Application of computer to monitoring and control of fermentation process: Microbial conversion of ML-236B Na to pravastatin

An automatic feeding process for microbial hydroxylation of ML236B sodium salt (ML-236B Na; compactin) by Streptomyces carbophilus SANK 62585 was developed. The hydroxylated product, pravastatin sodium salt (pravastatin; trade name Mevalotin), is an inhibitor of 3-hydroxy-3-methyglutaryl-coenzyme A reductase (HMG-CoA reductase) used as cholesterol-lowering drug. The hydroxylation activity of S. carbophilus was induced by the addition of ML236B Na to culture broth but inhibited by high concentration of ML236B Na. In order to obtain high conversion yield, it was necessary to maintain optimum ML236B Na concentration throughout the fermentation by continuous feeding. For this purpose, we developed an on-line monitoring method, which mainly consisted of a cross-flow filtration module, high-performance liquid chromatography (HPLC) analyzer, feed pump, and microcomputer for regulation of ML236B Na concentration. An algorithm for control of ML236B Na feed rate based on feedback and feed-forward control where conversion rate after Deltat was estimated by using regression analysis of the five latest values of conversion rate. In a fed-batch culture employing this system, the concentration of ML236B Na was maintained at optimum level during the fermentation and the productivity of pravastatin was increased threefold over that obtained in manual control culture. (c) 1993 John Wiley & Sons, Inc.     

A Two Step Stain for Normal and Leukemic Monocytes Using Two Different Dyes Applied in Sequence

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

Prostaglandins and thromboxanes in amniotic fluid during rivanol-induced abortion and labour

Abortion or delivery were induced by extra-amniotic instillation of Rivanol during the second trimester in twelve patients and during the third trimester in two patients with fetal death and one patient with fetal acrania. Serial sampling of amniotic fluid was performed through a transabdominal catheter and the levels of free arachidonic acid (AA), prostaglandin F₂α (PGF₂α), prostaglandin E₂ (PGE₂), 6-keto-prostaglandin F₁α (6-keto-PGF₁α) and thromboxane B₂ (TXB₂) were determined. The levels of AA, PGF₂α, PGE₂, 6-keto-PGF₁α and TXB₂ in amniotic fluid increased significantly during induction with the exception of AA in fetal death which was high and remained constant during induction. Furthermore, PGF₂α, 6-keto-PGF₁α and TXB₂ were all significantly correlated to AA.These observations suggested that free AA is released during Rivanol-induction of abortion and labour giving an increased synthesis of PGF₂α, PGE₂ prostacyclin and thromboxane A₂ in the fetal membranes and the decidua but not in the fetus. This increase might be relevant for the initiation and progress of abortion and labour in these patients.     

Induced pluripotent stem cell‐derived myeloid cells expressing OX40 ligand amplify antigen‐specific T cells in advanced melanoma

Immune checkpoint inhibitors improved the survival rate of patients with unresectable melanoma. However, some patients do not respond, and variable immune‐related adverse events have been reported. Therefore, more effective and antigen‐specific immune therapies are urgently needed. We previously reported the efficacy of an immune cell therapy with immortalized myeloid cells derived from induced pluripotent stem cells (iPS‐ML). In this study, we generated OX40L‐overexpressing iPS‐ML (iPS‐ML‐Zsgreen‐OX40L) and investigated their characteristics and in vivo efficacy against mouse melanoma. We found that iPS‐ML‐Zsgreen‐OX40L suppressed the progression of B16‐BL6 melanoma, and prolonged survival of mice with ovalbumin (OVA)‐expressing B16 melanoma (MO4). The number of antigen‐specific CD8⁺ T cells was higher in spleen cells treated with OVA peptide‐pulsed iPS‐ML‐Zsgreen‐OX40L than in those without OX40L. The OVA peptide‐pulsed iPS‐ML‐Zsgreen‐OX40L significantly increased the number of tumor‐infiltrating T lymphocytes (TILs) in MO4 tumor. Flow cytometry showed decreased regulatory T cells but increased effector and effector memory T cells among the TILs. Although we plan to use allogeneic iPS‐ML in the clinical applications, iPS‐ML showed the tumorgenicity in the syngeneic mice model. Incorporating the suicide gene is necessary to ensure the safety in the future study. Collectively, these results indicate that iPS‐ML‐Zsgreen‐OX40L therapy might be a new method for antigen‐specific cancer immunotherapy.     

Modifcation of the French Azure C Tissue Stain

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

Comparative analysis of circulating dendritic cell subsets in patients with atopic diseases and sarcoidosis

Background

Dendritic cells (DCs) are professional antigen-presenting cells that play a crucial role in the initiation and modulation of immune responses. Human circulating blood DCs are divided into two major subsets: myeloid DCs (mDCs); and plasmacytoid DCs (pDCs). Furthermore, mDCs are subdivided into two subsets: Th1-promoting mDCs (mDC1s); and Th2-promoting mDCs (mDC2s). Although CD1a, CD1c, and CD141 are generally used for classifying mDC subsets, their adequacy as a specific marker remains unclear. We performed this study to compare circulating mDC, pDC, mDC1, and mDC2 subsets between Th1- and Th2-mediated diseases using CD1a and CD141, and to analyze the adequacy of CD1a and CD141 as a marker for mDC1s and mDC2s, respectively.

Methods

Thirty patients with sarcoidosis, 23 patients with atopic diseases, such as atopic bronchial asthma, and 23 healthy subjects as controls were enrolled in this study. Peripheral blood DC subsets were analyzed with flow cytometry according to expressions of CD11c, CD123, CD1a, and CD141. For functional analysis, we measured interleukin (IL) 12p40 levels produced by the sorted mDC subsets.

Results

The sarcoidosis group showed decreased total DC (P < 0.05) and mDC counts (P < 0.05) compared to controls. The atopy group showed decreased CD1a⁺mDC count (P < 0.05), and increased CD1a^-mDC count (P < 0.05) compared to controls. CD141⁺mDC count in the atopy group was higher than controls (P < 0.05). Sorted CD1a⁺mDCs produced higher levels of IL-12p40 than CD1a^-mDCs (P = 0.025) and CD141⁺mDCs (P = 0.018).

Conclusions

We conclude that decreased count of CD1a⁺mDC and increased count of CD141⁺mDC may reflect the Th2-skewed immunity in atopic diseases. The results of IL-12 levels produced by the sorted mDC subsets suggested the adequacy of CD1a and CD141 as a marker for mDC1 and mDC2, respectively, in vivo.     

Chlorophyll fluorescence in the leaves of Tradescantia species of different ecological groups: Induction events at different intensities of actinic light

Chlorophyll fluorescence analysis is one of the most convenient and widespread techniques used to monitor photosynthesis performance in plants. In this work, after a brief overview of the mechanisms of regulation of photosynthetic electron transport and protection of photosynthetic apparatus against photodamage, we describe results of our study of the effects of actinic light intensity on photosynthetic performance in Tradescantia species of different ecological groups. Using the chlorophyll fluorescence as a probe of photosynthetic activity, we have found that the shade-tolerant species Tradescantia fluminensis shows a higher sensitivity to short-term illumination (≤20 min) with low and moderate light (≤200 μE m⁻² s⁻¹) as compared with the light-resistant species Tradescantia sillamontana. In T. fluminensis, non-photochemical quenching of chlorophyll fluorescence (NPQ) and photosystem II operational efficiency (parameter Φ_PSII) saturate as soon as actinic light reaches ≈200 μE m⁻² s⁻¹. Otherwise, T. sillamontana revealed a higher capacity for NPQ at strong light (≥800 μE m⁻² s⁻¹). The post-illumination adaptation of shade-tolerant plants occurs slower than in the light-resistant species. The data obtained are discussed in terms of reactivity of photosynthetic apparatus to short-term variations of the environment light.     

Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.     

Jumping and gliding rodents: Mitogenomic affinities of Pedetidae and Anomaluridae deduced from an RNA-Seq approach

An RNA-Seq strategy was used to obtain the complete set of protein-coding mitochondrial genes from two rodent taxa. Thanks to the next generation sequencing (NGS) 454 approach, we determined the complete mitochondrial DNA genome from Graphiurus kelleni (Mammalia: Rodentia: Gliridae) and partial mitogenome from Pedetes capensis (Pedetidae), and compared them with published rodent and outgroup mitogenomes. We finished the mitogenome sequencing by a series of amplicons using conserved PCR primers to fill the gaps corresponding to tRNA, rRNA and control regions. Phylogenetic analyses of the mitogenomes suggest a well-supported rodent phylogeny in agreement with nuclear gene trees. Pedetes groups with Anomalurus into the clade Anomaluromorpha, while Graphiurus branches within the squirrel-related clade. Moreover, Pedetes + Anomalurus branch with Castor into the mouse-related clade. Our study demonstrates the utility of NGS for obtaining new mitochondrial genomes as well as the importance of choosing adequate models of sequence evolution to infer the phylogeny of rodents.