Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker’s Yeast

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker’s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.Glutamine is one of the most important compounds in nitrogen metabolism; it is not only a constituent of proteins but is also a donor of the amino (amido) moiety in the biosynthesis of other amino acids, purines, pyrimidines, pyridine coenzymes, and complex carbohydrates. Glutamine is also used in the treatment of gastric ulcers and has been produced commercially by direct fermentation with certain bacteria (6–10).In recent years, enzymatic synthesis has come to rival direct fermentation as a means of producing amino acids. In the case of glutamine, however, the need for a stoichiometric supply of ATP for the endoergonic reaction of glutamine synthetase (GS) precludes the development of an economically valuable method, unless ATP can be regenerated and recycled.Processes for the production of various substances using dried yeast cells as an enzyme source were established by Tochikura and colleagues (2, 4, 16, 18–20). The processes are driven by the chemical energy of ATP released by the alcoholic fermentation by the yeast, which has been wasted in alcoholic brewing (17). Tochikura and colleagues also designed a process in which the yeast fermentation of sugar is combined with an endoergonic reaction catalyzed by an enzyme from a different microorganism (3). The results suggest that the process offers the possibility of producing many compounds at a high yield by using various biosynthetic reactions and high concentrations of substrates. Tochikura et al. introduced the general idea of coupled fermentation with energy transfer for the process; its principle is indicated in Fig. ​Fig.1,1, with glutamine production as an example. Open in a separate windowFIG. 1Scheme of glutamine production by the coupled fermentation with energy transfer method. ∗1, glycolytic pathway is abridged. ∗2, inorganic phosphate (P_i) is recycled.In the process of coupled fermentation with energy transfer, a catalytic amount of ATP is regenerated with the energy of sugar fermented by yeast, in the form of baker’s yeast (4, 16, 18, 19, 23). The energy-utilizing system for the synthesis can involve the enzyme(s) of yeast itself or those of other organisms. It should be noted that, from another point of view, the use of the energy-utilizing system results in ADP regeneration to complete the fermentation of glucose, and that, if there is no ADP regeneration, the yeast fermentation of sugar can proceed only as follows, in the presence of inorganic phosphate (the Harden-Young effect of inorganic phosphate [1]), 2 · glucose + 2 · inorganic phosphate → fructose 1,6-bisphosphate (FBP) + 2 · C₂H₅OH + 2 · CO₂ (Harden-Young equation), where ADP regeneration for the fermentation of 1 mol of glucose is carried out by the phosphorylation of another mole of glucose to FBP.We previously reported glutamine production, obtained by employing a combination of baker’s yeast cells and GS from Gluconobacter suboxydans, as the first application of the coupled fermentation with energy transfer method for the production of a nonphosphorylated compound (12, 13). In addition, we achieved high-yield glutamine production by using the Corynebacterium glutamicum (Micrococcus glutamicus) enzyme and larger amounts of the substrates (15). The maximum amounts of glutamine formed (23 to 25 g/liter) and the yield based on glutamate (50 to 100%) were to some extent satisfactory, but the yield based on the energy source (glucose) for ATP regeneration was not satisfactory (about 40% of the theoretical value; 2 mol of glutamine can be formed when 1 mol of glucose is consumed).In the present study, we examined the characteristics of glutamine production regarding product yield based on the energy source for ATP regeneration and regarding the reactivity of GS during glutamine production, which is closely related to the product yield. The results of preliminary attempts to improve glutamine production are also described. In these experiments, a yeast mutant which has a low assimilating ability for glycerol and/or ethanol was used.     

Induction of Bcl-xL Expression by Human T-Cell Leukemia Virus Type 1 Tax through NF-κB in Apoptosis-Resistant T-Cell Transfectants with Tax

Human T-cell leukemia virus type 1 (HTLV-1) Tax is thought to play a pivotal role in immortalization of T cells. We have recently shown that the expression of Tax protected the mouse T-cell line CTLL-2 against apoptosis induced by interleukin-2 (IL-2) deprivation and converted its growth from being IL-2 dependent to being IL-2 independent. In this study, we demonstrate that constitutive expression of bcl-xl but not bcl-2, bcl-xs, bak, bad, or bax was associated with apoptosis resistance after IL-2 deprivation in CTLL-2 cells that expressed Tax. Transient-transfection assays showed that bcl-x promoter was transactivated by wild-type Tax. Similar effects were observed in mutant Tax retaining transactivating ability through NF-kappaB. Deletion or substitution of a putative NF-kappaB binding site identified in the bcl-x promoter significantly decreased Tax-induced transactivation. This NF-kappaB-like element was able to form a complex with NF-kappaB family proteins in vitro. Furthermore, Tax-induced transactivation of the bcl-x promoter was also diminished by the mutant IkappaBalpha, which specifically inhibits NF-kappaB activity. Our findings suggest that constitutive expression of Bcl-x(L) induced by Tax through the NF-kappaB pathway contributes to the inhibition of apoptosis in CTLL-2 cells after IL-2 deprivation.     

Current knowledge of vector-borne zoonotic pathogens in Zambia: A clarion call to scaling-up “One Health” research in the wake of emerging and re-emerging infectious diseases

BackgroundAlthough vector-borne zoonotic diseases are a major public health threat globally, they are usually neglected, especially among resource-constrained countries, including those in sub-Saharan Africa. This scoping review examined the current knowledge and identified research gaps of vector-borne zoonotic pathogens in Zambia.Methods and findingsMajor scientific databases (Web of Science, PubMed, Scopus, Google Scholar, CABI, Scientific Information Database (SID)) were searched for articles describing vector-borne (mosquitoes, ticks, fleas and tsetse flies) zoonotic pathogens in Zambia. Several mosquito-borne arboviruses have been reported including Yellow fever, Ntaya, Mayaro, Dengue, Zika, West Nile, Chikungunya, Sindbis, and Rift Valley fever viruses. Flea-borne zoonotic pathogens reported include Yersinia pestis and Rickettsia felis. Trypanosoma sp. was the only tsetse fly-borne pathogen identified. Further, tick-borne zoonotic pathogens reported included Crimean-Congo Haemorrhagic fever virus, Rickettsia sp., Anaplasma sp., Ehrlichia sp., Borrelia sp., and Coxiella burnetii.ConclusionsThis study revealed the presence of many vector-borne zoonotic pathogens circulating in vectors and animals in Zambia. Though reports of human clinical cases were limited, several serological studies provided considerable evidence of zoonotic transmission of vector-borne pathogens in humans. However, the disease burden in humans attributable to vector-borne zoonotic infections could not be ascertained from the available reports and this precludes the formulation of national policies that could help in the control and mitigation of the impact of these diseases in Zambia. Therefore, there is an urgent need to scale-up “One Health” research in emerging and re-emerging infectious diseases to enable the country to prepare for future epidemics, including pandemics.     

Clinical Report on the First Prototype of a Photoacoustic Tomography System with Dual Illumination for Breast Cancer Imaging

Photoacoustic tomography is a recently developed imaging modality that can provide high spatial-resolution images of hemoglobin distribution in tissues such as the breast. Because breast cancer is an angiogenesis-dependent type of malignancy, we evaluated the clinical acceptability of breast tissue images produced using our first prototype photoacoustic mammography (PAM) system in patients with known cancer. Post-excisionally, histological sections of the tumors were stained immunohistochemically (IHC) for CD31 (an endothelial marker) and carbonic anhydrase IX (CAIX) (a marker of hypoxia). Whole-slide scanning and image analyses were used to evaluate the tumor microvessel distribution pattern and to calculate the total vascular perimeter (TVP)/area for each lesion. In this clinical study, 42 lesions were primarily scanned using PAM preoperatively, three of which were reported to be benign and were excluded from statistical analysis. Images were produced for 29 out of 39 cancers (visibility rate = 74.4%) at the median depth of 26.5 (3.25–51.2) mm. Age, menopausal status, body mass index, history of neoadjuvant treatment, clinical stage and histological tumor angiogenesis markers did not seem to affect the visibility. The oxygen saturation level in all of the measured lesions was lower than in the subcutaneous counterpart vessels (Wilcoxon test, p value<0.001), as well as in the counterpart contralateral normal breast region of interest (ROI) (Wilcoxon test, p value = 0.001). Although the oxygen saturation level was not statistically significant between CAIX-positive vs. -negative cases, lesional TVP/area showed a positive correlation with the oxygen saturation level only in the group that had received therapy before PAM. In conclusion, the vascular and oxygenation data obtained by PAM have great potential for identifying functional features of breast tumors.     

Thiazolidinediones increase arachidonic acid release and subsequent prostanoid production in a peroxisome proliferator-activated receptor gamma-independent manner

Thiazolidinedione, peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, has been used as an anti-diabetic drug and as an useful tool to elucidate multiple PPARgamma functions by in vitro and in vivo studies. We investigated the effects of thiazolidinediones on prostanoid production in lipopolysaccharide-stimulated cells. The high concentrations (>10 microM) of rosiglitazone and pioglitazone significantly increased lipopolysaccharide-stimulated prostanoid production such as thromboxane A2 and prostaglandin E2. However, PPARgamma antagonist could not inhibit them. In PPARgamma-deficient cells, thiazolidinediones increased prostaglandin E2 production. Thiazolidinediones increased arachidonic acid (AA) release from the cell membrane by not stimulating AA releasing process involving several phospholipase A2s but inhibiting AA reuptaking process. The expression of cyclooxygenase-1 and cyclooxygenase-2 were not affected by thiazolidinediones. In this study, we demonstrated that high concentrations of TZDs increased AA release by the inhibition of AA reuptaking process, leading to subsequent increase in the prostanoid production in a PPARgamma-independent manner. This mechanism provides useful information for the elucidation of multiple PPARgamma functions and diabetic drug therapy.     

Cellular basis of the role of diesel exhaust particles in inducing Th2-dominant response

There is growing evidence that diesel exhaust particles (DEP) can induce allergic diseases with increased IgE production and preferential activation of Th2 cells. To clarify the cellular basis of the role of DEP in the induction of Th2-dominant responses, we examined the effects of DEP on the cytokine production by T cells stimulated with anti-CD3/CD28 Ab and on that by monocyte-derived dendritic cells (MoDCs) stimulated with CD40L and/or IFN-gamma. We examined IFN-gamma, IL-4, IL-5, IL-8, and IL-10 produced by T cells and TNF-alpha, IL-1beta, IL-10, and IL-12 produced by MoDCs using real-time PCR analysis or by ELISA. To highlight the effects of DEP, we compared the effects of DEP with those of dexamethasone (DEX) and cyclosporin A (CyA). DEP significantly suppressed IFN-gamma mRNA expression and protein production, while it did not affect IL-4 or IL-5 mRNA expression or protein production. The suppressive effect on IFN-gamma mRNA expression was more potent than that of DEX and comparable at 30 mug/ml with 10(-7) M CyA. The suppressive effect on IFN-gamma production was also more potent than that of either DEX or CyA. DEP suppressed IL-12p40 and IL-12p35 mRNA expression and IL-12p40 and IL-12p70 production by MoDCs, while it augmented IL-1beta mRNA expression. Finally, by using a thiol antioxidant, N-acetyl cysteine, we found that the suppression of IFN-gamma production by DEP-treated T cells was mediated by oxidative stress. These data revealed a unique characteristic of DEP, namely that they induce a Th2 cytokine milieu in both T cells and dendritic cells.     

Intragenomic heterogeneity and intergenomic recombination among haloarchaeal rRNA genes

More than one copy of rRNA operons, which code for both the small-subunit (SSU) and large-subunit (LSU) rRNA, are often found in prokaryotes. It is generally assumed that all rRNA operons within a single cell are almost identical. A notable exception is the extremely halophilic archaeal genus Haloarcula, most species of which are known to harbor highly divergent rRNA operons that differ at approximately 5% of the nucleotide positions in the SSU gene and at 1 to 2% of the nucleotide positions in the LSU gene. We report that such intragenomic heterogeneity is not unique to Haloarcula, as high levels of intragenomic sequence variation have been observed for the SSU genes of two other genera of extreme halophiles, Halosimplex and Natrinema. To investigate this in detail, the two rRNA operons of Halosimplex carlsbadense and the four operons of Natrinema sp. strain XA3-1 were cloned and completely sequenced. The SSU and LSU genes of H. carlsbadense show the highest levels of intragenomic heterogeneity observed so far in archaea (6.7 and 2.6%). The operons of Natrinema sp. strain XA3-1 have additional unusual characteristics, such as identical internal transcribed spacers, while one of four SSU genes is 5% divergent and all LSU genes differ from each other by 0.9 to 1.9%. The heterogeneity among the Natrinema sp. strain XA3-1 LSU genes is localized in hot spots, and one of these regions is shown to be the result of a recombination event with a distantly related halophile. This is the first example of interspecies recombination between rRNA genes in archaea, and the recombination occurred over one of the largest phylogenetic distances ever reported for such an event. We suggest that intragenomic heterogeneity of rRNA operons is an ancient and stable trait in several lineages of the Halobacteriales. The impact of this phenomenon on the taxonomy of extremely halophilic archaea is discussed.