Characteristics of the energy-transducing NADH-quinone oxidoreductase ofParacoccus denitrificans as revealed by biochemical,biophysical, and molecular biological approaches

A comparison of the mitochondrial NADH-ubiquinone oxidoreductase and the energy-transducing NADH-quinone oxidoreductase (NDH-1) ofParacoccus denitrificans revealed that both systems have similar electron-transfer and energy-transduction pathways. In addition, both complexes are sensitive to the same inhibitors and contain similar electron carriers, suggesting that theParacoccus NDH-1 may serve as a useful model system for the study of the human enzyme complex. The gene cluster encoding theParacoccus NDH-1 has been cloned and sequenced. It is composed of 18,106 base pairs and contains 14 structural genes and six unidentified reading frames (URFs). The structural genes, URFs, and their polypeptides have been characterized. We also discuss nucleotide sequences which are believed to play a role in the regulation of the NDH-1 gene cluster andParacoccus NDH-1 subunits which may contain the binding sites of substrates and/or electron carriers.     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

The Structure and Characterization of the Surface Protein Layer of a Comamonas-Like Organism

The regular surface layer of a strain of a Comamonas-like organism was examined by electron microscopy. The surface layer protein was easily extracted from the cell surface by a 2.5 M solution of lithium chloride. The protein subunit has a molecular size of 32,000 daltons, but usually forms a large aggregate of more than 1,200,000 daltons. In the extract it formed a regular array of p4 symmetry and was observed to be intimately associated with fragments of lipopolysaccharide. The size of a subunit determined by the negative staining method and the image processing method measured 5.2 × 6.4 nm (width and length), was arranged in a cobblestone-like pattern, and was located in a lattice space measuring 13.0 nm square.     

The Purification of a GroEL-Like Stress Protein from Aerobically Adapted Campylobacter jejuni

From plate cultures of Campylobacter jejuni grown in room air a particulate protein of 62 kDa was isolated by ion-exchange chromatography. The protein had a square shape from the side view but when viewed from the top it had a star-shaped structure. The molecular size of the whole particle determined by gel filtration was 850 kDa which suggested the presence of 14 subunits of 62 kDa in each particle. The N-terminal 37 amino residues showed more than 80% homology with the sequence of these heat shock protein (HSP) 60 homologs of Chlamydia trachomatis, Helicobacter pylori, and Escherichia coli (GroEL). This protein is immunologically cross-reactive with the antiserum for the 60-kDa HSP of Yersinia enterocolitica. Production of the 62-kDa protein increased under heat stress and growth in an aerobic atmospheric environment. From these observations we concluded that the 62-kDa protein is a Campylobacter stress protein (Cj62) which belongs to the HSP 60 family.     

Expression of rice OSH1 gene is localized in developing vascular strands and its ectopic expression in transgenic rice causes altered morphology of leaf

Transgenic rice plants (Oryza sativa cv. Nipponbare) carrying 1 or 2 copies of a rice homeobox gene, OSH1, under the control of the CaMV 35S promoter were generated. The transgene caused altered morphology of leaf, such as ligule-replacement and abnormal division of sclerenchyma cells. The phenotype of these leaves resembles that of maize leaf morphological mutant, Knotted 1, which is caused by duplication of the KN1 gene (Veit et al., 1990). The in situ hybridization analysis has revealed that the expression of endogenous OSH1 is mainly localized in developing vascular strands of stem. We have discussed the biological roles of OSH1 in rice based on these results.     

Structural characterization of neutral glycosphingolipids using high-performance liquid chromatography-electrospray ionization mass spectrometry with a repeated high-speed polarity and MS<Superscript>n</Superscript> switching system

Four types of neutral glycosphingolipids (LacCer, Gb₃Cer, Gb₄Cer, and IV³αGalNAc-Gb₄Cer; 10 pmol each) were analyzed using high-performance liquid chromatography (HPLC)-electrospray ionization quadrupole ion trap time-of-flight (ESI-QIT-TOF) mass spectrometry (MS) with a repeated high-speed polarity and MSⁿ switching system. This system can provide six types of mass spectra, including positive and negative ion MS, MS², and MS³ spectra, within 1 s per cycle. Using HPLC with a normal-phase column, information on the molecular weights of major molecular species of four neutral glycosphingolipids was obtained by detecting [M+Na]⁺ in the positive ion mode mass spectra and [M?H]^? in the negative ion mode mass spectra. Sequences of glycosphingolipid oligosaccharide were obtained in the negative ion MS² spectra. In addition, information on the ceramide structures was clearly obtained in the negative ion MS³ mass spectra. GlcCer molecular species were analyzed by HPLC-ESI-QIT-TOF MS with a reversed-phase column using 1 pmole of GlcCer. The structures of the seven molecular species of GlcCer, namely, d18:1-C16:0, d18:1-C18:0, d18:1-C20:0, d18:1-C22:0, d18:1-C23:0, d18:1-C24:1, and d18:1-C24:0, were characterized using positive ion MS and negative ion MS, MS², and MS³. The established HPLC-ESI-QIT-TOF MS with MSⁿ switching and a normal phase column has been successfully applied to the structural characterization of LacCer and Gb₄Cer in a crude mixture prepared from human erythrocytes.     

Peptidoglycan‐induced T helper 2 immune response in mice involves interleukin‐10 secretion from Langerhans cells

Patients with atopic dermatitis (AD) have superficial skin colonization with Staphylococcus aureus and an increased number of T helper (Th)2 cells in their peripheral blood. The purpose of this study was to clarify the involvement of interleukin (IL)‐10 secretion from Langerhans cells (LCs) in staphylococcal peptidoglycan (PEG)‐induced Th2 immune responses in mice. Mice were primed with LCs pulsed with PEG (or LPS) and ovalbumin (OVA) and then given a booster OVA injection 2 days later in the hind footpad. Five days after the OVA injection, cytokine responses in the draining popliteal lymph nodes were investigated by RT‐PCR and ELISA. Production of both IL‐10 and IL‐12 by cultured LCs was detected by ELISA. Administration of PEG‐ or LPS‐stimulated LCs into the hind footpads of the mice induced Th2‐prone and Th1‐prone immune responses, respectively, as represented by expression of IL‐4 and interferon ‐γ . In vitro experiments showed that PEG induced greater production of IL‐12 p40 from LCs than did LPS, whereas LPS induced greater production of IL‐12 p70 from LCs than did PEG. Furthermore, it was found that PEG‐stimulated LCs induced greater production of IL‐10 than did LPS‐stimulated LCs, and that neutralization of IL‐10 augmented IL‐12 p70 production and inhibited Th2 development by PEG‐stimulated LCs. These results suggest that PEG can induce Th2 development through down‐regulation of IL‐12 p70 production by LCs in an IL‐10 production‐dependent manner and would explain the role of S. aureus colonization in patients with AD.     

Circadian Pattern of Wheel‐Running Activity of a South American Subterranean Rodent (Ctenomys cf knightii)

Circadian rhythms are regarded as essentially ubiquitous features of animal behavior and are thought to confer important adaptive advantages. However, although circadian systems of rodents have been among the most extensively studied, most comparative biology is restricted to a few related species. In this study, the circadian organization of locomotor activity was studied in the subterranean, solitary north Argentinean rodent, Ctenomys knightii. The genus, Ctenomys, commonly known as Tuco‐tucos, comprises more than 50 known species over a range that extends from 12°S latitude into Patagonia, and includes at least one social species. The genus, therefore, is ideal for comparative and ecological studies of circadian rhythms. Ctenomys knightii is the first of these to be studied for its circadian behavior. All animals were wild caught but adapted quickly to laboratory conditions, with clear and precise activity‐rest rhythms in a light‐dark (LD) cycle and strongly nocturnal wheel running behavior. In constant dark (DD), the rhythm expression persisted with free‐running periods always longer than 24 h. Upon reinstatement of the LD cycle, rhythms resynchronized rapidly with large phase advances in 7/8 animals. In constant light (LL), six animals had free‐running periods shorter than in DD, and 4/8 showed evidence of “splitting.” We conclude that under laboratory conditions, in wheel‐running cages, this species shows a clear nocturnal rhythmic organization controlled by an endogenous circadian oscillator that is entrained to 24 h LD cycles, predominantly by light‐induced advances, and shows the same interindividual variable responses to constant light as reported in other non‐subterranean species. These data are the first step toward understanding the chronobiology of the largest genus of subterranean rodents.