NKT cells play a limited role in the neutrophilic inflammatory responses and host defense to pulmonary infection with Pseudomonas aeruginosa

CD1d-restricted NKT cells are reported to play a critical role in the host defense to pulmonary infection with Pseudomonas aeruginosa. However, the contribution of a major subset expressing a Valpha14-Jalpha18 gene segment remains unclear. In the present study, we re-evaluated the role of NKT cells in the neutrophilic inflammatory responses and host defense to this infection using mice genetically lacking Jalpha18 or CD1d (Jalpha18KO or CD1dKO mice). These mice cleared the bacteria in lungs at a comparable level to wild-type (WT) mice. There was no significant difference in the local neutrophilic responses, as shown by neutrophil counts and synthesis of MIP-2 and TNF-alpha, in either KO mice from those in WT mice. Administration of alpha-galactosylceramide, a specific activator of Valpha14+ NKT cells, failed to promote the bacterial clearance and neutrophilic responses, although the same treatment increased the synthesis of IFN-gamma, suggesting the involvement of this cytokine downstream of NKT cells. In agreement against this notion, these responses were not further enhanced by administration of recombinant IFN-gamma in the infected Jalpha18KO mice. Our data indicate that NKT cells play a limited role in the development of neutrophilic inflammatory responses and host defense to pulmonary infection with P. aeruginosa.     

Construction and in vitro characterization of a chimeric simian and human immunodeficiency virus with the RANTES gene

Chimeric simian-human immunodeficiency virus (SHIV) containing the env gene of HIV-1 infects macaque monkeys and provides basic information that is useful for the development of HIV-1 vaccines. Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper type-1 responses against HIV-1. With the final goal of testing the adjuvant effects of RANTES in SHIV-macaque models, we constructed a SHIV having the RANTES gene (SHIV-RANTES) and characterized its properties in vitro. SHIV-RANTES replicated both in human and monkey T cell lines. Along with SHIV-RANTES replication, RANTES was detected in the supernatant of human and monkey cell cultures, at maximal levels of 98.5 and 4.1 ng/ml, respectively. A flow cytometric analysis showed that the expressed RANTES down-modulated CC-chemokine receptor 5 (CCR5) on PM1 cells, which was restored by adding anti-RANTES antibody. UV-irradiated culture supernatants from the SHIV-RANTES-infected cells suppressed replication of CCR5-tropic HIV-1 BaL in PM-1 cells. Differentiating real-time RT-PCR showed that pre-infection of SHIV-RANTES in C8166 cells expressing CCR5 suppressed the replication of HIV-1 BaL. Biological activity of the expressed RANTES and the inserted RANTES gene in SHIV-RANTES remained stable after 10 passages. These results suggest that SHIV-RANTES is worth testing in macaque models.     

Molecular Characterization of a Deep-Sea Methanotrophic Mussel Symbiont that Carries a RuBisCO Gene

In our previous investigation on the genes of 1,5-ribulose bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39) in deep-sea chemoautotrophic and methanotrophic endosymbioses, the gene encoding the large subunit of RuBisCO form I (cbbL) had been detected in the gill of a mussel belonging to the genus Bathymodiolus from a western Pacific back-arc hydrothermal vent. This study further examined the symbiont source of the RuBisCO cbbL gene along with the genes of 16S ribosomal RNA (16S rDNA) and particulate methane monooxygenase (EC 1.14.13.25; pmoA) and probed for the presence of the ATP sulfurylase gene (EC 2.7.7.4; sopT). The 16S rDNA sequence analysis indicated that the mussel harbors a monospecific methanotrophic Gammaproteobacterium. This was confirmed by amplification and sequencing of the methanotrophic pmoA, while thiotrophic sopT was not amplified from the same symbiotic genome DNA. Fluorescence in situ hybridization demonstrated simultaneous occurrence of the symbiont-specific 16S rDNA, cbbL and pmoA, but not sopT, in the mussel gill. This is the first molecular and visual evidence for a methanotrophic bacterial endosymbiont that bears the RuBisCO cbbL gene relevant to autotrophic CO₂ fixation.     

Silicon intake to vertebral columns of mice after dietary supply

Vertebral columns were dissected and analyzed after birth with oral administration of silicon for 4 wk and for 8 wk. The silicon level was lower (20 μg/g) at the beginning. It remains unchanged after 4 wk and then increases twice as much as that for those mice bred for 8 wk than those bred for 4 wk. This increase depends remarkably on the mass ratio of Si/Ca (M/M). The ratio increases to three times higher than that of the control at the beginning of the experiments (5 wk after birth). Although the S and P contents appeared to be lower, these increased when Si was administered in combination with phosphopeptide. Other elements, such as Ca, Mg, Fe, and Zn, appeared to be unchanged as the weeks proceeded. These findings seem to support a proposal that silicon is necessary for the growth of backbones in mice.     

Importance of Trp59 and Trp60 in chitin-binding, hydrolytic, and antifungal activities of Streptomyces griseus chitinase C

The chitin-binding domain of Streptomyces griseus chitinase C (ChBD_ChiC) belongs to CBM family 5. Only two exposed aromatic residues, W59 and W60, were observed in ChBD_ChiC, in contrast to three such residues on CBD_Cel5 in the same CBM family. To study importance of these residues in binding activity and other functions of ChBD_ChiC, site-directed mutagenesis was carried out. Single (W59A and W60A) and double (W59A/W60A) mutations abolished the binding activity of ChiC to colloidal chitin and decreased the hydrolytic activity toward not only colloidal chitin but also a soluble high Mr substrate, glycol chitin. Interaction of ChBD_ChiC with oligosaccharide was eliminated by these mutations. The hydrolytic activity toward oligosaccharide was increased by deletion of ChBD but not affected by these mutations, indicating that ChBD interferes with oligosaccharide hydrolysis but not through its binding activity. The antifungal activity was drastically decreased by all mutations and significant difference was observed between single and double mutants. Taken together with the structural information, these results suggest that ChBD_ChiC binds to chitin via a mechanism significantly different from CBD_Cel5, where two aromatic residues play major role, and contributes to various functions of ChiC. Sequence comparison indicated that ChBD_ChiC-type CBMs are dominant in CBM family 5.     

Sex difference in body weight gain and leptin signaling in hypocretin/orexin deficient mouse models

Recent studies in human and animal models of narcolepsy have suggested that obesity in narcolepsy may be due to deficiency of hypocretin signaling, and is also under the influence of environmental factors and the genetic background. In the current study, using two hypocretin/orexin deficient narcoleptic mouse models (i.e. preproorexin knockout (KO) and orexin/ataxin-3 transgenic (TG) mice) with cross-sectional assessments, we have further analyzed factors affecting obesity. We found that both KO and TG narcoleptic mice with mixed genetic backgrounds (N4-5, 93.75-96.88% genetic composition of C57BL/6) tended to be heavier than wild type (WT) mice of 100-200 days old. The body weight of heterozygous mice was intermediate between those of KO and WT mice. Obesity was more prominent in females in both KO and TG narcoleptic mice and was associated with higher serum leptin levels, suggesting a partial leptin resistance. Obesity is less prominent in the congenic TG narcoleptic mice, but is still evident in females. Our results confirmed that hypocretin/orexin ligand deficiency is one of the critical factors for the obese tendency in narcolepsy. However, multiple factors are also likely to affect this phenotype, and a sex difference specific alteration of leptin-hypocretin signaling may be involved.     

Clozapine but not haloperidol suppresses the changes in the levels of neuropeptides in MK-801-treated rat brain regions

Noncompetitive NMDA receptor antagonist (+)MK-801 is known to induce neurotoxicity and schizophrenia-like symptomatology where atypical neuroleptic clozapine is effective in contrast to typical neuroleptic, haloperidol. Although neuropeptides are implicated in memory and cognition, their roles in schizophrenia are not well understood. In the present study, we therefore examined the possible roles of neuropeptides, cholecystokinin (CCK) and somatostatin (SS) in the posterior cingulate/retrosplenial cortices (PC/RSC), frontal cortex, and hippocampus of a MK-801-induced schizophrenia-like model rat brain. This study further investigated the pretreated effect of atypical versus typical neuroleptics on the peptidergic system. SS mRNA and peptide levels significantly decreased in the PC/RSC and hippocampus but not in the frontal cortex 3 days after 0.5 mg/kg MK-801 treatment whereas CCK mRNA and peptide levels significantly decreased in all of the brain regions examined. Pretreatment with clozapine but not haloperidol completely recovered the changes in both mRNA and peptide levels of SS and CCK in those brain regions. These data suggest that peptidergic system in the brain presumably plays an important role in the control of negative schizophrenia.     

Behavioral responses to the alarm pheromone of the ant Camponotus obscuripes (Hymenoptera: Formicidae)

The alarm pheromone of the ant Camponotus obscuripes (Formicinae) was identified and quantified by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Comparisons between alarm pheromone components and extracts from the major exocrine gland of this ant species revealed that the sources of its alarm pheromone are Dufour's gland and the poison gland. Most components of Dufour's gland were saturated hydrocarbons. n-Undecane comprised more than 90% of all components and in a single Dufour's gland amounted to 19 microg. n-Decane and n-pentadecane were also included in the Dufour's gland secretion. Only formic acid was detected in the poison gland, in amounts ranging from 0.049 to 0.91 microl. This ant species releases a mixture of these substances, each of which has a different volatility and function. When the ants sensed formic acid, they eluded the source of the odor; however, they aggressively approached odors of n-undecane and n-decane, which are highly volatile. In contrast, n-pentadecane, which has the lowest volatility among the identified compounds, was shown to calm the ants. The volatilities of the alarm pheromone components were closely related to their roles in alarm communication. Highly volatile components vaporized rapidly and spread widely, and induced drastic reactions among the ants. As these components became diluted, the less volatile components calmed the excited ants. How the worker ants utilize this alarm communication system for efficient deployment of their nestmates in colony defense is also discussed herein.     

Mechanism of plasma glutathione peroxidase production in bovine adipocytes

CD97, an epidermal growth factor (EGF)-TM7 receptor, is not restricted to hematopoetic and carcinoma cells but is also found on smooth muscle cells (SMC). We have examined its location and biochemical structure in various normal and tumorigenic SMC-containing tissues. SMC of the urinary bladder, lung bronchi and bronchioles, myometrium, and gastrointestinal tract were immunohistologically stained by using monoclonal antibodies (mabs) to the CD97 stalk region (CD97^stalk). Mabs directed against an N-glycosylation-dependent epitope within the EGF-domains (CD97^EGF) did not bind to normal SMC. Vascular SMC, which was also CD97^EGF-negative, showed further CD97 heterogeneity. Only a few, if any, SMC from the aorta or elastic arteries of the systemic circulation were positive for CD97 mRNA and therefore also for CD97^stalk. CD97^stalk-positive SMC were slightly more numerous in muscular and peripheral arteries. In contrast, most venous SMC expressed CD97^stalk. A comparison with other SMC molecules revealed a similar but not identical staining pattern for CD97^stalk and desmin. Further CD97 heterogeneity was observed during SMC transformation. All leiomyomas (n=5) and nine out of 21 leiomyosarcomas were positive for both CD97^stalk and CD97^EGF. As expected, CD97^EGF-positive SMC tumors expressed partly N-glycosylated CD97. Seven out of 21 leiomyosarcomas were completely devoid of CD97. Thus, CD97 showed variable expression in vascular and biochemical modification in tumorigenic SMC, suggesting that the function of the molecule is specific for the SMC subtype. This study was supported by a joint grant from the German Research Council (DFG; project AU 132/3-1) and by the Interdisziplinary Center of Clinical Research (IZKF) Leipzig at the Faculty of Medicine, University of Leipzig (project D6). E. Wandel is a fellow of the IZKF.