Unidentified Target Research Articles

Pharmacokinetics of artemisinin-type compounds.

Various compounds of the artemisinin family are currently used for the treatment of patients with malaria worldwide. They are characterised by a short half-life and feature the most rapidly acting antimalarial drugs to date. They are increasingly being used, often in combination with other drugs, although our knowledge of their main pharmacological features (including their absorption, distribution, metabolism and excretion) is still incomplete. Such data are particularly important in the case of combinations. Artemisinin derivatives are converted primarily, but to different extents, to the bioactive metabolite artenimol after either parenteral or gastrointestinal administration. The rate of conversion is lowest for artelinic acid (designed to protect the molecule against metabolism) and highest for the water-soluble artesunate. The absolute and relative bioavailability of these compounds has been established in animals, but not in humans, with the exception of artesunate. Oral bioavailability in animals ranges, approximately, between 19 and 35%. A first-pass effect is highly probably for all compounds when administered orally. Artemisinin compounds bind selectively to malaria-infected erythrocytes to yet unidentified targets. They also bind modestly to human plasma proteins, ranging from 43% for artenimol to 81.5% for artelinic acid. Their mode of action is still not completely understood, although different theories have been proposed. The lipid-soluble artemether and artemotil are released slowly when administered intramuscularly because of the 'depot' effect related to the oil formulation. Understanding the pharmacokinetic profile of these 2 drugs helps us to explain the characteristics of the toxicity and neurotoxicity. The water-soluble artesunate is rapidly converted to artenimol at rates that vary with the route of administration, but the processes need to be characterised further, including the relative contribution of pH and enzymes in tissues, blood and liver. This paper intends to summarise contemporary knowledge of the pharmacokinetics of this class of compounds and highlight areas that need further research.

The Chemotactic Action of Urokinase on Smooth Muscle Cells Is Dependent on Its Kringle Domain: CHARACTERIZATION OF INTERACTIONS AND CONTRIBUTION TO CHEMOTAXIS

Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His(204) to Gln) (r-uPA(H/Q)), urokinase with mutation of His(204) to Gln together with a deletion of growth factor-like domain (r-uPA(H/Q)-GFD), the catalytic domain of urokinase (r-uPA(LMW)), and its kringle domain (r-KD) were expressed in Escherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPA(H/Q), and r-uPA(H/Q)-GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC(50)) were apparent at approximately 2 nm with all the uPA variants. The kringle domain induced cell migration with an EC(50) of about 6 nm, whereas the denaturated r-KD and r-uPA(LMW) were without effect. R-uPAwt-induced chemotaxis was dependent on an association with uPAR and a uPA-kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA-kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r-uPA(H/Q)-GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC. uPAR-deficient HEK 293 cells specifically bound r-uPAwt and r-uPA(H/Q)-GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPA(H/Q)-GFD and uPAwt, but not r-uPA(LMW). HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPA(H/Q)-GFD. Together, these findings indicate that uPA-induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR.

The F-actin cytoskeleton modulates slow secretory components rather than readily releasable vesicle pools in bovine chromaffin cells

Adrenal chromaffin cells were used to test the role of the peripheral cytoskeleton of F-actin in controlling different vesicle pools. Phorbol 12-myristate 13-acetate and calyculin A, two substances affecting phosphorylation–dephosphorylation cycles, produced different degrees of F-actin reorganization, inducing the partial and the almost total disassembly of this structure, respectively, as visualized using rhodamine–phalloidin staining. Consequently, electron microscopy studies revealed the higher efficiency of calyculin-A over phorbol 12-myristate 13-acetate in promoting vesicle access to the plasmalemma boundary. Surprisingly, only the phorbol ester enhanced fast kinetics and the population of rapidly releasable vesicle pools as studied by single-cell amperometry, whereas both agents, as well as the F-actin severing compound, Latrunculin A, promoted an increase in the population of vesicles recruited in response to prolonged or repetitive stimulations. Taken together, our data support the notion that the F-actin peripheral barrier controls primary granule recruitment from reserve vesicle pools, whereas the phorbol ester effect on the rapidly releasable pools might be related to the alteration of late secretory stage through protein kinase C-dependent phosphorylation of an unidentified target.

Structure−Activity Relationships of (1‘S)-1‘-Acetoxychavicol Acetate, a Major Constituent of a Southeast Asian Condiment Plant Languas galanga, on the Inhibition of Tumor-Promoter-Induced Epstein−Barr Virus Activation

The structure-activity relationships of (1'S)-1'-acetoxychavicol acetate (ACA), a cancer chemopreventive agent of food origin, were investigated in an inhibitory test of tumor promoter teleocidin B-4-induced Epstein-Barr virus (EBV) activation in Raji cells. Through a test of 16 derivatives, the structural factors regulating activity were found to be as follows: (1) the absolute configuration at the 1'-position does not affect activity; (2) hydrogenation of the terminal methylene group abolishes activity; (3) both the phenolic and alcoholic hydroxyl groups are compulsorily acetylated, and it is necessary that the former is oriented only at the position para to the side chain; (4) an additional acetoxyl group is allowed to locate at the ortho or meta position; and (5) substitution of the hydrogen atom at the 1'-position by a methyl group reduces activity. Upon esterase blockade in Raji cells, (1'R,S)-ACA suppressed EBV activation, the extent of which was the same as tested in the control, suggesting that ACA bearing two acetoxyl groups is an intracellular structure prerequisite for activity exhibition. The present study suggests that nucleophilic attack to the 3'-position is important and involved in the interaction of ACA with an unidentified target molecule(s) participating in the process of EBV activation.

Poliovirus 2A protease induces apoptotic cell death.

A cell line was generated that expresses the poliovirus 2A protease in an inducible manner. Tightly controlled expression was achieved by utilizing the muristerone A-regulated expression system. Upon induction, cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI) and eIF4GII is observed, with the latter being cleaved in a somewhat slower kinetics. eIF4G cleavage was accompanied by a severe inhibition of protein synthesis activity. Upon induction of the poliovirus 2A protease, the cells displayed fragmented nuclei, chromatin condensation, oligonucleosome-size DNA ladder, and positive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining; hence, their death can be characterized as apoptosis. These results indicate that the expression of the 2A protease in mammalian cells is sufficient to induce apoptosis. We suggest that the poliovirus 2A protease induces apoptosis either by arresting cap-dependent translation of some cellular mRNAs that encode proteins required for cell viability, by preferential cap-independent translation of cellular mRNAs encoding apoptosis inducing proteins, or by cleaving other, yet unidentified cellular target proteins.

Unique biochemical, cytotoxic, and antitumor activity of camptothecin and 4β-amino-4′- O-demethylepipodophyllotoxin conjugates

Two compounds having a camptothecin (CPT) analog conjugated to the 4β-amino-4′- O-demethylepipodophyllotoxin analog were evaluated for their biochemical and biological activities. W1 [camptothecin-( para)-4β-amino-4′- O-demethylepipodophyllotoxin] had no activity against topoisomerase II (TOP II), but inhibited topoisomerase I (TOP I) with an ic 50 value 2-fold higher than CPT. W2 [camptothecin-( ortho)-4β-amino-4′- O-demethylepipodophyllotoxin] had inhibitory activity against TOP I and TOP II with ic 50 values 1.5-fold higher than either CPT or etoposide (VP-16). Both conjugates had similar cytotoxicity against the KB cell line, although the protein-linked DNA breaks (PLDBs) generated by W2 in KB cells were about 4-fold more than those of W1. No cross-resistance with the two conjugates was seen in a VP-16-resistant KB subline, which showed down-regulation of TOP II and overexpression of the multiple drug resistance-associated protein, or in a vincristine-resistant KB subline with overexpression of gp-170/ mdr-1. The CPT-resistant KB variant (KB CPT 100), which has a reduction in TOP I content and another mechanism that occurs post-PLDB formation, was partially resistant to both compounds. W1 was not affected by this post-PLDB resistance mechanism. Cell cycle analysis demonstrated that W1 and W2 had similar cell cycle effects on KB and KB CPT 100 cells, which accumulated in S-phase upon drug treatment. These results suggested that W1 and W2 exerted their cytotoxicity through TOP I. In CPT-resistant cells, however, an unidentified target also may be involved in the cytotoxic action of W1, and TOP II may still be a target for W2. In vivo, W1 was more effective against the growth of human prostate cancer cells in nude mice than VP-16, CPT, or W2. Given its antitumor activity and unique biochemical mechanism of action, W1 warrants exploration as an antitumor compound.

The role of mitogen-activated protein kinases in stimulation of the smooth muscle cell migration by urokinase

The urokinase-type plasminogen activator, or urokinase, stimulates proliferation, adhesion, and migration of different type cells both due to its proteolytic activity and by activation of intracellular transduction pathways after interaction with the external cell surface. It is suggested that activation of p42/p44erk1,2 MAP-kinases in response to specific receptor binding to the urokinase N-terminal domain is the key event in initiation of cell migration. However, we have found that the central kringle-domain of urokinase has its own target on the cell surface, and that its binding causes a migration response of human smooth muscle cells (SMCs). In the present study, we have shown that the urokinase kringle-domain is required to activate the p38 MAP kinase cascade, and that its inhibition leads to suppression of the migration response of SMCs. On the contrary, stimulation of the p42/p44erk1,2 MAP-kinase cascade is determined only by proteolytic activity of urokinase and does not depend on its binding to SMCs. Selective inhibition of the p42/p44erk1,2 MAP-kinase cascade produced a depression of the SMC migration induced by catalytically active urokinase, but did not affect the migration induced by non-active urokinase. It is concluded that binding of the urokinase kringle-domain to a yet unidentified target at the SMC surface is required for activation of the p38 MAP-kinase cascade and of the cell migration. Urokinase was shown to stimulate phosphorylation and activation of regulatory light myosin chains that are required to increase the cytoskeleton dynamics and SMC motility. The participation of p42/p44erk1,2 and p38 MAP-kinase cascade in the realization of this effect is discussed.

Insect peptides with improved protease-resistance protect mice against bacterial infection.

At a time of the emergence of drug-resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as-yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against gram-negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia coli infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.

Selective Effects ofneuronal-synaptobrevinMutations on Transmitter Release Evoked by Sustained Versus Transient Ca2+Increases and by cAMP

Synaptobrevin is a key constituent of the synaptic vesicle membrane. The neuronal-synaptobrevin (n-syb) gene in Drosophila is essential for nerve-evoked synaptic currents, but miniature excitatory synaptic currents (mESCs) remain even in the complete absence of this gene. To further characterize the defect in these mutants, we have examined conditions that stimulate secretion. Despite the inability of an action potential to trigger fusion, high K+ saline could increase the frequency of mESCs 4- to 17-fold in a Ca2+-dependent manner, and the rate of fusion approached 25% of that seen in wild-type synapses under the same conditions. Similarly, the mESC frequency in n-syb null mutants could be increased by a Ca2+ ionophore, A23187, and by black widow spider venom. Thus, the ability of the vesicles to fuse in response to sustained increases in cytosolic Ca2+ persisted in the absence of this protein. Tetanic stimulation could also increase the frequency of mESCs, particularly toward the end of a train and after the train of stimuli. In contrast, these mutants did not respond to an elevation of cAMP induced by an activator of adenylyl cyclase, forskolin, or a membrane-permeable analog of cAMP, dibutyryl cAMP, which in wild-type synapses causes a marked increase in the mESC frequency even in the absence of external Ca2+. These results are discussed in the context of models that invoke a special role for n-syb in coupling fusion to the transient, local changes in Ca2+ and an as yet unidentified target of cAMP.

Mapping membrane potential transients in crayfish (Procambarus clarkii) optic lobe neuropils with voltage-sensitive dyes.

Voltage-sensitive dyes NK 2761 and RH 155 were employed (in conjunction with a 12 x 12 photodiode array) to study membrane potential transients in optic lobe neuropils in the eye stalk of the crayfish Procambarus clarkii. By this means we investigated a pathway linking deutocerebral projection neurons, via hemiellipsoid body local interneurons, to an unidentified target (most likely neurons processing visual information) in the medulla terminalis. Rapid (10- to 20-ms duration), transient changes in absorption with the characteristics of action potentials were recorded from the optic nerve and the region occupied by deutocerebral projection neurons after stimulation of the olfactory globular tract in the optic nerve and were blocked by 1 microM tetrodotoxin. Action potentials appeared to propagate to the glomerular layer of the hemiellipsoid body where synaptic responses were recorded from a restricted region of the hemiellipsoid body occupied by dendrites of hemiellipsoid body neurons. Action potentials were also recorded from processes of hemiellipsoid body neurons located in the medulla terminalis. Synaptic responses in the hemiellipsoid body and medulla terminalis were eliminated by addition to the saline of 500 microM Cd2+ or 20 mM Co2+, whereas the action potential attributed to branches of deutocerebral projection neurons in the hemiellipsoid body remained unaffected. Action potentials of hemiellipsoid body neurons in the medulla terminalis evoked postsynaptic potentials (50- to 200-ms duration) with an unidentified target in the medulla terminalis. Transient absorption signals were not detected in either the internal or external medulla nor were they recorded from other parts of the optic lobes in response to electrical stimulation of axons of the deutocerebral projection neurons. Functional maps of optical activity, together with electrophysiological and pharmacological findings, suggest that gamma-aminobutyric acid affects synaptic transmission in glomeruli of the hemiellipsoid body. Synapses of the olfactory pathway located in the medulla terminalis may act as a "filter," modifying visual information processing during olfactory stimulation.

Illusory Tip-of-the-tongue States

The tip-of-the-tongue state (TOT) is the phenomenological experience that a target word is on the verge of being recalled. An illusory TOT occurs when a person experiences a TOT, but the actual target is either unavailable, forgotten, or never learned. Illusory TOTs were induced by asking participants to answer questions that did not have correct answers. In Experiment 1, an episodic-memory paradigm, participants were shown fictional animals, some of which were accompanied by the animal's name (identified targets) and some of which were not (unidentified targets). Some participants experienced TOTs for unidentified targets. In Experiment 2, a semantic-memory paradigm, participants were asked generalinformation questions, some of which were questions with no correct answer. Every one of the 31 participants experienced at least one illusory TOT. The characteristics of illusory TOTs are discussed in light of inferential and directaccess views of TOTS.

Definition of the transcriptional activation domains of three human HOX proteins depends on the DNA-binding context.

Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions which ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analysis. We report the identification of the transcriptional activation domains of three human Hox proteins, HOXB1, HOXB3, and HOXD9, which interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery depending on the structure of their target regulatory elements.

Specificity of bactericidal antibody response to serogroup B meningococcal strains in Brazilian children after immunization with an outer membrane vaccine.

Pre- and postvaccination serum samples from 77 children aged 2 to 6 years, who received the Cuban BC vaccine (B:4:P1.15), were analyzed for bactericidal antibodies against a local B:4:P1.15 strain (N44/89). Sera from 16 individuals with bactericidal antibodies against the B:4:P1.15 strain were tested against 23 Brazilian isolates. These include B:4 strains of distinct serosubtypes: P1.15, P1.7,1, P1.3, P1.9, P1.nt, and a B:8,19,23:P1.16 strain. A Cuban B:4:P1.15 strain (Cu385/83) was also included in the study. The specificities of bactericidal antibodies were analyzed by using mutant strains lacking a class 1 protein (PorA protein) or a class 5 protein or both. The results indicated that PorA and class 5 proteins are the main targets recognized by the bactericidal antibodies of vaccinees. Nonetheless, a complex pattern of recognition by bactericidal antibodies was found, and vaccinees were grouped according to antibody specificity. Antibodies from some individuals recognized PorA of serosubtype P1.15. However, antibodies from these individuals could not kill all P1.15 strains tested. Antibodies from a second group recognized both PorA and class 5 proteins, and antibodies from a third group recognized an as yet unidentified target antigen. The results demonstrate the importance of determining the fine epitope specificity of bactericidal antibodies to improve the existing vaccines against B meningococci.

Targeting transposition: at home in the genome.

The completion of the Saccharomyces genome sequence in 1996 signaled the beginning of a new chapter in repetitive element sequence analysis (Cherry et al. 1997). The genomic analysis in the article by Kim et al. (1998) in this issue provides new insights into the nature and distribution of repetitive DNA sequences in the Saccharomyces genome. It not only assesses the repetitive sequences present in the genome but also clarifies what is not found. Saccharomyces has five long terminal repeat (LTR)– retrotransposon families (Ty elements) but no nuclear LINE-like or SINE retroelements and no identifiable DNA-based transposable elements. Remarkably, each Ty family displays insertion specificity. The parallels and differences between the organization of transposable elements in the Saccharomyces genome, and what is thus far known of the organization of elements in the genomes of other eukaryotes, suggest that this chapter will have many interesting sequelae. The genomic analysis by Kim et al. (1998) further defines the relationships of the Ty families of yeast comprised of four copia-like (Ty1, Ty2, Ty4, and Ty5) and one gypsy-like (Ty3) families. The copia-like families have been divided further based on similarity of encoded proteins and LTRs. Ty1 and Ty2 share significant similarity, particularly in the capsid domain, and share a common LTR, called d. In addition, Kim et al. showed for the first time, that Ty1 and Ty2 d elements are distinct—differing consistently at a single position. Unlike retrovirus LTRs, yeast element LTRs flanking the internal domain undergo recombination, thereby deleting one copy of the LTR and the internal domain sequence. Interestingly, among the families, there are widely differing copy numbers of complete elements and LTRs: 32 Ty1 elements and 217 Ty1-type d elements; 13 Ty2 elements and 34 Ty2type d elements; two Ty3 elements and 41 s elements; 3 Ty4 elements and 32 t elements; and one Ty5 element and seven v elements. Thus, ratios of LTR sequences to complete elements range from 7 for Ty1 to 21 for Ty3. Because there are many more Ty1 insertions than Ty3 insertions and because the Ty1-type d elements are more degenerate than the s elements, Ty1 probably represents an older class of elements within yeast. The fact that the ratio of LTR to complete copies is so much higher for Ty3 suggests that generation of isolated LTRs occurs more frequently for some sequences than for others. With stunning completeness, the genome sequence reveals the nonrandom nature of the retroelement insertions for each family (see Fig. 1) (Chalker and Sandmeyer 1992; Devine and Boeke 1996; Zou et al. 1996; Bryk et al. 1997; Smith and Boeke 1997). In addition to tRNA genes, 5S, U6, and PI RNA genes were also found associated with insertions. The Tyl, Ty2, Ty3, and Ty4 elements target the upstream region of RNA polymerase 111-transcribed genes: 196 of 217 Ty1; 28 of 34 Ty2; 40 of 41 Ty3; and 30 of 32 Ty4 insertions are within 750 bp of a gene transcribed by RNA polymerase III. This listing is conservative because it does not exclude the possibility that some of the seemingly exceptional insertions are associated with unidentified targets, such as unknown or degenerate polymerase IIItranscribed genes. Ty5 does not target tRNA genes. Instead, Ty5 inserts into silenced regions of the genome, the telomeres and mating type loci. In addition to these important insights, the analysis of Kim et al. (1998) raises provocative questions concerning the relationship between repetitive elements and their habitats: Why is Saccharomyces apparently lacking major classes of elements, including quite ancient ones found in bacteria; what is the impact of these elements on genome maintenance; and why do these elements target particular genomic regions? These questions are addressed in the remainder of this article.

Insulin-like Growth Factor-I Receptor and Insulin Receptor Association with a Src Homology-2 Domain-containing Putative Adapter

Insulin receptor (IR) and the related insulin-like growth factor-I (IGF-I) receptor (IGF-IR) mediate a variety of metabolic and mitogenic cellular responses, some of which may involve unidentified receptor targets. A Src homology-2 (SH2) domain-coding region of a mouse protein was cloned based on its interaction with IR. It was designated mSH2-B based on its high similarity to an earlier reported rat sequence SH2-B. A role of mSH2-B in IGF-I and insulin action was suggested by the interaction of the SH2 domain with activated IGF-IR and IR catalytic fragments but not with an inactive IR catalytic fragment in the yeast two-hybrid system in vivo and by the hormone-dependent association of a glutathione S-transferase (GST) SH2 domain fusion protein of mSH2-B with both receptors in cell extracts. A comparison of IGF-IR and IR mutants lacking individual Tyr autophosphorylation sites for association with GST mSH2-B showed that homologous juxtamembrane (IR960/IGF-IR950) and C-terminal (IR1322/IGF-IR1316) receptor motifs were required. Synthetic phosphopeptides representing IR960 and IR1322 competed for GST mSH2-B binding to the receptor, suggesting that both motifs participate in the association with mSH2-B. Antibodies raised against GST mSH2-B identified a cellular protein of 92 kDa that was not found to be phosphorylated on Tyr. It co-immunoprecipitated with IGF-IR or IR, which was strictly dependent on receptor activation. IR and IGF-IR Tyr phosphorylation motifs were not identified in the complete SH2-B primary structure, suggesting that it may participate as an adapter rather than a substrate in the IGF-I and insulin signaling pathways.

The held out wings (how) Drosophila gene encodes a putative RNA-binding protein involved in the control of muscular and cardiac activity.

In an attempt to identify genes that are involved in Drosophila embryonic cardiac development, we have cloned and characterized a gene whose function is required late in embryogenesis to control heart rate and muscular activity. This gene has been named held out wings (how) because hypomorphic mutant alleles produce adult animals that have lost their ability to fly and that keep their wings horizontal at a 90 degree angle from the body axis. In contrast to the late phenotype observed in null mutants, the How protein is expressed early in the invaginating mesoderm and this expression is apparently under the control of twist. When the different mesodermal lineages segregate, the expression of How becomes restricted to the myogenic lineage, including the cardioblasts and probably all the myoblasts. Antibodies directed against the protein demonstrate that How is localized to the nucleus. how encodes a protein containing one KH-domain which has been implicated in binding RNA. how is highly related to the mouse quaking gene which plays a role at least in myelination and that could serve to link a signal transduction pathway to the control of mRNA metabolism. The properties of the how gene described herein suggest that this gene participates in the control of expression of as yet unidentified target mRNAs coding for proteins essential to cardiac and muscular activity.

Activation of Mitochondrial 2-Oxoacid Dehydrogenases by Thioredoxin

The regulation of mitochondrial dehydrogenases of 2-oxoacids by thioredoxin is established. It is found that at low NAD+ and saturating concentrations of 2-oxoacids and CoA, inactivation of 2-oxoacid dehydrogenase complexes takes place, preventing NAD+ reduction under such conditions. However, addition of oxidized E. coli thioredoxin to the reaction medium without dithiothreitol allows effective NAD+ reduction at this substrate ratio. Product accumulation curves show that thioredoxin activates the complexes by protecting them from the inactivation observed in the conditions when the complex-bound dihydrolipoate is accumulated. Disappearance of the activatory effect of thioredoxin after its treatment with SH-specific reagents indicates the involvement of the redox-active cysteine couple of thioredoxin in its activation of 2-oxoacid dehydrogenase complexes. The redox-inactive thioredoxin not only shows no activation, but in fact exerts an inhibitory effect. The inhibition manifests the complex formation between SH-modified thioredoxin and dehydrogenase systems, involving amino acid residues of thioredoxin other than cysteine. High efficiency of thioredoxin from E. coli as compared to chloroplast thioredoxin f and glutathione disulfide is revealed. This indicates the importance of specific protein structure also for the influence of the redox-active thioredoxin upon the 2-oxoacid dehydrogenase complexes. The results obtained suggest that these key enzyme systems of mitochondrial metabolism represent previously unidentified targets for the action of mitochondrial thioredoxin, which is known to resemble the E. coli counterpart studies in this work.

Direct Association between the Ret Receptor Tyrosine Kinase and the Src Homology 2-containing Adapter Protein Grb7

Adapter proteins containing Src homology 2 (SH2) domains link transmembrane receptor protein-tyrosine kinases to downstream signal transducing molecules. A family of SH2 containing adapter proteins including Grb7 and Grb10 has been recently identified. We had previously shown that Grb10 associates with Ret via its SH2 domain in an activation-dependent manner (Pandey, A., Duan, H., Di Fiore, P.P., and Dixit, V.M. (1995) J. Biol, Chem. 270, 21461-21463). We now demonstrate that the related adapter molecule Grb7 also associates with Ret in vitro and in vivo, and that the binding of the SH2 domain of Grb7 to Ret is direct. This binding is dependent upon Ret autophosphorylation since Grb7 is incapable of binding a kinase-defective mutant of Ret. Thus two members of the Grb family, Grb7 and Grb10, likely relay signals emanating from Ret to other, as yet, unidentified targets within the cell.

Role of EGR1 in Regulation of Stimulus-Dependent CD44 Transcription in B Lymphocytes

The immediate-early gene egr-1 encodes a transcription factor (EGR1) that links B-cell antigen receptor (BCR) signals to downstream activation events through the regulation of previously unidentified target genes. Here we identify the gene encoding the lymphocyte homing and migration protein CD44 as a target of EGR1 regulation in B cells. BCR-induced increases in CD44 mRNA expression and transcription levels are shown to occur in EGR1-expressing but not in nonexpressing subclones of the B-cell line WEHI-231. Kinetics of egr-1 transcription and the appearance of nuclear EGR1 protein precede CD44 induction and occur within 30 min after stimulation in the EGR1-expressing subclone. A single EGR1 binding motif is demonstrated at bp -301 of the human CD44 promoter. Cotransfection of a CD44 promoter-chloramphenicol acetyltransferase reporter construct with an egr-1 expression vector resulted in a 6.5- to 8.5-fold induction of transcriptional activity relative to an empty expression vector. The EGR1 binding motif was shown to be necessary for stimulus-induced expression of a CD44 promoter-chloramphenicol acetyltransferase reporter construct in nontransformed B lymphocytes and was required for transactivation by an EGR1 expression vector in a B-cell line. These studies identify EGR1 as an intermediary linking BCR-derived signals to the induction of CD44. The relevance of these molecular events to BCR signal transduction and antigen-stimulated B-cell-mediated immune responses is discussed.

Conformational changes and calcium binding by calretinin and its recombinant fragments containing different sets of EF hand motifs.

Four recombinant fragments, representing different sets of EF-hand motifs of rat calretinin (CR) (I-II, I-III, III-VI, IV-VI), were prepared, and their Ca2+ -induced conformational changes were compared with those of full-length recombinant CR. All fragments were able to bind calcium ions as shown by 45Ca2+ overlay method on nitrocellulose and fluorescence measurements. The intrinsic tryptophan fluorescence intensity (FI) of apo-CR reversibly increased about 3-fold upon addition of calcium, indicating a change of conformation. The FI of fragments I-II (Trp 25) and I-III (Trp 25 and 116) increased about 1.4-fold on calcium binding, but that of fragment III-VI (Trp 116) increased 3.5-fold. Calcium titration of CR monitored by Trp fluorescence intensity showed that recombinant CR and some fragments bound Ca2+ with high affinity (Kd below 0.4 microM) and with high cooperativity. An apparent Hill coefficient for Ca2+ -induced fluorescence changes in CR was about 3.7. CR bound to organomercurial-agarose Cys 101 and 266 did not form cystine. The fluorescence intensities of cysteine-linked fluorescent probes 5-iodoacetamidofluorescein and N-(1-pyreneiodoacetamide) were increased approximately 1.3-fold upon calcium binding by CR. These data indicate that CR binds Ca2+ with high affinity and cooperativity and that this binding induces a change of conformation that involves the interaction of different parts of the molecule. Taken together, our results suggest that CR works as an on/off switch within a narrow range of free Ca2+ by interacting with as yet unidentified targets.