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Biomedical subjects

K D O'Neal

Publications and source records attributed to K D O'Neal.

12 recordsLinked to original sources
PubMed1999

Total upper eyelid reconstruction with mucosalized tarsal graft and overlying bipedicle flap.

Total or near-total upper eyelid reconstruction with a mucosalized tarsal graft and overlying bipedicled myocutaneous flap was performed on 12 patients. This technique offers several advantages over other reconstructive methods: (1) the opportunity for immediate visual rehabilitation through a single-staged procedure without obscuration of the visual axis, (2) preservation of eyelid blinking and closure by relocation of functional orbicularis muscle, (3) excellent eyelid contour, and (4) use of tissue physiologically similar to that removed. The technique and clinical results are presented herein.

Aged↗
PubMed1997

Template-based docking of a prolactin receptor proline-rich motif octapeptide to FKBP12: implications for cytokine receptor signaling.

A conserved proline-rich motif (PRM) in the cytoplasmic domain of cytokine receptors has been suggested to be a signaling switch regulated by the action of the FK506 binding protein (FKBP) family of peptidylprolyl isomerases (O'Neal KD, Yu-Lee LY, Shearer WT, 1995, Ann NY Acad Sci 766:282-284). We have docked the prolactin receptor PRM (Ile1-Phe2-Pro3-Pro4-Val5-Pro6-Gly7-Pro8) to the ligand binding site of FKBP12. The procedure involved conformational search restricted by NMR restraints (O'Neal KD et al., 1996, Biochem J 315:833-844), energy minimization of the octapeptide conformers so obtained, template-based docking of a selected conformer to FKBP12, and energy refinement of the resulting complex. The template used was the crystal structure of a cyclic FK506-peptide hybrid bound to FKBP12. Val5-Pro6 of the PRM was taken to be the biologically relevant Xaa-Pro bond. The docked conformer is stabilized by two intramolecular hydrogen bonds, N7H7-->O4 and N2H2-->O8, and two intermolecular ones, Ile56; N-H-->O = C:Pro6 and Tyr82:O-H-->O = C:Gly7. This conformer features a Type I beta-turn and has extensive hydrophobic contacts with the FKBP12 binding surface. The observed interactions support the hypothesis that FKBP12 catalyzes cis-trans isomerization in the PRM when it is part of the longer cytoplasmic domain of a cytokine receptor, and suggest a significant role for the PRM in signal transduction.

Amino Acid Sequence↗
PubMed1997

Multiple prolactin (PRL) receptor cytoplasmic residues and Stat1 mediate PRL signaling to the interferon regulatory factor-1 promoter.

The Nb2 PRL receptor (PRL-R) is known to mediate PRL signaling to the interferon (IFN) regulatory factor-1 (IRF-1) gene via the family of signal transducers and activators of transcription or Stats. To analyze the components of the PRL-R/Stat/IRF-1 signaling pathway, various PRL-R, Stat, and IRF-1-CAT reporter constructs were transiently cotransfected into COS cells. First, mutations in the IFNgamma-activated sequence (GAS), either multimerized or in the context of the 1.7-kb IRF-1 promoter, failed to mediate a PRL response, showing that the IRF-1 GAS is a target of PRL signaling. Next, pairwise alanine substitutions into conserved residues in the proline-rich motif or Box 1 region and two tyrosine mutations, Y308F and Y382F, in the PRL-R intracellular domain all impaired PRL signaling to multimerized GAS or to the 1.7-kb IRF-1 promoter. Furthermore, these PRL-R mutants mediated reduced Stat1 binding to the IRF-1 GAS. Transfection of Stat1 further enhanced PRL signaling to the IRF-1 promoter, suggesting that Stat1 is a positive mediator of PRL action. These studies show that both membrane proximal and distal residues of the PRL-R are involved in signaling to the IRF-1 gene. Further, Stat1 and the GAS element are important for PRL activation of the IRF-1 gene.

Amino Acid Sequence↗
PubMed1996

Multiple cis-trans conformers of the prolactin receptor proline-rich motif (PRM) peptide detected by reverse-phase HPLC, CD and NMR spectroscopy.

An eight-amino-acid synthetic peptide (IIe1-Phe2-Pro3-Pro4-Val5-Pro6-Gly7-Pro8) corresponding to the conserved proline-rich motif (PRM) of the intracellular domain of the prolactin receptor (PRL-R) was studied by one- and two-dimensional (1D and 2D) proton NMR spectroscopy in water and DMSO in order to characterize its conformational dynamics. The purified PRL-R PRM peptide eluted as two partially resolved peaks in equilibrium on reverse-phase HPLC (RP-HPLC) at 20 degrees C with a ratio of 60:40. At 30 degrees C, the two peaks coalesced into a single peak The two RP-HPLC peaks correspond to two peptide conformers resulting from the slow cis-trans isomerization of one of the four proline amide bonds. Although the peptide has only three amide (NH) protons, its ID NMR spectrum in water contains approximately 15 discernible NH region peaks, providing evidence for multiple conformers. The amide resonances were assigned on the basis of 2D-COSY spectra, chemical shift values resonance splitting patterns and temperature coefficients. The cis:trans ratio for each proline in water, calculated from integrated intensities and/or peak heights of the appropriate resonances, were Phe2-Pro3 (35:65), Pro3-Pro4 (40:60), Val5-Pro6 (70:30), and Gly7-Pro8 (30:70). Temperature studies (25-70 degrees C) were used to semi-quantitatively estimate the rates of isomerization for the different prolines. In water, Pro8 undergoes rapid isomerization; Pro3 isomerizes at an intermediate rate; while Pro4 and Pro6 both appear to isomerize very slowly since no coalescence of amide resonances was observed. In DMSO, only Pro4 displayed slow isomerization. Slow kinetics combined with a similar 60:40 ratio of conformers determined by RP-HPLC and NMR suggests that isomerization of the Pro3-Pro4 bond generates the two RP-HPLC peaks. Both proximal and distal proline isomerization effects were observed in NMR experiments. All of the 16 theoretical (24 = 16) proline configurations appear to exist in equilibrium in water The predominant (19%) conformation, trans3-trans4-cis6-trans8, may reflect the configuration of the PRM prolines in the native PRL-R. Isomerization of Pro6 from cis to trans generates an interaction between the peptide N-and C-termini, suggesting an overall pseudo-cyclic conformation. This all-trans proline configuration may play an important biochemical role in the function of cytokine/haematopoietin receptors. A model is proposed which suggests that isomerization of the PRM by an immunophilin such as the FK 506-binding protein (FKBP) serves as an on-off switch for cytokine receptor activation.

Amides↗
PubMed1995

Characterization of a prolactin-inducible gene, clone 15, in T cells.

To examine how PRL regulates lymphocyte proliferation, a number of PRL-activated genes were identified from a PRL-dependent rat T lymphoma cell line, Nb2. One of the downstream genes in the PRL signaling cascade was identified as clone 15 (c15). PRL stimulation of quiescent Nb2 T cells results in the expression of a 1.7-kilobase c15 mRNA, which reaches maximum levels between 8 and 10 h after stimulation. Corresponding [3H]thymidine incorporation experiments show that the maximum level of c15 mRNA expression correlates with the G1/S transition phase of the cell cycle. Sequencing of approximately 1.3-kilobase cDNA revealed one open reading frame that predicts a 332-amino acid protein. In vitro transcription/translation of c15 cDNA resulted in the production of a 45-kilodalton protein. Sequence analysis revealed that the c15 open reading frame contains a potential nuclear localization signal, a very acidic region, and a carboxy-terminal region of 94 amino acids which are 68% identical and 78% similar to the nuclear movement protein, NUDC, found in Aspergillus nidulans. Such a high degree of conservation suggests that the NUDC-like motif in c15 has been conserved through evolution for an important structure and/or function.

Amino Acid Sequence↗
PubMed1994

Differential signal transduction of the short, Nb2, and long prolactin receptors. Activation of interferon regulatory factor-1 and cell proliferation.

An Nb2 prolactin receptor (PRL-R) cDNA has been cloned from the PRL-dependent Nb2-11C cell line, and the protein-coding region is identical to that of the PRL-R isolated from the PRL-independent cell line Nb2-Sp. Short, Nb2, and long forms of the PRL-R were analyzed for signal transduction to the immediate-early gene, interferon regulatory factor-1 (IRF-1) and for cellular proliferation. Receptor and IRF-1-CAT reporter constructs were transiently cotransfected into the interleukin-3-dependent cell lines FDC-P1 and BaF3. The Nb2 PRL-R induced IRF-1-CAT 14.3-fold on addition of PRL, while the long PRL-R induced IRF-1-CAT 5.6-fold in FDC-P1 cells. The short PRL-R did not activate the IRF-1 promoter. Stable transfectants were also generated by selecting for growth in PRL. Only the Nb2 and long forms were able to convert the IL-3-dependent cells to PRL-dependence. IRF-1-CAT was induced in these cell lines by the Nb2 PRL-R 10- to 12-fold and long PRL-R 3- to 3.5-fold. Overall, the Nb2 form is more efficient than the long form by about 3-fold at inducing IRF-1-CAT. A PRL dose-response growth curve showed that the Nb2 form requires 20-fold less PRL for half maximal growth than the long form. A PRL dose-response for IRF-1-CAT activity gave similar results, indicating a tight correlation between IRF-1 induction and cell proliferation. These results show that the short PRL-R does not signal to IRF-1 or for growth, and that the Nb2 PRL-R signals more efficiently than the long PRL-R.

Base Sequence↗
PubMed1993

The proline-rich motif (PRM): a novel feature of the cytokine/hematopoietin receptor superfamily.

Members of the cytokine receptor superfamily have been grouped together by function and by the presence of conserved amino acids in the extracellular domain, including four cysteine residues and the Trp-Ser-X-Trp-Ser (WSXWS) motif. However, no consensus sequence motif has been described in the intracellular domain of the cytokine receptors. We now report the presence of a proline-rich consensus sequence motif, eight amino acids in length, which is found in the intracellular domain of all the cytokine receptors. The proline-rich motif (PRM) can be divided into two complementary families that have superimposable consensus sequences. The consensus sequences were found by allowing similar amino acids (aliphatic = Al, aromatic = Ar) to be grouped together. The first motif (PRM1) has the sequence Al-Ar-Pro-X-Al-Pro-X-Pro, while the second (PRM2) is Ar-X-X-X-Al-Pro-X-Pro. An overall consensus sequence for the PRM (PRM1 and PRM2) is derived by allowing aromatic and aliphatic residues to be considered hydrophobic (psi): psi-X-X-X-Al-Pro-X-Pro. Several alternative cytokine receptor isoforms contain two copies of the PRM within the same intracellular domain. The conservation of the proline-rich motif in cytokine receptors suggests that it plays a critical role in receptor function and defines a new feature of the cytokine receptor superfamily.

Amino Acid Sequence↗
PubMed1992

Solution structure and dynamics of epidermal growth factor and transforming growth factor alpha.

Circular dichroism (CD) and Fourier transform infrared spectroscopic studies have shown that the secondary structure of transforming growth factor alpha (TGF-alpha) is very similar to that of epidermal growth factor (EGF). The infrared spectra revealed a minor difference between the two proteins, in particular in the beta-sheet structure. A large difference was observed with CD between the two proteins in the apparent conformation each adopts when the disulfide bonds are reduced. Reduced TGF-alpha showed a distinct alpha-helical conformation only at a high trifluoroethanol concentration, whereas reduced EGF assumed an alpha-helical conformation in the absence of trifluoroethanol. This indicates that these two proteins adopt different secondary structures in the absence of disulfide bonds, although they assume similar folding structures in their presence. These data suggest that the disulfide bonds to a large degree dictate the conformation of these two proteins. Additionally, differences in the dynamic behavior between EGF and TGF-alpha were also observed. Infrared experiments showed that the hydrogen-deuterium exchange rate is much higher for TGF-alpha than for EGF, indicating that TGF-alpha is a more flexible molecule. The rate of reduction of the disulfide bonds by dithiothreitol was also faster for TGF-alpha. Therefore, it can be concluded that although EGF and TGF-alpha have a similar overall conformation, TGF-alpha is a more flexible molecule than EGF.

Circular Dichroism↗
PubMed1992

Prolactin gene expression in human thymocytes.

Recent evidence suggests that lymphocytes produce prolactin (PRL). Here, we report the cDNA cloning and expression of PRL from normal human thymocytes. Sequence analysis showed that the thymocyte cDNA encodes a 23 kDa protein which is identical to pituitary PRL. RNA blot analysis showed that the thymocyte PRL mRNA is approximately 170 nucleotides larger than the pituitary PRL message. PRL message was also detected in several non-pituitary human cell lines including Jurkat T, HeLa, and JEG cells. Furthermore, PRL gene expression in JEG cells was inhibited by glucocorticoid treatment. Our data support the hypothesis that PRL is a T cell-derived cytokine.

Base Sequence↗
PubMed1991

Prolactin receptor gene expression in lymphoid cells.

To understand the role of pituitary prolactin (PRL) and its receptor (PRL-R) in the growth and differentiation of lymphoid cells, PRL-R gene expression was analyzed in various lymphoid tissues and in a rat T lymphoma cell line, Nb2, which requires PRL for growth. The technique of reverse transcription coupled to polymerase chain reaction (RT-PCR) was used to detect the low abundance PRL-R transcripts. Within 30 min to 1 h, PRL stimulates a rapid but transient increase in PRL-R mRNA levels in Nb2 T cells. By 4 h, PRL-R mRNA returned to near basal levels and then gradually declined to a new steady-state level by 12 h. Significant increases in receptor RNA levels were observed in the presence of protein synthesis inhibitors, which suggests that PRL-R mRNA levels are under negative regulation. PRL-R gene expression was also demonstrated in normal mouse thymocytes, splenocytes, and in several lymphoid cell lines. The expression of the PRL-R gene in stimulated lymphoid cells provides additional evidence for the role of PRL as an immunomodulatory molecule.

Animals↗