Transport regulation of recombinant gene expression in E. coli and B. subtilis

Expression kinetics of the lactose (lac) operon in Escherichia coli are reviewed for both wild-type and recombinant cell cultures under chemostatic conditions. A unified model which involves regulation of active inducer (lactose) transport, promoter-operator regulated expression of the lac operon, glucose-mediated inducer exclusion, and catabolite repression is summarized and supporting data is shown to verify its accuracy. The synthesis of alpha-amylase with a recombinant form of Bacillus subtilis is also reviewed to point out generic features in transport regulation, the lac operon model providing a point of departure. While there are many similarities in the influence of transport on both regulating models, there are also important differences. In a chemostat system, the synthesis of alpha-amylase is nongrowth associated, while beta-galactosidase is a growth-associated enzyme. Nevertheless, transport regulation is an important feature in both instances.     

Species differences in the binding kinetics of 25-hydroxyvitamin D3 to vitamin D binding protein

The specific binding of 25-hydroxyvitamin D3 to its binding protein was studied in serum of the human, rhesus monkey, cow, horse, and rat. The free fraction of 25-hydroxyvitamin D3 in the rat was 0.34 +/- 0.15 pmol free/nmol total (+/- SD) and this was lower than in any of the other species (p less than 0.01). In the human, the free fraction was 1.5 +/- 0.32 pmol free/nmol total, which was higher than in any of the other species (p less than 0.001). The differences in the free fraction were mainly due to differences in dissociation constant. The relative levels of free 25-hydroxyvitamin D should be taken into account when extrapolating findings about vitamin D metabolism in animals to the human. A technical outcome of this study is that of the species tested, vitamin D binding protein from rat serum is the most suitable as a reagent component for methods used to measure total 25-hydroxyvitamin D by competitive protein binding assay.     

Immobilization of Brevibacterium flavum cells on collagen for the production of glutamic acid in a recycle reactor

In this study, live cells of Brevibacterium flavum were immobilized for the production of glutamic acid. The reason for such a choice was that glutamic acid fermentation is an extensively studied fermentation and one which requires the viability of entire cellular faculties for the acid production. Brevibacterium flavum was chosen because it is an industrially used bacterium, and is very potent via a vis glutamic acid production. Studies were performed to find aeration and agitation conditions for optimal growth and glutamic acid productivity. Experiments were also done to find the optimum harvesting time. The cell activity peaks during the run of fermentation, and the time at which the peak occurs, was found. Conventional methods for immobilizing the cells on collagen were found to be lacking. The pH and drying were the two main reasons for loss of viability of the cells; the latter being more important. A modified immobilization procedure has been devised, which can immobilize live cells at any given pH and ionic strength, in contrast to the conventional method which requires the pH to be above 11 or below 3. This new method involves dialysis of collagen in suitable dialysis bags against water at pH7 (or buffer at any desired pH). The dialysed collagen blended at 20,000 rpm, resulted in a very smooth dispersion, unnoticeably different from collagen dispersion prepared at pH 11. The dispersed collagen was then cast and dried at an elevated temperature, and high air flow rate over the cast membrane, decreasing the time of drying from 6–8 hr ( in the conventional method) to 1.5–2 hr. The membrane has been tested for glutamic acid producing capabilities in a column reactor with the membrane spirally wound. The reactor has been operated under continuous conditions for 5–10 days with stable activities.     

Expression of chemerin and its receptors in the ovaries of prepubertal and mature gilts

Recent studies have demonstrated that chemerin participates in the regulation of female reproductive function at the level of the ovaries. Due to the lack of data concerning the presence of the chemerin system (chemerin and its receptors: CMKLR1, GPR1, CCRL2) in the ovaries of pigs, one of the most economically important livestock species, the aim of this study was to investigate the expression and localization of chemerin and its receptors in the ovaries of prepubertal and mature gilts. We also aimed to examine the concentrations of chemerin in the follicular fluid of prepubertal and mature animals. In the present study, we have demonstrated the expression patterns of chemerin system components in the porcine follicles of different sizes of prepubertal and mature animals, as well as in corpora lutea of mature gilts during the estrous cycle and early pregnancy. The obtained results suggest that the expression of chemerin system components is influenced by the reproductive stage, cell type, and the hormonal status of gilts (the estrous cycle/pregnancy). We have also presented the localization of the chemerin system components in various ovarian structures, and also showed changes in the concentration of chemerin in the follicular fluid of pigs. The presented findings not only confirm that chemerin is produced locally in the porcine ovary but they also demonstrate that chemerin directly affects ovarian cells, as confirmed by the presence of chemerin receptors in all ovarian structures. Therefore, chemerin appears to be an important intra‐ovarian factor that could regulate ovary function in pigs.     

Synthesis and in vitro evaluation of antiviral and cytostatic properties of novel 8-triazolyl acyclovir derivatives

Abstract

As a part of the research aimed on identification of new nucleobase derivatives with improved biological properties, a series of novel 8-substituted acyclovir derivatives were synthesized. The 8-azidoguanosine 4 and novel 8-azidoacyclovir 9 were synthesized from commercially available guanosine 1 and acyclovir 6 which were transformed into 8-bromopurine derivatives 2 and 7 and hydrazine derivatives 3 and 8, respectively. 8-Triazolylguanosine 5 and 8-triazolylacyclovir analogs 10–12 were successfully synthesized via the Cu(I) catalyzed 1,3-dipolar cycloaddition reaction of azides 4 and 9 with propargyl alcohol, 4-pentyn-1-ol and 5-hexyn-1-ol. The novel 1,4-disubstituted 1,2,3-triazolyl compounds 5, 10–12 were evaluated for antiviral activity against selected DNA and RNA viruses and cytostatic activity against normal Madine Darby canine kidney (MDCK I) cells, and seven tumor cell lines (HeLa, CaCo-2, NCI-H358, Jurkat, K562, Raji and HuT78). While tested compounds exerted no antiviral activity at nontoxic concentrations, the 8-triazolyl acyclovir derivative 10, with the shortest alkyl substituent at the C-4 of triazole ring, was found to be the most active against the CaCo-2 cell line.     

High Content Screening as High Quality Assay for Biological Evaluation of Photosensitizers In Vitro

A novel single step assay approach to screen a library of photdynamic therapy (PDT) compounds was developed. Utilizing high content analysis (HCA) technologies several robust cellular parameters were identified, which can be used to determine the phototoxic effects of porphyrin compounds which have been developed as potential anticancer agents directed against esophageal carcinoma. To demonstrate the proof of principle of this approach a small detailed study on five porphyrin based compounds was performed utilizing two relevant esophageal cancer cell lines (OE21 and SKGT-4). The measurable outputs from these early studies were then evaluated by performing a pilot screen using a set of 22 compounds. These data were evaluated and validated by performing comparative studies using a traditional colorimetric assay (MTT). The studies demonstrated that the HCS assay offers significant advantages over and above the currently used methods (directly related to the intracellular presence of the compounds by analysis of their integrated intensity and area within the cells). A high correlation was found between the high content screening (HCS) and MTT data. However, the HCS approach provides additional information that allows a better understanding of the behavior of these compounds when interacting at the cellular level. This is the first step towards an automated high-throughput screening of photosensitizer drug candidates and the beginnings of an integrated and comprehensive quantitative structure action relationship (QSAR) study for photosensitizer libraries.     

Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3

Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.     

A different methylation profile of circadian genes promoter in breast cancer patients according to clinicopathological features

ABSTRACT

One of the supposed mechanisms that may lead to breast cancer (BC) is an alteration of circadian gene expression and DNA methylation. We undertook an integrated approach to identify methylation pattern of core circadian promoter regions in BC patients with regard to clinical features. We performed a quantitative methylation-specific real-time PCR analysis of a promoter methylation profile in 107 breast tumor and matched non-tumor tissues. A panel of circadian genes CLOCK, BMAL1, PERIOD (PER1, 2, 3), CRYPTOCHROME (CRY1, 2) and TIMELESS as well as their association with clinicopathological characteristics were included in the analysis. Three out of the eight analyzed genes exhibited marked hypermethylation (PER1, 2, 3), whereas CLOCK, BMAL1, CRY2 showed significantly lower promoter CpG methylation in the BC tissues when compared to the non-tumor tissues. Among variously methylated genes we found an association between the elevated methylation level of PERs promoter region and molecular subtypes, histological subtypes and tumor grading of BC. Methylation status may be associated with a gene expression level of circadian genes in BC patients. An aberrant methylation pattern in circadian genes in BC may provide information that could be used as novel biomarkers in clinics and molecular epidemiology as well as play an important role in BC etiology.