splenectomised for thalassaemia did not occur after embolisation, presumably owing to preservation of some immune function by the splenic remnant. By contrast with the change in platelet counts seen after splenectomy, platelet counts remained normal after partial splenic embolisation, so reducing the risk of thromboses. On the other hand, pre-existing leucopenia and thrombocytopenia were corrected after embolisation. It is concluded that partial splenic embolisation provides an alternative to splenectomy for thalassaemia major and is equally effective and much safer.

Adolescent↗