Pressure activates rat pancreatic stellate cells

Pancreatic stellate cells (PSCs) play a central role in development of pancreatic fibrosis. In chronic pancreatitis, pancreatic tissue pressure is higher than that of the normal pancreas. We here evaluate the effects of pressure on the activation of rat PSCs. PSCs were isolated from the pancreas of Wistar rat using collagenase digestion and centrifugation with Nycodenz gradient. Pressure was applied to cultured rat PSCs by adding compressed helium gas into the pressure-loading apparatus to raise the internal pressure. Cell proliferation rate was assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation. MAPK protein levels and alpha-smooth muscle actin (alpha-SMA) expression were evaluated by Western blot analysis. Concentration of activated transforming growth factor-beta1 (TGF-beta1) secreted from PSCs into culture medium was determined by ELISA. Collagen type I mRNA expression and collagen secretion were assessed by quantitative PCR and Sirius red dye binding assay, respectively. Application of pressure significantly increased BrdU incorporation and alpha-SMA expression. In addition, pressure rapidly increased the phosphorylation of p44/42 and p38 MAPK. Treatment of PSCs with an MEK inhibitor and p38 MAPK inhibitor suppressed pressure-induced cell proliferation and alpha-SMA expression, respectively. Moreover, pressure significantly promoted activated TGF-beta1 secretion, collagen type I mRNA expression, and collagen secretion. Our results demonstrate that pressure itself activates rat PSCs and suggest that increased pancreatic tissue pressure may accelerate the development of pancreatic fibrosis in chronic pancreatitis.     

New highly active taxoids from 9 beta-dihydrobaccatin-9,10-acetals. Part 5

To improve the metabolic stability of 3, which exhibited both in vitro antitumor activity and in vivo efficacy by both iv and po administration, we designed and synthesized new taxane analogues. Most of the synthetic compounds maintained excellent antitumor activity and were scarcely metabolized by human liver microsomes. And some compounds exhibited potent antitumor effects against B16 melanoma BL6 in vivo by both iv and po administration similarly to 3.     

Acoustic stiffness and change in plug cartilage over time after autologous osteochondral grafting: correlation between ultrasound signal intensity and histological score in a rabbit model

We investigated quantitative changes over time in ultrasound signal intensity (an index of stiffness), signal duration (an index of surface irregularity), and interval between signals (an index of thickness) of plug cartilage in an animal model of autologous osteochondral grafting. A full-thickness osteochondral plug was surgically removed and replaced in male Japanese white rabbits (n = 22). Specimens obtained at day 0 and weeks 2, 4, 8, 12 and 24 postoperatively were assessed using an ultrasound system and by macroscopic and histological evaluation (modified Mankin's score). Histology revealed that the plug sank until 2 weeks postoperatively, and that newly formed cartilage-like tissue covered the plug, but at 24 weeks the tissue detached. The plug itself survived well throughout the period of observation. Although the signal intensity at the plug site was same as that in the sham operated contralateral knee at day 0, from 2 to 24 weeks postoperatively it was less than that in the sham knee. At 8 weeks, this difference was significant (P < 0.05). Modified Mankin's score revealed early degenerative changes at the site, but macroscopic examination did not. Signal intensity correlated significantly with score (both at day 0 and at the five postoperative time points [P < 0.05, r = -0.91] and as a whole [P < 0.05, r = -0.36]). Signal intensity also significantly correlated with the individual subscores for 'cartilage structure' (P < 0.05, r = -0.32) and 'cartilage cells' (P < 0.05, r = -0.30) from the modified Mankin's score, but not significantly with subscores for 'staining' and 'tidemark'. Signal duration correlated significantly with total score (as a whole [P < 0.05, r = 0.34]), but not significantly with the score for cartilage structure (P = 0.0557, r = 0.29). The interval between signals reflected well the actual thickness of the plug site. The significant relationships between ultrasound signal intensity and scores suggest that early degenerative changes in plug cartilage and cartilage-like tissue, especially in the superficial layer, are detectable by high-frequency ultrasound assessment.     

Macrophage-Specific Overexpression of Human Matrix Metalloproteinase-12 in Transgenic Rabbits

Increased matrix metalloproteinase-12 (MMP-12) has been implicated in atherosclerosis and many other inflammatory processes. To define MMP-12 functions in vivo, we generated transgenic rabbits that expressed human (h) MMP-12 gene under the control of a macrophage-specific promoter, the human scavenger receptor promoter. Two transgenic founder rabbits were found to have hMMP-12 transgene integration by Southern blot analysis. hMMP-12 mRNA was expressed in peritoneal and alveolar macrophages, and in tissues enriched in macrophages in transgenic rabbits. High levels of hMMP-12 protein were detected in the conditioned media of cultured peritoneal and alveolar macrophages from transgenic rabbits. Zymography showed that hMMP-12 secreted from macrophages possessed enzymatic activity toward β-casein. To evaluate the expression of hMMP-12 in inflammatory sites, we used carrageenan-induced granulomas as an in vivo model for tissue macrophages and foam cells. Granuloma size in transgenic rabbits was significantly increased compared to that in control rabbits, and histological examination revealed that granulomas of transgenic rabbits were enriched in macrophages associated with increased hMMP-12 expression. We believe that this transgenic rabbit model with increased expression of hMMP-12 may become a useful model for further mechanistic studies of MMP-12 in inflammatory diseases and cancer invasion; it is also an ideal model for testing the in vivo action of MMP-12 inhibitors.     

Potent and orally active ET(A) selective antagonists with 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acid structures

The synthesis and structure-activity relationships of a series of 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids are described. Our efforts have been focused on modification of the aryl ring at the 5-position and the alkyl substituent at the 2-position of the bottom 4-methoxyphenyl ring in an effort to develop orally available ET(A) selective antagonists with safer profiles in terms of the P-450 enzyme inhibitory activity. Incorporation of a hydroxymethyl group as an alkyl substituent in methylenedioxyphenyl and 6-dihydrobenzofuran derivatives led to the identification of orally bioavailable ET(A) selective antagonists 1f and 7f. These compounds also showed not only excellent binding affinity (IC(50) < 0.10nM, more than 800-fold selectivity for the ET(A) receptor over the ET(B) receptor) but also sufficient oral bioavailability, 48% and 56%, respectively, in rats. Furthermore, these compounds did not exhibit either competitive or mechanism-based inhibition of human cytochrome P450 enzymes.     

Neuroprotective Mechanisms of Antiparkinsonian Dopamine D2-Receptor Subfamily Agonists

Numerous studies have shown that endogenous and/or environmental neurotoxins and oxidative stress may participate in the pathogenesis of Parkinson's disease (PD), but the detailed mechanisms are still unclear. While dopamine (DA) replacement therapy with L-DOPA (levodopa) improves PD symptoms, it does not inhibit the degeneration of DA neurons in the substantia nigra. Recently, bromocriptine, pramipexole and several other agonists of the dopamine D₂-receptor subfamily (including D₂, D₃ and D₄-subtypes) have been shown to have neuroprotective effects in parkinsonian models in vitro and in vivo. Their neuroprotective effects may be mediated directly and/or indirectly by antioxidant effects, mitochondrial stabilization or induction of the antiapoptotic Bcl-2 family.     

Measurement of fasting serum apoB-48 levels in normolipidemic and hyperlipidemic subjects by ELISA

The present study was designed to evaluate the metabolism of chylomicron and chylomicron remnants by measuring serum apolipoprotein B-48 (apoB-48) levels in 335 normolipidemic and 253 hyperlipidemic subjects using a novel ELISA system. The distribution of fasting serum apoB-48 levels in normolipidemic subjects varied widely, ranging from <1 to >24 microgram/ml (mean, 5.2 +/- 3.8 microgram/ml; median, 3.9 microgram/ml). Serum apoB-48 levels correlated with serum triglyceride (TG) concentrations (r = 0.45, P < 0.001), but not with total cholesterol levels. Serum apoB-48 levels were 7 to 18 times higher in patients with Type I, Type V, and Type III hyperlipidemia, and only slightly higher in patients with Type IIa, Type IIb, and Type IV hyperlipidemia, compared with normolipidemic subjects. The calculated apoB-48/TG ratio was elevated only in patients with dysbetalipoproteinemia (apoE2/2 phenotype). In normolipidemic subjects, oral fat loading resulted in about 2-fold increase in serum apoB-48 levels, with a peak level recorded at 3-4 h postloading, and then returned to the baseline level within 6 h. On the other hand, in patients with dysbetalipoproteinemia, serum apoB-48 levels did not change considerably. Our results indicate that serum apoB-48 is a very useful parameter for evaluating lipoprotein metabolism in exogenous pathways.     

Interisland Mutation of a Novel Phospholipase A<Subscript>2</Subscript> from <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom and Evolution of Crotalinae Group II Phospholipases A<Subscript>2</Subscript>

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A₂ (PLA₂). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA₂, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD₅₀ for mice was 1.34 µg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA₂s based on the amino acid sequences revealed that neurotoxic PLA₂s including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA₂ groups such as PLA₂ type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA₂s showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA₂s, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA₂s evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation. The nucleotide sequences reported in this paper are available from the GenBank/EMBL/DDBJ databases under accession numbers AB102728 and AB102729.     

Treponemal phospholipids inhibit innate immune responses induced by pathogen-associated molecular patterns

Host innate immune responses to microbial components, known as pathogen-associated molecular patterns (PAMPs), are regulated and modified by cellular receptors and serum proteins, including Toll-like receptors (TLRs), CD14, and LPS-binding protein (LBP). We demonstrated that a treponemal membrane lipid inhibited PAMPs-induced immune responses. The chemical structure of the lipid was elucidated as a phosphatidylglycerol (PG) derivative, which is scarce in most mammalian tissues, but relatively abundant in treponemal membrane lipids. Natural and synthetic PG counterparts as well as related natural anionic phospholipids, phosphatidylinositol, phosphatidylserine, and cardiolipin, also demonstrated an inhibitory effect. Further, we noted that PG inhibited PAMPs-induced immune responses by blocking the binding of PAMPs with LBP and CD14. In addition, PG decreased proinflammatory cytokine production in serum of LPS-injected mice and depressed abscess formation in mice infected with treponemes. These results suggest that treponemal phospholipid interfere the function of LBP/CD14 and act as a modulator of innate immune responses.