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Biomedical subjects

M Silverman

Publications and source records attributed to M Silverman.

At least 73 records · Page 4Linked to original sources
PubMed1998

Functional studies of the rabbit intestinal Na+/glucose carrier (SGLT1) expressed in COS-7 cells: evaluation of the mutant A166C indicates this region is important for Na+-activation of the carrier.

We have exploited two mutants of the rabbit intestinal Na+/glucose carrier SGLT1 to explore the structure/function relationship of this Na+/glucose transporter in COS-7 cells. A functional N-terminal myc-epitope-tagged SGLT1 protein was constructed and used to determine the plasma-membrane localization of SGLT1. The kinetic and specificity characteristics of the myc-tagged SGLT1 mutant were identical with those of wild-type SGLT1. Immunogold labelling and electron microscopy confirmed the topology of the N-terminal region to be extracellular. Expression of the SGLT1 A166C mutant in these cells showed diminished levels of Na+-dependent alpha-methyl-d-glucopyranoside transport activity compared with wild-type SGLT1. For SGLT1 A166C, Vmax was 0.92+/-0.08 nmol/min per mg of protein and Km was 0.98+/-0.13 mM; for wild-type SGLT1, Vmax was 1.98+/-0.47 nmol/min per mg of protein and Km was 0.36+/-0.16 mM. Significantly, phlorrhizin (phloridzin) binding experiments confirmed equal expression of Na+-dependent high-affinity phlorrhizin binding to COS-7 cells expressing SGLT1 A166C or wild-type SGLT1 (Bmax 1.55+/-0.18 and 1.69+/-0.57 pmol/mg of protein respectively); Kd values were 0.46+/-0.15 and 0.51+/-0.11 microM for SGLT1 A166C and wild-type SGLT1 respectively. The specificity of sugar interaction was unchanged by the A166C mutation. We conclude that the replacement of an alanine residue by cysteine at position 166 has a profound effect on transporter function, resulting in a decrease in transporter turnover rate by a factor of 2. Taken as a whole the functional changes observed by SGLT1 A166C are most consistent with the mutation having caused an altered Na+ interaction with the transporter.

Animals↗
PubMed1998

Tyrosine kinase cell signaling pathways of rat mesangial cells in 3-dimensional cultures: response to fetal bovine serum and platelet-derived growth factor-BB.

Cells grown in 3-dimensional collagen gels adopt a nonproliferative, contractile phenotype which is more characteristic of cells in vivo than cells grown in 2-dimensional culture. The floating collagen gel contraction assay is a well-defined system used to study cell-extracellular matrix interactions grown in 3-dimensional culture. Although the cell biology of this system is well defined, the cell signaling associated with gel contraction has not been well characterized. In this study we demonstrate that fetal bovine (FBS) and platelet-derived growth factor (PDGF)-induced mesangial cell-collagen gel contraction is associated with increased tyrosine phosphorylation of a number of proteins including focal adhesion kinase (FAK) and the 42-kDa isoform of MAPK (ERK2). FBS-induced gel contraction is not affected by the presence of the MEK inhibitor PD098059. Low concentrations of PDGF-BB (10 ng/ml) induce gel contraction; however, at higher PDGF-BB concentrations (80 ng/ml) gel contraction is not observed. PDGF-BB-induced gel contraction as well as tyrosine phosphorylation of FAK are inhibited in the presence of the PI-3 kinase inhibitor wortmanin. Minimal autophosphorylation of the PDGF-beta receptor is observed under 3-dimensional culture conditions following PDGF-BB stimulation; however, when mesangial cells grown in 2-dimensional culture are exposed to PDGF-BB, the PDGF-beta receptor was prominently phosphorylated. We conclude that induction of collagen gel contraction by FBS and PDGF-BB is associated with tyrosine kinase phosphorylation and that these responses differ substantially from what occurs in 2-dimensional cultures in the presence of the same agonists.

Androstadienes↗
PubMed1998

Replacement of Ala-166 with cysteine in the high affinity rabbit sodium/glucose transporter alters transport kinetics and allows methanethiosulfonate ethylamine to inhibit transporter function.

An alanine to cysteine mutation at position 166 has been introduced by site-directed mutagenesis into the rabbit sodium/glucose transporter (rSGLT1). When expressed in Xenopus laevis oocytes, this mutant transporter (A166C rSGLT1) demonstrates a significantly lower apparent affinity for alpha-methyl glucoside (alphaMG) compared with the wild-type transporter (apparent Km = 0.8 versus 0.15 mM). Using the two-electrode voltage clamp technique, transient currents have also been measured, and for the mutant transporter, the transients induced by large depolarizations exhibit longer time constants than those for wild type. Moreover, the substitution of Ala-166 with a cysteine allows the sulfydryl specific reagent, methanethiosulfonate ethylamine (MTSEA), to react with and alter the function of the transporter. Whereas the wild-type transporter is unaffected by reaction with MTSEA, A166C rSGLT1 has its steady-state currents induced by 1 mM alphaMG inhibited 83% within a minute of exposure to MTSEA. Furthermore, the pre-steady-state transients of the A166C mutant after MTSEA exposure demonstrate much shorter time constants than before while the total amount of charge transferred is only slightly diminished. These results together provide evidence that position 166 is situated in a region critical to the functioning of rSGLT1.

Alanine↗
PubMed1998

Maintenance of mobility in residents of an Alzheimer special care facility.

The cognitive and functional decline of demented residents in a specialized Alzheimer's facility was compared to that of demented residents living in a traditional nursing home. All residents met DSM-III-R criteria for dementia. The evaluation consisted of a clinical interview with patients and collateral assessments of cognition, general health, problem behaviors, depression, and a broad range of activities of daily living (ADLs) including mobility. Residents were evaluated at baseline and 6-month intervals over 18 months. Similar rates of decline were found in cognition and overall ADLs in both groups. However, the most striking finding was preserved mobility in residents of the Alzheimer's facility. This finding suggests that specialized facilities do not halt or slow the overall progression of cognitive and functional decline seen in Alzheimer's disease; however, residents of such facilities may show slower decline in mobility.

Accidental Falls↗
PubMed1998

Cloning of a novel gene in the human kidney homologous to rat munc13s: its potential role in diabetic nephropathy.

Glomerular mesangial cells (MC) are believed to play a pivotal role in development of diabetic nephropathy. We employed differential display reverse transcription polymerase chain reaction (DDRT-PCR) comparing human MC grown under 25 mM and 5.5 mM D-glucose and osmolarity control as a first step to identify possible candidate genes regulated by hyperglycemia. This strategy resulted in cloning of a novel gene in human MC, human munc13 (hmunc13), a human homologue of rat munc13s with the N-terminal segment similar to munc13-1 and the C-terminal segment more similar to munc13-2. Hmunc13 is also expressed in human kidney cortical epithelial cells. By using relative RT-PCR and Northern blot, we have confirmed that expression of hmunc13 in MC is up-regulated by high D-glucose treatment. Together with previous reports that munc13s binds to diacylglycerol (DAG) and that hyperglycemia increases DAG levels, these findings point to a potential role of hmunc13 in mediating some of the acute and chronic changes in MC produced by exposure to hyperglycemia.

Amino Acid Sequence↗
PubMed1998

Anterior cervical osteophytes as a cause of pharyngeal stage dysphagia.

OBJECTIVE: To describe the association of anterior cervical osteophytes impairing epiglottic movement as an etiology of pharyngeal stage dysphagia. DESIGN: A retrospective chart review. SETTING: An academic affiliated Veterans Affairs Medical Center. PATIENTS: Veterans 60 years of age or older referred to speech pathology for dysphagia evaluation. RESULTS: Of 376 veterans evaluated consecutively since 1990, 40 patients had dysphagia determined to be secondary to cervical bony protuberances. The evaluation and management of three cases with functional consequences at level C3-5 with no dysphagia etiology present are described. CONCLUSION: Cervical osteophytes need to be considered in the differential diagnosis of dysphagia in older people.

Aged↗
PubMed1998

Soluble L-selectin concentration in bronchoalveolar lavage fluid obtained from infants who develop chronic lung disease of prematurity.

AIMS: To explore the changes in neutrophil adhesion molecule expression and release into bronchoalveolar lavage fluid (BAL) obtained from infants who developed chronic lung disease (CLD). METHODS: BAL fluid was obtained from 37 infants: 18 (median gestation 26 weeks, birthweight 835 g) who developed CLD, 12 (29 weeks, 1345 g) with respiratory distress syndrome (RDS) and seven control infants (33 weeks, 2190 g). RESULTS: Soluble L-selectin (sL-selectin) in BAL fluid from the CLD and non-CLD groups was similar immediately after birth, but in infants who subsequently developed CLD, sL-selectin remained persistently increased (at day 7: CLD 42.6 vs RDS 6.0 ng/ml, p < 0.05; CLD vs controls 1.5 ng/ml; p < 0.05). CD11b/CD18 expression on neutrophils obtained by BAL increased with time to reach a maximum at 17 days of age in infants who developed CLD. CONCLUSIONS: These results suggest that leucocyte traffic persists in infants who develop CLD and may have an important part to play in the pathogenesis of CLD.

Analysis of Variance↗
PubMed1998

Arachidonic acid potentiates the feedback response of mesangial BKCa channels to angiotensin II.

The influence of arachidonic acid (AA) on the feedback regulation of mesangial contraction by large Ca(2+)-activated K+ channels (BKCa) was determined through single-channel analysis using the patch clamp method. The mesangial BKCa is a low-gain negative feedback inhibitor of contraction that is activated in response to agonist-induced Ca2+ transients and membrane depolarization. AA activated BKCa in cell-attached patches in a dose-dependent manner with a maximal effect at 400 nM and a half-maximal response at 49 nM. In inside-out patches, AA directly activated BKCa with a maximal effect at 400 nM. BKCa was activated significantly in response to addition of 100 nM ANG II in the presence but not the absence of AA. Since it was shown previously that fatty acids stimulated both soluble and membrane-bound guanylyl cyclase, we determined whether AA activated BKCa by interfering with cGMP-mediated signal transduction pathways. It was previously shown that 10 microM cGMP, via cGMP-dependent protein kinase, activated BKCa in a biphasic manner with an early increase in probability of a channel existing in an open state (Po) and a subsequent inactivation mediated by protein phosphatase 2A (PP2A). We found that 10 microM dibutyryl-cGMP enhanced BKCa activity in an additive manner with saturating concentrations (400 nM) of AA. Moreover, the inactivation phase mediated by PP2A was not abolished. Thus AA does not affect the phosphorylation/dephosphorylation regulatory cycle for BKCa. It is concluded that AA potentiates the ANG II feedback response of BKCa by a mechanism that is independent of the phosphorylation cycle.

Angiotensin II↗
PubMed1998

Irregularities and power law distributions in the breathing pattern in preterm and term infants.

Unlike older children, young infants are prone to develop unstable respiratory patterns, suggesting important differences in their control of breathing. We examined the irregular breathing pattern in infants by measuring the time interval between breaths ("interbreath interval"; IBI) assessed from abdominal movement during 2 h of sleep in 25 preterm infants at a postconceptional age of 40.5 +/- 5.2 (SD) wk and in 14 term healthy infants at a postnatal age of 8.2 +/- 4 wk. In 10 infants we performed longitudinal measurements on two occasions. We developed a threshold algorithm for the detection of a breath so that an IBI included an apneic period and potentially some periods of insufficient tidal breathing excursions (hypopneas). The probability density distribution (P) of IBIs follows a power law, P(IBI) approximately IBI-alpha, with the exponent alpha providing a statistical measurement of the relative risk of insufficient breathing. With maturation, alpha increased from 2.62 +/- 0.4 at 41. 2 +/- 3.6 wk to 3.22 +/- 0.4 at 47.3 +/- 6.4 wk postconceptional age, indicating a decrease in long hypopneas (for paired data P = 0.002). The statistical properties of IBI were well reproduced in a model of the respiratory oscillator on the basis of two hypotheses: 1) tonic neural inputs to the respiratory oscillator are noisy; and 2) the noise explores a critical region where IBI diverges with decreasing tonic inputs. Accordingly, maturation of infant respiratory control can be explained by the tonic inputs moving away from this critical region. We conclude that breathing irregularities in infants can be characterized by alpha, which provides a link between clinically accessible data and the neurophysiology of the respiratory oscillator.

Abdominal Muscles↗
PubMed1998

High-frequency respiratory impedance measured by forced-oscillation technique in infants.

Measurements of respiratory input impedance (Zin) in infants using forced oscillations at the airway opening up to 256 Hz have been shown to include a first antiresonance (ar,1). We wondered whether features derived from high-frequency Zin change during methacholine-induced airway obstruction in infants, whether those changes could be explained by a lumped parameter model as in dogs (providing a value for respiratory resistance [Rrs]), or whether they are similar to Zin data in human adults with airway obstruction. In 13 wheezy infants (age 58 +/- 19 wk) Zin(omega) was assessed at baseline, and in nine infants after methacholine challenge, using a provocation dose defined by fall of more than 30% in VmaxFRC (rapid chest-compression technique). Following methacholine challenge, VmaxFRC decreased significantly (p < 0.0005), the frequency at which ar,1 occurred ( far,1) increased significantly (p = 0.0007), and the relative maximum in the real part at far,1 [Zinre( far,1)] increased significantly (p = 0.02), whereas Rrs did not change. We conclude that in wheezy infants ar,1 is highly sensitive to changes in lung mechanics. Although ar,1 cannot be explained by a simple lumped-parameter model, it is likely due to wave propagation phenomena, as in human adults. In either case, far,1 potentially contains information about airway wall compliance, which is important for the understanding of flow limitation in infant wheezing disorders.

Airway Resistance↗
PubMed1998

Differences in airway wall compliance as a possible mechanism for wheezing disorders in infants.

High-frequency input impedance measurements (Z(f)) provide useful noninvasive information on airway geometry and especially airway wall mechanics in the canine and human adult respiratory system. Using the high-speed interrupter technique (HIT), we have shown that it is possible to measure high-frequency Z(f) in infants up to 900 Hz, including antiresonant phenomena which are known to be related to wave propagation velocity. This implies that the first antiresonant frequency (far,1) is a function of airway wall compliance. Since, airway wall mechanics are particularly important for the flow limitation phenomena, we wondered whether we could find evidence that airway wall properties were important for the occurrence of flow limitation during incremental methacholine challenge in infants. We measured Z(f) from 32-900 Hz and maximal flow at functional residual capacity (V'maxFRC) by the rapid chest compression technique in 10 infants (aged 36-81 weeks) with wheezing disorders. far,1 increased significantly at very low doses of mch before any decline could be detected in V'maxFRC. We hypothesize that these changes in far,1 are determined by mch-induced decrease in airway wall compliance. High-speed interrupter technique in combination with rapid chest compression technique can be used to study developmental differences in airway function (particularly of airway wall properties) and their contribution to airway disease and response to bronchodilator therapy in infants.

Adult↗
PubMed1998

High-frequency respiratory input impedance measurements in infants assessed by the high speed interrupter technique.

High-frequency input impedance (Z(f)) measurements, including antiresonances, provide useful noninvasive information on airway geometry and especially airway wall mechanics in the canine and human adult respiratory system. A knowledge of airway wall mechanics would be particularly important in understanding flow limitation phenomena in infants. High-frequency Z(f) has not been measured in infants above 256 Hz, because the high impedance of the infantile respiratory system would be expected to result in low amplitudes of oscillatory flow at higher frequencies. The aim of this study was to develop a technique to measure high-frequency Z(f) in infants and to elucidate the nature of the antiresonance phenomena in the Z(f) spectrum in infants. Z(f) was measured from 32-900 Hz during rapid airflow interruption by the high-speed interrupter technique (HIT) in 18 infants (aged 24-149 weeks) with wheezing disorders. The HIT enables the excitement of higher flow amplitudes at high frequencies using a pseudostep forcing function. In all infants Z(f) showed a mean (SD) first antiresonance (far,1) of 172 (35) Hz (real part of Z(f) at far,1 (Z(f)re(far,1)): 4.9 (1.1) kPa x L(-1) x s) and in five infants a second antiresonance (far,2) of 564 (51) Hz (Z(f)re(far,2): 2.0 (0.7) kPa x L(-1) x s). The antiresonances were found to be related to wave propagation in the airways (acoustic antiresonances), because they increased by a factor of approximately 2 when He-O2 was inhaled. This implies that far,1 and its harmonics are a function of airway wall compliance. In conclusion, the first and second antiresonances may be helpful in understanding flow limitation in wheezing disorders in infants, because flow limitation is related not only to airway diameter but also to airway wall compliance.

Adult↗
PubMed1998

Issues relating to children born prematurely.

Preterm infants suffer increased risk of acute and chronic respiratory disorders. In patients with chronic lung disease or severe bronchopulmonary dysplasia, long-term respiratory morbidity and pulmonary dysfunction into late childhood and early adulthood have been reported. This includes symptomatic morbidity (recurrent cough and/or wheeze) and lung function abnormalities such as increased resistance to airflow, airway hyperresponsiveness, and increased propensity to air-trapping. To date, no clinically significant association between prematurity and classical atopic asthma has been demonstrated. Therefore, interventions should primarily focus on the reduction of wheezing disorders and lung function abnormalities in children born prematurely. In order to design interventions in the foetal and early neonatal period or during childhood the potential risk factors for long-term morbidity need to be carefully identified at different age groups: factors which affect pre- and postnatal lung growth, airway inflammation, viral infections, air pollution and others. Future research issues should include well-conducted prospective follow-up programmes which will identify major risk factors in specific populations. Early intervention will provide information on disease mechanisms and on new prophylactic as well as therapeutic strategies.

Bronchial Hyperreactivity↗