Reversible alteration of morphology in an invertebrate erythrocyte: properties of the natural inducer and the cellular response

The normal shape of the erythrocytes of the bivalves known as blood clams is maintained by a marginal band (MB) of microtubules. When hemolymph (or "blood") is withdrawn from the animal, its erythrocytes change, within minutes, from the normal smooth-surfaced, flattened ellipsoids (N-cells) to spheroids with folded surfaces (X-cells). This alteration can be prevented by rapidly diluting the hemolymph with physiological medium, yielding N-cells for use in studying the transformation to X-cells. Bioassays showed that shape transformation was induced by a hemolymph activity (Hx) and was a function, in part, of cell responsiveness to this activity. Eventually the shape of the cells spontaneously returned to normal, at a rate dependent upon the concentration of the cells and of Hx; recovery was correlated with loss of Hx. The X-cells contained an intact but highly deformed MB, but this was not the effector of the transformation. Erythrocytes made to lack MBs still changed shape, although they did not recover as completely as did the MB-containing controls. When clams were cooled before hemolymph was withdrawn, the concentration of Hx was reduced. Hx was retained after dialysis of hemolymph, and initial filtration and chromatography indicated that its Mr was greater than 500,000. Shape transformation was blocked by EGTA, by serine protease inhibitors, and by sodium azide; the last indicates ATP-dependence. Although the mechanism responsible for shape transformation remains to be determined, the data suggest that the change is triggered by a coagulation-related activity in response to the removal of hemolymph from the animal.     

3-hydroxyisobutyrate dehydrogenase-I from Pseudomonas denitrificans ATCC 13867 degrades 3-hydroxypropionic acid

This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDHI) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant (k _cat/K _m) of 604.1 ± 71.1 mM/S on (S)-3-hydroxyisobutyrate, but no detectable activity on (R)-3-hydroxyisobutyrate. 3HIBDH-I preferred NAD⁺ over NADP⁺ as a cofactor for its catalytic activity. The k _cat/K _m determined for 3-HP was 15.40 ± 1.43 mM/S in the presence of NAD⁺ at 37°C and pH 9.0. In addition to (S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized l-serine, methyl-d,l-serine, and methyl-(S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the k _cat/K _m values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn²⁺ increased the activity of 3HIBDHI significantly, whereas the presence of Fe²⁺, Hg²⁺ and Ag⁺ in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation.     

Molecular Basis of Glycosaminoglycan Heparin Binding to the Chemokine CXCL1 Dimer

Glycosaminoglycan (GAG)-bound and soluble chemokine gradients in the vasculature and extracellular matrix mediate neutrophil recruitment to the site of microbial infection and sterile injury in the host tissue. However, the molecular principles by which chemokine-GAG interactions orchestrate these gradients are poorly understood. This, in part, can be directly attributed to the complex interrelationship between the chemokine monomer-dimer equilibrium and binding geometry and affinities that are also intimately linked to GAG length. To address some of this missing knowledge, we have characterized the structural basis of heparin binding to the murine CXCL1 dimer. CXCL1 is a neutrophil-activating chemokine and exists as both monomers and dimers (K_d = 36 μm). To avoid interference from monomer-GAG interactions, we designed a trapped dimer (dCXCL1) by introducing a disulfide bridge across the dimer interface. We characterized the binding of GAG heparin octasaccharide to dCXCL1 using solution NMR spectroscopy. Our studies show that octasaccharide binds orthogonally to the interhelical axis and spans the dimer interface and that heparin binding enhances the structural integrity of the C-terminal helical residues and stability of the dimer. We generated a quadruple mutant (H20A/K22A/K62A/K66A) on the basis of the binding data and observed that this mutant failed to bind heparin octasaccharide, validating our structural model. We propose that the stability enhancement of dimers upon GAG binding regulates in vivo neutrophil trafficking by increasing the lifetime of “active” chemokines, and that this structural knowledge could be exploited for designing inhibitors that disrupt chemokine-GAG interactions and neutrophil homing to the target tissue.     

LED-Fluorescence Microscopy for Diagnosis of Pulmonary Tuberculosis under Programmatic Conditions in India

Background

Light-emitting diode fluorescence microscopy (LED-FM) has been shown to be more sensitive than conventional bright field microscopy using Ziehl-Neelsen (ZN) stain in detecting sputum smear positive tuberculosis in controlled laboratory conditions. In 2012, Auramine O staining based LED-FM replaced conventional ZN microscopy in 200 designated microscopy centres (DMC) of medical colleges operating in collaboration with India’s Revised National Tuberculosis Control Programme. We aimed to assess the impact of introduction of LED-FM services on sputum smear positive case detection under program conditions.

Methods

This was a before and after comparison study. In 15 randomly selected medical college DMCs, all presumptive TB patients who underwent sputum smear examination in the years 2011 (before LED-FM) and 2012 (after LED-FM) were compared. An additional 15 comparable DMCs that implemented conventional ZN sputum smear microscopy were also selected for comparison between 2011 and 2012.

Results

The proportion of presumptive TB patients (PTP)found sputum smear positive increased by 30%- from 13.6% (3432/25159) in 2011 to 17.8% (4706/26426) in 2012 (P value <0.01) in the sites that implemented LED-FM microscopy, whereas in DMCs where the ZN staining procedure is followed the proportion of sputum smear positive had remained unchanged (13.0%versus 12.6%;P value0.31).

Conclusion

Use of LED-FM significantly increased the proportion of smear positive cases among presumptive TB patients under routine program conditions in high workload laboratories. The study provides operational evidence needed to scale-up the use of LED-FM in similar settings in India and beyond.     

Transgenic Overexpression of Ephrin B1 in Bone Cells Promotes Bone Formation and an Anabolic Response to Mechanical Loading in Mice

To test if ephrin B1 overexpression enhances bone mass, we generated transgenic mice overexpressing ephrin B1 under the control of a 3.6 kb rat collagen 1A1 promoter (Col3.6-Tg^efnb1). Col3.6-Tg^efnb1 mice express 6-, 12- and 14-fold greater levels of full-length ephrin B1 protein in bone marrow stromal cells, calvarial osteoblasts, and osteoclasts, respectively. The long bones of both genders of Col3.6-Tg^efnb1 mice have increased trabecular bone volume, trabecular number, and trabecular thickness and decreased trabecular separation. Enhanced bone formation and decreased bone resorption contributed to this increase in trabecular bone mass in Col3.6-Tg^efnb1 mice. Consistent with these findings, our in vitro studies showed that overexpression of ephrin B1 increased osteoblast differentiation and mineralization, osterix and collagen 1A1 expression in bone marrow stromal cells. Interaction of ephrin B1 with soluble clustered EphB2-Fc decreased osteoclast precursor differentiation into multinucleated cells. Furthermore, we demonstrated that the mechanical loading-induced increase in EphB2 expression and newly formed bone were significantly greater in the Col3.6-Tg^efnb1 mice than in WT littermate controls. Our findings that overexpression of ephrin B1 in bone cells enhances bone mass and promotes a skeletal anabolic response to mechanical loading suggest that manipulation of ephrin B1 actions in bone may provide a means to sensitize the skeleton to mechanical strain to stimulate new bone formation.     

Inhibitory insulin-like growth factor-binding protein: cloning, complete sequence, and physiological regulation.

In this study we report the preparation of a human osteosarcoma cell cDNA library and describe the isolation and sequence determination of a clone encoding the complete sequence of a novel human insulin-like growth factor (IGF)-binding protein (hIGFBP-4). Previous work indicated that hIGFBP-4 is the predominant IGFBP expressed by human osteoblast-like cells, and that IGFBP-4 binds and inhibits the mitogenic activities of IGF-I and IGF-II. Sequence determination revealed that hIGFBP-4 is a unique gene product with significant amino- and carboxy-terminal sequence similarity to three other known IGFBPs. Identical alignment of 18 cysteines in IGFBP-4 and the three other IGFBPs is a key structural feature of this protein family. In vitro studies of human osteoblast-like cells suggest that PTH regulates the expression of hIGFBP-4 and that the PTH effect is mediated through a cAMP mechanism. hIGFBP-4 mRNA was also expressed in skin fibroblasts, and thus, this inhibitory IGFBP could be an important physiological regulator of IGF actions in bone cells and other cell types as well.     

Caspase-2 triggers Bax-Bak-dependent and -independent cell death in colon cancer cells treated with resveratrol

Polyphenol phytoalexin (resveratrol), found in grapes and red wine is a strong chemopreventive agent with promising safety records with human consumption and unique forms of cell death induction in a variety of tumor cells. However, the mechanism of resveratrol-induced apoptosis upstream of mitochondria is still not defined. The results from this study suggest that caspase-2 activation occurs upstream of mitochondria in resveratrol-treated cells. The upstream activation of caspase-2 is not dependent on its antioxidant property or NF-kappaB inhibition. The activated caspase-2 triggers mitochondrial apoptotic events by inducing conformational changes in Bax/Bak with subsequent release of cytochrome c, apoptosis-inducing factor, and endonuclease G. Caspase-8 activation seems to be independent of these events and does not appear to be mediated by classical death receptor processing or downstream caspases. Both caspase-2 and caspase-8 contribute toward the mitochondrial translocation of Bid, since neither caspase-8 inhibition nor caspase-2 inhibition could prevent translocation of Bid DsRed into mitochondria. Caspase-2 inhibitors or antisense silencing of caspase-2 prevented cell death induced by resveratrol and partially prevented processing of downstream caspases, including caspase-9, caspase-3, and caspase-8. Studies using mouse embryonic fibroblasts deficient for both Bax and Bak indicate the contribution of both Bax and Bak in mediating cell death induced by resveratrol and the existence of Bax/Bak-independent cell death possibly through caspase-8- or caspase-2-mediated mitochondria-independent downstream caspase processing.     

Pigmented pheochromocytoma: report of a case with diagnosis by fine needle aspiration

BACKGROUND: Pheochromocytoma is a common tumor of the adrenal medulla, but its pigmented variant is rare. CASE REPORT: A 38-year-old woman presented with complaints of abdominal pain. Ultrasound revealed a right suprarenal mass. Fine needle aspiration (FNA) smears showed the characteristic cytomorphology of pheochromocytoma, with melanin pigment in the cytoplasm. Melanin was differentiated from lipofuschin and hemosiderin by various histochemical stains. Histopathologic findings and immunohistochemical stains confirmed the diagnosis. To our knowledge, this is the first reported case of pigmented pheochromocytoma diagnosed on FNA. CONCLUSION: FNA has proven to be a rapid and conclusive method of diagnosing pigmented pheochromocytoma, with no complications.     

The protein tyrosine phosphatase SHP-1 modulates the suppressive activity of regulatory T cells

The importance of regulatory T cells (Tregs) for immune tolerance is well recognized, yet the signaling molecules influencing their suppressive activity are relatively poorly understood. In this article, through in vivo studies and complementary ex vivo studies, we make several important observations. First, we identify the cytoplasmic tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1) as an endogenous brake and modifier of the suppressive ability of Tregs; consistent with this notion, loss of SHP-1 expression strongly augments the ability of Tregs to suppress inflammation in a mouse model. Second, specific pharmacological inhibition of SHP-1 enzymatic activity via the cancer drug sodium stibogluconate potently augmented Treg suppressor activity both in vivo and ex vivo. Finally, through a quantitative imaging approach, we directly demonstrate that Tregs prevent the activation of conventional T cells and that SHP-1-deficient Tregs are more efficient suppressors. Collectively, our data reveal SHP-1 as a critical modifier of Treg function and a potential therapeutic target for augmenting Treg-mediated suppression in certain disease states.     

Predicting CO2 adsorption and reactivity on transition metal surfaces using popular density functional theory methods

ABSTRACT

In this work, with Ni (110) as a model catalyst surface and CO₂ as an adsorbate, a performance study of Density Functional Theory methods (functionals) is performed. CO being a possible intermediate in CO₂ conversion reactions, binding energies of both, CO₂ and CO, are calculated on the Ni surface and are compared with experimental data. OptPBE-vdW functional correctly predicts CO₂ binding energy on Ni (?62?kJ/mol), whereas CO binding energy is correctly predicted by the rPBE-vdW functional (?138?kJ/mol). The difference in computed adsorption energies by different functionals is attributed to the calculation of gas phase CO₂. Three alternate reaction systems based on a different number of C=O double bonds present in the gas phase molecule are considered to replace CO₂. The error in computed adsorption energy is directly proportional to the number of C=O double bonds present in the gas phase molecule. Additionally, both functionals predict similar carbon–oxygen activation barrier (40?kJ/mol) and equivalent C1s shifts for probe species (?2.6?eV for CCH₃ and +1.5?eV CO₃^?), with respect to adsorbed CO₂. Thus, by including a correction factor of 28?kJ/mol for the computed CO₂ gas phase energy, we suggest using rPBE-vdW functional to investigate CO₂ conversion reactions on different metals.