Machinability Analysis and Optimization in Wire EDM of Medical Grade NiTiNOL Memory Alloy.

NiTiNOL (Nickel-Titanium) shape memory alloys (SMAs) are ideal replacements for titanium alloys used in bio-medical applications because of their superior properties like shape memory and super elasticity. The machining of NiTiNOL alloy is challenging, as it is a difficult to cut material. Hence, in the current research the experimental studies on machinability aspects of medical grade NiTiNOL SMA during wire electric discharge machining (WEDM) using zinc coated brass wire as electrode material have been carried out. Pulse time (Ton), pause time (Toff), wire feed (WF), and servo voltage (SV) are chosen as varying input process variables and the effects of their combinational values on output responses such as surface roughness (SR), material removal rate (MRR), and tool wear rate (TWR) are studied through response surface methodology (RSM) based developed models. Modified differential evolution (MDE) optimization technique has been developed and the convergence curve of the same has been compared with the results of differential evolution (DE) technique. Scanning electron microscopy (SEM) and energy dispersive X-ray spectrography (EDS) analysis are carried out to study the surface morphology of the machined alloy. SV is found to be more influential process parameter for achieving better MRR with minimal SR and TWR, followed by Ton, Toff, and WF. The WF has good impact on reduced SR and TWR responses and found to be least significant in maximizing MRR.

A Study about Co-Infection of Fungal Pathogens in Active Tuberculosis Patients.

The diagnosis of mycotic lung infection in pulmonary TB patients remains misdiagnosed because of its non-specific clinical manifestations which mimics the symptoms of TB. Physicians have to rely on the investigation but as radiology and pathology cannot probe the appropriate diagnosis, conventional microbiology or PCR testing continue as an essential mode for the diagnosis. In developing country like India PCR is not cost effective. Thus, Direct microscopy by KOH (10%), Gram's staining & Culture remains only option for identification. A three-year cross-sectional study was carried out in the Department of Microbiology, Maharishi Markandeshwar Institute of Medical Science & Research, Mullana, India from August 2015 to August 2018. On 300 LED positive sputum samples collected from previously treated cases of pulmonary TB. Early morning sputum was collected and subjected to KOH 10%, Gram's staining afterwards cultured on Sabouraud Dextrose Agar and species identification was done by LPCB preparation. In 300 LED smear positive samples, the dominant pathogens were C. albicans (43.3%), followed by C. non-albicans (26.7%), A. fumigatus (21.7%) etc. ATT administration for 5-8 months' duration of illness showed highest fungal infection (45%) and maximum growth of fungus was seen in the Autumn season (45%). The co-occurrence of fungi with tubercle bacteria adds fatal consequences thus routine screening is recommended for proper diagnosis and early treatment of mycotic infection in the patients of Pulmonary TB on ATT.

Semaphorin 3A upregulates FOXO 3a-dependent MelCAM expression leading to attenuation of breast tumor growth and angiogenesis.

Semaphorin 3A (Sema 3A), a member of semaphorin family, serves as a guidance clue during embryonic development and is known as a candidate tumor suppressor that attenuates breast tumor progression by binding with its co-receptor, neuropilin-1 (NRP-1). However, the underlying mechanism by which Sema 3A suppresses breast tumor growth is still unexplored. In this study, we report that Sema 3A regulates phosphorylation and nuclear translocation of phosphatase and tensin homolog (PTEN) and FOXO 3a. Moreover, Sema 3A controls NRP-1-mediated PTEN-dependent FOXO 3a activation. Overexpression of PTEN and FOXO 3a enhances Sema 3A-induced attenuation of breast cancer cell migration. Chromatin immunoprecipitation and electrophoretic mobility shift assay data revealed that FOXO 3a regulates MelCAM at the transcriptional level. Furthermore, Sema 3A induces NRP-1-mediated MelCAM expression through PTEN and FOXO 3a. The data also showed that vascular endothelial growth factor-induced angiogenesis is inhibited by Sema 3A. Loss of or gain in function study revealed that Sema 3A modulates phosphorylation of PTEN and FOXO 3a and expression of MelCAM, leading to suppression of tumor growth and angiogenesis using in vivo mice model. Clinical specimen analysis revealed that reduced expression of Sema 3A and p-PTEN are correlated with enhanced breast cancer progression, further strengthening our in vitro and in vivo findings. Correlation of relapse-free survival of breast cancer patients (n=2878) with expression levels of Sema 3A, NRP-1, FOXO 3a and MelCAM were studied by Kaplan-Meier analysis. Statistical analysis revealed a close association between reduced expression of Sema 3A and MelCAM with that of poor patient's survival. Our study demonstrated a novel mechanism of regulation of tumor suppression by Sema 3A in coordination with a chain of tumor-suppressor genes, which in turn inhibits breast cancer cell migration, tumor growth and angiogenesis.

Hypoxia-driven osteopontin contributes to breast tumor growth through modulation of HIF1α-mediated VEGF-dependent angiogenesis.

Hypoxia is a salient feature of most solid tumors, and hypoxic adaptation of cancer cells has crucial implications in propagation of malignant clonal cell population. Osteopontin (OPN) has been identified as a hypoxia-responsive gene, but the mechanistic and regulatory role of OPN under hypoxia is less characterized. The present study identifies the existence of a positive inter-regulatory loop between hypoxia and OPN. We have shown that hypoxia induces OPN expression in breast cancer cells; however, the expression was found to be HIF1α independent. OPN enabled transcriptional upregulation of HIF1α expression both under normoxia and hypoxia, whereas stability of HIF1α protein in breast cancer cells remained unaffected. Moreover, we have shown that OPN induces integrin-linked kinase (ILK)/Akt-mediated nuclear factor (NF)-κB p65 activation leading to HIF1α-dependent vascular endothelial growth factor (VEGF) expression and angiogenesis in response to hypoxia. These in vitro data are biologically important as OPN expressing cells induce greater tumor growth and angiogenesis through enhanced expressions of proangiogenic molecules as compared with control. Immunohistochemical analysis of human breast cancer specimens revealed significant correlation between OPN and HIF1α but not HIF2α. Elevated expression of HIF1α and OPN was observed in pre-neoplastic and early stage infiltrating ductal carcinoma implicating the role of these proteins in neoplastic progression of breast cancer. Together, our results substantiate the prime role of OPN in cellular adaptation through ILK and NF-κB-mediated HIF1α-dependent VEGF expression in response to hypoxia that ultimately controls breast cancer progression and angiogenesis. Our study reinforces the fact that targeting OPN and its regulated signaling network hold important therapeutic implications.

Facial wrigglies: live extralymphatic filarial infestation in subcutaneous tissues of the head and neck.

We report a rare case of a 32-year-old male with live extralymphatic filarial infestation presenting as a facial subcutaneous soft-tissue swelling. To the best of our knowledge these imaging findings have not been previously reported in the head and neck region in the existing English language literature. Real-time high-resolution ultrasonography revealed a solitary well-defined subcutaneous cystic lesion over the right zygomatic arch. It showed multiple linear, echogenic, undulating structures exhibiting a persistent twirling motion during the examination. This typical ultrasonographic appearance was consistent with the filarial dance sign (FDS) of live adult filarial worms. Subsequent MRI confirmed the cystic and solitary nature of the lesion. Complete excision of the cyst was performed, which revealed intracystic straw-coloured fluid and multiple white-coloured adult worms within the lesion. Histopathological examination confirmed multiple adult filarial worms with surrounding reactive inflammatory changes. In an endemic region, identification of the FDS in any normal anatomical structure or abnormal swelling, however remote or unusual the location within the body, should strongly suggest the diagnosis of live active filarial infestation. In view of the increasing migratory trends in the global population, it is imperative for radiologists in all countries to be aware of the typical imaging findings of this disease to arrive at the correct diagnosis.

Analysis of sulfated peptides using positive electrospray ionization tandem mass spectrometry.

Presented is a method for analyzing sulfated peptides, and differentiating the post-translational modification (PTM) from its isobaric counterpart phosphorylation, using quadrupole time-of-flight (Qq/TOF) mass spectrometry (MS) and positive ion nanoelectrospray MS/MS. A set of commercially available sulfo- and phosphopeptide standards was analyzed via in-source dissociation and MS/MS to generate fragmentation signatures that were used to characterize and differentiate the two modifications. All of the phosphorylated peptides retained their +80 Da modifications under collision-induced decomposition (CID) conditions and peptide backbone fragmentation allowed for the site-specific identification of the modification. In sharp contrast, sulfated peptides lost SO3 from the precursor as the collision energy (CE) was increased until only the non-sulfated form of the peptide was observed. The number of 80 Da losses indicated the number of sulfated sites. By continuing to ramp the CE further, it was possible to fragment the non-sulfated peptides and obtain detailed sequence information. It was not possible to obtain site-specific information on the location of the sulfate moieties using positive ion MS/MS as none of the original precursor ions were present at the time of peptide backbone fragmentation. This method was applied to the analysis of recombinant human B-domain deleted factor VIII (BDDrFVIII), which has six well-documented sulfation sites and several potential phosphorylation sites located in two of the sulfated regions of the protein. Seven peptides with single and multiple +80 Da modifications were isolated and analyzed for their respective PTMs. The fragmentation patterns obtained from the BDDrFVIII peptides were compared with those obtained for the standard peptides; and in all cases the peptides were sulfated. None of the potential phosphorylation sites were found to be occupied, and these results are consistent with the literature.

Reversible inactivation of AT(2) angiotensin II receptor from cysteine-disulfide bond exchange.

Dithiothreitol (DTT) treatment of angiotensin II (Ang II) type 2 (AT(2)) receptor potentiates ligand binding, but the underlying mechanism is not known. Two disulfide bonds proposed in the extracellular domain were examined in this report. Based on the analysis of ligand affinity of cysteine (Cys, C) to alanine (Ala, A) substitution mutants, we provide evidence that Cys(35)-Cys(290) and Cys(117)-Cys(195) disulfide bonds are formed in the wild-type AT(2) receptor. Disruption of the highly conserved Cys(117)-Cys(195) disulfide bond linking the second and third extracellular segments leads to inactivation of the receptor. The Cys(35)-Cys(290) bond is highly sensitive to DTT. Its breakage results in an increased binding affinity for both Ang II and the AT(2) receptor-specific antagonist PD123319. Surprisingly, in the single Cys mutants, C35A and C290A, a labile population of receptors is produced which can be re-folded to high-affinity state by DTT treatment. These results suggest that the free -SH group of Cys(35) or Cys(290) competes with the disulfide bond formation between Cys(117) and Cys(195). This Cys-disulfide bond exchange results in production of the inactive population of the mutant receptors through formation of a non-native disulfide bond.

Ligand-independent signals from angiotensin II type 2 receptor induce apoptosis.

Conventional models of ligand-receptor regulation predict that agonists enhance the tone of signals generated by the receptor in the absence of ligand. Contrary to this paradigm, stimulation of the type 2 (AT(2)) receptor by angiotensin II (Ang II) is not required for induction of apoptosis but the level of receptor protein expression is critical. We compared Ang II-dependent and -independent AT(2) receptor signals involved in regulating apoptosis of cultured fibroblasts, epithelial cells and vascular smooth muscle cells. We found that induction of apoptosis-blocked by pharmacological inhibition of p38 mitogen-activated protein kinase and caspase 3-is a constitutive function of the AT(2) receptor. Biochemical and genetic studies suggest that the level of AT(2) receptor expression is critical for physiological ontogenesis and its expression is restricted postnatally, coinciding with cessation of developmental apoptosis. Re-expression of the AT(2) receptor in remodeling tissues in the adult is linked to control of tissue growth and regeneration. Therefore, we propose that overexpression of the AT(2) receptor itself is a signal for apoptosis that does not require the renin-angiotensin system hormone Ang II.

Angiotensin II type 1 receptor-function affected by mutations in cytoplasmic loop CD.

To explore peptide hormone-induced conformational changes, we attempted to engineer a metal-ion binding site between the cytoplasmic loops CD and EF in the angiotensin II type 1 (AT(1)) receptor. We constructed 12 double and six triple histidine mutant receptors, and tested the ability of each mutant and the wild-type to activate inositol phosphate (IP) production with and without ZnCl(2). Inhibition by ZnCl(2) in the double and triple His mutant receptors was not significant, but these mutations directly decreased the IP production. Systematic analysis of single His mutants demonstrated that the loop CD-mutants displayed 52-74% inhibition of IP production, whereas the loop EF-mutants did not affect IP production. These results indicate that the cytoplasmic loop CD-segment from Tyr(127) to Ile(130) is important for G(q/11) activation by the AT(1) receptor.

Agonist-induced phosphorylation of the angiotensin II (AT(1A)) receptor requires generation of a conformation that is distinct from the inositol phosphate-signaling state.

G protein-coupled receptors are thought to isomerize between distinct inactive and active conformations, an idea supported by receptor mutations that induce constitutive (agonist-independent) activation. The agonist-promoted active state initiates signaling and, presumably, is then phosphorylated and internalized to terminate the signal. In this study, we examined the phosphorylation and internalization of wild type and constitutively active mutants (N111A and N111G) of the type 1 (AT(1A)) angiotensin II receptor. Cells expressing these receptors were stimulated with angiotensin II (AngII) and [Sar(1),Ile(4),Ile(8)]AngII, an analog that only activates signaling through the constitutive receptors. Wild type AT(1A) receptors displayed a basal level of phosphorylation, which was stimulated by AngII. Unexpectedly, the constitutively active AT(1A) receptors did not exhibit an increase in basal phosphorylation nor was phosphorylation enhanced by AngII stimulation. Phosphorylation of the constitutively active receptors was unaffected by pretreatment with the non-peptide AT(1) receptor inverse agonist, EXP3174, and was not stimulated by the selective ligand, [Sar(1),Ile(4),Ile(8)]AngII. Paradoxically, [Sar(1),Ile(4), Ile(8)]AngII produced a robust ( approximately 85% of AngII), dose-dependent phosphorylation of the wild type AT(1A) receptor at sites in the carboxyl terminus similar to those phosphorylated by AngII. Moreover, internalization of both wild type and constitutive receptors was induced by AngII, but not [Sar(1),Ile(4),Ile(8)]AngII, providing a differentiation between the phosphorylated and internalized states. These data suggest that the AT(1A) receptor can attain a conformation for phosphorylation without going through the conformation required for inositol phosphate signaling and provide evidence for a transition of the receptor through multiple states, each associated with separate stages of receptor activation and regulation. Separate transition states may be a common paradigm for G protein-coupled receptors.

Role of transmembrane helix IV in G-protein specificity of the angiotensin II type 1 receptor.

G-protein activation by G-protein coupled receptors (GPCRs) is accomplished through proper interaction with the cytoplasmic loops rather than through sequence-specific interactions. However, the mechanism by which a specific G-protein is selected by a GPCR is not known. In the current model of GPCR activation, agonist binding modulates helix-helix interactions, which is necessary for fully determining G-protein specificity and stimulation of GDP/GTP exchange. In this study, we report that a single-residue deletion in transmembrane helix IV leads the angiotensin II type 1 (AT(1)) receptor chimera CR17 to retain GTP-sensitive high affinity for the agonist angiotensin II but results in complete inactivation of intracellular inositol phosphate production. The agonist dissociation profile of CR17 in the presence of guanosine 5'-3-O-(thio)triphosphate suggests that the activation-induced conformational changes of the chimeric receptor itself remain intact. Insertion of an alanine at position 149 (CR17triangle down149A) in this chimera rescued the inactive phenotype, restoring intracellular inositol phosphate production by the chimera. This finding suggests that in the wild-type AT(1) receptor the orientation of transmembrane helix IV-residues following Cys(149) is a key determinant for effectively distinguishing among various structurally similar G-proteins. The results emphasize that the contacts within the membrane-embedded portion of transmembrane helix IV in the AT(1) receptor is important for specific G-protein selection.

Angiotensin II type 1 and type 2 receptors bind angiotensin II through different types of epitope recognition.

OBJECTIVE: This study was designed to demonstrate that the principle of molecular recognition underlying high-affinity binding of angiotensin II to the type 2 (AT2) receptor is distinct from that of the type 1 (AT1) receptor. In general, the same functional pharmacophores in hormones are used to bind and activate different subtypes of cell surface receptors. However, the binding of angiotensin II to the AT2 receptor is distinct from that of the AT1 receptor. DESIGN AND METHODS: To systematically evaluate the effect of modification of angiotensin II side chains on binding to both the receptors, several analogs of angiotensin II were synthesized. Rat AT1 or AT2 receptors expressed in COS1 cell membranes were used to determine the affinity of analogs using radioligand competition binding experiments under equilibrium conditions. RESULTS: Modifications of all angiotensin II side chains affected binding to the AT2 receptor to nearly similar extents. In contrast, binding to the AT1 receptor was significantly affected by modifications at side chain positions 2, 4, 6 and 7. In accordance with previous observations that Tyr4- or Phe8-modified angiotensin II analogs antagonized vasoconstriction mediated exclusively by the AT1 receptor, binding to the AT1 receptor was significantly dependent on Tyr4 or Phe8 of angiotensin II whereas binding to the AT2 receptor was not. Rather surprisingly, the affinity profile of several angiotensin II analogs towards the AT2 receptor was similar to the measured affinity of the constitutively active N111G mutant AT1 receptor. CONCLUSIONS: These results suggest that the AT2-receptor pharmacophore is very distinct from that of the AT1 receptor. The AT1 receptor is in a constrained conformation and is activated only when bound to angiotensin II. In contrast, the AT2 receptor is 'relaxed' in that no single interaction is critical for binding, like the N111G mutant AT1 receptor, which is constitutively active.

Role of aromaticity of agonist switches of angiotensin II in the activation of the AT1 receptor.

We have shown previously that the octapeptide angiotensin II (Ang II) activates the AT1 receptor through an induced-fit mechanism (Noda, K., Feng, Y. H., Liu, X. P., Saad, Y., Husain, A., and Karnik, S. S. (1996) Biochemistry 35, 16435-16442). In this activation process, interactions between Tyr4 and Phe8 of Ang II with Asn111 and His256 of the AT1 receptor, respectively, are essential for agonism. Here we show that aromaticity, primarily, and size, secondarily, of the Tyr4 side chain are important in activating the receptor. Activation analysis of AT1 receptor position 111 mutants by various Ang II position 4 analogues suggests that an amino-aromatic bonding interaction operates between the residue Asn111 of the AT1 receptor and Tyr4 of Ang II. Degree and potency of AT1 receptor activation by Ang II can be recreated by a reciprocal exchange of aromatic and amide groups between positions 4 and 111 of Ang II and the AT1 receptor, respectively. In several other bonding combinations, set up between Ang II position 4 analogues and receptor mutants, the gain of affinity is not accompanied by gain of function. Activation analysis of position 256 receptor mutants by Ang II position 8 analogues suggests that aromaticity of Phe8 and His256 side chains is crucial for receptor activation; however, a stacked rather than an amino-aromatic interaction appears to operate at this switch locus. Interaction between these residues, unlike the Tyr4:Asn111 interaction, plays an insignificant role in ligand docking.

Goblet cell-specific expression mediated by the MUC2 mucin gene promoter in the intestine of transgenic mice.

The regulation of MUC2, a major goblet cell mucin gene, was examined by constructing transgenic mice containing bases -2864 to +17 of the human MUC2 5'-flanking region fused into the 5'-untranslated region of a human growth hormone (hGH) reporter gene. Four of eight transgenic lines expressed reporter. hGH message expression was highest in the distal small intestine, with only one line expressing comparable levels in the colon. This contrasts with endogenous MUC2 expression, which is expressed at its highest levels in the colon. Immunohistochemical analysis indicated that goblet cell-specific expression of reporter begins deep in the crypts, as does endogenous MUC2 gene expression. These results indicate that the MUC2 5'-flanking sequence contains elements sufficient for the appropriate expression of MUC2 in small intestinal goblet cells. Conversely, elements located outside this region appear necessary for efficient colonic expression, implying that the two tissues utilize different regulatory elements. Thus many, but not all, of the elements necessary for MUC2 gene regulation reside between bases -2864 and +17 of the 5'-flanking region.

Mechanism of constitutive activation of the AT1 receptor: influence of the size of the agonist switch binding residue Asn(111).

The AT1 receptor is a G-protein-coupled receptor (GPCR); its activation from the basal state (R) requires an interaction between Asn111 in transmembrane helix III (TM-III) of the receptor and the Tyr4 residue of angiotensin II (Ang II). Asn111 to Gly111 mutation (N111G) results in constitutive activation of the AT1 receptor (Noda et al. (1996) Biochemistry, 35, 16435-16442). We show here that replacement of the AT1 receptors TM-III with a topologically identical 16-residue segment (Cys101-Val116) from the AT2 receptor induces constitutive activity, although Asn111 is preserved in the resulting chimera, CR18. Effects of CR18 and N111G mutations are neither additive nor synergistic. The conformation(s) induced in either mutant mimics the partially activated state (R'), and transition to the fully activated R conformation in both no longer requires the Tyr4 of Ang II. Both the R state of the receptor and the Tyr4 Ang II dependence of receptor activation can be reinstated by introduction of a larger sized Phe side chain at the 111 position in CR18, suggesting that the CR18 mutation generated an effect similar to the reduction of side chain size in the N111G mutation. Consistently in the native AT1 receptor, R' conformation is generated by replacement with residues smaller but not larger than the Asn111. However, size substitution of several other TM-III residues in both receptors did not affect transitions between R, R', and R states. Thus, the property responsible for Asn111 function as a conformational switch is neither polarity nor hydrogen bonding potential but the side chain size. We conclude that the fundamental mechanism responsible for constitutive activation of the AT1 receptor is to increase the entropy of the key agonist-switch binding residue, Asn111. As a result, the normally agonist-dependent R --> R' transition occurs spontaneously. This mechanism may be applicable to many other GPCRs.

Biochemical characterization of recombinant factor IX.

Mature human factor IX is a 55,000-d glycoprotein with a modular domain structure and numerous posttranslational modifications. A recombinant form of human factor IX (rFIX) has been produced from a Chinese hamster ovary cell line that was engineered for high-level protein processing and expression. To ensure that the recombinant molecule contains the requisite structural and functional features of the plasma-derived form, rFIX was subjected to detailed biochemical and biophysical characterization. The laboratory studies showed that the posttranslational modifications and primary, secondary, and tertiary structures of rFIX were similar to those of plasma-derived factor IX (pdFIX). In addition, rFIX displayed a high degree of purity and a product release specification for specific activity that is > or = 200 IU/mg.

Transducin-alpha C-terminal peptide binding site consists of C-D and E-F loops of rhodopsin.

The binding of heterotrimeric GTP-binding proteins (G-proteins) to serpentine receptors involves several independent contacts. We have deduced the points of interaction between mutant bovine rhodopsins and alphat-(340-350), a peptide corresponding to the C terminus of the alpha subunit (alphat) of bovine retinal G-protein, transducin. Direct binding of alphat-(340-350) to rhodopsin stabilizes the activated metarhodopsin II state (M II), consequently uncoupling the rhodopsin-transducin interaction. This peptide action requires two segments on the cytoplasmic domain of rhodopsin: the Tyr136-Val137-Val138-Val139 sequence on the C-D loop and the Glu247-Lys248-Glu249-Val250-Thr251 sequence on the E-F loop. We propose that a tertiary interaction of these two loop regions forms a pocket for binding the alphat C terminus of the transducin during light transduction in vivo. In most G-proteins, the C termini of alpha subunits are important for interaction with receptors, and, in several serpentine receptors, regions similar to those in rhodopsin are essential for G-protein activation, indicating that the interaction described here may be a generally applicable mode of G-protein binding in signal transduction.

Distinct multisite synergistic interactions determine substrate specificities of human chymase and rat chymase-1 for angiotensin II formation and degradation.

Human chymase and rat chymase-1 are mast cell serine proteases involved in angiotensin II (Ang II) formation and degradation, respectively. Previous studies indicate that both these enzymes have similar P1 and P2 preferences, which are the major determinants of specificity. Surprisingly, despite the occurrence of optimal P2 and P1 residues at the Phe8 downward arrow and Tyr4 downward arrow bonds (where downward arrow, indicates the scissile bond in peptide substrates) in Ang I (DRVYIHPFHL), human chymase cleaves the Phe8 downward arrow bond with an approximately 750-fold higher catalytic efficiency (kcat/Km) than the Tyr4 downward arrow bond in Ang II (DRVYIHPF), whereas rat chymase-1 cleaves the Tyr4 downward arrow bond with an approximately 20-fold higher catalytic efficiency than the Phe8 downward arrow bond. Differences in the acyl groups IHPF and DRVY at the Phe8 downward arrow and Tyr4 downward arrow bonds, respectively, are chiefly responsible for the preference of human chymase for the Phe8 downward arrow bond. We show that the IHPF sequence forms an optimal acyl group, primarily through synergistic interactions between neighboring acyl group residues. In contrast to human chymase, rat chymase-1 shows a preference for the Tyr4 downward arrow bond, mainly because of a catalytically productive interaction between the enzyme and the P'1 Ile5. The overall effect of this P'1 Ile interaction on catalytic efficiency, however, is influenced by the structure of the acyl group and that of the other leaving group residues. For human chymase, the P'1 Ile interaction is not productive. Thus, specificity for Ang II formation versus Ang II degradation by these chymases is produced through synergistic interactions between acyl or leaving group residues as well as between the acyl and leaving groups. These observations indicate that nonadditive interactions between the extended substrate binding site of human chymase or rat chymase-1 and the substrate are best explained if the entire binding site is taken as an entity rather than as a collection of distinct subsites.

Selective reporter expression in mast cells using a chymase promoter.

Primate alpha-chymases are mast cell neutral proteases that are involved in regulating several regulatory peptides including angiotensin II. Because of significant substrate specificity differences among the chymase group of enzymes, animal models that overexpress primate chymases are crucial for delineating the in vivo function of these enzymes. Activation of alpha-prochymase requires processing enzymes and proteoglycans found in mast cell secretory granules. Thus, the development of models overexpressing active primate chymase requires a mast cell-specific promoter. We show that the 571-base pair (bp) 5'-upstream sequence of the baboon chymase gene, which encodes an alpha-chymase, coupled to the prokaryotic lacZ gene allows the targeting of beta-galactosidase to mast cells in transgenic mice. Tissue expression of the transgene is similar to the expression of the endogenous mouse alpha-chymase mouse mast cell protease-5. A mouse mast cell line that endogenously expresses mouse mast cell protease-5 (JKras mast cells) also selectively supports the expression of this transgene. In vitro transcription studies in JKras mast cells shows the critical role of a GATA cis-regulatory motif in baboon chymase promoter, located approximately 430-bp upstream of the transcription start site. These results suggest that the 571-bp domain of the baboon chymase promoter contains most, if not all, of the mast cell-specific region of the promoter. We describe here for the first time a promoter that directs expression of transgenes specifically to mouse mast cells. This promoter should be generally applicable for dominant expression of mast cell regulatory proteins.