Molecular Characterization and Subtyping of Blastocystis Species in Irritable Bowel Syndrome Patients from North India

Blastocystis species has been extensively studied in recent few years to establish its pathogenecity. Present study was designed to identify and examine the association of Blastocystis sp. and its subtypes with Irritable Bowel Syndrome (IBS).Blastocystis sp. detected using wet-mount microscopy, trichrome staining, in-vitro culture and Polymerase Chain Reaction (PCR) assay in a cohort of IBS patients (n = 150) and healthy controls (n = 100). Isolates of Blastocystis sp.were subtyped using Sequence Tagged Site and representative samples were sequenced at SSUrRNA locus.A total of sixty five isolates of Blastocystis sp. were identified [IBS (n = 50); Controls (n = 15)] of which 91% belonged to ST3 and 9% belonged to ST1. No other subtypes could be identified. Statistically significant association was observed between Blastocystis sp. and IBS patients; however no particular subtype could be ascertained to any particular clinical type of IBS.The frequency of occurrence of Blastocystis sp. was more in IBS patients as compared to the controls and ST3 being the most prevalent subtype. The genetic polymorphism of SSU-rRNA gene amongst the different Blastocystis sp.isolates found in this study reinforces the fact that these organisms are genetically highly divergent.     

Cytodifferentiation of striated duct cells and secretory cells of the convoluted granular tubules of the rat submandibular gland.

The structural and functional development of the striated ducts and convoluted granular tubules (CGT) of the rat submandibular gland (SMG) were studied by electron microscopy and alkaline protease chemistry. Development of the SMG was followed from 14 days of gestation through 30 weeks of age. The specialized morphology of the basal aspect of the striated duct cells arises from cellular extensions which are first seen at 20 days of gestation. These processes elongate and intertwine with similar processes from adjacent cells, and as the cells enlarge the processes are compressed together giving the appearance of "infolding" of the basal plasma membrane. Mitochondria migrate to the basal part of the cell and are seen in close relationship to the cellular extensions throughout the development of these cells. Development of the striated duct is complete by one week after birth. The CGT develop from the proximal portions of intralobular striated ducts. At one week after birth, cells of the proximal striated duct demonstrate apical vacuoles. By two weeks after birth these vacuoles are replaced by distinct zymogen-like granules. There is a progressive accumulation of large numbers of secretory granules in the CGT cells as the animals age. However, rough endoplasmic reticulum is a relatively inconspicuous cellular component throughout development. The accumulation of alkaline protease activity in the gland closely parallels the pattern of granule accumulation.     

Effects of LF-14, THA and physostigmine in rat hippocampus and cerebral cortex

The effects of physostigmine, tetrahydroaminoacridine (THA) and LF-14 [3,3-dimethyl-1(4- amino-3-pyridyl)urea], a 3,4-diaminopyridine derivative, were compared on inhibition of acetyl- cholinesterase (AChE) activity, and release of [³H]acetylcholine (ACh) from rat brain cortical and hippocampal slices. All three compounds caused a concentration dependent inhibition of AChE, with an order of potency physostigmine > THA > LF-14. The electrically stimulated release of ACh from hippocampal and cortical slices was decreased by 10⁻⁵M physostigmine, although the effect was significant only in cortex. THA (5 × 10⁵M) caused a slight, but not significant, decrease in ACh release from both tissues. In contrast, LF-14 (5 × 10⁻⁵ M) caused an approx. 3-fold enhancement of stimulated release. When AChE was inhibited by prior addition of physostigmine, THA caused only a slight enhancement of ACh release, whereas LF-14 greatly increased release. ACh release was also reduced by stimulation of presynaptic muscarinic receptors with oxotremorine. In this case, THA had no effect on ACh release, while LF-14 was able to reverse the inhibition. This study suggests that LF-14 acts to promote ACh release through blocking K⁺ channels, and has a less potent AChE inhibitory effect. It is possible that a compound like LF-14 could be useful in treating diseases of cholinergic dysfunction such as Alzheimer's disease, by both promoting the release of ACh and inhibiting its breakdown.     

On the Use of Infinite Elements for the Determination of Optimal Closure Patterns based on Stress Analysis

Solid infinite elements are used in conjunction with finite elements to compute the stress and displacement distribution resulting from the suturing of wounds of symmetric and nonsymmetric shapes in orthotropic, abdominal human skin. The optimal pattern of suturing of wounds are investigated from a stress perspective. Highly accurate, quantitative and qualitative improvements over the use of finite elements to approximate distant boundaries are obtained. Numerical results quantitatively agree with analytic results computed using complex analysis techniques. The technique used and the results obtained will aid surgeons in closing nonsymmetrical wounds on regions of the body that exhibit orthotropy.     

Casein kinase I in bovine sperm: purification and characterization

A highly purified preparation of sperm casein kinase I was obtained by sequential chromatography with phosphocellulose, gel filtration on sephacryl S-300, Affi-gel blue and DEAE-Cellulose. The chromatographic behavior and properties of the enzyme suggest that the sperm enzyme is similar to casein kinase I from other tissues. Antibodies against calf thymus casein kinase I cross-react with the sperm enzyme. A special feature of the sperm enzyme is that the activity is stimulated by spermine.     

Benefits and limitations of isolated floral patches in a pollinator restoration project in Arizona

This study examined invertebrate floral visitor responses to floral richness, floral abundance, and distance between floral patches within a newly planted pollinator restoration habitat in an arid ecosystem in central Arizona, United States. We created a pollinator habitat experiment consisting of a large central garden (11‐m diameter) surrounded by concentric rings of smaller habitat patches (1‐m diameter), separated from one another by 1, 8, 13, and 21 m, respectively, and including four flowering species. We observed plant and visitor interactions via structured 10‐minute flower visitation observations over a 3‐month period. Key findings included: (1) each plant species interacted with a variety of flower visitors, but flower visitor groups differed only marginally among the plant species; (2) floral patches outside the central garden exhibited reduced quantities of floral structures; and (3) number of floral structures per patch, but not isolation of floral patches within the habitat, affected the number of visitors and visitor taxa richness. For practitioners and land managers looking to restore pollination systems in arid ecosystems with low establishment via seeding, the results of this study suggest that installing species‐rich and florally abundant patches of flowering plant species within a habitat could efficiently support plant‐pollinator interactions.     

Prostate apoptosis response 4 (Par-4), a novel substrate of caspase-3 during apoptosis activation

Prostate apoptosis response 4 (Par-4) is a ubiquitously expressed proapoptotic tumor suppressor protein. Here, we show for the first time, that Par-4 is a novel substrate of caspase-3 during apoptosis. We found that Par-4 is cleaved during cisplatin-induced apoptosis in human normal and cancer cell lines. Par-4 cleavage generates a C-terminal fragment of ~25 kDa, and the cleavage of Par-4 is completely inhibited by a caspase-3 inhibitor, suggesting that caspase-3 is directly involved in the cleavage of Par-4. Caspase-3-deficient MCF-7 cells do not show Par-4 cleavage in response to cisplatin treatment, and restoration of caspase-3 in MCF-7 cells produces a decrease in Par-4 levels, with the appearance of a cleaved fragment. Additionally, knockdown of Par-4 reduces caspase-3 activation and apoptosis induction. Site-directed mutagenesis reveals that Par-4 cleavage by caspase-3 occurs at an unconventional site, EEPD(131)↓G. Interestingly, overexpression of wild-type Par-4 but not the Par-4 D131A mutant sensitizes cells to cisplatin-induced apoptosis. Upon caspase-3 cleavage, the cleaved fragment of Par-4 accumulates in the nucleus and displays increased apoptotic activity. Overexpression of the cleaved fragment of Par-4 inhibits IκBα phosphorylation and blocks NF-κB nuclear translocation. We have identified a novel specific caspase-3 cleavage site in Par-4, and the cleaved fragment of Par-4 retains proapoptotic activity.     

pH-dependent modulation of alkaline phosphatase activity in Serratia marcescens

Serratia marcescens is an opportunistic pathogen responsible for causing nosocomial infections, corneal ulcer, necrotizing fasciitis, cellulites, and brain abscess. Alkaline phosphatase (APase) is believed to play an important role in the survival of several intracellular pathogens and their adaptation. We have studied the effect of low phosphate concentration and acid pH on the APase activities of S. marcescens. In a low phosphate medium, some strains of S. marcescens synthesize two different types of APases, a constitutive (CAPase) and an inducible (IAPase). Both the CAPase and IAPase isoenzymes completely lost their enzyme activities at pH 2.3, within 10 min of incubation at 0°C. Acid-treated IAPase isoenzymes I, II, III, and IV solutions when adjusted to pH 7.8 showed recovery of 70%, 52%, 72%, and 60% of the lost activities, respectively. When the pH of the CAPase reaction mixture was raised to pH 7.8, the enzyme activity regained only 5% of its initial activity. Variations in protein concentration also affected the pH-dependent reversible changes of the IAPase activity. The higher the protein concentration, the faster the inactivation of enzyme activity observed at acidic pH at 0°C. Conversely, the lower the protein concentration, the higher the rate of reactivation of enzyme activity observed for IAPase at alkaline pH. Protein interaction studies revealed a lack of similarity between CAPase and IAPase, suggesting separate genetic origin of these potentially virulent genes of S. marcescens. Received: 4 December 2001 / Accepted: 7 January 2002