Temperature-sensitive random insulin granule diffusion is a prerequisite for recruiting granules for release

Glucose-evoked insulin secretion exhibits a biphasic time course and is associated with accelerated intracellular granule movement. We combined live confocal imaging of EGFP-labelled insulin granules with capacitance measurements of exocytosis in clonal INS-1 cells to explore the relation between distinct random and directed modes of insulin granule movement, as well as exocytotic capacity. Reducing the temperature from 34 degrees C to 24 degrees C caused a dramatic 81% drop in the frequency of directed events, but reduced directed velocities by a mere 25%. The much stronger temperature sensitivity of the frequency of directed events (estimated energy of activation approximately 135 kJ/mol) than that of the granule velocities (approximately 22 kJ/mol) suggests that cooling-induced suppression of insulin granule movement is attributable to factors other than reduced motor protein adenosine 5'-triphosphatase activity. Indeed, cooling suppresses random granule diffusion by approximately 50%. In the single cell, the number of directed events depends on the extent of granule diffusion. Finally, single-cell exocytosis exhibits a biphasic pattern corresponding to that observed in vivo, and only the component reflecting 2nd phase insulin secretion is affected by cooling. We conclude that random diffusive movement is a prerequisite for directed insulin granule transport and for the recruitment of insulin granules released during 2nd phase insulin secretion.     

Phylogenetic position of the parasitoid nanoflagellate Pirsonia inferred from nuclear-encoded small subunit ribosomal DNA and a description of Pseudopirsonia n. gen. and Pseudopirsonia mucosa (Drebes) comb. nov

Sequences of the nuclear encoded small subunit (SSU) rRNA were determined for Pirsonia diadema, P. guinardiae, P. punctigerae, P. verrucosa, P. mucosa and three newly isolated strains 99-1, 99-2, 99-S. Based on phylogenetic analysis all Pirsonia strains, except P. mucosa, clustered together in one clade, most closely related to Hyphochytrium catenoides within the group of stramenopiles. However, P. mucosa was most closely related to Cercomonas sp. SIC 7235 and Heteromita globosa and belongs to the heterogenic group of Cercozoa. In addition to the SSU rDNA sequences, P. mucosa differs from the stramenopile Pirsonia species in some characteristics and was therefore redescribed in this paper as Pseudopirsonia mucosa. The three newly isolated strains 99-1, 99-2, and 99-S differed by 28 bp in their SSU rDNA sequences from their closest neighbour P. diadema and only 1 to 3 bp among themselves. These base differences and a host range similar to P. formosa were sufficient to assign them as new strains of P. formosa.     

Multi-modality imaging identifies key times for annexin V imaging as an early predictor of therapeutic outcome

Radiolabeled annexin V may provide an early indication of the success or failure of anticancer therapy on a patient-by-patient basis as an in vivo marker of tumor cell killing. An important question that remains is when, after initiation of treatment, should annexin V imaging be performed. To address this issue, we obtained simultaneous in vivo measurements of tumor burden and uptake of radiolabeled annexin V in the syngeneic orthotopic murine BCL1 lymphoma model using in vivo bioluminescence imaging (BLI) and small animal single-photon emission computed tomography (SPECT). BCL1 cells labeled for fluorescence and bioluminescence assays (BCL1-gfp/luc) were injected into mice at a dose that leads to progressive disease within two to three weeks. Tumor response was followed by BLI and SPECT before and after treatment with a single dose of 10 mg/kg doxorubicin. Biodistribution analyses revealed a biphasic increase of annexin V uptake within the tumor-bearing tissues of mice. An early peak occurring before actual tumor cells loss was observed between 1 and 5 hr after treatment, and a second longer sustained rise from 9 to 24 hr after therapy, which heralds the onset of tumor cell loss as confirmed by BLI. Multimodality imaging revealed the temporal patterns of tumor cell loss and annexin V uptake revealing a better understanding of the timing of radiolabeled annexin V uptake for its development as a marker of therapeutic efficacy.     

NTP pattern of avian embryonic red cells: role of RNA degradation and AMP deaminase/5'-nucleotidase activity

During terminal erythroid differentiation, degradation of RNA is a potential source for nucleotide triphosphates (NTPs) that act as allosteric effectors of hemoglobin. In this investigation, we assessed the developmental profile of RNA and purine/pyrimidine trinucleotides in circulating embryonic chick red blood cells (RBC). Extensive changes of the NTP pattern are observed which differ significantly from what is observed for adult RBC. The biochemical mechanisms have not been identified yet. Therefore, we studied the role of AMP deaminase and IMP/GMP 5'-nucleotidase, which are key enzymes for the regulation of the purine nucleotide pool. Finally, we tested the effect of major NTPs on the oxygen affinity of embryonic/adult hemoglobin. The results are as follows. 1) Together with ATP, UTP and CTP serve as allosteric effectors of hemoglobin. 2) Degradation of erythroid RNA is apparently a major source for NTPs. 3) Developmental changes of nucleotide content depend on the activities of key enzymes (AMP deaminase, IMP/GMP 5'-nucleotidase, and pyrimidine 5'-nucleotidase). 4) Oxygen-dependent hormonal regulation of AMP deaminase adjusts the red cell ATP concentration and therefore the hemoglobin oxygen affinity.     

The UCS domain protein She4p binds to myosin motor domains and is essential for class I and class V myosin function

BACKGROUND: Myosins are motor proteins involved in processes like cell motility, vesicle transport, or cytokinesis. In a variety of organisms, a novel group of proteins forming the UCS (UNC-45/CRO1/SHE4) domain-containing family are essential for proper myosin function. The Saccharomyces cerevisae UCS domain protein She4p is involved in two myosin-requiring events, endocytosis and mRNA localization. RESULTS: In contrast to UCS domain proteins from other organisms that interact with class II myosins, we demonstrate that She4p associates with yeast class I and class V myosins. She4p binds to motor domains of class V myosin Myo4p and class I myosin Myo5p, and this binding depends on She4p's UCS domain. In vivo, She4p is essential for the function and localization of Myo3p, Myo4p, and Myo5p (but not of Myo2p) and for colocalization of class I myosins with cortical actin patches. In vitro, She4p stimulates binding of Myo5p to filamentous actin. Wild-type She4p, but not a mutant lacking the UCS domain, accumulates in a cap-like structure at the bud tip. This localization requires Myo2p and actin, suggesting a Myo2-dependent mechanism by which She4p is targeted to the bud cap. Localization of She4p is essential for proper positioning and myosin-actin association of cortical Myo5p. CONCLUSIONS: Our results suggest that She4p is a novel myosin motor domain binding protein and operates as a localized regulator of myosin function of class I and likely class V myosins.     

Army ants: an evolutionary bestseller?

Army ants are characterized by a complex combination of behavioral and morphological traits. Molecular data now indicate that army ant behavior has a unique evolutionary origin and has been conserved for over more than 100 million years.     

Crystal structure of Schizosaccharomyces pombe riboflavin kinase reveals a novel ATP and riboflavin-binding fold

The essential redox cofactors riboflavin monophosphate (FMN) and flavin adenine dinucleotide (FAD) are synthesised from their precursor, riboflavin, in sequential reactions by the metal-dependent riboflavin kinase and FAD synthetase. Here, we describe the 1.6A crystal structure of the Schizosaccharomyces pombe riboflavin kinase. The enzyme represents a novel family of phosphoryl transferring enzymes. It is a monomer comprising a central beta-barrel clasped on one side by two C-terminal helices that display an L-like shape. The opposite side of the beta-barrel serves as a platform for substrate binding as demonstrated by complexes with ADP and FMN. Formation of the ATP-binding site requires significant rearrangements in a short alpha-helix as compared to the substrate free form. The diphosphate moiety of ADP is covered by the glycine-rich flap I formed from parts of this alpha-helix. In contrast, no significant changes are observed upon binding of riboflavin. The ribityl side-chain might be covered by a rather flexible flap II. The unusual metal-binding site involves, in addition to the ADP phosphates, only the strictly conserved Thr45. This may explain the preference for zinc observed in vitro.     

A long-term porcine model for measurement of gastrointestinal motility

Animal models have become an essential tool in the investigations of gut motility under experimental conditions. To determine the influence of various anaesthetic drugs on the motility pattern of the gastroduodenal tract, a new long-term model has had to be developed for allowing measurements in conscious and unrestrained as well as in sedated and analgosedated pigs. Since mechanical ventilation influences gut motility, it was necessary that this animal model enabled the investigation of the effect of drugs causing sedation and analgosedation during spontaneous breathing. Seven male, castrated pigs, German landrace, 32-40 kg bodyweight (BW) were investigated in this study. After habituation of the pigs to local housing conditions over 5 days, the animals were trained over 4 days to prepare for experimental situations and investigators. Pigs were inserted with a central venous catheter and with percutaneous enterogastrostomy (PEG) under general anaesthesia. Intestinal motility was measured by intraluminal impedancometry. The catheter was introduced over the PEG into the stomach and positioned into the duodenum by duodenoscopy. Measurements were done in conscious, unrestrained pigs and with sedated, and analgosedated animals on subsequent days. The habituation and training of the pigs to the investigators and for the laboratory conditions took between 7 and 9 days. The initial anaesthesia protocol for the instrumentation using remifentanil/propofol led to pyloric spasm and was thus unsuitable for duodenal intubation with an endoscope. In contrast, a combination of ketamine/propofol enabled this procedure. It was practicable to measure gut motility in conscious, unrestrained pigs. Spontaneous breathing was sufficient under propofol sedation and analgosedation using fentanyl-propofol. Systematically local application of polividon iodine in the area of the subcutaneous catheters avoided the necessity of using systemic prophylactic antibiotics. In conclusion, the habituation and training for 9 days enabled the measurement of gut motility by intraluminal impedancometry in conscious pigs. The insertion of the catheter was done during general anaesthesia using a combination of propofol and ketamine. For the future determination of gut motility performed under general anaesthesia, each sedation and analgosedation concept has to be evaluated to see whether it allows spontaneous breathing or whether mechanical ventilation is necessary.