Massive calcium-activated endocytosis without involvement of classical endocytic proteins

We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4-64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP(2)) synthesis: PIP(2) substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5'-[γ-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP(2) induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP(2) when cytoplasmic free Ca is clamped to 10 μM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP(2) and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief β-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A(2) inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side.     

Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains

The pore-lining amino acids of ion channel proteins reside on the interface between a polar (the pore) and a nonpolar environment (the rest of the protein). The structural dynamics of this region, which physically controls ionic flow, are essential components of channel gating. Using large-conductance, Ca(2+)-dependent K(+) (BK) channels, we devised a systematic charge-substitution method to probe conformational changes in the pore region during channel gating. We identified a deep-pore residue (314 in hSlo1) as a marker of structural dynamics. We manipulated the charge states of this residue by substituting amino acids with different valence and pKa, and by adjusting intracellular pH. We found that the charged states of the 314 residues stabilized an open state of the BK channel. With models based on known structures of related channels, we postulate a dynamic rearrangement of the deep-pore region during BK channel opening/closing, which involves a change of the degree of pore exposure for 314.     

The geographical distribution of lymphatic filariasis infection in Malawi

Background

Papua New Guinea is the only endemic country in the Western Pacific Region that has not yet introduced a countrywide programme to eliminate lymphatic filariasis. However, on Misima Island in Milne Bay Province, government and private sectors have collaborated to implement a pilot elimination programme. Although interim evaluation indicated that the programme has been parasitologically successful, an appreciation that sustainable health gains depend on understanding and accommodating local beliefs prompted this qualitative study.

Methods

We investigated Misima community members knowledge and attitudes about lymphatic filariasis and the elimination programme. A combination of focus groups and key informant interviews were used to explore participants perceptions of health; knowledge of the aetiology and symptoms of filariasis, elephantiasis and hydrocele; attitudes towards the disease and mass drug distribution; and the social structure and decision-making protocols within the villages.

Results

Focus group discussions proved inferior to key informant interviews for gathering rich data. Study participants did not consider lymphatic filariasis ("pom") a major health problem but were generally positive about mass drug administration campaigns. A variety of conditions were frequently and incorrectly attributed to filariasis. Participants expressed the belief that individuals infected with filariasis always had visible manifestations of disease. A common misconception was that taking drugs during campaigns provided long-term immunity against disease. The role of mosquito vectors in transmission was not generally appreciated and certain clinical presentations, particularly hydrocele, were associated with supernatural forces. Multiple adverse events were associated with mass drug administration campaigns and most study participants mentioned community members who did not participate in campaigns.

Conclusion

Important issues requiring educational intervention and elimination activity modification in the Misima region were identified during this study. Research outcomes should assist Papua New Guinea in developing and implementing a national elimination strategy and inform discussions regarding the appropriateness of current elimination strategies.     

Seropositivity is associated with insulin resistance in patients with early inflammatory polyarthritis: results from the Norfolk Arthritis Register (NOAR): an observational study

Introduction  

Cardiovascular disease (CVD) is the leading cause of death in patients with inflammatory polyarthritis (IP), especially in seropositive disease. In established rheumatoid arthritis (RA), insulin resistance (IR) is increased and associated with CVD. We investigated factors associated with IR in an inception cohort of patients with early IP.