Successive phase-transition phenomena and phase diagram of the phosphatidylcholine-water system as revealed by differential scanning calorimetry

A completely dehydrated dipalmitoylphosphatidylcholine (DPPC) was prepared with dehydration under high vacuum and at a temperature above its main transition temperature. Thermal analyses on about forty different samples of the DPPC-water system indicated that the main transition temperature decreased stepwise with an increase in the water content to the limiting temperature at 42.6°C, reflecting the thermal behaviors of a total of five endothermic peaks. The pretransition appeared at a water content above 17 g%, and the predominant role of ‘newly incorporated water’ between the bilayers of DPPC molecules at the pretransition was made evident.     

Fibroblast growth factor-2 inhibits CD40-mediated periodontal inflammation

Fibroblast growth factor-2 (FGF-2) stimulates periodontal regeneration by a broad spectrum of effects on periodontal ligament (PDL) cells, such as proliferation, migration, and production of extracellular matrix. A critical factor in the success of periodontal regeneration is the rapid resolution of inflammatory responses in the tissue. We explored an anti-inflammatory effect of FGF-2 during periodontal regeneration and healing. We found that FGF-2 on mouse periodontal ligament cells (MPDL22) markedly downregulated CD40 expression, a key player of inflammation. In addition, FGF-2 inhibited CD40 signaling by the non-canonical nuclear factor-kappa B2 (NFκB2) pathway, resulting in decreased production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which have the potential to recruit immune cells to inflamed sites. Furthermore, in vivo treatment of FGF-2 enhanced healing of skin wounds by counteracting the CD40-mediated inflammation. These results reveal that FGF-2 has an important function as a negative regulator of inflammation during periodontal regeneration and healing.     

Differential pharmacological properties of GABAA receptors in axon terminals and soma of dentate gyrus granule cells

Although it has been well established that GABA_A receptors are molecular targets of a variety of allosteric modulators, such as benzodiazepines, the pharmacological properties of presynaptic GABA_A receptors are poorly understood. In this study, the effects of diazepam and Zn²⁺ on presynaptic GABA_A receptors have been investigated by measuring the GABA_A receptor-mediated facilitation of spontaneous glutamate release in mechanically dissociated rat CA3 pyramidal neurons. Diazepam significantly enhanced the muscimol-induced facilitation (particularly at submicromolar concentrations) of spontaneous glutamate release and shifted the concentration–response relationship for muscimol toward the left, whereas Zn²⁺ (≤ 100 μM) had little effect on the muscimol-induced facilitation of spontaneous glutamate release. In contrast, Zn²⁺ significantly suppressed the muscimol-induced currents mediated by GABA_A receptors expressed on dentate gyrus granule cells, which are parent neurons of mossy fibers, whereas the effect of diazepam on GABA_A receptors expressed on dentate gyrus granule cells was lesser than that on presynaptic GABA_A receptors. The results suggest that the pharmacological properties of GABA_A receptors differ considerably between presynaptic (axon terminals) and somatic regions in the same granule cell and that presynaptic GABA_A receptors should be considered as one of the important pharmacological targets of many drugs affecting GABA_A receptors.     

Regression of Albuminuria and Hypertension and Arrest of Severe Renal Injury by a Losartan-Hydrochlorothiazide Association in a Model of Very Advanced Nephropathy

Treatments that effectively prevent chronic kidney disease (CKD) when initiated early often yield disappointing results when started at more advanced phases. We examined the long-term evolution of renal injury in the 5/6 nephrectomy model (Nx) and the effect of an association between an AT-1 receptor blocker, losartan (L), and hydrochlorothiazide (H), shown previously to be effective when started one month after Nx. Adult male Munich-Wistar rats underwent Nx, being divided into four groups: Nx+V, no treatment; Nx+L, receiving L monotherapy; Nx+LH, receiving the L+H association (LH), and Nx+AHHz, treated with the calcium channel blocker, amlodipine, the vascular relaxant, hydralazine, and H. This latter group served to assess the effect of lowering blood pressure (BP). Rats undergoing sham nephrectomy (S) were also studied. In a first protocol, treatments were initiated 60 days after Nx, when CKD is at a relatively early stage. In a second protocol, treatments were started 120 days after Nx, when glomerulosclerosis and interstitial fibrosis are already advanced. In both protocols, L treatment promoted only partial renoprotection, whereas LH brought BP, albuminuria, tubulointerstitial cell proliferation and plasma aldosterone below pretreatment levels, and completely detained progression of renal injury. Despite normalizing BP, the AHHz association failed to prevent renal damage, indicating that the renoprotective effect of LH was not due to a systemic hemodynamic action. These findings are inconsistent with the contention that thiazides are innocuous in advanced CKD. In Nx, LH promotes effective renoprotection even at advanced stages by mechanisms that may involve anti-inflammatory and intrarenal hemodynamic effects, but seem not to require BP normalization.     

Detection of Verocytotoxin from Stool and Serological Testing of Patients with Diarrhea Caused by Escherichia coli O157: H 7

The detecion of verocytotoxin (VT) in stool and measurement of antibodies against VT and three antigens (unheated-antigen, LPS, and flagellin) of Escherichia coli O157: H7 in the serum of patients with diarrhea were examined. Five of 14 inpatients during an outbreak had fecal VT2 in stool taken within 5 days of onset to hospitalization. Among these 5, 3 of them also had fecal VT-producing E. coli (VTEC) serotype O157: H7, whereas the other 2 did not. In the passive hemagglutination (PHA) test with formalinized sheep red blood cells sensitized with theee VTEC O157: H7 antigens, 49 (74.2%) of 66 outbreak patients and 3 of 3 sporadic cases had antibodies against both or one of unheated-antigen and LPS of E. coli O157, but none had antibody against flagellin. In addition, anti-VT2 antibody was demonstrated in serum samples from 15 (94%) of 16 inpatients and 2 (4%) of 50 outpatients in an outbreak by a VT-enzyme-linked immunosorbent assay (VT-ELISA). These results showed that serological assay particularly for antibodies against VT and unheated-antigen or LPS of VTEC O157 may provide a useful tool for diagnosis of infection with VTEC O157.     

Cytoprotective Effect of Short-Term Pretreatment with Proanthocyanidin on Human Gingival Fibroblasts Exposed to Harsh Environmental Conditions

Our previous study showed that exposing mouse fibroblasts to proanthocyanidin (PA) for only 1 min accelerated cell proliferation in a concentration-dependent manner. In this study, exposing human gingival fibroblasts (HGFs) to PA for 1 min similarly accelerated the proliferative response of the cells. Besides the accelerated proliferative response, PA showed a cytoprotective effect on HGFs exposed to harsh environmental conditions; short-term exposure of HGFs in the mitotic phase to pure water or physiological saline resulted in a lower recovery of viable cells. Pretreatment and concomitant treatment with PA improved the low recovery of cells exposed to pure water or physiological saline. In addition, HGFs exposed to PA for 1 min proliferated well even after being cultured in serum-free medium. In 100% confluent HGFs, being cultured in serum-free medium resulted in a high intracellular reactive oxygen species (ROS) level, but pretreatment with PA prevented the cells from increasing intracellular ROS. Thus, the results suggest that a short-term PA treatment exerts cytoprotective effects on HGFs exposed to harsh environmental conditions by improving the intracellular oxidative stress response.     

Purification and chemical and physical characterisation of an antitumour polysaccharide from the brown seaweed Sargassum fulvellum

A polysaccharide isolated from a hot-water extract of Sargassum fulvellum and purified by gel-filtration chromatography on Sepharose 4B inhibited the growth of subcutaneous Sarcoma-180 in mice. The purified, active substance appeared to be sodium alginate having a mol. wt. of 33,400 and a molar ratio of mannuronic acid to guluronic acid of 2.78.     

Generation of Live Piglets for the First Time Using Sperm Retrieved from Immature Testicular Tissue Cryopreserved and Grafted into Nude Mice

Cryopreservation of immature testicular tissues is essential for increasing the possibilities of offspring generation by testicular xenografting for agricultural or medical purposes. However, successful production of offspring from the sperm involved has never been reported previously. In the present study, therefore, using intracytoplasmic sperm injection (ICSI), we examined whether xenogeneic sperm obtained from immature pig testicular tissue after cryopreservation would have the capacity to produce live piglets. Testicular fragments from 9- to 11-day-old piglets were vitrified after 10- or 20-min immersion in vitrification solution containing ethylene glycol (EG), polyvinyl pyrrolidone (PVP) and trehalose as cryoprotectants, and then stored in liquid nitrogen for more than 140 days. Thirty nude mice were assigned to each immersion-time group. Testicular fragments were transplanted under the back skin of castrated mice immediately after warming and removal of the cryoprotectants. Blood and testicular grafts were then recovered from the recipient mice on days 60, 120, 180 and 230−350 (day 0 =  grafting). Histological assessment of the testicular grafts and analyses of inhibin and testosterone production revealed no significant differences between the two immersion-time groups, indicating equal growth activity of the cryopreserved tissues. A single sperm obtained from a mouse in each group on day 230−350 was injected into an in vitro-matured porcine oocyte, and then the ICSI oocytes were transferred to the oviducts of estrus-synchronized recipient gilts. One out of 4 gilts that had received oocytes fertilized using sperm from the 10-min immersion group delivered 2 live piglets, and one of another 4 gilts from the 20-min group delivered 4 live piglets. Thus, we have successfully generated porcine offspring utilizing sperm from immature testicular tissues after cryopreservation and transplantation into nude mice. The present model using pigs will be applicable to many large animals, since pigs are phylogenetically distant from the murine recipients.