Comparative effects of GLP-1 and GIP on cAMP production, insulin secretion, and in vivo antidiabetic actions following substitution of Ala8/Ala2 with 2-aminobutyric acid

The two major incretin hormones, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP), are currently being considered as prospective drug candidates for treatment of type 2 diabetes. Interest in these gut hormones was initially spurred by their potent insulinotropic activities, but a number of other antihyperglycaemic actions are now established. One of the foremost barriers in progressing GLP-1 and GIP to the clinic concerns their rapid degradation and inactivation by the ubiquitous enzyme, dipeptidyl peptidase IV (DPP IV). Here, we compare the DPP IV resistance and biological properties of Abu(8)/ Abu(2) (2-aminobutyric acid) substituted analogues of GLP-1 and GIP engineered to impart DPP IV resistance. Whereas (Abu(8))GLP-1 was completely stable to human plasma (half-life > 12h), GLP-1, GIP, and (Abu(2))GIP were rapidly degraded (half-lives: 6.2, 6.0, and 7.1 h, respectively). Native GIP, GLP-1, and particularly (Abu(8))GLP-1 elicited significant adenylate cyclase and insulinotropic activity, while (Abu(2))GIP was less effective. Similarly, in obese diabetic (ob/ob) mice, GIP, GLP-1, and (Abu(8))GLP-1 displayed substantial glucose-lowering and insulin -releasing activities, whereas (Abu(2))GIP was only weakly active. These studies illustrate divergent effects of penultimate amino acid Ala(8)/Ala(2) substitution with Abu on the biological properties of GLP-1 and GIP, suggesting that (Abu(8))GLP-1 represents a potential candidate for future therapeutic development. (C) 2004 Elsevier Inc. All rights reserved.

Degradation, cyclic adenosine monophosphate production, insulin secretion, and glycemic effects of two novel N-terminal Ala(2)-substituted analogs of glucose-dependent insulinotropic polypeptide with preserved biological activity in vivo

Glucose-dependent insulinotropic polypeptide (GIP) has significant potential in diabetes therapy due to its ability to serve as a glucose-dependent activator of insulin secretion. However, its biological activity is severely compromised by the ubiquitous enzyme dipeptidylpeptidase IV (DPP IV), which removes the N-terminal Tyr(1)-Ala(2) dipeptide from GIP. Therefore, 2 novel N-terminal Ala(2)-substituted analogs of GIP, with Ala substituted by 2-aminobutyric acid (Abu) or sarcosine (Sar), were synthesized and tested for metabolic stability and biological activity both in vitro and in vivo. Incubation with DPP IV gave half-lives for degradation of native GIP, (Abu(2))GIP, and (Sar(2))GIP to be 2.3, 1.9, and 1.6 hours, respectively, while in human plasma, the half-lives were 6.2, 7.6, and 5.4 hours, respectively. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, native GIP, (Abu(2))GIP, and (Sar(2))GIP dose-dependently stimulated cyclic adenosine monophosphate (camp) production with EC50 values of 18.2, 38.5, and 54.6 nmol/L, respectively. In BRIN-BD11 cells, both (Abu(2))GIP and (Sar(2))GIP (10(-13) to 10(-8) mol/L) dose-dependently stimulated insulin secretion with significantly enhanced effects at 16.7 mmol/L compared with 5.6 mmol/L glucose. In obese diabetic (ob/ob) mice, GIP and (Sar(2))GIP significantly increased (1.4-fold to 1.5-fold; P <.05) plasma insulin concentrations, whereas (Abu(2))GIP exerted only minor effects. Changes in plasma glucose were small reflecting the severe insulin resistance of this mutant. The present data show that substitution of the penultimate N-terminal Ala(2) in GIP by Abu or Sar results in analogs with moderately reduced metabolic stability and biological activity in vitro, but with preserved biological activity in vivo. (C) 2003 Elsevier Inc. All rights reserved.

Clinical and immunohistochemical assessment of vulval intraepithelial neoplasia following photodynamic therapy using a novel bioadhesive patch-type system loaded with 5-aminolevulinic acid

Background: The work in this study appraised photodynamic treatment (PDT) as a treatment method for vulval intraepithelial neoplasia (VIN) using a novel bioadhesive patch to deliver aminolevulinic acid. An analysis of changes in expression of apoptotic and cell cycle proteins (p53, p21, Mdm2, Blc-2, Bax, Ki-67) in response to PDT was evaluated. Methods: PDT was performed using non-laser light, either as a one or two-cycle treatment, with clinical and pathological assessment following after 6 weeks. Twenty-three patients with 25 VIN lesions underwent 49 cycles of PDT Patches were designed to conform to uneven vulval skin and contained 38 mg cm(-2) aminolevulinic acid. Assessment was carried out at 6 weeks post-treatment. Patient-based treatment assessment, along with clinical and pathological changes, were monitored. Immunohistochemical staining was used to elucidate a possible biomolecular basis for induced cellular changes. Results: Most patients (52%) reported a symptomatic response, with normal pathology restored in 38% of lesions. The patch was easy to apply and remove, causing minimal discomfort. Fluorescence inspection confirmed protoporphyrin accumulation. Pain during implementation of PDT was problematic, necessitating some form of local analgesia. Changes in expression of cell cycle and apoptotic-related proteins suggested involvement of apoptotic pathways. Down regulation of p21 and inverse changes in Bcl-2 and Bax were key findings. Conclusion: Treatment of VIN lesions using a novel bioadhesive patch induced changes in cell cycle and apoptotic proteins in response to PDT with possible utilisation of apoptotic pathways. The efficacy of PDT in treating VIN could be improved by a better understanding of these apoptotic mechanisms, the influence of factors, such as HPV status, and of the need for effective pain management.

Partial agonism, neutral antagonism, and inverse agonism at the human wild-type and constitutively active cholecystokinin-2 receptors

Cholecystokinin receptor-2 (CCK2R) is a G protein receptor that regulates a number of physiological functions. Activation of CCK2R and/or expression of a constitutively active CCK2R variant may contribute to human diseases, including digestive cancers. Search for antagonists of the CCK2R has been an important challenge during the last few years, leading to discovery of a set of chemically distinct compounds. However, several early-discovered antagonists turned out to be partial agonists. In this context, we carried out pharmacological characterization of six CCK2R antagonists using COS-7 cells expressing the human CCK2R or a CCK2R mutant having a robust constitutive activity on inositol phosphates production, and we investigated the molecular mechanisms which, at a CCK2R binding site, account for these features. Results indicated that three compounds, 3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3- yl)-N'-(3-methylphenyl)urea (L365,260), 4-{[2-[[3-(lH-indol-3-yl)-2- methyl-1-oxo-2-[[[1.7.7-trimethyl-bicyclo[2.2.1]hept-2-yl)-oxy]carbonyl]amino] propyl]amino]-1-phenylethyl]amino-4-oxo-[lS-la.2[S*(S*)]4a]} -butanoate N-methyl-D-glucamine (PD135, 158), and (R)-1-naphthalenepropanoic acid, b-[2-[[2-(8-azaspiro-[4.5]dec-8-ylcarbonyl)-4,6-dimethylphenyl]amino]-2- oxoethyl] (CR2945), were partial agonists; one molecule, 1-[(R)-2,3-dihydro-1- (2,3-dihydro-1-(2-methylphenacyl)-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl] -3-(3-methylphenyl)urea (YM022), was a neutral antagonist; and two compounds, N-(+)-[1-(adamant-1-ylmethyl)-2,4-dioxo-5-phenyl2,3,4,5-tetrahydro-1H-1, 5-benzodiazepin-3-yl]-N'-phenylurea (GV150,013X) and ([(N-[methoxy-3 phenyl] N-[N-methyl N-phenyl carbamoylmethyl], carbomoylmethyl)-3 ureido]-3-phenyl)2-propionic acid (RPR101,048), were inverse agonists. Furthermore, target- and pharmacophore-based docking of ligands followed by molecular dynamic simulation experiments resulted in consistent motion of aromatic residues belonging to a network presumably important for activation, thus providing the first structural explanations for the different pharmacological profiles of tested compounds. This study confirms that several referenced so-called antagonists are in fact partial agonists, and because of this undesired activity, we suggest that newly generated molecules should be preferred to efficiently block CCK2R-related physiological effects. Furthermore, data on the structural basis for the different pharmacological features of CCK2R ligands will serve to further clarify CCK2R mechanism of activation. Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics.

Novel hapten synthesis for antibody production and development of an enzyme-linked immunosorbent assay for determination of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ)

A heterologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of the furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) was developed. AMOZ was derivatised with 2-(4-formylphenoxy) acetic acid or 2-(3-formylphenoxy) acetic acid to obtain two novel immunizing haptens. The ability of these haptens in producing specific polyclonal antibodies against the nitrophenyl derivative of AMOZ (NPAMOZ) was compared with that of traditional immunizing haptens (derivatised AMOZ with 3-carboxybenzaldehyle or 4-carboxybenzaldehyle). The results indicated that the novel immunizing haptens were able to produce antibodies with almost a two-fold improvement in sensitivity of the ciELISA for NPAMOZ in comparison with the existing antibody based ELISAs. The differences in sensitivity were explained by the molecular modeling of the lowest energy conformations of NPAMOZ and the haptens. Another novel hapten, derivatised AMOZ with 2-oxoacetic acid, was synthesized and used as a heterologous coating hapten. The results showed that this strategy of using only a partial structure of the target molecule as the coating hapten was able to obtain a two to three-fold improvement in sensitivity. This study provided a modern approach for the development of an immunoassay with improved sensitivity for the metabolites of nitrofuran antibiotics. © 2012 Elsevier B.V. All rights reserved.

Taguchi optimisation approach for production of activated carbon from phosphoric acid impregnated palm kernel shell by microwave heating

Taguchi method was applied to investigate the optimal operating conditions in the preparation of activated carbon using palm kernel shell with quadruple control factors: irradiation time, microwave power, concentration of phosphoric acid as impregnation substance and impregnation ratio between acid and palm kernel shell. The best combination of the control factors as obtained by applying Taguchi method was microwave power of 800 W, irradiation time of 17 min, impregnation ratio of 2, and acid concentration of 85%. The noise factor (particle size of raw material) was considered in a separate outer array, which had no effect on the quality of the activated carbon as confirmed by t-test. Activated carbon prepared at optimum combination of control factors had high BET surface area of 1,473.55 m² g-1 and high porosity. The adsorption equilibrium and kinetic data can satisfactorily be described by the Langmuir isotherm and a pseudo-second-order kinetic model, respectively. The maximum adsorbing capacity suggested by the Langmuir model was 1000 mg g-1.

Electrical activity induced by nitric oxide in canine colonic circular muscle.

Nitric oxide generates slow electrical oscillations (SEOs) in cells near the myenteric edge of the circular muscle layer, which resemble slow waves generated by interstitial cells of Cajal (ICCs) at the submucosal edge of this muscle. The properties of SEOs were studied to determine whether these events are similar to slow waves. Rapid frequency membrane potential oscillations (MPOs; 16 +/- 1 cycles/min and 9.6 +/- 0.2 mV) were recorded from control muscles near the myenteric edge. Sodium nitroprusside (0.3 microM) reduced MPOs and initiated SEOs (1.3 +/- 0.3 cycles/min and 13.4 +/- 1.4 mV amplitude). SEOs were abolished by the guanylate cyclase inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxaline-1-one (10 microM). MPOs were abolished by nifedipine (1 microM), whereas SEO frequency increased and the amount of depolarization decreased. BAY K 8644 (1 microM) prolonged SEOs and reduced their frequency. SEOs were abolished by Ni(2+) (0.5 mM), low Ca(2+) solution (0.1 mM Ca(2+)), cyclopiazonic acid (10 microM), and the mitochondrial uncouplers antimycin (10 microM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (1 microM). Oligomycin (10 microM) was without effect. These effects are similar to those described for colonic slow waves. Our results suggest that nitric oxide-induced SEOs are similar in mechanism to slow waves, an activity not previously thought to be generated by myenteric pacemakers.

New ways to break an old bond: the bacterial carbon-phosphorus hydrolases and their role in biogeochemical phosphorus cycling

Phosphonates are organophosphorus molecules that contain the highly stable C-P bond, rather than the more common, and more labile, C-O-P phosphate ester bond. They have ancient origins but their biosynthesis is widespread among more primitive organisms and their importance in the contemporary biosphere is increasingly recognized; for example phosphonate-P is believed to play a particularly significant role in the productivity of the oceans. The microbial degradation of phosphonates was originally thought to occur only under conditions of phosphate limitation, mediated exclusively by the poorly characterized C-P lyase multienzyme system, under Pho regulon control. However, more recent studies have demonstrated the Pho-independent mineralization by environmental bacteria of three of the most widely distributed biogenic phosphonates: 2-aminoethylphosphonic acid (ciliatine), phosphonoacetic acid, and 2-amino-3-phosphonopropionic acid (phosphonoalanine). The three phosphonohydrolases responsible have unique specificities and are members of separate enzyme superfamilies; their expression is regulated by distinct members of the LysR family of bacterial transcriptional regulators, for each of which the phosphonate substrate of the respective degradative operon serves as coinducer. Previously no organophosphorus compound was known to induce the enzymes required for its own degradation. Whole-genome and metagenome sequence analysis indicates that the genes encoding these newly described C-P hydrolases are distributed widely among prokaryotes. As they are able to function under conditions in which C-P lyases are inactive, the three enzymes may play a hitherto-unrecognized role in phosphonate breakdown in the environment and hence make a significant contribution to global biogeochemical P-cycling.

Volatile compounds in Bacillus-fermented soybeans

Thua nao, a traditional, proteolytic, fermented soybean condiment of northern Thailand, was prepared from cooked whole soybeans by natural flora fermentation. The microbial flora during the fermentation was dominated by Bacillus species. The formation of volatile compounds during the fermentation was studied. In addition, the volatile compounds of two samples of commercial dried thua nao and two samples of commercial Japanese natto were analysed. Fermentation led to a large increase in the concentration of total volatile compounds, from 35 mug kg(-1) wet weight in cooked soybeans to 3500 mug kg(-1) wet weight in 72h fermented material. The major volatile compounds in fermented beans were 3-hydroxybutanone (acetoin), 2-methlybutanoic acid, pyrazines, dimethyl disulphide and 2-pentylfuran. Sun drying of 72 h fermented material resulted in the loss of 65% of total volatiles, including important aroma compounds. The commercial dried thua nao samples had low concentrations of total volatile compounds (380 mug kg(-1) wet weight). It is suggested that improved drying/preservation methods are needed to retain aroma compounds in the traditional products. The natto samples were devoid of aldehydes, aliphatic acids and esters, and sulphur compounds, whereas the thua nao samples contained a diversity of these compounds. Previous investigators have reported these compounds in natto and it is not possible to suggest the existence of systematic differences between the volatile compounds in traditional thua nao prepared with an undefined, mixed microbial flora and those in natto fermented with Bacillus subtilis. (C) 2001 Society of Chemical Industry.

Ca2+-activated Cl- current in retinal arteriolar smooth muscle.

PURPOSE: To characterize the biophysical, pharmacologic, and functional properties of the Ca(2+)-activated Cl(-) current in retinal arteriolar myocytes. METHODS: Whole-cell perforated patch-clamp recordings were made from myocytes within intact isolated arteriolar segments. Arteriolar tone was assessed using pressure myography. RESULTS: Depolarizing of voltage steps to -40 mV and greater activated an L-type Ca(2+) current (I(Ca(L))) that was followed by a sustained current. Large tail currents (I(tail)) were observed on stepping back to -80 mV. The sustained current and I(tail) reversed close to 0 mV in symmetrical Cl(-) concentrations. The ion selectivity sequence for I(tail) was I(-)> Cl(-)> glucuronate. Outward I(tail) was sensitive to the Cl(-) channel blockers 9-anthracene-carboxylic acid (9-AC; 1 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 1 mM), and disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS; 1 mM), but only DIDS produced a substantial (78%) block of inward tail currents at -100 mV. I(tail) was decreased in magnitude when the normal bathing medium was substituted with Ca(2+)-free solution or if I(Ca(L)) was inhibited by 1 microM nimodipine. Caffeine (10 mM) produced large transient currents that reversed close to the Cl(-) equilibrium potential and were blocked by 1 mM DIDS or 100 microM tetracaine. DIDS had no effect on basal vascular tone in pressurized arterioles but dramatically reduced the level of vasoconstriction observed in the presence of 10 nM endothelin-1. CONCLUSIONS: Retinal arteriolar myocytes have I(Cl(Ca)), which may be activated by Ca(2+) entry through L-type Ca(2+) channels or Ca(2+) release from intracellular stores. This current appears to contribute to agonist-induced retinal vasoconstriction.

Studies on the biodegradation of fosfomycin: Growth of Rhizobium huakuii PMY1 on possible intermediates synthesised chemically

The first step of the mineralisation of fosfomycin by R. huakuii PMY1 is hydrolytic ring opening with the formation of (1R, 2R)-1,2-dihydroxypropylphosphonic acid. This phosphonic acid and its three stereoisomers were synthesised by chemical means and tested as their ammonium salts for mineralisation as evidenced by release of P-i. Only the (1R, 2R)-isomer was degraded. A number of salts of phosphonic acids such as (+/-)-1,2-epoxybutyl-, (+/-)-1,2-dihydroxyethyl-, 2-oxopropyl-, (+/-)-2-hydroxypropyl-, (+/-)-1-hydroxypropyl- and (+/-)-1-hydroxy-2-oxopropylphosphonic acid were synthesised chemically, but none supported growth. In vitro C-P bond cleavage activity was however detected with the last phosphonic acid. A mechanism involving phosphite had to be discarded as it could not be used as a phosphorus source. R. huakuii PMY1 grew well on (R)- and ( S)- lactic acid and hydroxyacetone, but less well on propionic acid and not on acetone or (R)- and (+/-)-1,2-propanediol. The Pi released from (1R, 2R)-1,2-dihydroxypropylphosphonic acid labelled with one oxygen-18 in the PO3H2 group did not stay long enough in the cells to allow complete exchange of O-18 for O-16 by enzymic turnover.

Inward rectifier potassium channels in the HL-1 cardiomyocyte-derived cell line.

HL-1 is a line of immortalized cells of cardiomyocyte origin that are a useful complement to native cardiomyocytes in studies of cardiac gene regulation. Several types of ion channel have been identified in these cells, but not the physiologically important inward rectifier K(+) channels. Our aim was to identify and characterize inward rectifier K(+) channels in HL-1 cells. External Ba(2+) (100?µM) inhibited 44?±?0.05% (mean?±?s.e.m., n?=?11) of inward current in whole-cell patch-clamp recordings. The reversal potential of the Ba(2+)-sensitive current shifted with external [K(+)] as expected for K(+)-selective channels. The slope conductance of the inward Ba(2+)-sensitive current increased with external [K(+)]. The apparent Kd for Ba(2+) was voltage dependent, ranging from 15?µM at -150 ?mV to 148?µM at -75 ?mV in 120 ?mM external K(+). This current was insensitive to 10?µM glybenclamide. A component of whole-cell current was sensitive to 150?µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), although it did not correspond to the Ba(2+)-sensitive component. The effect of external 1 mM Cs(+) was similar to that of Ba(2+). Polymerase chain reaction using HL-1 cDNA as template and primers specific for the cardiac inward rectifier K(ir)2.1 produced a fragment of the expected size that was confirmed to be K(ir)2.1 by DNA sequencing. In conclusion, HL-1 cells express a current that is characteristic of cardiac inward rectifier K(+) channels, and express K(ir)2.1 mRNA. This cell line may have use as a system for studying inward rectifier gene regulation in a cardiomyocyte phenotype.

Phosphonoacetate biosynthesis: In vitro detection of a novel NADP(+)-dependent phosphonoacetaldehyde-oxidizing activity in cell-extracts of a marine Roseobacter

A novel phosphonoacetaldehyde-oxidizing activity was detected in cell-extracts of the marine bacterium Roseovarius nubinhibens ISM grown on 2-aminoethylphosphonic acid (2-AEP; ciliatine). Extracts also contained 2-AEP transaminase and phosphonoacetate hydrolase activities. These findings indicate the existence of a biological route from 2-AEP via phosphonoacetaldehyde for the production of phosphonoacetate, which has not previously been shown to be a natural product. The three enzymes appear to constitute a previously-unreported pathway for the mineralization of 2-AEP which is a potentially important source of phosphorus in the nutrient-stressed marine environment.