Novel insights into diminished cardiac reserve in non-obstructive hypertrophic cardiomyopathy from four-dimensional flow cardiac magnetic resonance component analysis.

AIMS: Hypertrophic cardiomyopathy (HCM) is characterized by hypercontractility and diastolic dysfunction, which alter blood flow haemodynamics and are linked with increased risk of adverse clinical events. Four-dimensional flow cardiac magnetic resonance (4D-flow CMR) enables comprehensive characterization of ventricular blood flow patterns. We characterized flow component changes in non-obstructive HCM and assessed their relationship with phenotypic severity and sudden cardiac death (SCD) risk. METHODS AND RESULTS: Fifty-one participants (37 non-obstructive HCM and 14 matched controls) underwent 4D-flow CMR. Left-ventricular (LV) end-diastolic volume was separated into four components: direct flow (blood transiting the ventricle within one cycle), retained inflow (blood entering the ventricle and retained for one cycle), delayed ejection flow (retained ventricular blood ejected during systole), and residual volume (ventricular blood retained for >two cycles). Flow component distribution and component end-diastolic kinetic energy/mL were estimated. HCM patients demonstrated greater direct flow proportions compared with controls (47.9 ± 9% vs. 39.4 ± 6%, P = 0.002), with reduction in other components. Direct flow proportions correlated with LV mass index (r = 0.40, P = 0.004), end-diastolic volume index (r = -0.40, P = 0.017), and SCD risk (r = 0.34, P = 0.039). In contrast to controls, in HCM, stroke volume decreased with increasing direct flow proportions, indicating diminished volumetric reserve. There was no difference in component end-diastolic kinetic energy/mL. CONCLUSION: Non-obstructive HCM possesses a distinctive flow component distribution pattern characterised by greater direct flow proportions, and direct flow-stroke volume uncoupling indicative of diminished cardiac reserve. The correlation of direct flow proportion with phenotypic severity and SCD risk highlight its potential as a novel and sensitive haemodynamic measure of cardiovascular risk in HCM.

Molecular characterization and In silico analysis of Sorghum Panallergens: Profilin and Polcalin.

In India, 20-30% of the human population suffer from allergic rhinitis and 15% of them develop asthma. Plant pollens are one of the causative aeroallergens and are mixture of a number of molecules including major and minor allergens (Panallergens). Profilin and polcalcin are the known pollen specific panallergens. Allergenicity of the Sorghum plant in Andhra Pradesh was found to be 54.9%. But the allergens responsible have not been characterized well. This study highlights identification and molecular characterization of Sorghum bicolor profilin (Sorb PF) and S. bicolor polcalcin (Sorb PC) allergen genes based on homology. The coding sequences of the two genes were PCR amplified from the cDNA constructed from Sorghum pollen total RNA. The gene sequences were deposited in NCBI, KC427126 and KC427125. Recombinantly expressed histidine tag (His-tag) purified Sorghum polcalcin and profilin confirmed 9 and 14 kDa proteins, respectively. Based on multiple sequence alignment and phylogenetic analysis, Sorghum polcalcin and profilin were found to be closely related with Cynodon dactylon, Phleum pratense and Oryza sativa grass species. In silico Algpred based screening of SorbPF and SorbPC showed an allergenicity score of 1.149 and 0.879, respectively. The structure of two Ef-hand sequences (DTNGDGKISLSEL and DTDGDGFIDFNEF) of SorbPC showed an exact match with Phlp7. It is concluded that Sorghum recombinant profilin and polcalcin proteins can be of potential use in developing diagnostic kits for allergenicity to Sorghum pollen grains.

Clopidogrel "resistance": pre- vs post-receptor determinants.

The clinical efficacy of the P2Y12 receptor antagonist clopidogrel as an agent to prevent thrombotic events predominantly reflects its anti-aggregatory effects. Stent thrombosis in particular is more likely to occur in patients in whom clopidogrel effect is limited. "Resistance" to clopidogrel in general should theoretically arise either because of a reduction in plasma concentration of the active metabolite and/or of the downstream intracellular biochemical changes underlying antiplatelet effects. We therefore postulate that "resistance" to clopidogrel arises via a combination of pharmacogenetic, pharmacokinetic and intracellular biochemical mechanisms. Considerable attention has been so far directed to the finding that stent thrombosis occurs more frequently in patients with loss-of-function mutations of CYP2C19, thus limiting clopidogrel bioactivation. Furthermore, a number of drug-drug interactions may marginally impair responsiveness to clopidogrel, largely via impairment of bioactivation. However, population data also suggest that clopidogrel "resistance" occurs more frequently in patients with acute coronary syndromes than in normal subjects, and that "resistance" is particularly common in obese subjects and with diabetes. Here we critically review available literature and speculate on the possibility that non-genetic causes of clopidogrel "resistance" may arise from impairments of the intracellular signaling cascade initiated by P2Y12 receptor inhibition. In such cases, "resistance" to clopidogrel may also theoretically occur with other P2Y12 receptor antagonists, irrespective of the need for bioactivation. Delineation of this non-genetic component of "resistance" to P2Y12 inhibitors may facilitate the development of optimal therapeutic strategies for high-risk cardiovascular patients.

[Comparison of the depth predictability of intra corneal ring segment implantation by mechanical versus femtosecond laser-assisted techniques using optical coherence tomography (OCT Visante(®))]. / Comparaison en tomographie en cohérence optique (OCT Visante(®)) de la prédictibilité de la profondeur d'implantation des anneaux intracornéens après tunnélisation mécanique versus tunnélisation assistée au laser femtoseconde.

UNLABELLED: Comparison of the depth predictability of intracorneal ring segment implantation by mechanical versus femtosecond laser-assisted techniques using optical coherence tomography (OCT Visante(®)). PURPOSE: To compare the depth predictability of intracorneal ring segment implantation by mechanical versus femtosecond laser-assisted techniques using OCT Visante(®). METHODS: This observational prospective study included, after patients' consent, 76 keratoconic eyes, of which 31 eyes (group 1) were operated using the mechanical technique and 45 eyes (group 2) with the femtosecond laser-assisted technique. The target depth was two-thirds of the peripheral corneal thickness, ranging from 5 to 7 mm in diameter. Every patient underwent high-resolution anterior segment OCT (OCT Visante(®)) measurement preoperatively and for the implant depth, 1 month postoperatively. Then two different sites were used to determine the segment depth, at the segment site and tangentially, at a distance of 700 µm central to the segment's inner edge. RESULTS: Both measurement techniques demonstrated that targeted implantation depth of 66% of the corneal thickness was not observed in either group. The mean difference between the preoperative expected depth and final segment implantation was 76.64 ± 48.76 µm in the manual technique and 85.85 ± 33.02 µm in the femtosecond-assisted technique, with no statistically significant difference between the groups. Comparison between the measurement sites showed implantation depth to be shallower, 54.93±6.03%, at the tangential site compared to 55.14 ± 8.08% at the implant site in group 1 and 56.17 ± 5.82% versus 58.88 ± 6.06%, respectively, in group 2. CONCLUSION: Both mechanical and femtosecond laser-assisted techniques showed a more superficial Intacs(®) placement than predicted. No statistically significant difference was observed in implantation depth between the two groups.

Development and Validation of New RP-HPLC Method for the Determination of Dexrazoxane.

A new sensitive, precise, rapid and linear RP-HPLC method was developed and validated for the determination of dexrazoxane in formulations and human serum samples. Good chromatographic separation of dexrazoxane was achieved by using Kromasil C18 column. The system was operated at ambient temperature using a mobile phase consisting of methanol, 5% ortho phosphoric acid, 0.01M ammonium dihydrogen phosphate and tetrahydrofuran, pH 4.2 (10:40:30:20, v/v) isocratically at a flow rate of 1 ml/min. The method showed high sensitivity with good linearity (r(2)=0.9998) over the tested concentration range of 0.1 to 0.9 mg/ml. Detection was carried out at 272 nm and retention time was 7.005 min. The accuracy, formulation assay and percentage of RSD were 100.03, 97.48 and 0.03184, respectively with tailing factor (1.84). This method can be used for the routine quality control analysis.

Arginine hydrochloride enhances the dynamics of subunit assembly and the chaperone-like activity of alpha-crystallin.

PURPOSE: Alpha-crystallin, a major eye lens protein, bears homology with small heat shock proteins (sHsps) and exhibits molecular chaperone-like activity. Structural perturbation by temperature or low concentrations of denaturants leads to enhancement of its chaperone-like activity. We have earlier demonstrated similar enhancement of chaperone-like activity using biologically compatible solutes such as arginine hydrochloride and aminoguanidine. The purpose of the present study is to get an insight into the mechanism of the arginine induced enhancement of chaperone-like activity of alpha-crystallin. METHODS: The effect of arginine hydrochloride on the chaperone-like activity of alpha-crystallin at 25 degrees C was studied using DTT induced aggregation of insulin as a model system. Changes in the accessibility of the thiol group near the end of the alpha-crystallin domain in the absence and the presence of arginine hydrochloride were studied using dithiobisnitrobenzoic acid. Fluorescence resonance energy transfer studies were performed to investigate changes in the dynamics of the subunit assembly. Urea induced denaturation studies of alpha-crystallin were carried out to investigate structural destabilization of alpha-crystallin, if any, in the presence of arginine hydrochloride. RESULTS: Arginine hydrochloride increases the chaperone-like activity of alpha-crystallin several fold towards DTT induced aggregation of insulin at room temperature. Our study shows that both the extent and the rate of accessibility of the thiol group are increased in the presence of arginine. Fluorescence resonance energy transfer experiments show that arginine hydrochloride significantly increases the subunit exchange between the oligomers of alpha-crystallin. Arginine induced structural perturbation and loosening of subunit assembly of alpha-crystallin leads to overall destabilization of the protein as reflected by the urea denaturation study. CONCLUSIONS: Arginine perturbs the tertiary and quaternary structure of alpha-crystallin and enhances the dynamics of the subunit assembly leading to enhanced chaperone-like activity. Thus, in addition to size, surface hydrophobicity, and charge distribution, the dynamics of the subunit assembly appears to be one of the critical factors that can modulate the chaperone activity.

Pseudotumor cerebri with transient oculomotor palsy.

Pseudotumor cerebri is a clinical syndrome characterized by raised intracranial pressure with normal ventricular size, anatomy and position. Headache, vomiting and diplopia are the most common symptoms. Signs include those of raised intracranial pressure including papilledema and absence of focal neurological signs. A secondary cause is identifiable in 50% of children; the most common predisposing conditions are otitis media, viral infection and medications. Management is mainly directed towards identifying and treating the cause and measures to reduce the raised intracranial pressure. Though it is mostly a self limited condition, optic atrophy and blindness can occur. Oculomotor nerve palsy is very rarely associated with pseudotumor cerebri. We report a unique case of pseudotumor cerebri who had left Oculomotor palsy with sparing of the pupillary fibres, which resolved following treatment with oral acetazolamide.

Enteromorpha compressa (L.) Greville an edible green alga as a source of antiallergic principle (S).

Enteromorpha compressa a marine green algal species grows extensively in North coastal Andhra Pradesh. Besides its nutritional importance it has also been identified as source of anti-anaphylactic compound(s).E. compressa extracts alleviated the IgE levels raised against ovalbumin and other allergens in mice. Further,Enteromorpha extract also significantly down regulated the serum IgE levels in different murine models irrespective of their genetic background. The results obtained in this study suggest thatE. compressa extract has compound(s), which inhibit IgE immune response and may have potential in curing various types of allergies.

Aortoiliac angulation and the need for secondary procedures to secure stent graft fixation: which angle is important?

BACKGROUND: The purpose of this study was to quantify the degree of aortoiliac tortuosity and determine the relationship between aortoiliac angulation and the need for a secondary procedure following endovascular repair. METHODS: Among 206 patients treated with the AneuRx stent graft, 3-year follow up data were available in 71 patients. Twenty eight patients without duplex and CT angiograms (CT angiography) on follow-up were excluded. The anatomy of the preoperative proximal aortic neck was evaluated using 3D-CT angiography reconstructed images in: a) Group I: 15 patients who required secondary procedures and b) Group II: 18 patients without any endovascular leak during follow up. The groups did not differ in age (72.9+/-6.1 versus 73.3+/-9.1) or aneurysm diameter (60.1+/-9.1 versus 60.5+/-10.1). In order to determine the aortoiliac tortuosity, we measured: a) the suprarenal aorta-infrarenal aortic neck angle: angle of the aorta at the level of the renal arteries, b) infrarenal aortic neck-aneurysm angle: angle of the aorta at the start of aneurysm, c) right iliac angle, d) left iliac angle, e) aortic neck length, f) aortic neck diameter. RESULTS: Computer-based measurements on 3D-CT angiography reconstructed images were: a) suprarenal aorta-infrarenal aortic neck angle: group I: (22.6+/-16.2), group II: (11.9+/-6.9), p<0.05; b) infrarenal aortic neck-aneurysm angle: group I: 17.6+/-12.4, group II: 18.8+/-9.4, p=NS; c) right iliac angle: group I: 22.9+/-12.6, group II: 20.4+/-9.5, p=NS; d) left iliac angle: group I: 22.4+/-10.5, group II: 19.1+/-12.2, p=NS; e) aortic neck length: group I: 18.9+/-5.3 mm, group II: 20.4+/-5.3 mm, p=NS; f) aortic neck diameter: group I: 24.1+/-1.0 mm, group II: 23.3+/-1.6, p=NS. CONCLUSIONS: Aortoiliac angulation can be defined and quantified. In patients requiring secondary procedures, there is an increased angulation at the proximal aortic neck angle.

Unfolding and refolding of a quinone oxidoreductase: alpha-crystallin, a molecular chaperone, assists its reactivation.

alpha-Crystallin, a member of the small heat-shock protein family and present in vertebrate eye lens, is known to prevent the aggregation of other proteins under conditions of stress. However, its role in the reactivation of enzymes from their non-native inactive states has not been clearly demonstrated. We have studied the effect of alpha-crystallin on the refolding of zeta-crystallin, a quinone oxidoreductase, from its different urea-denatured states. Co-refolding zeta-crystallin from its denatured state in 2.5 M urea with either calf eye lens alpha-crystallin or recombinant human alpha B-crystallin could significantly enhance its reactivation yield. alpha B-crystallin was found to be more efficient than alpha A-crystallin in chaperoning the refolding of zeta-crystallin. In order to understand the nature of the denatured state(s) of zeta-crystallin that can interact with alpha-crystallin, we have investigated the unfolding pathway of zeta-crystallin. We find that it unfolds through three distinct intermediates: an altered tetramer, a partially unfolded dimer, which is competent to fold back to its active state, and a partially unfolded monomer. The partially unfolded monomer is inactive, exhibits highly exposed hydrophobic surfaces and has significant secondary structural elements with little or no tertiary structure. This intermediate does not refold into the active state without assistance. alpha-Crystallin provides the required assistance and improves the reactivation yield several-fold.

N(omega)-arginine dimethylation modulates the interaction between a Gly/Arg-rich peptide from human nucleolin and nucleic acids.

We studied the interaction between a synthetic peptide (sequence Ac-GXGGFGGXGGFXGGXGG-NH(2), where X = arginine, N(omega),N(omega)-dimethylarginine, DMA, or lysine) corresponding to residues 676-692 of human nucleolin and several DNA and RNA substrates using double filter binding, melting curve analysis and circular dichroism spectroscopy. We found that despite the reduced capability of DMA in forming hydrogen bonds, N(omega),N(omega)-dimethylation does not affect the strength of the binding to nucleic acids nor does it have any effect on stabilization of a double-stranded DNA substrate. However, circular dichroism studies show that unmethylated peptide can perturb the helical structure, especially in RNA, to a much larger extent than the DMA peptide.

Interaction of human recombinant alphaA- and alphaB-crystallins with early and late unfolding intermediates of citrate synthase on its thermal denaturation.

We have investigated the role of recombinant human alphaA- and alphaB-crystallins in the heat-induced inactivation and aggregation of citrate synthase. Homo-multimers of both alphaA- and alphaB-crystallins confer protection against heat-induced inactivation in a concentration-dependent manner and also prevent aggregation. Interaction of crystallins with early unfolding intermediates of citrate synthase reduces their partitioning into aggregation-prone intermediates. This appears to result in enhanced population of early unfolding intermediates that can be reactivated by its substrate, oxaloacetate. Both these homo-multimers do not form a stable complex with the early unfolding intermediates. However, they can form a soluble, stable complex with aggregation-prone late unfolding intermediates. This soluble complex formation prevents aggregation. Thus, it appears that the chaperone activity of alpha-crystallin involves both transient and stable interactions depending on the nature of intermediates on the unfolding pathway; one leads to reactivation of the enzyme activity while the other prevents aggregation.

Redox-regulated chaperone function and conformational changes of Escherichia coli Hsp33.

We have studied the chaperone activity and conformation of Escherichia coli heat shock protein (Hsp)33, whose activity is known to be switched on by oxidative conditions. While oxidized Hsp33 completely prevents the heat-induced aggregation of zeta-crystallin at 42 degrees C at a ratio of 1:1 (w/w), the reduced form exhibits only a marginal effect on the aggregation. Far UV-circular dichroism (CD) spectra show that reduced Hsp33 contains a significant alpha-helical component. Oxidation results in significant changes in the far UV-CD spectrum. Near UV-CD spectra show changes in tertiary structural packing upon oxidation. Polarity-sensitive fluorescent probes report enhanced hydrophobic surfaces in the oxidized Hsp33. Our studies show that the oxidative activation of the chaperone function of Hsp33 involves observable conformational changes accompanying increased exposure of hydrophobic pockets.

Detection and assay of proteases using calf lens beta-crystallin aggregate as substrate.

The eye lens protein, betaL-crystallin, aggregates and yields a turbid solution upon refolding from its denatured state. We have observed that the addition of trace amounts of protease results in clearing of this turbidity. Based on this observation, we have developed a simple and rapid method for the detection and assay of proteases. This assay can be performed in the pH range of 6.0-9.0. We could assay the activity of trypsin at a concentration as low as 5 microg/ml.

Artificial chaperoning of insulin, human carbonic anhydrase and hen egg lysozyme using linear dextrin chains--a sweet route to the native state of globular proteins.

Linear dextrins (alpha-1,4-D-glucopyranoside chains) are known to possess amphiphilic surfaces and solubilize lipophilic compounds. We have assessed the ability of this amphiphilic surface of dextrin to inhibit the self-aggregation and assist the refolding of proteins. Addition of decameric dextrin, or dextrin-10, in the renaturation buffer improves the refolding yield of human carbonic anhydrase from its guanidinium chloride-induced denatured state. It is also seen to inhibit the self-aggregation of insulin. The ability of dextrin-10 to interact with cetyltrimethylammonium bromide and postpone its critical micellar concentration allows the use of dextrin-10 as a 'detergent stripping agent' in a novel artificial chaperoning process described earlier. The aggregation of human carbonic anhydrase and lysozyme upon refolding is prevented by cetyltrimethylammonium bromide due to the formation of a protein-detergent complex; dextrin-10 strips off the detergent from the complex and allow the proteins to fold, thus increasing the renaturation yield. Dextran-4 (the alpha-1,6-D-glucopyranoside chain), which does not exhibit amphiphilic properties, does not help in such artificial chaperoning.

The chaperone-like alpha-crystallin forms a complex only with the aggregation-prone molten globule state of alpha-lactalbumin.

The chaperone-like alpha-crystallin prevents aggregation of several proteins by interacting with their non-native states. Alpha-Lactalbumin adopts different non-native states under different experimental conditions. We have investigated the interaction of alpha-crystallin with three non-identical non-native states, using fluorescence, circular dichroism, and gel filtration chromatography. The compact molten globule state of apo-alpha-lactalbumin in tris buffer does not interact with alpha-crystallin. The expanded, flexible molten globule-like state of reduced apo-alpha-lactalbumin (formed at pH 7.2) also does not interact with alpha-crystallin. Only the aggregation-prone non-native state of reduced apo-alpha-lactalbumin formed at pH 6.0 interacts with alpha-crystallin to form a stable complex. The alpha-crystallin bound reduced apo-alpha-lactalbumin exhibits properties similar to those of a molten globule. Our results show that alpha-crystallin interacts only with the aggregation prone molten globule state of reduced apo-alpha-lactalbumin but not with the other non-aggregating molten globule states of the protein.

Separation and estimation of seven vasodilators using packed column supercritical fluid chromatography.

This paper reports a method for an isocratic separation and simultaneous estimation of seven vasodilators: isosorbide mononitrate (ISMN), isosorbide dinitrate (ISDN), cyclandelate, nimodipine, amlodipine, pentifylline and pentoxifylline using packed column supercritical fluid chromatography (SFC). An arbitrary choice of vasodilatory compounds with respect to their chemical structures was made to examine the viability of this technique for analysis of drugs and pharmaceuticals. Elution was performed on a RP-C18 column. SFC offers several degrees of freedom: temperature, pressure and modifier concentration to attain optimum resolution and sensitivity. The effects of these parameters on retention time have been studied using methanol modified carbon dioxide. The analytes were identified and measured by UV-detection. The chromatographic points of merit have been listed. Detection limits appear to be similar to those found in liquid chromatography. Modifier concentration does generally make major changes in retention and selectivity. A full scale validation for the seven vasodilators has been attempted and the statistical quality evaluated.

Structural perturbation of alpha-crystallin and its chaperone-like activity.

alpha-Crystallin is a multimeric lenticular protein that has recently been shown to be expressed in several non-lenticular tissues as well. It is shown to prevent aggregation of non-native proteins as a molecular chaperone. By using a non-thermal aggregation model, we could show that this process is temperature-dependent. We investigated the chaperone-like activity of alpha-crystallin towards photo-induced aggregation of gamma-crystallin, aggregation of insulin and on the refolding induced aggregation of beta- and gamma-crystallins. We observed that alpha-crystallin could prevent photo-aggregation of gamma-crystallin and this chaperone-like activity of alpha-crystallin is enhanced several fold at temperatures above 30 degrees C. This enhancement parallels the exposure of its hydrophobic surfaces as a function of temperature, probed using hydrophobic fluorescent probes such as pyrene and 8-anilinonaphthalene-1-sulfonate. We, therefore, concluded that alpha-crystallin prevents the aggregation of other proteins by providing appropriately placed hydrophobic surfaces; a structural transition above 30 degrees C involving enhanced or re-organized hydrophobic surfaces of alpha-crystallin is important for its chaperone-like activity. We also addressed the issue of conformational aspects of target proteins and found that their aggregation prone molten globule states bind to alpha-crystallin. We trace these developments and discuss some new lines that suggest the role of tertiary structural aspects in the chaperone process.