Glomerular bypass shunts and distribution of glomeruli in the kidney of the lesser spotted dogfish,Scyliorhinus caniculus

Summary Scanning electron microscopy of corroded resin casts of the renal vasculature of Scyliorhinus caniculus has revealed a novel vascular pathway arising from the afferent arteriole and bypassing the glomerulus. This glomerulus bypass shunt occurred in 36% of the glomerular casts examined. The shunt ran to join a peritubular network of capillaries and thereby offers the potential to vary the degree of glomerular perfusion and control the proportion of active glomeruli. In 29% of glomeruli two efferent arterioles drained the capillary knot. Glomeruli were located close to the dorsal margin of the posterior mass of the kidney, and towards the lateral edge of the anterior lobes of the kidney of female dogfish. In male dogfish, glomeruli were evenly distributed through the posterior mass of kidney, while in female dogfish 89% of glomeruli occurred in the posterior mass and 11% of glomeruli were located within the small anterior lobes.     

Identification of the posttranslational modifications of bovine lens alpha B-crystallins by mass spectrometry.

A combination of mass spectrometric techniques has been used to investigate the amino acid sequence and post-translational modifications of alpha B-crystallin isolated from bovine lenses by gel filtration chromatography and reversed-phase high performance liquid chromatography. Chromatographic fractions were analyzed by electrospray ionization mass spectrometry to determine the homogeneity and molecular weights of proteins in the fractions. The alpha B-crystallin primary gene product, its mono- and diphosphorylated forms, its N- and C-terminal truncated forms, as well as other lens proteins unrelated to the alpha B-crystallins were identified by their molecular weights. Detailed information about the sites of phosphorylation, as well as evidence supporting reassignment of Asn to Asp at position 80, was obtained by analyzing proteolytic digests of these proteins by fast atom bombardment mass spectrometry. Results of this investigation indicate that alpha B-crystallin is phosphorylated in vivo at Ser 45, Ser 59, and either Ser 19 or 21. From the specificity of phosphorylation of alpha-crystallins, it appears that there may be two different kinases responsible for their phosphorylation.     

Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator

The blood anion nitrite contributes to hypoxic vasodilation through a heme-based, nitric oxide (NO)-generating reaction with deoxyhemoglobin and potentially other heme proteins. We hypothesized that this biochemical reaction could be harnessed for the treatment of neonatal pulmonary hypertension, an NO-deficient state characterized by pulmonary vasoconstriction, right-to-left shunt pathophysiology and systemic hypoxemia. To test this, we delivered inhaled sodium nitrite by aerosol to newborn lambs with hypoxic and normoxic pulmonary hypertension. Inhaled nitrite elicited a rapid and sustained reduction ( approximately 65%) in hypoxia-induced pulmonary hypertension, with a magnitude approaching that of the effects of 20 p.p.m. NO gas inhalation. This reduction was associated with the immediate appearance of NO in expiratory gas. Pulmonary vasodilation elicited by aerosolized nitrite was deoxyhemoglobin- and pH-dependent and was associated with increased blood levels of iron-nitrosyl-hemoglobin. Notably, from a therapeutic standpoint, short-term delivery of nitrite dissolved in saline through nebulization produced selective, sustained pulmonary vasodilation with no clinically significant increase in blood methemoglobin levels. These data support the concept that nitrite is a vasodilator acting through conversion to NO, a process coupled to hemoglobin deoxygenation and protonation, and evince a new, simple and inexpensive potential therapy for neonatal pulmonary hypertension.     

Ordering the Cytochrome c–initiated Caspase Cascade: Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9–dependent Manner

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1–mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c–inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.     

Diversity of Sulfate-Reducing Bacteria in Oxic and Anoxic Regions of a Microbial Mat Characterized by Comparative Analysis of Dissimilatory Sulfite Reductase Genes

Sequence analysis of genes encoding dissimilatory sulfite reductase (DSR) was used to identify sulfate-reducing bacteria in a hypersaline microbial mat and to evaluate their distribution in relation to levels of oxygen. The most highly diverse DSR sequences, most related to those of the Desulfonema-like organisms within the δ-proteobacteria, were recovered from oxic regions of the mat. This observation extends those of previous studies by us and others associating Desulfonema-like organisms with oxic habitats.     

Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa

Enteric neurotransmitters can modulate the biodefensive functions of the intestinal mucosa, but their role in mucosal interactions with enteropathogens is not well defined. Here we tested the hypothesis that norepinephrine (NE) modulates interactions between enterohemorrhagic Escherichia coli O157:H7 (EHEC) and the colonic epithelium. Mucosal sheets from porcine distal colon were mounted in Ussing chambers. Drugs and an inoculum of either Shiga toxin-negative or -positive EHEC were added to the contraluminal and luminal bathing medium, respectively. After 90 min, adherent bacteria were quantified by an adherence assay and by immunohistochemical methods; short-circuit current (I(sc)) was measured continuously to assess changes in active ion transport. NE-treated tissues exhibited concentration-dependent increases in I(sc) and EHEC adherence. NE did not alter adherence of a rodent-adapted, noninfectious E. coli strain or two porcine-adapted non-O157 E. coli strains. The actions of NE on EHEC adherence but not I(sc) were prevented by the alpha-adrenergic antagonist yohimbine and the PKA activator Sp-8-bromoadenosine-3',5'-cyclic monophosphorothioate. Like NE, the PKA inhibitor Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate or indirectly acting sympathomimetic agents increased EHEC adherence. Nerve fibers immunoreactive for the NE-synthesizing enzymes tyrosine hydroxylase and dopamine beta-hydroxylase appeared to innervate the colonic epithelium. EHEC-like immunoreactivity on the colonic surface had the appearance of bacterial microcolonies and increased after NE treatment by a phentolamine-sensitive mechanism. Through interactions with alpha(2)-adrenergic receptors, NE appears to increase EHEC adherence to the colonic mucosa. Changes in sympathetic neural outflow may alter intestinal susceptibility to infection.     

Upregulation of osteopontin expression is involved in the development of nonalcoholic steatohepatitis in a dietary murine model

The pathogenesis of nonalcoholic steatohepatitis (NASH) is poorly defined. Feeding mice a diet deficient in methionine and choline (MCD diet) induces experimental NASH. Osteopontin (OPN) is a Th1 cytokine that plays an important role in several fibroinflammatory diseases. We examined the role of OPN in the development of experimental NASH. A/J mice were fed MCD or control diet for up to 12 wk, and serum alanine aminotransferase (ALT), liver histology, oxidative stress, and the expressions of OPN, TNF-alpha, and collagen I were assessed at various time points. MCD diet-fed mice developed hepatic steatosis starting after 1 wk and inflammation by 2 wk; serum ALT increased from day 3. Hepatic collagen I mRNA expression increased during 1-4 wk, and fibrosis appeared at 8 wk. OPN protein expression was markedly increased on day 1 of MCD diet and persisted up to 8 wk, whereas OPN mRNA expression was increased at week 4. TNF-alpha expression was increased from day 3 to 2 wk, and evidence of oxidative stress did not appear until 8 wk. Increased expression of OPN was predominantly localized in hepatocytes. Hepatocytes in culture also produced OPN, which was stimulated by transforming growth factor-beta and TNF-alpha. Moreover, MCD diet-induced increases in serum ALT levels, hepatic inflammation, and fibrosis were markedly reduced in OPN(-/-) mice when compared with OPN(+/+) mice. In conclusion, our results demonstrate an upregulation of OPN expression early in the development of steatohepatitis and suggest an important role for OPN in signaling the onset of liver injury and fibrosis in experimental NASH.     

Recipes for reconstituting skin

Reconstituted Living Skin Equivalent (LSE) is made up of a dermal equivalent (DE) on which keratinocytes are plated where they give rise to a multilayered differentiated epidermis. The dermal equivalent develops through interactions between fibroblasts and collagen fibrils that begin to form after the cell-matrix precursor is cast. The gel that forms as a result of collagen polymerization and fluid trapping is contracted uniformly in all dimensions. By securing it at ends and edges in the mold in which it is cast, the final dimensions, strength and morphology of the forming tissue are altered. The same phenomena are seen in casting tubular tissues for the fabrication of small caliber blood vessel equivalents. The cells of the dermal equivalent are biosynthetically active and enrich the matrix to different degrees with secretory products, depending on how the cells are stimulated and on the presence or absence of an epidermis. Collagen biosynthesis by dermal cells in the DE is sensitive to growth factors, ascorbate concentrations and amino acid pools. Both ascorbate and TGF beta 1 increase total collagen biosynthesis at least two-fold by one week after tissue formation. With TGF beta 1 present, the capacity of cells in the DE to synthesize collagen increases with time, over a two-week period. If ascorbate (200 micrograms/ml) is added just after the tissue is cast and daily thereafter, contraction lattice is blocked, and collagen biosynthesis is enhanced relative to contracted controls that had received 200 micrograms/ml ascorbate once. The increase was nearly an order of magnitude over that of controls and was coordinate with a comparable increase in hyaluronate and sulfated glycosaminoglycan (GAG) production as shown by TCA-precipitable glucosamine in the intercellular matrix of the DE. Both the LSE and the Living Dermal Equivalent (LDE) exhibit complex responses to UV radiation and to various chemicals that are greatly different from responses given by monolayered cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Patient-specific modeling of dyssynchronous heart failure: A case study

The development and clinical use of patient-specific models of the heart is now a feasible goal. Models have the potential to aid in diagnosis and support decision-making in clinical cardiology. Several groups are now working on developing multi-scale models of the heart for understanding therapeutic mechanisms and better predicting clinical outcomes of interventions such as cardiac resynchronization therapy. Here we describe the methodology for generating a patient-specific model of the failing heart with a myocardial infarct and left ventricular bundle branch block. We discuss some of the remaining challenges in developing reliable patient-specific models of cardiac electromechanical activity, and identify some of the main areas for focusing future research efforts. Key challenges include: efficiently generating accurate patient-specific geometric meshes and mapping regional myofiber architecture to them; modeling electrical activation patterns based on cellular alterations in human heart failure, and estimating regional tissue conductivities based on clinically available electrocardiographic recordings; estimating unloaded ventricular reference geometry and material properties for biomechanical simulations; and parameterizing systemic models of circulatory dynamics from available hemodynamic measurements.