No abstract

The Src homology 2 (SH2) domain-containing protein SH2-B␤ binds to and is a substrate of the growth hormone (GH) and cytokine receptor-associated tyrosine kinase JAK2. SH2-B␤ also binds, via its SH2 domain, to multiple activated growth factor receptor tyrosine kinases. We have previously implicated SH2-B␤ in GH and platelet-derived growth factor regulation of the actin cytoskeleton. We extend these findings by establishing a potentiating effect of SH2-B␤ on GH-dependent cell motility and defining regions of SH2-B␤ required for this potentiation. Time-lapse video microscopy, phagokinetic, and/or wounding assays demonstrate reduced movement of cells overexpressing SH2-B␤ lacking an intact SH2 domain because of a point mutation or a C-terminal truncation. An N-terminal proline-rich domain (amino acids 85-106) of SH2-B␤ is required for inhibition of cellular motility by SH2 domain-deficient mutants. Co-immunoprecipitation experiments indicate that Rac binds to this domain. GH is shown to activate endogenous Rac, and dominant negative mutants of SH2-B␤ are shown to inhibit membrane ruffling induced by constitutively active Rac. These findings suggest that SH2-B␤ is an adapter protein that facilitates actin rearrangement and cellular motility by recruiting Rac and potentially Rac-regulating, Rac effector, or other actinregulating proteins to activated cytokine (e.g. GH) and growth factor receptors.Cell migration is critical for many vital biological functions, including embryonic development, the inflammatory immune response, wound repair, tumor formation and metastasis, and tissue remodeling and growth. The actin cytoskeleton provides both the protrusive and contractile forces required for cell migration via a combination of actin polymerization and depolymerization, actin filament cross-linking, and the interaction of myosin-based motors with actin filaments (1). The complexity of cell motility and the fact that it is regulated by many hormones, cytokines, and growth factors, including growth hormone (GH)

The Src homology-2 (SH2) domain-containing protein SH2-B␤ is a substrate of the growth hormone (GH) receptor-associated tyrosine kinase JAK2. Here we tested whether SH2-B␤ is involved in GH regulation of the actin cytoskeleton. Based on cell fractionation and confocal microscopy, we find SH2-B␤ present at the plasma membrane and in the cytosol. SH2-B␤ colocalized with filamentous actin in GH and platelet-derived growth factor (PDGF)-induced membrane ruffles. To test if SH2-B␤ is required for actin reorganization, we transiently overexpressed wild-type or mutant SH2-B␤ in 3T3-F442A cells and assayed for GH-and PDGF-induced membrane ruffling and fluid phase pinocytosis. Overexpression of wild-type SH2-B␤ enhanced ruffling and pinocytosis produced by submaximal GH but not submaximal PDGF. Point mutant SH2-B␤ (R555E) and truncation mutant ⌬C555, both lacking a functional SH2 domain, inhibited membrane ruffling and pinocytosis induced by GH and PDGF. Mutant ⌬N504, which possesses a functional SH2 domain and enhances JAK2 kinase activity in overexpression systems, also inhibited GH-stimulated membrane ruffling. ⌬N504 failed to inhibit GH-induced nuclear localization of Stat5B, indicating JAK2 is active in these cells. Taken together, these results show that SH2-B␤ is required for GH-induced actin reorganization by a mechanism discrete from the action of SH2-B␤ as a stimulator of JAK2 kinase activity.

The objective of this study was to determine whether neuropeptide Y (NPY) and recombinant human interleukin-1 receptor antagonist (IL-1ra) would: first, increase food intake; secondly, decrease concentrations of GH; thirdly, reduce GHRH-induced release of GH; and fourthly, reduce changes to concentrations of IGF-I in plasma during experimental endotoxemia in sheep.Six treatments were given to six castrated male sheep in a 6 6 Latin square treatment order. Osmotic minipumps were implanted at 0 h and a jugular vein was cannulated. Each sheep was continuously infused with saline (0·9%) or lipopolysaccharide (LPS) (20 µg/kg per 24 h, s.c.) at 10 µl/h for 72 h via the osmotic mini-pumps. Blood samples (3 ml) were collected at 15-min intervals from 24 to 33 h. At 26 h, one of three treatments (artificial cerebrospinal fluid, NPY or IL-1ra) was injected i.c.v. within 30 s (0·3 µg/kg), then infused i.c.v. from 26 to 33 h (600 µl/h) at 0·3 µg/kg per h. GHRH was injected i.v. (0·075 µg/kg) at 32 h after which blood samples were collected at 5, 10, 15, 30, 45 and 60 min. Feed intake was reduced up to 50% for 48 h in LPS-treated compared with non-LPS-treated sheep.NPY restored feed intake in LPS-treated sheep and induced hyperphagia in non-LPS-treated sheep from 24 to 48 h. In contrast, IL-1ra did not affect appetite. Injection of NPY increased concentrations of GH from 26 to 27 h, while IL-1ra had no effect. Infusion of NPY suppressed GHRH-induced release of GH. However, no treatment altered pulse secretion parameters of GH. Concentrations of IGF-I were 20% higher at 72 h in LPS-treated sheep given NPY than in sheep treated with LPS alone, and this may reflect increased appetite from 24 to 48 h.We concluded that reduced appetite during endotoxemia is due to down-regulation of an NPY-mediated mechanism. Furthermore, NPY stimulates release of GH in healthy sheep, does not reduce pulse secretion parameters of GH, but does suppress GHRH-induced release of GH in endotoxic sheep. Therefore, NPY may be an important neurotransmitter linking appetite with regulation of GH during endotoxemic and healthy states in sheep.

OBJECTIVE. Theaimof thisstudywastoassess therolesof transrectal colorDoppler andgray-scale sonography in revealing prostatic cancer,using biopsy as the referencestandard. SUBJECTSAND METHODS. Twohundred fifty-sixpatients referred forurologic studies underwent transrectal sonography using gray-scale and color Doppler scanning. All abnor mal areas shown on gray-scale or color Doppler sonography or both were targeted and biopsies were performed. The patients also underwent random sextant biopsies. All biopsies were mdi viduallycorrelatedwith histopathologic findingsandall resultswereanalyzed. RESULTS.Cancer wasfoundonbiopsyin 100patients (39%),andequivocal sonographicresults or prostatic intraepithelial neoplasia was found in 22 other patients (9%). In 16 of the patients in whom cancer was detected, the tumors were correctly revealed only with color Dop pIer sonography.These 16patients had a meanGleason scoreof 6.4 (range, 5â€"8). Biopsy find ings in these 16 patients showed eight patients with extensive lesions, three with moderate lesions, and five with minimal lesions. However, in nine other patients with cancer (9% of can cers detected), both gray-scale and color Doppler sonography failed to reveal lesions that were found on sextant biopsy. An analysis showed that, although highly sensitive, color Doppler sonography wassomewhatlessspecificthangray-scalesonography. CONCLUSION. ColorDopplersonography should become a routinepartof transrectalsonography of theprostateglandto improvedetectionandtargetingoflesions.The practiceof performing random sextant biopsies should also continue. P rostatic cancer is the most common malignancy in the American male population. Although widely ac ceptedand usedin the diagnosisof prostatic cancer, transrectal sonography has limitations.Prostaticcarcinomasmay be isoechoicI 1] and consequently not visualized on gray-scale sonography. Conversely, the most common sonographic appearance for prostate carci nomaâ€"theperipheral zone hypoechoic le sionâ€"canalso, on histopathologic examination of biopsy specimens, be found to represent a be nign lesion [2, 31.Limitations such as these may be partly over come by advances in technique. Several reports To evaluate the role of color Doppler transrectal sonography in detecting prostatic cancer in referred urologic patients, we pro spectively compared its usefulness with that of gray-scale imaging (keeping in mind the role of sextant biopsies) and correlated the results with biopsy findings. Subjects and MethodsThe population in this study consisted of 256 consecutive patients referred by urologists during theperiodbetween April andSeptember 1996. The patientswere40â€"84 yearsold (meanage,64years). Forty-threepatients(17%) were referredbecauseof an abnormality found on digital rectal examination, I 17 (46%) because of a raised (or rising) level of prostate-specific antigen (PSA), 90 (35%) because of botha raisedlevelof PSAandanabnormality on