A dual participation of ZAP-70 and scr protein tyrosine kinases is required for TCR-induced tyrosine phosphorylation of Sam68 in Jurkat T cells

Sam68 has been initially described as a substrate of src kinases during mitosis in fibroblasts. Recent evidence suggests that in T lymphocytes Sam68 may act as an adaptor protein and participate in the early biochemical cascade triggered after CD3 stimulation. A direct interaction between Sam68 and the two src kinases involved in T cell activation, p59^fyn and p56^lck, as well as a partnership of Sam68 with various key downstream signaling molecules, like phospholipase Cγ-1 and Grb2, has been shown. In this study we analyze the contribution of p56^lck, as well as the role of ZAP-70, the second class of protein tyrosine kinase involved in T cell activation, in Sam68 tyrosine phosphorylation in the human Jurkat T cell line. Using the src inhibitor PP1 [4-amino-5-(4-methylphenyl)7-(t-butyl) pyrazolo [3,4-d] pyrymidine] and cell variants with defective expression of p56^lck or expressing a dominant negative form of ZAP-70, we demonstrate that, while both p56^lck and ZAP-70 are dispensable for the low constitutive phosphorylation of Sam68 observed in Jurkat cells, a cooperation between the two kinases is required to increase its rapid phosphorylation observed in vivo after CD3 stimulation. We also show that recombinant forms of both p56^lck and ZAP-70 phosphorylate Sam68 in vitro. However, using CD2 stimulated cells, we observe that p56^lck activation by itself does not induce Sam68 tyrosine phosphorylation. We conclude that p59^lck and p56^lck differently participate in regulating the phosphorylation state of Sam68 in T cells and that ZAP-70 may contribute to Sam68 tyrosine phosphorylation and to the specific recruitment of this molecule after CD3 stimulation.     

An inhibitor of cytotoxic functions produced by CD8+CD57+ T lymphocytes from patients suffering from AIDS and immunosuppressed bone marrow recipients

An inhibitor of the cytotoxic functions (ICF) mediated by human immunodeficiency virus (HIV)- or HLA-specific cytotoxic T lymphocytes, natural killer and lymphokine-activated killer (LAK) cells is secreted by CD8⁺CD57^? T lymphocytes, a subset expanded during infection with HIV and after bone marrow transplantation. We previously showed an apparent molecular mass of 20–30 kDa for this soluble glycosylated concanavalin A-binding inhibitor which is distinct from known cytokines. Here, we report a characterization of the mechanism of action of this CD8⁺CD57⁺ ICF. We show that the ICF-induced inhibition of LAK cell cytolytic activity is transient, with a spontaneous recovery of cytolytic potential after 18 h. When testing interactions of ICF with a large set of cytokines we found that the ICF-mediated inhibition of cytotoxic functions is antagonized by two cytokines: recombinant interleukin (rIL)-4 and recombinant interferon (rIFN)-γ. Finally, we show that ICF acts at the level of cytolytic effector cells, where it induces a significant increase of cyclic AMP (cAMP) level. In contrast, no modification of either cell surface antigen expression or of target/effector cell conjugate formation could be evidenced. Addition of rIL-4 and rIFN-γ reverses such an increase of cAMP levels and in parallel restores the cytolytic activity. Altogether, these data demonstrate that the glycoprotein ICF produced by CD8⁺CD57⁺ cells (1) inhibits cell-mediated cytotoxicity by sensitizing cytolytic effector cells to the cAMP pathway, and (2) is part of a cytokine network controlling cell-mediated cytotoxic functions.     

A comparison of three methods of scoring penile circumference changes

There are presently three widely used methods of scoring penile circumference data. The present experiment attempted to determine the ability of each method to explain the variance within a data set. A total of 19 subjects were presented with 20 photographic slides assigned to five categories: neutral, female adult, female adolescent, male adult, and male adolescent. Erectile responses to each slide were recorded and the data analyzed in terms of the raw scores, percentage of full erection, and a z-score transformation. Results indicated that the z scores captured the highest proportion of the variance (52.7%), followed by the percentage scores (32.5%), and the raw scores (30.1%). Findings are discussed in terms of their research and clinical implications.     

Agonist-mediated conformational changes of β-adrenoceptors could occur independent of functional coupling to Ns

Summary Agonist and antagonist binding characteristics of -adrenoceptors in turkey erythrocyte ghosts were determined at different temperatures ranging between 7°C and 42°C. [³H]-DHA saturation binding experiments revealed that the antagonist-receptor interaction is entropy-driven with a small enthalpic contribution. Isoproterenol/[³H]-DHA competition binding followed the law of mass action at all the investigated temperatures. The agonist-receptor interaction is enthalpy driven with a small unfavorable decrease in entropy. This is consistent with the agonist's ability to favor an endoenergetic transconformation of the receptors.Only part of the agonist-bound receptors can undergo functional coupling to the stimulatory component of the adenylate cyclase system (Ns). This coupling process is associated with locking-in of the agonist and becomes persistent in the presence of the alkylating reagent N-ethylmaleimide. The number of agonist/N-ethylmaleimide-sensitive sites (i.e. coupling-prone receptors) increases with the temperature until it reaches a plateau value of 50% between 27–32°C. Qualitatively similar data were obtained for rat lung and turkey erythrocyte membranes. These observations suggest that the whole receptor population can undergo agonist-mediated conformational changes but that only part of them can couple to Ns.     

Anatomical Mapping of the External Obturator Footprint: A Study In Cadavers with Implications for Direct Anterior THA

BackgroundThe external obturator footprint in the trochanteric fossa has been suggested as a potential landmark for stem depth in direct anterior THA. Its upper border can be visualized during surgical exposure of the femur. A recent study reported that the height of the tendon has little variability (6.4 ± 1.4 mm) as measured on CT scans and that the trochanteric fossa is consistently visible on conventional pelvic radiographs. However, it is unclear where exactly the footprint of this tendon should be templated during preoperative planning so that it can be useful intraoperatively.Questions/purposesIn this study, we sought: (1) to provide instructions on exactly where to template the external obturator footprint on a preoperative planning radiograph, and (2) to confirm the small variability in height of the external obturator footprint found on CT scans in a cadaver study.MethodsTwo-dimensional (2-D) and three-dimensional (3-D) imaging was used to map the anatomy of the external obturator footprint. This dual approach was chosen because of their complementarity; conventional 2-D radiographs translate to clinical practice but 3-D navigation-based digitalization combined with CT allows for a better understanding of the cortical lines that comprise the outline of the trochanteric fossa. In 12 (four males, mean age 80 years, range 69 to 88) formalin-treated cadaveric lower extremities including the pelvis, the external obturator tendon was dissected, and the top and bottom end of its footprint marked with two small needles, and calibrated radiographs were taken. For another five (three males, mean age 75.7 years, range 61 to 91) fresh-frozen cadaveric lower extremities, including femoral reflective marker frames, CT scans were obtained and the exact location of the external obturator footprint was recorded using 3-D navigation-based digitalization. Qualitative analysis of both imaging modalities was used to develop instructions on where the external obturator footprint should be templated on a preoperative planning radiograph. Quantitative analysis of the dimensions of the external obturator footprint was performed.ResultsThe lowest point of the external obturator footprint was consistently found (± 1 mm) at the intersection of the vertical line comprised of the lateral wall of the trochanteric fossa and the oblique line formed by the intertrochanteric crest and therefore allows templating of this structure on the preoperative planning radiograph. The median (range) height of the footprint measured 6.4 mm and demonstrated small variability (4.7 to 7.6).ConclusionsWe suggest templating a 6.4-mm circle with its bottom on the intersection described above.Clinical RelevanceThe distance between the templated shoulder of the stem and the top of the circle can be used intraoperatively for guidance. Discrepancy should lead to re-evaluation of stem depth and leg length. Future work will investigate the usability, validity, and reliability of the proposed methodology in daily clinical practice.     

Gene Replacement Strategies for Lung Cancer

Advances in our understanding of the molecular genetics of cancer present an opportunity to develop prevention and treatment strategies based on the reversal of specific genetic lesions. This strategy is analogous to the classic concept of gene therapy for replacement of defective or nonfunctioning genes. The gene families implicated in carcinogenesis include dominant oncogenes and tumor suppressor genes. Regional administration of viral vectors expressing wildtype p53 and antisense K-ras prevents tumor growth for tumors with the specific genetic lesions in orthotopic tumor models and mediates regression of large established tumors. These studies provide a rationale for a new clinical protocol recently approved by the National Institutes of Health Recombinant DNA Advisory Committee and Food and Drug Administration to replace a defective p53 gene with intratumor injection of recombinant retrovirus expressing wild-type p53 or elimination of activated K-ras by expression of antisense K-ras messenger RNA. If these agents are efficacious, their lack of toxicity may provide a sufficiently high therapeutic index such that they could be used as an adjuvant to surgery to treat patients with earlier stages of cancer or as prevention for second primary cancers for individuals with genetic abnormalities in premalignant lesions. Although much research needs to be done, the possibility of specific gene targeting with a high therapeutic index makes this a promising area of investigation.     

Intraoperative clonidine enhances postoperative morphine patient-controlled analgesia

In this prospective study, the postoperative analgesic effects of intraoperative iv clonidine were evaluated. Two hundred consecutive patients undergoing major abdominal surgery were randomly assigned to either balanced anaesthesia with iv clonidine (Group 1) or balanced anaesthesia alone (Group 2). A PCA infuser was connected immediately after tracheal extubation. It was programmed to deliver morphine "on demand" iv boluses at doses of 1 mg for patients greater than 65 yr and 1.5 mg for women or 2 mg for men less than 65 yr old. A blinded observer assessed postoperative analgesia by recording the analgesic demands (both met and unmet), patient pain scores, sedation scores, and any side effects during the first 36 hr after surgery. Intraoperative clonidine reduced the number of analgesic demands during the observation period (45 +/- 27 demands in Group 1 vs 81 +/- 60 in Group 2, P = 0.0001). This resulted in a reduction in morphine delivered (55.4 +/- 30.6 mg vs 67.1 +/- 45.1 mg, P less than 0.05), mainly during the first 12 hr (19.7 +/- 11.1 mg vs 27.6 +/- 18.1, mg P less than 0.001) and the unmet demand rate was also reduced at all time intervals (P less than 0.01). Clonidine did not exacerbate sedation or side effects. However, clonidine provided better analgesia in men and in patients less than 65 yr of age. Intraoperative iv clonidine enhances morphine analgesia after abdominal surgery.     

Laser angioplasty in peripheral arterial disease

Laser angioplasty was performed in 66 patients with total occlusion of the ilio-femoral artery. The system used consisted of a pulsed dye laser operating at 480 nm, 50 mJ pulse^–1, 2s pulse^–1 and 5 Hz. The treatment laser was coupled into a 200m optical fibre wrapped around a coil for improved flexibility and radiopacity. The treatment laser was connected with a diagnostic laser consisting of a continuous wave helium-cadmium laser operating at 325 nm, 50 ms and 3 mW for tissue detection using fluorescence spectroscopy. The primary success rate was 82%, the complication rate was 15% without any clinical sequelae and the 18 month follow-up patency rate was 64%. Due to the narrow pilot channel created by laser angioplasty, in each case a complementary balloon angioplasty was required to restore the arterial lumen. The success rate did not depend upon the length of occlusion but was rather related to the extent of calcification. Laser angioplasty guided by spectroscopy is effective and safe in patients with totally occluded peripheral arteries in whom mechanical devices failed to cross the obstruction.