The characterisation and function of the polysaccharidases of human synovial fluid in rheumatoid and osteoarthritis.

A potential enzymic mechanism for the degradation of glycosaminogly cans was characterised using enzymes found in rheumatoid synovial fluid from the knee joint. This mechanism involves a true hyluronidase together with the concerted action of beta-glucuronidase and beta-N-acetylhexosaminidase. The contribution of the exopolysaccharidases to hyaluronate degradation was demonstrated by the use of specific inhibitors, while the distinct identity of a true hyaluronidase was shown by ammonium sulphate and agarose gel column fractionations. Only the hyluronidase fraction was capable of degrading high molecular weight hyaluronate. The exopolysaccharidase activities were shown to be markedly elevated in rheumatoid as compared to osteoarthritic synovial fluid and also normal serum. On the other hand, hyluronidase was similarly active in rheumatoid and osteoarthritic synovial fluids; both these levels were lower than that of normal human serum. Hyaluronidase in synovial fluid may thus be derived by diffusion from serum, since it is of relatively low molecular weight (60 000). The pH requirements of this enzyme system and the strong inhibition of hyaluronidase by synovial fluid make it unlikely that the mechanism operates extracellularly. It is proposed that as a lysosomal mechanism, however, it is an important contributing factor in the chronic erosion process characteristic of rheumatoid arthritis.     

Studies on the binding of 1-anilino-8-naphthalene sulfonate to very low density and high density himan serum lipoproteins.

Very low density and high density lipoproteins have been isolated from human plasma and their interaction with 1-anilin0-8-naphthalene sulfonate has been studied under different conditions of pH and added salt. Intrinsic fluorescence of bound 1-anilino-8-naphthalene sulfonate was higher for high density lipoproteins then for very low density lipoproteins, but was unaffected by salt in both systems. Binding of 1-anilino-8-naphthalene sulfonate by both these lipoproteins was saturable and was higher in the presence of added NaCl or CaCl2, Ca2+ having a greater effect than Na+ in enhancing fluorescence. The binding data were analyzed by Scatchard plots; the number of binding sites and the affinity of 1-anilino-8-naphthalene sulfonate for the site increased with increasing salt concentration. Fluorescence pH curves were similar to those published for phospholipids. From these and previous observations it is suggested that the phospholipids probably represent the major binding sites for 1-anilino-8-naphthalene sulfonate.     

Effect of 3-(3,4-dihydro-6-methoxy-2-naphthyl)2,2-dimethyl pentanoic acid--an antifertility agent--on in vitro uptake of estradiol 17beta-6, 7(3)-H in rat uterus.

The effect of 3-(3,4-dihydro-6-methoxy-2-naphthyl)-2,2-dimethyl-pentanoic acid, a potent nonsteroidal antifertility compound, on the uptake of estradiol-17beta-6, 7-tritiated in vitro in the rat uterus was studied. The estradiol uptake of estrogen-primed and compound-treated groups were the same. When estradiol and the compound were present in medium at the same time, estradiol uptake was significantly (p less than .01) increased. The results indicate that the compound synergizes the effect of estradiol.     

A Highlights from MBoC Selection: Fast and parallel nanoscale three-dimensional tracking of heterogeneous mammalian chromatin dynamics

Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel three-dimensional (3D) tracking of multiple chromosomal loci of choice over many thousands of frames on various timescales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking both of fast dynamics (50 Hz) and over timescales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent α and a transition to more-isotropic dynamics in 3D after such treatment. We think that in the future our methodology will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function.     

Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly

We recently reported that serine–arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65 and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.     

Production of monoclonal antibodies to estriol and their application in the development of a sensitive nonisotopic immunoassay

The development of highly specific monoclonal antibodies to estriol and a nonisotopic immunoassay (EIA) for unconjugated estriol based on the use of these monoclonal antibodies have been described. The monoclonal antibodies show little cross reactivity with other steroids and steroid conjugates and can be used directly in immunoassays without any purification. The EIA described here can be performed in 96-well microtiter plates or polystyrene tubes that have been coated with estriol-bovine serum albumin conjugate. In this assay, estriol in the standard or clinical samples (serum or saliva) competes with the immobilized steroid on the plate or the tube for binding with the antibody. The assay shows good agreement with radioimmunoassay (RIA) and is highly sensitive and reliable. Since no prior processing or extraction of the clinical samples is necessary, the method is potentially applicable for routine use in fetal monitoring as well as in a steroid laboratory.     

Molecular detection of Candidatus Phytoplasma spp. causing witches’ broom disease of acid lime (Citrus aurantifolia) in India

Phytoplasma infected acid lime plants in India develop characteristic symptoms like small chlorotic leaves, multiple sprouting and shortened internodes. Leaves drop prematurely and infected branches have distorted twigs resembling witches’ broom appearance which eventually show die-back symptoms. During its first report in 1999, witches’ broom disease identification was made on the basis of symptomatology and electron microscopy. However, molecular techniques have proved to be more accurate and reliable for phytoplasma detection than the conventional methods. During survey in the year 2010 six samples were collected from infected acid lime plants showing typical field symptoms from Vidarbha region of Maharastra. Initially, phytoplasma bodies were observed in phloem tissues of all six symptomatic samples under JEM 100S transmission electron microscope and all these six samples were subsequently screened using different set of phytoplasma specific universal primers by nested PCR, a widely recommended molecular technique for phytoplasma detection. In the present study P1/P7 “universal” phytoplasma-primer set was used for first round of PCR and amplified products were processed separately for nested PCR with three different nested primer pairs viz. R16F2n/R16R2, R16mF2/R16mR1 and fU5/rU3. The presence of phytoplasma was confirmed in all six suspected samples and one representative ~1.2 kb size amplicon was sequenced and deposited in GenBank as Candidatus Phytoplasma species AL-M (JQ808143). This is the first report of PCR based molecular detection of phytoplasma-induced witches’ broom disease of acid lime (WBDL) in India. Further molecular evaluation to determine the identity to the species level is in progress.     

Influence of Critical Parameters of Nanosuspension Formulation on the Permeability of a Poorly Soluble Drug through the Skin—A Case Study

In transdermal drug delivery systems, it is always a challenge to achieve stable and prolonged high permeation rates across the skin since the concentrations of the drug dissolved in the matrix have to be high in order to maintain zero order release kinetics. Several attempts have been reported to improve the permeability of poorly soluble drug compounds using supersaturated systems. However, due to thermodynamic challenges, there was a high tendency for the drug to nucleate immediately after formulating or even during storage. The present study focuses on the efficiency of nanoparticles and influence of different concentrations of solubilizer such as vitamin E TPGS (d-a-tocopheryl polyethylene glycol 1000 succinate) to improve the permeation rate through the skin. Effects of several formulation factors were studied on the nanosuspension systems using ibuprofen as a model drug. The overall permeation enhancement process through the skin was influenced mostly by the solubilizer and also by the size of nanoparticles. The gel formulation developed with vitamin E TPGS + HPMC nanosuspension, consequently represent a promising approach aiming to improve the permeability performance of a poorly water soluble drug candidate.KEY WORDS: dermal drug delivery, human skin, nanosuspension, permeation rate, porcine skin, vitamin E TPGS     

Joint Modeling for Cognitive Trajectory and Risk of Dementia in the Presence of Death

Summary .  Dementia is characterized by accelerated cognitive decline before and after diagnosis as compared to normal aging. It has been known that cognitive impairment occurs long before the diagnosis of dementia. For individuals who develop dementia, it is important to determine the time when the rate of cognitive decline begins to accelerate and the subsequent gap time to dementia diagnosis. For normal aging individuals, it is also useful to understand the trajectory of cognitive function until their death. A Bayesian change-point model is proposed to fit the trajectory of cognitive function for individuals who develop dementia. In real life, people in older ages are subject to two competing risks, e.g., dementia and dementia-free death. Because the majority of people do not develop dementia, a mixture model is used for survival data with competing risks, which consists of dementia onset time after the change point of cognitive function decline for demented individuals and death time for nondemented individuals. The cognitive trajectories and the survival process are modeled jointly and the parameters are estimated using the Markov chain Monte Carlo method. Using data from the Honolulu Asia Aging Study, we show the trajectories of cognitive function and the effect of education, apolipoprotein E 4 genotype, and hypertension on cognitive decline and the risk of dementia.