RESUMEN

Axl is a receptor tyrosine kinase (RTK) with oncogenic potential and transforming activity. Since Axl bears structural similarities to cell adhesion molecules such as neural cell adhesion molecule (NCAM) (FNIII domains), it is thought that Axl might play a role in adhesion. In this study, we have analysed the expression of the Axl protein and its ligand, Gas6, in human lung cancer cell lines of different histological origin. Axl expression occurred in approximately 60% of non-small cell lung cancer (NSCLC) cell lines, which grow adherently, and in normal bronchial epithelial cells (NHBE), but not in cell lines of small cell lung cancer origin (SCLC), which grow in suspension. A number of SCLC sublines, which could be selected spontaneously or after oncogene transfection for adherent growth, all expressed Axl protein. Overexpression of Axl per se, however, did not induce any change in the adhesion phenotype. All Axl-expressing cell lines demonstrated a membrane-bound 140 kD form, as well as a soluble 85 kD form, detectable in supernatant, of Axl-RTK. Expression of the Axl ligand Gas6 was detected in approximately 80% of all cell lines investigated. We conclude from these data that loss of Axl expression is a feature of SCLC tumour cells. Axl expression appears to be a consequence of cellular adhesion and possibly influences differentiation in human lung cancers.

Asunto(s)

RESUMEN

The individual zinc endoproteinases of the tissue degrading matrix metalloproteinase (MMP) family share a common catalytic architecture but are differentiated with respect to substrate specificity, localization, and activation. Variation in domain structure and more subtle structural differences control their characteristic specificity profiles for substrates from among four distinct classes (Nagase, H., and Woessner, J. F. J. (1999) J. Biol. Chem. 274, 21491-21494). Exploitation of these differences may be decisive for the design of anticancer or other drugs, which should be highly selective for their particular MMP targets. Based on the 1.8-A crystal structure of human neutrophil collagenase (MMP-8) in complex with an active site-directed inhibitor (RO200-1770), we identify and describe new structural determinants for substrate and inhibitor recognition in addition to the primary substrate recognition sites. RO200-1770 induces a major rearrangement at a position relevant to substrate recognition near the MMP-8 active site (Ala206-Asn218). In stromelysin (MMP-3), competing stabilizing interactions at the analogous segment hinder a similar rearrangement, consistent with kinetic profiling of several MMPs. Despite the apparent dissimilarity of the inhibitors, the central 2-hydroxypyrimidine-4,6-dione (barbiturate) ring of the inhibitor RO200-1770 mimics the interactions of the hydroxamate-derived inhibitor batimastat (Grams, F., Reinemer, P., Powers, J. C., Kleine, T., Pieper, M., Tschesche, H., Huber, R., and Bode, W. (1995) Eur. J. Biochem. 228, 830-841) for binding to MMP-8. The two additional phenyl and piperidyl ring substituents of the inhibitor bind into the S1' and S2' pockets of MMP-8, respectively. The crystal lattice contains a hydrogen bond between the O(gamma) group of Ser209 and N(delta)1 of His207 of a symmetry related molecule; this interaction suggests a model for recognition of hydroxyprolines present in physiological substrates. We also identify a collagenase-characteristic cis-peptide bond, Asn188-Tyr189, on a loop essential for collagenolytic activity. The sequence conservation pattern at this position marks this cis-peptide bond as a determinant for triple-helical collagen recognition and processing.

Asunto(s)

RESUMEN

The Escherichia coli ribosomal protein L7/L12 is central to the translocation step of translation, and it is known to be flexible under some conditions. The assignment of electron density to L7/L12 was not possible in the recent 2.4 A resolution x-ray crystallographic structure (Ban, N., Nissen, P., Hansen, J., Moore, P. B., and Steitz, T. A. (2000) Science 289, 905-920). We have localized the two dimers of L7/L12 within the structure of the 70 S ribosome using two reconstitution approaches together with cryo-electron microscopy and single particle reconstruction. First, the structures were determined for ribosomal cores from which protein L7/L12 had been removed by treatment with NH(4)Cl and ethanol and for reconstituted ribosomes in which purified L7/L12 had been restored to core particles. Difference mapping revealed that the reconstituted ribosomes had additional density within the L7/L12 shoulder next to protein L11. Second, ribosomes were reconstituted using an L7/L12 variant in which a single cysteine at position 89 in the C-terminal domain was modified with Nanogold (Nanoprobes, Inc.), a 14 A gold derivative. The reconstruction from cryo-electron microscopy images and difference mapping placed the gold at four interfacial positions. The finding of multiple sites for the C-terminal domain of L7/L12 suggests that the conformation of this protein may change during the steps of elongation and translocation.

Asunto(s)

RESUMEN

BACKGROUND: Recent preclinical studies have shown that lithium (Li) robustly increases the levels of the major neuroprotective protein, bcl-2, in rat brain and in cells of human neuronal origin. These effects are accompanied by striking neuroprotective effects in vitro and in the rodent central nervous system in vivo. We have undertaken the present study to determine if lithium exerts neurotrophic/ neuroprotective effects in the human brain in vivo. METHODS: Using quantitative proton magnetic resonance spectroscopy, N-acetyl-aspartate (NAA) levels (a putative marker of neuronal viability and function) were investigated longitudinally in 21 adult subjects (12 medication-free bipolar affective disorder patients and 9 healthy volunteers). Regional brain NAA levels were measured at baseline and following 4 weeks of lithium (administered in a blinded manner). RESULTS: A significant increase in total brain NAA concentration was documented (p < .0217). NAA concentration increased in all brain regions investigated, including the frontal, temporal, parietal, and occipital lobes. CONCLUSIONS: This study demonstrates for the first time that Li administration at therapeutic doses increases brain NAA concentration. These findings provide intriguing indirect support for the contention that chronic lithium increases neuronal viability/function in the human brain, and suggests that some of Li's long-term beneficial effects may be mediated by neurotrophic/neuroprotective events.

Asunto(s)

RESUMEN

OBJECTIVE: To determine whether the currently available evidence supports the hypothesis that antidepressants and mood stabilizers may bring about some of their long-term therapeutic effects by regulating signal transduction pathways and gene expression in the central nervous system. METHODS: To address this question, we reviewed the evidence showing that chronic administration of antidepressants and mood stabilizers involves alterations in signaling pathways and gene expression in the central nervous system. RESULTS: A large body of data has shown that lithium and valproate exert effects on the protein kinase C signaling pathway and the activator protein 1 family of transcription factors; in contrast, antidepressants affect the cyclic adenosine monophosphate pathway and may bring about their therapeutic effects by modulating cyclic adenosine monophosphate-regulated gene expression in the central nervous system. CONCLUSIONS: Given the key roles of these signaling cascades in the amplification and integration of signals in the central nervous system, the findings have clear implications not only for research into the etiology and pathophysiology of the severe mood disorders but also for the development of novel and innovative treatment strategies.

Asunto(s)

Asunto(s)

RESUMEN

In an attempt to find the key to reducing the excessive morbidity and mortality seen with mood disorders, our laboratory has been extensively investigating lithium's mechanisms of action in an integrated series of clinical and preclinical studies. We have found that the chronic administration of the 2 structurally highly dissimilar agents, lithium and valproate, brings about a strikingly similar reduction in protein kinase C (PKC) alpha and epsilon isozymes in rat frontal cortex and hippocampus. In view of PKC's critical role in regulating neuronal excitability and neurotransmitter release, we have postulated that PKC inhibition may have antimanic efficacy. In a small study, we have found that tamoxifen (which, in addition to its estrogen receptor blockade, is also a PKC inhibitor) has marked antimanic efficacy. These exciting preliminary results suggest that PKC inhibitors may represent a novel class of improved therapeutic agents for bipolar disorder, and this is under further investigation. The beneficial effects of mood stabilizers require a lag period for onset of action and are generally not immediately reversed upon drug discontinuation; such patterns of effects suggest alterations at the genomic level. We have therefore undertaken a series of studies to investigate the effects of these agents on the AP-1 family of transcription factors and have found that both drugs increase AP-1 DNA binding activity in areas of rodent brain ex vivo and in human neuronal cells in culture. Both treatments also increase the expression of a reporter gene driven by an AP-1-containing promoter, and mutations in the AP-1 sites of the reporter gene promoter markedly attenuate these effects. Both treatments also increase the expression of several endogenous proteins, whose genes are known to be regulated by AP-1. Although the precise mechanisms have not been fully elucidated, preliminary results suggest that these effects may be mediated, in part, by mitogen-activating protein kinases and glycogen synthase kinase 3beta. We have also utilized mRNA reverse transcription-polymerase chain reaction (RT-PCR) differential display to identify concordant changes in gene expression induced by the chronic administration of both lithium and valproate. We have identified concordant changes in a number of cDNA bands by both lithium and valproate. Cloning and characterizing of these genes is currently underway. The identification of the functions of these genes offers the potential not only for improved therapeutics for reducing the morbidity and mortality associated with mood disorders, but may also provide important clues about the underlying pathophysiology.

Asunto(s)

RESUMEN

Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are generally regarded as eosinophil-specific proteins. We tested whether EDN and ECP are present in mature neutrophils. By indirect immunofluorescence, both eosinophils and neutrophils stained with antibodies to EDN and ECP. Lysates of purified (<0.1% eosinophil contamination) neutrophils contained EDN, 112+/-4 ng/10(6) cells, and ECP, 163+/-2 ng/10(6) cells, whereas eosinophil major basic protein (MBP) was not detectable. Electron microscopic examination of immunogold-labeled buffy coat cells stained with EDN antibody showed that EDN is localized to neutrophil granules. Finally, EDN mRNA was detected in lysates of highly purified neutrophils (0.001% eosinophil contamination) by the reverse transcription-polymerase chain reaction. We conclude that proteins that are either identical to or immunologically cross-reactive with EDN and ECP are present in neutrophils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exercised in interpreting the presence of EDN and ECP as specific markers of eosinophil-associated inflammation in human disease.

Asunto(s)

RESUMEN

Significant amounts of ribonuclease inhibitor protein are present in human and rat erythrocytes, cells that are essentially devoid of ribonuclease or functional RNA. The protein from human erythrocytes is indistinguishable from human placental ribonuclease inhibitor protein by immunological and biochemical criteria. Each inhibitor forms an equimolar complex with bovine pancreatic ribonuclease A and is inactivated by treatment with the sulfhydryl reagent p-(hydroxymercuri)benzoate. Amino acid composition and several cycles of amino acid sequence analysis also showed apparent identify of the erythrocyte and placental proteins. We calculate a level of 1.5-3.5 x 10(4) molecules of active inhibitor per erythrocyte, most or all of which occurs in an uncomplexed form since inactivation of the inhibitor revealed barely detectable levels of RNase activity. Immunogold localization showed a high level of labeling and a uniform distribution of gold particles in the cytoplasm of erythrocytes, while little inhibitor activity was found in association with isolated red blood cell membranes. Oxidative stress on isolated red cells resulted in a decrease in the level of reduced glutathione and a gradual and irreversible loss of inhibitor activity; inhibitor disappeared from the cytosol and became associated with nascent Heinz bodies. We suggest a role for this protein in the metabolism and aging process of the erythrocyte.

Asunto(s)

RESUMEN

Escherichia coli ribosomal protein L7/L12 occurs on the large subunit as two dimers: one dimer is extended and comprises the stalk, while the second dimer is folded and occupies a site on the subunit body. A variant protein, in which all 18 amino acids of the flexible hinge region that links separate N-terminal and C-terminal domains of L7/L12 has been deleted, binds the subunit as a single dimer and does not generate stalks that are visible in electron micrographs. Monoclonal antibodies directed against each domain of the protein have been used to localize the variant in electron micrographs of 50S subunits. Both C-terminal domains are seen at a shoulder of the subunit, near its edge as viewed in the most common quasisymmetric projection. N-terminal domains are placed on the subunit body, about 50 A from the C-terminal domains. The antibody to the N-terminal domain also causes dissociation of the variant dimer from the particle and the formation of oligomeric antibody-protein dimer complexes. Similar complexes were seen previously (Olson HM et al (1986) J Biol Chem 261, 6924-6936) when this antibody induced dissociation of one dimer of the native protein. We conclude that the shortened variant most probably occupies the lower-affinity site on the subunit that is normally filled by the stalk dimer.

Asunto(s)

RESUMEN

Escherichia coli ribosomal protein L23 was derivatized with [3H]2, 4-dinitrofluorobenzene both at the N terminus and at internal lysines. Dinitrophenyl-L23 (DNP-L23) was taken up into 50 S subunits from a reconstitution mixture containing rRNA and total 50 S protein depleted in L23. Unmodified L23 competed with DNP-L23 for uptake, indicating that each protein form bound in an identical or similar position within the subunit. Modified L23, incorporated at a level of 0.7 or 0.4 DNP groups per 50 S, was localized by electron microscopy of subunits complexed with antibodies to dinitrophenol. Antibodies were seen at two major sites with almost equal frequency. One site is beside the central protuberance, in a region previously identified as the peptidyltransferase center. The second location is at the base of the subunit, in the area of the exit site from which the growing peptide leaves the ribosome. Models derived from image reconstruction show hollows or canyons in the subunit and a tunnel that links the transferase and exit sites. Our results indicate that L23 is at the subunit interior, with separate elements of the protein at the subunit surface at or near both ends of this tunnel.

Asunto(s)

RESUMEN

Pre-mRNA splicing occurs on spliceosomes, a family of ribonucleoprotein particles. Spliceosome assembly on exogenous adenovirus pre-mRNA was blocked at the A-complex (or pre-spliceosome) stage, either by destruction of the small nuclear ribonucleoproteins (snRNPs) that comprise the U4/U5/U6 tri-snRNP complex, or by interference in tri-snRNP assembly and interactions. The A-complex was isolated by size exclusion chromatography; homogeneity was shown by electrophoresis in nondenaturing polyacrylamide gels, gradient sedimentation, and electron microscopy. Northern hybridization showed U1 and U2 snRNAs to be present in the preparation, but not U4, U5, or U6. Antibodies specific for a component of the U1 snRNP or for a component that is common to all snRNPs (except U6) each precipitated an A-complex containing pre-mRNA, U1 and U2 snRNPs. Electron micrographs showed 230 x 270-A particles whose two components appear similar to individual U1 and U2 snRNPs. Electron micrographs of an A-complex-5'-biotinyl oligonucleotide-streptavidin-gold composite allowed identification of the U2 snRNP within the structure and the localization of the 5'-segment of U2 snRNA at a unique site in the A-complex. This region of U2 RNA is adjacent to the developing catalytic center of the spliceosome.

Asunto(s)

RESUMEN

Oligodeoxynucleotide models of mRNA were used to determine the ribosomal site of specific nucleotides 3' to the initiation codon. Each mRNA analog had a 5'-terminal 9-base Shine-Dalgarno sequence, a 7-nucleotide spacer, and an ATG initiation signal, followed by up to 31 nucleotides, one of which carried an antibody-recognizable marker. All probes bound efficiently to activated Escherichia coli 30 S ribosomal subunits. Complexes were formed using ribosomal subunits, initiator tRNA, an oligodeoxynucleotide probe, and antibodies. Electron microscopy was then used to place specific positions in the mRNA analog on the subunit and thus to trace the pathway of the messenger. As viewed from the cytoplasmic side of the subunit, the 5' segment of the mRNA lies on the left, along the inner surface of the platform. The initiation codon and the next 9 nucleotides are located in the cleft between the subunit platform and body; within this segment the mRNA makes a U turn and emerges from the cleft at the left of the neck that separates the subunit head and body. The mRNA then loops around the neck to the right, along the cytoplasmic surface of the subunit and toward the site of the 7-methylguanosine residue of the 16 S ribosomal RNA.

Asunto(s)

RESUMEN

This is the third paper in a series (Olah, T. V., Olson, H. M., Glitz, D. G., and Cooperman, B. S. (1988) J. Biol. Chem. 263, 4795-4800; Olson, H. M., Olah, T., Cooperman, B. S., and Glitz, D. G. (1988) J. Biol. Chem. 263, 4801-4806) describing the use of 2,4-dinitrophenyl (DNP) derivatives of Escherichia coli 30 S ribosomal proteins to locate the positions of these proteins within the 30 S subunit by immune electron microscopy. In it we describe the derivatization of proteins S6, S13, S16, and S18 with [3H]2,4-dinitrofluorobenzene, identify the nature of the derivatized amino acids within each protein, and demonstrate that each DNP protein, denoted DNP-Sx, can be taken up into a reconstituted 30 S subunit when added to a reconstitution mixture containing 16 S rRNA and total 30 S protein depleted in Sx. We further demonstrate that each DNP-Sx binds within the 30 S subunit in a position identical or similar to that of the unmodified Sx protein, as judged by its meeting one or more of the following three criteria: (i) unmodified Sx competes with the uptake of DNP-Sx into 30 S subunits; (ii) DNP-Sx restores functional activity to those single protein omission reconstitution particles lacking full activity; (iii) DNP-Sx induces the uptake of proteins into 30 S subunits that depend on the presence of Sx. The fourth paper in this series (Montesano-Roditis, L., McWilliams, R., Glitz, D. G., Olah, T. V., Perrault, A. R., and Cooperman, B. S. (1993) J. Biol. Chem. 268, 18701-18709), which follows this one, describes the localization of the DNP-Sx proteins within the 30 S subunit by immune electron microscopy.

Asunto(s)

RESUMEN

Purified Escherichia coli ribosomal proteins S6, S13, S16, and S18 were dinitrophenylated at their amino termini and/or at one or more internal lysine residues. Each dinitrophenyl protein was then separately incorporated into reconstituted small ribosomal subunits. Modified proteins were localized on the 30 S subunit surface by electron microscopy of reconstituted subunits complexed with antibodies to dinitrophenol (DNP). DNP protein S13 was placed on the subunit head above the platform and on the surface that faces the large subunit. DNP-S18 was localized to the subunit platform below the tip and in a region associated with binding to 50 S subunits. DNP proteins S6 and S16 were both localized near the junction of the subunit body and platform; DNP-S6 was available to antibody in 70 S ribosomes and was placed on the cytoplasm-facing side of the subunit in an area that overlaps the platform and body of the particle. DNP-S16 in 70 S ribosomes was not bound by antibody. It was localized to the 30 S body near its junction with the platform and on the surface facing the 50 S particle. The results complement and clarify data obtained using other approaches.

Asunto(s)

RESUMEN

We have previously found decreased standard deviations and mean consecutive differences of R-R intervals in panic disorder patients in standing posture, compared with control subjects. In the present study, we used spectral analysis of heart rate variability to examine autonomic function in 21 panic disorder patients and 21 normal control subjects. Patients had a significantly lower standard deviation of heart rate in supine as well as standing postures. Absolute low frequency power (0.01-0.05 Hz) was also significantly lower in panic disorder patients in standing postures. Upon standing, the panic disorder patients had significantly higher relative mid-frequency power (0.07-0.15 Hz). During a standing deep-breathing condition at six breaths per minute, the patients had a significantly decreased absolute and relative mid-frequency (0.07-0.15 Hz) power compared with control subjects. These findings suggest a decrease in cholinergic and a relative increase in adrenergic responsiveness in panic disorder patients compared with control subjects.

Asunto(s)

RESUMEN

Eosinophil-derived neurotoxin (EDN) and human liver RNase were found to be indistinguishable from each other but distinct from the pancreatic ribonucleases in their nucleolytic activity on polynucleotides or small defined substrates. Antibodies to EDN and liver RNase showed identical cross-reactivities in assays of nuclease inhibition and in a radioimmunoassay. In each instance, EDN and liver RNase were easily distinguished from bovine or human pancreatic RNase. When injected intrathecally into rabbits, 5-10 micrograms of EDN or liver RNase each was neurotoxic as judged by induction of the Gordon phenomenon. Human pancreatic RNase was less neurotoxic, and up to 20-fold higher levels of bovine pancreatic RNase showed no effect. Treatment of EDN, liver RNase, and eosinophil cationic protein with iodoacetic acid at pH 5.5 resulted in inactivation of their RNase activity and also destroyed their neurotoxicity. EDN conformation was not greatly affected by iodoacetate treatment since interaction of the modified protein with antibodies was only slightly altered. We conclude that RNase activity is necessary but not sufficient to induce neurotoxic action.

Asunto(s)

RESUMEN

Human papillomavirus type 16 (HPV-16) transcription was analysed in one squamous cervical carcinoma by cDNA cloning and DNA sequencing, and in eight additional squamous cervical carcinomas and 11 precancerous lesions by RNA-RNA in situ hybridization. The nucleotide sequences of the cDNA clones revealed structures of early HPV-16 mRNAs (E6*-E7-E1 E4-E5) in agreement with data reported for other premalignant and malignant tumours. cDNA clones possibly representing viral RNA of antisense orientation were also detected. These RNAs included sequences of the upstream regulatory region, part of the early and the late region of the genome. In three of eight squamous cervical carcinomas examined by in situ hybridization, signals specific for viral antisense RNA were also found. The antisense RNAs had a predominantly nuclear localization. Viral antisense RNA could not be detected in any of 11 HPV-16-positive premalignant lesions. The expression of HPV antisense RNA is likely to be linked to viral integration into the host genome. The possible effects of viral antisense transcription with regard to tumour progression remain to be determined.

Asunto(s)

RESUMEN

We examined adrenergic regulation in patients with panic disorder by challenging 10 patients and 14 age-matched and sex-matched controls with intravenous infusions of clonidine hydrochloride (2 micrograms/kg), an alpha 2-adrenoreceptor agonist. Growth hormone, 3-methoxy-4-hydroxyphenylglycol (MHPG), blood pressure, heart rate, and behavioral (anxiety, sedation) responses were monitored. The data replicated the previously reported finding of blunted growth hormone (GH) responses to clonidine in patients with panic disorder. Reported abnormalities in MHPG, cardiovascular, and behavioral responses of panic patients to clonidine infusion were not replicated. The robustly blunted GH response to clonidine in panic patients supports the adrenergic dysregulation hypothesis of panic disorder, but alternative interpretations of this finding are available and further study is needed.

Asunto(s)

RESUMEN

We have analyzed human papillomavirus (HPV) type 16 RNA expression in premalignant cervical lesions of different severity and in squamous cervical cancers by RNA-RNA in situ hybridization in order to find differences in the topographic distribution of viral RNA, which might correlate with the severity of disease. In the basal layer of low-grade squamous intraepithelial lesions (SIL) only weak transcription of viral early genes was observed. Signal intensity increased strongly in the more differentiated cells accompanied by high levels of HPV DNA replication. This pattern of viral gene expression, together with the onset of viral late transcription in the upper differentiated layer of the epithelium, most likely reflects the productive phase of viral infection. In contrast, in high-grade SIL viral transcription was comparatively strong in basal cells and evenly distributed throughout the undifferentiated epithelium. This difference of viral transcription in the basal layer of the respective lesions points to an altered regulation of viral gene expression which may be causally linked to the progression of precursor lesions. Evidence for disrupted expression of 3' early genes (E2, E4, and E5), analogous to the situation in HPV-DNA containing cervical carcinoma-derived cell lines, was not found in any of the HPV-16-positive premalignant lesions nor in the majority of cancers. The similarity of the viral transcription pattern of high-grade SIL and cancers suggests that additional host gene alterations are necessary for malignant progression.

Asunto(s)