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T Trenkle

Publications and source records attributed to T Trenkle.

10 recordsLinked to original sources
PubMed2000

Major transcript variants of VAV3, a new member of the VAV family of guanine nucleotide exchange factors.

VAV3 is a new member of the VAV oncogene family with a strong homology to VAV and VAV2. A conceptual translation of the cDNA indicates that VAV3 is between 40 and 77% identical to VAV and VAV2 at the amino acid level in all identified functional motifs. This homology suggests that VAV3 occupies a similar position in signal transduction as the other family members. A major variant transcript, VAV3.1, found in both humans and mice, appears to encode only the 3' SH3-SH2-SH3 region, which suggests that it may substitute for the full-length isoform in functions mediated by this domain, or compete with the full-length isoform in functions mediated by more N-terminal motifs. VAV3.1 either is a partly unspliced mRNA or originates from a different promoter. VAV3 transcripts are found in cells of hematopoietic origin, where VAV is primarily expressed. However, unlike, VAV, the VAV3 and VAV3.1 transcripts are also found at varying levels in a wide variety of other tissues and cell lines. TGF-beta and EGF reversibly down-regulate the abundance of the VAV3. 1 transcript in HaCaT keratinocytes, representing the first observation of transcript regulation of a member of the VAV family by a growth factor.

Amino Acid Sequence↗
PubMed2000

Vav3 mediates receptor protein tyrosine kinase signaling, regulates GTPase activity, modulates cell morphology, and induces cell transformation.

A recently reported new member of the Vav family proteins, Vav3 has been identified as a Ros receptor protein tyrosine kinase (RPTK) interacting protein by yeast two-hybrid screening. Northern analysis shows that Vav3 has a broad tissue expression profile that is distinct from those of Vav and Vav2. Two species of Vav3 transcripts, 3.4 and 5.4 kb, were detected with a differential expression pattern in various tissues. Transient expression of Vav in 293T and NIH 3T3 cells demonstrated that ligand stimulation of several RPTKs (epidermal growth factor receptor [EGFR], Ros, insulin receptor [IR], and insulin-like growth factor I receptor [IGFR]) led to tyrosine phosphorylation of Vav3 and its association with the receptors as well as their downstream signaling molecules, including Shc, Grb2, phospholipase C (PLC-gamma), and phosphatidylinositol 3 kinase. In vitro binding assays using glutathione S-transferase-fusion polypeptides containing the GTPase-binding domains of Rok-alpha, Pak, or Ack revealed that overexpression of Vav3 in NIH 3T3 cells resulted in the activation of Rac-1 and Cdc42 whereas a deletion mutant lacking the N-terminal calponin homology and acidic region domains activated RhoA and Rac-1 but lost the ability to activate Cdc42. Vav3 induced marked membrane ruffles and microspikes in NIH 3T3 cells, while the N-terminal truncation mutants of Vav3 significantly enhanced membrane ruffle formation but had a reduced ability to induce microspikes. Activation of IR further enhanced the ability of Vav3 to induce membrane ruffles, but IGFR activation specifically promoted Vav3-mediated microspike formation. N-terminal truncation of Vav3 activated its transforming potential, as measured by focus-formation assays. We conclude that Vav3 mediates RPTK signaling and regulates GTPase activity, its native and mutant forms are able to modulate cell morphology, and it has the potential to induce cell transformation.

3T3 Cells↗
PubMed1999

Differential display probes for cDNA arrays.

PCR with a combination of one arbitrary and one oligo(dT) anchor primer can be used to generate an effective probe for cDNA arrays. The method uses less than 1/200 of the amount of RNA used in some other array hybridization methods. Each fingerprint detects approximately 5% of the transcribed mRNAs, sampled almost independent of abundance, using inexpensive E. coli colony arrays of expressed sequence tag (EST) clones. It proved necessary to alter the differential display (DD) protocol to generate a sufficient mass of PCR products for use as a probe. The use of different oligo(dT) anchor primers with the same arbitrary primer resulted in considerable overlap among the genes sampled by each probe. This can be avoided by using different arbitrary primers with each oligo(dT) anchor primer. Four genes not previously known to be regulated by epidermal growth factor (EGF) and three genes known to be regulated by EGF in other cell types were characterized using DD fingerprints as probes for arrays. It should be possible to convert archived DD fingerprints into effective probes for arrays, allowing thousands of experiments that have already been performed to yield further information. The use of DD fingerprints as probes should increase the rate of identification of differentially regulated genes several fold while obviating the need for cloning and sequencing.

Cell Line↗
PubMed1998

Non-stoichiometric reduced complexity probes for cDNA arrays.

A method is presented in which the reduced complexity and non-stoichiometric amplification intrinsic to RNA arbitrarily primed PCR fingerprinting (RAP-PCR) is used to advantage to generate probes for differential screening of cDNA arrays. RAP-PCR fingerprints were converted to probes for human cDNA clones arrayed as Escherichia coli colonies on nylon membranes. Each array contained 18 432 cDNA clones from the IMAGE consortium. Hybridization to approximately 1000 cDNA clones was detected using each RAP-PCR probe. Different RAP-PCR fingerprints gave hybridization patterns having very little overlap (<3%) with each other or with hybridization patterns from total cDNA probes. Consequently, repeated application of RAP-PCR probes allows a greater fraction of the message population to be screened on this type of array than can be achieved with a radiolabeled total cDNA probe. This method was applied to RNA from HaCaT keratinocytes treated with epidermal growth factor. Two RAP-PCR probes detected hybridization to 2000 clones, from which 22 candidate differentially expressed genes were observed. Differential expression was tested for 15 of these clones using RT-PCR and 13 were confirmed. The use of this cDNA array to analyze RAP-PCR fingerprints allowed for an increase in detection of 10-20-fold over the conventional denaturing polyacrylamide gel approach to RAP-PCR or differential display. Throughput is vastly improved by the reduction in cloning and sequencing afforded by the use of arrays. Also, repeated cloning and sequencing of the same gene or of genes already known to be regulated in the system of interest is minimized. The procedure we describe uses inexpensive arrays of plasmid clones spotted as E.coli colonies to detect differential expression, but these reduced complexity probes should also prove useful on arrays of PCR-amplified fragments and on oligonucleotide chips. Genesobserved in this manuscript: H11520, U35048, R48633, H28735, M13918, H12999, H05639, X79781, M31627, H23972, AB000712, R75916, U66894, AF067817.

DNA, Complementary↗
PubMed1998

Estrogen-responsive RING finger mRNA induction in gastrointestinal carcinoma cells following bile acid treatment.

The underlying molecular mechanisms of the tumor-promoting activity of bile acids such as chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) and the protective effect of ursodeoxycholic acid (UDCA) remain largely unclear. Using RNA arbitrarily primed PCR (RAP-PCR) for differential display, we identified, cloned and sequenced differentially expressed transcripts after treating gastric carcinoma cells (St 23132) with the bile acids CDCA, DCA and UDCA. One of these transcripts was identified to be an estrogen-responsive RING finger protein (efp) mRNA. The differential expression of efp in gastric cancer cells was confirmed by low stringency RT-PCR. efp mRNA levels were induced 3-fold in gastric carcinoma cells after CDCA and DCA treatment, whereas no change in expression was detected after UDCA treatment. Finally, treatment of the colon carcinoma cell line HT 29 with DCA resulted in a 2- to 5-fold induction of efp mRNA levels whereas UDCA did not induce efp. As expected, efp expression was also increased after 24 h of estrogen treatment. In summary, a synergy or a common pathway of tumor enhancement of bile acids and estrogen via efp in gastrointestinal carcinogenesis can be envisioned.

Bile Acids and Salts↗
PubMed1998

GeneUp: a program to select short PCR primer pairs that occur in multiple members of sequence lists.

A computer program is presented that selects a small set of short primer pairs for PCR to sample all the sequences in a user-specified list of mRNAs. Such primer pairs could be used to increase the probability of sampling mRNAs of particular interest in differential display and to generate simplified hybridization probes for DNA chips or arrays. The program uses simulated PCR to find pairs of primers that sample more than one sequence in the list. A small set of such primer pairs is selected that give maximal coverage of the sequences in the list. Primer pairs are excluded that: (i) generate simulated PCR products of the same size from a number of sequences in the list, (ii) can easily form primer dimers, (iii) are outside a specified range of G + C content or (iv) occur in another list of undesirable sequences, such as rRNAs and Alu repeats. Five lists consisting of from 48-285 cDNA sequences were used to test the program. A small number of pairs of primers, 8-10 bases in length, were selected that fit the above criteria and that generate one or more simulated PCR products in all or most of the cDNAs in each list.

Base Sequence↗
PubMed1998

Expression of the ERBB2/neu and neurofibromatosis type 1 gene products in reactive and neoplastic schwann cell proliferation.

In the present study we investigated the expression of the ERBB2 protein and neurofibromin in human benign and malignant Schwann cell tumors, traumatic neuromas and peripheral nerves without pathological findings. By immunohistochemistry and Western analysis ERBB2 expression was not detectable in normal nerves but in proliferating Schwann cells of traumatic neuromas. While none of the malignant schwannomas exhibited ERBB2 expression, weak expression was seen in a small proportion of the benign schwannomas. Low levels of neurofibromin were detectable in normal nerves but not in traumatic neuromas or tumors. These data indicate an inverse expression pattern of ERBB2 and neurofibromin in human non-neoplastic Schwann cells, but not in neurinoma cells.

Adult↗
PubMed1997

Growth inhibition by dominant-negative mutations of the neu-encoded oncoprotein.

In the present study, kinase-deficient mutants of the neu gene were constructed in order to generate dominant-negative receptor molecules, which should abolish phosphorylation of receptor complexes. One construct carried a mutation of the putative ATP-binding site (K758M), while the other mutant was generated by deletion of the kinase domain (ID400). Neither receptor showed phosphorylation by in vitro kinase assay. When NIH3T3 fibroblasts were co-transfected by the oncogenic neu gene and one of either construct, the transforming effect could be partially reversed. Therefore, kinase-negative mutations of the neu-encoded receptor seemed to have a dominant-negative effect on the action of the activated protein. To test this hypothesis, rat neurinoma cell lines containing oncogenic neu genes were transfected with the constructs. Expression of the kinase-defective mutants and reduced phosphorylation could be detected in different clones derived from single transfected cells. Striking growth inhibition and reduction of colony formation in soft agar were observed in these cell lines when compared with untransfected cells. Thus, kinase-deficient mutants exert a dominant-negative effect on phosphorylation of receptor complexes, resulting in a reversion of the transformed phenotype.

3T3 Cells↗