Alkylamides from Echinacea are a new class of cannabinomimetics. Cannabinoid type 2 receptor-dependent and -independent immunomodulatory effects

Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor alpha mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB2) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563-569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB2 receptor more strongly than the endogenous cannabinoids. The Ki values of A1 and A2 (CB2 approximately 60 nM; CB1 >1500 nM) were determined by displacement of the synthetic high affinity cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB2, directed by H-bonding and pi-pi interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2+ in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist SR144528. At 50 nM, A1, A2, and the endogenous cannabinoid anandamide (CB2 Ki >200 nM) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB2-dependent manner. A1, A2, anandamide, the CB2 antagonist SR144528 (Ki <10 nM), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor alpha, IL-1beta, and IL-12p70 expression (5-500 nM) in a CB2-independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB2 binding.

The differential effects of thiazolidindiones on atherogenic dyslipidemia in type 2 diabetes: what is the clinical significance?

Diabetic dyslipidemia is typically characterized by an increase in plasma triglycerides, a decrease in high-density lipoprotein cholesterol and a concomitant increase in atherogenic small dense low-density lipoproteins. Thiazolidindiones are able to lower the levels of fasting glucose and glycated hemoglobin significantly by improving insulin sensitivity, as well as improving some aspects of diabetic dyslipidemia: total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol tend to increase while triglycerides are generally decreased.

Effect of 6-month supervised exercise on low-density lipoprotein apolipoprotein B kinetics in patients with type 2 diabetes mellitus

Although low-density lipoprotein (LDL) cholesterol is often normal in patients with type 2 diabetes mellitus, there is evidence for a reduced fractional catabolic rate and consequently an increased mean residence time (MRT), which can increase atherogenic risk. The dyslipidemia and insulin resistance of type 2 diabetes mellitus can be improved by aerobic exercise, but effects on LDL kinetics are unknown. The effect of 6-month supervised exercise on LDL apolipoprotein B kinetics was studied in a group of 17 patients with type 2 diabetes mellitus (mean age, 56.8 years; range, 38-68 years). Patients were randomized into a supervised group, who had a weekly training session, and an unsupervised group. LDL kinetics were measured with an infusion of 1-(13)C leucine at baseline in all groups and after 6 months of exercise in the patients. Eight body mass index-matched nondiabetic controls (mean age, 50.3 years; range, 40-67 years) were also studied at baseline only. At baseline, LDL MRT was significantly longer in the diabetic patients, whereas LDL production rate and fractional clearance rates were significantly lower than in controls. Percentage of glycated hemoglobin A(1c), body mass index, insulin sensitivity measured by the homeostasis model assessment, and very low-density lipoprotein triglyceride decreased (P < .02) in the supervised group, with no change in the unsupervised group. After 6 months, LDL cholesterol did not change in either the supervised or unsupervised group; but there was a significant change in LDL MRT between groups (P < .05) that correlated positively with very low-density lipoprotein triglyceride (r = 0.51, P < .04) and negatively with maximal oxygen uptake, a measure of fitness (r = -0.51, P = .035), in all patients. The LDL production and clearance rates did not change in either group. This study suggests that a supervised exercise program can reduce deleterious changes in LDL MRT.

Synthesis and in vitro characterization of radioiodinatable benzodiazepines selective for type 1 and type 2 cholecystokinin receptors

Radiolabeled antagonists of specific peptide receptors identify a higher number of receptor binding sites than agonists and may thus be preferable for in vivo tumor targeting. In this study, two novel radioiodinated 1,4-benzodiazepines, (S)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (9) and (R)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (7), were developed. They were characterized in vitro as high affinity selective antagonists at cholecystokinin types 1 and 2 (CCK(1) and CCK(2)) receptors using receptor binding, calcium mobilization, and internalization studies. Their binding to human tumor tissues was assessed with in vitro receptor autoradiography and compared with an established peptidic CCK agonist radioligand. The (125)I-labeled CCK(1) receptor-selective compound 9 often revealed a substantially higher amount of CCK(1) receptor binding sites in tumors than the agonist (125)I-CCK. Conversely, the radioiodinated CCK(2) receptor-selective compound 7 showed generally weaker tumor binding than (125)I-CCK. In conclusion, compound 9 is an excellent radioiodinated nonpeptidic antagonist ligand for direct and selective labeling of CCK(1) receptors in vitro. Moreover, it represents a suitable candidate to test antagonist binding to CCK(1) receptor-expressing tumors in vivo.

Long-term cardiovascular and non-cardiovascular mortality in women and men with type 1 and type 2 diabetes mellitus: a 30-year follow-up in Switzerland

While studies from other countries have shown an excess mortality in diabetic individuals when compared with the general population, comparable long-term data is not available for Switzerland.

[Type 2 diabetes among elderly people]

Type 2 Diabetes is frequent among elderly people. The appropriate target for HbA1c in elderly patients ( > 70 years or life expectancy < 10 years) should be around 7.0% (maximally 8%). In patients with multiple co-morbidities, the goal must be an improvement of symptoms and preservation of weight, especially muscle mass. In the setting of an uncontrolled symptomatic diabetes with concomitant catabolism, insulin is the most effective therapy and, therefore, the treatment of choice. The prevention of hypoglycemia must be a major aim. A balanced and regular food intake facilitates therapy and improves quality of life. The priorities of the management of cardiovascular risk factors should be based upon the individual's overall health condition.

[Diabetes update: preventing type 2 diabetes. Individualized stepwise therapy (oral antidiabetic agents). Multifactorial intervention]

Preclinical disorders of glucose metabolism should be systematically included in the high-risk group for diabetes mellitus and affected individuals provided with preventive measures. Their underlying insulin resistance is determined with the help of a checklist and a method called homeostasis model assessment (HOMA). Patients with impaired fasting glucose (IFG) must change their lifestyles. If this does not lead to a response or the patient is unable to modify behavior, medication is required. In the case of manifest type 2 diabetes mellitus, a graded schedule is used for differential management, which should be based on nutritional and exercise therapy. Oral medication with metformin is probably the drug of choice in both obese and non-obese patients. It is crucial not to delay raising the level of treatment until HbA1c has fallen to within an unsatisfactory range (wait-and-see strategy). Rather, the level should be intensified when persistent exacerbation starts to become apparent (proactive therapy). In diabetes mellitus, the same guidelines for secondary prevention apply to the associated cardiovascular risk factors as with coronary heart disease. An intensified and, especially, early treatment is to be preferred over a conservative, wait-and-see approach, in this case as well.

Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

Effects of anticonvulsants on human p450c17 (17alpha-hydroxylase/17,20 lyase) and 3beta-hydroxysteroid dehydrogenase type 2.

PURPOSE Women with epilepsy apparently have a higher incidence of polycystic ovary syndrome (PCOS) than do women without epilepsy. Whether the underlying disease or the antiepileptic drug (AED) treatment is responsible for this increased risk is unknown, although clinical reports implicate valproic acid (VPA) as a potential cause. The steroidogenic enzymes 3beta HSDII (3beta-hydroxysteroid dehydrogenase) and P450c17 (17alpha-hydroxylase/17,20 lyase) are essential for C19 steroid biosynthesis, which is enhanced during adrenarche and in PCOS. METHODS To determine whether the AEDs VPA, carbamazepine (CBZ), topiramate (TPM), or lamotrigine (LYG) directly affect the activities of human 3beta HSDII and P450c17, we added them to yeast expressing human P450c17 or 3beta HSDII and assayed enzymatic activities in the microsomal fraction. RESULTS Concentrations of VPA < or = 10 mM had no effect on activities of P450c17; however, VPA inhibited 3beta HSDII activity starting at 0.3 mM (reference serum unbound concentration, 0.035-0.1 mM) with an IC50 of 10.1 mM. CBZ, TPM, and LTG did not influence 3beta HSDII or P450c17 activities at typical reference serum unbound concentrations, but did inhibit 3beta HSDII and P450c17 at concentrations >10-fold higher. CONCLUSIONS None of the tested AEDs influenced 3beta HSDII or P450c17 activities at concentrations normally used in AED therapy. However, VPA started to inhibit 3beta HSDII activity at concentrations 3 times above the typical reference serum unbound concentration. Because inhibition of 3beta HSDII activity will shift steroidogenesis toward C19 steroid production when P450c17 activities are unchanged, very high doses of VPA may promote C19 steroid biosynthesis, thus resembling PCOS. CBZ, TPM, and LTG influenced 3beta HSDII and P450c17 only at toxic concentrations.

Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c.

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially.

Furosemide inhibits 11beta-hydroxysteroid dehydrogenase type 2.

11Beta-hydroxsteroid dehydrogenase 2 (11beta-OHSD2) protects the nonselective renal mineralocorticoid receptor from the endogenous glucocorticoid cortisol. Thus, drugs inhibiting 11beta-OHSD2 might enhance urinary loss of potassium. As diuretics influence the renal handling of potassium, we analyzed the impact of 13 commonly used diuretics on 11beta-OHSD2. Furosemide was the only inhibitor. Its inhibition constant (Ki) was 30 micromol when extracts from COS-1 cells transfected with human 11beta-OHSD2 were used as an enzyme source. The type of inhibition was competitive. To establish whether furosemide inhibits 11beta-OHSD2 and 11beta-OHSD1 in the renal target tissue, isolated tubular segments from rats were analyzed. Furosemide decreased the oxidative activity of 11beta-OHSD2 in intact distal tubules and 11beta-OHSD1 in proximal convoluted tubules. For the assessment of furosemide on the excretion of corticosterone metabolites in vivo, rats were given furosemide i.p., and the ratio of tetrahydrocorticosterone plus 5alpha-tetrahydrocorticosterone to 11-dehydrotetrahydrocorticosterone was determined in urine. This ratio increased after the administration of furosemide in all animals, indicating inhibition of the oxidative activity of 11beta-OHSD. Thus, furosemide inhibits the 11beta-OHSD2 enzyme in the target tissue and might by that mechanism enhance the mineralocorticoid effect of 11beta-hydroxyglucocorticoids.