Probing the coupling of Ca2+ and rigor activation of rabbit psoas myofibrillar ATPase with ethylene glycol

We have exploited solvent perturbation to probe the coupling of Ca²⁺ and rigor activation of the ATPase of myofibrils from rabbit psoas. Three techniques were used: overall myofibrillar ATPases by the rapid-flow quench method; kinetics of the interaction of ATP with myofibrils by fluorescence stopped-flow; and myofibrillar shortening by optical microscopy. Because of its extensive use with muscle systems, ranging from myosin subfragment-1 to muscle fibres, we chose 40% ethylene glycol as the relaxing agent. At 4°C, the glycol had little effect on the myofibrillar ATPase at low [Ca²⁺], but at high [Ca²⁺] the activity was reduced 50-fold, close to the level found under relaxing conditions, and there was no shortening. However, the ATPase of chemically cross-linked myofibrils (permanently activated even without Ca²⁺) was reduced only 3–4-fold. The lesser reduction of the ATPase of permanently activated myofibrils was also observed in single turnover experiments in which activation occurs by a few heads in the rigor state activating the remaining heads. The addition of ADP, which also promotes strong head-thin filament interactions, also activated the ATPase but only in the presence of Ca²⁺. Further experiments revealed that in 40% ethylene glycol, Ca²⁺ does initiate shortening but only with the aid of strong interactions and at temperatures above 15°C. This confirms that in the organized and intact myofibril, Ca²⁺ and rigor activation are coupled, as proposed previously for regulated actomyosin subfragment-1. This revised version was published online in September 2006 with corrections to the Cover Date.     

Lymphoid cells in afferent and efferent intestinal lymph: lymphocyte subpopulations and cell migration.

Gut wall emigrating cells have been characterized in the intestinal lymph. The intestinal lymph duct was cannulated in 6-month-old minipigs. Under non-restraining conditions the efferent lymph from the mesenteric lymph nodes was collected in seven normal animals. Lymph coming directly from the gut (afferent lymph) was also collected in 18 pigs after resection of the mesenteric lymph node chains 3 months previously. The intestinal lymph flow was similar in both groups (around 18 ml/h). The lymphoid cell yield was 1.2 +/- 1.0 x 10(6)/h in control animals, while in mesenteric lymph node resected pigs it was around 20 times higher (26.2 +/- 17.6 x 10(6)/h). In the gut-derived lymph 76.5 +/- 8.8% T lymphocytes were observed (CD4+, 48.1 +/- 15.5%; CD8+, 53.6 +/- 12.7%). The percentage of immunoglobulin-positive cells was lower (IgM+, 10.1 +/- 4.5; IgA+, 1.7 +/- 1.1). In 14 mesenteric lymph node resected pigs a mean of 5.6 +/- 3.1 x 10(8) lymphocytes from the gut lymph were labelled in vitro with a fluorescent dye and retransfused. The labelling index of fluorescent cells in the intestinal lymph increased rapidly and remained at a high level until 44 h after cell transfusion. A four-to-ten times lower labelling index was found in the spleen, various lymph nodes and Peyer's patches. Most of the recovered lymphocytes were T cells. This model provides access to the cell pool leaving the gut wall, thus allowing an examination of its role in the gastrointestinal tract and other mucosal-lined organs.     

Epitope definition by proteomic similarity analysis: identification of the linear determinant of the anti-Dsg3 MAb 5H10

Cell-based bioassays have been suggested for screening of hormones and drug bioactivities. They are a plausible alternative to animal based methods. The technique used is called receptor/reporter system. Receptor/reporter system was initially developed as a research technique to understand gene function. Often reporter constructs containing viral promoters were used because they could be expressed with very 'high' magnitude in a variety of cell types in the laboratory. On the other hand mammalian genes are expressed in a cell/tissue specific manner, which makes them (i.e. cells/tissues) specialized for specific function in vivo. Therefore, if the receptor/reporter system is to be used as a cell-based screen for testing of hormones and drugs for human therapy then the choice of cell line as well as the promoter in the reporter module is of prime importance so as to get a realistic measure of the bioactivities of 'test' compounds. We evaluated two conventionally used viral promoters and a natural mammalian promoter, regulated by steroid hormone progesterone, in a cell-based receptor/reporter system. The promoters were spliced into vectors expressing enzyme CAT (chloramphenicol acetyl transferase), which served as a reporter of their magnitudes and consistencies in controlling gene expressions. They were introduced into breast cell lines T47D and MCF-7, which served as a cell-based source of progesterone receptors. The yardstick of their reliability was highest magnitude as well as consistency in CAT expression on induction by sequential doses of progesterone. All the promoters responded to induction by progesterone doses ranging from 10^-12 to 10^-6 molar by expressing CAT enzyme, albeit with varying magnitudes and consistencies. The natural mammalian promoter showed the most coherence in magnitude as well as dose dependent expression profile in both the cell lines. Our study casts doubts on use of viral promoters in a cell-based bioassay for measuring bioactivities of drugs and hormones for human therapy and suggests caution regardingtranslation in toto, of a research technique as a cell-based bioassay for drug screening.     

AIDS precautions in practice

Are labs taking appropriate measures to prevent HIV transmission? MLO's national survey indicates that efforts are admirable overall, although follow-up can be inconsistent.     

Juvenile dermatomyositis associated with autoantibodies to small ubiquitin-like modifier activating enzyme: a report of 4 cases from North India and a review of literature

Immunologic Research - Juvenile dermatomyositis (JDM) is the commonest inflammatory myositis in children. The clinical phenotype is often characterized by the presence of myositis-specific...     

Bilateral hamartoma of tonsils bearing ectopic teeth: a case report

Ectopic teeth presenting in bath the tonsils with hatnartomatous lesion has not been reported earlier. We present here a case of benign hamartoma of both the tonsils bearing 7 teeth in a 13 year old girl and also discuss about unusual Ectopic sites of tooth eruption as well as benign lesions of tonsil.     

Clinically lesser known entity in India: A Report of two cases of Melioidosis

Melioidosis is endemic in the South Asian regions, like Thailand, Singapore Malaysia and Australia. The disease is more pronounced in the southern part of the country. It is caused by Burkholderia pseudomallei which causes systemic involvement, morbidity and mortality associated with the disease is high. Due to highly varied clinical presentation, and low general awareness this infection is largely underdiagnosed and under reported in our country. Most laboratories in the country still rely on conventional culturing methods with their low sensitivity, adding to the under reporting. To enhance physician awareness we describe here two cases who presented to our institute after months of misdiagnosis.     

New Characterization Measures of Pore Shape and Connectivity Applied to Coatings used for Controlled Drug Release

Pore geometry characterization-methods are important tools for understanding how pore structure influences properties such as transport through a porous material. Bottlenecks can have a large influence on transport and related properties. However, existing methods only catch certain types of bottleneck effects caused by variations in pore size. We here introduce a new measure, geodesic channel strength, which captures a different type of bottleneck effect caused by many paths coinciding in the same pore. We further develop new variants of pore size measures and propose a new way of visualizing 3-D characterization results using layered images. The new measures together with existing measures were used to characterize and visualize properties of 3-D FIB-SEM images of three leached ethyl-cellulose/hydroxypropyl-cellulose films. All films were shown to be anisotropic, and the strongest anisotropy was found in the film with lowest porosity. This film had very tortuous paths and strong geodesic channel-bottlenecks, while the paths through the other two films were relatively straight with well-connected pore networks. The geodesic channel strength was shown to give important new visual and quantitative insights about connectivity, and the new pore size measures provided useful information about anisotropies and inhomogeneities in the pore structures. The methods have been implemented in the freely available software MIST.