Regulation of Sos activity by intramolecular interactions

The guanine nucleotide exchange factor Sos mediates the coupling of receptor tyrosine kinases to Ras activation. To investigate the mechanisms that control Sos activity, we have analyzed the contribution of various domains to its catalytic activity. Using human Sos1 (hSos1) truncation mutants, we show that Sos proteins lacking either the amino or the carboxyl terminus domain, or both, display a guanine nucleotide exchange activity that is significantly higher compared with that of the full-length protein. These results demonstrate that both the amino and the carboxyl terminus domains of Sos are involved in the negative regulation of its catalytic activity. Furthermore, in vitro Ras binding experiments suggest that the amino and carboxyl terminus domains exert negative allosteric control on the interaction of the Sos catalytic domain with Ras. The guanine nucleotide exchange activity of hSos1 was not augmented by growth factor stimulation, indicating that Sos activity is constitutively maintained in a downregulated state. Deletion of both the amino and the carboxyl terminus domains was sufficient to activate the transforming potential of Sos. These findings suggest a novel negative regulatory role for the amino terminus domain of Sos and indicate a cooperation between the amino and the carboxyl terminus domains in the regulation of Sos activity.     

Activation of T lymphocytes by syngeneic murine intestinal smooth muscle cells

BACKGROUND & AIMS: Intestinal smooth muscle cells (ISMCs) express major histocompatibility complex II (MCH II) and intercellular adhesion molecule 1 (ICAM-1) after exposure to interferon gamma (IFN-gamma). T lymphocytes invade the intestinal musculature during Crohn's disease or pseudoobstruction. The aim of this study was to determine whether ISMCs activate syngeneic T cells via MHC II and ICAM-1. METHODS: Cultured murine ISMCs were exposed to IFN-gamma for 72 hours and analyzed for Mac-1 (CD11B CD18) antigen, MHC II, and ICAM-1 expression using enzyme-linked immunosorbent assay and fluorescence-activated cell sorter scan. T lymphocytes from mesenteric lymph nodes of ovalbumin-sensitized mice were examined for their ability to proliferate after coculture with IFN-gamma-pretreated and ovalbumin-pretreated ISMCs using [3H]thymidine incorporation. RESULTS: ISMCs expressed smooth muscle alpha-actin before and after IFN-gamma exposure. No macrophages were identified in these cultures. Exposure to IFN-gamma and ovalbumin for 72 hours induced MHC II and ICAM-1 expression; these treated ISMCs induced T-cell proliferation, whereas untreated ISMCs did not. T-cell proliferation was markedly enhanced by adding interleukin 2 and was blocked by antibodies against MHC II and ICAM-1. CONCLUSIONS: ISMCs activate T lymphocytes in an MHC II-linked manner and thus possess the ability to modulate immune function in the gut.     

Nasopharyngeal colonization with nontypeable Haemophilus influenzae in chinchillas

Colonization of the nasopharynx by a middle ear pathogen is the first step in the development of otitis media in humans. The establishment of an animal model of nasopharyngeal colonization would therefore be of great utility in assessing the potential protective ability of candidate vaccine antigens (especially adhesins) against otitis media. A chinchilla nasopharyngeal colonization model for nontypeable Haemophilus influenzae (NTHI) was developed with antibiotic-resistant strains. This model does not require coinfection with a virus. There was no significant difference in the efficiency of NTHI colonization between adult (1- to 2-year-old) and young (2- to 3-month-old) animals. However, the incidence of middle ear infection following nasopharyngeal colonization was significantly higher in young animals (83 to 89%) than in adult chinchillas (10 to 30%). Chinchillas that had recovered either from a previous middle ear infection caused by NTHI or from an infection by intranasal inoculation with NTHI were completely protected against nasopharyngeal colonization with a homologous strain and were found to be the best positive controls in protection studies. Systemic immunization of chinchillas with inactivated whole-cell preparations significantly protected animals not only against homologous NTHI colonization but also partially against heterologous NTHI infection. In all protected animals, significant serum anti-P6 and anti-HMW antibody responses were observed. The outer membrane P6 and high-molecular-weight (HMW) proteins appear to be promising candidate vaccine antigens to prevent nasopharyngeal colonization and middle ear infection caused by NTHI.     

Expression of interleukin-6 in polymorphic reticulosis--immunohistochemical study of 5 cases

Peripheral T cell lymphoma encompasses lymphomas with a variety of histologic appearances and clinical patterns. Recently, it has been suggested that almost all of the histologic features described under the name of polymorphic reticulosis(PR), lethal midline granuloma, and midline malignant reticulosis can be included in those generally described for malignant lymphomas of peripheral T cell origin(PTCL). There have been few studies of pathogenesis or tissue damage mechanism in PR patients. The need for a precise mechanism for tissue damage has important therapeutic implications. Using immunohistochemical methods with polyclonal anti IL-6 antibody, the authors describe 5 cases of PR with clinically and pathologically typical PR demonstrating a high expression of IL-6. According to classification, 2 cases of grade 1 PR showed the highest expressions, and 2 cases of grade 2 PR with atypical lymphoid cells showed moderate activity, but one case progressed into frank lymphoma(grade 3) and lost IL-6 expression. This strongly implies that some cases of PR have a different mechanism of tissue damage from frank PTCL, despite the one disease spectrum. Further studies on more cases may help clarify the pathogenesis.     

Synergistic interaction of 3 M KCl-extracted donor antigens (e-HAg) with cyclosporine or cyclosporine/sirolimus for prolongation of rat heart allograft survival

This article examines the attaching and detaching experience of a mother encountering the perinatal death of a twin. Her experience is related to the relevant theoretical and research literature pertaining to prebirth and postbirth maternal-infant attachment and detachment (grieving). Literature for both single infants and twins is considered. The experience of this mother suggests that elements of postbirth attachment may have been accelerated into the prebirth period. In addition, her postbirth attaching and detaching experience suggests that an attachment and detachment to the twins as a unit preceded detachment from the twin who died. The health care provider's role in promoting maternal well-being, and indirectly the well-being of the surviving infant, is described.     

Thoracoscopic T2-sympathetic block by clipping--a better and reversible operation for treatment of hyperhidrosis palmaris: experience with 326 cases

Although thoracoscopic sympathectomy or sympathicotomy is the best treatment for hyperhidrosis palmaris, a new approach of clipping only without transection of T2-sympathetic trunk is just as effective. Aside from the guaranteed cure of hyperhidrosis, this new method has fewer complications and has the advantage of recovery of the sympathetic tone in the hands if the procedure is reversed by the removal of the clips. Between March 18 and September 30 of 1996, 326 patients (190 female and 136 male with a mean age of 20.5 years) underwent thoracoscopic T2-sympathetic block by clipping to treat hyperhidrosis. Good results and few complications were noted during follow up six months to one year postoperatively. Five of the 326 patients, all female, had the operation reversed because of intolerable compensatory sweating. Three recovered from the compensatory sweating within two months and had less palmar sweating than before their sympathetic block; the fourth achieved relief of compensatory sweating after nine months, and the fifth reported no improvement.     

Treatment of vancomycin-resistant Enterococcus faecium infections with an investigational streptogramin antibiotic (quinupristin/dalfopristin): a report of fifteen cases

OBJECTIVE: Previous studies of surgical treatment for acromegaly have used varied criteria for 'cure', but elevated GH levels are considered to be associated with continuing disease activity. We wished to analyse the results of transsphenoidal pituitary surgery for acromegaly and assess the longer-term outcome for patients not offered further treatment when post-operative levels of GH < 5 mU/l were achieved. DESIGN: We studied a retrospective group of patients who underwent transsphenoidal surgery for acromegaly at St Bartholomew's Hospital between 1985 and 1993. PATIENTS: One hundred consecutive patients (53 male, mean age 46 years, range 18-68 years) undergoing transsphenoidal surgery for acromegaly were assessed. The patients were followed for a mean of 3.8 years (range 0.5-8 years) after operation. MEASUREMENTS: GH levels are represented as a mean value from a four-point day curve taken at 0830, 1300, 1700 and 1900 h. ACTH reserve was assessed basally and, if this was normal, with the insulin tolerance or glucagon tests. TSH, T4, PRL, LH, FSH, testosterone or oestradiol and plasma and urine osmolality were also measured. RESULTS: Post-operatively, 42% of patients achieved a mean GH level of < 5 mU/l. The success of surgery was related to the preoperative GH level; 65% of the patients with preoperative GH levels < 20 mU/l but only 18% of the patients with GH levels > 100 mU/l achieved post-operative GH values < 5 mU/l. In addition, tumour size influenced the outcome of surgery with 61% of patients with a microadenoma but only 23% of patients with a macroadenoma achieving post-operative GH levels of < 5 mU/l. Of the 42 patients considered in remission post-operatively (mean GH < 5 mU/l), 32 were available for long-term follow-up and were not offered any further treatment: only one of these has shown evidence of mild biochemical recurrence after a mean follow-up of 3.8 years (range 0.5-8). There were no peri-operative deaths. Two patients required surgical repair for CSF leaks and there were eight documented cases of meningitis. Permanent diabetes insipidus was noted in eight patients post-operatively. New anterior pituitary deficiency occurred in 21% of patients following surgery; 73% had unaltered pituitary function and in 6% recovery of partial hypopituitarism was noted. CONCLUSIONS: The stated outcome of surgery depends on the criteria adopted. Safe GH levels (mean levels < 5 mU/l) can be achieved in 42% of an unselected series of patients with acromegaly and if the tumour is a microadenoma this figure rises to 61%. Based on the current evidence it is safe not to offer further treatment to those patients in whom post-operative GH < 5 mU/l are achieved.     

GABAergic neurons in the embryonic olfactory pit/vomeronasal organ: maintenance of functional GABAergic synapses in olfactory explants

In previous work, we showed a robust gamma-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic mice in vivo and study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures. In vivo, glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around -50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABAA) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl- conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pit in vivo.