Biomedical subjects

H Shapira

Publications and source records attributed to H Shapira.

At least 19 recordsLinked to original sources

PubMed2004

Expression of tetraspanins in peripheral blood leukocytes: a comparison between normal and infectious conditions.

The role of tetraspanins is undefined, despite their detection in diverse cell types and functions. This study addresses the characterization of tetraspanin expression levels in normal peripheral blood leukocytes (PBL) and in patients with bacterial infection. Membranal and cytoplasmic expression of CD9, CD53, CD63, CD81, CD82 and CD151 in polymorphonuclears (PMN), monocytes, B and T lymphocytes was assessed using flow cytometry. Results suggested that for normal PBL, PMN are distinguished by dominant cytoplasmic CD63; monocytes and B cells prevailingly express CD53; CD82 is primarily expressed on T-cell membranes. However, a major trend of downregulation was demonstrated for the examined tetraspanins, except CD63, in all patients' PBL subtypes. Therefore, tetraspanin modulation in infections may be attributed to elevated leukocyte motility in immune reactions and this is compatible with the previous publications of tetraspanins as metastasis suppressors. This work represents the first comprehensive baseline of tetraspanin expression in normal PBL and in infectious disorders.

Adolescent↗

PubMed2001

Postpartum chills phenomenon: is it a feto-maternal transfusion reaction?

OBJECTIVE: To examine the theory that the postpartum shivering phenomenon is related to feto-maternal bleed during the third stage of labor. METHODS: One hundred laboring low-risk women who had a normal vaginal delivery were observed for the presence of postpartum chills. The duration of the first and second stages of labor changes in body temperature, maternal and fetal blood types and the use of epidural anesthesia were recorded. Following the delivery maternal blood was examined for the presence of fetal red blood cells using the Kleihauer-Betke stain. RESULTS: Complete data was available in 97 patients. Post-partum chills occurred in 31 of them (32%). Women with and without chills were similar in their maternal and gestational age, the use of epidural anesthesia, and length of second stage of labor. Women with chills delivered smaller babies but the difference did not reach significance. Maternal-fetal blood group incompatibility was significantly more common among shivering than non-shivering women (48% vs. 20% respectively, p=0.006). Kleihauer-Betke test was positive in 11 women. The only two women in this group who experienced chills had maternal-fetal blood group incompatibility. CONCLUSION: Post-partum chills are a common phenomenon. It may be the clinical presentation of feto-maternal transfusion reaction. The small number of positive Kleihauer-Betke tests may reflect its low sensitivity in the detection of small feto-maternal bleeds.

ABO Blood-Group System↗

PubMed1998

Galpha14 and Galphaq mediate the response to trypsin in Xenopus oocytes.

Xenopus oocytes respond to trypsin with a characteristic chloride current, virtually indistinguishable from responses mediated by a large number of native and expressed G protein-coupled receptors. We studied the involvement of G proteins of the Galphaq family as possible mediators of this and other G protein-coupled receptor-mediated responses in Xenopus oocytes. We have cloned the third member of the Galphaq family, Xenopus Galpha14, in addition to the previously cloned Xenopus Galphaq and Galpha11 (Shapira, H., Way, J., Lipinsky, D., Oron, Y., and Battey, J. F. (1994) FEBS Lett. 348, 89-92). Amphibian Galpha14 is 354 amino acids long and is 93% identical to its mammalian counterpart. Based on the Galpha14 cDNA sequence, we designed a specific antisense DNA oligonucleotide (antiGalpha14) that, together with antiGalphaq and antiGalpha11, was used in antisense depletion experiments. 24 h after injection into oocytes, either antiGalphaq or antiGalpha14 reduced the response to 1 microg/ml trypsin by 70%, whereas antiGalpha11 had no effect. A mixture of antiGalphaq and antiGalpha14 virtually abolished the response. These data strongly suggest that Galphaq and Galpha14 are the exclusive mediators of the trypsin-evoked response in Xenopus oocytes. Similar experiments with the expressed gastrin-releasing peptide receptor and muscarinic m1 receptor revealed the coupling of Galphaq and Galpha11, but not Galpha14, to these receptors in oocytes. These results confirm the hypothesis that endogenous members of the Galphaq family discriminate among different native receptors in vivo.

Amino Acid Sequence↗

PubMed1998

A human gene encodes a putative G protein-coupled receptor highly expressed in the central nervous system.

The mammalian bombesin (Bn)-like neuropeptide receptors gastrin-releasing peptide receptor (GRP-R) and neuromedin B receptor (NMB-R) transduce a variety of physiological signals that regulate secretion, growth, muscle contraction, chemotaxis and neuromodulation. We have used reverse transcription-polymerase chain reaction (PCR) to isolate a cDNA from human brain mRNA, GPCR/CNS, that encodes a putative G protein-coupled receptor (GPCR) based upon the presence of the paradigmatic seven heptahelical transmembrane domains in its predicted amino acid sequence. Analysis of the deduced protein sequence of GPCR/CNS reveals this putative receptor to be 98% identical to the deduced amino acid sequence of a recently reported gene product and minimally identical (approximately 23%) to both murine GRP-R and human endothelin-B (ET-B) receptor. Our deduced protein sequence differs at 12 positions, scattered throughout the open reading frame, relative to the original sequence. A 3.7 kb GPCR/CNS mRNA species is expressed in vivo in a tissue-specific manner, with highest levels detected in brain and spinal cord, lower levels found in testis, placenta and liver, but no detectable expression observed in any other tissue. Analysis of GPCR/CNS genomic clones reveals that the human gene contains one intron that is about 21 kb in length that divides the coding region into two exons and maps to human chromosome 7q31. No specific binding is observed with either a newly identified ligand (DTyr6, beta Ala11, Phe13, Nle14]Bn-(6-14)) having high affinity for all Bn receptor subtypes or Bn after GPCR/CNS is stably expressed in fibroblasts. No elevation in inositol trisphosphate is observed after the application of micromolar levels of either DPhe6, beta Ala11, Phe13, Nle14]Bn-(6-14) or Bn, a concentration of agonist known to activate all four known Bn receptor subtypes. When GPCR/CNS is expressed in Xenopus oocytes, no activation of the calcium-dependent chloride channel is detected despite the addition of micromolar levels of Bn peptide agonists. We conclude that the natural ligand for this receptor is none of the known naturally occurring Bn-like peptides and the true agonist for GPCR/CNS remains to be elucidated.

3T3 Cells↗

PubMed1996

Pseudohypoparathyroidism, a novel mutation in the betagamma-contact region of Gsalpha impairs receptor stimulation.

Pseudohypoparathyroidism, type Ia (PHP-Ia), is a dominantly inherited endocrine disorder characterized by resistance to hormones that act by stimulating adenylyl cyclase. It is caused by inheritance of an autosomal mutation that inactivates the alpha subunit (alphas) of Gs, the stimulatory regulator of adenylyl cyclase. In three members of a family, the PHP-Ia phenotype is associated with a mutation (R231H) that substitutes histidine for an arginine at position 231 in alphas. We assessed signaling function of alphas-WT versus alphas-R231H transiently transfected in HEK293 cells. Hormone receptor-dependent stimulation of cAMP accumulation in cells expressing alphas-R231H is reduced by approximately 75% in comparison to cAMP accumulation in cells expressing alphas-WT. A second mutation, alphas-R201C, inhibits the GTPase turnoff reaction of alphas, thus producing receptor-independent stimulation of cAMP accumulation. The double mutant, alphas-R231H/R201C, stimulates cAMP accumulation almost as well (approximately 80%) as does alphas-R201C itself, indicating that the R231H mutation selectively impairs receptor-dependent signaling. In three-dimensional structures of G protein heterotrimers, Arg-231 is located in a region, switch 2, that is thought to interact with the betagamma subunit rather than with the hormone receptor. Thus, the R231H phenotype suggests that switch 2 (perhaps in concert with betagamma) mediates G protein activation by receptors at a site distant from the receptor-G protein contact surface.

Adenylyl Cyclases↗

PubMed1996

Pseudohypoparathyroidism type Ia: two new heterozygous frameshift mutations in exons 5 and 10 of the Gs alpha gene.

Pseudohypoparathyroidism type Ia (PHP-Ia) is a hereditary disease characterized by resistance to PTH and other hormones that act via cAMP. Patients have deficient activity of Gs alpha, the alpha subunit of the G protein, which couples hormone receptors to stimulation of adenylate cyclase. We describe two new mutations discovered in two sporadic patients with PHP-Ia. Using genomic DNA, we have amplified exons 2-13 of the Gs alpha gene (GNAS1) by PCR, and sequenced the resulting products. Both patients had Albright's hereditary osteodystrophy, resistance to multiple hormones, and deficient Gs alpha activity. In the first patient, a deletion of a C in exon 5 at codon 115 was found. In the second patient, an insertion of a C in exon 10 at codon 267 was detected. Both these heterozygous mutations cause frameshift, and predict decreased production of Gs alpha. This report adds two new Gs alpha mutations to the known ten mutations recently described.

Adolescent↗

PubMed1996

Agonist-evoked calcium efflux from a functionally discrete compartment in Xenopus oocytes.

Agonist-induced calcium (Ca) mobilization is accompanied by Ca efflux, presumably reflecting the rise in Ca concentration at the cytosolic surface of the cell membrane. We studied the relationship between Ca efflux and intracellular Ca mobilization in Xenopus oocytes. Elevation of cytosolic Ca by a direct injection of 1 nmol 45CaCl2 resulted in a typical Ca-activated chloride current, but not in 45Ca efflux. This demonstrated that a Ca rise at the cytoplasmic surface of the membrane is not sufficient to produce an increased efflux. Co-injection of inositol 1,4,5-trisphosphate (InsP3), to prevent rapid Ca sequestration, also failed to cause Ca efflux. Smaller amounts of labelled Ca (0.05 nmol) equilibrated with Ca stores in a time-dependent pattern with an optimum at 2 h after injection. In contrast, Ca taken up from the medium was immediately available for agonist- or InsP3-induced efflux. Emptying the agonist-sensitive stores with thapsigargin (TG) did not affect chloride currents induced by Ca injection, indicating that these currents were due to direct elevation of Ca at the plasma membrane, rather than Ca-induced Ca release from InsP3-sensitive stores. Agonist-induced depletion of Ca stores enhanced uptake from the extracellular medium and the subsequent release of the label by an agonist. Similar protocol when the label was injected into the oocytes, failed to affect agonist induced efflux. We suggest that, under physiological conditions, agonist-dependent Ca extrusion or uptake in oocytes is executed exclusively via a functionally restricted compartment, which is closely associated with both agonist-sensitive Ca stores and the plasma membrane.

Acetylcholine↗

PubMed1996

Familial Albright's hereditary osteodystrophy with hypoparathyroidism: normal structural Gs alpha gene.

Albright's hereditary osteodystrophy (AHO) is a characteristic skeletal phenotype, including short stature, obesity, round face, and brachydactyly. AHO appears in patients with pseudohypoparathyroidism (PHP) who have resistance to PTH and in their eumetabolic family members who have pseudopseudohypoparathyroidism (PPHP). The differential diagnosis of AHO in families without PHP includes brachydactyly E, whose existence as a distinct entity has been questioned. We studied a patient with familial AHO who presented with hypocalcemia. To our surprise, PTH levels were low, and the response to PTH administration was normal. This is the first case of familial AHO with hypoparathyroidism. The proband's family included 22 affected subjects spanning 3 generations, who had variable degrees of AHO manifestations, with an autosomal dominant inheritance trait. The metacarpophalangeal pattern profile was typical of that of PHP-PPHP. As deficient activity and inactivating mutations of Gs alpha were described in PHP as well as in PPHP, we measured the biological activity of Gs in family members, which was normal. To exclude subtle abnormalities in the Gs alpha gene, we sequenced the entire coding region of Gs alpha in the propositus, which was normal. We conclude that hypocalcemia should be adequately evaluated even in the presence of familial AHO, and that familial AHO can occur with a normal coding structural Gs alpha gene. Identification of the molecular defect in familial AHO without PHP will shed light on the pathogenesis of AHO in general.

Adult↗

PubMed1994

Neuromedin B receptor, expressed in Xenopus laevis oocytes, selectively couples to G alpha q and not G alpha 11.

G-proteins of the q family have been implicated as mediators of bombesin receptors action. We cloned Xenopus G alpha q and G alpha 11 and specifically disrupted the synthesis of either protein with selective antisense oligonucleotides. G alpha q antisense inhibited responses mediated by neuromedin B receptor (NMB-R) by 74%, though not by gastrin-releasing peptide receptor (GRP-R). G alpha 11 antisense had little effect on either GRP-R- or NMB-R-mediated responses. This suggests that NMB-R couples to G alpha q, and that GRP-R and NMB-R show distinct G-protein coupling preferences in the Xenopus oocyte.

Amino Acid Sequence↗

PubMed1993

The fifth transmembrane segment of the neuromedin B receptor is critical for high affinity neuromedin B binding.

The two bombesin receptor subtypes, neuromedin B (NMB-R) and gastrin releasing peptide (GRP-R) receptors, bind their respective ligands with high affinity. To identify molecular components mediating high affinity NMB binding, four mutant receptors were constructed, in which different parts of the NMB-R were replaced with the corresponding regions of the GRP-R. When stably expressed in Balb 3T3 fibroblasts, all four NMB-R/GRP-R chimeras were functional and showed NMB-induced stimulation of inositol phosphate (IP) formation. Results of 125I-[D-Tyr0]NMB displacement assays using unlabeled NMB for competition indicated that high affinity NMB binding was determined by amino acid sequences in transmembrane domain V (TM-V) of the NMB-R. To identify which amino acid(s) in TM-V of NMB-R contributed to high affinity NMB binding, four additional NMB-R mutants were constructed where non-conserved amino acids in TM-V of NMB-R were replaced by the corresponding GRP-R amino acids. Three of the mutations, TyrPheLeu220-222-->PheTyrVal, Ile230-->Val, and His234-->Phe, did not affect high affinity NMB binding. The Ile216-->Ser substitution, however, abolished high affinity NMB binding and severely impaired the ability of the mutant receptor to stimulate NMB-dependent inositol phosphate formation. These results suggest that ILe216 in TM-V of NMB-R may be critical for high affinity NMB binding.

3T3 Cells↗

PubMed1993

BRS-3: a novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells.

The bombesin (BN)-like peptides mediate a diverse spectrum of biological activities and have been implicated as autocrine growth factors in the pathogenesis and progression of some human small cell lung carcinoma tumors. Previously, two mammalian BN-like peptide receptor subtypes, gastrin-releasing peptide receptor and neuromedin-B receptor, have been cloned and characterized. In this study, we have isolated and characterized human genomic and complementary DNA (cDNA) clones encoding a new BN-like peptide receptor subtype, BN receptor subtype 3 (BRS-3). Expression of BRS-3 cDNA in Xenopus oocytes encodes a functional receptor that is specifically activated by BN-like peptides. Chromosome mapping studies indicate that the BRS-3 gene is located on human chromosome X. BRS-3 mRNA expression in rat tissues is limited to secondary spermatocytes in testis. In contrast, BRS-3 mRNA is widely expressed in a panel of human cell lines from all histological types of lung carcinoma. These results suggest a role for BN-like peptides and their receptors in mammalian reproductive physiology and also indicate that BRS-3 could serve as a potential therapeutic target for human lung carcinoma.

Amino Acid Sequence↗

PubMed1992

Cloning and expression of a novel angiotensin II receptor subtype.

Angiotensin II (AII) is a major regulator of cardiovascular function and fluid homeostasis. Recently, the cDNA for an AII receptor (AT1) was cloned from rat smooth muscle and bovine adrenal. To search for AII receptor subtypes, we amplified rat adrenal cortex cDNA by PCR using primers based on the AT1 receptor. The product was distinct from the AT1 receptor as indicated by restriction enzyme analysis and DNA sequencing. A full-length cDNA clone (2.2 kilobase pairs) encoding a novel AII receptor (AT3) was obtained by screening an adrenal cortex library. The AT3 cDNA encodes a Mr 40,959 protein with 95% amino acid identity to the rat smooth muscle receptor, but the overall nucleotide similarity is 71% due to low homology in the 5'- (58%) and 3'- (62%) untranslated regions. Expressed AT3 receptors in Xenopus oocytes and COS-7 cells mediate agonist-induced Ca2+ mobilization but are pharmacologically distinct from the AT1 receptors. AT3 mRNA is most abundant in the adrenal cortex and pituitary and differs from AT1 mRNA in its tissue distribution. The structural features of the AT3 receptor, including two additional potential phosphorylation sites for protein kinase C, could be related to the distinctive binding properties of the adrenal and vascular receptors and to their differential regulation during altered sodium intake.

Amino Acid Sequence↗

PubMed1992

Purification, molecular cloning, and functional expression of the cholecystokinin receptor from rat pancreas.

The cholecystokinin (CCK) family of peptides and their receptors are widely distributed throughout the gastrointestinal and central nervous systems where they regulate secretion, motility, growth, anxiety, and satiety. The CCK receptors can be subdivided into at least two subtypes, CCKA and CCKB on the basis of pharmacological studies. We report here the purification of the CCKA receptor to homogeneity from rat pancreas by using ion-exchange and multiple affinity chromatographic separations. This allowed partial peptide sequencing after chemical/enzymatic cleavage and synthesis of degenerate oligonucleotide primers. These primers were used for initial cloning of the cDNA from rat pancreas by PCR. The predicted protein sequence of the cDNA clone contained the five partial peptide sequences obtained from the purified protein. Seven putative transmembrane domains suggest its membership in the guanine nucleotide-binding regulatory protein-coupled receptor superfamily. In vitro transcripts of the cDNA clone were functionally expressed in Xenopus oocytes and displayed the expected agonist and antagonist specificity.

Amino Acid Sequence↗

PubMed1992

Inositol trisphosphate may access calcium from stores not coupled to muscarinic receptors in Xenopus oocytes.

Oocytes of a large fraction of Xenopus females exhibit a complex response to acetylcholine (ACh) consisting of rapid, transient and prolonged, slow chloride currents. Frequent consecutive challenges or a single prolonged challenge with ACh result in a marked decrease in response amplitudes, i.e. refractoriness. In ACh-refractory oocytes, the response to injected inositol 1,4,5-trisphosphate (InsP3), the intracellular mediator of the ACh response, is not affected. Similarly, InsP3-evoked responses were obtained in oocytes that lacked muscarinic response or that lost their responsiveness as a result of progesterone-induced maturation. To investigate the mechanism of this phenomenon, we have depleted intracellular calcium stores by repeated challenges with ACh in calcium-free medium. Disappearance of the ACh response through depletion of the ACh-coupled calcium store did not prevent a subsequent response to InsP3. These results imply that InsP3 can mobilize calcium from other stores, not depleted by previous exposure to ACh. This finding is further reinforced by our results that demonstrate that ACh causes 45Ca efflux in responsive oocytes, while InsP3 in supramaximal concentrations does not induce 45Ca efflux. Indeed, InsP3 can induce 45Ca efflux only when more than 2 pmol/oocyte is injected. This is also the concentration of InsP3 that desensitizes the InsP3 response. These data suggest that InsP3 also releases cellular calcium from stores different from those mobilized by ACh.

Acetylcholine↗

PubMed1992

Molecular genetic analysis of two distinct receptors for mammalian bombesin-like peptides.

The mammalian bombesin-like peptides are known to be growth factors for certain cells with high-affinity bombesin receptors and have been implicated as autocrine growth factors influencing the pathogenesis and progression of a subset of human small-cell lung carcinomas. Thus, antagonists that interfere with bombesin receptor-ligand interaction might prove to be of value in treatment of gastrin-releasing peptide (GRP)-responsive tumors. A precise definition of the structure and properties of the bombesin receptors found on human lung cancer cells would provide important information for the design and rational application of such antagonists. Recently, we isolated cDNA clones encoding two distinct receptors for the mammalian bombesin-like peptides, GRP, and neuromedian B (NMB). The two receptors show 56% amino acid identity, encode seven putative transmembrane domains, and are members of the G-protein-coupled receptor superfamily. Ligand-binding studies show that while both receptors can be activated by either GRP or NMB, one receptor has a higher affinity for GRP than for NMB (GRP-R), while the other has a higher affinity for NMB than for GRP (NMB-R). A different spectrum of antagonists is needed to block responses from the two different receptors. These studies indicate that it will be critical in future studies to define which bombesin receptor subtypes are present on a given tumor to optimize the potential therapeutic benefit of antagonists in blocking growth.

Amino Acid Sequence↗

PubMed1991

Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells.

Bombesin-like peptides have been implicated as autocrine growth factors influencing the pathogenesis and progression of some human lung carcinoma cells. To determine the pharmacologic and structural properties of the bombesin receptors expressed in human lung carcinoma cells, cDNA clones encoding a human gastrin-releasing peptide receptor (GRP-R) and a pharmacologically distinct neuromedin-B preferring bombesin-receptor (NMB-R) were isolated from a human small cell lung carcinoma cell line (NCI-H345). After expression in Xenopus oocytes, a GRP-R-specific antagonist was effective in blocking responses elicited from the cloned GRP-R, but not the NMB-R. Both GRP-R and NMB-R mRNA expression was detected at varying levels in a panel of human lung cancer cell lines. These results indicate heterogeneity of bombesin receptor subtypes exists in human lung carcinoma cells and should be considered in the design of bombesin receptor antagonists intended to inhibit tumor cell growth.

Amino Acid Sequence↗

PubMed1991

Plasmin cleavage of vitronectin. Identification of the site and consequent attenuation in binding plasminogen activator inhibitor-1.

Plasmin is shown to specifically cleave vitronectin at the Arg361-Ser362 bond, 18 amino acid residues upstream from the site of the endogenous cleavage which gives rise to the two-chain form of vitronectin in plasma. The cleavage site is established using the exclusive phosphorylation of Ser378 with protein kinase A. As a result of the plasmin cleavage, the affinity between vitronectin and the type-1 inhibitor of plasminogen activator (PAI-1) is significantly reduced. This cleavage is stimulated by glycosaminoglycans, which are known to anchor vitronectin to the extracellular matrix. A mechanism is proposed through which plasmin can arrest its own production by feedback signalling, unleashing PAI-1 from the immobilized vitronectin found in the vascular subendothelium, which becomes exposed at the locus of a hemostatic event.

Amino Acid Sequence↗

PubMed1991

Distinguishing bombesin receptor subtypes using the oocyte assay.

Physiological responses to mammalian bombesin-like peptides were studied in Xenopus oocytes injected with mRNA isolated from Swiss 3T3 cells and rat esophagus in order to identify and characterize bombesin receptor subtypes. Both groups respond similarly to either gastrin releasing peptide or neuromedin B, but only the response to neuromedin B in oocytes expressing the esophagus mRNA is not blocked by a specific gastrin releasing peptide receptor antagonist, des-Met-[D-Phe6]Bn(6-13) ethyl ester. Complete desensitization of gastrin releasing peptide-evoked responses in oocytes expressing esophagus mRNA does not abolish neuromedin B-evoked responses. A single application of neuromedin B abolishes responses to subsequently applied gastrin releasing peptide in oocytes expressing esophagus, but not Swiss 3T3, mRNA. RNA blot hybridization studies using a Swiss 3T3 gastrin releasing peptide receptor cDNA probe show no detectable hybridization in esophagus mRNA samples. These data suggest that a gastrin releasing peptide receptor is expressed in the esophagus and that it is distinct from that expressed in Swiss 3T3 cells and may represent a third subtype of mammalian bombesin receptor.

Animals↗