Purification and properties of molecular-weight variants of human placental alkaline phosphatase

1. Alkaline phosphatase of human placenta was purified by a procedure involving homogenization with tris buffer, pH8.6, extraction with butanol, ammonium sulphate fractionation, exposure to heat, ethanol fractionation, gel filtration, triethylaminoethylcellulose anion-exchange chromatography, continuous curtain electrophoresis on paper and equilibrium dialysis. Methods for both laboratory-scale and large-scale preparation were devised. 2. Two major molecular-weight variants designated A and B were separated by molecular sieving with Sephadex G-200 and variant A was purified 4000-fold. 3. Variant B, which comes off the Sephadex G-200 column before variant A, is the electrophoretically slower-moving species on starch gel and is quite heterogeneous. 4. Purified variant A was fairly homogeneous on the basis of electrophoretic studies on starch gel and Sephadex gel, ultracentrifugation and immunodiffusion. 5. The respective molecular weights for variants A and B were 70000 and over 200000 on the basis of sucrose-density-gradient ultracentrifugation. Variant A exhibited a sedimentation coefficient of 4.2s. 6. Crystalline variant B could be converted into fast-moving variant A and vice versa. 7. Kinetic studies indicated no difference between the two variants. These include linear rates of hydrolysis, pH optimum, Michaelis constants and uncompetitive stereospecific l-phenylalanine inhibition. 8. The amino acid compositions of variants A and B and of placental albumin were determined.     

Epistatic role of base excision repair and mismatch repair pathways in mediating cisplatin cytotoxicity

Base excision repair (BER) and mismatch repair (MMR) pathways play an important role in modulating cis-Diamminedichloroplatinum (II) (cisplatin) cytotoxicity. In this article, we identified a novel mechanistic role of both BER and MMR pathways in mediating cellular responses to cisplatin treatment. Cells defective in BER or MMR display a cisplatin-resistant phenotype. Targeting both BER and MMR pathways resulted in no additional resistance to cisplatin, suggesting that BER and MMR play epistatic roles in mediating cisplatin cytotoxicity. Using a DNA Polymerase β (Polβ) variant deficient in polymerase activity (D256A), we demonstrate that MMR acts downstream of BER and is dependent on the polymerase activity of Polβ in mediating cisplatin cytotoxicity. MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin. Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing. In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.     

Biophysical factors in the regulation of asymmetric division of stem cells

Stem cells are a promising cell source for regenerative medicine due to their characteristics of self‐renewal and differentiation. The intricate balance between these two cell fates is maintained by precisely controlled symmetric and asymmetric cell divisions. Asymmetric division has a fundamental importance in maintaining tissue homeostasis and in the development of multi‐cellular organisms. For example, during development, asymmetric cell divisions are responsible for the formation of the body axis. Mechanistically, mitotic spindle dynamics determine the assembly and separation of chromosomes and regulate the orientation of cell division. Interestingly, symmetric and asymmetric cell division is not mutually exclusive and a range of factors are involved in such cell‐fate decisions, the measurement of which can provide efficient and reliable information on the regenerative potential of a cell. The balance between self‐renewal and differentiation in stem cells is controlled by various biophysical and biochemical cues. Although the role of biochemical factors in asymmetric stem cell division has been widely studied, the effect of biophysical cues in stem‐cell self‐renewal is not comprehensively understood. Herein, we review the biological relevance of stem‐cell asymmetric division to regenerative medicine and discuss the influences of various intrinsic and extrinsic biophysical cues in stem‐cell self‐renewal. This review particularly aims to inform the clinical translation of efforts to control the self‐renewal ability of stem cells through the tuning of various biophysical cues.     

Characterization of monosaccharides in human placental alkaline phosphatase by gas—liquid chromatography

Gas—liquid chromatography of hydrolysates of highly purified human placental alkaline phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1) demonstrated the presence of monosaccharide residues, mannose, galactose, glucose and fucose. This enzyme, therefore, is a sialoglycoprotein.     

Aeromycoflora in the Central Milk Dairy of Calcutta, India

Intramural aeromycological survey was performed at the Central Milk Dairy, Calcutta, covering eight locations within the Dairyusing Burkard personal volumetric air sampler. The locations were butter cold storage (−2 °C), cold store (8 °C), packaging section (23 °C), milk processing section (24 °C), reconstituent of skimmed milk (24 °C), quality control lab (25 °C), raw milk reception (28 °C) and loading dock (26 °C). A number of fungal spores, conidia and mycelia were recorded in different rooms: the highest spore quantity was recorded in the packaging section (23 °C) and the minimum at the butter cold store (−2 °C). The dominant spores consisted of Aspergillus niger, A flavus,Cladosporium sp., Fusarium sp., Curvularia sp.,Alternaria sp., Torula sp., Myrotheciumsp., Helminthosporium sp., Periconia sp.,Nigrospora sp. and Pithomyces sp. This revised version was published online in June 2006 with corrections to the Cover Date.     

Hyperglycaemia Enhances Nitric Oxide Production in Diabetes: A Study from South Indian Patients

Background

We have previously reported that increased glucose levels were associated with higher serum nitric oxide (NO) levels in fructose-fed insulin resistant rats. However, the relationship between hyperglycemia and serum NO level was not clear. Therefore, the present study was designed to find the association between hyperglycemia and serum NO levels in Type 2 diabetic (T2DM) patients and T2DM with cardiovascular complication.

Methods

Endothelial cells (HUVEC) were treated with of D-glucose (10-100mM), and NO levels and NOS gene expression was measured. Hyperglycaemia was induced in Sprague-Dawley rats, and serum NO levels were measured after 8 weeks. For clinical evaluation, five groups of patients were recruited: Control (CT, n=48), Type 2 diabetes (T2DM, n=26), T2DM with hypertension (DMHT, n=46), Coronary artery diseases (CAD, n=29) and T2DM with coronary artery diseases (DMCD, n=38). NO (nitrite + nitrate) levels were measured from human serum.

Results

We found a significant (p<0.05) and dose-dependent increase in NO levels in HUVEC cells after 4 hours of high glucose exposure. eNOS and iNOS gene expression was increased in HUVEC cells after different concentrations and time periods of glucose treatment. We also observed significant (149.1±25μM, p<0.01) increase in serum NO levels in hyperglycaemic rats compared to control (76.6±13.2μM). Serum NO level was significantly higher in T2DM (111.8 μM (81.7-122.4), p<0.001) and DMCD patients ((129.4 μM (121.2-143.5), p <0.001) but not in CAD patients (76.4 μM (70.5-87)), as compared to control (68.2 μM (56.4-82.3)). We found significantly lower NO levels (83.5 μM (60.5-122.9)) in subjects suffering from diabetes since more than 5 years, compared to subjects (115.3 μM (75.2-127.1), p<0.001) with less than 5 years.

Conclusion

In conclusion, high NO levels were observed in South Indian diabetic patients. Higher glucose levels in serum might be responsible for activation of endothelial cells to enhance NO levels.     

Evaluation of Acute Toxicity Levels and Ethological Responses Under Tetrachlorocatechol Exposure in Common Carp,Cyprinus carpio (Linnaeus)

Tetrachlorocatechol (TCC) is one of the most toxic chlorinated catechol produced by the chlorobleaching of pulp and frequently found in the kraft pulp mill effluents. Toxicity of TCC to common carp, Cyprinus carpio and their ethological changes were evaluated in the present study. The 24, 48, 72 and 96 h LC₅₀ values of TCC to C. carpio were 4.73, 3.95, 3.20 and 2.43 mg/l respectively. The mortality rate of C. carpio showed significant relationship (p < 0.05) at different exposure times (24, 48, 72 and 96 h) in different treatments of TCC (2, 3, 4, 5 and 7 mg/l). On the other hand, a significant correlation (p < 0.01) was observed between the mortality rate of fish with all the concentrations of TCC. Mean opercular movement in the fish increased significantly with the increasing dose and time of exposure to TCC indicating acute respiratory distress. The jerky movement, somersaulting, circular movement and mucus secretion of fish increased gradually with the increasing concentrations and exposure times of TCC for adapting a compensatory mechanism to derive energy in order to avoid stress due to toxicity.     

ApoTome to visualize E-cadherin and p63 expression in oral pre-cancer

Precise histopathological localization of E-cadherin and p63 is of immense importance in understanding the integrity of oral mucosal stratified epithelium in normal and diseased conditions. Necessarily immunohistochemical imaging should have minimum bleaching impact on the dyes and ability to produce clear and crisp images. Here ApoTome provides an alternative with metal halide light source and structured illumination under the assistance of grids, along with integrated image processing modality to generate crisp images with digital interface. The current study demonstrates the applicability of such microscopic system in capturing fluorescence images of immunohistochemical sections of normal and precancerous biopsies in respect to the expression of p63 and E-cadherin in the epithelial cells. The ApoTome images localize the nuclear and membranous expressions of p63 and E-cadherin, respectively, with remarkable specificity. The findings on E-cadherin expression have enormous diagnostic significance as these images clearly differentiate the early and advanced stages of oral submucous fibrosis based on their cytoplasmic and membranous location. Thus, this study clearly depicts a remarkable performance of ApoTome with diagnostic significance.     

Essential steps in bioprinting: From pre- to post-bioprinting

An increasing demand for directed assembly of biomaterials has inspired the development of bioprinting, which facilitates the assembling of both cellular and acellular inks into well-arranged three-dimensional (3D) structures for tissue fabrication. Although great advances have been achieved in the recent decade, there still exist issues to be addressed. Herein, a review has been systematically performed to discuss the considerations in the entire procedure of bioprinting. Though bioprinting is advancing at a rapid pace, it is seen that the whole process of obtaining tissue constructs from this technique involves multiple-stages, cutting across various technology domains. These stages can be divided into three broad categories: pre-bioprinting, bioprinting and post-bioprinting. Each stage can influence others and has a bearing on the performance of fabricated constructs. For example, in pre-bioprinting, tissue biopsy and cell expansion techniques are essential to ensure a large number of cells are available for mass organ production. Similarly, medical imaging is needed to provide high resolution designs, which can be faithfully bioprinted. In the bioprinting stage, compatibility of biomaterials is needed to be matched with solidification kinetics to ensure constructs with high cell viability and fidelity are obtained. On the other hand, there is a need to develop bioprinters, which have high degrees of freedom of movement, perform without failure concerns for several hours and are compact, and affordable. Finally, maturation of bioprinted cells are governed by conditions provided during the post-bioprinting process. This review, for the first time, puts all the bioprinting stages in perspective of the whole process of bioprinting, and analyzes their current state-of-the art. It is concluded that bioprinting community will recognize the relative importance and optimize the parameter of each stage to obtain the desired outcomes.