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Biomedical subjects

R A Montgomery

Publications and source records attributed to R A Montgomery.

At least 37 records · Page 2Linked to original sources
PubMed1998

Multiple molecular mechanisms underlying subdiagnostic variants of Marfan syndrome.

Mutations in the FBN1 gene, which encodes fibrillin-1, cause Marfan syndrome (MFS) and have been associated with a wide range of milder, overlap phenotypes. The factors that modulate phenotypic severity, both between and within families, remain to be determined. This study examines the relationship between the FBN1 genotype and phenotype in families with extremely mild phenotypes and in those that show striking clinical variation among apparently affected individuals. In one family, clinically similar but etiologically distinct disorders are segregating independently. In another, somatic mosaicism for a mutant FBN1 allele is associated with subdiagnostic manifestations, whereas germ-line transmission of the identical mutation causes severe and rapidly progressive disease. A third family cosegregates mild mitral valve prolapse syndrome with a mutation in FBN1 that can be functionally distinguished from those associated with the classic MFS phenotype. These data have immediate relevance for the diagnostic and prognostic counseling of patients and their family members.

Adult↗
PubMed1997

Semi-quantitative polymerase chain reaction analysis of cytokine and cytokine receptor gene expression during thymic ontogeny.

Semi-quantitative, polymerase chain reaction (PCR) is used to uncover the patterns of cytokine transcription in the mouse thymus from day 14 to day 20 of gestation, a time period which includes many of the important events in thymic ontogeny. Interleukin 4 (IL-4), IL-7 and interferon gamma (IFN-gamma) mRNA is abundant from fetal day (Fd) 14-16, corresponding with the period of rapid proliferation of immature thymocytes in vivo. As the level of mRNA for these cytokines diminishes, the induction and increased expression of IL-3 and IL-2 occurs. The transcription of these cytokines correlates temporally with the period of proliferation-dependent phenotypic differentiation between Fd 16 and 20. The thymic epithelium (TE)-derived cytokines including IL-1alpha, IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) begin to be transcribed between Fd 14-15 and show peak mRNA abundance from Fd 16-20. IL-5, tumour necrosis factor alpha (TNF-alpha) and LT (lymphotoxin or TNF-beta) constitute a fourth group of cytokines, along with the IL-4 receptor (IL-4R), which are transcribed at an even level throughout the fetal period. The IL-2 receptor beta chain (IL-2Rbeta) and IL-10 show abundant mRNA from Fd 14-20 and have a peak level of mRNA content on Fd 16. Taken together, these studies uncover complex, overlapping patterns of cytokine gene expression. The mRNA abundance and pattern of expression of each cytokine or cytokine receptor may indicate the relative contribution that it makes to different stages of fetal thymic ontogeny.

Animals↗
PubMed1997

Inhibition of fibrillin 1 expression using U1 snRNA as a vehicle for the presentation of antisense targeting sequence.

This study examines whether the mimicking of selected properties of naturally occurring antisense RNAs in prokaryotes allows efficient inhibition of gene expression by in situ-expressed recombinant molecules in mammalian cells. Prokaryotic regulatory transcripts are expressed at high levels and have hairpin structures at their termini, features reminiscent of small nuclear RNAs (snRNAs) which are abundant and stable in the nucleus of all mammalian cells. A sequence complementary to fibrillin-1 (FBN1) mRNA, interrupted in its center by a hammerhead ribozyme, was substituted for the Sm protein binding site between the stem-loop structures of U1 snRNA. Expression of the chimeric antisense RNA resulted in dramatic inhibition of expression of fibrillin-1 message and protein in stably transfected cultured cells. The inhibitory effect was localized to the nucleus. The biological properties of U1 snRNA may provide a widely applicable vehicle for the in vivo delivery of antisense targeting sequences.

Base Sequence↗
PubMed1996

Glanzmann thrombasthenia. Cooperation between sequence variants in cis during splice site selection.

Glanzmann thrombasthenia (GT), an autosomal recessive bleeding disorder, results from abnormalities in the platelet fibrinogen receptor, GP(IIb)-IIIa (integrin alpha(IIb)beta3). A patient with GT was identified as homozygous for a G-->A mutation 6 bp upstream of the GP(IIIa) exon 9 splice donor site. Patient platelet GP(IIIa) transcripts lacked exon 9 despite normal DNA sequence in all of the cis-acting sequences known to regulate splice site selection. In vitro analysis of transcripts generated from mini-gene constructs demonstrated that exon skipping occurred only when the G-->A mutation was cis to a polymorphism 116 bp upstream, providing precedence that two sequence variations in the same exon which do not alter consensus splice sites and do not generate missense or nonsense mutations, can affect splice site selection. The mutant transcript resulted from utilization of a cryptic splice acceptor site and returned the open reading frame. These data support the hypothesis that pre-mRNA secondary structure and allelic sequence variants can influence splicing and provide new insight into the regulated control of RNA processing. In addition, haplotype analysis suggested that the patient has two identical copies of chromosome 17. Markers studied on three other chromosomes suggested this finding was not due to consanguinity. The restricted phenotype in this patient may provide information regarding the expression of potentially imprinted genes on chromosome 17.

Adult↗
PubMed1995

A novel method for incorporation of ion channels into a planar phospholipid bilayer which allows solution changes on a millisecond timescale.

We have developed a method of rapidly changing the solutions on one side of a planar phospholipid bilayer. Bilayers can be painted on glass pipettes of tip diameter > or = 50 microns. By modifying an established method for rapid exchange of solutions bathing excised membrane patches, solution changes can be made at the bilayer within 10 ms. After incorporation of channels into the bilayer, the bilayer is moved into one of two parallel streams of solution flowing from a length of double-barrelled glass theta tubing. Activation of a solenoid system rapidly moves the theta tubing so that the bilayer is in the flow of the adjacent solution. For various reasons, the single-channel gating mechanisms of many channels are studied in planar bilayer systems. The conventional bilayer technique only allows for steady-state single-channel gating to be monitored. This novel method now allows the effects of rapid changes in modulators of channels incorporated into planar phospholipid bilayers to be measured.

Animals↗
PubMed1995

New insights into the gating mechanisms of cardiac ryanodine receptors revealed by rapid changes in ligand concentration.

We have developed a novel technique for incorporation of sheep cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channels into planar phospholipid bilayers in order to investigate changes in [Ca2+] on a physiological time scale and have investigated whether the rate of change of cytosolic [Ca2+] has a direct effect on the gating of the cardiac SR Ca(2+)-release channel. Vesicles of heavy SR were incorporated into planar phospholipid bilayers painted on glass pipettes, and an established technique for rapid solution exchanges at excised membrane patches was modified to allow solution changes to be made at the bilayer within 10 ms. For a given change in [Ca2+], we demonstrate that the open probability (Po) is similar whether the cytosolic [Ca2+] is increased rapidly (10 ms) or slowly (1 s) and appears to be no different from the Po measured under steady state conditions that were recorded by using conventional bilayer techniques. We also demonstrate that no desensitization or inactivation occurs at -40 mV when the channel is activated by Ca2+ alone or in the presence of other channel activators, ATP or EMD 41000. However, at +40 mV, rapid channel activation followed by inactivation was observed. The probability of such voltage-dependent inactivation appears to depend on the mechanism of channel activation.

Animals↗
PubMed1991

Analysis of cytokine gene expression during fetal thymic ontogeny using the polymerase chain reaction.

The expression of 12 cytokine and cytokine receptor genes during mouse thymic ontogeny has been studied using the polymerase chain reaction. The data reveal that IL-2 gene transcription does not begin in the thymus until fetal day (Fd) 16. However, IL-2R beta-chain mRNA was detectable throughout the period of investigation, from Fd 14 to 20. This suggests that the potential for high or intermediate affinity IL-2 binding is present in the thymus before the IL-2 gene is actually expressed. Overall, cytokine gene induction occurred in two distinct patterns. In the first group, represented by IL-1 beta, IL-4, IL-5, IL-6, IL-7, IFN-gamma, and lymphotoxin (lymphotoxin or TNF-beta) there was detectable mRNA present from Fd 14 to 20. IL-1 alpha, IL-2, IL-3, and GM-CSF followed a second pattern in which gene induction was delayed until Fd 16. The expression of IL-7 and IL-2 genes showed an interesting temporal relationship. IL-7 mRNA transcription occurred before IL-2 but diminished from Fd 16 after the onset of IL-2 gene induction. Because these data reflect the in vivo progression of gene expression during T cell ontogeny, they provide a guideline for assessing the physiologic significance of thymocyte responses to cytokines at different stages of fetal development.

Animals↗
PubMed1991

Cytokine gene expression: analysis using northern blotting, polymerase chain reaction and in situ hybridization.

We describe here the use of northern blotting, PCR and in situ hybridization for the analysis of cytokine gene expression. These techniques, each with their advantages and disadvantages, have been used to monitor cytokine gene expression in sites of immune reactivity and in the developing thymus. Whilst expression of a gene usually correlates well with protein production from that gene, this may not always be the case. The development of methods to analyze protein production in situ, for instance by immunohistochemistry, together with analysis of mRNA expression will allow us to begin to understand the role of cytokines within the immune system of the intact animal.

Animals↗
PubMed1991

Sheep cardiac sarcoplasmic reticulum calcium-release channels: modification of conductance and gating by temperature.

1. The gating and conduction properties of single calcium-release channels of sheep isolated cardiac junctional sarcoplasmic reticulum membranes incorporated into planar phospholipid bilayers were investigated under voltage clamp conditions at temperatures between 4 and 32 degrees C. 2. Single channel conductance was reduced linearly when temperature was decreased from 32 to 5 degrees C with a Q10 value of 1.5 between 10 and 20 degrees C. The apparent activation enthalpy for conductance between 32 and 5 degrees C was 6.16 +/- 1.2 kcal/mol. 3. Cooling the channel increased open probability (Po) when activating cytosolic calcium concentrations were varied within the range 0.1-100 microM. At an activating free calcium concentration of 10 microM, channel Po increased from 0.13 +/- 0.05 at 23 degrees C to 0.69 +/- 0.07 at 5-10 degrees C. 4. At sub-activating calcium concentrations (100 pM) or high concentrations of calcium (greater than or equal to 1000 microM), the calcium-release channel remained closed at 23 degrees C. Cooling the channel under these conditions did not increase Po. 5. Lifetime analysis indicates that with calcium as the sole activating ligand, the cooling-induced increase in Po results from an increase in channel open lifetimes with no significant alteration in the frequency of channel opening. At 23 degrees C, the open and closed lifetime distributions of the calcium-activated channel are best described by two and three exponentials respectively. At reduced temperatures (5-10 degrees C), both open and closed lifetime distributions were best described by three exponentials. 6. At sub-activating calcium concentrations, calcium-independent channel openings could be induced by sulmazole (AR-L 115 BS, 0.5-10 mM). At 23 degrees C, with sulmazole as the sole activating ligand, the best fits to both open and closed lifetime distributions were obtained with three exponentials. At reduced temperatures (5-10 degrees C), Po was increased. Gating was characterized by long open events, however the open and closed lifetime distributions were still best described by three exponentials. 7. The net effect of temperature reduction is an increase in calcium current through the channel. This finding is consistent with the suggestion that calcium release from the SR is a major factor in the initiation of rapid cooling contractures of mammalian cardiac muscle preparations.

Animals↗
PubMed1989

Lymphoscintigraphy to confirm the clinical diagnosis of lymphedema.

Confirmation of the diagnosis of lymphedema often requires lymphangiography, a procedure that is painful for the patient and technically demanding. Radioisotope lymphoscintigraphy is a relatively new technique that uses technetium 99 m antimony trisulfide colloid to produce a diagnostic image similar to a lymphangiogram. The procedure requires a single subcutaneous injection in the involved extremity, and images are obtained 3 hours later. It is technically easy to perform, produces minimal discomfort for the patient, and has no adverse effects. We have recently used radioisotope lymphoscintigraphy to evaluate 17 patients with extremity edema. These patients initially had a presumed diagnosis of lymphedema involving the upper or lower extremity. Lymphoscintigraphy confirmed the diagnosis of lymphedema in 12 (70.6%) patients. In five of the 17 patients (29.4%) the clinical impression of lymphedema was not supported by lymphoscintigraphy, leading to alternative diagnoses such as lipomatosis, venous insufficiency (two patients), congestive heart failure, and disuse edema. In all patients with secondary lymphedema the lymphatic system in the involved extremity could be partially visualized. Conversely, three of four patients with primary lymphedema had no ascent of the tracer from the foot and no lymphatic channels could be visualized. Lymphoscintigraphy is relatively easy to perform, safe, minimally invasive, and not uncomfortable for the patient. It is useful in differentiating lymphedema from other causes of extremity edema, allowing institution of appropriate therapy.

Adolescent↗
PubMed1988

An improved apparatus for the optical recording of contraction of single heart cells.

An optical system for measuring changes in cell length during unloaded contractions of cardiac myocytes is described. A one-dimensional video "image" of a cell is obtained every 4 ms with a linear photodiode array, which is aligned with the longitudinal axis of the cell. The circuit used to process the image from the photodiode array has a variety of features to aid in the accurate determination of the distance between the ends of the cell, i.e. the cell length. First, the video image of the cell is divided into two "windows", one encompassing the "front" edge of the cell, the other encompassing the "rear" edge. Other cells or debris beyond the cell edges are excluded. Changes in the general light level, for example as a result of debris floating above the cell, have little effect because within the windows the "background light level" is subtracted from the signals before they are processed further. To detect the cell edges, the system determines when the signals within the windows exceed (front edge) or drop below (rear edge) chosen thresholds, which are different for the front and rear edges. The system has "memory" and it identifies the rear edge of the cell as the last time the signal falls below the threshold; because of this "bright spots" within the cell are not mistaken for the end of the cell. The system has "hysteresis", which enables it to ignore small fluctuations in brightness around the threshold. The system is easy to use, accurate, readily calibrated, and it has good spatial and time resolution (about 0.25 micron and 4 ms respectively).

Animals↗
PubMed1988

A cDNA for the estradiol-regulated 24K protein: control of mRNA levels in MCF-7 cells.

We have previously demonstrated an estradiol-regulated 24 kDa (24K) protein in human breast cancer tissue culture cells and human tumor biopsies. The presence of 24K correlates well with the presence of steroid hormone receptors. In order to further study the hormonal regulation of the 24K protein and gene, we have isolated cDNA clones corresponding to the 24K mRNA. Poly(A)+ RNA isolated from the MCF-7 human breast cancer cell line was translated in a cell-free translation system containing [35S]-methionine. The translation products were immunoprecipitated with a 24K monoclonal antibody, and the in vitro synthesis of 24K protein was confirmed by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. The same poly(A)+ RNA was used to construct an oligo(dT)-primed cDNA library in the lambda gt11 expression vector system. The library was screened with a highly specific polyclonal antibody raised against 24K protein purified by immunoaffinity chromatography. Four recombinant clones reacting with the antibody by virtue of antigen expression were isolated and three were used in hybridization-selected translation. Three clones were able to hybridize specifically to a messenger RNA (mRNA) that yielded a Mr 24,000 protein when translated in vitro and analyzed by SDS/polyacrylamide gel electrophoresis. This protein was also immunoprecipitable by the 24K monoclonal antibody. MCF-7 mRNA size fractionated by formaldehyde-agarose gel electrophoresis was transferred to nitrocellulose paper and hybridized to a nick-translated 24K cDNA clone. A single band of hybridization corresponding to a mRNA size of approximately 0.9-1.0 kilobase (kb) was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Structural aspects of the sarcoplasmic reticulum K+ channel revealed by gallamine block.

We have studied single-channel conductance fluctuations of K+ channels present in the sarcoplasmic reticulum (SR) membrane systems of rabbit cardiac and skeletal muscle. K+ conductance through the channels is reversibly blocked by gallamine. Conductance block occurs only from the trans side of the channel and is resolved as a smooth reduction in the open state conductance. At a fixed K+ concentration, conduction decreases with increasing gallamine concentration and the data can be fitted to a single-site inhibition scheme. The degree of block seen at a constant gallamine concentration decreases as K+ concentration is increased, indicating competition between gallamine and K+. Gallamine block is voltage dependent, the degree of block increasing with increasing negative holding potential. Quantitative analysis of block yields a zero voltage dissociation constant of 55.3 +/- 16 microM and an effective valence of block of 0.93 +/- 0.12. We conclude that gallamine blocks by interacting with a site or sites located at an electrical distance 30-35% into the voltage drop from the trans side of the channel. This site must have a cross-sectional area of at least 1.2 nm2. The results of this study have been used to modify and extend our view of the structure of the channel's conduction pathway.

Animals↗