Conjugation of α-amylase with dextran for enhanced stability: Process details, kinetics and structural analysis

The influence of enzyme polysaccharide interaction on enzyme stability and activity was elucidated by covalently binding dextran to a model enzyme, α-amylase. The conjugation process was optimized with respect to concentration of oxidizing agent, pH of enzyme solution, ratio of dextran to enzyme concentration, temperature and time of conjugate formation, and was found to affect the stability of α-amylase. α-Amylase conjugated under optimized conditions showed 5% loss of activity but with enhanced thermal and pH stability. Lower inactivation rate constant of conjugated α-amylase within the temperature range of 60-80°C implied its better stability. Activation energy for denaturation of α-amylase increased by 8.81kJ/mol on conjugation with dextran. Analysis of secondary structure of α-amylase after covalent binding with dextran showed helix to turn conversion without loss of functional properties of α-amylase. Covalent bonding was found to be mandatory for the formation of conjugate.     

Collection devices for cervicovaginal cytology: a comparison

OBJECTIVE: To compare cervicovaginal smears obtained by a cotton-tipped swab with those obtained by cervix brush and modified Ayre spatula. STUDY DESIGN: A combined cervicovaginal smear was collected from 100 women using 3 different collection devices: cotton-tipped swab, cervix brush, and modified Ayre spatula. In each patient a set of 3 smears was collected by the same cytotechnologist using all 3 devices in random order. Smears were evaluated using parameters mandatory for an optimal smear: evenly dispersed, well-preserved, adequate cells from the transformation zone. The cost and availability of the collection devices were also considered. RESULTS: The swab was the most effective device in obtaining thin, evenly spread, adequate, well-preserved smears as against the cervix brush and Ayre spatula. The pickup of abnormal cells was similar with the cotton-tipped swab and cervix brush, while the Ayre spatula failed to yield high grade squamous intraepithelial lesions, atypical glandular cells of undetermined significance and adenocarcinoma cells. The cotton-tipped swab proved to be the most cost effective. CONCLUSION: A properly prepared cotton-tipped swab is an inexpensive, readily available, nontraumatic collection device that yields smear of optimal quality.     

Activation of Autophagic Flux against Xenoestrogen Bisphenol-A-induced Hippocampal Neurodegeneration via AMP kinase (AMPK)/Mammalian Target of Rapamycin (mTOR) Pathways

The human health hazards related to persisting use of bisphenol-A (BPA) are well documented. BPA-induced neurotoxicity occurs with the generation of oxidative stress, neurodegeneration, and cognitive dysfunctions. However, the cellular and molecular mechanism(s) of the effects of BPA on autophagy and association with oxidative stress and apoptosis are still elusive. We observed that BPA exposure during the early postnatal period enhanced the expression and the levels of autophagy genes/proteins. BPA treatment in the presence of bafilomycin A₁ increased the levels of LC3-II and SQSTM1 and also potentiated GFP-LC3 puncta index in GFP-LC3-transfected hippocampal neural stem cell-derived neurons. BPA-induced generation of reactive oxygen species and apoptosis were mitigated by a pharmacological activator of autophagy (rapamycin). Pharmacological (wortmannin and bafilomycin A₁) and genetic (beclin siRNA) inhibition of autophagy aggravated BPA neurotoxicity. Activation of autophagy against BPA resulted in intracellular energy sensor AMP kinase (AMPK) activation, increased phosphorylation of raptor and acetyl-CoA carboxylase, and decreased phosphorylation of ULK1 (Ser-757), and silencing of AMPK exacerbated BPA neurotoxicity. Conversely, BPA exposure down-regulated the mammalian target of rapamycin (mTOR) pathway by phosphorylation of raptor as a transient cell''s compensatory mechanism to preserve cellular energy pool. Moreover, silencing of mTOR enhanced autophagy, which further alleviated BPA-induced reactive oxygen species generation and apoptosis. BPA-mediated neurotoxicity also resulted in mitochondrial loss, bioenergetic deficits, and increased PARKIN mitochondrial translocation, suggesting enhanced mitophagy. These results suggest implication of autophagy against BPA-mediated neurodegeneration through involvement of AMPK and mTOR pathways. Hence, autophagy, which arbitrates cell survival and demise during stress conditions, requires further assessment to be established as a biomarker of xenoestrogen exposure.     

The N-acylethanolamine-mediated regulatory pathway in plants

While cannabinoids are secondary metabolites synthesized by just a few plant species, N-acylethanolamines (NAEs) are distributed widely in the plant kingdom, and are recovered in measurable, bioactive quantities in many plant-derived products. NAEs in higher plants are ethanolamides of fatty acids with acyl-chain lenghts of C12-C(18) and zero to three C=C bonds. Generally, the most-abundant NAEs found in plants and vertebrates are similar, including NAE 16 : 0, 18 : 1, 18 : 2, and 18 : 3. Like in animal systems, NAEs are formed in plants from N-acylphosphatidylethanolamines (NAPEs), and they are hydrolyzed by an amidase to yield ethanolamine and free fatty acids (FFA). Recently, a homologue of the mammalian fatty acid amide hydrolase (FAAH-1) was identified in Arabidopsis thaliana and several other plant species. Overexpression of Arabidopsis FAAH (AtFAAH) resulted in plants that grew faster, but were more sensitive to biotic and abiotic insults, suggesting that the metabolism of NAEs in plants resides at the balance between growth and responses to environmental stresses. Similar to animal systems, exogenously applied NAEs have potent and varied effects on plant cells. Recent pharmacological approaches combined with molecular-genetic experiments revealed that NAEs may act in certain plant tissues via specific membrane-associated proteins or by interacting with phospholipase D-alpha, although other, direct targets for NAE action in plants are likely to be discovered. Polyunsaturated NAEs can be oxidized via the lipoxygenase pathway in plants, producing an array of oxylipin products that have received little attention so far. Overall, the conservation of NAE occurrence and metabolic machinery in plants, coupled with the profound physiological effects of elevating NAE content or perturbing endogenous NAE metabolism, suggest that an NAE-mediated regulatory pathway, sharing similarities with the mammalian endocannabinoid pathway, indeed exists.     

Peganine hydrochloride dihydrate an orally active antileishmanial agent

Protozoic infections caused by genus Leishmania pose an enormous public health threat in developing countries, compounded by the toxicity and resistance to current therapies. Under the aegis of our ongoing program on drug discovery and development on antileishmanial agents from plants, we carried out bioassay guided fractionation on Peganum harmala seeds which resulted in the isolation of 1 as an antileishmanial agent. 2D-NMR spectral data and single crystal X-ray crystallography data indicated 1 as peganine hydrochloride in dihydrated form. The compound 1 exhibited in-vitro activity against both extracellular promastigotes as well as intracellular amastigotes residing within murine macrophages in Leishmania donovani. Furthermore, 1 also exhibited in-vivo activity, 79.6 (±8.07)% against established VL in hamsters at a dose of 100 mg/kg b.wt.     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Integration of cell line and process development to overcome the challenge of a difficult to express protein

This case study addresses the difficulty in achieving high level expression and production of a small, very positively charged recombinant protein. The novel challenges with this protein include the protein's adherence to the cell surface and its inhibitory effects on Chinese hamster ovary (CHO) cell growth. To overcome these challenges, we utilized a multi‐prong approach. We identified dextran sulfate as a way to simultaneously extract the protein from the cell surface and boost cellular productivity. In addition, host cells were adapted to grow in the presence of this protein to improve growth and production characteristics. To achieve an increase in productivity, new cell lines from three different CHO host lines were created and evaluated in parallel with new process development workflows. Instead of a traditional screen of only four to six cell lines in bioreactors, over 130 cell lines were screened by utilization of 15 mL automated bioreactors (AMBR) in an optimal production process specifically developed for this protein. Using the automation, far less manual intervention is required than in traditional bench‐top bioreactors, and much more control is achieved than typical plate or shake flask based screens. By utilizing an integrated cell line and process development incorporating medium optimized for this protein, we were able to increase titer more than 10‐fold while obtaining desirable product quality. Finally, Monte Carlo simulations were performed to predict the optimal number of cell lines to screen in future cell line development work with the goal of systematically increasing titer through enhanced cell line screening. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1201–1211, 2015