Insulin-mimetic and insulin-sensitizing activities of a pentacyclic triterpenoid insulin receptor activator

Five pentacyclic triterpenoids isolated from Campsis grandiflora were tested for insulin-mimetic and insulin-sensitizing activity. The compounds enhanced the activity of insulin on tyrosine phosphorylation of the IR (insulin receptor) beta-subunit in CHO/IR (Chinese-hamster ovary cells expressing human IR). Among the compounds tested, CG7 (ursolic acid) showed the greatest enhancement and CG11 (myrianthic acid) the least. We characterized the effect of CG7 further, and showed that it acted as an effective insulin-mimetic agent at doses above 50 mug/ml and as an insulin-sensitizer at doses as low as 1 mug/ml. Additional experiments showed that CG7 increased the number of IRs that were activated by insulin. This indicates that a major mechanism by which CG7 enhances total IR auto-phosphorylation is by promoting the tyrosine phosphorylation of additional IRs. CG7 not only potentiated insulin-mediated signalling (tyrosine phosphorylation of the IR beta-subunit, phosphorylation of Akt and glycogen synthase kinase-3beta), but also enhanced the effect of insulin on translocation of glucose transporter 4 in a classical insulin-sensitive cell line, 3T3-L1 adipocytes. The results of the present study demonstrate that a specific pentacyclic triterpenoid, CG7, exerts an insulin-sensitizing effect as an IR activator in CHO/IR cells and adipocytes. The enhancement of insulin activity by CG7 may be useful for developing a new class of specific IR activators for treatment of Type 1 and Type 2 diabetes.     

A synthetic de-greening gene circuit provides a reporting system that is remotely detectable and has a re-set capacity

Plants have evolved elegant mechanisms to continuously sense and respond to their environment, suggesting that these properties can be adapted to make inexpensive and widely used biological monitors, or sentinels, for human threats. For a plant to be a sentinel, a reporting system is needed for large areas and widespread monitoring. The reporter or readout mechanism must be easily detectable, allow remote monitoring and provide a re-set capacity; all current gene reporting technologies fall short of these requirements. Chlorophyll is one of the best-recognized plant pigments with an already well-developed remote imaging technology. However, chlorophyll is very abundant, with levels regulated by both genetic and environmental factors. We designed a synthetic de-greening circuit that produced rapid chlorophyll loss on perception of a specific input. With induction of the de-greening circuit, changes were remotely detected within 2 h. Analyses of multiple de-greening circuits suggested that the de-greening circuit functioned, in part, via light-dependent damage to photosystem cores and the production of reactive oxygen species. Within 24–48 h of induction, an easily recognized white phenotype resulted. Microarray analysis showed that the synthetic de-greening initiated a process largely distinct from normal chlorophyll loss in senescence. Remarkably, synthetically de-greened white plants re-greened after removal of the inducer, providing the first easily re-settable reporter system for plants and the capacity to make re-settable biosensors. Our results showed that the de-greening circuit allowed chlorophyll to be employed as a simple but powerful reporter system useful for widespread areas.     

Time-course alterations of Toll-like receptor 4 and NF-κB p65, and their co-expression in the gerbil hippocampal CA1 region after transient cerebral ischemia

Innate immune system is very important to modulate the host defense against a large variety of pathogens. Toll-like receptors (TLRs) play a key role in controlling innate immune response. Among TLRs, TLR4 is a specific receptor for lipopolysaccharide and associated with the release of pro-inflammatory cytokines. In the present study, we investigated ischemia-related changes of TLR4 immunoreactivity and its protein level, and nuclear factor κB (NF-κB) p65 immunoreactivity regarding inflammatory responses in the hippocampal CA1 region after 5 min of transient cerebral ischemia to identify the correlation between transient ischemia and inflammation. In the sham-operated group, TLR4 immunoreactivity was easily detected in pyramidal neurons of the hippocampal CA1 region (CA1). TLR4 immunoreactivity in pyramidal neurons was distinctively decreased after ischemia/reperfusion (I/R); instead, based on double immunofluorescence study, TLR4 immunoreactivity was expressed in non-pyramidal neurons and astrocytes from 2 days postischemia. In addition, TLR4 protein level was lowest at 1 day postischemia and highest 4 days after I/R. On the other hand, NF-κB p65 immunoreactivity was not detected in the CA1 of the sham-operated group, and NF-κB p65 immunoreactivity was not observed until 1 day after I/R. However, NF-κB p65 immunoreactivity began to be expressed in astrocytes at 2 days postischemia, and the immunoreactivity was strong 4 days postischemia. Our results indicate that TLR4 and NF-κB p65 immunoreactivity are changed in CA1 pyramidal neurons and newly expressed in astrocytes, not in microglia, in the CA1 region after transient cerebral ischemia.     

The role ofH-2 regions in overcoming tissue incompatibility

Neonatal transplantation tolerance to the products of theH-2 ^b complex was induced in B10.A (H-2 ^a ) mice. On the basis of the survival of skin allografts it was found that antigens determined by theD region of theH-2 ^b complex (of the B10.A(2R) strain) were most easily overcome and that tolerance to the products of theD end of theH-2 complex (of the B10.A(4R) strain) was also easy to induce. The antigens produced by theK end ofH-2 (of the B10.A(5R) and B10.A(3R) strains) represented a stronger incompatibility barrier and a difference in the entireH-2 ^b complex caused strongest resistance to tolerance induction. When tolerance to the products of the entireH-2 ^b complex was induced in newborn B10.A mice, and the neonatally treated animals were grafted simultaneously with five different grafts, those disparate at theK end ofH-2 and in the entireH-2 region were rejected in some animals, while the grafts disparate at theD end of H-2 remained intact in the same mice. No dependence on theI-J subregion was observed in this system. Furthermore, tolerance was more easily inducible in male than in female B10.A mice.     

Comparison of Expression of Inflammatory Cytokines in the Spinal Cord Between Young Adult and Aged Beagle Dogs

Aging is an inevitable process that occurs in the whole body system accompanying with many functional and morphological changes. Inflammation is known as one of age-related factors, and inflammatory changes could enhance mortality risk. In this study, we compared immunoreactivities of inflammatory cytokines, such as interleukin (IL)-2 (a pro-inflammatory cytokine), its receptor (IL-2R), IL-4 (an anti-inflammatory cytokine), and its receptor (IL-4R) in the cervical and lumbar spinal cord of young adult (2–3 years old) and aged (10–12 years old) beagle dogs using immunohistochemistry and western blotting. IL-2 and IL-2R-immunoreactive nerve cells were found throughout the gray matter of the cervical and lumbar spinal cord of young adult and aged dogs. In the spinal cord neurons of the aged dog, immunoreactivity and protein levels were apparently increased compared with those in the young adult dog. Change patterns of IL-4- and IL-4R-immunoreactive cells and their protein levels were also similar to those in IL-2 and IL-2R; however, IL-4 and IL-4R immunoreactivity in the periphery of the neuronal cytoplasm in the aged dog was much stronger than that in the young adult dog. These results indicate that the increase of inflammatory cytokines and their receptors in the aged spinal cord might be related to maintaining a balance of inflammatory reaction in the spinal cord during normal aging.     

A Randomised Trial Evaluating the Safety and Immunogenicity of the Novel Single Oral Dose Typhoid Vaccine M01ZH09 in Healthy Vietnamese Children

Background

The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile.

Objectives

We conducted a randomised placebo controlled, single-blind trial to evaluate the safety and immunogenicity of M01ZH09 in healthy Vietnamese children aged 5 to 14 years.

Methods

Subjects were randomly assigned to receive either a nominal dose of 5×10⁹ CFU of M01ZH09 or placebo and were followed up for 28 days. The primary safety outcome was the proportion of subjects with any adverse event attributed to M01ZH09. The primary immunogenicity endpoint was the proportion of subjects who showed a positive immune response to M01ZH09 in the Salmonella Typhi lipopolysaccharide (LPS) specific serum IgA and IgG ELISA.

Principal Findings

One hundred and fifty-one children were enrolled, 101 subjects received M01ZH09 and 50 subjects received placebo. An intention to treat analysis was conducted. There were no serious adverse events and no bacteraemias. In the M01ZH09 group, 26 (26%; 95% CI, 18–5%) of 101 subjects experienced adverse events compared to 11 (22%; 95% CI, 12–36%) of 50 subjects in the placebo group (odds ratio (OR) [95%CI]  = 1.23 [0.550–2.747]; p = 0.691). Faecal shedding of S. Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 was detected in 51 (51%; 95% CI, 41–61%) of 100 M01ZH09 subjects. No shedding was detected beyond day 3. A positive immune response, defined as 70% increase (1.7 fold change) in LPS specific serum IgG (day 14 or 28) and/or 50% increase (1.5 fold change) in LPS specific serum IgA (day 7 or 14) from baseline was detected in 98 (97%; 95% CI, 92–99%) of 101 M01ZH09 recipients and 8 (16%; 95% CI, 7–29%) of 50 placebo recipients. Twenty-eight (100%; 95% CI, 88–100%) of 28 vaccine recipients who were evaluated in the LPS specific IgA ELISPOT assay showed a positive response compared to none of the 14 placebo recipients tested.

Conclusions

This was the first phase II trial of a novel oral candidate typhoid vaccine in children in an endemic country. M01ZH09 had an appropriate safety profile and was immunogenic in children.

Trial Registration

Controlled-trials.comISRCTN91111837     

Safety assessment of potential lactic acid bacteria Bifidobacterium longum SPM1205 isolated from healthy Koreans

The safety assessment of Bifidobacterium longum SPM1205 isolated from healthy Koreans and this strain's inhibitory effects on fecal harmful enzymes of intestinal microflora were investigated. The overall safety of this strain was investigated during a feeding trial. Groups of SD rats were orally administered a test strain or commercial reference strain B. longum 1 x 10(9) CFU/kg body weight/day for four weeks. Throughout this time, their feed intake, water intake and live body weight were monitored. Fecal samples were periodically collected to test harmful enzyme activities of intestinal microflora. At the end of the four-week observation period, samples of blood, liver, spleen, kidney, and gut tissues were collected to determine for hematological parameters and histological differences. The results obtained in this experiment demonstrated that four weeks of consumption of this Bifidobacterium strain had no adverse effects on rat's general health status, blood biochemical parameters or histology. Therefore, it is likely to be safe for human use. Fecal harmful enzymes such as beta-glucosidase, beta-glucuronidase, tryptophanase and urease, were effectively inhibited during the administration of the B. longum SPM1205. These results suggested that this B. longum SPM 1205 could be used for humans as a probiotic strain.     

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in ¹. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases ². The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands ³. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways ³.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.     

Vertical distribution of Microcystis population in the regulated Nakdong River, Korea

The vertical distribution of a bloom-forming Microcystis population was studied based on the relevant limnological parameters obtained from the lower Nakdong River (Mulgum) during the summer of 1994. Over three months (late June to late September), a high abundance of Microcystis population (mean ± SD, 2.9 ± 8.4 × 10⁵ cells ml⁻¹, n = 40) and algal biomass (mean ± SD, chlorophyll a, 131 ± 346 μg l⁻¹, n = 31) was persistent throughout the entire water column (0–5 m, n = 11). The vertical distribution of carbon content was uneven, with a high concentration near the surface zone (mean ± SD, total, 7.9 ± 7.8; Microcystis, 5.2 ± 8.3 μg C ml⁻¹, n = 15). Incorporating limnological and meteorological factors, a diel study of the vertical distribution of Microcystis showed that the chlorophyll a concentration was highest near the surface zone on a calm night (wind velocity, <2 m s⁻¹, 2300–700) but was evenly distributed on a windy day (>4 m s⁻¹, 1100–1900). Among many possible factors, wind velocity may have played an important role in controlling the vertical distribution of Microcystis in the lower Nakdong River. Received: July 12, 1999 / Accepted: November 15, 1999