BiP-dependent export of cholera toxin from endoplasmic reticulum-derived microsomes

Cholera toxin (CT) is transported from the cell surface to the endoplasmic reticulum (ER) from where it is translocated to the cytosol in a process depending on ATP and luminal ER proteins. To test whether the molecular chaperone BiP (heavy chain binding protein), which is an ER-luminal ATPase, was one of the required proteins the export of CT was analyzed using ER-derived CT-loaded microsomes. The resubstitution of extracted export-incompetent microsomes with purified BiP was sufficient to restore the export of CT. As BiP protected CT from aggregation it is proposed that BiP maintains CT in a soluble, export-competent state.     

Correlation between the Paleocene/Eocene boundary and the Ilerdian at Campo, Spain

The Ilerdian is a well-established Tethyan marine stage, which corresponds to an important phase in the evolution of larger foraminifera not represented in the type-area of the classical Northwest-European stages. This biostratigraphic restudy of its parastratotype in the Campo Section (northeastern Spain) based on planktic foraminifera, calcareous nannofossils, dinoflagellate cysts and the distribution of the stable isotopes ∂¹³C and ∂¹⁸O is an attempt to correlate the Paleocene/Eocene boundary based on a characteristic carbon isotope excursion (CIE) marking the onset of the Initial Eocene Thermal Maximum (IETM) and the Ilerdian stage. The base of this ∂¹³C excursion has been chosen as the criterion for the recent proposal of the Global Stratotype Section and Point (GSSP) of the base of the Eocene (= base of the Ypresian) in the Dababiya Section (Egypt) to which an age of 54.9 Ma has been attributed. This level is also characterized by a marked extinction among the deep-water benthic foraminifera (Benthic Foraminifera Extinction Event, BFEE), a flood of representatives of the planktic foraminiferal genus Acarinina and the acme of dinoflagellate cysts of the genus Apectodinium. In the Campo Section, detailed biozonations (planktic foraminifera, calcareous nannofossils, dinoflagellate cysts) are recognized in the Lower and Middle Ilerdian. The correlation with the Ypresian stratotype is based on dinoflagellate cysts and calcareous nannofossils. The base of the Ilerdian is poor in planktic microfossils and its precise correlation with the redefined Paleocene/Eocene boundary remains uncertain.     

Neck length and processivity of myosin V

Myosin V is an unconventional myosin that transports cargo such as vesicles, melanosomes, or mRNA on actin filaments. It is a two-headed myosin with an unusually long neck that has six IQ motifs complexed with calmodulin. In vitro studies have shown that myosin V moves processively on actin, taking multiple 36-nm steps that coincide with the helical repeat of actin. This allows the molecule to "walk" across the top of an actin filament, a feature necessary for moving large vesicles along an actin filament bound to the cytoskeleton. The extended neck length of the two heads is thought to be critical for taking 36-nm steps for processive movements. To test this hypothesis we have expressed myosin V heavy meromyosin-like fragments containing 6IQ motifs, as well as ones that shorten (2IQ, 4IQ) or lengthen (8IQ) the neck region or alter the spacing between 3rd and 4th IQ motifs. The step size was proportional to neck length for the 2IQ, 4IQ, 6IQ, and 8IQ molecules, but the molecule with the altered spacing took shorter than expected steps. Total internal reflection fluorescence microscopy was used to determine whether the heavy meromyosin IQ molecules were capable of processive movements on actin. At saturating ATP concentrations, all molecules except for the 2IQ mutant moved processively on actin. When the ATP concentration was lowered to 10 microm or less, the 2IQ mutant demonstrated some processive movements but with reduced run lengths compared with the other mutants. Its weak processivity was also confirmed by actin landing assays.     

Recombinant canarypox vaccine-elicited CTL specific for dominant and subdominant simian immunodeficiency virus epitopes in rhesus monkeys

Since virus-specific CTL play a central role in containing HIV replication, a candidate AIDS vaccine should generate virus-specific CTL responses. In this study, the ability of a recombinant canarypox virus expressing SIV Gag-Pol-Env (ALVAC/SIV gag-pol-env) was assessed for its ability to elicit both dominant and subdominant epitope-specific CTL responses in rhesus monkeys. Following a series of five immunizations, memory CTL responses specific for a dominant Gag epitope could be demonstrated in the peripheral blood of vaccinated monkeys. Memory CTL responses to a subdominant Pol epitope were undetectable in these animals. Following challenge with SIVmac251, the experimentally vaccinated animals developed high frequency CTL responses specific for the dominant Gag epitope that emerged in temporal association with the early containment of viral replication. Interestingly, the experimentally vaccinated, but not the control vaccinated animals, developed CTL responses to the subdominant Pol epitope that were detectable only after containment of early viremia. Thus, recombinant canarypox vaccination elicited low frequency, but durable memory CTL populations. The temporal association of the emergence of the dominant epitope-specific response with early viral containment following challenge suggests that this immune response played a role in the accelerated clearing of early viremia in these animals. The later emerging CTL response specific for the subdominant epitope may contribute to the control of viral replication in the setting of chronic infection.     

Human insulin release processes measured by intraportal sampling

Insulin is secreted as a series of punctuated secretory bursts superimposed on variable basal insulin release. The contribution of these secretory bursts to overall insulin secretion has been estimated on the basis of peripheral vein sampling in humans to encompass > or =75% of overall insulin release. A similar contribution of the pulsatile mode of release was inferred in a canine model by use of portal vein sampling. The primary regulation of insulin secretion is through perturbation of the mass and frequency of these secretory bursts. The mode of delivery of insulin into the circulation seems important for insulin action; therefore, physiological conditions that alter the pattern of insulin release may affect insulin action through this mechanism. Transhepatic intraportal shunt in humans may provide access to portal vein samples, thus potentially improving the sensitivity of detecting and quantitating the frequency, mass, and amplitude of secretory bursts along with basal release and the regularity of these variables. To establish the insulin-secretory mechanism in nondiabetic humans by the use of portal vein sampling, we here assessed the mass, frequency, amplitude, and overall contribution of pulsatile insulin secretion by deconvolution analysis of portal vein insulin profiles. We find that, in nondiabetic humans fasted overnight, the portal vein insulin concentration oscillates at a periodicity of 4.1 +/- 0.2 min/pulse and with secretory peak amplitudes averaging 660% of basal (interpulse) release. The frequency was confirmed by spectral and autocorrelation analyses. The punctuated insulin-secretory bursts partially overlap and are responsible for the majority (70 +/- 4%) of insulin release. After ingestion of a mixed meal, the insulin release was increased through amplification of the secretory burst mass (507 +/- 104 vs. 1,343 +/- 211 pmol x l(-1) x min(-1), P < 0.001), whereas frequency (4.4 +/- 0.2 vs. 4.3 +/- 0.2, P = 0.86) and basal secretion (62 +/- 14 vs. 91 +/- 22 pmol x l(-1) x min(-1), P = 0.33) were unaffected. One subject with diabetes and cirrhosis had a similar insulin-secretory pattern, whereas a subject with insulin-dependent diabetes mellitus and minimal insulin release had preserved pulsatile release. A single subject was entrained to show agreement between entrained frequency and portal vein insulin oscillations. We conclude that insulin release in the human portal vein occurs at a mean periodicity of 4.4 +/- 0.2 min with a high signal-to-noise ratio (pulse amplitude 660% of basal). The impact of noise on the detected high frequency cannot be excluded.     

Prohibitin and prohibitone are contained in high-molecular weight complexes and interact with alpha-actinin and annexin A2

The closely related proteins prohibitin (p32) and prohibitone (p37) are evolutionarily conserved with homologues found from cyanobacteria to man. They are thought to be exclusively mitochondrial and have been assigned many-rather different-functions, ranging from a role in lifespan, in mitochondrial inheritance and as chaperones of mitochondrial proteases in yeast. Evidence for a localisation outside of mitochondria has been brought forward in mammalian cells, where they influence cell-cycle progression and are found in association with cell surface receptors. We have employed a yeast two-hybrid screen to identify other interacting proteins and have identified alpha-actinin and annexin A2 as binding partners for prohibitin and prohibitone. Coprecipitation experiments supported the putative binding between prohibitin and prohibitone on the one hand and annexin A2 or alpha-actinin on the other hand in intact cells. Surface plasmon resonance analysis was used to determine relative affinities between prohibitin and alpha-actinin and between prohibitone and annexin A2 and alpha-actinin, respectively. We further show that prohibitin and prohibitone can also form homomeric (preferentially tetrameric) and heteromultimeric complexes, with significant affinities.     

Authors' index

Authors Index

Author's Index