Enigma Prevents Cbl-c-Mediated Ubiquitination and Degradation of RETMEN2A

The Cbl proteins (Cbl, Cbl-b, and Cbl-c) are a highly conserved family of RING finger ubiquitin ligases (E3s) that function as negative regulators of tyrosine kinases in a wide variety of signal transduction pathways. In this study, we identify a new Cbl-c interacting protein, Enigma (PDLIM7). This interaction is specific to Cbl-c as Enigma fails to bind either of its closely related homologues, Cbl and Cbl-b. The binding between Enigma and Cbl-c is mediated through the LIM domains of Enigma as removal of all three LIM domains abrogates this interaction, while only LIM1 is sufficient for binding. Here we show that Cbl-c binds wild-type and MEN2A isoforms of the receptor tyrosine kinase, RET, and that Cbl-c enhances ubiquitination and degradation of activated RET. Enigma blocks Cbl-c-mediated RETMEN2A ubiquitination and degradation. Cbl-c decreased downstream ERK activation by RETMEN2A and co-expression of Enigma blocked the Cbl-c-mediated decrease in ERK activation. Enigma showed no detectable effect on Cbl-c-mediated ubiquitination of activated EGFR suggesting that this effect is specific to RET. Through mapping studies, we show that Cbl-c and Enigma bind RETMEN2A at different residues. However, binding of Enigma to RETMENA prevents Cbl-c recruitment to RETMEN2A. Consistent with these biochemical data, exploratory analyses of breast cancer patients with high expression of RET suggest that high expression of Cbl-c correlates with a good outcome, and high expression of Enigma correlates with a poor outcome. Together, these data demonstrate that Cbl-c can ubiquitinate and downregulate RETMEN2A and implicate Enigma as a positive regulator of RETMEN2A through blocking of Cbl-mediated ubiquitination and degradation.     

Conversion of Neuropeptide K to Neurokinin A and Vesicular Colocalization of Neurokinin A and Substance P in Neurons of the Guinea Pig Small Intestine

The highest concentration of neurokinin A-like immunoreactivity and substance P-like immunoreactivity in the guinea pig small intestine was associated with the myenteric plexus-containing longitudinal muscle layer. Chromatographic analysis of extracts of this tissue demonstrated the presence of neurokinin A and neuropeptide K but the probable absence of neurokinin B. A fraction of synaptic vesicles of density 1.133 +/- 0.003 g/ml was prepared from the myenteric plexus-containing tissue by density gradient centrifugation in a zonal rotor and was enriched 29 +/- 12-fold in the concentration of neurokinin A-like immunoreactivity and 43 +/- 13-fold in the concentration of substance P-like immunoreactivity. This fraction was separated from the fraction of vasoactive intestinal peptide-containing vesicles (density, 1.154 +/- 0.009 g/ml). Chromatographic analysis of lysates of the vesicles indicated the presence of neurokinin A but not neuropeptide K. It is postulated that beta-pre-protachykinin is processed to substance P, neurokinin A, and neuropeptide K in the cell bodies of myenteric plexus neurons but that conversion of neuropeptide K to neurokinin A takes place during packaging into storage vesicles for axonal transport. The data are consistent with the proposal that neurokinin A and substance P are stored in the same synaptic vesicle, but the possibility of cosedimentation of different vesicles of very similar density cannot be excluded.     

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.     

Separation of beta-casein peptides through UF inorganic membranes.

Ultrafiltration of peptide mixtures is studied under various operating conditions (transmembrane pressure, tangential flow-rate) using two ultrafiltration inorganic membranes M5 and M1 with molecular weight cut-offs, MWCO 10 and 70 kD, respectively. It is shown that the separation of peptides is controlled by a dual mechanism: size exclusion and electrostatic repulsion. When the ionic strength is high enough to screen out the electrostatic interactions, experimental data are in good agreement with a sieving model developed to estimate the intrinsic transmission from the molecular weight of a component and from the MWCO of the membranes. Although the transmission so found is altered by concentration polarisation and pore blocking mechanisms, the results explain the apparent low transmission of peptides by ultrafiltration membranes. If the ionic strength of the fluid is low, electrostatic interactions can influence the transport phenomena, provided that the molecules are highly charged (at pHs away from the pI). For attractive interactions, an apparent partition coefficient larger than 1 is observed. Otherwise, the transmission is lower than predicted by the sieving theoretical equation, as if the partition coefficient were smaller than 1.     

Selective separation of tryptic beta-casein peptides through ultrafiltration membranes: Influence of ionic interactions

Peptide separation by selective membrane filtration has numerous potential applications such as production of peptides with biological activities or spectific enrichment in compounds acting as flavoring agents or as growth factors required by the fermentation industry. The retention of peptides arising from tryptic hdroysis of beta-casein using an M5 Carbosep membrane (molecular wieght cutoll = 10,000 D) has been studied. The peptides with known sequences were characterized by their molecular weight, isoelectric point, and hydrophobicity. Our experiments highlighted that their transmission involves mechanisms other than size exclusion as developed elsewhere. The effect of ionic interactions between peptides and membrance has been investgated by vrying pH, ionic strength of bulk, and electric potential of filtering material. The charge of both peptides and membrane plays an important role in the transmission, particularly with small size and high or lkow isoelectric point. Then, peptides with the same sign as the membrane have lower transmission than expected from the size xclusion model, whereas peptides with opposite sign have higher trnsmission than expected, and even higher than 1 with some of them. (c) 1995 John Wiley & Sons, Inc.     

Spina bifida aperta induced by valproic acid and by all-trans-retinoic acid in the mouse: distinct differences in morphology and periods of sensitivity.

The antiepileptic drug valproic acid (VPA) has been implicated as a human teratogen causing spina bifida aperta. Recently, we developed a mouse model inducing spina bifida aperta with VPA. To elucidate the pathogenesis of VPA-induced spina bifida aperta we now investigated the anatomy and histology of this defect in the mouse. The morphology of spina bifida aperta induced by all-trans-retinoic acid (RA) was used for comparison. Various doses of VPA and RA were administered at different times to determine the periods of sensitivity for inducing spina bifida aperta with these drugs. Each administration regimen consisted of three doses applied at intervals of 6 hr. RA induced spina bifida aperta during an earlier developmental period (day 8 of gestation) than VPA (day 9 of gestation). The most effective regimens for induction of spina bifida aperta in mice were injections of 3 x 500 mg VPA-Na/kg body weight (b.w.) intraperitoneally on day 9 of gestation at 0, 6, and 12 hr; RA (12.5 mg/kg b.w.) was given orally on day 8 of gestation at 12 and 18 hr, day 9 at 0 hr. VPA did not induce spina bifida aperta on day 8 of gestation and RA did not induce this effect on day 9 of gestation. Histological studies of day 18 fetuses carrying spina bifida aperta were performed. The spina bifida aperta induced by VPA shows a disorganized and necrotic spinal cord. In the vertebral canal were observed cell debris, blood cells, capillaries, macrophages, and rests of meninges. These results indicate that the spinal cord is almost destroyed at the affected section. In contrast, the spina bifida aperta induced by RA demonstrates a spinal cord organized in the gray and white matter, the dorsal and ventral horn. But the neural canal does not exist, only a layer of ependymal cells lies on the surface of the spinal cord. Our results indicate that the morphology of spina bifida aperta induced by VPA differed distinctly from that induced by RA in the mouse fetus. Moreover VPA produced a spina bifida aperta with a specific morphology. Also the period of sensitivity for induction of this lesion differed and occurred earlier for RA than for VPA. VPA and RA may possibly induce spina bifida aperta via different mechanisms in the mouse.     

Gas chromatography-mass spectrometry for probing the structure and mechanism of action of enzyme active sites. The role of Glu-270 in carboxypeptidase A.

A new technique for the study of the mechanism of enzymes has been developed. An enzyme, modified by an active-site directed reagent, is digested by one or more proteases. The resulting mixture of oligopeptides is then analyzed directly by gas chromatography-mass spectrometry without the use of separation or isolation procedures. A comparison with unmodified enzyme identifies the modified residue as well as quantifies the reaction. This approach has been applied to the identification of Glu-270 in the active site of carboxypeptidase A using a carbodiimide as modification reagent. Studies on the possible incorporation of 18O (from 18O-enriched water) into Glu-270 or other acidic residues near the active site of carboxypeptidase A show that the oxygens of the carboxyl groups of these residues are not exchangeable.     

Corynebacterium jeikeium jk0268 Constitutes for the 40 Amino Acid Long PorACj,Which Forms a Homooligomeric and Anion-Selective Cell Wall Channel

Corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. jeikeium K411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. A gene coding for a 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium is discussed.     

Anthocyanin contribution to chlorophyll meter readings and its correction

Leaf chlorophyll content is an important physiological parameter which can serve as an indicator of nutritional status, plant stress or senescence. Signals proportional to the chlorophyll content can be measured non-destructively with instruments detecting leaf transmittance (e.g., SPAD-502) or reflectance (e.g., showing normalized differential vegetation index, NDVI) in red and near infrared spectral regions. The measurements are based on the assumption that only chlorophylls absorb in the examined red regions. However, there is a question whether accumulation of other pigments (e.g., anthocyanins) could in some cases affect the chlorophyll meter readings. To answer this question, we cultivated tomato plants (Solanum lycopersicum L.) for a long time under low light conditions and then exposed them for several weeks (4 h a day) to high sunlight containing the UV-A spectral region. The senescent leaves of these plants evolved a high relative content of anthocyanins and visually revealed a distinct blue color. The SPAD and NDVI data were collected and the spectra of diffusive transmittance and reflectance of the leaves were measured using an integration sphere. The content of anthocyanins and chlorophylls was measured analytically. Our results show that SPAD and NDVI measurement can be significantly affected by the accumulated anthocyanins in the leaves with relatively high anthocyanin content. To describe theoretically this effect of anthocyanins, concepts of a specific absorbance and a leaf spectral polarity were developed. Corrective procedures of the chlorophyll meter readings for the anthocyanin contribution are suggested both for the transmittance and reflectance mode.     

Low diversity and abundance of root endophytes prevail throughout the life cycle of an annual halophyte

Plants growing in highly saline soils harbor unique communities of fungal root endophytes. We aimed to gain insight into how these communities are established in natural plant populations. We used cultivation-based and molecular approaches to examine root-endophytic colonization in the annual halophyte Salicornia patula at three time points over a 5-month period, from establishment to flowering. At the last sampling, the endophytic community of S. patula was compared to that in the related but perennial halophyte Arthrocnemum macrostachyum. The presence of root endophytes in S. patula was negligible at the first two sampling times, and remained low at the last sampling compared to A. macrostachyum. The latter species showed a well-established endophytic community in its roots that differed from that in S. patula, which was dominated by members of Pleosporales. Although such differences could be partially due to the host lifestyle, the possibility of a strong effect of the substratum could not be excluded. Altogether, our data indicate that the fungal endophytic colonization of roots is a slow process under salt stress. Therefore, we suggest that, in contrast to what is proposed for other systems, endophyte symbioses are unlikely to impact the development of the short-life-cycled S. patula living in these environments.