Rat liver adenyl cyclase activity in various thyroid states

Thyroidectomized and euthyroid rats were injected with three doses of triiodothyronine (T(3)) or of the diluent over a 6 day period, and liver homogenates were assayed for basal, epinephrine-stimulated, and NaF-stimulated adenyl cyclase activity. Based on NaF-stimulated levels, total adenyl cyclase activity, expressed per milligram of liver protein, was increased after thyroidectomy. Administration of T(3) to either hypothyroid or euthyroid rats, however, had no effect on the NaF-stimulated levels. Basal and epinephrine-stimulated enzyme activities were the same in hypothyroid, euthyroid, and hyperthyroid (euthyroid + T(3)) liver homogenates. In contrast, injections of T(3) in hypothyroid rats increased the activities of basal and epinephrine-stimulated adenyl cyclase. In view of the findings in euthyroid and hyperthyroid liver, it is possible that this effect is transient. In general, no correlation was found between the effects of thyroid hormone on respiration and on adenyl cyclase activity of the rat liver. These results imply that the hepatic thermogenic response to thyroid hormone is not mediated by stimulation of adenyl cyclase activity with the possible exception of the early effects of T(3) in the athyroid rat.     

Left and right ventricular diastolic dysfunction and diastolic heart failure: does one lead to the other?

Background and Objective Diastolic dysfunction of the left ventricle is a mechanical abnormality diagnosed primarily by echocardiogram, and can be distinguished into three separate degrees based on the severity of reduction in passive compliance and active myocardial relaxation. Methods A literature search was performed for basic science studies, clinical studies and major practice guidelines on the subject of diastolic dysfunction and diastolic heart failure. Important findings were analyzed and correlated with regard to clinical relevance. Results Left ventricular diastolic dysfunction appears to compromise exercise tolerance and is believed to contribute to the pathophysiology in patients with diastolic heart failure. In the clinical setting, however, oftentimes no clear distinction is made between echocardiographically diagnosed diastolic dysfunction and diastolic heart failure, and adequate treatment recommendations are sparse and aimed to prevent worsening and progression of clinical symptoms. To date, there is a lack of high powered trials assessing the possible progression rate from echocardiographically diagnosed diastolic dysfunction to the clinical diagnosis of diastolic heart failure. Furthermore, there are no solid indices to assess the degree of severity of diastolic dysfunction or its progression. Pure right ventricular diastolic dysfunction appears to be even less understood and under-recognized, although it may play a role in the development of both right and left heart failure. Currently there are few but interesting data on the possible interaction between ventricles with diastolic dysfunction and the overall affect on the development of heart failure. Conclusions The timeline and progression of diastolic dysfunction to diastolic heart failure have not been well established and warrant further investigation.     

Engineering Approaches Toward Deconstructing and Controlling the Stem Cell Environment

Stem cell-based therapeutics have become a vital component in tissue engineering and regenerative medicine. The microenvironment within which stem cells reside, i.e., the niche, plays a crucial role in regulating stem cell self-renewal and differentiation. However, current biological techniques lack the means to recapitulate the complexity of this microenvironment. Nano- and microengineered materials offer innovative methods to (1) deconstruct the stem cell niche to understand the effects of individual elements; (2) construct complex tissue-like structures resembling the niche to better predict and control cellular processes; and (3) transplant stem cells or activate endogenous stem cell populations for regeneration of aged or diseased tissues. In this article, we highlight some of the latest advances in this field and discuss future applications and directions of the use of nano- and microtechnologies for stem cell engineering.     

NOD2 exonic variations in Iranian Crohn's disease patients

Purpose

The NOD2 gene is known to have a strong association with Crohn??s disease, but different trends were reported in occurrence of NOD2 variants in distinct ethnicities. The aim of this study was to assess all exonic sequences of the NOD2 gene in Iranian Crohn's disease patients and healthy controls to identify any existing variation and evaluate their association with Crohn's disease.

Methods

A total of 90 non-related Crohn's disease patients and 120 sex- and age-matched healthy controls of Iranian origin were enrolled in this study. The participants were referred to a tertiary center in a 2-year period (2006?C2008). The exonic regions of the NOD2 gene were amplified by polymerase chain reaction and evaluated by direct sequencing.

Results

A total of 21 sequence variations were identified among all exonic regions of the NOD2 gene, of which eight had an allele frequency of more than 5%. Eight new mutations (one in exon 2 and seven in exon 4) were observed. The three main variants (R702W, G908R, and 1007fs) showed allele frequencies of 13.3%, 2.2%, and 1.7%, respectively. Three new variations (P371T, A794P, and Q908H) and R702W mutation were significantly more frequent in Crohn's disease patients compared to controls.

Conclusions

Eight novel mutations were identified in the NOD2 exons, but the pathophysiological importance of these variants remains unclear. Iranian patients with their different genetic reservoirs may demonstrate some novel characteristics for disease susceptibility.     

Protective hinge in insulin opens to enable its receptor engagement

Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal B-chain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated “microreceptors” that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of Phe^B24 to a 60° rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptor''s L1–β₂ sheet. Opening of this hinge enables conserved nonpolar side chains (Ile^A2, Val^A3, Val^B12, Phe^B24, and Phe^B25) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptor-bound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.How insulin engages the insulin receptor has inspired speculation ever since the structure of the free hormone was determined by Hodgkin and colleagues in 1969 (1, 2). Over the ensuing decades, anomalies encountered in studies of analogs have suggested that the hormone undergoes a conformational change on receptor binding: in particular, that the C-terminal β-strand of the B chain (residues B24–B30) releases from the helical core to expose otherwise-buried nonpolar surfaces (the detachment model) (3–6). Interest in the B-chain β-strand was further motivated by the discovery of clinical mutations within it associated with diabetes mellitus (DM) (7). Analysis of residue-specific photo–cross-linking provided evidence that both the detached strand and underlying nonpolar surfaces engage the receptor (8).The relevant structural biology is as follows. The insulin receptor is a disulfide-linked (αβ)₂ receptor tyrosine kinase (Fig. 1A), the extracellular α-subunits together binding a single insulin molecule with high affinity (9). Involvement of the two α-subunits is asymmetric: the primary insulin-binding site (site 1^*) comprises the central β-sheet (L1–β₂) of the first leucine-rich repeat domain (L1) of one α-subunit and the partially helical C-terminal segment (αCT) of the other α-subunit (Fig. 1A) (10). Such binding initiates conformational changes leading to transphosphorylation of the β-subunits’ intracellular tyrosine kinase (TK) domains. Structures of wild-type (WT) insulin (or analogs) bound to extracellular receptor fragments were recently described at maximum resolution of 3.9 Å (11), revealing that hormone binding is primarily mediated by αCT (receptor residues 704–719); direct interactions between insulin and L1 were sparse and restricted to certain B-chain residues. On insulin binding, αCT was repositioned on the L1–β₂ surface, and its helix was C-terminally extended to include residues 711–714. None of these structures defined the positions of C-terminal B-chain residues beyond B21. Support for the detachment model was nonetheless provided by entry of αCT into a volume that would otherwise be occupied by B-chain residues B25–B30 (i.e., in classical insulin structures; Fig. 1B) (11).Open in a separate windowFig. 1.Insulin B-chain C-terminal β-strand in the μIR complex. (A) Structure of apo-receptor ectodomain. One monomer is in tube representation (labeled), the second is in surface representation. L1, first leucine-rich repeat domain; CR, cysteine-rich domain; L2, second leucine-rich repeat domain; FnIII-1, -2 and -3; first, second and third fibronectin type III domains, respectively; αCT, α-subunit C-terminal segment; coral disk, plasma membrane. (B) Insulin bound to μIR; the view direction with respect to L1 in the apo-ectodomain is indicated by the arrow in A. Only B-chain residues indicated in black were originally resolved (11). The brown tube indicates classical location of residues B20-B30 in free insulin, occluded in the complex by αCT. (C) Orthogonal views of unmodeled 2F_obs-F_calc difference electron density (SI Appendix), indicating association of map segments with the αCT C-terminal extension (transparent magenta), insulin B-chain C-terminal segment (transparent gray), and Asn^A21 (transparent yellow). Difference density is sharpened (B_sharp = −160 Ų). (D–F) Refined models of respective segments insulin B20–B27, αCT 714–719, and insulin A17-A21 within postrefinement 2F_obs-F_calc difference electron density (B_sharp = −160 Ų). D is in stereo.We describe here the structure and interactions of the detached B-chain C-terminal segment of insulin on its binding to a “microreceptor” (μIR), an L1–CR domain-minimized version of the α-subunit (designated IR310.T) plus exogenous αCT peptide 704–719 (11). Our analysis defines a hinge in the B chain whose opening is coupled to repositioning of αCT between nonpolar surfaces of L1 and the insulin A chain. To understand the role of this hinge in holoreceptor binding and signaling, we designed three insulin analogs containing structural constraints (d-Ala^B20, d-Ala^B23]-insulin, ∆Phe^B25-insulin, and ∆Phe^B24-insulin, where ∆Phe is (α,β)-dehydrophenylalanine (Fig. 2) (12). The latter represents, to our knowledge, the first use of ∆Phe—a rigid “β-breaker” with extended electronic conjugation between its side chain and main chain (SI Appendix, Fig. S1)—as a probe of induced fit in macromolecular recognition. In addition, a fourth analog, active but with anomalous flexibility in the B chain (5, 6) (

Membrane receptors and their redistribution in lymphoproliferative disorders.

Lymphoid cells from 20 patients with lymphoproliferative disorders, including chronic lymphocytic leukemia, hairy cell leukemia, Sezary syndrome, lymphoma, and lymphadenitis, were studied for redistribution of surface membrane immunoglobulins (SmIg) and concanavalin A (Con-A) receptors. Fluorescein-labeled polyvalent goat anti-human immunoglobulin and fluoresceinated concanavalin A were used as ligands. Results were similar with both ligands. The highest percentage of capping of ligand-membrane receptors was noted in mononuclear cells from patients with "hairy" cell leukemia: from 24% to 90%. These cells showed moderate to marked fluorescein activity and were able to cap within 15 min at 4 degrees C. Chronic lymphocytic leukemia cells showed a weak fluorescein stain with a very low percentage of cells (0%--16%) capping. Lymph node cells from patients with lymphoma demonstrated moderate to strong fluorescein activity with only an average of 3% of the cells capping; while lymphoid cells from patients with lymphaedenitis showed an average of 27.5% capping and moderate fluorescein activity. Capping of Con-A receptors in mononuclear cells from patients with Sezary syndrome was poor (0%--14%) with moderate fluorescein intensity. This report demonstrates difference in density and mobility of binding sites for SmIg and Con-A on the surface membrane of lymphoid cells from various subclasses of lymphoproliferative disorders. These differences may assist in the differential diagnosis and classification of these conditions.     

Histological response and antigen transmission in the lymph nodes of athymic nu/nu mice inoculated with Crohn's disease tissue filtrates.

Lymph node histology and antigen transmission in the nu/nu mouse in response to animal inoculation with Crohn's disease tissue filtrates were re-evaluated. We found that a hyperplastic lymph node response in nu/nu mice occurred with Crohn's disease (CD), ulcerative colitis (UC), or other intestinal disease (OID) tissue inoculations. In addition, antigen transmission to lymph nodes as detected by indirect immunofluorescence using CD sera was observed in all inoculation groups. The immunofluorescent reaction also occurred independently of lymph node histology. Thus, we confirm that CD sera recognize an antigen(s) expressed in lymph nodes of athymic mice inoculated with CD tissue filtrates. The antibody (or antibodies) in CD sera was not specific for this 'CD antigen or antigens', however, as tested in the nu/nu mouse system, because the CD sera antibodies also recognised antigens in UC inoculated and OID inoculated animals.