Tyrosine 474 of ZAP-70 is required for association with the Shc adaptor and for T-cell antigen receptor-dependent gene activation

The protein tyrosine kinase ZAP-70 plays a central role in T-cell activation. Following receptor engagement, ZAP-70 is recruited to the phosphorylated subunits of the T-cell antigen receptor (TCR). This event results in ZAP-70 activation and in association of ZAP-70 with a number of signaling proteins. Among these is the Shc adaptor, which couples the activated TCR to Ras. Shc interaction with ZAP-70 is mediated by the Shc PTB domain. The inhibitory effect of a Shc mutant containing the isolated PTB domain suggests that Shc interaction with ZAP-70 might be required for TCR signaling. Here, we show that a point mutation (Phe474) of the putative Shc binding site on ZAP-70, spanning tyrosine 474, prevented ZAP-70 interaction with Shc and the subsequent binding of Shc to phospho-zeta. Neither ZAP-70 catalytic activity nor the pattern of protein phosphorylation induced by TCR triggering was affected by this mutation. However expression of the Phe474 ZAP-70 mutant resulted in impaired TCR-dependent gene activation. ZAP-70 could effectively phosphorylate Shc in vitro. Only the CH domain, which contains the two Grb2 binding sites on Shc, was phosphorylated by ZAP-70. Both Grb2 binding sites were excellent substrates for ZAP-70. The data show that Tyr474 on ZAP-70 is required for TCR signaling and suggest that Shc association with ZAP-70 and the resulting phosphorylation of Shc might be an obligatory step in linking the activated TCR to the Ras pathway.     

A comparison of intra- and interpersonal interlimb coordination: coordination breakdowns and coupling strength

Intra- and interpersonal interlimb coordination of pendulums swung from the wrist was investigated. For both kinds of coordination, the steady state and breakdown of bimanual rhythmic coordination as indexed by the time series of the relative phase angle phi were studied under the manipulation of coordination mode, frequency of oscillation, and the difference in the eigenfrequencies (preferred tempos) of the individual oscillating limbs. The properties observed for both intra- and interpersonal coordination were those predicted by a dynamical model of rhythmic coordination that considers the coordinated limbs coupled to be nonlinear oscillators. Using a regression method, the coupling strengths of the coupled system were recovered. As predicted by the dynamical model, the strength of the dynamic was generally greater for the in-phase than the anti-phase mode and decreased with increasing frequency. Further, the strength of the interpersonal interlimb coupling was weaker than that of intrapersonal interlimb coupling.     

Change in environment of the P1 side chain upon progression from the Michaelis complex to the covalent serpin-proteinase complex

Serpins inhibit proteinases by forming a kinetically trapped intermediate during a suicide substrate inhibition reaction. To determine whether the kinetic trap involves a repositioning of the P1 side chain of the serpin following formation of the initial Michaelis complex, we used the tryptophan of a P1 M-->W variant of human alpha1-proteinase inhibitor as a fluorescent reporter group of the environment of the P1 side chain. The P1W variant was a valid model serpin and formed SDS-stable complexes with both trypsin and chymotrypsin with a stoichiometry of inhibition close to 1.0. Rates of inhibition of chymotrypsin for wild-type and variant alpha1-proteinase inhibitor differred only approximately 1.8-fold. Rates of inhibition of trypsin were, however, 25-fold lower for the variant than for the wild-type inhibitor. Steady-state fluorescence spectra showed a change in environment for the P1 side chain upon forming both covalent complex with trypsin or chymotrypsin and noncovalent complex with anhydrochymotrypsin. The P1 environments in the chymotrypsin and anhydrochymotrypsin complexes were, however, different. Fluorescence quenching studies confirmed the burial of the P1 side chain upon formation of both the noncovalent and covalent complexes, but were not able to discriminate between the solvent accessibility in these complexes. Stopped-flow fluorescence measurements resolved the covalent intramolecular reaction that led to covalent complex and showed that, during the course of the covalent reaction, the environment of the P1 side chain changed consistent with a repositioning relative to residues of the proteinase active site as part of formation of the trap. This repositioning is likely to be a crucial part of the trapping mechanism.     

Pharmacotherapy of acid-peptic disorders

In the past two decades, there have been tremendous changes in the understanding and therapy of acid-peptic diseases. Currently there are many new and effective approaches to the treatment of dyspeptic symptoms and the ulcerative and erosive diseases of the upper gastrointestinal tract. Application of an appropriate therapeutic strategy requires understanding of the pathophysiology of the disease process as well as the pharmacology of the available medications. In general, medications to neutralize gastric acid or suppress acid secretion are selected relative to the severity of the acid-mediated injury. Antacids are ideal for suppression of sporadic symptoms. H2-receptor antagonists are useful for the treatment of dyspepsia or uncomplicated ulcers. For the most severe injuries, proton pump inhibitors provide both relief of symptoms and healing of damaged mucosa.     

Diffusion of nitric oxide and scavenging by blood in the vasculature

It has been deduced (Lancaster, Proc. Natl. Acad. Sci. USA 91 (1994) 8137-8141), from a consideration of Fick's law of diffusion, that the very effective scavenging of nitric oxide (NO) by haemoglobin in red blood cells prevents any NO from endothelial cells migrating outwards into vascular smooth muscle. This conclusion has led some authors to suggest that endothelium-derived relaxing factor (EDRF) is not free NO. We have reconsidered the application of Fick's law to the migration of NO in the vasculature, making allowance for the reaction of NO with guanylate cyclase and for the layer of red blood-free plasma next to the endothelium. The source of NO is taken as an infinite cylinder. Calculations for vessels of various diameters indicate that a substantial amount of NO migrates outwards in spite of very effective scavenging by haemoglobin and that the relative amount of NO migrating outwards depends upon the radius of the vessel. The view that locally produced NO is not responsible for vascular dilation has not been sustained.     

Characterisation of the glass transition of an amorphous drug using modulated DSC

PURPOSE: The use of modulated differential scanning calorimetry (MDSC) as a novel means of characterising the glass transition of amorphous drugs has been investigated, using the protease inhibitor saquinavir as a model compound. In particular, the effects of measuring variables (temperature cycling, scanning period, heating mode) have been examined. METHODS: Saquinavir samples of known moisture content were examined using a TA Instruments 2920 MDSC at a heating rate of 2 degrees C/min and an amplitude of +/-0.159 degrees C with a period of 30 seconds. These conditions were used to examine the effects of cycling between - 50 degrees C and 150 degrees C. A range of periods between 20 and 50 seconds were then studied. Isothermal measurements were carried out between 85 degrees C and 120 degrees C using an amplitude of +/-0.159 degrees C with a period of 30 seconds. RESULTS: MDSC showed the glass transition of saquinavir (0.98 +/- 0.05%w/w moisture content) in isolation from the relaxation endotherm to give an apparent glass transition temperature of 107.0 degrees C +/- 0.4 degrees C. Subsequent temperature cycling gave reproducible glass transition temperatures of approximately 105 degrees C for both cooling and heating cycles. The enthalpic relaxation peak observed in the initial heating cycle had an additional contribution from a Tg "shift" effect brought about by the difference in response to the glass transition of the total and reversing heat flow signals. Isothermal studies yield a glass transition at 105.9 degrees C +/- 0.1 degrees C. CONCLUSIONS: MDSC has been shown to be capable of separating the glass transition of saquinavir from the relaxation endotherm, thereby facilitating measurement of this parameter without the need for temperature cycling. However, the Tg "shift" effect and the number of modulations through the transition should be taken into account to avoid drawing erroneous conclusions from the experimental data. MDSC has been shown to be an effective method of characterising the glass transition of an amorphous drug, allowing the separate characterisation of the Tg and endothermic relaxation in the first heating cycle.     

Ronald Ross: a century of the transfer of malaria by mosquitoes

Ronald Ross is a brilliant and polyvalent mind. When orientated towards medicine he took the training amateurishly and ended up with a limited qualification. After 2 years as a ship doctor, he attended the compulsory complementary training in order to be admissible in the IMS, the garrison life left him with plenty of time to engage in his hobby's: painting for a short while, writing, poetry and mathematics. By the end of his first term he questioned the sense of his medical activities and decided, with a view to his career, to acquire a Public Health diploma and some complementary bacteriology. During his second term the malaria problem drew his attention. As he was unable to detect the parasite of Laveran in the blood of patients with malarial fevers he concluded that the parasite had been some lucky microscopic finding without any value and turned this parasite into ridicule. During his leave in 1894 he met Manson, who showed him the technique to put the parasite in evidence and convinced him to search for its vector which, to his opinion, should be a mosquito. Ross decided to follow this lead. With his minimal parasitological knowledge and an entomological background limited to the external appearance of mosquitoes he endeavoured to establish the life cycle of the malarial parasites. Notwithstanding some service bounded transfers, he followed the fate of the filaments of the crescents and discovered the parasite on the stomachwall of dapled-winged mosquitoes. During his special mission in Calcutta and in the absence of suitable malaria infections in man he shifted to bird proteosoma (now P. relictum) with a grey mosquito (culex) as vector. He demonstrated the complete life-cycle ending in the salivary glands of the mosquitoes and succeeded in transmitting this infection by mosquito-bites in healthy birds. This climax in his research was crowned by the attribution in 1902 of the Nobel Prize for Medicine. In the mean time he resigned from the IMS and was appointed as "Lecturer on Tropical Diseases" at the Liverpool School. He reoriented his activities to the prevention of malaria by control of the vector in its aquatic larval stage, which he tried out and promoted during his journeys to the West African Coast and other countries. His current were variegated but without salient details. Through the survey of the parasite index and the assessment of the spleen rate in children he founded the malariometry as a epidemiological tool, focussed attention on the relation of malaria and the community and on the complexity of the transmission dynamics. By handing in his resignation at the Liverpool School and moving to London he hampered further his scientific productivity. The tardy foundation of the Ross Institute did not stimulate a new impetus. He suffered a stroke which left him partially crippled an he died in his Institute.     

Cervical carcinoma metastatic to para-aortic nodes: extended field radiation therapy with concomitant 5-fluorouracil and cisplatin chemotherapy: a Gynecologic Oncology Group study

PURPOSE: A multicenter trial of chemoradiation therapy to evaluate the feasibility of extended field radiation therapy (ERT) with 5-fluorouracil (5-FU) and cisplatin, and to determine the progression-free interval (PFI), overall survival (OS), and recurrence sites in patients with biopsy-confirmed para-aortic node metastases (PAN) from cervical carcinoma. METHODS AND MATERIALS: Ninety-five patients with cervical carcinoma and PAN metastases were entered and 86 were evaluable: Stage I--14, Stage II--40, Stage III--27, Stage IVA--5. Seventy-nine percent of the patients were followed for 5 or more years or died. ERT doses were 4500 cGy (PAN), 3960 cGy to the pelvis (Stages IB/IIB), and 4860 cGy to the pelvis (Stages IIIB/IVA). Point A intracavitary (IC) doses were 4000 cGy (Stages IB/IIB), and 3000 cGy (Stages IIIB/IVA). Point B doses were raised to 6000 cGy (ERT + IC) with parametrial boost. Concomitant chemotherapy consisted of 5-FU 1000 mg/m2/day for 96 hours and cisplatin 50 mg/m2 in weeks 1 and 5. RESULTS: Eighty-five of 86 patients completed radiation therapy and 90% of patients completed both courses of chemotherapy. Gynecologic Oncology Group (GOG) grade 3-4 acute toxicity were gastrointestinal (18.6%) and hematologic (15.1%). Late morbidity actuarial risk of 14% at 4 years primarily involved the rectum. Initial sites of recurrence were pelvis alone, 20.9%; distant metastases only, 31.4%; and pelvic plus distant metastases, 10.5%. The 3-year OS and PFI rate were 39% and 34%, respectively, for the entire group. OS was Stage I--50%, Stage II--39%, and Stage III/IVA--38%. CONCLUSIONS: Extended field radiation therapy with 5-FU and cisplatin chemotherapy was feasible in a multicenter clinical trial. PFI of 33% at 3 years suggests that a proportion of patients achieve control of advanced pelvic disease and that not all patients with PAN metastases have systemic disease. This points to the importance of assessment and treatment of PAN metastases.     

A prospective study to assess the effect of ambulatory teaching on patient satisfaction

PURPOSE: To assess the effect of ambulatory teaching on patients' satisfaction. METHOD: In 1996, 103 adult patients presenting to the Walter Reed General Medicine Walk-in Clinic completed a patient-satisfaction questionnaire immediately following their visits, during which they were initially seen by a trainee (third-year medical student or intern) and then seen by a faculty preceptor. The questionnaire included five items from the validated Medical Outcomes Study (MOS)-9 questionnaire as well as two open-ended questions. Fourteen staff physicians, 13 students (49% of the visits), and 11 interns (51% of the visits) participated in the study. Satisfaction was analyzed by level of training, and the responses from the study patients were compared with the responses from 372 usual-care (i.e., non-teaching) patients from the same clinic, using the chi-squared test. RESULTS: The study patients were typically pleased with their encounters, rating their overall satisfaction as excellent (61%), very good (29%), or good (9%). Nearly two thirds of the patients rated their satisfaction with waiting time to be very good or excellent. Compared with the usual-care patients, the study patients reported equal or greater satisfaction for all five MOS-9 items. Ninety-five percent of the study patients said they would be willing to be seen by a trainee-staff team on future visits. There was no difference in patient satisfaction by trainee level. The study patients cited enhanced interaction (45%), enhanced education (34%), and improved care (26%) as benefits of trainee-involved care, and increased waiting time (18%) and worse care (5%) as drawbacks. CONCLUSION: The results of this study suggest that ambulatory teaching does not adversely affect patient satisfaction, regardless of trainee level, and that patients who have been seen by trainee-staff teams are willing to experience such encounters again.