Coronary perfusion and muscle lengthening increase cardiac contraction: different stretch-triggered mechanisms

An increase in coronary perfusion, transversal stretch of the myocardium, increases developed force (F(dev)) (Gregg effect) through activation of stretch-activated ion channels (SACs). Lengthening of the muscle, longitudinal stretch of the myocardium, causes an immediate increase in F(dev) followed by a slow F(dev) increase (Anrep effect). In isometrically contracting perfused papillary muscles of Wistar rats, we investigated whether both effects were based on similar stretch-induced mechanisms by measuring F(dev) and intracellular Ca(2+) concentration ([Ca(2+)](i)) after a muscle length increase from 85% to 95% L(max) (length at which maximal isometric force develops) at low and high coronary perfusion before and after inhibition of SACs with gadolinium (10 micromol/l Gd(3+)). The increase of F(dev) and peak [Ca(2+)](i) by the Gregg effect was of similar magnitude as the Anrep effect (from 3.5 +/- 0.8 to 3.9 +/- 1.2 mN/mm(2) and from 3.0 +/- 0.7% to 3.8 +/- 0.9% normalized [Ca(2+)](i), means +/- SE). SAC blockade completely blunted the increase of F(dev) and peak [Ca(2+)](i) by the Gregg effect; however, it did not affect the Anrep effect. The slow force response, but not the calcium response, was augmented by an increase in coronary perfusion. Therefore, increased coronary perfusion, transversal stretch of the myocardium, and muscle lengthening, longitudinal stretch of the myocardium, increase myocardial contraction in the rat through different stretch-triggered mechanisms.     

Nuclear localization of non-receptor protein tyrosine phosphatase epsilon is regulated by its unique N-terminal domain

Precise subcellular localization is an important factor in regulation of the functions of protein tyrosine phosphatases. The non-receptor form of protein tyrosine phosphatase epsilon (cyt-PTP(epsilon)) can be found in cell nuclei, among other cellular locations, while p67 PTP(epsilon), a naturally occurring isoform which lacks the 27 N terminal residues of cyt-PTP(epsilon), is exclusively cytosolic. Using deletion and scanning mutagenesis we report that the first 10 amino acid residues of cyt-PTP(epsilon), in particular residues R4, K5, and R9, are critical components for its nuclear localization. We also establish that increased oxidative stress enhances accumulation of cyt-PTP(epsilon) in cell nuclei. Of the four known protein forms of PTP(epsilon), cyt-PTP(epsilon) is the only one which includes the extreme N-terminal sequence containing R4, K5, and R9. The role of the unique N terminus of cyt-PTP(epsilon) is therefore to regulate its subcellular localization. The existence of naturally occurring forms of PTP(epsilon) which lack this sequence and which are generated by translational and posttranslational mechanisms, suggests that nuclear localization of cyt-PTP(epsilon) can be actively regulated by cells.     

Biomass production and assimilation of dissolved organic matter by SAR11 bacteria in the Northwest Atlantic Ocean

Members of the SAR11 clade often dominate the composition of marine microbial communities, yet their contribution to biomass production and the flux of dissolved organic matter (DOM) is unclear. In addition, little is known about the specific components of the DOM pool utilized by SAR11 bacteria. To better understand the role of SAR11 bacteria in the flux of DOM, we examined the assimilation of leucine (a measure of biomass production), as well as free amino acids, protein, and glucose, by SAR11 bacteria in the Northwest Atlantic Ocean. We found that when SAR11 bacteria were >25% of total prokaryotes, they accounted for about 30 to 50% of leucine incorporation, suggesting that SAR11 bacteria were major contributors to bacterial biomass production and the DOM flux. Specific growth rates of SAR11 bacteria either equaled or exceeded growth rates for the total prokaryotic community. In addition, SAR11 bacteria were typically responsible for a greater portion of amino acid assimilation (34 to 61%) and glucose assimilation (45 to 57%) than of protein assimilation (< or = 34%). These data suggest that SAR11 bacteria do not utilize various components of the DOM pool equally and may be more important to the flux of low-molecular-weight monomers than to that of high-molecular-weight polymers.     

Meconium ileus caused by mutations in GUCY2C, encoding the CFTR-activating guanylate cyclase 2C

Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage.     

Oxytocin knockout mice demonstrate enhanced intake of sweet and nonsweet carbohydrate solutions

Oxytocin knockout (OT KO) mice display enhanced intake of nutritive and nonnutritive sweet solutions (i.e., sucrose and saccharin) compared with wild-type (WT) mice of the same C57BL/6 background strain. The present study further investigated the differential behavioral response of OT KO and WT mice to sucrose solutions and also examined intake preferences of OT KO and WT mice for palatable but nonsweet isocaloric solutions of carbohydrate and fat. A progressive ratio operant licking procedure demonstrated that OT KO and WT mice display a similar motivational drive to consume 10% sucrose. A series of two-bottle intake tests revealed that OT KO mice consume significantly larger amounts of both sweet and nonsweet carbohydrate solutions (i.e., sucrose, Polycose, and cornstarch) compared with WT cohorts. Intake pattern analyses revealed that OT KO mice overconsume carbohydrate solutions by initiating more drinking bouts compared with WT mice; bout sizes did not differ between the genotypes. In contrast, OT KO and WT mice did not differ in their intake of Intralipid, a palatable soybean oil emulsion. These findings indicate that the absence of OT in mice does not affect their appetitive drive to consume palatable sucrose solutions. Instead, the absence of OT may increase daily intake of palatable sweet and nonsweet solutions of carbohydrate (but not fat) by selectively blunting or masking processes that contribute to postingestive satiety.     

The Spatiotemporal Role of COX-2 in Osteogenic and Chondrogenic Differentiation of Periosteum-Derived Mesenchymal Progenitors in Fracture Repair

Periosteum provides a major source of mesenchymal progenitor cells for bone fracture repair. Combining cell-specific targeted Cox-2 gene deletion approaches with in vitro analyses of the differentiation of periosteum-derived mesenchymal progenitor cells (PDMPCs), here we demonstrate a spatial and temporal role for Cox-2 function in the modulation of osteogenic and chondrogenic differentiation of periosteal progenitors in fracture repair. Prx1Cre-targeted Cox-2 gene deletion in mesenchyme resulted in marked reduction of intramembraneous and endochondral bone repair, leading to accumulation of poorly differentiated mesenchyme and immature cartilage in periosteal callus. In contrast, Col2Cre-targeted Cox-2 gene deletion in cartilage resulted in a deficiency primarily in cartilage conversion into bone. Further cell culture analyses using Cox-2 deficient PDMPCs demonstrated reduced osteogenic differentiation in monolayer cultures, blocked chondrocyte differentiation and hypertrophy in high density micromass cultures. Gene expression microarray analyses demonstrated downregulation of a key set of genes associated with bone/cartilage formation and remodeling, namely Sox9, Runx2, Osx, MMP9, VDR and RANKL. Pathway analyses demonstrated dysregulation of the HIF-1, PI3K-AKT and Wnt pathways in Cox-2 deficient cells. Collectively, our data highlight a crucial role for Cox-2 from cells of mesenchymal lineages in modulating key pathways that control periosteal progenitor cell growth, differentiation, and angiogenesis in fracture repair.     

Rescue of syringyl lignin and sinapate ester biosynthesis in Arabidopsis thaliana by a coniferaldehyde 5-hydroxylase from Eucalyptus globulus

Key message

The gene coding for F5H from Eucalyptus globulus was cloned and used to transform an f5h -mutant of Arabidopsis thaliana , which was complemented, thus verifying the identity of the cloned gene.

Abstract

Coniferaldehyde 5-hydroxylase (F5H; EC 1.14.13) is a cytochrome P450-dependent monooxygenase that catalyzes the 5-hydroxylation step required for the production of syringyl units in lignin biosynthesis. The Eucalyptus globulus enzyme was characterized in vitro, and results showed that the preferred substrates were coniferaldehyde and coniferyl alcohol. Complementation experiments demonstrated that both cDNA and genomic constructs derived from F5H from E. globulus under the control of the cinnamate 4-hydroxylase promoter from Arabidopsis thaliana, or a partial F5H promoter from E. globulus, can rescue the inability of the A. thaliana fah1-2 mutant to accumulate sinapate esters and syringyl lignin. E. globulus is a species widely used to obtain products that require lignin removal, and the results suggest that EglF5H is a good candidate for engineering efforts aimed at increasing the lignin syringyl unit content, either for kraft pulping or biofuel production.     

Acute and specific collagen type I degradation increases diastolic and developed tension in perfused rat papillary muscle

Collagen degradation is suggested to be responsible for long-term contractile dysfunction in different cardiomyopathies, but the effects of acute and specific collagen type I removal (main type in the heart muscle) on tension have not been studied. We determined the diastolic and developed tension length relations in isometric contracting perfused rat papillary muscles (perfusion pressure 60 cmH(2)O) before and after acute and specific removal of small collagen struts with the use of purified collagenase type I. At 95% of the maximal length (95%L(max)), diastolic tension increased 20.4 +/- 8.1% (P < 0.05, n = 6) and developed tension increased 15.0 +/- 6.7% after collagenase treatment compared with time controls. Treatment increased the diastolic muscle diameter by 7.1 +/- 3.4% at 95%L(max), whereas the change in diameter due to contraction was not changed. Diastolic coronary flow and normalized coronary arterial flow impediment did not change after collagenase treatment. Electron microscopy revealed that the number of small collagen struts, interconnecting myocytes, and capillaries was reduced to approximately 32% after treatment. We conclude that removal of the small collagen struts by acute and specific collagen type I degradation increases diastolic and developed tension in perfused papillary muscle. We suggest that diastolic tension is increased due to edema, whereas developed tension is increased because the removal of the struts poses a lower lateral load on the cardiac myocytes, allowing more myocyte thickening.     

Evidence for intracellular spatial separation of hexokinases and fructokinases in tomato plants

Four hexokinase (LeHXK1–4) and four fructokinase (LeFRK1–4) genes were identified in tomato plants. Previous GFP fusion studies indicate that the gene product of LeHXK3 is associated with the mitochondria while that of LeHXK4 is located within plastids. In this study we found that the enzyme encoded by the fructokinase gene LeFRK3 is also located within plastids. The presence of LeFrk3 enzyme in plastids raises the question of the origin of fructose in these organelles. The other three FRKs enzymes, LeFrk1&2&4, are located in the cytosol. Unlike LeFrk1&2&4, the two additional HXKs, LeHxk1&2, share a common membrane anchor domain and are associated with the mitochondria similar to LeHxk3. The difference in the locations of the cytoplasmic FRK and HXK isozymes suggests that glucose phosphorylation is confined to defined special intracellular localizations while fructose phosphorylation is less confined.Contribution from the Agriculture Research Organization, The Volcani Center, Bet Dagan, Israel, No. 126/2006 series.