Mechanism of induction ofBar-like eye malformation by transient overexpression ofBar homeobox genes inDrosophila melanogaster

TheBar locus ofDrosophila is known to be a small complex consisting of two similar homeobox genes,BarH1 andBarH2. Usingegr as an ommatidium marker, possible mechanisms of formation of malformed eyes were examined. As in the case ofBarH1, overexpression ofBarH2 was found to be capable of inducingBar-like eye malformation. It was suggested that suppression of the anterior progression of the morphogenetic furrow and inhibition of reinitiation of normal ommatidial differentiation were mandatory to formation of the reduced eye morphology inBar mutants. These authors equally contributed to the present paper.     

Membrane-bound Bacillus cytochromes c and their phylogenetic position among bacterial class I cytochromes c

Abstract Gram-positive bacteria lack a periplasmic compartment and contain only membrane-bound cytochromes c . There are at least two types. One is found in subunit II of cytochrome oxidase, and the other is small cytochrome c which is also membrane-bound because of an unprocessed signal sequence or post-translational acylation at the N-terminal end of the protein. These Bacillus cytochromes c are compared with known class I cytochromes c , and a phylogenetic tree has been constructed by the neighbour-joining method.     

Synergistic induction of lymphokine (IL-2)-activated killer activity by IL-2 and the polysaccharide lentinan,and therapy of spontaneous pulmonary metastases

Summary Spleen cells of C57BL/6N mice bearing lung metastases were induced to the cytotoxic state by subcutaneous injection of recombinant human interleukin-2 (IL-2) at a minimum dose of 5×10⁴ U/mouse three times a day for 3 consecutive days. A single intraperitoneal injection of lentinan alone at concentrations of up to 10 mg/kg body weight did not render spleen cells cytotoxic to P-29 cells, but a combination of subthreshold doses of these agents (5×10⁴ U/ml IL-2 and 5 mg/kg lentinan) induced significant in vivo lymphokine-activated killer activity in spleen cells of tumor-bearing mice. Similarly, spleen cells from mice treated i.p. with lentinan became cytotoxic on in vitro treatment with IL-2. The in vitro responsiveness of spleen cells to IL-2 was maximal 3 days after i.p. injection of lentinan. Synergism between IL-2 and lentinan was also observed in mice bearing spontaneous lung micrometastases: neither IL-2 (<5×10⁴ U/mouse) nor lentinan (<2.5 mg/kg) alone had a therapeutic effect, but multiple injections of IL-2 with a single injection of lentinan resulted in significant inhibition of spontaneous pulmonary metastases. From these results we conclude that IL-2 and lentinan in combination are more effective than either one alone for inducing destruction of pulmonary metastases.     

Effector mechanism of human monocyte-mediated cytotoxicity: role of a new tumor cytotoxic factor distinct from interleukin 1 and tumor necrosis factorα

Human blood monocytes activated to the tumoricidal state were previously found to release a factor(s) responsible for tumor cell killing. The activity of the tumor cytotoxic factor(s) (TCF) was determined by release assay of radioactivity from human A375 melanoma cells. On fractionation of the supernatant of activated monocytes by Ultrogel AcA34 and TSK-G3000SW gel chromatographies two major peaks of the material with TCF activity with MWs of 30,000 and 15,000, called TCF-I and TCF-11, respectively were obtained. TCF-II could be neutralized by polyclonal anti-IL-1 antiserum, but anti-IL-1 antiserum did not neutralize either factor. TCF-I was separated by ampholine column electrofocusing into three major fractions with TCF activity at pI 5, 6 and 6.8, named TCF-1, TCF-1 and TCF-1, respectively. The cytotoxic and IL-1 activities of TCF-1 were neutralized by anti-IL-1 serum, whereas those of TCF-1 and TCF-1 were not completely neutralized by anti-IL-1 or anti-IL-1 antiserum. On DEAE ion-exchange chromatography (TSK DEAE 5PW) TCF-I gave two peaks with TCF activity (TCF-I1 and TCF-I2). TCF-I1 was slightly neutralized by anti-TNF antibody, but TCF-I2 was not affected by antisera against IL-1 and IL-1, or anti-TNF antibody, thus ruling out the possibility that tumor necrosis factor (TNF) might be involved in tumor cell killing mediated by TCF-I2. These results indicate that human monocyte-mediated cytotoxicity against human A375 melanoma cells is mediated in part by a tumor cytotoxic factor (TCF; MW, 30,000; pI 6), differing from IL-1 and TNF.     

Detection of immunoreactive endothelin in plasma of hemodialysis patients

Two types of radioimmunoassay (RIA) methods for measuring endothelin (ET) in human plasma were developed. One was an extraction procedure using a Sep-Pak C18 cartridge, the other being a direct method. By the extraction method, plasma ET levels were lower than the detectable limit (7 pg/ml) in normal subjects and elevated in hemodialysis patients. The absolute values obtained via the direct method were 20-times higher than those from extraction. Gel-filtration experiments revealed that this discrepancy was mainly due to immunoreactive (IR-) endothelin-like substances of high molecular mass near 11.6 kDa (large IR-ET). Extraction of the peptide by the C18 cartridge could eliminate interference by large IR-ET and is important in the accurate measurement of ET concentrations in plasma.     

Cloning and sequence analysis of the arginine deiminase gene from Mycoplasma arginini

Summary A deletion mutant of Rhizobium leguminosarum biovar viciae lacking the host-specific nodulation (nod) gene region (nodFEL nodMNT and nodO) but retaining the other nod genes (nodD nodABCIJ) was unable to nodulate peas or Vicia hirsuta, although it did induce root hair deformation. The mutant appeared to be blocked in its ability to induce infection threads and could be rescued for nodulation of V. hirsuta in mixed inoculation experiments with an exopolysaccharide deficient mutant (which is also Nod^–). The nodulation deficiency of the deletion mutant strain could be partially restored by plasmids carrying the nodFE, nodFEL or nodFELMNT genes but not by nodLMN. Surprisingly, the mutant strain could also be complemented with a plasmid that did not carry any of the nodFELMNT genes but which did carry the nodO gene on a 30 kb cloned region of DNA. Using appropriate mutations it was established that nodO is essential for nodulation in the absence of nodFE. Thus, either of two independent nod gene regions can complement the deletion mutant for nodulation of V. hirsuta. Similar observations were made for pea nodulation except that nodL was required in addition to nodO for nodulation in the absence of the nodFE genes. These observations show that nodulation can occur via either of two pathways encoded by non-homologous genes.Dedicated to the memory of the late Dr. David Goodchild     

Did Archaeal and Bacterial Cells Arise Independently from Noncellular Precursors? A Hypothesis Stating That the Advent of Membrane Phospholipid with Enantiomeric Glycerophosphate Backbones Caused the Separation of the Two Lines of Descent

One of the most remarkable biochemical differences between the members of two domains Archaea and Bacteria is the stereochemistry of the glycerophosphate backbone of phospholipids, which are exclusively opposite. The enzyme responsible to the formation of Archaea-specific glycerophosphate was found to be NAD(P)-linked sn-glycerol-1-phosphate (G-1-P) dehydrogenase and it was first purified from Methanobacterium thermoautotrophicum cells and its gene was cloned. This structure gene named egsA (enantiomeric glycerophosphate synthase) consisted of 1,041 bp and coded the enzyme with 347 amino acid residues. The amino acid sequence deduced from the base sequence of the cloned gene (egsA) did not share any sequence similarity except for NAD-binding region with that of NAD(P)-linked sn-glycerol-3-phosphate (G-3-P) dehydrogenase of Escherichia coli which catalyzes the formation of G-3-P backbone of bacterial phospholipids, while the deduced protein sequence of the enzyme revealed some similarity with bacterial glycerol dehydrogenases. Because G-1-P dehydrogenase and G-3-P dehydrogenase would originate from different ancestor enzymes and it would be almost impossible to interchange stereospecificity of the enzymes, it seems likely that the stereostructure of membrane phospholipids of a cell must be maintained from the time of birth of the first cell. We propose here the hypothesis that Archaea and Bacteria were differentiated by the occurrence of cells enclosed by membranes of phospholipids with G-1-P and G-3-P as a backbone, respectively. Received: 24 March 1997 / Accepted: 21 May 1997     

Biostratigraphy and palaeobiogeography of Lower Permian (lower Kungurian) conodonts from the Tak Fa Formation (Saraburi Limestone), Thailand

Lower Permian (lower Kungurian) conodonts are reported from the Indochina Block of Southeast Asia. The fauna from the Tak Fa Formation of the Saraburi Limestone Group exposed in limestone hills NNW of Khok Samrong, Thailand, includes Sweetognathus subsymmetricus Wang, Ritter and Clark (early forms) and Pseudosweetognathus costatus Wang, Ritter and Clark. The co-occurrence of these species indicates a Lower Permian age (upper half of the lower Kungurian) for the sampled limestones representative of the Mesogondolella siciliensis–S. subsymmetricus Zone of South China. Sweetognathus and Pseudosweetognathus are for the first time reported from the Indochina terrane located in the palaeoequatorial belt in the Kungurian. Pseudosweetognathus appears restricted to the Kungurian of the South China and Indochina terranes thus supporting palaeogeographic reconstructions that isolate these terranes within the Palaeo-Tethys during the Kungurian.     

Potential of selected strains of lactic acid bacteria to induce a Th1 immune profile

Lactic acid bacteria (LAB) might switch the Th2 biased immune response in allergic patients towards a balanced Th1/Th2 immune profile, leading to amelioration of allergy. To select strains of LAB that could be of potential application for foods in controlling allergy, 35 bacterial strains were screened in vitro using murine splenocytes and peritoneal exudate cells (PECs). Streptococcus thermophilus AHU1838 (FERM AP-21009), and Lactobacillus paracasei subsp. casei AHU1839 (FERM AP-21010) enhanced the secretion of Th1 cytokines such as interferon-gamma (IFN-gamma) and interleukin-12 (IL-12). The two strains of LAB also up-regulated the expression of CD40, and CD86 in dendritic cells (DCs), and activated cytotoxic T lymphocytes (CTL). These two strains could therefore be used in producing fermented food products that can enhance the Th1 immune profile which is important in ameliorating allergy.     

Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: eukaryotic clones containing two and three ORF multi-gene cassettes expressed from a single promoter

Two types of eukaryotic operon-type Expression clones were constructed using the Multisite Gateway system employing six types of att signals. These clones harbored a DNA cassette containing two heterologous ORFs (cDNAs) or three heterologous ORFs in tandem downstream of a single promoter. The most promoter-proximal ORF was translated via a Kozak signal and the downstream one or two ORF(s) were translated as directed by internal ribosome entry site(s) (IRES). These clones were observed to produce two or three different proteins at levels that depended on the activities of the translational initiation signals used. With the intention of modulating the expression level of the first ORF, the translational initiation signals including a Kozak sequence and 11 different IRESs were investigated for their efficiency using a single ORF. The translational activity of these signals varied within a 10-fold magnitude. Using these results, expression at pre-described relative levels was achieved from the optional IRES of the respective ORFs in the cassette. Controllable expression at desired levels of two different ORFs directed by optional IRESs on a bicistronic construct, transcribed from a single promoter, was demonstrated.