Microbial Synthesis of Antiviral Nucleosides Using Escherichia coli BL21 as Biocatalyst

Experimental conditions such as shaking (aeration) rate, concentration of reagents and extent of culture growth for the optimal synthesis of adenosine using Escherichia coli BL21 as biocatalyst were assessed, achieving 95% yield in 30 min of reaction using microorganisms harvested from late exponential phase. The ability of E. coli BL21 to synthesise purine nucleosides containing sugar residues such as 2'-deoxyribose, 2',3'-dideoxyribose and arabinose was also verified. 2'-Deoxyribo- and arabinonucleosides could be prepared in high yield, while the results obtained with 2',3'-dideoxyribonucleosides were not satisfactory. In the case of 2'-deoxyadenosine, using thymidine as a starting material, a yield of 94% was achieved at 45°C.     

Human mesenchymal stromal cell therapy for damaged cochlea repair in nod-scid mice deafened with kanamycin

Background

Kanamycin, mainly used in the treatment of drug-resistant-tuberculosis, is known to cause irreversible hearing loss. Using the xeno-transplant model, we compared both in vitro and in vivo characteristics of human mesenchymal stromal cells (MSCs) derived from adult tissues, bone marrow (BM-MSCs) and adipose tissue (ADSCs). These tissues were selected for their availability, in vitro multipotency and regenerative potential in vivo in kanamycin-deafened nod-scid mice.

Acute myocardial infarction: Circadian, weekly, and seasonal patterns of occurrence

Convincing evidence has demonstrated that cardiovascular diseases do not occur randomly throughout the day, the week, or the year but show a well defined temporal organization. This article will review circadian, weekly, and seasonal patterns of occurrence of acute myocardial infarction, along with their underlying pathophysiological triggering factors.     

CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels

Despite being one of the most studied proteases in bacteria, very little is known about the role of ClpXP in mitochondria. We now present evidence that mammalian CLPP has an essential role in determining the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly. Through a proteomic approach and the use of a catalytically inactive CLPP, we produced the first comprehensive list of possible mammalian ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation, and a number of metabolic pathways. We further show that the defect in mitoribosomal assembly is a consequence of the accumulation of ERAL1, a putative 12S rRNA chaperone, and novel ClpXP substrate. The presented data suggest that the timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.     

Induction by Hypoxia of Heterologous-Protein Production with the KlPDC1 Promoter in Yeasts

The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the KlPDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1β was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The lacZ gene from Escherichia coli was cloned downstream of the KlPDC1 promoter in order to get β-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii, and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii; thus, this promotor constitutes a possible inducible system for this new nonconventional host.     

Isolation, Nucleotide Sequence, and Physiological Relevance of the Gene Encoding Triose Phosphate Isomerase from Kluyveromyces lactis

Lack of triose phosphate isomerase activity (TIM) is of special interest because this enzyme works at an important branch point of glycolytic flux. In this paper, we report the cloning and sequencing of the Kluyveromyces lactis gene encoding TIM. Unlike Saccharomyces cerevisiae ΔTPI1 mutants, the K. lactis mutant strain was found to be able to grow on glucose. Preliminary bioconversion experiments indicated that, like the S. cerevisiae TIM-deficient strain, the K. lactis TIM-deficient strain is able to produce glycerol with high yield.