Characterization of RyR1-slow, a ryanodine receptor specific to slow-twitch skeletal muscle

Two distinct skeletal muscle ryanodine receptors (RyR1s) are expressed in a fiber type-specific manner in fish skeletal muscle (11). In this study, we compare [(3)H]ryanodine binding and single channel activity of RyR1-slow from fish slow-twitch skeletal muscle with RyR1-fast and RyR3 isolated from fast-twitch skeletal muscle. Scatchard plots indicate that RyR1-slow has a lower affinity for [(3)H]ryanodine when compared with RyR1-fast. In single channel recordings, RyR1-slow and RyR1-fast had similar slope conductances. However, the maximum open probability (P(o)) of RyR1-slow was threefold less than the maximum P(o) of RyR1-fast. Single channel studies also revealed the presence of two populations of RyRs in tuna fast-twitch muscle (RyR1-fast and RyR3). RyR3 had the highest P(o) of all the RyR channels and displayed less inhibition at millimolar Ca(2+). The addition of 5 mM Mg-ATP or 2.5 mM beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) to the channels increased the P(o) and [(3)H]ryanodine binding of both RyR1s but also caused a shift in the Ca(2+) dependency curve of RyR1-slow such that Ca(2+)-dependent inactivation was attenuated. [(3)H]ryanodine binding data also showed that Mg(2+)-dependent inhibition of RyR1-slow was reduced in the presence of AMP-PCP. These results indicate differences in the physiological properties of RyRs in fish slow- and fast-twitch skeletal muscle, which may contribute to differences in the way intracellular Ca(2+) is regulated in these muscle types.     

Differential nuclear localization and nuclear matrix association of the splicing factors PSF and PTB

A monoclonal antibody raised against nuclear matrix proteins detected a protein of basic pI in human nuclear matrix protein samples of various cellular origin. The ubiquitously occurring (common) nuclear matrix protein was identified as splicing factor PSF (PTB associated splicing factor). The interaction between the splicing factors PSF and PTB/hnRNP I was confirmed by co-immunoprecipitation from nuclear salt extracts. However, the nuclear localization of PSF and PTB and their distribution in subnuclear fractions differed markedly. Isolated nuclear matrices contained the bulk of PSF, but only minor amounts of PTB. In confocal microscopy both proteins appeared in speckles, the majority of which did not co-localize. Removing a large fraction of the soluble PTB structures by salt extraction revealed some colocalization of the more stable PTB fraction with PSF. These PTB/PSF complexes as well as the observed PSF-PTB interaction may reflect the previously reported presence of PTB and PSF in spliceosomal complexes during RNA processing. The present data, however, point to different cellular distribution and nuclear matrix association of the majority of PSF and PTB.     

Analysis of complex physiological systems by information flow: a time scale-specific complexity assessment.

In the last two decades conventional linear methods for biosignal analysis have been substantially extended by non-stationary, non-linear, and complexity approaches. So far, complexity is usually assessed with regard to one single time scale, disregarding complex physiology organised on different time scales. This shortcoming was overcome and medically evaluated by information flow functions developed in our research group in collaboration with several theoretical, experimental, and clinical partners. In the present work, the information flow is introduced and typical information flow characteristics are demonstrated. The prognostic value of autonomic information flow (AIF), which reflects communication in the cardiovascular system, was shown in patients with multiple organ dysfunction syndrome and in patients with heart failure. Gait information flow (GIF), which reflects communication in the motor control system during walking, was introduced to discriminate between controls and elderly patients suffering from low back pain. The applications presented for the theoretically based approach of information flow confirm its value for the identification of complex physiological systems. The medical relevance has to be confirmed by comprehensive clinical studies. These information flow measures substantially extend the established linear and complexity measures in biosignal analysis.     

Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells

Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell.     

Two domains in dihydropyridine receptor activate the skeletal muscle Ca(2+) release channel

The II-III cytoplasmic loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha(1)-subunit is essential for skeletal-type excitation-contraction coupling. Single channel and [(3)H]ryanodine binding studies with a full-length recombinant peptide (p(666-791)) confirmed that this region specifically activates skeletal muscle Ca2+ release channels (CRCs). However, attempts to identify shorter domains of the II-III loop specific for skeletal CRC activation have yielded contradictory results. We assessed the specificity of the interaction of five truncated II-III loop peptides by comparing their effects on skeletal and cardiac CRCs in lipid bilayer experiments; p(671-680) and p(720-765) specifically activated the submaximally Ca2+-activated skeletal CRC in experiments using both mono and divalent ions as current carriers. A third peptide, p(671-690), showed a bimodal activation/inactivation behavior indicating a high-affinity activating and low-affinity inactivating binding site. Two other peptides (p(681-690) and p(681-685)) that contained an RKRRK-motif and have previously been suggested in in vitro studies to be important for skeletal-type E-C coupling, failed to specifically stimulate skeletal CRCs. Noteworthy, p(671-690), p(681-690), and p(681-685) induced similar subconductances and long-lasting channel closings in skeletal and cardiac CRCs, indicating that these peptides interact in an isoform-independent manner with the CRCs.     

Calmodulin binding and inhibition of cardiac muscle calcium release channel (ryanodine receptor)

Metabolically (35)S-labeled calmodulin (CaM) was used to determine the CaM binding properties of the cardiac ryanodine receptor (RyR2) and to identify potential channel domains for CaM binding. In addition, regulation of RyR2 by CaM was assessed in [(3)H]ryanodine binding and single-channel measurements. Cardiac sarcoplasmic reticulum vesicles bound approximately four CaM molecules per RyR2 tetramer in the absence of Ca(2+); in the presence of 100 microm Ca(2+), the vesicles bound 7.5 CaM molecules per tetramer. Purified RyR2 bound approximately four [(35)S]CaM molecules per RyR tetramer, both in the presence and absence of Ca(2+). At least four CaM binding domains were identified in [(35)S]CaM overlays of fusion proteins spanning the full-length RyR2. The affinity (but not the stoichiometry) of CaM binding was altered by redox state as controlled by the presence of either GSH or GSSG. Inhibition of RyR2 activity by CaM was influenced by Ca(2+) concentration, redox state, and other channel modulators. Parallel experiments with the skeletal muscle isoform showed major differences in the CaM binding properties and regulation by CaM of the skeletal and cardiac ryanodine receptors.     

Characterization of diabetes following pancreatic surgery in patients with congenital hyperinsulinism

Background

Congenital hyperinsulinism (CHI) is the most common cause of persistent hypoglycaemia in infancy that leads to unfavourable neurological outcome if not treated adequately. In patients with severe diffuse CHI it remains under discussion whether pancreatic surgery should be performed or intensive medical treatment with the acceptance of recurrent episodes of mild hypoglycaemia is justified. Near-total pancreatectomy is associated with high rates of insulin-dependent diabetes mellitus and exocrine pancreatic insufficiency. Little is known about the management and long-term glycaemic control of CHI patients with diabetes after pancreatic surgery. We searched the German/Austrian DPV database and compared the course of 42 CHI patients with diabetes to that of patients with type 1 diabetes mellitus (T1DM). Study groups were compared at diabetes onset and after a follow-up period of 6.1 [3.3–9.7] (median [interquartile range]) years.

Results

The majority of CHI patients with diabetes were treated with insulin (85.2% [70.9–99.5] at diabetes onset, and 90.5% [81.2–99.7] at follow-up). However, compared to patients with T1DM, significantly more patients in the CHI group with diabetes were treated with conventional insulin therapy (47.8% vs. 24.4%, p?=?0.03 at diabetes onset, and 21.1% vs. 6.4% at follow-up, p?=?0.003), and only a small number of CHI patients were treated with insulin pumps. Daily insulin dose was significantly lower in CHI patients with diabetes than in patients with T1DM, both at diabetes onset (0.3 [0.2–0.5] vs. 0.6?IE/kg/d [0.4–0.8], p?=?0.003) and follow-up (0.8 [0.4–1.0] vs. 0.9 [0.7–1.0] IE/kg/d, p?=?0.02), while daily carbohydrate intake was comparable in both groups. Within the first treatment year, HbA1c levels were significantly lower in CHI patients with diabetes (6.2% [5.5–7.9] vs. 7.2% [6.5–8.2], p?=?0.003), but increased to a level comparable to that of T1DM patients at follow-up. Interestingly, in CHI patients, the risk of severe hypoglycaemia tends to be higher only at diabetes onset (14.8% vs. 5.8%, p?=?0.1).

Conclusions

In surgically treated CHI patients insulin treatment needs to be intensified in order to achieve good glycaemic control. Our data furthermore emphasize the need for improved medical treatment options for patients with diazoxide- and/or octreotide-unresponsive CHI.

     

Changes in Khasi pine (Pinus kesiya Royle ex Gordon) tree stands affected by Dimorphic Egret Egretta dimorpha colony at Madagascar

Establishing plantations of quick growing introduced trees, combined with effect of a bird colony, affect habitat conditions. This causes alterations in spatial structure of a phytocenosis, its species composition and size of plants in herb layer. In the studied Khasi pine (PinuskesiyaRoyle ex Gordon) stand in central Madagascar the shrub layer was found only under egrets' nests, while it did not occur outside the colony. We observed four times higher number of species, and much greater coverage of shrub and herb layers in the colony than outside, which reflects the increased soil trophy in the colony. One of the common species of herb layer was Commelinaafricana. Leaf blades of its exemplars growing under the egret nests were on average larger, which provided greater surface of the assimilative apparatus, than of those growing outside the colony. All differences between the in the studied tree stands seem to reflect soil enrichment in nutrients caused by the excretions from egrets. Such changes in phytocoenoses caused by effect of bird colonies have been studied so far only in cold and temperate climates. Our results confirm a similar effect of birds' breeding on a plant community in a tropical climate.     

Bhargavaea indica sp. nov., a member of the phylum Firmicutes, isolated from Arabian Sea sediment

A Gram-positive, aerobic, coccoid-rod shaped, non-motile, catalase- and oxidase-positive bacterium, designated strain KJW98^T, was isolated from the marine sediment of Karwar jetty, west coast of India. The strain was β-haemolytic, non-endospore-forming and grew with 0–8.5% (w/v) NaCl, at 15–48°C and at pH 6.5–9.0, with optimum growth with 0.5% (w/v) NaCl, at 42°C and at pH 7.0–8.0. Phylogenetic analyses based on 16S rRNA and gyrB gene sequences showed that strain KJW98^T forms a lineage within the genus Bhargavaea. The G+C content of the genomic DNA was 55 mol%. The DNA-DNA relatedness values of strain KJW98^T with B. beijingensis DSM 19037^T, B. cecembensis LMG 24411^T and B. ginsengi DSM 19038^T were 43.2, 39 and 26.5%, respectively. The major fatty acids were anteiso-C_15:0 (37.7%), iso-C_15:0 (19.7%), anteiso-C_17:0 (17.0%) and iso-C_16:0 (11.1%). The predominant menaquinone was MK-8 and the cell-wall peptidoglycan was of A4α type with L-lysine as the diagnostic diamino acid. The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The phenotypic, genotypic and DNA-DNA relatedness data indicate that strain KJW98^T should be distinguished from the members of the genus Bhargavaea, for which the name Bhargavaea indica sp. nov. is proposed with the type strain KJW98^T (=KCTC 13583^T =LMG 25219^T).