Identification of the minimal requirements for binding to the human epidermal growth factor (EGF) receptor using chimeras of human EGF and an EGF repeat of Drosophila Notch

Many proteins contain so-called epidermal growth factor (EGF)-like domains that share the characteristic spacing of cysteines and glycines with members of the EGF family. They are, however, functionally unrelated, despite the fact that the three-dimensional structure of these EGF-like domains, also, is often very similar to that of the EGF receptor agonists. In the present study, we linked an EGF-like repeat from the Drosophila Notch protein to the N- and C-terminal linear tail sequences of human EGF (hEGF), and we showed that this chimera (E1N6E) is unable to bind or activate the hEGF receptor. This recombinant protein was then used as a basic construct for identifying the minimal requirements for high affinity EGF receptor binding and activation. We selectively reintroduced a limited number of important hEGF-derived residues, and by using this unique approach, we were able to make hEGF/Notch chimeras that, compared with wild type hEGF, showed nearly 100% binding affinity and mitogenic activity on HER-14 cells expressing the hEGF receptor.     

Midwall fractional shortening is an independent predictor of left ventricular diastolic dysfunction in asymptomatic patients with systemic hypertension

Conventional measures of left ventricular (LV) systolic performance suggest that diastolic dysfunction precedes the development of systolic dysfunction in hypertension. Midwall fractional shortening is a new measure of systolic function that identifies hypertensive patients who have evidence of target-organ damage, impaired contractile reserve, and increased mortality. We therefore sought to determine whether depressed midwall fiber shortening is associated with abnormal diastolic function. Echocardiograms were obtained in 102 otherwise healthy hypertensive patients without treatment with normal conventional measures of systolic function. Of these, 15 had depressed midwall shortening based on previously described normative relations. Patients with depressed midwall shortening had slightly higher blood pressure. Abnormal diastolic function, defined as late (A) LV inflow velocity greater than early (E) velocity, was observed in 33% of those with normal midwall shortening but in 60% of those with depressed shortening (p <0.05). Patients with A/E >1 had lower absolute midwall fiber shortening (15 +/- 3% vs 18 +/- 3%, p <0.0001) but similar endocardial shortening. Patients with abnormal midwall shortening had higher A/E and longer isovolumic relaxation times (both p <0.05). In multivariate analysis, midwall fractional shortening, age, and heart rate were independent predictors (p <0.01) of A/E in a model including blood pressure, LV mass, and endocardial shortening. We conclude that subnormal midwall shortening predicts LV diastolic abnormalities in this population of hypertensive patients with otherwise normal measures of LV systolic function. Contrary to our previous understanding, depressed LV systolic performance, when identified with this newer method, occurs coincidentally with impaired diastolic function.     

Adoptive immunotherapy for leukemia: donor lymphocytes transduced with the herpes simplex thymidine kinase gene for remission induction. HGTRI 0103

This study will evaluate the safety and efficacy of allogenic donor lymphocyte infusions in patients who have relapsed hematologic malignancies after allogeneic bone marrow transplantation (BMT). Donor lymphocyte transfusions have resulted in the cure of some patients with relapsed leukemia or lymphoproliferative disorder after allogeneic BMT, but has been complicated by the development of graft versus host disease (GvHD). We hypothesize that a retroviral vector containing the Herpes simplex thymidine kinase (HStk) gene will allow for retention of the anti-leukemia response of transfused donor lymphocytes while allowing for the adverse effects of GVHD to be mitigated. Patients with relapsed hematologic malignancies after allogeneic BMT will be infused with ex vivo gene modified donor lymphocytes. The Herpes Simplex thymidine kinase (HStk) gene will be transduced into the cells ex vivo using LTKOSN. 1 vector supernate. Insertion of the HStk gene into lymphocytes confers a sensitivity to the anti-herpes drug ganciclovir (GCV). This selective destruction of donor lymphocytes in situ will be used to abrogate the effect of graft versus host disease, if it develops.     

Differential response of cortical plate and ventricular zone cells to GABA as a migration stimulus

A microdissection technique was used to separate differentiated cortical plate (cp) cells from immature ventricular zone cells (vz) in the rat embryonic cortex. The cp population contained >85% neurons (TUJ1(+)), whereas the vz population contained approximately 60% precursors (nestin+ only). The chemotropic response of each population was analyzed in vitro, using an established microchemotaxis assay. Micromolar GABA (1-5 microM) stimulated the motility of cp neurons expressing glutamic acid decarboxylase (GAD), the rate-limiting enzyme in GABA synthesis. In contrast, femtomolar GABA (500 fM) directed a subset of GAD- vz neurons to migrate. Thus, the two GABA concentrations evoked the motility of phenotypically distinct populations derived from different anatomical regions. Pertussis toxin (PTX) blocked GABA-induced migration, indicating that chemotropic signals involve G-protein activation. Depolarization by micromolar muscimol, elevated [K+]o, or micromolar glutamate arrested migration to GABA or GABA mimetics, indicating that migration is inhibited in the presence of excitatory stimuli. These results suggest that GABA, a single ligand, can promote motility via G-protein activation and arrest attractant-induced migration via GABAA receptor-mediated depolarization.     

Incidence of and risk factors for hepatitis B virus and hepatitis C virus infection among haemodialysis and CAPD patients: evidence for environmental transmission

Hepatitis B virus (HBV) serum markers (HBsAg, anti-HBs, anti-HBc) and antihepatitis C antibody (anti-HCV) were prospectively followed in haemodialysis and CAPD patients. From January 1987 to January 1990, 185 patients on haemodialysis and 124 on CAPD were analysed. Among patients susceptible to HBV (69 on haemodialysis and 70 on CAPD), there were 17 HBsAg seroconversions on haemodialysis (0.19/patient-year) and 1 on CAPD (0.01/patient-year). A Cox proportional hazards model showed that haemodialysis treatment was the only risk factor significantly associated with HBV infection, thus suggesting transmission through the environment. Regarding hepatitis C, 83 anti-HCV-negative patients on haemodialysis and 46 on CAPD were followed. There were 18 seroconversions on haemodialysis (0.15/patient-year) and two seroconversions on CAPD (0.03/patient-year). Haemodialysis treatment was also the only risk factor significantly associated with a higher risk of HCV infection. The hazard ratio for HCV infection in haemodialysis patients was 5.7 compared to CAPD patients. Nevertheless, for one patient on CAPD treatment transfusions were the only possible source of HCV infection. In conclusion, both viruses were transmitted mainly through the haemodialysis environment, but the role of transfusions could not be excluded.     

Retinal vessels before and after photocoagulation in diabetic retinopathy. Determining the diameter using digitized color fundus slides

BACKGROUND: Retinal vessel diameter is an important parameter in blood flow analysis. Despite modern digital image technology, most clinical studies investigate diameters subjectively using projected fundus slides or negatives. In the present study we used a technique to examine vessel diameters by digital image analysis of color fundus slides. We investigated in a retrospective manner diameter changes in twenty diabetic patients before and after panretinal laser coagulation. MATERIAL AND METHODS: Color fundus slides were digitized by a new high resolution scanning device. The resulting images consisted in three channels (red, green, blue). Since vessel contrast was the highest in the green channel, we assessed grey value profiles perpendicular to the vessels in the green channel. Diameters were measured at the half-height of the profile. RESULTS: After panretinal laser coagulation, average venous diameter was decreased, whereas arterial diameter remained unchanged. There was no significant relation between the diameter change and the number of laser burns or the presence of neovascularization. CONCLUSIONS: Splitting digitized images into color planes enables objective measurements of retinal diameters in conventional color slides.