Inorganic phosphate speeds loaded shortening in rat skinned cardiac myocytes

Force generation in striated muscle is coupled with inorganic phosphate (P_i) release from myosin, because force falls with increasing P_i concentration ([P_i]). However, it is unclear which steps in the cross-bridge cycle limit loaded shortening and power output. We examined the role of P_i in determining force, unloaded and loaded shortening, power output, and rate of force development in rat skinned cardiac myocytes to discern which step in the cross-bridge cycle limits loaded shortening. Myocytes (n = 6) were attached between a force transducer and position motor, and contractile properties were measured over a range of loads during maximal Ca²⁺ activation. Addition of 5 mM P_i had no effect on maximal unloaded shortening velocity (V_o) (control 1.83 ± 0.75, 5 mM added P_i 1.75 ± 0.58 muscle lengths/s; n = 6). Conversely, addition of 2.5, 5, and 10 mM P_i progressively decreased force but resulted in faster loaded shortening and greater power output (when normalized for the decrease in force) at all loads greater than 10% isometric force. Peak normalized power output increased 16% with 2.5 mM added P_i and further increased to a plateau of 35% with 5 and 10 mM added P_i. Interestingly, the rate constant of force redevelopment (k_tr) progressively increased from 0 to 10 mM added P_i, with k_tr 360% greater at 10 mM than at 0 mM added P_i. Overall, these results suggest that the P_i release step in the cross-bridge cycle is rate limiting for determining shortening velocity and power output at intermediate and high relative loads in cardiac myocytes. muscle mechanics; force-velocity relationship; cross-bridge cycle     

Intracellular positioning of isoforms explains an unusually large adenylate kinase gene family in the parasite Trypanosoma brucei

Adenylate kinases occur classically as cytoplasmic and mitochondrial enzymes, but the expression of seven adenylate kinases in the flagellated protozoan parasite Trypanosoma brucei (order, Kinetoplastida; family, Trypanosomatidae) easily exceeds the number of isoforms previously observed within a single cell and raises questions as to their location and function. We show that a requirement to target adenylate kinase into glycosomes, which are unique kinetoplastid-specific microbodies of the peroxisome class in which many reactions of carbohydrate metabolism are compartmentalized, and two different flagellar structures as well as cytoplasm and mitochondrion explains the expansion of this gene family in trypanosomes. The three isoforms that are selectively built into either the flagellar axoneme or the extra-axonemal paraflagellar rod, which is essential for motility, all contain long N-terminal extensions. Biochemical analysis of the only short form trypanosome adenylate kinase revealed that this enzyme catalyzes phosphotransfer of gamma-phosphate from ATP to AMP, CMP, and UMP acceptors; its high activity and specificity toward CMP is likely to reflect an adaptation to very low intracellular cytidine nucleotide pools. Analysis of some of the phosphotransfer network using RNA interference suggests considerable complexity within the homeostasis of cellular energetics. The anchoring of specific adenylate kinases within two distinct flagellar structures provides a paradigm for metabolic organization and efficiency in other flagellates.     

Conserved signature proposed for folding in the lipocalin superfamily

We systematically identify a group of evolutionarily conserved residues proposed for folding in a model beta-barrel superfamily, the lipocalins. The nature of conservation at the structural level is defined and we show that the conserved residues are involved in a network of interactions that form the core of the fold. Exploratory kinetic studies are conducted with a model superfamily member, human serum retinol-binding protein, to examine their role. The present results, coupled with key experimental studies conducted with another lipocalin beta-lactoglobulin, suggest that the evolutionarily conserved regions fold on a faster folding time-scale than the non-conserved regions.     

High-pressure freezing, cellular tomography, and structural cell biology

Structural cell biology, which we define as electron microscopic analysis of intact cells, suffered a loss of interest and activity following the advances in light microscopy beginning in the 1990s. Interestingly, it is the wealth of detailed observation in the light microscope that is one of the driving forces for the current renewed interest in electron microscopy (EM). A great many cellular details are simply beyond the resolving power of the light microscope. In this article, we describe how electron microscopists are responding to the demands for better preservation of cells and for ways to view cell ultrastructure in three dimensions at high resolution. We discuss how low temperature methods, especially high-pressure freezing and freeze substitution, reduce the artifacts of conventional EM specimen preparation. We also give a brief introduction to cellular electron tomography, a powerful analytical method that can give near-atomic resolution of cell ultrastructure in three-dimensional (3-D) models.     

P-selectin and CD63 use different mechanisms for delivery to Weibel-Palade bodies

The biogenesis of endothelial-specific Weibel-Palade bodies (WPB) is poorly understood, despite their key role in both haemostasis and inflammation. Biogenesis of specialized organelles of haemopoietic cells is often adaptor protein complex 3-dependent (AP-3-dependent), and AP-3 has previously been shown to play a role in the trafficking of both WPB membrane proteins, P-selectin and CD63. However, WPB are thought to form at the trans Golgi network (TGN), which is inconsistent with a role for AP-3, which operates in post-Golgi trafficking. We have therefore investigated in detail the mechanisms of delivery of these two membrane proteins to WPB. We find that P-selectin is recruited to forming WPB in the trans-Golgi by AP-3-independent mechanisms that use sorting information within both the cytoplasmic tail and the lumenal domain of the receptor. In contrast, CD63 is recruited to already-budded WPB by an AP-3-dependent route. These different mechanisms of recruitment lead to the presence of distinct immature and mature populations of WPB in human umbilical vein endothelial cells (HUVEC).     

N-terminal region of gelsolin induces apoptosis of activated hepatic stellate cells by a caspase-dependent mechanism

Activated hepatic stellate cells (HSCs) are the major source for alteration of extracellular matrix in fibrosis and cirrhosis. Conditioned medium (CM) collected from immortalized human hepatocytes (IHH) have earlier been shown to be responsible for apoptosis of HSCs. In this study, we have shown that antibodies raised against a peptide derived from a linear B-cell epitope in the N-terminal region of gelsolin identified a gelsolin fragment in IHH CM. Analysis of activated stellate cell death by CM collected from Huh7 cells transfected with plasmids encoding gelsolin deletion mutants suggested that the N-terminal half of gelsolin contained sequences which were responsible for stellate cell death. Further analysis determined that this activity was restricted to a region encompassing amino acids 1-70 in the gelsolin sequence; antibody directed to an epitope within this region was able to neutralize stellate cell death. Gelsolin modulation of cell death using this fragment involved upregulation of TRAIL-R1 and TRAIL-R2, and involved caspase 3 activation by extrinsic pathway. The apoptotic activity of N-terminal gelsolin fragments was restricted to activated but not quiescent stellate cells indicating its potential application in therapeutic use as an anti-fibrotic agent. Gelsolin fragments encompassing N-terminal regions in polypeptides of different molecular sizes were detected by N-terminal peptide specific antiserum in IHH CM immunoprecipitated with chronically HCV infected patient sera, suggesting the presence of autoantibodies generated against N-terminal gelsolin fragments in patients with chronic liver disease.     

Pulsatile urea excretion in the gulf toadfish: mechanisms and controls

Opsanus beta expresses a full complement of ornithine–urea cycle (OUC) enzymes and is facultatively ureotelic, reducing ammonia-N excretion and maintaining urea-N excretion under conditions of crowding/confinement. The switch to ureotelism is keyed by a modest rise in cortisol associated with a substantial increase in cytosolic glutamine synthetase for trapping of ammonia-N and an upregulation of the capacity of the mitochondrial OUC to use glutamine-N. The entire day's urea-N production is excreted in 1 or 2 short-lasting pulses, which occur exclusively through the gills. The pulse event is not triggered by an internal urea-N threshold, is not due to pulsatile urea-N production, but reflects pulsatile activation of a specific branchial excretion mechanism that rapidly clears urea-N from the body fluids. A bidirectional facilitated diffusion transporter, with pharmacological similarity to the UT-A type transporters of the mammalian kidney, is activated in the gills, associated with an increased trafficking of dense-cored vesicles in the pavement cells. An 1814 kB cDNA (‘tUT’) coding for a 475–amino acid protein with approximately 62% homology to mammalian UT-A's has been cloned and facilitates phloretin-sensitive urea transport when expressed in Xenopus oocytes. tUT occurs only in gill tissue, but tUT mRNA levels do not change over the pulse cycle, suggesting that tUT regulation occurs at a level beyond mRNA. Circulating cortisol levels consistently decline prior to a pulse event and rise thereafter. When cortisol is experimentally clamped at high levels, natural pulse events are suppressed in size but not in frequency, an effect mediated through glucocorticoid receptors. The cortisol decline appears to be permissive, rather than the actual trigger of the pulse event. Fluctuations in circulating AVT levels do not correlate with pulses; and injections of AVT (at supraphysiological levels) elicit only minute urea-N pulses. However, circulating 5-hydroxytryptamine (5-HT) levels fluctuate considerably and physiological doses of 5-HT cause large urea-N pulse events. When the efferent cranial nerves to the gills are sectioned, natural urea pulse events persist, suggesting that direct motor output from the CNS to the gill is not the proximate control.     

Do Low-Mercury Terrestrial Resources Subsidize Low-Mercury Growth of Stream Fish? Differences between Species along a Productivity Gradient

Low productivity in aquatic ecosystems is associated with reduced individual growth of fish and increased concentrations of methylmercury (MeHg) in fish and their prey. However, many stream-dwelling fish species can use terrestrially-derived food resources, potentially subsidizing growth at low-productivity sites, and, because terrestrial resources have lower MeHg concentrations than aquatic resources, preventing an increase in diet-borne MeHg accumulation. We used a large-scale field study to evaluate relationships among terrestrial subsidy use, growth, and MeHg concentrations in two stream-dwelling fish species across an in-stream productivity gradient. We sampled young-of-the-year brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar), potential competitors with similar foraging habits, from 20 study sites in streams in New Hampshire and Massachusetts that encompassed a wide range of aquatic prey biomass. Stable isotope analysis showed that brook trout used more terrestrial resources than Atlantic salmon. Over their first growing season, Atlantic salmon tended to grow larger than brook trout at sites with high aquatic prey biomass, but brook grew two-fold larger than Atlantic salmon at sites with low aquatic prey biomass. The MeHg concentrations of brook trout and Atlantic salmon were similar at sites with high aquatic prey biomass and the MeHg concentrations of both species increased at sites with low prey biomass and high MeHg in aquatic prey. However, brook trout had three-fold lower MeHg concentrations than Atlantic salmon at low-productivity, high-MeHg sites. These results suggest that differential use of terrestrial resource subsidies reversed the growth asymmetry between potential competitors across a productivity gradient and, for one species, moderated the effect of low in-stream productivity on MeHg accumulation.     

First record of Renibacterium salmoninarum in the sea lamprey (Petromyzon marinus)

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a widespread problem with major implications for salmonid fish species. The mechanisms by which the bacterium has reached high levels of infection previously unrecorded in the Laurentian Great Lakes are presently unknown. Research involving reservoirs and mechanisms of R. salmoninarum transmission in fish is lacking because of the ecologic complexity of heterogeneous habitats and the lack of adequate funding. Herein, we report on the isolation of R. salmoninarum from the kidneys of the sea lamprey (Petromyzon marinus). The bacterium was cultured from kidneys of 16% and 4% of lampreys collected from two locations within the Lake Ontario watershed in 2003 and 2004, respectively. The identity of bacterial colonies was verified with the nested polymerase chain reaction and quantitative enzyme-linked immunosorbent assay.