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Biomedical subjects

M Grabe

Publications and source records attributed to M Grabe.

At least 19 recordsLinked to original sources
PubMed2002

Biosynthesis and secretion of pituitary hormones: dynamics and regulation.

Production and secretion of hormones by the pituitary involve highly orchestrated intracellular transport and sorting steps. Hormone precursors are routed through a series of compartments before being packaged in secretory granules. These highly dynamic carriers play crucial roles in both prohormone processing and peptide exocytosis. We have employed the ACTH-secreting AtT-20 cell line to study the membrane sorting events that confer functionality (prohormone activation and regulated exocytosis) to these secretory carriers. The unique ability of granules to promote prohormone processing is attributed to their acidic interior. Using a novel avidin-targeted fluorescence ratio imaging technique, we have found that the trans-Golgi of live AtT-20 cells maintains a mildly acidic (approximately pH 6.2) interior. Budding of secretory granules causes the lumen to acidify to <pH 6.0, which is both necessary and sufficient to trigger SPC3-mediated proteolytic conversion of proopiomelanocortin to ACTH. Investigation of the pH regulatory mechanism indicates that the trans-Golgi and secretory granules maintain different pH values by distinct sorting of key membrane transporters. Mathematical modeling of our data suggests that the decreasing pH values of organelles of the regulated secretory pathway is established by gradually increasing the density of active H+ pumps from the ER to Golgi while concomitantly decreasing the H+ permeability from ER to Golgi to secretory granules. An in vitro assay was developed to study the formation of processing-competent secretory granules from their processing-incompetent precursor trans-Golgi compartment. Our data suggest that ARF1-mediated sorting of proton pumps and leaks during early stages of granule formation confers processing competency to the resulting organelle. Once formed, these young granules continue to undergo membrane remodeling which results in dynamic changes in their exocytotic behavior. Two SNAREs, VAMP4 and synaptotagmin IV, enter newly formed granules but are removed from the maturing granule membrane by vesicle budding. Sorting of these proteins is correlated with the acquisition of Ca2+-triggered exocytosis and a decrease in unregulated exocytotic rate. Thus, biosynthesis and secretion of pituitary hormones are dynamically regulated by intracellular sorting events that govern the functions of their secretory carriers.

Animals↗
PubMed2001

Mechanisms of pH regulation in the regulated secretory pathway.

A precise pH gradient between organelles of the regulated secretory pathway is required for sorting and processing of prohormones. We studied pH regulation in live endocrine cells by targeting biotin-based pH indicators to cellular organelles expressing avidin-chimera proteins. In AtT-20 cells, we found that steady-state pH decreased from the endoplasmic reticulum (ER) (pH(ER) = 7.4 +/- 0.2, mean +/- S.D.) to Golgi (pH(G) = 6.2 +/- 0.4) to mature secretory granules (MSGs) (pH(MSG) = 5.5 +/- 0.4). Golgi and MSGs required active H(+) v-ATPases for acidification. ER, Golgi, and MSG steady-state pH values were also dependent upon the different H(+) leak rates across each membrane. However, neither steady-state pH(MSG) nor rates of passive H(+) leak were affected by Cl(-)-free solutions or valinomycin, indicating that MSG membrane potential was small and not a determinant of pH(MSG). Therefore, our data do not support earlier suggestions that organelle acidification is primarily regulated by Cl(-) conductances. Measurements of H(+) leak rates, buffer capacities, and estimates of surface areas and volumes of these organelles were applied to a mathematical model to determine the H(+) permeability (P(H+)) of each organelle membrane. We found that P(H+) decreased progressively from ER to Golgi to MSGs, and proper acidification of Golgi and MSGs required gradual decreases in P(H+) and successive increases in the active H(+) pump density.

Animals↗
PubMed2001

Regulation of organelle acidity.

Intracellular organelles have characteristic pH ranges that are set and maintained by a balance between ion pumps, leaks, and internal ionic equilibria. Previously, a thermodynamic study by Rybak et al. (Rybak, S., F. Lanni, and R. Murphy. 1997. Biophys. J. 73:674-687) identified the key elements involved in pH regulation; however, recent experiments show that cellular compartments are not in thermodynamic equilibrium. We present here a nonequilibrium model of lumenal acidification based on the interplay of ion pumps and channels, the physical properties of the lumenal matrix, and the organelle geometry. The model successfully predicts experimentally measured steady-state and transient pH values and membrane potentials. We conclude that morphological differences among organelles are insufficient to explain the wide range of pHs present in the cell. Using sensitivity analysis, we quantified the influence of pH regulatory elements on the dynamics of acidification. We found that V-ATPase proton pump and proton leak densities are the two parameters that most strongly influence resting pH. Additionally, we modeled the pH response of the Golgi complex to varying external solutions, and our findings suggest that the membrane is permeable to more than one dominant counter ion. From this data, we determined a Golgi complex proton permeability of 8.1 x 10(-6) cm/s. Furthermore, we analyzed the early-to-late transition in the endosomal pathway where Na,K-ATPases have been shown to limit acidification by an entire pH unit. Our model supports the role of the Na,K-ATPase in regulating endosomal pH by affecting the membrane potential. However, experimental data can only be reproduced by (1) positing the existence of a hypothetical voltage-gated chloride channel or (2) that newly formed vesicles have especially high potassium concentrations and small chloride conductance.

Acid-Base Equilibrium↗
PubMed2001

Perioperative antibiotic prophylaxis in urology.

This report reviews recent studies on infectious complications and antibiotic prophylaxis in common urological instrumentation. As a result of variations in the definitions of infectious complications and inconsistencies in study design and risk factor analysis there is presently limited clear-cut evidence for giving definite standards regarding antibiotic prophylaxis for most urological interventions. The consequences are that patients may be exposed to unnecessary hazards and the healthcare system to additional costs. Nonetheless, most authors agree that patients should have sterile urine at urological instrumentation and that any other detected risk factor should be controlled. When antibiotic prophylaxis is considered, it should be timed properly before the intervention, which varies with the type of intervention and the choice of antibiotic, and should last for a limited period of time. In most common urological manipulations, correctly administered oral prophylaxis has been shown to be as effective as intravenous prophylaxis. A series of guidelines aimed at keeping the rates of healthcare-associated infections and the level of bacterial resistance as low as possible should, in combination with the rational use of antibiotics, be one of several marks of quality of a urological centre. To achieve this goal, new well-designed studies considering different regimens, risk factor analysis and economical analysis should be encouraged.

Antibiotic Prophylaxis↗
PubMed2001

Proton leak and CFTR in regulation of Golgi pH in respiratory epithelial cells.

Work addressing whether cystic fibrosis transmembrane conductance regulator (CFTR) plays a role in regulating organelle pH has remained inconclusive. We engineered a pH-sensitive excitation ratiometric green fluorescent protein (pHERP) and targeted it to the Golgi with sialyltransferase (ST). As determined by ratiometric imaging of cells expressing ST-pHERP, Golgi pH (pH(G)) of HeLa cells was 6.4, while pH(G) of mutant (DeltaF508) and wild-type CFTR-expressing (WT-CFTR) respiratory epithelia were 6.7-7.0. Comparison of genetically matched DeltaF508 and WT-CFTR cells showed that the absence of CFTR statistically increased Golgi acidity by 0.2 pH units, though this small difference was unlikely to be physiologically important. Golgi pH was maintained by a H(+) vacuolar (V)-ATPase countered by a H(+) leak, which was unaffected by CFTR. To estimate Golgi proton permeability (P(H(+))), we modeled transient changes in pH(G) induced by inhibiting the V-ATPase and by acidifying the cytosol. This analysis required knowing Golgi buffer capacity, which was pH dependent. Our in vivo estimate is that Golgi P(H(+)) = 7.5 x 10(-4) cm/s when pH(G) = 6.5, and surprisingly, P(H(+)) decreased as pH(G) decreased.

Animals↗
PubMed2001

Cystic fibrosis transmembrane conductance regulator and H+ permeability in regulation of Golgi pH.

This paper reviews experiments from this lab that have tested the hypothesis that pH of the Golgi (pH(G)) of cystic fibrosis (CF) airway epithelial cells is alkaline compared to normal, that this altered pH affects sialyltransferase and other Golgi enzymes controlling biochemical composition of the plasma membrane and that altered surface biochemistry increases bacterial binding. We generated a plasmid encoding a modified green fluorescence protein-sialyltransferase (GFP-ST) chimera protein that was pH-sensitive and localized to the Golgi when transfected into HeLa cells and also CF and normal or cystic fibrosis transmembrane conductance regulator- (CFTR)-corrected airway epithelial cells. Digital imaging microscopy of these Golgi-localized probes showed that there was no correlation between pH(G) (6.4-7.0) and the presence of CFTR, whether cells were in HCO(3)(-)/CO(2)-containing or in HCO(3)(-)/CO(2)-free solutions. Activation of CFTR by raising cell [cAMP] had no effect on pH(G). Thus, CFTR seemed not to be involved in controlling pH(G). Experiments on HeLa cells using an avidin-sialyltransferase chimera in combination with a pH-sensitive fluorescent biotin indicated that even in cells that do not express CFTR, Cl(-) and K(+) conductances of the Golgi and other organelle membranes were large and that pH(G) was controlled solely by the H(+) v-ATPase countered by a H(+) leak. A mathematical model was applied to these and other published data to calculate passive H(+) permeability (P(H+)) of the Golgi, endoplasmic reticulum, trans-Golgi network, recycling endosomes and secrety granules from a variety of cells. An organelle's acidity was inversely correlated to its calculated P(H+). We conclude that the CFTR plays a minor role in organelle pH regulation because other (Cl(-) and K(+)) channels are present in sufficient numbers to shunt voltages generated during H(+) pumping. Acidity of the Golgi (and perhaps other organelles) appears to be determined by the activity of H(+) pumps countered by H(+) leaks.

Cystic Fibrosis Transmembrane Conductance Regulato↗
PubMed2000

How Fo-ATPase generates rotary torque.

The F-ATPases synthesize ATP using a transmembrane ionmotive force (IMF) established by the electron transport chain. This transduction involves first converting the IMF to a rotary torque in the transmembrane Fo portion. This torque is communicated from Fo to the F1 portion where the energy is used to release the newly synthesized ATP from the catalytic sites according to Boyer's binding change mechanism. Here we explain the principle by which an IMF generates this rotary torque in the Fo ion engine.

Amino Acid Sequence↗
PubMed2000

The mechanochemistry of V-ATPase proton pumps.

The vacuolar H(+)-ATPases (V-ATPases) are a universal class of proton pumps that are structurally similar to the F-ATPases. Both protein families are characterized by a membrane-bound segment (V(o), F(o)) responsible for the translocation of protons, and a soluble portion, (V(1), F(1)), which supplies the energy for translocation by hydrolyzing ATP. Here we present a mechanochemical model for the functioning of the V(o) ion pump that is consistent with the known structural features and biochemistry. The model reproduces a variety of experimental measurements of performance and provides a unified view of the many mechanisms of intracellular pH regulation.

Adenosine Triphosphate↗
PubMed1999

Energy transduction in the sodium F-ATPase of Propionigenium modestum.

The F-ATPase of the bacterium Propionigenium modestum is driven by an electrochemical sodium gradient between the cell interior and its environment. Here we present a mechanochemical model for the transduction of transmembrane sodium-motive force into rotary torque. The same mechanism is likely to operate in other F-ATPases, including the proton-driven F-ATPases of Escherichia coli.

Bacterial Proteins↗
PubMed1996

The use of a water extract from the bark of Choerospondias axillaris in the treatment of second degree burns.

Burns are common in Vietnam, and because of economic constraints and limited resources for the import of appropriate treatments, the health authorities are obliged to rely on traditional herbal remedies. It is therefore essential to evaluate current drugs, one of which is the water extract of the bark of the tree Choerospondias axillaris. It has been used for many years in the Vietnam-Sweden hospital at Uong Bi in northern Vietnam. We assessed the efficacy of the remedy in an open, randomised controlled clinical trial, in which 20 patients with second degree burns were treated with the extract of the Choerospondias axillaris and 19 with saline gauze. The mean healing time was significantly shorter for patients treated with Choerospondias axillaris (11 days) compared with patients treated with saline gauze (17 days) (p < 0.01), and the number of wound infections was significantly lower in the Choerospondias axillaris group (7/20 compared with 16/19, p = 0.003). The bark extract was easy to apply and additional wound care was not usually necessary, while the treatment with saline gauze was laborious for both patients and staff and was much more expensive. The extract from Choerospondias axillaris is a convenient treatment for second degree burns in both children and adults.

Adolescent↗
PubMed1995

[Antibiotic prophylaxis in diagnostic and therapeutic urological interventions].

An enquiry into the use of antibiotic prophylaxis in conjunction with diagnostic or therapeutic urological procedures at hospitals in four Scandinavian countries showed manifest national differences to exist for most procedures. In transurethral resection, for instance, antibiotic cover was used at 79 percent of Finnish hospitals, but at only nine percent of Danish hospitals. Not only were dosage regimens characterized by wide national variation, but also the spectrum of antibiotics used, quinolones being most frequently used in Sweden, but ampicillin and pivampicillin in Denmark. For some procedures policy was more uniform in all countries, antibiotic cover rarely being used in connection with ureterocystoscopy (5 percent of hospitals), but often in conjunction with percutaneous stone surgery (72 per cent). In certain procedures where there is strong evidence suggesting the necessity of antibiotic prophylaxis, it was not always used-e.g., in transrectal prostate biopsy where it was used at only 62 per cent of hospitals. The interpretation of published findings and clinical experience would appear to differ markedly, and local traditions would seem to be strong determinants of clinical routines. The wide variation suggests that all patients do not receive optimal treatment. To improve routines, our knowledge of antibiotic preparations needs to be expanded by well executed studies, followed by general implementation of the results at the various centres. A series of consensus conferences should be arranged and the recommendations published as a first step toward a more uniform and probably better use of antibiotic prophylaxis in conjunction with diagnostic and therapeutic urological procedures.

Anti-Bacterial Agents↗
PubMed1992

Studies on the individual and combined diuretic effects of four Vietnamese traditional herbal remedies (Zea mays, Imperata cylindrica, Plantago major and Orthosiphon stamineus).

Herbal remedies are widely used in Vietnam alongside modern drugs. We assessed the diuretic effect of four traditional Vietnamese herbal remedies from Zea mays, Imperata cylindrica, Plantago major and Orthosiphon stamineus, all claimed to produce an increase of diuresis. No influence was recorded for the 12- and 24-h urine output or on the sodium excretion for any of the drugs when tested under standardized conditions in a placebo controlled double-blind crossover model. The present study indicates the need for critical review of the present recommendations regarding therapy with plant materials in countries relying on empiric traditions.

Adult↗
PubMed1991

Survey on immunization, diarrhoeal disease and mortality in Quang Ninh Province, Vietnam.

A survey on EPI, CDD, child morbidity, and mortality was conducted in 1988 in one province of northeastern Vietnam. Thirty out of 114 communes were randomly selected for the survey and the interviews were made by 60 students from the provincial nursing school, supervised by 10 teachers. It was found that 23 per cent of the children (n = 211) were fully vaccinated, while 54 per cent had partial coverage. Lack of information or ignorance were the main causes of vaccination failure. The adjusted yearly diarrhoeal rate was 1.6 per child under 5 years of age (n = 9.691). Infant mortality and under-five mortality rates were found to be 28 per thousand live births (95 per cent confidence interval: 21-35; n = 2.321) and 44 (36-52), respectively. High literacy among mothers, good breast-feeding practices, low mortality due to diarrhoea, malaria, and measles, and a well-functioning rural health care system were considered to be the main contributing factors to the low infant mortality. The results also point out the weaknesses in the existing reporting system and indicates the need for follow-up studies.

Cause of Death↗
PubMed1991

Long term survival after transurethral resection of the prostate. Influence of preoperative bacteriuria and indwelling catheter treatment on late mortality.

In this report we have analysed the long term survival after transurethral resection of the prostate in patients with cancer and benign hyperplasia, with special reference to the effect of bacteriuria. One hundred and eighty-nine men were followed for seven years after operation. Life tables according to the Kaplan-Meier method indicated a decreased survival rate for patients with preoperative catheter treatment and/or bacteriuria (p = 0.004 and p = 0.013, respectively). In order to evaluate the influence on the long-term survival of each of these factors alone as well as of other factors like diagnosis, age at operation and perioperative antibiotic treatment, a multivariate analysis, according to Cox proportional hazards method was made. This displayed a two-fold increase of mortality in the patients attributed to the catheter treatment per se, whereas bacteriuria alone was not associated with an increased risk of earlier death.

Aged↗