Structural characterization and study of immunoenhancing and antioxidant property of a novel polysaccharide isolated from the aqueous extract of a somatic hybrid mushroom of Pleurotus florida and Calocybe indica variety APK2

A water-soluble polysaccharide was isolated from the aqueous extract of the fruit bodies of somatic hybrid PCH9FB, obtained through intergeneric protoplast fusion between the strains Pleurotus florida and Calocybe indica var. On the basis of total acid hydrolysis, the polysaccharide was found to contain galactose, fucose, and glucose in a molar ratio of nearly 2:1:2. Methylation analysis and NMR experiments ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC) showed that the structure of the repeating unit present in the polysaccharide was This molecule showed macrophage, splenocyte, thymocyte activation as well as antioxidant property.     

Changes in biochemical composition of follicular fluid during reproductive acyclicity in water buffalo (Bubalus bubalis)

This study describes the changes in biochemical composition of follicular fluid during reproductive acyclicity in buffalo. A total of 73 pairs of ovaries collected from 26 reproductively acyclic and 47 reproductively cyclic buffaloes were used in the investigation. Ovarian follicles were classified into small (5.0-6.9 mm), medium (7.0-9.9 mm) and large (≥10.0 mm) sized categories depending upon their diameter. Follicular fluid was aspirated, processed and assayed for glucose, cholesterol, total protein, acid phosphatase and alkaline phosphatase. Glucose concentration was lesser in reproductively acyclic compared to cyclic buffaloes (19.3 ± 2.59 mg/dl compared to 32.6 ± 2.60 mg/dl; P<0.05), mainly due to difference in concentration between small sized follicles (12.4 ± 2.59 mg/dl compared to 28.0 ± 3.32 mg/dl; P<0.05). Cholesterol concentration was also lesser in reproductively acyclic compared to cyclic buffaloes (32.2 ± 2.14 mg/dl compared to 35.5 ± 2.16 mg/dl; P<0.05) and this was related to the lesser concentration found in large follicles (13.8 ± 3.45 mg/dl compared to 37.2 ± 4.10mg/dl; P<0.001). Total protein and acid phosphatase levels were not affected by either the reproductive cyclicity status or the follicular size (4.9 ± 1.07 g/dl to 6.0 ± 0.28 g/dl and 1.2 ± 0.17 U/dl to 2.5 ± 1.22 U/dl, respectively). An increased alkaline phosphatase activity was, however, observed in reproductively acyclic compared to cyclic buffaloes (27.5 ± 3.08 U/dl compared to 14.0 ± 1.09 U/dl; P<0.0001). In conclusion, results of the present study indicate an alteration in the biochemical composition of follicular fluid during reproductive acyclicity in buffalo. The findings provide further support to the notion that poor nutrition is an important factor triggering reproductive acyclicity in buffalo.     

Abnormality of circadian rhythm of serum melatonin and other biochemical parameters in fibromyalgia syndrome

Fibromyalgia syndrome (FMS) is a complex chronic condition causing widespread pain and variety of other symptoms. It produces pain in the soft tissues located around joints throughout the body. FMS has unknown etiology and its pathophysiology is not fully understood. However, abnormality in circadian rhythm of hormonal profiles and cytokines has been observed in this disorder. Moreover, there are reports of deficiency of serotonin, melatonin, cortisol and cytokines in FMS patients, which are fully regulated by circadian rhythm. Melatonin, the primary hormone of the pineal gland regulates the body's circadian rhythm and normally its levels begin to rise in the mid-to-late evening, remain high for most of the night, and then decrease in the early morning. FMS patients have lower melatonin secretion during the hours of darkness than the healthy subjects. This may contribute to impaired sleep at night, fatigue during the day and changed pain perception. Studies have shown blunting of normal diurnal cortisol rhythm, with elevated evening serum cortisol level in patients with FMS. Thus, due to perturbed level of cortisol secretion several symptoms of FMS may occur. Moreover, disturbed cytokine levels have also been reported in FMS patients. Therefore, circadian rhythm can be an important factor in the pathophysiology, diagnosis and treatment of FMS. This article explores the circadian pattern of abnormalities in FMS patients, as this may help in better understanding the role of variation in symptoms of FMS and its possible relationship with circadian variations of melatonin, cortisol, cytokines and serotonin levels.     

Low dose Estrogen Prevents Neuronal Degeneration and Microglial Reactivity in an Acute Model of Spinal Cord Injury: Effect of Dosing,Route of Administration,and Therapy Delay

Spinal cord injury (SCI), depending on the severity of injury, leads to neurological dysfunction and paralysis. Methylprednisolone, the only currently available therapy renders limited protection in SCI. Therefore, other therapeutic agents must be tested to maximize neuroprotection and functional recovery. Previous data from our laboratory indicate that estrogen (17β-estradiol) at a high dose may attenuate multiple damaging pathways involved in SCI and improve locomotor outcome. Since use of high dose estrogen may have detrimental side effects and therefore may never be used in the clinic, the current study investigated the efficacy of this steroid hormone at very low doses in SCI. In particular, we tested the impact of dosing (1–10 μg/kg), mode of delivery (intravenous vs. osmotic pump), and delay in estrogen application (15 min–4 h post-SCI) on microgliosis and neuronal death in acute SCI in rats. Treatment with 17β-estradiol (1–10 μg/kg) significantly reduced microglial activation and also attenuated apoptosis of neurons compared to untreated SCI animals. The attenuation of cell death and inflammation by estrogen was observed regardless of mode and time of delivery following injury. These findings suggest estrogen as a potential agent for the treatment of individuals with SCI.     

A cytotoxic type-2 ribosome inactivating protein (from leafless mistletoe) lacking sugar binding activity

Articulatin-D, a 66 kDa ribosome inactivating protein (RIP) comprised of 29 kDa A-chain linked to 35 kDa B-chain, is purified from leafless mistletoe (Viscum articulatum) parasitic on Dalbergia sp. from Western Ghats (India). N-terminal sequence and LC-MS/MS analyses of A- and B-chain confirmed that articulatin-D is a type-2 RIP having high homology with other mistletoe lectins. Translation inhibition and diagnostic N-glycosidase activity of articulatin-D illustrate the presence of catalytically active A-chain. Its inability to: (i) bind to acid treated Sepharose CL-6B column, (ii) agglutinate trypsin-treated and untreated RBCs of human (A, B, O, AB), mice, rat, rabbit, buffalo, porcine, pigeon, cock, fish, sheep and goat even with 10 mg/ml of purified articulatin-D, (iii) show change in circular dichroism spectra after addition of sugar to the native protein, (iv) bind to different sugars (galactose, lactose, gal-NAc, rhamnose, arabinose, fucose and mannose) immobilized on Sepharose 4B matrix, and (v) show change in enthalpy during titration with galactose confirm that the B-chain of articulatin-D lacks sugar binding activity. Despite this, articulatin-D is highly toxic as characterized with low IC₅₀ against different cancer cell lines (Jurkat: 0.31 ± 0.02 nM, MOLT-4: 0.51 ± 0.03 nM, U-937: 0.64 ± 0.07 nM, HL-60: 0.79 ± 0.11 nM, Raji: 1.45 ± 0.09 nM). Toxicity of RIPs has been ascribed to the absence/presence of B-chain with sugar binding activity. Identification of articulatin-D, the first cytotoxic RIP with B-chain lacking sugar binding activity opens new vistas in understanding cytotoxic action of RIPs.     

Simulation of LV pacemaker lead in marginal vein: potential risk factors for acute dislodgement

Although left ventricular (LV) coronary sinus lead dislodgement remains a problem, the risk factors for dislodgement have not been clearly defined. In order to identify potential risk factors for acute lead dislodgement, we conducted dynamic finite element simulations of pacemaker lead dislodgement in marginal LV vein. We considered factors such as mismatch in lead and vein diameters, velocity of myocardial motion, branch angle between the insertion vein and the coronary sinus, degree of slack, and depth of insertion. The results show that large lead-to-vein diameter mismatch, rapid myocardial motion, and superficial insertion are potential risk factors for lead dislodgement. In addition, the degree of slack presents either a positive or negative effect on dislodgement risk depending on the branch angle. The prevention of acute lead dislodgment can be enforced by inducing as much static friction force as possible at the lead-vein interface, while reducing the external force. If the latter exceeds the former, dislodgement will occur. The present findings underscore the major risk factors for lead dislodgment, which may improve implantation criterion and future lead design.     

Viral infection and neural stem/progenitor cell's fate: implications in brain development and neurological disorders

Viral infections in the prenatal (during pregnancy) and perinatal period have been a common cause of brain malformation. Besides the immediate neurological dysfunctions, virus infections may critically affect CNS development culminating in long-term cognitive deficits. Most of these neurotropic viruses are most damaging at a critical stage of the host, when the brain is in a dynamic stage of development. The neuropathology can be attributed to the massive neuronal loss induced by the virus as well as lack of CNS repair owing to a deficit in the neural stem/progenitor cell (NSPC) pool or aberrant formation of new neurons from NSPCs. Being one of the mitotically active populations in the post natal brain, the NSPCs have emerged as the potential targets of neurotropic viruses. The NSPCs are self-renewing and multipotent cells residing in the neurogenic niches of the brain, and, therefore, hampering the developmental fate of these cells may adversely affect the overall neurogenesis pattern. A number of neurotropic viruses utilize NSPCs as their cellular reservoirs and often establish latent and persistent infection in them. Both HIV and Herpes virus infect NSPCs over long periods of time and reactivation of the virus may occur later in life. The virus infected NSPCs either undergoes cell cycle arrest or impaired neuronal or glial differentiation, all of which leads to impaired neurogenesis. The disturbances in neurogenesis and CNS development following neurotropic virus infections have direct implications in the viral pathogenesis and long-term neurobehavioral outcome in infected individuals.     

Requirement of rRNA methylation for 80S ribosome assembly on a cohort of cellular internal ribosome entry sites

Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation.     

Membrane topology of NAADP-sensitive two-pore channels and their regulation by N-linked glycosylation

Two-pore channels (TPCs) localize to the endolysosomal system and have recently emerged as targets for the Ca(2+)-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, their membrane topology is unknown. Using fluorescence protease protection assays, we show that human TPC1 and TPC2 possess cytosolic N and C termini and therefore an even number of transmembrane regions. Fluorophores placed at position 225 or 347 in TPC1, or 339 in TPC2 were also cytosolic, whereas a fluorophore at position 628 in TPC1 was luminal. These data together with sequence similarity to voltage-gated Ca(2+) and Na(+) channels, and unbiased in silico predictions are consistent with a topology in which two homologous domains are present, each comprising 6 transmembrane regions and a re-entrant pore loop. Immunocytochemical analysis of selectively permeabilized cells using antipeptide antibodies confirmed that the C-terminal tails of recombinant TPCs are cytosolic and that residues 240-254 of TPC2 prior to putative pore 1 are luminal. Both TPC1 and TPC2 are N-glycosylated with residues 599, 611, and 616 contributing to glycosylation of TPC1. This confirms the luminal position of these residues, which immediately precede the putative pore loop of the second domain. Mutation of all three glycosylation sites in TPC1 enhances NAADP-evoked cytosolic Ca(2+) signals. Our data establish essential features of the topology of two-pore channels.