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Comp Biochem Physiol C Toxicol Pharmacol. 2000 Aug;127(1):49-59.
Inhibition of collagen biosynthesis and increases in low molecular weight IGF-I binding proteins in the skin of fasted rats.

Cechowska-Pasko M, Palka J.

Department of Biochemistry, Medical Academy of Bialystok, Poland. pamb.ac.bialystok.pl

Insulin-like growth factor-I (IGF-I) is an important stimulator of collagen biosynthesis and prolidase activity in connective tissue cells. The disturbances in skin collagen metabolism (reflected by significant decrease in skin collagen content, collagen biosynthesis and prolidase activity) in fasted rats were accompanied by decrease in serum IGF-I level. Fasted rat serum was found to contain about 58% of IGF-I (101.6 +/- 15.4 ng/ml) as compared to control rat serum (175.7 +/- 19.8 ng/ml), while the skin of control and fasted rats contained similar concentrations of IGF-I (about 77 ng/g tissue). The insulin-like growth factor binding proteins (IGFBPs) of sera and tissue extracts (known to regulate IGF-I activity) were analysed by ligand blotting. In the serum of control rats one IGFBP band of about 46 kDa (corresponding to the acid-dissociated IGFBP-3) was detected. In the serum of fasted rats the 46 kDa IGFBP was not observed, however, an other IGFBP of about 30 kDa (corresponding to low molecular weight IGFBPs, e.g. IGFBP-1 or IGFBP-2) was found. The intensity of IGF-I binding to the 30 kDa IGFBP was much higher than that of IGFBP-3, found in control rat serum. Control and fasted rat skin contained similar IGFBPs, however their IGF-I binding abilities were much lower, compared to their serum counterparts. It was found that 46 kDa and 30 kDa proteins, observed in ligand blotting represent IGFBP-3 and IGFBP-1 or IGFBP-2. respectively as demonstrated by western immunoblot analysis. An increase in IGF-binding to 30 kDa IGFBP-1 and/or IGFBP-2 (known as an inhibitors of IGF-dependent functions) in the skin of fasted rats may explain the mechanism of reduced collagen biosynthesis and deposition in tissues during fasting.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11081412&dopt=Abstract

Fish Shellfish Immunol. 2000 Oct;10(7):623-30.
Identification of viral induced genes in Ig+ leucocytes of Japanese flounder Paralichthys olivaceus, by differential hybridisation with subtracted and un-subtracted cDNA probes.

Aoki T, Hirono I, Kim MG, Katagiri T, Tokuda Y, Toyohara H, Yamamoto E.

Department of Aquatic Biosciences, Tokyo University of Fisheries, Minato, Japan. aokokyo-u-fish.ac.jp

Up-regulated genes of leucocytes expressing immunoglobulin (Ig+ leucocytes) of hirame rhabdovirus (HRV)-infected Japanese flounder were identified by differential hybridisation, using subtracted and un-subtracted cDNA probes. Ig+ leucocytes were separated from apparently healthy and HRV-infected Japanese flounder by the magnetic beads antibody method using mouse anti-Japanese flounder Ig monoclonal antibody (mab). A cDNA library was constructed from HRV-infected Japanese flounder leucocytes, and was screened with subtracted cDNA probes enriched in genes up-regulated by HRV infection. Fifty cDNAs were isolated for further analysis. These included cDNAs coding for homologues of interferon-inducible 56K protein (IFI56), Stat3, CEF-10, RGS5, inducible poly(A) binding protein, prolylcarboxylpeptidase, basigin III (Ig superfamily), MUC-18 (Ig superfamily), proteasome-nexin 1 (SERPIN), herpes virus entry mediator (TNFR family), collagenase III, gelatinase-b, megakaryocyte stimulating factor, Rab8-interacting protein, IgM, IgD and 20 unknown cDNA clones. The majority of these identified genes are reported for the first time in fish. From leucocytes mRNA for homologues of IFI56, CEF-10, Stat3, SERPIN and inducible poly (A) binding protein expression was shown to increase following HRV infection.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11081439&dopt=Abstract

Br J Pharmacol. 2000 Nov;131(6):1195-203.
The pharmacology of nucleotide receptors on primary rat brain endothelial cells grown on a biological extracellular matrix: effects on intracellular calcium concentration.

Sipos I, Domotor E, Abbott NJ, Adam-Vizi V.

Department of Medical Biochemistry, Semmelweis University, Budapest, H-1444, P.O. Box 262, Hungary.

1. Brain capillary endothelial cells express a variety of nucleotide receptors, but differences have been reported between culture models. This study reports examination of nucleotide receptors on primary cultured rat brain capillary endothelial cells (RBCEC) grown on a biological extracellular matrix (ECM) to produce a more differentiated phenotype. 2. Fura-2 fluorescence ratio imaging was used to monitor intracellular free calcium concentration [Ca(2+)](i). ATP, UTP, and 2-methylthioATP (2-MeSATP) increased [Ca(2+)](i) to similar levels, while 2-MeSADP, ADP and adenosine gave smaller responses. 3. Removal of extracellular calcium caused no significant change in the [Ca(2+)](i) response to 2-MeSATP, evidence that the response was mediated by a metabotropic (P2Y) receptor. 4. All cells tested responded to ATP, UTP, 2-MeSATP and ADP, while 63% responded to adenosine and 50% to 2-MeSADP. No cells responded to alpha, beta-methyleneATP. Cells grown on rat tail collagen instead of ECM gave smaller and less uniform [Ca(2+)](i) responses, suggesting that the differentiating effect of the ECM contributed to a more uniform receptor profile. 5. The [Ca(2+)](i) response to the P2Y(1)-selective agonist 2-MeSADP was abolished in the presence of the subtype-selective antagonist adenosine 3'-phosphate 5'-phosphosulphate (PAPS). 6. The P2Y(2) antagonist suramin completely blocked the response to ATP and inhibited the response to UTP by 66%. 7. The A(1) subtype-selective adenosine receptor agonist N(6)-Cyclopentyladenosine (CPA) gave a small but characteristic [Ca(2+)](i) response, while A(2A) and A(2B) subtype-selective agonists failed to generate [Ca(2+)](i) changes. 8. The results are consistent with the presence on RBCEC of a P2Y(2)-like receptor coupled to phospholipase C, and a P2Y(1)-like receptor mobilizing intracellular Ca(2+). The role of multiple nucleotide receptors in the function of the brain endothelium is discussed.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11082128&dopt=Abstract

Br J Pharmacol. 2000 Nov;131(6):1204-10.
Effect of neutral endopeptidase inhibitor on endogenous atrial natriuretic peptide as a paracrine factor in cultured cardiac fibroblasts.

Maki T, Horio T, Yoshihara F, Suga S, Takeo S, Matsuo H, Kangawa K.

Research Institute, National Cardiovascular Center, 5-7-1, Fujishirodai, Suita, Osaka 565-8565, Japan.

1. Cardiac remodelling is a fundamental response to hypertension, myocardial infarction and chronic heart failure, and involves cardiac fibroblast proliferation and production of extracellular matrix components such as collagen. The present study was performed to examine the role of endogenous atrial natriuretic peptide (ANP) as a possible paracrine factor for cardiac fibroblasts, and to examine the effects of three neutral endopeptidase (NEP) inhibitors, thiorphan, phosphoramidon and ONO-BB-039-02 (ONO-BB) on endogenous ANP-induced changes in collagen synthesis by cultured neonatal rat cardiac fibroblasts. 2. Each NEP inhibitor singly had no significant effect on collagen synthesis by cardiac fibroblasts, except for maximum concentration (10(-3) M) of thiorphan. 3. Exogenous ANP inhibited collagen synthesis in a concentration-dependent manner (10(-8) - 10(-6) M). Thiorphan (10(-4) and 10(-3) M) and phosphoramidon (10(-5) and 10(-4) M) enhanced the ANP (10(-7) M)-induced decrease in collagen synthesis. ONO-BB (10(-5) and 10(-4) M) slightly enhanced the ANP-induced decrease in collagen synthesis. 4. Myocyte-conditioned medium (MC-CM), as well as exogenous ANP, inhibited collagen synthesis dose-dependently. The decrease in collagen synthesis at 100% MC-CM was augmented by thiorphan (10(-3) M), phosphoramidon (10(-4) M) and ONO-BB (10(-4) M). 5. HS-142-1, a natriuretic peptide receptor antagonist, significantly reduced the MC-CM plus thiorphan- and MC-CM plus ONO-BB-induced decrease in collagen synthesis, by 92 and 62%, respectively and showed a tendency to attenuate the MC-CM plus phosphoramidon-induced decrease in collagen synthesis by 40%. 6. Our observations suggested that endogenous ANP released from cardiomyocytes inhibited collagen synthesis as a paracrine factor and that NEP inhibitors enhanced the activity of this peptide in cardiac fibroblasts.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11082129&dopt=Abstract

Hypertension. 2000 Nov;36(5):845-50.
Angiotensin II type 2 receptors stimulate collagen synthesis in cultured vascular smooth muscle cells.

Mifune M, Sasamura H, Shimizu-Hirota R, Miyazaki H, Saruta T.

Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.

Previously, we and others have shown that angiotensin II enhances vascular smooth muscle cell extracellular matrix synthesis via stimulation of the angiotensin II type 1 (AT(1)) receptor. Recently, expression of the type 2 (AT(2)) receptor has been confirmed in the adult vasculature, but its role has not yet been fully defined. The aim of the present study was to examine the effects of stimulation of AT(2) receptors on collagen synthesis in vascular smooth muscle cells. Retroviral gene transfer was used to supplement adult vascular smooth muscle cells with AT(2) receptors to mimic the vasculature in vivo. The treatment of these cells with the AT(2) receptor agonist CGP42212A (10(-7) mol/L) alone did not cause a significant change in p42/p44 MAP kinase activity but caused a modest (30% to 50%) decrease in protein tyrosine phosphatase activity. Treatment with CGP42112A also caused a dose- and time-dependent increase in both cell-associated and secretory collagen synthesis (148+/-17% of control at 48 hours, P<0.05), which was completely inhibited by the AT(2) receptor antagonist PD123319, unaffected by the AT(1) receptor antagonist losartan, and attenuated by treatment with pertussis toxin or G(alpha)(i) antisense oligonucleotides. Interestingly, studies in other cell lines demonstrated that CGP42112A caused similar results in transfected mesangial cells but had essentially opposite effects in fibroblasts (NIH-3T3-AT(2)). These results suggest that AT(2) receptor stimulation can increase collagen synthesis in vascular smooth muscle cells via a G(alpha)(i)-mediated mechanism and provide evidence for heterogeneity in the effects of AT(2) receptor stimulation in different tissues.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11082154&dopt=Abstract

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