Development of an in vitro Bioassay for Recombinant Human Erythropoietin (rHuEPO) Based on Proliferative Stimulation of an Erythroid Cell Line and Analysis of Sialic Acid Dependent Microheterogeneity: UT-7 Cell Bioassay

Determination of biological activity and its comparison with clinical behavior is important in the quality assessment of therapeutic glycoproteins. In vivo studies are usually employed for evaluating bioactivity of these glycomolecules. However, alternative methods are required to simplify the bioassay and avoid ethical issues associated with in vivo studies. Negatively charged sialic acid residues are known to be critical for in vivo bioactivity of rHuEPO. To address this need, we employed the human acute myeloid leukemia cell line UT-7 for the determination of proliferative stimulation induced by rHuEPO. Relative potencies of various intact and sugar-trimmed rHuEPO preparations were estimated using the International Standard for Human r-DNA derived EPO (87/684) as a reference for bioactivity. The cellular response was measured with a multi-channel photometer using a colorimetric microassay, based on the metabolism of the Resazurin sodium by cell viability. For a resourceful probing of physiological features of rHuEPO with significance, we obtained partly or completely desialylated rHuEPO digested by the neuraminidase enzyme without degradation of carbohydrates. Two-fold higher specific activity was shown by asialoerythropoietin in in vitro analysis compared with the sialoerythropoietin. Further, computational studies were also carried out to construct the 3D model of the erythropoietin (EPO) protein structure using standard comparative modeling methods. The quality of the model was validated using Procheck and protein structure analysis (ProSA) server tools. N–glycan units were constructed; moreover, EPO protein was glycosylated at potential glycosylation amino acid residue sites. The method described should be suitable for potency assessments of pharmaceutical formulations of rHuEPO (European Pharmacopeia, 2016).     

Evolution of an intermediate C4 photosynthesis in the non-foliar tissues of the Poaceae

Photosynthesis Research - Carbon concentrating mechanisms (CCMs) in plants are abaptive features that have evolved to sustain plant growth in unfavorable environments, especially at low atmospheric...     

Increased degradation of dermal collagen in diabetic rats.

The effect of alloxan induced diabetes on the dermal collagen content of albino rats was studied in relation to few lysosomal enzymes. Diabetes decreased the dermal collagen content. The specific activities of the lysosomal enzymes studied in the diabetic rat skin were elevated. It has been established that lysosomal enzymes degrade the connective tissue components. Thus, it may be suggested that the increase in the lysosomal enzymes studied should have facilitated the decrease in dermal collagen content of diabetic rats by increasing the degradation of dermal collagen.     

Prostaglandin release by zygotes and endometria of pregnant rabbits

When 4-day rabbit zygotes were incubated for 1 h at 37 degrees C in vitro, very little prostaglandin (PG) was released into the medium, and the concentration of PGs in the zygotes after incubation was also low. The release of prostaglandin E (PGE) and prostaglandin F (PGF) into the medium, and their concentration in the zygotes after incubation, increased sharply on Days 6 and 7 of pregnancy, reaching, by Day 7, values close to 200 ng of each PG released in 1 h per mg of protein. By contrast, endometrial samples on Days 4 and 5 of pregnancy released more PGF and less PGE than the zygotes of the same ages on a per mg of protein basis, and on Days 6 and 7, less of both PGs. Furthermore, endometrial concentrations of PGs after incubation, except for PGF on Day 4, were always lower than values for zygotes. Endometrial concentrations of PGs on Day 6 were lower before than after incubation. Although there was a slight upward trend in PG release by endometrial samples with increasing length of pregnancy, the changes were minimal and, in the case of PGE, none of the mean values exceeded 1 ng per mg of protein. In 7-day blastocysts, high levels of both PGF and PGE were found in the blastocoelic fluid, and these did not change during the 1-h incubation. The release of PGF and PGE during in vitro incubation of ruptured and washed Day 6 blastocysts was stimulated by arachidonic acid, and that of PGF, but not PGE, inhibited by indomethacin. The release of PGE, but not of PGF, from Day 6 blastocysts was inhibited by low temperature, and the same conditions inhibited release of both PGF and PGE from endometrial cell suspensions. It seems that both blastocysts and endometria have capability to synthesize PGs, the blastocysts being particularly active in this regard on Days 6 and 7 of pregnancy. It is hypothesized that, in vivo, Day 6 and 7 blastocysts release large quantities of PGs which trigger some of the local endometrial changes associated with pregnancy.     

Molecular characterization of reciprocal crosses of Aerides vandarum and Vanda stangeana (Orchidaceae) at the protocorm stage

Aerides vandarum and Vanda stangeana are two rare and endangered vandaceous orchids with immense floricultural traits. The intergeneric hybrids were synthesized by performing reciprocal crosses between them. In vitro germination response of the immature hybrid embryos was found to be best on half-strength Murashige and Skoog medium supplemented with 20% (v/v) coconut water/liquid endosperm from tender coconut. Determination of hybridity was made as early as the immature seeds or embryos germinated in vitro, using randomly amplified polymorphic DNA (RAPD) markers. Out of 15 arbitrarily chosen decamer RAPD primers, two were found to be useful in amplification of polymorphic bands specific to the parental species and their presence in the reciprocal crosses. However, a decisive profile that can identify the reciprocal crosses could not be provided by RAPD. Amplification of the trnL-F non-coding regions of chloroplast DNA of the parent species and hybrids aided easy identification of the reciprocal crosses from the fact that maternal inheritance of chloroplast DNA held true for these intergeneric hybrids. Subsequent restriction digestion of the polymerase chain reaction (PCR) amplified trnL-F non-coding regions of chloroplast DNA also consolidated the finding. Such PCR-based molecular markers could be used for early determination of hybridity and easy identification of the reciprocal crosses.     

Yolk partitioning in embryos of the lizard, Calotes versicolor: maximize body size or save energy for later use?

The hatchlings of Calotes versicolor and other congeners retain residual yolk for immediate post-hatching needs. Excision of 8% yolk (approximately equal residual yolk) from the eggs resulted in smaller hatchlings when compared to those emerging from sham operated eggs. However, hatchlings in both groups retained the same amount of residual yolk. The findings suggest that residual yolk in hatchlings of C. versicolor is an important part of the energy budget set aside by developing embryos and that the advantages of large hatchling size are counter balanced by selection for residual yolk.     

Does ichthyosis uteri have malignant potential? : A case report of squamous cell carcinoma of endometrium associated with extensive ichthyosis uteri

This short report discusses a case of solitary colonic polypoid ganglioneuroma associated with melanosis coli in a woman with no systemic manifestations. To our knowledge this is the first ganglioneuroma reported in the literature in association with melanosis coli. The nature and significance of this event remains unclear, although this may be coincidental due to the laxative intake. Further investigation is necessary to clarify this point. The interest of this case lies moreover in the rarity of this entity and its endoscopic and histologic resemblance to sessile polyps frequent in the clinical practice.     

Genetic Variation at Selected SNPs in the Leptin Gene and Association of Alleles with Markers of Kidney Disease in a Xhosa Population of South Africa

Background

Chronic kidney disease (CKD) is a significant public health problem that leads to end-stage renal disease (ESRD) with as many as 2 million people predicted to need therapy worldwide by 2010. Obesity is a risk factor for CKD and leptin, the obesity hormone, correlates with body fat mass and markers of renal function. A number of clinical and experimental studies have suggested a link between serum leptin and kidney disease. We hypothesised that variants in the leptin gene (LEP) may be associated with markers of CKD in indigenous black Africans.

Methodology/Principal Findings

Black South Africans of Xhosa (distinct cultural Bantu-speaking population) descent were recruited for the study and four common polymorphisms of the LEP (rs7799039, rs791620, rs2167270 and STS-{"type":"entrez-nucleotide","attrs":{"text":"U43653","term_id":"1226243","term_text":"U43653"}}U43653 [ENSSNP5824596]) were analysed for genotype and haplotype association with urine albumin-to-creatinine ratio (UACR), estimated glomerular filtration rate (eGFR), Serum creatinine (Scr) and serum leptin level. In one of the four single nucleotide polymorphisms (SNPs) we examined, an association with the renal phenotypes was observed. Hypertensive subjects with the T allele (CT genotype) of the ENSSNP5824596 SNP had a significantly higher eGFR (p = 0.0141), and significantly lower Scr (p = 0.0137). This was confirmed by haplotype analysis. Also, the haplotype GAAC had a modest effect on urine albumin-to-creatinine ratio in normotensive subjects (p = 0.0482).

Conclusions/Significance

These results suggest that genetic variations of the LEP may be associated with phenotypes that are markers of CKD in black Africans.     

Characterization of a Novel Cr6+ Reducing Pseudomonas sp. with Plant Growth–Promoting Potential

The isolate RNP4 obtained from a long-term tannery waste contaminated soil was characterized and presumptively identified as Pseudomonas sp. The strain RNP4 tolerated concentrations up to 450 mg Cr⁶⁺/L on a Luria-Bartani (LB) agar medium and reduced a substantial amount of Cr⁶⁺ to Cr³⁺ in the LB liquid medium. The ability of performing multifarious activities in tandem suggested the uniqueness of isolate RNP4. The strain produced a substantial amount of indole acetic acid (IAA) in tryptophan-supplemented medium. The strain also exhibited the production of siderophore and solubilization of phosphorus in mineral salt medium and SRS1 medium, respectively. Concurrent production of IAA and siderophore and the solubilization of phosphorus revealed its plant growth promotion potential. Furthermore, the strain was able to promote the growth of black gram, Indian mustard, and pearl millet in the presence of Cr⁶⁺. Thus, the innate capability of this novel isolate for parallel bioremediation and plant growth promotion has significance in the management of environmental and agricultural problems.