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S F Hamm-Alvarez

Publications and source records attributed to S F Hamm-Alvarez.

26 records · Page 2Linked to original sources
PubMed1997

Cholinergic stimulation of lacrimal acinar cells promotes redistribution of membrane-associated kinesin and the secretory protein, beta-hexosaminidase, and increases kinesin motor activity.

The role of the microtubule-based motor, kinesin, in membrane trafficking has been investigated in resting and stimulated acinar cells from rabbit lacrimal gland, a cholinergically controlled secretory tissue. Microtubule-dependent motors from extracts of control and carbachol-treated acini were isolated by microtubule-affinity purification and their activity was determined using a video-enhanced differential interference contrast microscopy assay for microtubule gliding. The observation that carbachol treatment resulted in a 2.2-fold stimulation of the frequency of GTP-dependent microtubule gliding in fractions isolated by microtubule-affinity purification and GTP release suggested that kinesin was a target of carbachol-induced stimulation. Resolution of membranes from resting cells by fractionation on a sorbitol density gradient followed by partitioning analysis in a dextran-polyethyleneglycol two-phase system revealed that membrane-associated kinesin codistributed with Golgi-derived membranes, a post-Golgi secretory compartment designated Hex1, membranes from a trans Golgi network-like compartment, endoplasmic reticulum and a group of putative lysosomal membranes containing cathepsin B. Comparable fractionation of carbachol-treated acini showed that stimulation caused redistributions of membrane-associated kinesin, the secretory enzyme beta-hexosaminidase, and galactosyltransferase that appeared to reflect both a reorganization within the Golgi complex and a return of material to the Golgi complex from the secretory pathway. Our findings that carbachol promotes activation of lacrimal acinar kinesin as well as major shifts in kinesin-membrane association within the secretory pathway suggests that kinesin plays a major role in secretory vesicle assembly, apical secretion, and/or secretory vesicle membrane recycling in the lacrimal gland.

Animals↗
PubMed1997

Involvement of phosphorylation in doxorubicin-mediated myofibril degeneration. An immunofluorescence microscopy analysis.

Loss of myofilaments has been observed in both adaptive cardiac responses (i.e., hypertrophy) as well as in chemotheraputic use of antineoplastic drugs with cardiotoxic side effects (i.e., doxorubicin). An understanding of the degenerative process is a prerequisite for determining approaches to limit the cardiomyopathic changes associated with chronic heart disease or long-term chemotheraputic treatments. However, little is known about the specific events and molecular changes that initiate the degenerative process. To study this process, neonatal rat cardiomyocytes were treated with doxorubicin, which induced rapid and widespread thin-filament degeneration as observed by fluorescence confocal microscopy. Which demonstrated deterioration of sarcomeric thin-filament structure. Changes in the spontaneous beating of cardiomyocytes corresponding with myofibrillar degeneration were apparent using differential interference contrast video microscopy. After finding induction of kinase activity by doxorubicin in cultured cardiomyocytes, the protective effects of specific inhibitors of kinase activity were assessed for their ability to inhibit doxorubicin-induced myofibrillar break-down. Doxorubicin-induced changes appeared similar to the degeneration observed after treatment with a protein kinase activator (phorbol 12-myristate 13-acetate) or a serine-threonine protein phosphatase inhibitor (okadaic acid). Collectively, these results indicate that activation of protein kinase is an important event in the initiation of myofibrillar degeneration by doxorubicin. Further analyses of myofibrillar proteins with respect to biochemical modifications will be necessary to determine if phosphorylation events transmit signal(s) to initiate degeneration.

Animals↗
PubMed1996

Paclitaxel and nocodazole differentially alter endocytosis in cultured cells.

PURPOSE: Microtubule-based transport facilitates the endocytosis of exogenous macromolecules. We have determined how microtubule accumulation and disassembly alter endocytosis. METHODS: The effects of paclitaxel, which promotes microtubule assembly, and nocodazole, which promotes microtubule disassembly, on fluid-phase and receptor-mediated endocytosis were measured using uptake of horseradish peroxidase and 125I-transferrin, respectively. Changes in membrane and microtubule organization were examined by fluorescence microscopy. RESULTS: Neither paclitaxel (4 microM, 60 min pretreatment) nor nocodazole (1 microgram/ml, 60 min pretreatment) significantly inhibited fluid-phase endocytosis. However, paclitaxel caused a redistribution of fluorescent fluid-phase marker to the periphery. Both paclitaxel and nocodazole treatment significantly (p < or = 0.05) reduced the initial uptake of 125I-transferrin at 5 min to approximately 50% of control. Despite the similarity of the effects on initial endocytic uptake, the effects on steady state accumulation of 125I-transferrin were quite distinct. Exposure of CV-1 cells to paclitaxel for an additional 30, 60 or 90 min also showed reduced accumulation of 125I-transferrin up to a maximum significant (p < or = 0.05) inhibition of 48% +/- 10% of control at 90 min. In contrast, nocodazole caused an initial significant (p < or = 0.05) increase in 125I-transferrin accumulation after 30 min (159% +/- 13% of control), while by 90 min 125I-transferrin accumulation had returned to control levels. Microtubule content, particularly of stable microtubules, was increased in CV-1 cells by paclitaxel, but abolished by nocodazole treatment. CONCLUSIONS: Our data show that changes in the microtubule array can alter the dynamics of receptor movement through the endosomal pathway. However, microtubule assembly versus disassembly have different effects.

Animals↗
PubMed1996

Protein phosphatases independently regulate vesicle movement and microtubule subpopulations in hepatocytes.

To investigate the regulation of microtubule (MT)-based vesicle transport and the interphase MT array in hepatocytes, we have used okadaic acid (OKA) and microcystin (MCYST), two toxins that inhibit serine-threonine protein phosphatases (PP) 1 and 2A, to alter cellular phosphorylation. Video-enhanced differential interference contrast microscopy analysis revealed that both toxins inhibited the frequency, velocity, and run length of MT-dependent vesicle movements dose dependently between 50 and 500 nM. At our maximum dose of 500 nM, both toxins significantly decreased PP2A activity (OKA to 45 +/- 12% and MCYST to 57 +/- 2%), whereas PP1 was inhibited only by MCYST. Because no additional effects on vesicle movements were caused by MCYST over the changes caused by OKA, these data implicate PP2A in the regulation of MT-dependent vesicle movement. To understand whether the changes in parameters of vesicle movements were due to changes in the MT array, the effects of these toxins on MT distribution were examined by immunofluorescence microscopy. Although lower doses of OKA produced no effects, treatment with 500 nM OKA altered MT organization and also caused fragmentation and loss of acetylated (stable) MTs. In contrast, MCYST concentrations up to 500 nM elicited no changes in MT organization in general or in the acetylated (stable) array. From these findings we conclude that inhibition of MT-dependent vesicle movement by the PP inhibitors, MCYST and OKA, in hepatocytes cannot result from changes or disruption in the MT array. Because OKA (an inhibitor of PP2A only in our system) at high doses caused loss of stable MTs, whereas MCYST (an inhibitor of both PP1 and PP2A) did not, we conclude that the control of the preservation of the stable MT array in hepatocytes is complex. Stable MTs require active PP2A for maintenance, but the disruption of the array through inhibition of PP2A can be prevented if PP1 is also inhibited, suggesting that the relative degree of phosphorylation of multiple cellular components is the determinant of MT stability.

Animals↗
PubMed1994

Coordinate depression of bradykinin receptor recycling and microtubule-dependent transport by taxol.

Significant cardiovascular side effects have limited the use of taxol as an anticancer drug. A link between decreased plasma membrane dynamics and taxol has been implied because taxol can inhibit intracellular vesicle movements. Reduced membrane recycling caused by taxol could inhibit agonist-evoked Ca2+ signaling within endothelial cells, resulting in endothelium-dependent vasodilation. Bradykinin and ATP are two agonists that evoke Ca2+ transients in endothelial cells. Since the bradykinin receptor-agonist complex is internalized and recycled whereas the ATP agonist-receptor complex is not, we expected that a taxol inhibition of recycling would decrease bradykinin but not ATP receptor activity. We found that taxol depresses (i) the frequency (to 41% of control) and velocity (to 55% of control) of microtubule-dependent vesicle transport and (ii) bradykinin-evoked cytosolic Ca2+ transients (to 76% of control) in bovine aortic endothelial cells. In studying bradykinin receptor desensitization, which reflects receptor recycling, we demonstrate that taxol inhibits bradykinin-evoked Ca2+ transients by 50%. Taxol did not significantly alter ATP-evoked Ca2+ transients in either single-exposure or desensitization experiments. We suggest that taxol's reduction of bradykinin-evoked Ca2+ transients is due to altered microtubule-dependent membrane recycling. This report describes taxol's ability to alter plasma membrane composition through effects on vesicle transport and membrane trafficking pathways. This finding provides a possible mechanism by which taxol can substantially alter cardiovascular function.

Adenosine Triphosphate↗
PubMed1993

Regulation of vesicle transport in CV-1 cells and extracts.

To investigate the regulation of microtubule-dependent vesicle motility, we have studied the effects of pharmacological agents on the frequency and velocity of small vesicle movements in intact CV-1 cells. Nocodazole, but not cytochalasin B or D, abolished vesicle movements, indicating that these movements were microtubule and not actin-dependent. The frequency of vesicle movements was stimulated maximally sixfold by okadaic acid from a resting value of 1.6 movements/min per microns2 in serum-starved CV-1 cells. Other activators in decreasing order of effectiveness are fetal calf serum, dibutyryl cAMP, cholera toxin, genistein, A23187, and trental. On the other side, taxol inhibited vesicle movements by a factor of four. The activators, okadaic acid, fetal calf serum, and dibutyryl cAMP, also increased vesicle velocity and run length, while taxol decreased vesicle velocity. Although modulation of the frequency of vesicle movements over a > 20-fold range was observed, under all conditions the fraction of vesicles moving inward versus outward did not significantly change. Only in the case of taxol was the distribution of microtubules altered within this same time period. Both inward and outward microtubule-dependent vesicle movements therefore appear to be coordinately regulated. The enhanced vesicle motility elicited by fetal calf serum in intact cells correlated with in vitro measurements of vesicle motility and velocity on purified microtubules using microtubule affinity-purified motors and carbonate-washed vesicles from cells treated with fetal calf serum. This suggests that the amount of vesicular intracellular membrane traffic is coordinately regulated with microtubule-dependent motor activity.

Adenylyl Cyclases↗
PubMed1990

The presence and distribution of reduced folates in Escherichia coli dihydrofolate reductase mutants.

Escherichia coli DNA photolyase was overproduced and purified from each of two mutant E. coli strains lacking dihydrofolate reductase. The extent of over-production in the mutants was comparable to that seen in the wild type strain. Examination of the isolated photolyase from these strains revealed that the folate cofactor, 5,10-methenyltetrahydrofolate, was present in these proteins at a level of 60-80% compared to that purified from the wild type strain. Further examination of the dihydrofolate reductase-deficient strains revealed the presence of other tetrahydrofolate derivatives. These findings demonstrate that dihydrofolate reductase is not essential for the production of tetrahydrofolates in E. coli.

Chromatography, High Pressure Liquid↗