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Biomedical subjects

L S Weinstein

Publications and source records attributed to L S Weinstein.

At least 37 records · Page 2Linked to original sources
PubMed1999

Management of bile duct stones in 1572 patients undergoing laparoscopic cholecystectomy.

Evidence of bile duct stones (BDSs) was identified on routine cholangiogram in 136 (8.7%) of 1572 patients undergoing laparoscopic cholecystectomy from March 1989 through March 1997. Forty-two (30.9%) were unsuspected. All patients with evidence of BDSs underwent laparoscopic bile duct exploration (LBDE). Initially, a standard choledochotomy with T-tube drainage as in the open approach was used. Later, transcystic duct exploration was added to the algorithm. The algorithm evolved into an ongoing treatment protocol study that was initiated in March 1992. Through March 1997, 100 patients underwent LBDE based on the protocol. The study is divided into two groups. Group A comprises the total 136 patients undergoing LBDE, including those in the protocol study. A subgroup, Group B, comprises only the 100 patients in the protocol study. In Group A, LBDE was successful in 114 patients (83.8%). Stones were missed in seven patients and left behind for spontaneous passage or later retrieval in six patients. Eleven patients (8.1%) were converted to open. There were 13 major complications (9.6%), including the seven missed stones and two deaths. In Group B, LBDE was successful in 94 per cent. Stones were missed in one patient and intentionally left behind in four patients. One patient was converted to open. There were seven major complications (7%), including one of the missed stones and one death. Using the protocol algorithm and the techniques described, BDSs can be effectively managed laparoscopically at the time of cholecystectomy in approximately 94 per cent of cases.

Adolescent↗
PubMed1998

A novel mutation in the switch 3 region of Gsalpha in a patient with Albright hereditary osteodystrophy impairs GDP binding and receptor activation.

Albright hereditary osteodystrophy (AHO), a disorder characterized by skeletal abnormalities and obesity, is associated with heterozygous inactivating mutations in the gene for Gsalpha. A novel Gsalpha mutation encoding the substitution of tryptophan for a nonconserved arginine within the switch 3 region (Gsalpha R258W) was identified in an AHO patient. Although reverse transcription-polymerase chain reaction studies demonstrated that mRNA expression from wild type and mutant alleles was similar, Gsalpha expression in erythrocyte membranes from the affected patient was reduced by 50%. A Gsalpha R258W cDNA, as well as one with arginine replaced by alanine (Gsalpha R258A), was generated, and the biochemical properties of in vitro transcription/translation products were examined. When reconstituted with cyc- membranes, both mutant proteins were able to stimulate adenylyl cyclase normally in the presence of guanosine- 5'-O-(3-thiotriphosphate) (GTPgammaS) but had decreased ability in the presence of isoproterenol or AlF4- (a mixture of 10 microM AlCl3 and 10 mM NaF). The ability of each mutant to bind and be activated by GTPgammaS or AlF4- was assessed by trypsin protection assays. Both mutants were protected normally by GTPgammaS but showed reduced protection in the presence of AlF4-. The addition of excess GDP (2 mM) was able to rescue the ability of AlF4- to protect the mutants, suggesting that they might have reduced affinity for GDP. A Gsalpha R258A mutant purified from Escherichia coli had decreased affinity for GDP and an apparent rate of GDP release that was 10-fold greater than that of wild type Gsalpha. Sucrose density gradient analysis demonstrated that both Gsalpha R258W and Gsalpha R258A were thermolabile at higher temperatures and that denaturation of both mutants was prevented by the presence of 0.1 mM GTPgammaS or 2 mM GDP. The crystal structure of Gsalpha demonstrates that Arg258 interacts with a conserved residue in the helical domain (Gln170). Arg258 substitutions would be predicted to open the cleft between the GTPase and helical domains, allowing for increased GDP release in the inactive state, resulting in enhanced thermolability and reduced AlF4--induced adenylyl cyclase stimulation and trypsin protection, since activation by AlF4- requires bound GDP.

Adenylyl Cyclases↗
PubMed1998

Variable and tissue-specific hormone resistance in heterotrimeric Gs protein alpha-subunit (Gsalpha) knockout mice is due to tissue-specific imprinting of the gsalpha gene.

Albright hereditary osteodystrophy (AHO), an autosomal dominant disorder characterized by short stature, obesity, and skeletal defects, is associated with heterozygous inactivating mutations of GNAS1, the gene encoding the heterotrimeric G protein alpha-subunit (Gsalpha) that couples multiple receptors to the stimulation of adenylyl cyclase. It has remained unclear why only some AHO patients present with multihormone resistance and why AHO patients demonstrate resistance to some hormones [e.g., parathyroid hormone (PTH)] but not to others (e.g., vasopressin), even though all activate adenylyl cyclase. We generated mice with a null allele of the mouse homolog Gnas. Homozygous Gs deficiency is embryonically lethal. Heterozygotes with maternal (m-/+) and paternal (+/p-) inheritance of the Gnas null allele have distinct phenotypes, suggesting that Gnas is an imprinted gene. PTH resistance is present in m-/+, but not +/p-, mice. Gsalpha expression in the renal cortex (the site of PTH action) is markedly reduced in m-/+ but not in +/p- mice, demonstrating that the Gnas paternal allele is imprinted in this tissue. Gnas is also imprinted in brown and white adipose tissue. The maximal physiological response to vasopressin (urinary concentrating ability) is normal in both m-/+ and +/p- mice and Gnas is not imprinted in the renal inner medulla (the site of vasopressin action). Tissue-specific imprinting of Gnas is likely the mechanism for variable and tissue-specific hormone resistance in these mice and a similar mechanism might explain the variable phenotype in AHO.

Alleles↗
PubMed1997

A novel mutation adjacent to the switch III domain of G(S alpha) in a patient with pseudohypoparathyroidism.

A novel G(S alpha) mutation encoding the substitution of arginine for serine 250 (G[S alpha] S250R) was identified in a patient with pseudohypoparathyroidism type Ia. Both G(S) activity and G(S alpha) expression were decreased by about 50% in erythrocyte membranes from the affected patient. The cDNA of this G(S alpha) mutant, as well as one in which the S250 residue is deleted (G[S alpha]-deltaS250), was generated, and the biochemical properties of the products of in vitro transcription/translation were examined. Both mutants had a sedimentation coefficient similar to that of wild type G(S alpha) (approximately 3.7S) when kept at 0 C after synthesis. However when maintained for 1-2 h at 30-37 C, both mutants aggregated to a material sedimenting at approximately 6.3S or greater (G[S alpha]-S250R to a greater extent than G(S alpha]-deltaS250), while wild type G(S alpha) sedimented at approximately 3.7S, suggesting that the mutants were thermolabile. Incubation in the presence of high doses of guanine nucleotide partially prevented heat denaturation of G(S alpha) deltaS250 but had no protective effect on G(S alpha-S250R. Sucrose density gradient centrifugation at 0 C in the presence and absence of beta gamma-dimers demonstrated that, in contrast to wild type G(S alpha) neither mutant could interact with beta gamma. Trypsin protection assays revealed no protection of G(S alpha)-S250R by GTPgammaS or AIF4- at any temperature. GTPgammaS conferred modest protection of G(S alpha)-deltaS250 (approximately 50% of wild-type G[S alpha]) at 30 C but none at 37 C, while AIF4- conferred slight protection at 20 C but none at 30 C or above. Consistent with this result, G(S alpha)-deltaS250 was able to stimulate adenylyl cyclase at 30 C when reconstituted with cyc- membranes in the presence of GTPgammaS but not in the presence of AIF4-. G(S alpha)-S250R showed no ability to stimulate adenylyl cyclase in the presence of either agent. Stable transfection of mutant and wild-type G(S alpha) into cyc- S49 lymphoma cells revealed that the majority of wild type G(S alpha) localized to membranes, while little or no membrane localization occurred for either mutant. Modeling of G(S alpha) based upon the crystal structure of G(t alpha) or G(i alpha) suggests that Ser250 interacts with several residues within and around the conserved NKXD motif, which directly interacts with the guanine ring of bound GDP or GTP. It is therefore possible that substitution or deletion of this residue may alter guanine nucleotide binding, which could lead to thermolability and impaired function.

Animals↗
PubMed1996

Regulation of the human Gi alpha-2 gene promotor activity in embryonic chicken cardiomyocytes.

Increased expression of the inhibitory G protein Gi alpha-2 is assumed to contribute to desensitization of adenylyl cyclase in human heart failure. The mechanisms of upregulation involve increases in myocardial Gi alpha-2 protein, mRNA and gene transcriptional activity. To elucidate these mechanisms in more detail, the 5' flanking region of the human Gi alpha-2 gene (-1214/+115 bp) was cloned upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene and transfected in embryonic chick cardiomyocytes. CAT activity was measured 48 h after transfection. Unstimulated activity of the -1214/+115 bp construct was about 10-fold higher than activity of the basal CAT-construct (pGEMCAT). 5' deletion from -1214/+115 to -85/+115 bp upstream of the transcriptional start site increased, further stepwise deletions to 46/+115 gradually decreased promotor activity. Deletion from -46/+115 to -33/+115 bp completely abolished promotor activity. Stimulation of cardiomyocytes that had been transfected with the -1214/+115 CAT-construct with isoprenaline (10 microM), forskolin (10 microM), forskolin (10 microM) plus IBMX (10 microM) or dibutyryl-cAMP (1 mM) for 24 h induced an increase in CAT activity to 139 +/- 12% (n = 9), 211 +/- 18% (n = 12), 256 +/- 20% (n = 5) and 198 +/- 28% (n = 7) of unstimulated values, respectively. We conclude: 1) In chicken cardiomyocytes a sequence element of 52 bp between -85 and -33 bp is necessary to provide basal Gi alpha-2 promotor activity. 2) Elevation of cAMP has a stimulatory effect on the human Gi alpha-2 promotor, thereby offering a mechanism for beta-adrenoceptor-mediated increases in Gi alpha-2 in the heart.

Animals↗
PubMed1995

Laparoscopic reconstruction of gastroesophageal anatomy for the treatment of reflux disease.

This paper presents the technique and results of an operation that restores normal anatomical and physiological antireflux mechanisms for the treatment of gastroesophageal reflux disease (GERD). The Hill procedure was modified beginning in 1973, evolving into an operation that has been standard in our practice since 1987. Major changes included total fixation of the abdominal esophagus and elimination of phrenoesophageal bundle plication. We began performing the procedure laparoscopically in 1991 and simultaneously began a study to look at our results. This is the first report of the first 44 patients operated on from October 1991 through March 1994. There was one operative complication. Mean follow-up was 14 months. One patient was lost to follow-up and one patient died. There were no long-term side effects. A Visick grading scale was designed to categorize results. Forty graded satisfactory (95%) and two unsatisfactory.

Adolescent↗
PubMed1995

Pseudohypoparathyroidism type Ib is not caused by mutations in the coding exons of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene.

Pseudohypoparathyroidism type Ib (PHP-Ib) is thought to be caused by a PTH/PTH-related peptide (PTHrP) receptor defect. To search for receptor mutations in genomic DNA from 17 PHP-Ib patients, three recently isolated human genomic DNA clones were further characterized by restriction enzyme mapping and nucleotide sequencing across intron/exon borders. Regions including all 14 coding exons and their splice junctions were amplified by polymerase chain reaction, and the products were analyzed by either temperature gradient gel electrophoresis or direct nucleotide sequencing. Silent polymorphisms were identified in exons G (1 of 17), M4 (1 of 17), and M7 (15 of 17). Two base changes were found in introns, 1 at the splice-donor site of the intron between exons E2 and E3 (1 of 17) and the other between exons G and M1 (2 of 17). Total ribonucleic acid from COS-7 cells expressing minigenes with or without the base change between exons E2 and E3 showed no difference by either Northern blot analysis or reverse transcriptase-polymerase chain reaction. Radioligand binding was indistinguishable for both transiently expressed constructs. A missense mutation (E546 to K546) in the receptor's cytoplasmic tail (3 of 17) was also found in 1 of 60 healthy individuals, and PTH/PTHrP receptors with this mutation were functionally indistinguishable from wild-type receptors. PHP-Ib thus appears to be rarely, if ever, caused by mutations in the coding exons of the PTH/PTHrP receptor gene.

Adolescent↗
PubMed1994

Inhibition of adenylate cyclase activity by galanin in rat insulinoma cells is mediated by the G-protein Gi3.

Galanin inhibits adenylate cyclase activity and insulin secretion and modulates ion channels in pancreatic beta-cells through pertussis-toxin-sensitive G-protein(s). Antibodies directed against the C-terminal region of specific G-protein alpha-subunits were used to determine which G-protein(s) couple galanin receptors to inhibition of adenylate cyclase in the rat insulinoma cell line RINm5F. Preincubation of membranes with EC antibody (anti-alpha i3) decreased the inhibition of forskolin-stimulated adenylate cyclase activity by galanin (100 nM) by 45% compared with control IgG (P < 0.05) whereas preincubation with AS (anti-alpha i1, alpha i2) or GO (anti-alpha o) antibodies had no significant effect. To confirm these results, RINm5F cells were exposed intermittently over a 4-day period to phosphorothioate oligodeoxynucleotides that were either sense or antisense to alpha i1, alpha i2, alpha i3 or alpha o. Oligodeoxynucleotides antisense to alpha i2, alpha i3 and alpha o specifically decreased the levels of the targeted alpha-subunit in membranes. alpha i1 was undetectable in these cells. Inhibition of adenylate cyclase activity by galanin was largely abolished in membranes from cells exposed to the oligodeoxynucleotide antisense to alpha i3, whereas all other oligodeoxynucleotides had no significant effect on this pathway. Indirect immunofluorescence and immunoblotting of specific membrane fractions with EC antibody show significant localization of alpha i3 to intracellular membrane compartments. These results suggest that Gi3 is the G protein that couples galanin receptors to inhibition of adenylate cyclase activity in RINm5F cells.

Adenylate Cyclase Toxin↗
PubMed1994

An activating Gs alpha mutation is present in fibrous dysplasia of bone in the McCune-Albright syndrome.

McCune-Albright syndrome (MAS) is a sporadic disease characterized by polyostotic fibrous dysplasia, café-au-lait spots, and multiple endocrinopathies. The etiology of fibrous dysplasia is unknown. Activating mutations of codon 201 in the gene encoding the alpha-subunit of Gs, the G-protein that stimulates adenylyl cyclase, have been found in all affected MAS tissues that have been studied. Initial attempts to amplify DNA from decalcified paraffin-embedded bone specimens from MAS patients were unsuccessful. Therefore, we analyzed DNA from frozen surgical bone specimens from five MAS patients using polymerase chain reaction and allele-specific oligonucleotide hybridization. Most of the cells in four specimens of dysplastic bone contained a heterozygous mutation encoding substitution of Arg201 of Gs alpha with His, but the mutation was barely detectable in peripheral blood specimens from the patients. Only a small amount of mutant allele was detected in a specimen of normal cortical bone from the fifth patient, although this patient had a high proportion of mutation in other, affected tissues. The mosaic distribution of mutant alleles is consistent with an embryological somatic cell mutation of the Gs alpha gene in MAS. The presence of an activating mutation of Gs alpha in osteoblastic progenitor cells may cause them to exhibit increased proliferation and abnormal differentiation, thereby producing the lesions of fibrous dysplasia.

Adenylyl Cyclases↗
PubMed1993

G protein mutations in human disease.

The heterotrimeric G proteins couple cell-surface receptors for extracellular signals to intracellular effectors that generate second messengers. Abnormal G protein signalling, resulting from posttranslational modifications by bacterial toxins, altered gene expression, or gene mutations, may lead to diverse biological consequences. Mutations within G protein alpha subunit genes that lead to either constitutive activation or loss of function have been identified. Such G protein mutations play a role in the pathogenesis of several human diseases, including sporadic endocrine tumors, McCune-Albright syndrome, and Albright hereditary osteodystrophy.

Animals↗
PubMed1993

Severe endocrine and nonendocrine manifestations of the McCune-Albright syndrome associated with activating mutations of stimulatory G protein GS.

McCune-Albright syndrome (MCAS) is a sporadic disease classically including polyostotic fibrous dysplasia, café au lait spots, sexual precocity, and other hyperfunctional endocrinopathies. An activating missense mutation in the gene for the alpha subunit of GS, the G protein that stimulates cyclic adenosine monophosphate formation, has been reported to be present in these patients. The mutation is found in variable abundance in different affected endocrine and nonendocrine tissues, consistent with the mosaic distribution of abnormal cells generated by a somatic cell mutation early in embryogenesis. We describe three patients with MCAS who had profound endocrine and nonendocrine disease and who died in childhood. Two of the patients were severely ill neonates whose complex symptoms did not immediately suggest MCAS. A mutation of residue Arg201 of GS alpha was found in affected tissues from all three children. A review of the literature and unpublished case histories emphasizes the existence of other patients with severe and unusual clinical manifestations. We conclude that the manifestations of MCAS are more extensive than is generally appreciated, and may include hepatobiliary disease, cardiac disease, other nonendocrine abnormalities, and sudden or premature death.

Adolescent↗
PubMed1992

A heterozygous 4-bp deletion mutation in the Gs alpha gene (GNAS1) in a patient with Albright hereditary osteodystrophy.

Several heterozygous mutations within the gene encoding the alpha-subunit of Gs (GNAS1), the G protein that stimulates adenylyl cyclase, have been previously identified in patients with Albright hereditary osteodystrophy (AHO). We have now identified a fourth GNAS1 mutation from an AHO patient. Amplification by the polymerase chain reaction (PCR) of a genomic fragment encompassing GNAS1 exons 7 and 8 from one patient resulted in a product with aberrant migration on nondenaturing polyacrylamide and agarose gels. Direct DNA sequencing identified a 4-bp deletion in one allele of exon 7 encoding a frameshift with a premature stop codon. Analysis of lymphocyte RNA by reverse transcription-PCR and direct sequencing showed that the GNAS1 allele bearing the mutation is not expressed as mRNA. Consistent with this, Northern analysis revealed an approximate 50% deficiency in steady-state levels of GNAS1 mRNA. These findings further illustrate the heterogeneity of GNAS1 gene defects in AHO.

Amino Acid Sequence↗