Atrioventricular nodal reentrant tachycardia (AVNRT), the most common form of supraventricular tachycardia, results from conduction through a reentrant circuit comprising fast and slow atrioventricular nodal pathways. Antiarrhythmic-drug therapy is not consistently successful in controlling this rhythm disturbance. Catheter ablation of the fast pathway with radiofrequency current eliminates AVNRT, but it can produce heart block. We hypothesized that catheter ablation of the site of insertion of the slow pathway into the atrium would eliminate AVNRT while leaving normal (fast-pathway) atrioventricular nodal conduction intact.

Eighty patients with symptomatic AVNRT were studied. Retrograde slow-pathway conduction (in which the earliest retrograde atrial potential was recorded at the posterior septum, close to the coronary sinus) was present in 33 patients. The retrograde atrial potential was preceded by a potential consistent with activation of the atrial end of the slow pathway (ASP). In 46 of the 47 patients without retrograde slow-pathway conduction, a potential with the same characteristics as the ASP potential was recorded during sinus rhythm. Radiofrequency current delivered through a catheter to the ASP site (in the posteroseptal right atrium or coronary sinus) abolished or modified slow-pathway conduction in 78 patients, eliminating AVNRT without affecting normal atrioventricular nodal conduction. In the single patient without ASP, the application of radiofrequency current to the proximal coronary sinus ablated the fast pathway and AVNRT: Atrioventricular block occurred in one patient (1.3 percent) with left bundle-branch block, after inadvertent ablation of the right bundle branch. AVNRT has not recurred in any patient during a mean (+/- SD) follow-up of 15.5 +/- 11.3 months. Electrophysiologic study 4.3 +/- 3.3 months after ablation in 32 patients demonstrated normal atrioventricular nodal conduction without AVNRT:

Catheter ablation of the atrial end of the slow pathway using radiofrequency current, guided by ASP potentials, can eliminate AVNRT with very little risk of atrioventricular block.

Thrombospondin is one of a class of adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions. We have used two monoclonal antibodies to isolate cDNA clones of thrombospondin from a human endothelial cell cDNA library and have determined the complete nucleotide sequence of the coding region. Three regions of known amino acid sequence of human platelet thrombospondin confirm that the clones are authentic. Three types of repeating amino acid sequence are present in thrombospondin. The first is 57 amino acids long and shows homology with circumsporozoite protein from Plasmodium falciparum. The second is 50-60 amino acids long and shows homology with epidermal growth factor precursor. The third occurs as a continuous eightfold repeat of a 38-residue sequence; structural homology with parvalbumin and calmodulin indicates that these repeats constitute the multiple calcium-binding sites of thrombospondin. The amino acid sequence arg-gly-asp-ala is included in the last type 3 repeat. This sequence is probably the site for the association of thrombospondin with cells. In addition, localized homologies with procollagen, fibronectin, and von Willebrand factor are present in one region of the thrombospondin molecule.

Thirteen mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa (ADRP) have been produced by transfection of cloned cDNA into tissue culture cells. Three mutants [class I: Phe-45----Leu, Gln-344----termination (deletion of C-terminal positions 344-348), and Pro-347----Leu] resemble wild-type rhodopsin in yield, regenerability with 11-cis-retinal, and plasma membrane localization. Ten mutants [class II: Thr-17----Met, Pro-23----His, Thr-58----Arg, Val-87----Asp, Gly-89----Asp, Gly-106----Trp, Arg-135----Leu, Arg-135----Trp, Tyr-178----Cys, and Asp-190----Gly] accumulate to significantly lower levels, regenerate with 11-cis-retinal variably or not at all, and are transported inefficiently to the plasma membrane, remaining primarily in the endoplasmic reticulum. These data suggest that there are at least two distinct biochemical defects associated with different rhodopsin mutants in ADRP.

Alpha-synucleinopathies (ASP) are neurodegenerative disorders, characterized by accumulation of misfolded α-synuclein, selective neuronal loss, and extensive gliosis. It is accepted that microgliosis and astrogliosis contribute to the disease progression in ASP. Toll-like receptors (TLRs) are expressed on cells of the innate immune system, including glia, and TLR4 dysregulation may play a role in ASP pathogenesis. In this study we aimed to define the involvement of TLR4 in microglial and astroglial activation induced by different forms of α-synuclein (full length soluble, fibrillized, and C-terminally truncated). Purified primary wild type (TLR4(+/+)) and TLR4 deficient (TLR4(-/-)) murine microglial and astroglial cell cultures were treated with recombinant α-synuclein and phagocytic activity, NFκB nuclear translocation, cytokine release, and reactive oxygen species (ROS) production were measured. We show that TLR4 mediates α-synuclein-induced microglial phagocytic activity, pro-inflammatory cytokine release, and ROS production. TLR4(-/-) astroglia present a suppressed pro-inflammatory response and decreased ROS production triggered by α-synuclein treatment. However, the uptake of α-synuclein by primary astroglia is not dependent on TLR4 expression. Our results indicate the C-terminally truncated form as the most potent inductor of TLR4-dependent glial activation. The current findings suggest that TLR4 plays a modulatory role on glial pro-inflammatory responses and ROS production triggered by α-synuclein. In contrast to microglia, the uptake of alpha-synuclein by astroglia is not dependent on TLR4. Our data provide novel insights into the mechanisms of α-synuclein-induced microglial and astroglial activation which may have an impact on understanding the pathogenesis of ASP.

Although validation studies show that theoretical models for predicting the pKas of ionizable groups in proteins are increasingly accurate, a number of important questions remain: (1) What factors limit the accuracy of current models? (2) How can conformational flexibility of proteins best be accounted for? (3) Will use of solution structures in the calculations, rather than crystal structures, improve the accuracy of the computed pKas? and (4) Why does accurate prediction of protein pKas seem to require that a high dielectric constant be assigned to the protein interior? This paper addresses these and related issues. Among the conclusions are the following: (1) computed pKas averaged over NMR structure sets are more accurate than those based upon single crystal structures; (2) use of atomic parameters optimized to reproduce hydration energies of small molecules improves agreement with experiment when a low protein dielectric constant is assumed; (3) despite use of NMR structures and optimized atomic parameters, pKas computed with a protein dielectric constant of 20 are more accurate than those computed with a low protein dielectric constant; (4) the pKa shifts in ribonuclease A that result from phosphate binding are reproduced reasonably well by calculations; (5) the substantial pKa shifts observed in turkey ovomucoid third domain result largely from interactions among ionized groups; and (6) both experimental data and calculations indicate that proteins tend to lower the pKas of Asp side chains but have little overall effect upon the pKas of other ionizable groups.

Common histidine-to-aspartate (His->>Asp) phosphorelay is a paradigm of signal transduction in both prokaryotes and eukaryotes for the propagation of certain environmental stimuli, in which histidine (His)-kinases play central roles as sensors for environmental signals. For the higher plant, Arabidopsis thaliana, it was recently suggested that the His-kinase (AHK4 / CRE1 / WOL) is a sensor for cytokinins, which are a class of plant hormones important for the regulation of cell division and differentiation. Interestingly, AHK4 is capable of functioning as a cytokinin sensor in the eubacterium, Escherichia coli (Suzuki et al. 2001, Plant Cell Physiol. 42: 107). Here we further show that AHK4 is a primary receptor that directly binds a variety of natural and synthetic cytokinins (e.g. not only N(6)-substituted aminopurines such as isopentenyl-adenine, trans-zeatin, benzyl-adenine, but also diphenylurea derivatives such as thidiazuron), in a highly specific manner (K(d) = 4.55+/-0.48x10(-9) M). AHK4 has a presumed extracellular domain, within which a single amino acid substitution (Thr-301 to Ile) was shown to result in loss of its ability to bind cytokinins. This particular mutation corresponds to the previously reported wol allele (wooden leg) that causes a striking phenotype defective in vascular morphogenesis. Collectively, evidence is presented that AHK4 and its homologues (AHK3 and possibly AHK2) are receptor kinases that can transduce cytokinin signals across the plasma membrane of A. thaliana.

Transport of membrane proteins between intracellular compartments requires specific sequences in the protein cytoplasmic domain to direct packaging into vesicle shuttles. A sequence that mediates export from the endoplasmic reticulum (ER) has proved elusive. A di-acidic signal (Asp-X-Glu, where X represents any amino acid) on the cytoplasmic tail of vesicular stomatitis virus glycoprotein (VSV-G) and other cargo molecules was required for efficient recruitment to vesicles mediating export from the ER in baby hamster kidney cells. The existence of such a signal provides evidence that export from the ER occurs through a selective mechanism.

Phage displaying an Arg-Gly-Asp (RGD)-containing peptide with a high affinity for alpha v integrins homed to tumors when injected intravenously into tumor-bearing mice. A substantially higher amount of alpha v-directed RGD phage than control phage was recovered from malignant melanomas and breast carcinoma. Antibodies detected the alpha v-directed RGD phage in tumor blood vessels, but not in several normal tissues. These results show that the alpha v integrins present in tumor blood vessels can bind circulating ligands and that RGD peptides selective for these integrins may be suitable tools in tumor targeting for diagnostic and therapeutic purposes.

Fibronectin type III domains are found in many different proteins including cell surface receptors and cell adhesion molecules. The crystal structure of one such domain from the extracellular matrix protein tenascin was determined. The structure was solved by multiwavelength anomalous diffraction (MAD) phasing of the selenomethionyl protein and has been refined to 1.8 angstrom resolution. The folding topology of this domain is identical to that of the extracellular domains of the human growth hormone receptor, the second domain of CD4, and PapD. Although distinct, this topology is similar to that of immunoglobulin constant domains. An Arg-Gly-Asp (RGD) sequence that can function for cell adhesion is found in a tight turn on an exposed loop.

Pancreatic lipase (triacylglycerol acyl hydrolase) fulfills a key function in dietary fat absorption by hydrolysing triglycerides into diglycerides and subsequently into monoglycerides and free fatty acids. We have determined the three-dimensional structure of the human enzyme, a single-chain glycoprotein of 449 amino acids, by X-ray crystallography and established its primary structure by sequencing complementary DNA clones. Enzymatic activity is lost after chemical modification of Ser 152 in the porcine enzyme, indicating that this residue is essential in catalysis, but other data are more consistent with a function in interfacial recognition. Our structural results are evidence that Ser 152 is the nucleophilic residue essential for catalysis. It is located in the larger N-terminal domain at the C-terminal edge of a doubly wound parallel beta-sheet and is part of an Asp-His-Ser triad, which is chemically analogous to, but structurally different from, that in the serine proteases. This putative hydrolytic site is covered by a surface loop and is therefore inaccessible to solvent. Interfacial activation, a characteristic property of lipolytic enzymes acting on water-insoluble substrates at water-lipid interfaces, probably involves a reorientation of this flap, not only in pancreatic lipases but also in the homologous hepatic and lipoprotein lipases.

This paper describes a general method to calculate the pKas of ionizable groups in proteins. Electrostatic calculations are carried out using the finite difference Poisson-Boltzmann (FDPB) method. A formal treatment of the calculation of pKas within the framework of the FDPB method is presented. The major change with respect to previous work is the specific incorporation of the complete charge distribution of both the neutral and charged forms of each ionizable group into the formalism. This is extremely important for the treatment of salt bridges. A hybrid statistical mechanical/Tanford-Roxby method, which is found to be significantly faster than previous treatments, is also introduced. This simplifies the problem of summing over the large number of possible ionization states for a complex polyion. Applications to BPTI and serine proteases suggest that the calculations can be quite reliable. However, the necessity of including bound waters in the treatment of the Asp-70... His-31 salt bridge in T4 lysozyme and experience with other proteins suggest that additional factors ultimately need to be considered in a comprehensive treatment of pKas in proteins.

The observation that TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF cytokine family, induces apoptosis in a number of different tumor cell types led us to compare the tumoricidal effects of TRAIL to those of other TNF family molecules on human melanoma cells. We found that a high proportion of the melanoma cell lines tested were killed by TRAIL, whereas all the melanoma lines were resistant to the other TNF family cytokines tested. TRAIL-induced death was characterized by caspase activation and cellular protein cleavage within minutes of TRAIL addition, and death could be completely inhibited by the caspase inhibitors Ile-Glu-Thr-Asp (IETD) and Val-Ala-Asp (VAD), indicating the presence of a TRAIL receptor signaling pathway similar to that identified for Fas and TNF receptors. Specific TRAIL receptor expression was determined by RT-PCR, and the presence of mRNA encoding the "protective" TRAIL receptors did not correspond to resistance or sensitivity to TRAIL-induced apoptosis. Addition of protein synthesis inhibitors to TRAIL-resistant melanomas rendered them sensitive to TRAIL, indicating that the presence or the absence of intracellular apoptosis inhibitors may mediate resistance or sensitivity to TRAIL-mediated apoptosis. Expression of one such inhibitor, FLICE-inhibitory protein (FLIP), was highest in the TRAIL-resistant melanomas, while being low or undetectable in the TRAIL-sensitive melanomas. Furthermore, addition of actinomycin D to TRAIL-resistant melanomas resulted in decreased intracellular concentrations of FLIP, which correlated with their acquisition of TRAIL sensitivity. Collectively, our results indicate that TRAIL-induced apoptosis occurs through a caspase signaling cascade and that resistance is controlled by intracellular regulators of apoptosis.

Release of cytochrome c is important in many forms of apoptosis. Recent studies of CD95 (Fas/APO-1)-induced apoptosis have implicated caspase-8 cleavage of Bid, a BH3 domain-containing proapoptotic member of the Bcl-2 family, in this release. We now demonstrate that both receptor-induced (CD95 and tumor necrosis factor) and chemical-induced apoptosis result in a similar time-dependent activation of caspases-3, -7, -8, and -9 in Jurkat T cells and human leukemic U937 cells. In receptor-mediated apoptosis, the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD. FMK), inhibits apoptosis prior to commitment to cell death by inhibiting the upstream activator caspase-8, cleavage of Bid, release of mitochondrial cytochrome c, processing of effector caspases, loss of mitochondrial membrane potential, and externalization of phosphatidylserine. However, Z-VAD.FMK inhibits chemical-induced apoptosis at a stage after commitment to cell death by inhibiting the initiator caspase-9 and the resultant postmitochondrial activation of effector caspases. Cleavage of Bid but not release of cytochrome c is blocked by Z-VAD.FMK demonstrating that in chemical-induced apoptosis cytochrome c release is caspase-independent and is not mediated by activation of Bid. We propose that caspases form an integral part of the cell death-inducing mechanism in receptor-mediated apoptosis, whereas in chemical-induced apoptosis they act solely as executioners of apoptosis.

A growing body of evidence supports a role for mitochondria and mitochondria-derived factors in the cell death process. In particular, much attention has focused on cytochrome c, a key component of the electron transport chain, that has been reported to translocate from the mitochondria to the cytosol in cells undergoing apoptosis. The mechanism for this release is, as yet, unknown. Here we report that ectopic expression of Bax induces apoptosis with an early release of cytochrome c preceding many apoptosis-associated morphological alterations as well as caspase activation and subsequent substrate proteolysis. A loss of mitochondrial transmembrane potential was detected in vivo, although no mitochondrial swelling or loss of transmembrane potential was observed in isolated mitochondria treated with Bax in vitro. Caspase inhibitors, such as endogenous XIAP and synthetic peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), although capable of altering the kinetics and perhaps mode of cell death, had no influence on this release, suggesting that if cytochrome c plays a role in caspase activation it must precede this step in the apoptotic process. Mitochondrial permeability transition was also shown to be significantly prevented by caspase inhibition, indicating that the translocation of cytochrome c from mitochondria to cytosol is not a consequence of events requiring mitochondrial membrane depolarization. In contrast, Bcl-xL was capable of preventing cytochrome c release while also significantly inhibiting cell death. It would therefore appear that the mitochondrial release of factors such as cytochrome c represents a critical step in committing a cell to death, and this release is independent of permeability transition and caspase activation but is inhibited by Bcl-xL.

Zinc is known to be indispensable to growth and development and transmission of the genetic message. It does this through a remarkable mosaic of zinc binding motifs that orchestrate all aspects of metabolism. There are now nearly 200 three dimensional structures for zinc proteins, representing all six classes of enzymes and covering a wide range of phyla and species. These structures provide standards of reference for the identity and nature of zinc ligands in other proteins for which only the primary structure is known. Three primary types of zinc sites are apparent from examination of these structures: structural, catalytic and cocatalytic. The most common amino acids that supply ligands to these sites are His, Glu, Asp and Cys. In catalytic sites zinc generally forms complexes with water and any three nitrogen, oxygen and sulfur donors with His being the predominant amino acid chosen. Water is always a ligand to such sites. Structural zinc sites have four protein ligands and no bound water molecule. Cys is the preferred ligand in such sites. Cocatalytic sites contain two or three metals in close proximity with two of the metals bridged by a side chain moiety of a single amino acid residue, such as Asp, Glu or His and sometimes a water molecule. Asp and His are the preferred amino acids for these sites. No Cys ligands are found in such sites. The scaffolding of the zinc sites is also important to the function and reactivity of the bound metal. The influence of zinc on quaternary protein structure has led to the identification of a fourth type of zinc binding site, protein inteface. In this case zinc sites are formed from ligands supplied from amino acid residues residing in the binding surface of two proteins. The resulting zinc site usually has the coordination properties of a catalytic or structural zinc binding site.

X-ray data have been extended to 1.7 A for a binary complex of Escherichia coli dihydrofolate reductase with methotrexate and a ternary complex of Lactobacillus casei dihydrofolate reductase with methotrexate and NADPH. Models for both structures have been refined to R factors of 0.15 and include parameters for fixed and liquid solvent. The two species of dihydrofolate reductase resemble one another even more closely than was thought to be the case prior to refinement. Several new structural features have also been discovered. Among them are a cis peptide linking Gly-97 and Gly-98 (L. Casei numbering) in both species, an alpha helix involving residues 43 through 50 in the E. coli enzyme, and the existence of what may be a specific hydration site on exposed alpha helices. Refinement has led to a revised description of the details of methotrexate binding. We now see that a fixed water molecule mediates the interaction between methotrexate's 2-amino group and Thr-116 (L. casei numbering) and that the inhibitor's 4-amino group makes two hydrogen bonds with the enzyme (instead of one). Other revisions are also discussed. A hypothetical model for substrate binding is proposed in which the pteridine ring is turned upside down while all protein and solvent atoms remain fixed. Asp-26 in this model is hydrogen bonded to the substrate's 2-amino group and to N3.

Water deficit and the resulting osmotic stress affect plant growth. To understand how plant cells monitor and respond to osmotic change from water stress, we isolated a cDNA from dehydrated Arabidopsis plants. This cDNA encodes a novel hybrid-type histidine kinase, ATHK1. Restriction fragment length polymorphism mapping showed that the ATHK1 gene is on chromosome 2. The predicted ATHK1 protein has two putative transmembrane regions in the N-terminal half and has structural similarity to the yeast osmosensor synthetic lethal of N-end rule 1 (SLN1). The ATHK1 transcript was more abundant in roots than other tissues under normal growth conditions and accumulated under conditions of high or low osmolarity. Histochemical analysis of beta-glucuronidase activities driven by the ATHK1 promoter further indicates that the ATHK1 gene is transcriptionally upregulated in response to changes in external osmolarity. Overexpression of the ATHK1 cDNA suppressed the lethality of the temperature-sensitive osmosensing-defective yeast mutant sln1-ts. By contrast, ATHK1 cDNAs in which conserved His or Asp residues had been substituted failed to complement the sln1-ts mutant, indicating that ATHK1 functions as a histidine kinase. Introduction of the ATHK1 cDNA into the yeast double mutant sln1Delta sho1Delta, which lacks two osmosensors, suppressed lethality in high-salinity media and activated the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK). These results imply that ATHK1 functions as an osmosensor and transmits the stress signal to a downstream MAPK cascade.

Previously we identified the highly conserved amino acids Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ecto-domain of gp41 as the epitope of a neutralizing monoclonal antibody (2F5) directed against human immunodeficiency virus type 1. In the present study, the sequence defining the epitope was introduced into the loop of antigenic site B of the influenza virus hemagglutinin. The resulting chimeric virus was able to elicit ELDKWA-specific immunoglobulins G and A in antisera of mice. Moreover, the distantly related human immunodeficiency virus type 1 isolates MN, RF, and IIIB were neutralized by these antisera. These data suggest that this conserved B-cell epitope is a promising candidate for inclusion in a vaccine against AIDS. The results also show that influenza virus can be used to effectively present the antigenic structure of this B-cell epitope.

Signal transduction in microorganisms and plants is often mediated by His-Asp phosphorelay systems. Two conserved families of proteins are centrally involved: histidine protein kinases and phospho-aspartyl response regulators. The kinases generally function in association with sensory elements that regulate their activities in response to environmental signals. A sequence analysis with 348 histidine kinase domains reveals that this family consists of distinct subgroups. A comparative sequence analysis with 298 available receiver domain sequences of cognate response regulators demonstrates a significant correlation between kinase and regulator subfamilies. These findings suggest that different subclasses of His-Asp phosphorelay systems have evolved independently of one another.

When beta-lactamase was fused with the signal peptide plus the amino-terminal 9 amino acid residues of the major outer membrane lipoprotein, the resultant lipo-beta-lactamase (LL-1) was shown to be localized to the outer membrane. However, when the 9 residue sequence was replaced with the amino-terminal 12 residue sequence of lipoprotein-28, an inner membrane protein, the resultant lipo-beta-lactamase (LL-2) was found exclusively in the inner membrane. The localization of LL-2 was shifted to the outer membrane simply by substituting the second amino acid residue (Asp) of LL-2 with Ser. Conversely, the alteration of the second residue (Ser) of LL-1 to Asp resulted in the localization of LL-1 to the inner membrane. These results suggest that the second amino acid residue of the lipoproteins plays a crucial role in determining their final locations in the E. coli envelope.

Phosphorylation of NF-kappaB p65(RelA) serine 536 is physiologically induced in response to a variety of proinflammatory stimuli, but the responsible pathways have not been conclusively unraveled, and the function of this phosphorylation is largely elusive. In contrast to previous studies, we found no evidence for a role of c-Jun N-terminal kinase, p38 kinase, extracellular signal-regulated kinase, or phosphatidylinositol 3-kinase in interleukin-1- or tumor necrosis factor-induced Ser-536 phosphorylation, as revealed by pharmacological inhibitors. We were not able to suppress Ser-536 phosphorylation by either RNA interference directed at IkappaB kinase (IKK)-alpha/beta (the best characterized Ser-536 kinases so far) or the IKKbeta inhibitor SC-514 or dominant negative mutants of either IKK. A green fluorescent protein p65 fusion protein was phosphorylated at Ser-536 in the absence of IKK activation, suggesting the existence of IKKalpha/beta-independent Ser-536 kinases. Chromatographic fractionation of cell extracts allowed the identification of two distinct enzymatic activities phosphorylating Ser-536. Peak 1 represents an unknown kinase, whereas peak 2 contained IKKalpha, IKKbeta, IKKepsilon, and TBK1. Overexpressed IKKepsilon and TBK1 phosphorylate Ser-536 in vivo and in vitro. Reconstitution of mutant p65 proteins in p65-deficient fibroblasts that either mimicked phosphorylation (S536D) or preserved a predicted hydrogen bond between Ser-536 and Asp-533 (S536N) revealed that phosphorylation of Ser-536 favors interleukin-8 transcription mediated by TATA-binding protein-associated factor II31, a component of TFIID. In the absence of phosphorylation, the hydrogen bond favors binding of the corepressor amino-terminal enhancer of split to the p65 terminal transactivation domain. Collectively, our results provide evidence for at least five kinases that converge on Ser-536 of p65 and a novel function for this phosphorylation site in the recruitment of components of the basal transcriptional machinery to the interleukin-8 promoter.

The transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src) appear to be protein kinases that phosphorylate tyrosine in a variety of protein substrates. In addition, pp60v-src and pp60-c-src are themselves phosphorylated on serine and tyrosine. It is likely that these phosphorylations serve to regulate the function(s) of pp60v-src and pp60c-src. We have therefore characterized the sites of tyrosine phosphorylation in the two proteins. Tyrosine phosphorylation of pp60v-src in infected cells occurs mainly (if not entirely) at residue 419 in the deduced amino acid sequence of the protein. Surrounding this residue is the sequence Leu-Ile-Glu-Asp-Asn-Glu-Tyr(P)-Thr-Ala-Arg. This peptide is distinguished by the fact that three out of the four amino acids that precede the phosphorylated tyrosine are acidic in nature. These results define what may prove to be a widely used site for tyrosine phosphorylation in the regulation of cellular function. The same site was phosphorylated when partially purified pp60v-src was used in a phosphotransfer reaction in vitro. The results with pp60c-src were more complex. The site of tyrosine phosphorylation in vitro appeared to be the same as that found in pp60v-src. By contrast, phosphorylation of pp60c-src in vivo apparently occurred at a different, and currently unidentified, tyrosine residue. It is therefore possible that pp60v-src and pp60c-src respond differently to regulatory influences in the intact cell.

True lipases attach triacylglycerols and act at an oil-water interface; they constitute a ubiquitous group of enzymes catalysing a wide variety of reactions, many with industrial potential. But so far the three-dimensional structure has not been reported for any lipase. Here we report the X-ray structure of the Mucor miehei triglyceride lipase and describe the atomic model obtained at 3.1 A resolution and refined to 1.9 A resolution. It reveals a Ser..His..Asp trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.

Transforming growth factor-beta (TGF-beta) is capable of affecting the proliferation of many cell types. To identify novel genes whose protein products may mediate cellular responses to this factor, a cDNA library was made from mRNA isolated from a human lung adenocarcinoma cell line (A549) that had been treated for 3 days with TGF-beta. The library was screened by differential hybridization and a cDNA clone, beta ig-h3, was isolated. This gene was induced up to 20-fold in A549 cells after 2 days of treatment with TGF-beta 1. It was also induced in several other cell lines, including PC-3 and H2981. DNA sequence analysis of beta ig-h3 indicated that it encoded a novel protein, beta IG-H3, of 683 amino acids, which contained an amino-terminal secretory sequence and a carboxy-terminal Arg-Gly-Asp (RGD) sequence that can serve as a ligand recognition site for several integrins. beta IG-H3 also contained short amino acid regions homologous to similar regions in Drosophila fasciclin-I and four homologous internal domains, which can be folded into a potential bivalent structure and could act as a bridge between cells expressing the appropriate ligand. beta ig-h3 RNA was detected in several cell lines and tissues. COS cells transfected with plasmids encoding beta IG-H3 secreted a major 68-kD protein that was detected by immunoblotting using antipeptide antibodies. Since beta ig-h3 is induced in several cell lines whose proliferation is affected by TGF-beta 1, it may be involved in mediating some of the signals of this multifunctional growth modulator.