Estimating the savings of a national project to prevent healthcare-associated infections in intensive care units.

BACKGROUND: Healthcare-associated infections (HAIs) have a significant impact on patients' morbidity and mortality, and have a detrimental financial impact on the healthcare system. Various strategies exist to prevent HAIs, but economic evaluations are needed to determine which are most appropriate. AIM: To present the financial impact of a nationwide project on HAI prevention in intensive care units (ICUs) using a quality improvement (QI) approach. METHODS: A health economic evaluation assessed the financial results of the QI initiative 'Saúde em Nossas Mãos' (SNM), implemented in Brazil between January 2018 and December 2020. Among 116 participating institutions, 13 (11.2%) fully reported the aggregate cost and stratified patients (with vs without HAIs) in the pre-intervention and post-intervention periods. Average cost (AC) was calculated for each analysed HAI: central-line-associated bloodstream infections (CLABSIs), ventilator-associated pneumonia (VAP) and catheter-associated urinary tract infections (CAUTIs). The absorption model and time-driven activity-based costing were used for cost estimations. The numbers of infections that the project could have prevented during its implementation were estimated to demonstrate the financial impact of the SNM initiative. RESULTS: The aggregated ACs calculated for each HAI from these 13 ICUs - US$8480 for CLABSIs, US$10,039 for VAP, and US$7464 for CAUTIs - were extrapolated to the total number of HAIs prevented by the project (1727 CLABSIs, 3797 VAP and 2150 CAUTIs). The overall savings of the SNM as of December 2020 were estimated at US$68.8 million, with an estimated return on investment (ROI) of 765%. CONCLUSION: Reporting accurate financial data on HAI prevention strategies is still challenging in Brazil. These results suggest that a national QI initiative to prevent HAIs in critical care settings is a feasible and value-based approach, reducing financial waste and yielding a significant ROI for the healthcare system.

Chronic dietary exposure to branched chain amino acids impairs glucose disposal in vegans but not in omnivores.

BACKGROUND/OBJECTIVES: Branched chain amino acids (BCAA) are among nutrients strongly linked with insulin sensitivity (IS) measures. We investigated the effects of a chronic increase of BCAA intake on IS in two groups of healthy subjects differing in their basal consumption of BCAA, that is, vegans and omnivores. SUBJECTS/METHODS: Eight vegans and eight matched omnivores (five men and three women in each group) received 15 g (women) or 20 g (men) of BCAA daily for 3 months. Anthropometry, blood analyses, glucose clamp, arginine test, subcutaneous abdominal adipose tissue (AT) and skeletal muscle (SM) biopsies (mRNA levels of selected metabolic markers, respiratory chain (RC) activity) were performed at baseline, after the intervention and after a 6 month wash-out period. RESULTS: Compared with omnivores, vegans had higher IS at baseline (GIR, glucose infusion rate: 9.6±2.4 vs 7.1±2.4 mg/kg/min, 95% CI for difference: 0.55 to 5.82) that declined after the intervention and returned to baseline values after the wash-out period (changes in GIR with 95% CI, 3-0 months: -1.64 [-2.5; -0.75] and 9-3 months: 1.65 [0.75; 2.54] mg/kg/min). No such change was observed in omnivores. In omnivores the intervention led to an increased expression of lipogenic genes (DGAT2, FASN, PPARγ, SCD1) in AT. SM RC activity increased in both groups. CONCLUSIONS: Negative impact of increased BCAA intake on IS was only detected in vegans, that is, subjects with low basal amino acids/BCAA intake, which appear to be unable to induce sufficient compensatory changes within AT and SM on a BCAA challenge.

Automated nonparametric method for detection of step-like features in biological data sets.

Experimental data from single-molecule DNA-protein experiments, such as experiments using optical traps or magnetic tweezers, typically contain steps, plateaus, or dwell regions that are obscured by thermal and other noise sources. We present a nonparametric method for detecting step-like features in noisy biological data sets. Our algorithm does not assume that the steps can be modeled as Heaviside functions or any particular parametric form. No assumptions about the noise source, such as whether the noise is Gaussian or colored, are made either. Instead, for detection of plateaus, the algorithm uses the novel method of analyzing a probability distribution function of the data values. The vast majority of previously published methods for step detection rely on statistical fitting of step functions with the flat segments linked by vertical segments. Our approach is intended for use on data which cannot be modelled as a series of step functions but applies to step functions as a special case. These type of data traces have, so far, been difficult to characterize effectively. We examine the performance of the algorithm through systematic simulation studies and illustrate the use of our algorithm to analyze single molecule DNA-protein micromanipulation experiments carried out by our laboratory. The simulation results and experimental validation suggest that our method is very robust, avoids overfitting, and functions effectively in the presence of noise sources characteristic of single molecule experiments.

The effect of vitamin E on plasmatic malondialdehyde levels during surgical removal of ovarian and endometrial carcinomas.

This study deals with the monitoring of plasmatic levels of malondialdehyde, as the main indicator of oxidative damage to biomembranes. Malondialdehyde is determined by high-performance liquid chromatography (HPLC) after derivatization employing 2,4-dinitrophenylhydrazine. A clinical study involving 20 female patients suffering from ovarian and endometrial carcinomas has demonstrated elevated levels of malondialdehyde (10.1 +/- 1.1 microM), compared with the control group (7.5 +/- 2.7 microM). It has been further verified that surgical removal of the tumor leads to an additional increase in the plasmatic malondialdehyde content. This unfavourable situation can be effectively eliminated by administration of a single dose of vitamin E prior to surgery.

Treatment of chronic hepatitis C in HIV-infected patients with compensated liver cirrhosis.

The greatest benefit of hepatitis C virus (HCV) therapy is seen in cirrhotics attaining sustained virological response (SVR). However, concerns about toxicity and poorer responses often discourage treatment of cirrhotics. This may be particularly relevant in HIV-HCV-coinfected patients, in whom progression of liver fibrosis is faster and treatment responses lower. This is a retrospective analysis of HIV-HCV-coinfected patients who had received peginterferon-ribavirin therapy at our institution. Individuals naïve for interferon in whom liver fibrosis had been assessed using elastometry within the year before being treated were chosen. Response rates and toxicities were compared in cirrhotics (>14.5 KPa) and noncirrhotics. Patients with previous liver decompensation were excluded. Overall, 41 cirrhotics and 190 noncirrhotics entered the study. Groups were similar in age, gender, HCV genotypes and baseline serum HCV-RNA. SVR occurred at similar rates in cirrhotic and noncirrhotics, either considered by intention-to-treat (39%vs 45%; P = 0.4) or as treated (50%vs 52%, P = 0.8). In multivariate analysis (odds ratio, 95% CI, P), SVR was associated with HCV genotypes 2-3 (5, 2.9-11, <0.01) and lower serum HCV-RNA (2, 1.4-3.03 for every log decrease, <0.01) but not with cirrhosis (1.2, 0.4-3.6, 0.6). Treatment discontinuations because of adverse events tended to be more common in cirrhotics than in noncirrhotics (17%vs 12%; P = 0.2), but only severe thrombocytopenia was more frequent in cirrhotics than in non-cirrhotics (20%vs 3% at week 24; P < 0.01). Response to peginterferon-ribavirin therapy is similar in HIV-HCV coinfected patients with and without liver cirrhosis. Therefore, treatment must be encouraged in all compensated cirrhotic patients, although closer monitoring and management of side effects, mainly thrombocytopenia, may be warranted.

Hepatitis C virus (HCV) treatment uptake and changes in the prevalence of HCV genotypes in HIV/HCV-coinfected patients.

The efficacy of current hepatitis C therapy in HIV/HCV-coinfected patients is largely dependent on HCV genotype. The annual prevalence of HCV genotypes/subtypes and their influence on HCV clearance with antiviral treatment were examined in a dynamic cohort of HIV/HCV-coinfected patients followed up in Madrid since 2000. Patients entered the cohort at first visit and left the cohort when HCV clearance was achieved with HCV therapy or when follow-up was interrupted for any reason, including death. A total of 672 HIV/HCV-coinfected patients constituted the cohort. The mean follow-up time was 5.5 years, corresponding to 4108 patient-years. Mean age at entry was 37 years, and 73% were men and 86% were intravenous drug users. Overall distribution of HCV genotypes was as follows: 57.1% HCV-1 (1a: 29.2%, 1b: 20.4%, unknown: 7.6%), 1.3% HCV-2, 25.4% HCV-3 and 15.9% HCV-4. A total of 274 (40.8%) patients were treated with peginterferon-ribavirin, of whom 116 (42.3%) achieved HCV clearance following 1-3 courses of therapy. The proportion of HCV-1/4 rose from 71.7% in 2000 to 76.8% in 2008, whereas the proportion of HCV-2/3 fell from 28.1% in 2000 to 23.2% in 2008. The yearly prevalence increased for HCV-1 (R(2) : 0.92, b: 0.59, P < 0.001) and HCV-4 (R(2) : 0.77, b: 0.33, P < 0.005) and conversely diminished for HCV-3 (R(2) : 0.94, b: -0.82, P < 0.001). In summary, the prevalence of HCV-1 and HCV-4 has increased over the last decade in HIV/HCV-coinfected patients, whereas conversely it has declined for HCV-3, in association with the wider use of HCV therapy (41%) in this population.

Risk factors for advanced liver fibrosis in HIV-infected individuals: role of antiretroviral drugs and insulin resistance.

Liver damage may result from multiple factors in HIV-infected patients. The availability of reliable noninvasive tools to measure liver fibrosis has permitted the screening of large patient populations. Cross-sectional study of all consecutive HIV outpatients who underwent examination by transient elastometry (FibroScan) at one HIV reference clinic during 2007. Advanced liver fibrosis (ALF) was defined as hepatic stiffness >9.5 kilopascals, which corresponds to Metavir stages F3-F4 in the liver biopsy. A total of 681 consecutive HIV-infected patients (64% injecting drug users; mean age 43; 78% male; 98% on antiretroviral therapy) had at least one valid FibroScan evaluation. ALF was diagnosed in 215 (32%) of them. In the univariate analysis, ALF was significantly associated with older age, low CD4 counts, chronic hepatitis C, past alcohol abuse, elevated ALT, high triglycerides, low cholesterol, high homeostasis model assessment (HOMA) index and exposure to didanosine and/or stavudine. In a multivariate model (OR, 95% CI), chronic hepatitis C (2.83, 1.57-5.08), past alcohol abuse (2.26, 1.37-3.74), exposure to didanosine and/or stavudine (1.85, 1.14-3.01), high HOMA index (1.25, 1.04-1.51), older age (1.09, 1.05-1.14) and elevated ALT (1.04, 1.03-1.06) remained as independently associated with ALF. Therefore, in addition to chronic hepatitis C and alcohol abuse, insulin resistance and/or exposure to dideoxy-nucleosides may contribute to ALF in HIV-infected patients.

[Use of capillary electrophoresis in the clinical research]. / Moznosti vyuzití kapilárni elektroforézy v klinickém výzkumu.

Capillary electrophoresis (CE) is a modern separation method, which represents a competent alternative to liquid chromatography. The CE separation is performed in very thin silica capillaries. Advantages of CE are the high separation efficiency, low sample consumption and short time of analysis compared to liquid chromatography. CE represents a strong tool for analysis of body fluids.

[Diuretics in therapy of "diuretic resistance" by patients with congestive heart failure]. / Diuretika v terapii "diuretické rezistence" u pacientu s mestnavou srdecní slabostí.

Loop diuretics are integral part of overall therapy of severe congestive heart failure. Approximately 10-20 % of patients with congestive heart failure (NYHA class III-IV) do not respond satisfactorily to diuretic treatment. Despite its frequency, the term "diuretic resistance" remains inadequately defined. In general, failure to decrease the extracellular fluid volume despite liberal use of diuretics is often termed "diuretic resistance". The combination of diuretics, particularly of loop diuretic with thiazide agents, is recommended for prevention as well as treatment of this complication. Effective management is also continuous infusion of loop diuretic. If it is impossible to achieve adequate response by combination of diuretics, increasing of its dosage or/and frequency or continuous infusion, then dialysis methods may be employed (however it is not intended to discuss this option in this article).

Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells.

Using a microinjection approach to study apical plasma membrane protein trafficking in hepatic cells, we found that specific inhibition of Vps34p, a class III phosphoinositide 3 (PI-3) kinase, nearly perfectly recapitulated the defects we reported for wortmannin-treated cells (Tuma, P.L., C.M. Finnegan, J.-H Yi, and A.L. Hubbard. 1999. J. Cell Biol. 145:1089-1102). Both wortmannin and injection of inhibitory Vps34p antibodies led to the accumulation of resident apical proteins in enlarged prelysosomes, whereas transcytosing apical proteins and recycling basolateral receptors transiently accumulated in basolateral early endosomes. To understand how the Vps34p catalytic product, PI3P, was differentially regulating endocytosis from the two domains, we examined the PI3P binding protein early endosomal antigen 1 (EEA1). We determined that EEA1 distributed to two biochemically distinct endosomal populations: basolateral early endosomes and subapical endosomes. Both contained rab5, although the latter also contained late endosomal markers but was distinct from the transcytotic intermediate, the subapical compartment. When PI3P was depleted, EEA1 dissociated from basolateral endosomes, whereas it remained on subapical endosomes. From these results, we conclude that PI3P, via EEA1, regulates early steps in endocytosis from the basolateral surface in polarized WIF-B cells. However, PI3P must use different machinery in its regulation of the apical endocytic pathway, since later steps are affected by Vps34p inhibition.

Vinorelbine, epirubicin, and methotrexate (VEM) as primary treatment in locally advanced breast cancer.

PURPOSE: This phase II trial of VEM (vinorelbine + epirubicine + methotrexate) in the treatment of locally advanced breast cancer was conducted to obtain downstaging to allow surgery and breast conservation. PATIENTS AND METHODS: This multicenter study recruited 58 patients with locally advanced breast cancer (two patients ineligible); 56 were evaluable for response and tolerance. RESULTS: Downstaging was obtained in 77% of the patients with a pathological complete response (pCR) rate of 9%. At 33 months of follow-up, median survival has not been reached. Neutropenia grade 3-4 was reported in 31% of cycles with 3% of cycles with infection grade 3. Alopecia grade 3 was noticed for 71% of patients. CONCLUSION: VEM represents an effective regimen for patients with locally advanced breast cancer, allowing an important pCR. Moreover, this regimen appears to be particularly well tolerated.

Isolation of rat hepatocyte plasma membrane sheets and plasma membrane domains.

This unit describes a method for isolation of plasma membrane sheets from rat liver. It also includes protocols for preparation of plasma membrane domains isolated from plasma membrane sheets and indirect immunofluorescence localization of marker proteins associated with plasma membrane sheets. The unit has been updated with assays for the marker enzymes alkaline phosphodiesterase I, 5' nucleotidase, and K+-stimulated.

Split-flow injector for capillary zone electrophoresis.

A simple construction of a split-flow injector eliminating some common problems connected with the use of such devices is described. It consists of a low-pressure pump, an injection valve and a delivery tube in which the separating capillary inlet is fixed. The sample is injected without moving the separating capillary inlet and without interrupting the applied voltage. The grounded electrophoretic electrode is close to the injection valve so that all metal parts of the injector are kept at a sufficiently low potential. Minimum length and small internal diameter of delivery tube minimizes additional sample zone broadening. The effects of some experimental parameters, such as the position of the separation capillary inlet with respect to the background solution flow direction and background solution flow-rate are experimentally studied. The injector was tested primarily for the electrokinetic injection.

VAP-33 localizes to both an intracellular vesicle population and with occludin at the tight junction.

Tight junctions create a regulated intercellular seal between epithelial and endothelial cells and also establish polarity between plasma membrane domains within the cell. Tight junctions have also been implicated in many other cellular functions, including cell signaling and growth regulation, but they have yet to be directly implicated in vesicle movement. Occludin is a transmembrane protein located at tight junctions and is known to interact with other tight junction proteins, including ZO-1. To investigate occludin's role in other cellular functions we performed a yeast two-hybrid screen using the cytoplasmic C terminus of occludin and a human liver cDNA library. From this screen we identified VAP-33 which was initially cloned from Aplysia by its ability to interact with VAMP/synaptobrevin and thus was implicated in vesicle docking/fusion. Extraction characteristics indicated that VAP-33 was an integral membrane protein. Antibodies to the human VAP-33 co-localized with occludin at the tight junction in many tissues and tissue culture cell lines. Subcellular fractionation of liver demonstrated that 83% of VAP-33 co-isolated with occludin and DPPIV in a plasma membrane fraction and 14% fractionated in a vesicular pool. Thus, both immunofluorescence and fractionation data suggest that VAP-33 is present in two distinct pools in the cells. In further support of this conclusion, a GFP-VAP-33 chimera also distributed to two sites within MDCK cells and interestingly shifted occludin's localization basally. Since VAP-33 has previously been implicated in vesicle docking/fusion, our results suggest that tight junctions may participate in vesicle targeting at the plasma membrane or alternatively VAP-33 may regulate the localization of occludin.

Evidence for apical endocytosis in polarized hepatic cells: phosphoinositide 3-kinase inhibitors lead to the lysosomal accumulation of resident apical plasma membrane proteins.

The architectural complexity of the hepatocyte canalicular surface has prevented examination of apical membrane dynamics with methods used for other epithelial cells. By adopting a pharmacological approach, we have documented for the first time the internalization of membrane proteins from the hepatic apical surface. Treatment of hepatocytes or WIF-B cells with phosphoinositide 3-kinase inhibitors, wortmannin or LY294002, led to accumulation of the apical plasma membrane proteins, 5'-nucleotidase and aminopeptidase N in lysosomal vacuoles. By monitoring the trafficking of antibody-labeled molecules, we determined that the apical proteins in vacuoles came from the apical plasma membrane. Neither newly synthesized nor transcytosing apical proteins accumulated in vacuoles. In wortmannin-treated cells, transcytosing apical proteins traversed the subapical compartment (SAC), suggesting that this intermediate in the basolateral-to-apical transcytotic pathway remained functional. Ultrastructural analysis confirmed these results. However, apically internalized proteins did not travel through SAC en route to lysosomal vacuoles, indicating that SAC is not an intermediate in the apical endocytic pathway. Basolateral membrane protein distributions did not change in treated cells, uncovering another difference in endocytosis from the two domains. Similar effects were observed in polarized MDCK cells, suggesting conserved patterns of phosphoinositide 3-kinase regulation among epithelial cells. These results confirm a long-held but unproven assumption that lysosomes are the final destination of apical membrane proteins in hepatocytes. Significantly, they also confirm our hypothesis that SAC is not an apical endosome.

Rectangular flaps technique for treatment of congenital hand syndactyly.

The authors analysed a series of 22 patients undergoing surgical correction of congenital hand syndactyly by the rectangular flap technique. Using our evaluation method, we found that good functional and aesthetic results were obtained in 77.3% of the patients, with a complication rate of 13.6%. We concluded that the rectangular flap technique has a simple design, is easily reproducible by in-training staff, has good results, and can be applied on the majority of the syndactyly cases.

Endogenous syntaxins 2, 3 and 4 exhibit distinct but overlapping patterns of expression at the hepatocyte plasma membrane.

To investigate the mechanisms regulating polarized vesicle delivery to the cell surface in hepatocytes, we have characterized the endogenous plasma membrane (PM)-associated syntaxins. These integral membrane proteins are components of the membrane docking/fusion apparatus and are thought to function as vesicle receptors at the PM. In hepatocytes, the PM is divided into two domains, the apical and basolateral. If syntaxins are mediating the specific recognition of vesicles delivered to either membrane surface, the simple prediction is that each domain expresses one syntaxin isoform. However, we report that rat hepatocytes express three endogenous PM-associated syntaxin isoforms, syntaxins 2, 3 and 4. By biochemical subfractionation, we determined that the syntaxins exhibit distinct, but overlapping patterns of expression among the PM domains. Syntaxin 4 is primarily expressed at the basolateral surface while syntaxins 2 and 3 are enriched at the apical PM. The immunolocalization of syntaxins 2 and 4 in rat hepatocytes and PM sheets revealed similarly complex patterns of PM expression with enhanced apical staining for both. A significant proportion of syntaxin 3 (25%) was detected in subcellular fractions containing transport vesicles. We have used quantitative immunoblotting to determine that the syntaxins are relatively abundant PM molecules (11-260 nM) in rat liver, spleen and kidney. Also, we determined that the syntaxin binding protein, Munc-18, is present at concentrations from 1.5-20 nM in the same tissues. Although this fundamental quantitative and morphological information is lacking in other systems, it is critical not only for defining syntaxin function, but also for predicting the specific mechanisms that regulate vesicle targeting in hepatocytes and other tissues.

Dynamin forms polymeric complexes in the presence of lipid vesicles. Characterization of chemically cross-linked dynamin molecules.

Dynamin is a GTP-binding protein that is involved in the release of coated endocytic vesicles from the plasma membrane. We have been characterizing the enzymatic properties of purified rat brain dynamin to better understand how GTP binding and hydrolysis relate to its proposed function. Previously, we have demonstrated that activation of dynamin GTPase results from positive cooperative associations between dynamin molecules as they are bound to a polymeric surface. Our present report has extended these studies and has examined the structural features of dynamin self-association. After treatment with the zero-length protein cross-linking reagent, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, dynamin in solution was found cross-linked into dimers. This homodimer likely reflects the native soluble state of the molecule. After binding to brain vesicles, dynamin was cross-linked into higher order oligomers of greater than 800 kDa. Dynamin, copurified on brain membranous organelles, also formed multimeric complexes when cross-linked suggesting dynamin exists in polymeric form in vivo. No cross-linked species other than homo-oligomers were observed, providing no evidence for close interactions between dynamin and membrane proteins. From experiments examining the effects of GTP, GDP, guanosine 5'-3-O-(thio)triphosphate, and 5'-guanylyl-beta,gamma-imidodiphosphate on cross-linking, we have determined that both dynamin membrane binding and self-association occur independently from the nucleotide-bound state of the enzyme. An 80-kDa dynamin fragment that is lacking its carboxyl-terminal domain is not cross-linked into higher order oligomers, suggesting that this domain is required for binding of dynamin to membranes and the subsequent enhancement of oligomerization. However, the dynamin fragment was found to form dimers indicating that this domain is not required for dynamin dimerization. Cross-linked dynamin was able to cooperatively bind microtubules, but did not exhibit GTPase activation. We propose that intramolecular cross-links in the dynamin monomer impart structural constraints that prevent the enhancement of GTP hydrolysis. We describe a model of the dynamin activation process to be considered in further investigations of the role for dynamin in endocytic vesicle formation.

Implementation of the patient self-determination act in a hospital setting. An initial evaluation.

BACKGROUND: The Patient Self-Determination Act aims to enhance patient awareness of advance directives by requiring health-care institutions to ask patients whether they have advance directives and to inform patients of their rights to prepare these documents. We investigated the following: (1) compliance of the hospital staff with implementing this act, (2) the effects of this act on the extent to which patients discuss and prepare advance directives, and (3) variables that might influence patient discussions on advance planning and preparation of advance directives. METHODS: We surveyed 219 patients from a university hospital that implemented a nurse-dependent advance directive program. We also conducted a telephone interview with 57% of these patients at least 6 months after hospital discharge. RESULTS: Nurses asked 70% of the patients about the existence of an advance directive and of these patients, only 57% remembered the inquiry. Only 57% of the patients received the brochure on advance directives and of these patients, only 55% read the brochure. Only 2% of the patients requested to receive additional information on advance directives. Less than one quarter of the patients had discussions on advance planning while in the hospital and of those patients who were contacted within 6 months after hospital discharge, 39% had discussions on advance planning and 15% prepared an advance directive. Race was an independent predictor for hospital discussions, and educational level was an independent predictor for discussions and preparation of advance directives after hospital discharge. CONCLUSIONS: To enhance the effectiveness of a nurse-dependent advance directive program, hospitals may need (1) to strengthen the quality of the patient-nurse encounter in which the issue of advance directives is raised to more effectively promote patient interest, discussions, and preparation of advance directives and (2) to account for the social diversity of their patient population.

Activation of dynamin GTPase is a result of positive cooperativity.

Dynamin is a GTP-binding protein thought to be involved in the early stages of endocytosis. Presently, it is not known how dynamin GTP binding and hydrolysis are related to its role in this process. We previously characterized the ability of acidic phospholipid vesicles and microtubules to strongly stimulate the GTPase activity of purified brain dynamin. In a further analysis of dynamin enzymatic properties, we have found that the increase of dynamin GTP hydrolysis in the presence of activating agent depends on enzyme concentration. At low enzyme concentration, little or no activation is observed. Plots of dynamin GTPase activity with increasing enzyme concentration in the presence of either activating agent are strongly sigmoidal, indicating that positive cooperativity is responsible for the increased activity observed. A Hill coefficient of 2.3 was determined, implying that at least two dynamin molecules associate for maximal GTPase activity. No cooperative effects in GTP binding were observed. Linear transformation of reaction velocity versus enzyme concentration data indicate an apparent Km for dynamin-dynamin interactions of 37 nM, which is significantly lower than the physiological concentration of dynamin in brain. These results suggest that cooperative interactions between dynamin molecules are responsible for the apparent activation of GTPase observed and are likely involved in dynamin function in vivo.