Kegg Pathway: Alkaloid biosynthesis II

Chirality. 2009 Nov 19;
Sun DL, Huang SD, Wu PS, Li J, Ye YJ, Jiang HD

Tetrahydropalmatine (THP) is one of the active Alkaloid ingredients of Rhizoma Corydalis. THP has a chiral center, and the stereoselective pharmacokinetics and tissue distribution have been reported. The aim of the present article is to study the stereoselective protein binding of THP using equilibrium dialysis followed by HPLC-UV analysis. The results showed that THP stereoselectively binds to human serum albumin (HSA), alpha(1)-acid glycoprotein (AGP), and proteins in human plasma. The fraction binding of (+)-THP was significantly higher than that of (-)-THP, whereas such stereoselectivity was not found in rat plasma. The affinity of HSA and AGP to (+)-THP, expressed as nK(A), were 9.0 x 10(3) M(-1) and 2.34 x 10(5) M(-1), respectively, which were notablely higher than to (-)-THP, with the nK(A) of 3.4 x 10(3) M(-1) and 1.44 x 10(5) M(-1), respectively. The binding site of HSA for (-)-THP was Site I, whereas for (+)-THP was both Site I and Site II. The F1/S variants of AGP were proved to be the key variants (-)- and (+)-THP binding to both. Finally, the AGP binding drugs, such as mifepristone, were demonstrated to reduce the fraction binding of (-)- and (+)-THP with pure AGP (1 mg/ml) but did not affect the fraction binding of both (-)- and (+)-THP with proteins in human plasma. It can be concluded that protein binding of THP is species dependent and stereoselective, both HSA and AGP contribute to the stereoselective binding to THP enatiomers, and AGP binding drugs may not cause the drug-drug interaction on THP in healthy human plasma. Chirality, 2010. (c) 2009 Wiley-Liss, Inc.

Rapid Commun Mass Spectrom. 2009 Nov 16; 23(24): 3907-3916
Xiong A, Yang L, He Y, Zhang F, Wang J, Han H, Wang C, Bligh SW, Wang Z

Hepatotoxic pyrrolizidine Alkaloid (HPA)-containing plants have always been a threat to human and livestock health worldwide. Adonifoline, a main HPA in Senecio scandens Buch.-Ham. ex D. Don (Qianli guang), was used officially as an infusion in cases of oral and pharyngeal infections in China. In this study in vivo metabolism of adonifoline was studied for the first time by identifying the metabolites of adonifoline present in bile, urine and feces of rats using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS(n)) (ion trap) as well as liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) (quadrupole-time of flight). In total 19 metabolites were identified and, among them, retronecine-N-oxides were confirmed by matching their fragmentation patterns with their fully characterized synthetic compounds. These metabolites are all involved in both phase I and phase II metabolic processes and the principal in vivo metabolism pathways of adonifoline were proposed. Copyright (c) 2009 John Wiley & Sons, Ltd.

Anal Bioanal Chem. 2009 Nov 10;
Philipp AA, Wissenbach DK, Weber AA, Zapp J, Zoerntlein SW, Kanogsunthornrat J, Maurer HH

The Thai medicinal plant Mitragyna speciosa (Kratom in Thai) is misused as a herbal drug of abuse. During studies on the main Kratom Alkaloid mitragynine (MG) in rats and humans, several dehydro analogs could be detected in urine of Kratom users, which were not found in rat urine after administration of pure MG. Questions arose as to whether these compounds are formed from MG only by humans or whether they are metabolites formed from the second abundant Kratom Alkaloid paynantheine (PAY), the dehydro analog of MG. Therefore, the aim of the presented study was to identify the phase I and II metabolites of PAY in rat urine after administration of the pure Alkaloid. This was first isolated from Kratom leaves. Liquid chromatography-linear ion trap mass spectrometry provided detailed structure information of the metabolites in the MS(n) mode particularly with high resolution. Besides PAY, the following phase I metabolites could be identified: 9-O-demethyl PAY, 16-carboxy PAY, 9-O-demethyl-16-carboxy PAY, 17-O-demethyl PAY, 17-O-demethyl-16,17-dihydro PAY, 9,17-O-bisdemethyl PAY, 9,17-O-bisdemethyl-16,17-dihydro PAY, 17-carboxy-16,17-dihydro PAY, and 9-O-demethyl-17-carboxy-16,17-dihydro PAY. These metabolites indicated that PAY was metabolized via the same pathways as MG. Several metabolites were excreted as glucuronides or sulfates. The metabolism studies in rats showed that PAY and its metabolites corresponded to the MG-related dehydro compounds detected in urine of the Kratom users. In conclusion, PAY and its metabolites may be further markers for a Kratom abuse in addition of MG and its metabolites.

Crit Care Med. 2009 Oct; 37(10): 2767-74
Roberts DJ, Goralski KB, Renton KW, Julien LC, Webber AM, Sleno L, Volmer DA, Hall RI

OBJECTIVE: In animals, central nervous system inflammation increases drug accumulation in the brain partly due to a loss of central nervous system drug efflux transporter function at the blood-brain barrier. To determine whether a similar loss of active drug efflux occurs in humans after acute inflammatory brain injury. DESIGN: Observational human pharmacokinetic study. SETTING: Medical-surgical-neurosurgical intensive care unit at a university-affiliated, Canadian tertiary care center. PATIENTS: Patients with acute inflammatory brain injury, including subarachnoid hemorrhage (n = 10), intracerebral and/or intraventricular hemorrhage (n = 4), or closed head trauma (n = 2) who received morphine intravenously after being fitted with cerebrospinal fluid ventriculostomy and peripheral arterial catheters. INTERVENTIONS: We correlated the cerebrospinal fluid distribution of morphine, morphine-3-glucuronide, and morphine-6-glucuronide with the cerebrospinal fluid and plasma concentration of the proinflammatory cytokine interleukin-6 and the passive marker of blood-brain barrier permeability, albumin. MEASUREMENTS AND MAIN RESULTS: Acute brain injury produced a robust inflammatory response in the central nervous system as reflected by the elevated concentration of interleukin-6 in cerebrospinal fluid. Penetration of morphine metabolites into the central nervous system increased in proportion to the neuroinflammatory response as demonstrated by the positive correlation between cerebrospinal fluid interleukin-6 exposure and the area under the curve cerebrospinal fluid/plasma ratio for morphine-3-glucuronide (r = .49, p < .001) and morphine-6-glucuronide (r = .51, p < .001). In contrast, distribution of morphine into the brain was not linked with cerebrospinal fluid interleukin-6 exposure (r = .073, p = .54). Albumin concentrations in plasma and cerebrospinal fluid were consistently in the normal range, indicating that the physical integrity of the blood-brain barrier was likely undisturbed. CONCLUSIONS: Our results suggest that central nervous system inflammation following acute brain injury may selectively inhibit the activity of specific drug efflux transporters within the blood-brain barrier. This finding may have significant implications for patients with neuroinflammatory conditions when administered centrally acting drugs normally excluded from the brain by such transporters.

Nature. 2009 Oct 1; 461(7264): 674-8
Cortes Ledesma F, El Khamisy SF, Zuma MC, Osborn K, Caldecott KW

Topoisomerases regulate DNA topology and are fundamental to many aspects of chromosome metabolism. Their activity involves the transient cleavage of DNA, which, if it occurs near sites of endogenous DNA damage or in the presence of topoisomerase poisons, can result in abortive topoisomerase-induced DNA strand breaks. These breaks feature covalent linkage of the enzyme to the DNA termini by a 3'- or 5'-phosphotyrosyl bond and are implicated in hereditary human disease, chromosomal instability and cancer, and underlie the clinical efficacy of an important class of anti-tumour poisons. The importance of liberating DNA termini from trapped topoisomerase is illustrated by the progressive neurodegenerative disease observed in individuals containing a mutation in tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme that cleaves 3'-phosphotyrosyl bonds. However, a complementary human enzyme that cleaves 5'-phosphotyrosyl bonds has not been reported, despite the effect of DNA double-strand breaks containing such termini on chromosome instability and cancer. Here we identify such an enzyme in human cells and show that this activity efficiently restores 5'-phosphate termini at DNA double-strand breaks in preparation for DNA ligation. This enzyme, TTRAP, is a member of the Mg(2+)/Mn(2+)-dependent family of phosphodiesterases. Cellular depletion of TTRAP results in increased susceptibility and sensitivity to topoisomerase-II-induced DNA double-strand breaks. TTRAP is, to our knowledge, the first human 5'-tyrosyl DNA phosphodiesterase to be identified, and we suggest that this enzyme is denoted tyrosyl DNA phosphodiesterase-2 (TDP2).

Biotechnol Appl Biochem. 2009 Nov; 54(3): 177-86
Kai G, Li L, Jiang Y, Yan X, Zhang Y, Lu X, Liao P, Chen J

Tropane Alkaloids are used medicinally as anticholinergic agents with increasing market demand, so the improvement and production of active components from medicinal plants using molecular biotechnology show great potential for applications that should benefit human healthcare. Two tropinone reductases constitute a branching point in the biosynthesis of tropane Alkaloids. In the present paper, we report for the first time the cloning and characterization of two fulllength cDNAs encoding TRI (tropinone reductase I) (GenBank accession number EU424321) and TRII (tropinone reductase II) (GenBank(R) accession number EU424322) from the solanaceous plant Anisodus acutangulus by rapid amplification of cDNA ends. Sequence comparison indicated that AaTRI (A. acutangulus TRI) and AaTRII (A. acutangulus TRII) had high homology with other tropinone reductases from Hyoscyamus niger, Datura stramonium etc., but AaTRI and AaTRII showed identity of only 60.8%. Phylogenetic-tree analysis showed that AaTRI and AaTRII belong to different clusters and have the closest relationship with H. niger TRI and TRII respectively. Expression-pattern analysis showed that AaTRI and AaTRII were expressed in all tissues tested, including root, stem and leaf, but the transcript level of AaTRI was much lower than AaTRII. Expression of AaTRI and AaTRII could be enhanced by methyl jasmonate, with a weak effect for AaTRI and a strong effect for AaTRII. AaTRI-transformed hairy-root lines were accompanied by a mean 1.87-fold higher level of hyoscyamine and a mean 8-fold higher level of scopolamine compared with control roots, indicating that AaTRI is a promising target for genetic engineering to increase tropane Alkaloid in A. acutangulus.

Am J Physiol Heart Circ Physiol. 2009 Nov; 297(5): H1837-44
Zhang Q, Yao F, O'Rourke ST, Qian SY, Sun C

Angiotensin II (ANG II) increases GABA(B) receptor expression in neuronal cultures from the nucleus tractus solitarII (NTS). In the present study, the chronic effects of ANG II on GABA(B) receptor expression and activity were examined in the NTS of Sprague-Dawley rats. Intracerebroventricular infusion of ANG II caused a significant elevation in blood pressure (BP) and an increase in GABA(B) receptor expression in the NTS. Conversely, chronic N(G)-nitro-l-arginine methyl ester (l-NAME) treatment also increased BP, but had no effect on GABA(B) receptor expression in the NTS. Next, we examined the BP response to the GABA(B) receptor agonist baclofen microinjected into the NTS of ANG II- or artificial cerebrospinal fluid (aCSF)-infused rats. NTS microinjection of baclofen increased BP in both groups of rats. However, the pressor response to baclofen was enhanced in ANG II-infused rats compared with aCSF-infused rats. In addition, bilateral microinjection of the GABA(B) receptor antagonist CGP-35348 into the NTS evoked a decrease in BP in both group of rats, and the depressor responses to CGP-35348 were enhanced in the ANG II-infused rats. In contrast, the pressor responses to the GABA(A) receptor agonist muscimol and the depressor responses to the GABA(A) receptor antagonist bicuculline were comparable between aCSF- and ANG II-infused rats. These results indicate that chronic ANG II infusion stimulates GABA(B) receptor expression and augments GABA(B) receptor-mediated responses in the NTS. This effect could contribute to the central nervous system actions of ANG II that result in dampening of baroreflexes and elevation in arterial BP.

Biochemistry. 2009 Nov 3; 48(43): 10255-66
Yagi YI, Abe K, Ikebukuro K, Sode K

Pseudohypoaldosteronism type II (PHAII) is caused by the mutation of two members of the WNK (with-no-K[Lys] kinase) kinase family. We describe here the development of an in vitro WNK1 microfluidic mobility shift assay for kinetic mechanism studies. Assays using capillary electrophoresis on a microfluidic chip are suitable for both compound selection and mechanistic studies, because of the robustness of this method, as well as its high-throughput feature and insensitivity to the ATP concentration. Double-reciprocal plots of the initial rates versus the concentration of the substrate revealed that the random sequential activity of WNK catalyzed OXSR1 (oxidative stress response kinase-1) phosphorylation. WNK1 inhibitors were then found from among 86 kinases in a commercially available library. Interestingly, the Hck, Lck, and Src inhibitors, PP1 and PP2, exhibited positive inhibition against WNK1. The inhibition mode of PP1 was analyzed to be pure ATP competition with a K(i) value of 12.7 microM, showing noncompetitive inhibition against the OXSR1 peptide. From the structure-based comparison, we found that, since the WNK1 enzymes are categorized as STEs (homologues of yeast Sterile 7, Sterile 11, and Sterile 20 kinases) and Hck belongs to the TK (tyrosine kinase) family on the basis of the results of the Human Kinome Project, the residues at the catalytic site of the WNK1 that interact with PP1 were well-conserved in Hck. We concluded that the compound-based structural alignment enabled us to find interesting relationships among the kinases. This information helps us to screen specific WNK1 therapeutic reagents with no inhibition of the Src, Hck, and Lck kinases for the treatment of hypertension.

Eur J Neurosci. 2009 Sep; 30(6): 1101-10
Bilecki W, Wawrzczak-Bargiela A, Przewłocki R

Persistent changes that take place during the development of opioid addiction are thought to be due to reorganization of synaptic connections in relevant brain circuits. This neuronal plasticity requires trafficking of signaling molecules that are controlled by kinesins. In neurons, kinesin light chain 1 (KLC1) acts as the primary regulator of kinesin action. We observed that KLC1 was enriched in sub-cortical regions of the brain in C57Bl/6J mice. KLC1 expression was especially enriched in the striatum, hippocampus and amygdala, which are known to be involved in opioid addiction. Our study revealed that conditioning of C57Bl/6J mice with morphine elevated KLC1 levels in the amygdala, frontal cortex and hippocampus, but not in the striatum. Further study revealed that alterations in KLC1 protein levels in the studied brain regions correlated with the expression of morphine-induced conditioned place preference. In the cortex, hippocampus and amygdala, KLC1 co-localized with calcium/calmodulin-dependent protein kinase II (CaMKII), suggesting that KLC1 was present in the cell bodies and dendrites of pyramidal neurons. Our findings indicate that KLC1, a molecule involved in dendritic and axonal transport in the brain, is affected during chronic morphine treatment and may be involved in the development of opioid addiction.

J Neurosci. 2009 Aug 26; 29(34): 10488-98
Kolpak AL, Jiang J, Guo D, Standley C, Bellve K, Fogarty K, Bao ZZ

Macropinocytosis is a type of poorly characterized fluid-phase endocytosis that results in formation of relatively large vesicles. We report that Sonic hedgehog (Shh) protein induces macropinocytosis in the axons through activation of a noncanonical signaling pathway, including Rho GTPase and nonmuscle myosin II. Macropinocytosis induced by Shh is independent of clathrin-mediated endocytosis but dependent on dynamin, myosin II, and Rho GTPase activities. Inhibitors of macropinocytosis also abolished the negative effects of Shh on axonal growth, including growth cone collapse and chemorepulsive axon turning but not turning per se. Conversely, activation of myosin II or treatment of phorbol ester induces macropinocytosis in the axons and elicits growth cone collapse and repulsive axon turning. Furthermore, macropinocytosis is also induced by ephrin-A2, and inhibition of dynamin abolished repulsive axon turning induced by ephrin-A2. Macropinocytosis can be induced ex vivo by high Shh, correlating with axon retraction. These results demonstrate that macropinocytosis-mediated membrane trafficking is an important cellular mechanism involved in axon chemorepulsion induced by negative guidance factors.

J Neurochem. 2009 Nov; 111(3): 766-76
Abdul-Hay SO, Luo J, Ashghodom RT, Thatcher GR

The non-steroidal anti-inflammatory drug flurbiprofen is a selective amyloid lowering agent which has been studied clinically in Alzheimer's disease. HCT-1026 is an ester prodrug of flurbiprofen incorporating a nitrate carrier moiety that in vivo provides NO bioactivity and an improved safety profile. In vitro, HCT-1026 retained the cyclooxygenase inhibitory and non-steroidal anti-inflammatory drug activity of flurbiprofen, but at concentrations at which levels of amyloid-beta 1-42 amino acid were lowered by flurbiprofen, amyloid-beta 1-42 amino acid levels were elevated 200% by HCT-1026. Conversely, at lower concentrations, HCT-1026 behaved as a selective amyloid lowering agent with greater potency than flurbiprofen. The difference in concentration-responses between flurbiprofen and HCT-1026 in vitro suggests different cellular targets; and in no case did a combination of nitrate drug with flurbiprofen provide similar actions. In vivo, HCT-1026 was observed to reverse cognitive deficits induced by scopolamine in two behavioral assays; activity that was also shown by a classical nitrate drug, but not by flurbiprofen. The ability to restore aversive memory and spatial working and reference memory after cholinergic blockade has been demonstrated by other agents that stimulate NO/cGMP signaling. These observations add positively to the preclinical profile of HCT-1026 and NO chimeras in Alzheimer's disease.

Transl Res. 2009 Sep; 154(3): 142-52
Harada N, Shimozawa N, Okajima K

Insulin-like growth factor-I (IGF-I) is an important cardioprotective substance. We previously reported that sensory neuron stimulation increases IGF-I production by releasing calcitonin gene-related peptide (CGRP) in spontaneously hypertensive rats (SHRs). Because angiotensin II (Ang II) inhibits sensory neuron activation by interacting with Ang II type 1 (AT(1)) receptors, it is possible that AT(1) receptor blockers (ARBs) increase IGF-I production in SHRs. We examined this possibility in the current study, using the ARBs olmesartan, valsartan, losartan, and telmisartan. Plasma, renal, and cardiac levels of CGRP and IGF-I in SHRs were significantly lower than those in normotensive Wistar Kyoto rats (WKYs) (P < 0.01), which increased to levels found in WKYs after the administration of ARBs. These ARB-induced increases in SHRs were completely reversed by pretreatment with capsazepine (CPZ), which is a specific vanilloid receptor-1 (VR-1) antagonist. The mean arterial blood pressure (MABP) was decreased after administration of ARBs in SHRs, and those decreases were reversed by pretreatment with CPZ. The administration of nifedipine decreased MABP but did not increase CGRP or IGF-I levels in SHRs. Baseline CGRP release and cellular cyclic adenosine 3',5'-monophosphate (cAMP) levels in dorsal root ganglion neurons (DRG) isolated from SHRs were significantly lower than those in DRG isolated from WKYs (P < 0.01). Although ARBs reversed decreases in CGRP release and cAMP levels in the presence of Ang II in DRG isolated from WKYs, they increased CGRP release and cAMP levels in the absence of Ang II in DRG isolated from SHRs. Cellular levels of Ang II were not detected in DRG isolated from WKYs or SHRs, but messenger RNA (mRNA) levels for angiotensin-converting enzyme in DRG were significantly higher in SHRs than in WKYs (P < 0.01). The expression of AT(1) receptors in DRG was not different between WKYs and SHRs. Thus, it is likely that decreases in CGRP release and cAMP levels in DRG isolated from SHRs are mainly caused by AT(1) receptor activation by Ang II through an autocrine mechanism. These observations suggest that ARBs might increase CGRP release from sensory neurons by sensitizing VR-1 activation through increases in cAMP levels, which thereby increased the production of IGF-I in SHRs. These activities of ARBs might at least partly explain their therapeutic effects in areas such as improving insulin resistance in patients with diabetes and hypertension.

Wien Med Wochenschr. 2009; 159(13-14): 317-26
Durrant-Finn U, Osten B, Mügge C, Nenoff P

The uremic pruritus is a very painful symptom suffered by chronic haemodialysis patients and is observed in 22 to 74% of the subjects. The causes of uremic pruritus have not yet been clarified. During the last 20 to 30 years it has been focused on altogether 5 different pathophysiological hypotheses: stimulating influences (e.g. calcium phosphate deposits in the epidermis), stimuli (e.g. secondary hyperparathyroidism), neuropathic injuries (e.g. disturbance of the cutaneous innervation in patients with uremic peripheral neuropathy), and central nervous changes (e.g. accumulation of endorphins in uremic patients which is associated with increasing pruritus), and immunologic conditions. The last mentioned immunological hypothesis has increasing importance, not at least based on the fact that the application of a topical calcineurin inhibitor (tacrolimus) improves the uremic pruritus. However, this fact could not be confirmed in a recent prospective placebo-controlled study from the USA. Only after kidney transplantation with a functioning transplant the uremic pruritus is stopped. That is why no causal therapy exists so far. Actually, the uremic pruritus has to be treated by topical and systemic means in a symptomatic and polypragmatic way only. Urea represents one of the most important "natural moisturizing factors" which are responsible for the hydration of the skin. It has been demonstrated that older patients have decreased urea levels within the stratum corneum of the epidermis, whereas in patients with terminal kidney insufficiency - despite dryness of the skin - as a paradox finding elevated levels of urea have been assessed in the stratum corneum. Because of this reason, the meaning of urea as part of the "natural moisturizing factors" system is not understood, until now. However, there are very promising results of clinical phase II studies showing a significant effect of topical application of 2.5% L-arginine hydrochlorid ointment - a semi-essential amino acid - on improvement of dryness and, in particular, on improvement of pruritus in haemodialysis patients.

Handb Exp Pharmacol. 2009; 25-58
Kiesman WF, Elzein E, Zablocki J

Intense efforts of many pharmaceutical companies and academicians in the A(1) adenosine receptor (AR) field have led to the discovery of clinical candidates that are antagonists, agonists, and allosteric enhancers. The A(1)AR antagonists currently in clinical development are KW3902, BG9928, and SLV320. All three have high affinity for the human (h) A(1)AR subtype (hA(1) K (i) < 10 nM), > 200-fold selectivity over the hA(2A) subtype, and demonstrate renal protective effects in multiple animal models of disease and pharmacologic effects in human subjects. In the A(1)AR agonist area, clinical candidates have been discovered for the following conditions: atrial arrhythmias (tecadenoson, selodenoson and PJ-875); Type II diabetes and insulin sensitizing agents (GR79236, ARA, RPR-749, and CVT-3619); and angina (BAY 68-4986). The challenges associated with the development of any A(1)AR agonist are to obtain tissue-specific effects but avoid off-target tissue side effects and A(1)AR desensitization leading to tachyphylaxis. For the IV antiarrhythmic agents that act as ventricular rate control agents, a selective response can be accomplished by careful IV dosing paradigms. The treatment of type II diabetes using A(1)AR agonists in the clinic has met with limited success due to cardiovascular side effects and a well-defined desensitization of full agonists in human trials (GR79236, ARA, and RPR 749). However, new partial A(1)AR agonists are in development, including CVT-3619 hA(1) AR K(i) = 55nM, hA(2A:hA2B:hA(3))1,000:20, CV Therapeutics), which have the potential to provide enhanced insulin sensitivity without cardiovascular side effects and tachyphylaxis. The nonnucleosidic A(1)AR agonist BAY 68-4986 (capadenoson) represents a novel approach to angina wherein both animal studies and early human studies are promising. T-62 is an A(1)AR allosteric enhancer that is currently being evaluated in clinical trials as a potential treatment for neuropathic pain. The challenges associated with developing A(1)AR antagonists, agonists, or allosteric enhancers for therapeutic intervention are now well defined in humans. Significant progress has been made in identifying A(1)AR antagonists for the treatment of edema associated with congestive heart failure (CHF), A(1)AR agonists for the treatment of atrial arrhythmias, type II diabetes and angina, and A(1)AR allosteric enhancers for the treatment of neuropathic pain.

J Pediatr Surg. 2009 Aug; 44(8): 1611-20
Xu C, Liu W, Chen Z, Wang Y, Xiong Z, Ji Y

PURPOSE: Tetrandrine (Tet) is a bisbenzylisoquinoline Alkaloid isolated from the root of Stephania tetrandra, which has been used in traditional Chinese medicine to treat patients with silicosis, asthma, and pulmonary hypertension, and others and can be used as a pulmonary therapeutic agent. We hypothesized that it can also improve the lung growth in congenital diaphragmatic hernia (CDH) for its multiple biological effects. There are increasing evidences that suggest transforming growth factor beta1(TGF-beta1) plays a crucial role in fetal lung growth and morphogenesis. The aim of this study was to evaluate the effect of prenatal administration of Tet and to investigate its possible mechanism on the expression of TGF-beta1 in the lung of nitrofen-induced CDH rat model. METHODS: A CDH model was induced in pregnant Sprague-Dawley rats by administration of nitrofen on day 9.5 of gestation (Ed9.5 term, day 22). Tetrandrine (30 mg/kg) was given through gavage (once a day, for 3 days) on Ed11.5. Accordingly, there were 3 groups as follows: control (n = 9), CDH (n = 9), and CDH + Tet (n = 9). All the fetuses were delivered by cesarean delivery on Ed16.5, 18.5, and 21.5, respectively, to check if diaphragmatic hernia existed on each fetus, then the lung tissue weight (LW) and body weight (BW) of each fetus were recorded. Histologic evaluations and TGF-beta1 immunohistochemistry staining in the lung sample were performed for image analysis. RESULTS: Diaphragmatic hernia was observed in 95 of the 112 rat fetuses in CDH and CDH + Tet groups on Ed18.5 and Ed21.5 (84.8%), the incidence between the 2 groups had no statistical significance (P = .642). Lung weight/body weight in the CDH group and the CDH + Tet group were lower than that in the control group (P < .01), and LW/BW in the CDH group was lower than that in the CDH + Tet group (P < .05). Observed under the light microscope and electron microscope, marked hypoplasia of the lungs in fetuses among the CDH groups was observed, in contrast to improvement of the lungs in CDH + Tet fetuses. Statistical differences in morphological parameters (percentage of alveoli area, counting bronchus) were found even on Ed16.5 when diaphragm had not closed (P < .01). The number of type II pneumocytes and lamellar bodies in each group had no significant difference (P > .05). The immunoreactivity of TGF-beta1 in CDH group and CDH + Tet group were markedly stronger than that in the control group (P < .01). In addition, TGF-beta1 expression in the CDH group was stronger than that in the CDH + Tet group (P < .01). CONCLUSION: Nitrofen can interfere with lung development early in the fetal rat development before and separate from diaphragm development, and increased expression of TGF-beta1 in the lung of CDH rat model may suppress lung growth and development. Prenatal treatment with Tet can improve the growth of the lung of the nitrofen-induced CDH fetuses and its mechanism seems to be involved in downregulating the expression of TGF-beta1. It is a likely new approach to treat CDH and its coexistent lung hypoplasia by maternal Tet administration.

J Physiol Pharmacol. 2009 Jun; 60(2): 41-7
Konturek PC, Brzozowski T, Engel M, Burnat G, Gaca P, Kwiecien S, Pajdo R, Konturek SJ

Ghrelin is a novel growth hormone (GH)-releasing and orexigenic peptide with anti-inflammatory activities. However, the role of ghrelin in the colonic inflammation is still controversial. The aim of the present study was: 1) to examine the expression of ghrelin and TNF-alpha mRNA in the inflamed colonic mucosa of patients with ulcerative colitis (UC), 2) to analyze the effect of treatment with exogenous ghrelin on the healing of trinitrobenze sulphonic acid (TNBS)-induced colitis in rats, and 3) to assess the effects of ghrelin treatment on mRNA expression for iNOS and protein expression for COX-2 and PPARalpha in intact colonic mucosa and in that with TNBS-induced colitis. Fifteen patients with UC and fifteen healthy controls were enrolled in this study. Expression of ghrelin and TNF-alpha was assessed by semi-quantitative RT-PCR in the colonic mucosal biopsies from UC patients and healthy controls. In addition, the effect of exogenous ghrelin on healing of TNBS colitis was tested in rats without or with capsaicin-induced functional ablation of sensory nerves. Patients with UC showed a significant upregulation of mRNA for ghrelin and TNF-alpha in colonic mucosa as compared to that observed in healthy controls. The expression of ghrelin correlated with the grade of inflammation and expression of TNF-alpha. In rats the exogenous ghrelin administered daily at a dose of 20 microg/kg i.p. significantly accelerated the healing of TNBS colitis and this effect was accompanied by an increase in mRNA expression for iNOS and protein expression for COX-2 in the colonic mucosa. The protein expression for PPARgamma, which was down-regulated in rat colonic mucosa after exposure to TNBS as compared to that in intact colonic mucosa, was not significantly influenced by ghrelin treatment. We conclude that 1) patients with UC show an increased mucosal expression of mRNA for ghrelin in the colonic mucosa which could trigger protective response in inflamed colon; and 2) exogenous ghrelin accelerates healing of colonic lesions in animal model of ulcerative colitis via increased release of NO and PGE(2) due to an increase in iNOS and COX-2 expression and stimulation of sensory neuropeptides such as CGRP released from sensory afferent endings.

Hypertension. 2009 Sep; 54(3): 668-75
Csiszar A, Labinskyy N, Olson S, Pinto JT, Gupte S, Wu JM, Hu F, Ballabh P, Podlutsky A, Losonczy G, de Cabo R, Mathew R, Wolin MS, Ungvari Z

Proliferation of pulmonary arterial smooth muscle cells, endothelial dysfunction, oxidative stress, and inflammation promotes the development of pulmonary hypertension. Resveratrol is a polyphenolic compound that exerts antioxidant and anti-inflammatory protective effects in the systemic circulation, but its effects on pulmonary arteries remain poorly defined. The present study was undertaken to investigate the efficacy of resveratrol to prevent pulmonary hypertension. Rats injected with monocrotaline progressively developed pulmonary hypertension. Resveratrol treatment (25 mg/kg per day, PO, from day 1 postmonocrotaline) attenuated right ventricular systolic pressure and pulmonary arterial remodeling, decreased expression of inflammatory cytokines (tumor necrosis factor-alpha, interleukin 1beta, interleukin 6, and platelet-derived growth factor-alpha/beta), and limited leukocyte infiltration in the lung. Resveratrol also inhibited proliferation of pulmonary arterial smooth muscle cells. Treatment of rats with resveratrol increased expression of endothelial NO synthase, decreased oxidative stress, and improved endothelial function in small pulmonary arteries. Pulmonary hypertension was associated with an upregulation of NAD(P)H oxidase in small pulmonary arteries, which was significantly attenuated by resveratrol treatment. Our studies show that resveratrol exerts anti-inflammatory, antioxidant, and antiproliferative effects in the pulmonary arteries, which may contribute to the prevention of pulmonary hypertension.

Hypertension. 2009 Sep; 54(3): 473-4
Chicoine LG, Stewart JA, Lucchesi PA

Br J Cancer. 2009 Jul 21; 101(2): 232-7
Tubiana-Mathieu N, Bougnoux P, Becquart D, Chan A, Conte PF, Majois F, Espie M, Morand M, Vaissiere N, Villanova G

BACKGROUND: This multicentre, international phase II trial evaluated the efficacy and safety profile of a first-line combination of oral vinorelbine plus capecitabine for women with metastatic breast cancer (MBC). METHODS: Patients with measurable, HER2-negative disease received, as a first line in metastatic setting, 3-weekly cycles of oral vinorelbine 80 mg m(-2) (after a first cycle at 60) on day 1 and day 8, plus capecitabine 1000 mg m(-2) (750 if >or=65 years of age) twice daily, on days 1-14. Treatment was continued until progression or unacceptable toxicity. RESULTS: A total of 55 patients were enrolled and 54 were treated (median age: 58.5 years). Most (78%) had visceral involvement and 63% had received earlier (neo)adjuvant chemotherapy. The objective response rate (RECIST) in 49 evaluable patients was 51% (95% confidence interval (CI), 36-66), including complete response in 4%. The clinical benefit rate (response or stable disease for >or=6 months) was 63% (95% CI, 48-77). The median duration of response was 7.2 months (95% CI, 6.4-10.2). After a median follow-up of 41 months, median progression-free survival was 8.4 months (95% CI, 5.8-9.7) and median overall survival was 29.2 months (95% CI, 18.2-40.1). Treatment-related adverse events were manageable, the main grade 3-4 toxicity was neutropaenia (49%); two patients experienced febrile neutropaenia and three patients had a neutropaenic infection (including one septic death). A particularly low rate of alopaecia was observed. CONCLUSION: These results show that the all-oral combination of oral vinorelbine and capecitabine is an effective and well-tolerated first-line regimen for MBC.

J Neurophysiol. 2009 Sep; 102(3): 1834-42
Hermes ML, Kolaj M, Doroshenko P, Coderre E, Renaud LP

The hypothalamic suprachiasmatic nucleus (SCN) harbors the master circadian pacemaker. SCN neurons produce the amino acid gamma-aminobutyric acid (GABA) and several peptide molecules for coordination and communication of their circadian rhythms. A subpopulation of SCN cells synthesizes vasoactive intestinal polypeptide (VIP) and provides a dense innervation of the subparaventricular zone (SPZ), an important CNS target of the circadian pacemaker. In this study, using patch-clamp recording techniques and rat brain slice preparations, the contribution of VIP to SCN efferent signaling to SPZ was evaluated by examining membrane responses of SPZ neurons to exogenous VIP receptor ligands. In approximately 50% of the SPZ neurons receiving monosynaptic GABAA receptor-mediated inputs from SCN, bath-applied VIP (0.5-1 microM) resulted in a membrane depolarization caused by tetrodotoxin-resistant inward currents reversing at approximately -23 mV. These data suggest the existence of postsynaptic receptors that activate a nonselective cationic conductance. In addition, a subset of SPZ neurons showed an increase in the amplitude of SCN-evoked GABAergic inhibitory postsynaptic currents (IPSCs) and a decrease in their paired-pulse ratios. This, together with an increase in frequency of spontaneous and miniature IPSCs, implies the presence of presynaptic receptors that facilitate GABA release from SCN and possibly other synaptic terminals. The effects occurred in separate neurons and could be mimicked by the selective VPAC2 receptor agonist BAY 55-9837 (0.2-0.5 microM) and partially blocked by the VIP receptor antagonist VIP(6-28) (5 microM). The results indicate that VIP acts via both post- and presynaptic VPAC2 receptors to differentially modulate SCN GABAergic signaling to distinct subpopulations of SPZ neurons.