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T W Beck

Publications and source records attributed to T W Beck.

At least 37 records · Page 2Linked to original sources
PubMed1990

Mutational activation of c-raf-1 and definition of the minimal transforming sequence.

A series of wild-type and mutant raf genes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine- and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.

Adenosine Triphosphate↗
PubMed1990

Molecular organization of the human Raf-1 promoter region.

A genomic DNA fragment containing the Raf-1 promoter region was isolated by using a cDNA extension clone. Nucleotide sequencing of genomic DNA clones, primer extension, and S1 nuclease assays have been used to identify the 5' ends of Raf-1 RNAs. Consistent with its ubiquitous expression, the Raf-1 promoter region had features of a housekeeping gene in that it was GC-rich (HTF-like), lacked TATA and CAAT boxes, and contained heterogeneous RNA start sites and four potential binding sites for the transcription factor SP1. In addition, an octamer motif (ATTTCAT), a potential binding site for the octamer family of transcription factors, was located at -734 base pairs. The Raf-1 promoter region drove reporter gene expression 30-fold over the promoterless reporter in Cos 7 cells.

Base Sequence↗
PubMed1989

Increased axonal transport of trigeminal ganglion proteins in an electrode model.

This investigation uses an electrical stimulation model of ocular herpes simplex virus (HSV) reactivation to elucidate the relationship between neuronal protein synthesis, axonal transport and electrical stimulation. In this study, healthy adult albino rabbits were implanted with electrodes over the trigeminal ganglion (TG) and stimulated chronically twice a week for 28-34 days (previously shown to induce HSV reactivation in infected rabbits). The TG was visualized surgically and injected with 3H-proline to label newly synthesized protein. After various times, the TG, corneas and ophthalmic nerve were analyzed for labeled proteins. In nonimplanted and implanted rabbits (whether stimulated or not), the rate of fast anterograde axonal transport in the ophthalmic nerve was 347 mm/day and 326 mm/day, respectively. A lag period of 45 min occurred between initiation of protein labeling and onset of axonal transport. Only a small percentage of newly synthesized neuronal proteins were transported in the ophthalmic nerve (1-4% in the proximal 10 mm) or deposited in the cornea (less than 0.5%) after 24 hr. Neither the rate of protein synthesis in the TG nor the rate of axonal transport was changed by electrical stimulation. However, the amount of radioactively labeled proteins undergoing axonal transport in the stimulated group was five times that of the sham control group. Thus, the routing and loading of TG proteins for axonal transport is enhanced in the electrical stimulation model.

Animals↗
PubMed1988

An amperometric needle-type glucose sensor tested in rats and man.

An amperometric glucose-measuring 25 gauge (0.5 mm diameter) needle-type sensor has been developed using a glucose oxidase and dimethyl ferrocene paste behind a semi-permeable membrane situated over a window in the needle. Electron transfer results in direct current generation. Sensors have been tested subcutaneously in the abdomen both in anaesthetized rats (40 sensors, 11 rats) and in normal, conscious man (20 sensors, 10 subjects). In rats the blood glucose was modulated by glucose and by insulin infusion. In man the glucose concentrations were rapidly changed by use of a glucose clamp at 12 mmol/l plasma concentration for 2 h, after which the glucose returned to normal. In rats the median correlation between glucose change was 0.83 with an interquartile range from 0.70 to 0.92, and in man the median correlation was 0.80 with an interquartile range 0.67 to 0.86. Hysteresis, a measure of the accuracy on the upswing and downswing, was not a problem and cross-correlation showed no phase-lag. There were quantitative differences between in vitro calibration and the performance in vivo, reflecting the different conditions of use. The current in response to a glucose concentration was stable over 6.0 h in rats and 4.5 h in man.

Adult↗
PubMed1987

The complete coding sequence of the human A-raf-1 oncogene and transforming activity of a human A-raf carrying retrovirus.

The complete 606 amino acid sequence of the human A-raf oncogene has been deduced from the 2453 nucleotide sequence of a human T cell cDNA. A cysteine-rich region located near the amino terminus, which is highly conserved in A-raf and c-raf, shows significant homology with protein kinase C. A 5' deleted fragment of the cDNA has been incorporated into a murine retrovirus which endows the virus with the ability to transform cells in vivo and in vitro. Functionally, human A-raf is similar to v-raf and v-mos in that transformation is independent of ras gene function.

Amino Acid Sequence↗
PubMed1986

Complementary DNA and amino acid sequence of rat liver microsomal, xenobiotic epoxide hydrolase.

The coding nucleotide sequence for rat liver microsomal, xenobiotic epoxide hydrolase was determined from two overlapping cDNA clones, which together contain 1750 nucleotides complementary to epoxide hydrolase mRNA. The single open reading frame of 1365 nucleotides codes for a 455 amino acid polypeptide with a molecular weight of 52,581. The deduced amino acid composition agrees well with those determined by direct amino acid analysis of the rat protein, and the amino acid sequence is 81% identical to that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and comparison to codon usage for NADPH-cytochrome P-450 oxidoreductase and cytochromes P-450b, P-450d, and P-450PCN, suggest that epoxide hydrolase is more conserved than cytochromes P-450b and P-450PCN; comparison of the extent of sequence conservation for 12 homologous proteins between the rat and rabbit, including cytochrome P-450b, supports this hypothesis, and indicates that much of epoxide hydrolase is constrained to maintain its hydrophobic character, consistent with its intramembranous location. The predicted membrane topology of epoxide hydrolase delineates 6 membrane-spanning segments, less than the 8 or 10 predicted for two cytochrome P-450 isozymes; the lower number of membrane-spanning segments predicted for epoxide hydrolase correlates with its lesser dependence on the membrane for maintenance of its tertiary structure and catalytic activity.

Amino Acid Sequence↗
PubMed1986

Transformation by raf and myc oncogenes.

raf oncogenes were shown to act synergistically with myc in transformation. The contribution of myc was identified as that of a "second messenger" in signal transduction of at least some, competence inducing, growth factors. The role of raf appears to be that of a cytosolic ser/thr specific protein kinase which was placed downstream of ras in the signal transduction of serum growth factors by ras and raf antibody microinjection experiments. Because of the inability of raf to abrogate a cells need for myc inducing competence factors, as well as its synergistic effect with myc, raf was placed downstream of ras in the progression pathway of cellular growth control. We speculate that the basis for synergism with myc might be the ability of raf to activate competence factor induced myc protein or a myc induced protein by phosphorylation. The role of raf in lung tumors was examined by the development of a high incidence mouse model system using ethylnitrosourea as carcinogen and butylated hydroxytoluene as promoter. raf proteins of normal size were expressed at high levels, raf protein vaccination was apparently effective in eliminating the promoted phase of tumor induction.

Amino Acid Sequence↗
PubMed1985

In vitro transcription of herpes simplex virus genes: identification of a new initiation site and second intervening sequence in the immediate-early RNA-5 gene.

We have used partially purified preparations of RNA polymerase II from mock-infected and herpes simplex virus type 1-infected HEp-2 cells to transcribe the herpes simplex virus type 1 strain F BamHI Z DNA fragment containing the promoter for immediate-early RNA-5 (alpha 47), a 1.8-kilobase, spliced RNA. In agreement with the in vivo transcriptional regulation of herpes simplex virus type 1 immediate-early genes, electrophoretic analyses of runoff and truncated transcripts from this template showed that RNA polymerase II from mock-infected cells initiates transcription more selectively than does that from the herpes virus-infected cells at the immediate-early RNA-5 promoter. S1 nuclease mapping of the 5' ends of in vivo- and in vitro-synthesized mRNA placed the initiation site ca. 110 base pairs upstream from the previously published site and also demonstrated the presence of a second, smaller intervening sequence between this new cap site and the previously characterized intervening sequence. S1 analyses also suggested that some splicing of the larger but not the smaller intervening sequence occurred in vitro.

Base Sequence↗
PubMed1982

Regulation of herpes simplex virus gene transcription in vitro.

We used partially purified RNA polymerase II from uninfected (Pol II) and from herpes simplex virus type 1 (HSV-1) infected HEp-2 cells (Pol II-H) to transcribe HSV-1 DNA in vitro. Gel electrophoretic analysis of the products produced from native HSV-1 DNA yielded weight average chain lengths of 4.0 and 3.5 kb for the Pol II and Pol II-H products, respectively. Blot hybridization analyses of the HSV DNA transcripts showed that both enzymes transcribed RNA from essentially all regions of the genome. However, Pol II preferentially transcribed regions coding for the immediate-early or alpha mRNAs, whereas Pol II-H preferentially copied regions coding for the early (beta) and late (gamma) gene products. Transcriptional analyses of the cloned HSV-1 Bam HI-Q fragment (containing the thymidine kinase (TK) gene) and its subfragments showed that (1) the major transcripts produced by Pol II-H were distinctly different from those produced by Pol II; (2) Pol II and Pol II-H utilized different promoters for the synthesis of major transcripts; (3) both enzymes produced three minor transcripts that were partially overlapping and in opposite direction to the TK gene; and (4) only Pol II-H initiated transcription from the TK promoter. In contrast, both Pol II and Pol II-H generated an identical set of transcripts from an adenovirus 2 early region DNA fragment. The sizes of the products suggest that RNA processing may be occurring in vitro. These results show that HSV-1 infection alters the in vitro transcriptional specificity of RNA polymerase II and demonstrate that this system should be useful for studying in vitro the regulation of gene transcription.

Adenoviruses, Human↗
PubMed1982

Endocrine response of postpartum anestrous beef cows to GnRH or PMSG.

Forty-one postpartum anestrous Hereford cows, maintained under range conditions, were used to determine the influence of gonadotropin releasing hormone (GnRH) or pregnant mare serum gonadotropin (PMSG) on ovarian function. Anestrous cows were identified by estrous detection with sterile bulls and concentrations of progesterone in plasma obtained weekly. At 45+/-2 days postpartum, cows were allotted to the following treatments: (1) control (saline), (2) 100 microg GnRH, (3) 200 microg GnRH, (4) 200 microg GnRH in carboxymethyl cellulose (CMC), (5) 500 IU PMSG, (6) 1,000 IU PMSG or (7) 2,000 IU PMSG. Cows were bled frequently the first day after treatment and then every other day until 85 days postpartum. The LH responses after 100 and 200 microg of GnRH were not significantly different and mixing 200 microg GnRH with CMC before injection did not significantly alter the LH response. During the first 20 days after treatment, neither GnRH nor 500 IU PMSG altered estradiol concentrations in plasma, but treatment of cows with 1,000 or 2,000 IU PMSG resulted in increased (P<0.01) concentrations of estradiol. The time postpartum required for concentrations of progesterone in plasma to exceed 1 ng/ml was reduced (P<0.05) by all treatments except 100 microg GnRH. These data indicate that GnRH causes LH release in anestrous range cows and that treatment with 1,000 or 2,000 IU PMSG initiates ovarian activity as evidenced by increased concentrations of estradiol in plasma.

Journal Article↗
PubMed1982

Influence of ambient temperature on prolactin concentrations in serum of Holstein and Brahman x Hereford heifers.

Four Holstein and four Brahman x Hereford heifers about 8 mo of age were used in a study to determine whether breed influences the effects of ambient temperature on concentrations' of prolactin in serum. Two heifers of each breed were stanchioned in each of two environmental chambers at 21 C for 7 d, after which chamber temperatures were changed to 7 or 31 C during 6 h. After 5 d at 7, 21 or 31 C, heifers were injected with 60 micrograms thyrotropin-releasing hormone (TRH). A switch-back design was used and each heifer was exposed to all treatments. Concentrations of prolactin in serum of heifers during exposure to 7, 21 or 31 C for 5 d were related to ambient temperature (9.0, 20.9 and 29.5 ng/ml, respectively; P less than .001), but the response was not influenced by breed. Heifers of both breeds responded similarly to treatment with TRH, and prolactin in serum increased (P less than .001) within 5 min from 7.0 +/- 3.2 to 45.7 +/- 8.2 ng/ml in heifers at 7 C, from 13.1 +/- 1.6 to 97.2 +/- 9.6 ng/ml in heifers at 21 C and from 18.2 +/- 3.5 to 96.2 +/- 11.3 ng/ml in heifers at 31 C. We conclude that concentrations of prolactin in serum of heifers are positively associated with ambient temperature and that the effects of temperature on basal and TRH-stimulated concentrations of prolactin do not differ significantly between Holstein and Brahman x Hereford heifers. Thus, differences in tolerance to heat were not related to differences in prolactin secretion.

Animals↗
PubMed1979

Influence of 48 hour calf separation on milk production and calf growth in range cows.

Hereford cows and their calves were either left together or separated for a 48-hr period between 50 and 80 days postpartum. Milk production and calf weights were determined 1 and 2 weeks prior to and 1 and 3 weeks after calf separation. Daily milk production of separated cows (5.6+/-.1 kg) was not different from that of control cows (5.3+/-.1 kg) at any sampling period. Similarly, calf growth was not affected (P > .10) by separation; both groups of calves gained .64 kg/day. Average 205-day adjusted weaning weights were also similar, for the control (173.5+/-4.6 kg) and separated calves (181.8+/-4.6 kg). These results indicate that 48-hr calf separation could be used in a treatment regime to decrease the postpartum anestrous interval in range cattle without detrimental effects on milk production, calf growth or 205-day adjusted weaning weight.

Journal Article↗