Targeted isolation of a designated region of the Bacillus subtilis genome by recombinational transfer

A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.     

Epinephrine-induced hyperpolarization of islet cells without KATP channels

ID-1101 (4-hydroxyisoleucine), an amino acid extracted from fenugreek seeds, exhibits an interesting glucose-dependent insulin-stimulating activity. The present study was undertaken to investigate a possible extrapancreatic effect of ID-1101 on insulin signaling and action besides its previously described insulinotropic action. Insulin-sensitizing effects of ID-1101 were investigated in rat in vivo by three different approaches: 1) using euglycemic hyperinsulinemic clamps in two different rat models of insulin resistance, i.e., Zucker fa/fa rats and rats fed a sucrose-lipid diet; 2) measuring liver and muscle phosphatidylinositol (PI) 3-kinase activity after an acute injection of ID-1101 in normal and insulin-resistant diabetic rats; and 3) after chronic treatment in two rat models of insulin resistance. Euglycemic hyperinsulinemic clamp experiments revealed that ID-1101 can improve insulin resistance through an increase of peripheral glucose utilization rate in sucrose-lipid-fed rats and by decreasing hepatic glucose production in Zucker fa/fa rats. Moreover, we demonstrated that a single injection of ID-1101 activates the PI 3-kinase activity in liver and muscle from normal rats but also in muscle from diabetic rats. Finally, chronic ID-1101 treatment significantly reduced insulinemia in type 2 diabetic rats and reduced the progression of hyperinsulinemia in insulin-resistant obese Zucker fa/fa rats. These findings clearly demonstrate that ID-1101 can reduce insulin resistance through activation of the early steps of insulin signaling in peripheral tissues and in liver. In summary, ID-1101, besides its insulinotropic effect, directly improves insulin sensitivity, making it a potentially very valuable therapeutic agent for diabetes treatment.     

The hypothalamus is involved in the anorexic effect of glucagon-like peptide-1 in chicks

The present study was carried out to investigate whether the hypothalamus is involved in the anorexic effect of glucagon-like peptide-1 (GLP-1) in chicks. To examine this, Fos expression in the chick hypothalamus were immunohistochemically detected after intracerebroventricular (ICV) injection of 30-pmol GLP-1. ICV injection of GLP-1 stimulated the expression of Fos-like immunoreactive (FLI) cells in the ventromedial hypothalamic nucleus (VMN). When 15-pmol GLP-1 was directly injected into the chick VMN, the chick's food intake was significantly decreased compared with the control treatment. Microinjection of GLP-1 into the (LHA) also inhibited feeding in chicks, although ICV injection of GLP-1 did not stimulate FLI expression in the brain area. These results suggest that VMN and some brain regions are involved in the anorexic effect of GLP-1 in chicks.     

Solution properties of gellan gum: change in chain stiffness between single- and double-stranded chains

Nine samples of gellan gum in the sodium form, ranging in weight-average molar mass from 3.47 x 10(4) to 1.15 x 10(5) at 40 degrees C, were investigated by static and dynamic light scattering and viscometry in 25 mM aqueous NaCl both at 40 and at 25 degrees C. The ratios of the molar mass at 25 degrees C (in the ordered state) to that at 40 degrees C (in the disordered state) were in the range of 1.99 to 2.07, supporting the scheme of the conformational transition of gellan gum between a disassociated single chain and an associated chain composed of two molecules. Focusing on the effects of polydispersity, the intrinsic viscosities, radii of gyration, and hydrodynamic radii were analyzed on the basis of unperturbed wormlike chain models. The persistence lengths were evaluated as 9.4 nm at 40 degrees C and 98 nm at 25 degrees C.     

Guanylyl cyclase and cGMP-specific phosphodiesterase participate in the acrosome reaction of starfish sperm

In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, i.e. ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for inducing the acrosome reaction. Experimentally, ARIS and asterosap are sufficient for the induction. However, when sperm are treated only with asterosap, they become unresponsive to the egg jelly to undergo the reaction. In this study, we analysed the mechanism of the acrosome reaction, using sperm inactivation by asterosap as a clue. Asterosap causes a rapid and transient increase in intracellular cGMP through the activation of the asterosap receptor, a guanylyl cyclase, and causes an increase in intracellular Ca(2+). When sperm were pretreated with asterosap, the guanylyl cyclase seemed to be inactivated irreversibly by dephosphorylation. They were still responsive to ARIS but no longer to asterosap. However, in the presence of IBMX or zaprinast, inhibitors against phosphodiesterases (PDEs), they retained their capacity to undergo the acrosome reaction in response to the egg jelly or ARIS alone. IBMX and zaprinast suppressed the intracellular catabolism of cGMP, but not of cAMP. These results suggest that guanylyl cyclase and cGMP-specific, IBMX- and zaprinast-susceptible PDEs are involved in the regulation of the acrosome reaction.     

Interferon-gamma: a key contributor to hyperoxia-induced lung injury in mice

Hyperoxia-induced lung injury complicates the care of many critically ill patients who receive supplemental oxygen therapy. Hyperoxic injury to lung tissues is mediated by reactive oxygen species, inflammatory cell activation, and release of cytotoxic cytokines. IFN-gamma is known to be induced in lungs exposed to high concentrations of oxygen; however, its contribution to hyperoxia-induced lung injury remains unclear. To determine whether IFN-gamma contributes to hyperoxia-induced lung injury, we first used anti-mouse IFN-gamma antibody to blockade IFN-gamma activity. Administration of anti-mouse IFN-gamma antibody inhibited hyperoxia-induced increases in pulmonary alveolar permeability and neutrophil migration into lung air spaces. To confirm that IFN-gamma contributes to hyperoxic lung injury, we then simultaneously exposed IFN-gamma-deficient (IFN-gamma-/-) mice and wild-type mice to hyperoxia. In the early phase of hyperoxia, permeability changes and neutrophil migration were significantly reduced in IFN-gamma-/- mice compared with wild-type mice, although the differences in permeability changes and neutrophil migration between IFN-gamma-/- mice and wild-type mice were not significant in the late phase of hyperoxia. The concentrations of IL-12 and IL-18, two cytokines that play a role in IFN-gamma induction, significantly increased in bronchoalveolar lavage fluid after exposure to hyperoxia in both IFN-gamma-/- mice and wild-type mice, suggesting that hyperoxia initiates upstream events that result in IFN-gamma production. Although there was no significant difference in overall survival, IFN-gamma-/- mice had a better early survival rate than did the wild-type mice. Therefore, these data strongly suggest that IFN-gamma is a key molecular contributor to hyperoxia-induced lung injury.     

Liposomes with differential lipid components exert differential adjuvanticity in antigen-liposome conjugates via differential recognition by macrophages

We previously reported that liposomes having differential lipid components displayed differential adjuvant effects when antigen was coupled with liposomes via glutaraldehyde. In the present study, antigen-liposome conjugates prepared using liposomes having differential lipid components were added to the macrophage culture, and phagocytosis and the antigen digest of liposome-coupled antigen by macrophages were then investigated. Antigen presentation by macrophages to an antigen-specific T-cell clone was further investigated using the same conjugates. Antigen-liposome conjugates which induced higher levels of antibody production in vivo were recognized more often, and the liposome-coupled antigen was digested to a greater degree by macrophages than antigen-liposome conjugates which induced lower levels of antibody production. These results correlated closely with those regarding antigen presentation by macrophages; when antigen was coupled to liposomes showing higher adjuvant effect, macrophages cocultured with antigen-liposome conjugates activated antigen-specific T-cells at a higher degree. The concentration of OVA in the macrophage culture added as antigen-liposome conjugates was approximately 32 microg/mL. However, the extent of T-cell activation was almost equal to that when 800 microg/mL of soluble OVA was added to the culture. The results of the present study demonstrated that the adjuvant activity of liposomes observed primary in vivo correlated closely with the recognition of antigen-liposome conjugates and antigen presentation of liposome-coupled antigen by macrophages, suggesting that the adjuvant effects of liposomes are exerted at the beginning of the immune response, i.e., recognition of antigen by antigen-presenting cells.     

Skewed X-chromosome inactivation causes intra-familial phenotypic variation of an EBP mutation in a family with X-linked dominant chondrodysplasia punctata

X-linked dominant chondrodysplasia punctata (CDPX2) is a skeletal dysplasia characterized by stippled epiphyses, cataracts, alopecia and skin lesions, including ichthyosis. CDPX2 exhibits a number of perplexing clinical features, such as intra- and inter-familial variation, anticipation, incomplete penetrance and possible gonadal and somatic mosaicism. Recently, mutations in the gene encoding Delta8,Delta7 sterol isomerase/emopamil-binding protein (EBP) have been identified in CDPX2. To better understand the genetics of CDPX2, we examined the entire EBP gene by direct sequencing in four CDPX2 patients. We found EBP mutations in all four patients, including three novel mutations: IVS3+1G>A, Y165C and W82C. Surprisingly, a known mutation (R147H) was identified in a patient and her clinically unaffected mother. Expression analysis revealed the mutant allele was predominantly expressed in the patient, while both alleles were expressed in the mother. Methylation analysis revealed that the wild-type allele was predominantly inactivated in the patient, while the mutated allele was predominantly inactivated in her mother. Thus, differences in expression of the mutated allele caused by skewed X-chromosome inactivation produced the diverse phenotypes within the family. Our findings could explain some of the perplexing features of CDPX2. The possibility that an apparently normal parent is a carrier should be considered when examining seemingly sporadic cases and providing genetic counseling to CDPX2 families.