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Biomedical subjects

R G Smith

Publications and source records attributed to R G Smith.

At least 235 records · Page 13Linked to original sources
PubMed1991

Overexpression of c-fos in a human pre-B cell acute lymphocytic leukemia derived cell line, SMS-SB.

The c-fos proto-oncogene is found to be overexpressed at least 30-fold in SMS-SB, a pre-B leukemic cell line compared to other cell types. No gross alteration of the c-fos gene structure in SMS-SB cells can be detected by karyotypic or Southern analyses. C-fos in SMS-SB cells can still be induced by serum, TPA and the calcium ionophore, A23187, and superinduced by the combination of serum and cycloheximide. The elevated levels of c-fos transcripts are not due merely to an increased life span of mRNA, as the half-life of steady state c-fos mRNA in SMS-SB cells is about 40 min. Nuclear run-on transcription assays demonstrate that the transcription rate of c-fos in SMS-SB cells is up-regulated about 30-fold as compared to another pre-B leukemic cell line, NALM-6. In order to determine whether this is a cis- or transacting effect, we sequenced the 550 base pairs upstream of the SMS-SB c-fos coding region and found no mutations upstream of the mRNA CAP site. However, two point mutations at positions +23 and +98, respectively, were found in the 5' noncoding region of the first exon. Consistent with the overexpression of c-fos mRNA, the 55 kD c-fos protein is present in SMS-SB cells through detection by immunoprecipitation, but could not be detected in NALM-6 cells. SMS-SB cells however, do not appear to contain more abundant AP-1 DNA binding activity than NALM-6 cells.

Base Sequence↗
PubMed1991

Current diagnosis and therapy of parotid tumours.

After reviewing a group of more than 400 patients with parotid tumours, we feel that clinical examination, screening ultrasound scanning and pre-operative CT-scan are our most important diagnostic tools. Our treatment concept is based on direct posterior dissection of the main trunk of the facial nerve, total conservative parotidectomy with ablation of all glandular tissue especially in pleomorphic adenomas so that recurrency can be avoided, intra- and postoperative histological examination and filling in the post-operative soft tissue depression in all cases where intra-operative histological examination excludes malignancy. Primary post-parotidectomy defect substitution techniques using the platysma (Roscic) and the sternocleidomastoid muscle (Rausch) as well as a SMAS- and temporoparietal fascia- or galea-flap are illustrated. We currently use the combination of the Rauch- and Roscic- techniques in a way that a sternocleidomastoideus muscle rotation-flap together with a platysma-SMAS-fat-flap entirely cover the defect. We believe that the substitution-plasty, primarily performed for aesthetic reasons, acts as a mechanical barrier for the misdirected regeneration of the nervs to the skin and the sweat glands, and thus tends to avoid Frey's Syndrome. Using our technique concept, we feel that any disadvantage is counter-balanced by the absence of recurrences, no permanent facial paralysis, relatively good aesthetic results and lower incidence of Frey's Syndrome.

Diagnostic Imaging↗
PubMed1991

The microsperm penetration assay: development of a sperm penetration assay suitable for oligospermic males.

OBJECTIVE: To develop a specialized sperm penetration assay (SPA) for the evaluation of sperm from oligospermic patients. DESIGN: The development of the assay is in four parts: determine optimal sperm number; demonstrate quality control; establish statistical limits for fertile population; compare results to in vitro fertilization (IVF) outcome. SETTING AND PATIENTS: A group of 63 patients with oligospermia and/or poor motility and a group of 17 fertile donors were compared using the optimized SPA and the micro-SPA. Sperm from a third group of 35 patients were simultaneously incubated with human ova (IVF) and hamster ova (micro-SPA). MAIN OUTCOME MEASURES: Both types of SPA scores are expressed as a sperm capacitation index (penetrations per ovum). Outcome of IVF is expressed as a percent of ova fertilized. RESULTS: Using 25,000 sperm was found to be optimal. The normal fertile range was statistically determined to have a lower limit (-2 SD) of 2.0 penetrations per ovum. When scores from 63 male factor patients were compared using the optimized SPA, only 43% had sufficient swim-up sperm. However, the micro-SPA could accurately test 100% of the samples because it requires only one tenth the number of sperm. CONCLUSION: The micro-SPA provides a valuable diagnostic test for the evaluation of the male factor patient.

Animals↗
PubMed1990

Detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin hypervariable region specific oligonucleotide probes.

To develop a sensitive and specific assay for minimal residual disease in acute lymphoblastic leukemia (ALL), we exploited the enormous diversity of genomic sequences created by immune receptor gene rearrangements. To isolate clone-specific sequences, we first synthesized oligonucleotides that match conserved variable (VH) and joining (JH) sequences flanking the third hypervariable region (HVR3) in the rearranged immunoglobulin heavy chain (IgH) locus. In polymerase chain reactions (PCR), these primers were then used to amplify the intervening HVR3 segments from leukemic DNA samples. Of 12 B-lineage ALLs studied, ten generated one or more fragments of the size expected for HVR3 gene segments. Thus, this single pair of amplimers was sufficient to isolate HVR3 sequences from a majority of acute lymphoblastic leukemias. To verify that the amplified fragments originated from HVR3 alleles and to assess their diversity, we sequenced 7 PCR products derived from 6 leukemias. In addition to elements of recognized D segments, each of the 7 fragments contained novel VH-D and D-JH junctional sequences, including N nucleotides, not known to be present in the germline. Each sequence was unique, and allele-specific oligonucleotide probes hybridized only to HVR3 segments from which the probes were derived. Therefore, as anticipated, these HVR3 segments appeared to possess the diversity required to serve as clonal markers for leukemic populations. To demonstrate that these amplified HVR3 alleles could serve as the basis for a sensitive and specific assay to detect rare leukemic cells, we analyzed in detail one pre-B leukemia that had rearranged 2 IgH alleles. The HVR3 sequences were shown to be linked to rearranged JH-containing restriction fragments in digests of genomic DNA, establishing their origin in the leukemic cells. We synthesized oligonucleotides corresponding to the unique junctional sequences in the HVR3 segments. Using these novel amplimers in an allele-specific amplification and hybridization procedure, we showed that this assay can detect 10 leukemic cells in a background of 10(6) normal blood mononuclear cells. In contrast, the leukemic HVR3 sequences were not detected in extracts of normal or unrelated remission leukemic leukocytes. We conclude that the assay for specific IgH HVR3 sequences is a realistic strategy for detection of minimal residual disease in B-lineage ALL.

Alleles↗
PubMed1990

Molecular cloning of protein phosphatase inhibitor-1 and its expression in rat and rabbit tissues.

A cDNA encoding the complete amino acid sequence of rat protein phosphatase inhibitor-1 was obtained by screening a skeletal muscle library. The coding region represents a 171-residue polypeptide which demonstrated 80% overall identity with the primary sequence of rabbit inhibitor-1. Sequence homology between the rat and rabbit proteins was particularly striking (98% identity) in the NH2-terminal 61 amino acids, which encompass the threonine phosphorylated by cyclic AMP-dependent protein kinase. This domain possesses full inhibitor activity against type-1 protein phosphatases. In contrast, a domain of similar size at the COOH terminus showed only 57% conservation of primary structure between the two proteins. This reflects a remarkable difference in evolutionary pressures experienced by these domains and may emphasize a lesser role for the COOH-terminal region in inhibitor-1 function. Northern hybridization analysis of RNA from rat and rabbit tissues indicated the presence of two mRNAs, a major 0.7-kilobase and a minor 1.8-kilobase mRNA. The highest expression of inhibitor-1 mRNA was noted in skeletal muscle from both species. Analysis of mRNA levels illustrates potential post-transcriptional mechanisms controlling inhibitor-1 expression in some mammalian tissues.

Amino Acid Sequence↗
PubMed1990

Receptor interconversion model of hormone action. 1. Purification of a factor involved in conferring estradiol binding properties to the estrogen receptor.

We previously demonstrated that the chick oviduct estrogen receptor exists in three interconvertible forms. Two of these forms bind estradiol with high but distinct affinities. A third form exists as a non-estrogen binding recyclable form, Rnb, which upon treatment with ATP/Mg2+ is quantitatively converted to the lower affinity estradiol binding form. We now describe the isolation from chick oviduct cytosol of a factor involved in this conversion and its 1100-fold purification by ammonium sulfate fractionation, DEAE ion-exchange chromatography, and size-exclusion HPLC. The factor elutes from the size-exclusion column with an apparent molecular weight of 40,000. This highly purified factor potentiates estradiol binding in a dose-dependent manner in the presence of ATP/Mg2+. Its activity is destroyed by heating or by trypsin treatment but is relatively stable to freezing and thawing and is inert to treatment with reducing agents. ATP is an essential nucleotide substrate; GTP and cyclic nucleotides are inactive. Studies of cation dependence demonstrate that Mg2+ is also essential; Ca2+ alone is completely ineffective in catalyzing receptor potentiation and does not synergize with Mg2+. In the presence of excess ATP/Mg2+ and a fixed concentration of Fy, the Km for potentiation of estradiol binding is approximately 0.4 nM.

Ammonium Sulfate↗
PubMed1990

Receptor interconversion model of hormone action. 2. Requirement of both kinase and phosphatase activities for conferring estrogen binding activity to the estrogen receptor.

Three interconvertible forms of the estrogen receptor have been identified in the oviduct of estrogen-stimulated chicks. The non-estradiol binding form (Rnb) can be converted to the lower affinity binding form (Ry, Kd = 0.8 nM) by a process requiring the gamma-phosphoryl moiety of ATP. The enzymatic activity (Fy) essential for this "receptor potentiation" has been isolated from oviduct cytosol using ammonium sulfate fractionation, DEAE chromatography, and HPLC size-exclusion chromatography. The potentiation appears to require both kinase and phosphatase activities. The Fy kinase characteristically phosphorylates casein, histones, and glycogen synthase. Comparison of the kinase with casein kinase II, which also phosphorylates casein and glycogen synthase, indicates that Fy represents a distinct protein kinase since its activity is not stimulated by spermine or inhibited by heparin. Fy-mediated conversion of Rnb to Ry is blocked by the phosphatase inhibitors vanadate, fluoride, and pyrophosphate. The substrate specificity of the Fy phosphatase activity is distinct from that of the two well-characterized protein phosphatases 1 and 2A. Moreover, the requirement for Fy phosphatase activity in converting Rnb to Ry could not be mimicked by its substitution with purified protein phosphatases 1 or 2A. The unique substrate specificity of the oviduct protein phosphatase and protein kinase, which are apparently necessary to confer estradiol binding characteristics to the receptor, implies that these enzymes play a key role in the control of the estrogen receptor in its function as a transcription factor.

Amino Acids↗
PubMed1990

Receptor interconversion model of hormone action. 3. Estrogen receptor mediated repression of reporter gene activity in A431 cells.

The chicken estrogen receptor exists in three interconvertible forms, two of which bind estradiol with high affinity and one which lacks the capacity to bind estradiol. Interconversion is regulated by reactions involving ATP/Mg2+. By cotransfecting into A431 cells estrogen receptor cDNA in an expression vector together with the pA2 (-821/-87) tk-CAT vitellogenin construct, we demonstrate that constitutive expression of chloramphenicol acetyltransferase (CAT) activity can be regulated either by selection of ligand or by modifying phosphorylation reactions in the recipient cells. In the presence of estrogen receptors, constitutive expression of CAT activity is inhibited in three situations: (i) in the absence of an estrogenic ligand; (ii) in the presence of an anti-estrogen; and (iii) in the presence of an estrogenic ligand together with 12-O-tetradecanoylphorbol 13-acetate (TPA). Estrogen receptor mediated repression of constitutive CAT activity is not observed with the pA2 (-331/-87) tk-CAT construct, indicating that DNA sequences required for repression are located between -821 and -331 base pairs upstream of the transcription initiation site.

Animals↗
PubMed1990

Mass spectral characterization of acyloxymethyl phosphates.

A series of organophosphates having one or two acyloxymethyl groups have been characterized by methane chemical ionization mass spectrometry: twelve are model compounds with phenyl and benzyl groups while four are derived from pyrimidine-3'-deoxyriboside-5'-monophosphates. Some are protonated cyclohexyl-ammonium salts. In addition the phenyl and benzyl phosphate spectra exhibit an abundance of fragmentation and rearrangement ions, the majority of which were products of three processes--expulsion of formaldehyde, loss of the acyloxymethyl group with transfer of hydrogen to the phosphate, and fission of the methylene-acyloxy bond. The pyrimidine-3'-deoxyriboside-5'-monophosphate esters give less fragmentation under similar circumstances; prominent ions in their spectra are the protonated molecular ions and losses of one and two molecules of formaldehyde from them and the protonated bases.

Biotechnology↗
PubMed1990

Several types of adherent cells elaborate a dialyzable lymphopoietic cofactor (Abelson Growth Promoter).

We have cloned a stromal cell from mouse bone marrow selected on the basis of its ability to promote the growth of an Abelson virus-transformed pre-B cell line. These stromal cells have smooth muscle features, and the ultrafiltrate of stromal cell-conditioned medium has proliferative effects on all feeder layer-responsive mouse pre-B cell lines tested, as well as on normal pre-B cells. One of these low MW substances is a protease-resistant factor with an MW of approximately 450 Da which we designate Abelson Growth Promoter (AGP). AGP was initially characterized as an activity which promotes the growth of Abelson virus-transformed mouse pre-B cells, but it also promotes the growth of a ras-transformed pre-B cell line as a single agent and in synergistic fashion with recombinant interleukin (IL) 7. AGP as a single agent has no effect on normal pre-B cells, which have a brisk response to IL-7. In contrast, transformed pre-B cells display a blunted response to IL-7. We propose that AGP plays a role in normal lymphopoiesis by expanding clones of pre-B cells which have been activated by other stromal cell-derived signals, such as IL-7, and directly promotes the growth of nascently transformed pre-B cells.

Abelson murine leukemia virus↗
PubMed1990

Evoked responses in patients with macular holes.

Thirty-two eyes with idiopathic macular holes and one eye with a traumatic macular hole were assessed by pattern-reversal electroretinography, ganzfeld electroretinography and pattern-reversal visual evoked potentials. Results were inspected for qualitative abnormalities and then measured in comparison with fellow eyes and 41 control eyes of similar age. Qualitative abnormalities occurred in some eyes with macular holes, most commonly a reduction in pattern-reversal electroretinogram or pattern-reversal visual evoked potential amplitude; 15' check amplitudes were significantly lower in eyes with macular holes than in control eyes, but no significant difference in latency was found. Control pattern-reversal electroretinogram and pattern-reversal visual evoked potential amplitudes were noted to decline with age, and paired t-tests on an age-matched subgroup of eyes with macular holes and control eyes showed appreciable differences only in the pattern-reversal electroretinogram q-r (N95) amplitude.

Aged↗
PubMed1990

Variable region primary structures of monoclonal anti-DNA autoantibodies from NZB/NZW F1 mice.

VH and VL region primary structures of five NZB/NZW F1 derived monoclonal anti-DNA autoantibodies were determined from cloned cDNA. Comparative analysis of VH genes showed that except for two VH genes that shared complete identity the overall VH gene usage was diverse. Comparison of VH genes with those utilized in a variety of antibody responses showed they were generally unique to the autoanti-DNA response although framework homologies allowed assignment of four of five VH genes to existing murine heavy chain gene families. Only one out of five D segments shared homology to existing germline D segments, and all were rearranged to JH3. V kappa genes showed restriction for four of five light chains to the V kappa 1 subgroup. The V kappa 1 subgroup has been shown previously to be utilized in several anti-DNA autoantibodies as well as a variety of antibodies to exogenous antigens. H and L chain amino acid residues associated with the active site of a ssDNA specific autoantibody, 04-01, are discussed based on recently obtained crystallographic data.

Amino Acid Sequence↗
PubMed1990

Cone receptive field in cat retina computed from microcircuitry.

The receptive-field profile of the cone in cat-retina was computed. The computation was based on (1) the known anatomical circuit connecting cones via narrow-field bipolar cells to the on-beta ganglion cell; (2) the known physiological receptive-field profile of the on-beta (X) cell at the corresponding eccentricity; and (3) a model in which the beta receptive field arises by linear superposition of cone receptive fields. The computed cone receptive field has a center/surround organization with a center almost as broad as that of the beta cell center. The cone surround is comparably broad to that of the beta cell but somewhat lower in peak amplitude. The problems to which the center/surround receptive field are the solution, namely, signal compression and noise reduction, apparently must be solved before the first synapse of the visual pathway.

Animals↗
PubMed1990

Visual performance in idiopathic macular holes.

Previously published reports on the clinical features of idiopathic macular holes highlight the predilection for post-menopausal women and implicate vitreomacular traction in the aetiology of these lesions. Relatively little attention, however, has been paid to the quality of visual loss in eyes with macular holes. Histological studies of full-thickness macular holes have shown loss of all retinal layers in the area of the hole, and this would be expected to produce a central absolute scotoma of the same diameter. The majority of patients with full-thickness holes in this series did not have an absolute scotoma large enough to be detected on the Amsler Chart or when reading. It is suggested that following formation of a macular hole, enlargement may occur without further loss of foveal tissue and without enlargement of the absolute scotoma, due to tangential vitreous traction or contraction of an associated epiretinal membrane.

Aged↗
PubMed1990

The incidence of ophthalmic problems in the contact lens wearing population.

In a prospective study all contact lens wearers presenting to the ophthalmic casualty department of the University Hospital Nottingham within a six month period were examined to determine the number and pattern of ophthalmic problems occurring in this group. This information was then compared with prescribing information from contact lens dispensers in the city. The incidence of problems associated with contact lens wear within the first six months of starting to use a new contact lens was found to be 0.9% for soft daily wear lenses and 0.8% for rigid lenses. Reservations about these figures are discussed. The similarity between the incidence for the two types of lenses appears to contradict previous suggestions that soft lens wearers are more at risk of developing complications. It may be that the early period of lens wear represents a favoured time with respect to soft lens induced disease while hard lens problems are more common at this time. Both giant papillary conjunctivitis and corneal ulceration were seen in significant numbers but neither were associated with one particular lens type.

Adolescent↗
PubMed1990

"Collective coding" of correlated cone signals in the retinal ganglion cell.

The signals in neighboring cones are partially correlated due to local correlations of luminance in the visual scene. By summing these partially correlated signals, the retinal ganglion cell improves its signal/noise ratio (compared to the signal/noise ratio in a cone) and expands the variance of its response to fill its dynamic range. Our computations prove that the optimal weighting function for this summation is dome-shaped. The computations also show that (assuming a particular space constant for the correlation function) ganglion cell collecting area and cone density are matched at all eccentricities such that the signal/noise ratio improves by a constant factor. The signal/noise improvement factor for beta ganglion cells in cat retina is about 4.

Animals↗
PubMed1990

The relationship between digital rectal examination and abdominal radiographs in elderly patients.

The relationship between digital rectal examination and the findings of faecal loading on abdominal radiograph was studied in elderly patients admitted to hospital. No significant association was found but there was a relationship between faecal loading of the recto-sigmoid, colonic and caecal areas of the radiograph. Soft faeces in the rectum were commoner in older patients and were associated with faecal loading in the caecal area. The digital rectal examination is useful for identifying anal and rectal lesions and faecal impaction but abdominal radiograph is required to assess faecal loading of the large bowel.

Aged↗