Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension

Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.     

Extending Serum Half-life of Albumin by Engineering Neonatal Fc Receptor (FcRn) Binding

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.     

Life-cycle modification in open oceans accounts for genome variability in a cosmopolitan phytoplankton

Emiliania huxleyi is the most abundant calcifying plankton in modern oceans with substantial intraspecific genome variability and a biphasic life cycle involving sexual alternation between calcified 2N and flagellated 1N cells. We show that high genome content variability in Emiliania relates to erosion of 1N-specific genes and loss of the ability to form flagellated cells. Analysis of 185 E. huxleyi strains isolated from world oceans suggests that loss of flagella occurred independently in lineages inhabiting oligotrophic open oceans over short evolutionary timescales. This environmentally linked physiogenomic change suggests life cycling is not advantageous in very large/diluted populations experiencing low biotic pressure and low ecological variability. Gene loss did not appear to reflect pressure for genome streamlining in oligotrophic oceans as previously observed in picoplankton. Life-cycle modifications might be common in plankton and cause major functional variability to be hidden from traditional taxonomic or molecular markers.     

Staff Perception on Biomedical or Health Care Waste Management: A Qualitative Study in a Rural Tertiary Care Hospital in India

Background

Health care or biomedical waste, if not managed properly, can be of high risk to the hospital staff, the patients, the community, public health and the environment, especially in low and middle income settings where proper disposal norms are often not followed. Our aim was to explore perceptions of staff of an Indian rural tertiary care teaching hospital on hospital waste management.

Method

A qualitative study was conducted using 10 focus group discussions (FGDs), with different professional groups, cleaning staff, nurses, medical students, doctors and administrators. The FGD guide included the following topics: (i) role of Health Care Waste Management (HCWM) in prevention of health care associated infections, (ii) awareness of and views about HCWM-related guidelines/legislation, (iii) current HCWM practices, (iv) perception and preparedness related to improvements of the current practices, and (v) proper implementation of the available guidelines/legislation. The FGDs were recorded, transcribed verbatim, translated to English (when conducted in Hindi) and analysed using content analysis.

Results

Two themes were identified: Theme (A), ‘Challenges in integration of HCWM in organizational practice,’ with the categories (I) Awareness and views about HCWM, (II) Organizational practices regarding HCWM, and (III) Challenges in Implementation of HCWM; and Theme (B), ‘Interventions to improve HCWM,’ with three categories, (I) Educational and motivational interventions, (II) Organizational culture change, and (III) Policy-related interventions.

Conclusion

A gap between knowledge and actual practice regarding HCWM was highlighted in the perception of the hospital staff. The participants suggested organizational changes, training and monitoring to address this. The information generated is relevant not merely to the microsystem studied but to other institutions in similar settings.     

Variation for resistance to aphids (Homoptera: Aphididae) among tomato inbred backcross lines derived from wild Lycopersicon species

Two tomato inbred backcross line (IBL) populations, derived from crosses between aphid-susceptible Lycopersicon esculentum Mill. 'Peto 95-43' X resistant wild L. pennellii Corr (D'arcy) accession LA716, and Peto 95-43 X resistant wild L. hirsutum f. glabratum Mull accession LA407, were evaluated in replicated field experiments for resistance to potato aphid, Macrosiphum euphorbiae (Thomas), and green peach aphid, Myzus persicae (Sulzer). Aphid infestation scores for each IBL and control (LA716, LA407, Peto 95-43, and susceptible 'Alta') plot were recorded weekly for 5 and 9 wk during the summers of 2000 and 2001, respectively. Aphid infestation scores from leaflets were used to calculate area under the infestation pressure curve (AUIPC), a measure of aphid infestation throughout the growing season, for each IBL and control. Score AUIPC was highly correlated with actual aphid count AUIPC, indicating that scores accurately reflected aphid infestation. Score AUIPC was also highly correlated across both years (2000 and 2001) and locations. Low score AUIPC was significantly correlated with larger plant size and sprawling, indeterminate plant growth habit. Seven IBLs, LA716, and LA407 were significantly more resistant to aphids (lower score AUIPC) than susceptible parent Peto 95-43 in both years. Two IBLs, 1034 and 1051, were not significantly different from resistant LA407 for score AUIPC in both years. The seven aphid-resistant IBLs identified here can be useful as donor parent material for resistance breeding efforts in cultivated tomato.     

Kinetics and thermodynamics of mandelate racemase catalysis

Mandelate racemase (EC 5.1.2.2) from Pseudomonas putida catalyzes the interconversion of the two enantiomers of mandelic acid with remarkable proficiency, producing a rate enhancement exceeding 15 orders of magnitude. The rates of the forward and reverse reactions catalyzed by the wild-type enzyme and by a sluggish mutant (N197A) have been studied in the absence and presence of several viscosogenic agents. A partial dependence on relative solvent viscosity was observed for values of kcat and kcat/Km for the wild-type enzyme in sucrose-containing solutions. The value of kcat for the sluggish mutant was unaffected by varying solvent viscosity. However, sucrose did have a slight activating effect on mutant enzyme efficiency. In the presence of the polymeric viscosogens poly(ethylene glycol) and Ficoll, no effect on kcat or kcat/Km for the wild-type enzyme was observed. These results are consistent with both substrate binding and product dissociation being partially rate-determining in both directions. The viscosity variation method was used to estimate the rate constants comprising the steady-state expressions for kcat and kcat/Km. The rate constant for the conversion of bound (R)-mandelate to bound (S)-mandelate (k2) was found to be 889 +/- 40 s(-1) compared with a value of 654 +/- 58 s(-1) for kcat in the same direction. From the temperature dependence of Km (shown to equal K(S)), k2, and the rate constant for the uncatalyzed reaction [Bearne, S. L., and Wolfenden, R. (1997) Biochemistry 36, 1646-1656], we estimated the enthalpic and entropic changes associated with substrate binding (DeltaH = -8.9 +/- 0.8 kcal/mol, TDeltaS = -4.8 +/- 0.8 kcal/mol), the activation barrier for conversion of bound substrate to bound product (DeltaH# = +15.4 +/- 0.4 kcal/mol, TDeltaS# = +2.0 +/- 0.1 kcal/mol), and transition state stabilization (DeltaH(tx) = -22.9 +/- 0.8 kcal/mol, TDeltaS(tx) = +1.8 +/- 0.8 kcal/mol) during mandelate racemase-catalyzed racemization of (R)-mandelate at 25 degrees C. Although the high proficiency of mandelate racemase is achieved principally by enthalpic reduction, there is also a favorable and significant entropic contribution.     

An efficient synthesis of GDP-hexanolamine,a key tool for the purification of fucosyltransferases

A new efficient synthesis of GDP-hexanolamine from hexanolamine is reported with an overall yield of 71%. The pyrophosphate formation, the key step of this preparation, was achieved through a sequential GMP activation procedure based on polytrifluoroacetylation of GMP followed by activation of the phosphate group by 1-methylimidazole.     

The levels of mature glycosylated nicastrin are regulated and correlate with gamma-secretase processing of amyloid beta-precursor protein

Nicastrin, a type-I transmembrane glycoprotein, is a necessary component of the high molecular weight presenilin (PS) complexes that mediate intramembranous cleavage of beta-amyloid precursor protein (betaAPP) and Notch. Nicastrin undergoes trafficking-dependent glycosylation maturation, and PS1 interacts preferentially with these maturely glycosylated forms of nicastrin. We investigated the effects of differing levels of the immature and mature endoglycosidase-H-resistant forms of nicastrin on Abeta40- and Abeta42-peptide secretion in several cell lines stably expressing a mutant nicastrin (D336A/Y337A) that increases Abeta secretion. There was no correlation between Abeta secretion and the level of over-expression of the immature forms of nicastrin. The total level of mature nicastrin remained constant, but mutant nicastrin replaced endogenous mature nicastrin in varying degrees. Differences in the levels of mature mutant nicastrin positively correlated with Abeta secretion, but did not influence either betaAPP trafficking or processing by alpha- and beta-secretases. Proper trafficking and terminal maturation of nicastrin is therefore a necessary event for the regulated intramembranous proteolysis of betaAPP.