Apoptosis in antibody-dependent monocyte-mediated cytotoxicity with monoclonal antibody 17-1A against human colorectal carcinoma cells: enhancement with interferon γ

 Antibody-dependent cell-mediated cytotoxicity (ADCC) has been considered to be one of the main effector mechanisms by which unconjugated monoclonal antibody (mAb) 17-1A can exert an antitumor effect in vivo. Since the apoptotic pathway as well as the necrotic pathway have been shown to be utilized in various cytotoxic effector mechanisms, we investigated the role of apoptosis in ADCC mediated by monocytes (ADMC) using mAb 17-1A as an antibody and the human colorectal carcinoma cell line, COLO205, as target cells in vitro. The implications of the apoptosis during ADMC was demonstrated by means of both a DNA fragmentation assay and a TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Furthermore, interferon γ (IFNγ) was also found to enhance the induction of apoptosis significantly. The addition of superoxide dismutase did not reduce the level of the apoptosis, although superoxide anion (O₂ ^–) was observed to be produced. However, the release of tumor necrosis factor α (TNFα) was significantly enhanced during ADMC, while, in addition, apoptosis was significantly inhibited by the addition of anti-TNFα antibody. These findings indicated that apoptosis might be implicated in ADMC with mAb 17-1A, which was augmented by IFNγ, while, in addition, TNFα may also be one of the major mediators of apoptosis. Received: 1 August 1996 / Accepted: 27 August 1996     

Multi-stage biofilm reactor for acetic acid production at high concentration

Summary Continuous production of acetic acid by liquid surface culture ofAcetobacter aceti M7 was investigated using a Multi-Stage Biofilm Reactor (MSBFR) composed of ten shallow flow horizontal reactors of laboratory scale. With varying dilution rate in the range from 0.049 to 0.2 h^–1, the maximum exit acetic acid concentration reached was as high as 98.0 g/l at the lowest dilution rate with step feed of ethanol-rich medium to stages 3, 5, and 7. The production rate (4.3 g/l/h) was rather high considering the inhibitory effect of high acetic acid concentration. This may be ascribed to non-homogeneous distribution of acetic acid concentration in the bioreactor and step feed of ethanol-rich medium.     

Ajuga Δ24-Sterol Reductase Catalyzes the Direct Reductive Conversion of 24-Methylenecholesterol to Campesterol

Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C₂₈- and C₂₉-Δ²⁴⁽²⁸⁾-olefinic sterols to 24-methyl- and 24-ethylcholesterols. Generally, the reaction proceeds in two steps via the Δ²⁴⁽²⁵⁾ intermediate. In this study, we characterized the ArDWF1 gene from an expression sequence tag library of Ajuga reptans var. atropurpurea hairy roots. The gene was functionally expressed in the yeast T21 strain. The in vivo and in vitro study of the transformed yeast indicated that ArDWF1 catalyzes the conversion of 24-methylenecholesterol to campesterol. A labeling study followed by GC-MS analysis suggested that the reaction proceeded with retention of the C-25 hydrogen. The 25-H retention was established by the incubation of the enzyme with (23,23,25-²H₃,28-¹³C)-24-methylenecholesterol, followed by ¹³C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a Δ²⁴⁽²⁵⁾ intermediate. This is the first characterization of such a unique DWF1 enzyme. For comparison purposes, Oryza sativa DWF1 (OsDWF1) was similarly expressed in yeast. An in vivo assay of OsDWF1 supported the generally accepted two-step mechanism because the C-25 hydrogen of 24-methylenecholesterol was eliminated during its conversion to 24-methylcholesterol. As expected, the 24-methylcholesterol produced by OsDWF1 was a mixture of campesterol and dihydrobrassicasterol. Furthermore, the 24-methylcholesterol contained in the Ajuga hairy roots was determined to be solely campesterol through its analysis using chiral GC-MS. Therefore, ArDWF1 has another unique property in that only campesterol is formed by the direct reduction catalyzed by the enzyme.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

It was confirmed that washed yeast cells produced isobutanol from α-acetolactic acid which was presumed as the intermediate in the synthetic pathway of isobutanol from alanine described in the previous report. At the same time α-ketoisovaleric acid was detected in the fermented solution, which seemed to support this scheme. The effects of various fermentation conditions upon the formation of isobutanol were discussed.     

Synthesis and Biological Evaluation of 1′-C-Cyano-Pyrimidine Nucleosides

Abstract

2′-Deoxy-, 2′-bromo-, and arabino-1′-C-cyano-pyrimidine nucleosides were synthesized from O² ,2′-cyclouridine. Incorporation of cyano group at the anomeric position was achieved by treatment of 1′,2′-unsaturated uridine with NBS in the presence of pivalic acid followed by TMS-cyanide and stannic chloride. Antineoplastic and antiviral activities of those compounds are also discussed.

     

Properties and Transglycosylation Reaction of a Chitinase from Nocardia orientalis

The hydrolytic products of a chitinase purified from Nocardia orientalis were examined on reduced (GIcNAc)_n(n = 2~6). The rate of hydrolysis on reduced (GlcNAc)₄^₆ increased with increasing chain-length of A-acetylglucosamine residues, but the enzyme did not act on reduced (G1cNAc)₂ or reduced (GlcNAc)₃. Based on the analysis of the frequency distribution of bond cleavage on PNP-(GIcNAc)?(n = 2 ~ 5) in the initial hydrolysis, the enzyme was shown to release predominantly (G1cNAc)₂ from the nonreducing end of each substrate. The enzyme, which is essentially a hydrolase, also catalyzed a transglycosylation reaction in an excess of (GlcNAc)₄ as an initial substrate. In this case, the addition of ammonium sulfate to the reaction system resulted in a significant increase in (G1cNAc)₆ production. The yield of the hexasaccharide was about 34% of the chitinase-catalyzed net decrease of (GlcNAc)₄. The rate of the transglycosylation in the presence of ammonium sulfate greatly depended on the salt concentration, the temperature, and the substrate concentration.     

Out of the Orient: Post‐Tethyan transoceanic and trans‐Arabian routes fostered the spread of Baorini skippers in the Afrotropics

The origin of taxa presenting a disjunct distribution between Africa and Asia has puzzled biogeographers for more than a century. This biogeographic pattern has been hypothesized to be the result of transoceanic long‐distance dispersal, Oligocene dispersal through forested corridors, Miocene dispersal through the Arabian Peninsula or passive dispersal on the rifting Indian plate. However, it has often been difficult to pinpoint the mechanisms at play. We investigate biotic exchange between the Afrotropics and the Oriental region during the Cenozoic, a period in which geological changes altered landmass connectivity. We use Baorini skippers (Lepidoptera, Hesperiidae) as a model, a widespread clade of butterflies in the Old World tropics with a disjunct distribution between the Afrotropics and the Oriental region. We use anchored phylogenomics to infer a robust evolutionary tree for Baorini skippers and estimate divergence times and ancestral ranges to test biogeographic hypotheses. Our phylogenomic tree recovers strongly supported relationships for Baorini skippers and clarifies the systematics of the tribe. Dating analyses suggest that these butterflies originated in the Oriental region, Greater Sunda Islands, and the Philippines in the early Miocene c. 23 Ma. Baorini skippers dispersed from the Oriental region towards Africa at least five times in the past 20 Ma. These butterflies colonized the Afrotropics primarily through trans‐Arabian geodispersal after the closure of the Tethyan seaway in the mid‐Miocene. Range expansion from the Oriental region towards the African continent probably occurred via the Gomphotherium land bridge through the Arabian Peninsula. Alternative scenarios invoking long‐distance dispersal and vicariance are not supported. The Miocene climate change and biome shift from forested areas to grasslands possibly facilitated geodispersal in this clade of butterflies.