Possible involvement of phosphatidylinositol 3-kinase in regulated exocytosis: studies in chromaffin cells with inhibitor LY294002

Several lines of evidence suggest that phosphorylated products of phosphatidylinositol play critical functions in the regulation of membrane trafficking along the secretory pathway. To probe the possible involvement of phosphatidylinositol 3-kinase (PI 3-kinase) in regulated exocytosis, we have examined its subcellular distribution in cultured chromaffin cells by immunoreplica analysis and confocal immunofluorescence. We found that the PI 3-kinase heterodimer consisting of the regulatory and catalytic subunits was associated essentially with the subplasmalemmal cytoskeleton in both resting and nicotine-stimulated chromaffin cells. Attempts to immunoprecipitate PI 3-kinase with anti-phosphotyrosine antibodies failed, suggesting that the activity of PI 3-kinase was not modulated by tyrosine phosphorylation and/or physical interaction with SH2-containing proteins in stimulated chromaffin cells. LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a potent inhibitor of PI 3-kinase, produced a dose-dependent inhibition of catecholamine secretion evoked by various secretagogues. Furthermore, cytochemical experiments with rhodamine-labeled phalloidin revealed that LY294002 blocked the disassembly of cortical actin in chromaffin cells stimulated by a depolarizing concentration of potassium. Our results suggest that PI 3-kinase may be one of the important regulatory exocytotic components involved in the signaling cascade controlling actin rearrangements required for catecholamine secretion.     

Postsurgical wound progression monitored by temporal changes in the expression of matrix metalloproteinase-9

It is important to monitor the early stages of postoperative wound repair in order to identify those problems associated with impaired healing. Many of the crucial cellular responses of early wound healing, such as inflammatory infiltration, angiogenesis and re-epithelialization, are made possible through the action of matrix metalloproteinases (MMPs). Expression of MMP-2 and MMP-9 is elevated in acute wounds, and still greater levels are found in chronic wounds, indicating that uncontrolled proteolysis is a characteristic of retarded healing. Therefore, comparative measurements of MMPs may be used to monitor the progression of early wound healing. To investigate this, wound fluids and sera were collected from mastectomy and colectomy patients throughout early stages of repair, and the temporal expression profile established. Wounds which were healing were expressed maximal levels of MMP-9 at 24 h, followed by a significant decline by 48 h. Persistent elevation of MMP-9 expression was associated with infected and chronic wounds, and was identified in postoperative wounds by the absence of the significant decline between 24 and 48 h. Measurement of MMP-9 in postoperative wound fluids, therefore, provides an early indicator of impaired healing, which may be evaluated non-invasively within 48 h of closure.     

Randomized trial of breast self-examination in Shanghai: methodology and preliminary results

BACKGROUND: The efficacy of breast self-examination in helping to reduce mortality from breast cancer has not been rigorously demonstrated. PURPOSE: To assess efficacy, a large, randomized trial was initiated in Shanghai, China. METHODS: From October 1989 to October 1991, 267040 current and retired female employees associated with 520 factories in the Shanghai Textile Industry Bureau were randomly assigned on the basis of factory to either a self-examination instruction group (133375 women) or a control group (133665 women). The women were born within the period from 1925 through 1958. Women in the instruction group were given intensive training in breast self-examination, including the use of silicone breast models and personalized instruction, plus two subsequent reinforcement sessions and multiple reminders to practice the technique. Women in the control group were asked to attend training sessions on the prevention of low back pain. All women have been followed for the development of breast diseases and for death from breast cancer. RESULTS: A high level of participation during the first 4-5 years of the trial was documented among women in the instruction group. Randomly sampled women in this group demonstrated greater proficiency in detecting lumps in breast models than did randomly sampled women in the control group. Approximately equal numbers of breast cancers were detected in the two groups (331 in the instruction group and 322 in the control group) through 1994, which is the last year for which case-finding efforts have been completed. The breast cancers detected in the instruction group were not diagnosed at an appreciably earlier stage or smaller size than those in the control group. More benign breast lesions were detected in the instruction group than in the control group (1457 versus 623, respectively), suggesting a higher index of suspicion for women who received training. Cumulative breast cancer mortality rates through 5 years from entry into the study were nearly equivalent for the two groups. CONCLUSIONS: Breast self-examination has not led to a reduction in mortality from breast cancer in this study cohort in the first several years since the trial began. A shift toward the diagnosis of disease at a less advanced stage in women given instruction has also not been demonstrated. Longer follow-up of participants in this trial is required before final assessment can be made of the efficacy of breast self-examination. IMPLICATIONS: At this time, there is insufficient evidence to recommend for or against the teaching of breast self-examination.     

Kinetic behaviour of muscle LDH immobilized in nylon tubing

Immobilization was carried out of the lactate dehydrogenase (LDH) from rabbit muscle (EC 1.1.1.27), cross-linked through the bifunctional reactive glutar-aldehyde on to nylon tubing (1 m long, 53cm² internal surface area). Immobilized LDH inactivation kinetics are of first order (t_1/2 = 3·6 years, k = 5·4,e^?4 day^?1 to 5°C). The smaller effect of pH on activity than in the case of LDH in solution can be explained on the basis of limitation to proton diffusion towards the support. A limiting effect to free external diffusion of the substrate towards and products from the support was also observed, an effect which seems to determine the effective kinetic behaviour of immobilized LDH. The apparent optimum temperature is centred around 40°C, observing a clear inactivation (thermal denaturation) above this temperature. In the temperature range studied (10–40°C), the co-existence was seen of a kinetic control accompanied by another control, involving diffusional transport of substrates and products, on the global activity of the immobilized enzyme. This makes the Arrhenius profiles curvilinear. Both graphic and statistical non-linear regression analysis of the kinetic data—rate, v, versus substrate concentration [S]—carried out under conditions in which the diffusional limitations can be considered negligible (high recirculation flow rate), permitted investigation of the intrinsic kinetic behaviour of immobilized LDH. In this sense, it can be deduced that the rate equation to which these data seem to be fitted is of the polynomial quotient type in [S] of minimum degree 2:2. Although the diffusional limitations have a marked effect on the type of global kinetics shown by immobilized LDH, temperature was not found to affect its v[S] behaviour. The experimental evidence obtained thus indicates that the rate equation in the 10-40°C temperature range continues to be a rational equation of at least degree 2:2 in [S].