Regulation of human liver delta-aminolevulinic acid synthase by bile acids

Aminolevulinic acid synthase 1 (ALAS1) is the rate-limiting enzyme of heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, we describe human hepatic ALAS1 as a new direct target of the bile acid-activated nuclear receptor farnesoid X receptor (FXR). Experiments in primary human hepatocytes and in human liver slices showed that ALAS1 messenger RNA (mRNA) and activity is increased upon exposure to chenodeoxycholic acid (CDCA), the most potent natural FXR ligand, or the synthetic FXR-specific agonist GW4064. Moreover, overexpression of a constitutively active form of FXR further increased ALAS1 mRNA expression. In agreement with these observations, an FXR response element was identified in the 5' flanking region of human ALAS1 and characterized in reporter gene assays. A highly conserved FXR binding site (IR1) within a 175-bp fragment at -13 kilobases upstream of the transcriptional start site was able to trigger an FXR-specific increase in luciferase activity upon CDCA treatment. Site-directed mutagenesis of IR1 abolished this effect. Binding of FXR/retinoid acid X receptor heterodimers was demonstrated by mobility gel shift experiments. Conclusion: These data strongly support a role of bile acid-activated FXR in the regulation of human ALAS1 and, consequently, hepatic porphyrin and heme synthesis. These data also suggest that elevated endogenous bile acids may precipitate neuropsychiatric attacks in patients with acute hepatic porphyrias.

Synergy between trovafloxacin and ceftriaxone against penicillin-resistant pneumococci in the rabbit meningitis model and in vitro

The bactericidal activities of monotherapy with trovafloxacin (-0.37 +/- 0.15 Delta log(10) CFU/ml. h), vancomycin (-0.32 +/- 0.12 Delta log(10) CFU/ml. h), and ceftriaxone (-0.36 +/- 0.19 Delta log(10) CFU/ml. h) for the treatment of experimental meningitis in rabbits due to a clinical penicillin-resistant pneumococcal strain (MIC, 4 mg/liter) were similar. The combination of ceftriaxone with trovafloxacin considerably improved the killing rates (-0.67 +/- 0.16 Delta log(10) CFU/ml. h) and was slightly superior to ceftriaxone with vancomycin (killing rate, -0.53 +/- 0. 22 Delta log(10) CFU/ml. h), the regimen most commonly used in clinical practice. In vitro, synergy was demonstrated between ceftriaxone and trovafloxacin by the checkerboard method (fractional inhibitory concentration index, 0.5) and by time-killing assays over 8 h.

Experimental arthritis: effect on growth parameters and total skeletal calcium

Aim of the study was to investigate the possible mechanisms leading to stunted growth and osteoporosis in experimental arthritis. Fourty-two female rats of 7-8 weeks of age were randomly assigned to three groups of 14 animals each: (a) controls; (b) adjuvant-inoculated (AA); and (c) adjuvant-inoculated rats receiving 10 mg cyclosporin A (CsA) orally for 30 days. Biological parameters studied were: hindpaw swelling; vertebral length progression expressed as Delta increments between days 1 and 30 as a parameter of skeletal growth, and estimation of total skeletal mineral content by dual energy X-ray absorptiometry (n=10 each group) on day 30. Endocrine parameters measured were pulsatile release of growth hormone (rGH) on day 30 following jugular cannulation and measurement of insulin-like growth factor (IGF-1) in pooled plasma from rGH profiles. Results can be summarized as follows: Untreated AA rats exhibited local signs of inflammation in comparison with controls (hindpaw diameter 8.1-8.9 mm vs. 5.3-5.6 mm in controls). Treatment with CsA normalized this parameter (4.9-5.6 mm). Vertebral growth was significantly retarded in AA rats in comparison with controls (214+/-32 vs. 473+/-33 microm; p<0.001). Administration of CsA normalized vertebral size increment with a clear tendency to overgrowth (523+/-43 microm, n.s.). There was also a marked reduction in total skeletal mineral content in diseased (AA) rats as compared to controls (5.8+/-0.1 vs. 7.5+/-0.1g [OH-apatite]; p<0.001), and a moderate but significant increment above controls in the group receiving CsA (8.0+/-0.1 vs. 7.5+/-0.1g [OH-appatite]; p<0.04). Integrated rGH profiles exhibited a significant fall in arthritic rats and were completely restored to normal under CsA treatment. A trend toward higher rGH values was observed in the latter group (2908+/-554 in AA vs. 8317+/-1492 ng/ml/240 min in controls; p<0.001, and 10940+/-222 ng/ml/240 min, n.s. in the CsA group). There was a good correlation between skeletal growth and rGH pulsatility (r=0.81; p<0.001). IGF-1 followed a similar pattern (630+/-44 in AA vs. 752+/-30 ng/ml in controls; p<0.04, and 769+/-59 ng/ml in the CsA group, n.s. vs. controls). Thus, a clear tendency to skeletal overgrowth following treatment was observed in agreement with the hormonal data. It can therefore be concluded that, in experimental arthritis, attenuated GH-spiking and reduced circulating IGF-1 appear to be causally related to growth retardation, probably mimicking signs and symptoms observed in juvenile arthritis. Therapy with CsA is followed by normalization of hormonal and biological parameters accompanied by a catch up phenomenon in skeletal growth which is also observed clinically in juvenile arthritis. Generalized osteopenia is a prominent feature seemingly connected with the growth abnormalities as they parallel each other during the evolution of the disease and respond equally to therapy.

Differentiating normal hyaline cartilage from post-surgical repair tissue using fast gradient echo imaging in delayed gadolinium-enhanced MRI (dGEMRIC) at 3 Tesla

The purpose was to evaluate the relative glycosaminoglycan (GAG) content of repair tissue in patients after microfracturing (MFX) and matrix-associated autologous chondrocyte transplantation (MACT) of the knee joint with a dGEMRIC technique based on a newly developed short 3D-GRE sequence with two flip angle excitation pulses. Twenty patients treated with MFX or MACT (ten in each group) were enrolled. For comparability, patients from each group were matched by age (MFX: 37.1 +/- 16.3 years; MACT: 37.4 +/- 8.2 years) and postoperative interval (MFX: 33.0 +/- 17.3 months; MACT: 32.0 +/- 17.2 months). The Delta relaxation rate (DeltaR1) for repair tissue and normal hyaline cartilage and the relative DeltaR1 were calculated, and mean values were compared between both groups using an analysis of variance. The mean DeltaR1 for MFX was 1.07 +/- 0.34 versus 0.32 +/- 0.20 at the intact control site, and for MACT, 1.90 +/- 0.49 compared to 0.87 +/- 0.44, which resulted in a relative DeltaR1 of 3.39 for MFX and 2.18 for MACT. The difference between the cartilage repair groups was statistically significant. The new dGEMRIC technique based on dual flip angle excitation pulses showed higher GAG content in patients after MACT compared to MFX at the same postoperative interval and allowed reducing the data acquisition time to 4 min.

T1(Gd) gives comparable information as Delta T1 relaxation rate in dGEMRIC evaluation of cartilage repair tissue

OBJECTIVES: To evaluate the relationship between T1 after intravenous contrast administration (T1Gd) and Delta relaxation rate (DeltaR1) = (1/T1(Gd) - 1/T1o) in the delayed Gadolinium-Enhanced MRI of cartilage (dGEMRIC) evaluation of cartilage repair tissue. MATERIALS AND METHODS: Thirty single MR examinations from 30 patients after matrix-associated autologous chondrocyte transplantations of the knee joint with different postoperative intervals were examined using an 8-channel knee-coil at 3T. T1 mapping using a 3D GRE sequence with a 35/10 degrees flip angle excitation pulse combination was performed before and after contrast administration (dGEMRIC technique). T1 postcontrast (T1(Gd)) and the DeltaR1 (relative index of pre- and postcontrast R1 value) were calculated for repair tissue and the weight-bearing normal appearing control cartilage. For evaluation of the different postoperative intervals, MR exams were subdivided into 3 groups (up to 12 months, 12-24 months, more than 24 months). For statistical analysis Spearman correlation coefficients were calculated. RESULTS: The mean value for T1 postcontrast was 427 +/- 159 ms, for DeltaR1 1.85 +/- 1.0; in reference cartilage 636 +/- 181 ms for T1 postcontrast and 0.83 +/- 0.5 for DeltaR1.The correlation coefficients were highly significant between T1 (Gd) and DeltaR1 for repair tissue (0.969) as well as normal reference cartilage (0.928) in total, and for the reparative cartilage in the early, middle postoperative, and late postoperative interval after surgery (R values: -0.986, -0.970, and -0.978, respectively). Using either T1(Gd) or DeltaR1, the 2 metrics resulted in similar conclusions regarding the time course of change of repair tissue and control tissue, namely that highly significant (P > 0.01) differences between cartilage repair tissue and reference cartilage were found for all follow-up groups. Additionally, for both metrics highly significant differences (P < 0.01) between early follow up and the 2 later postoperative groups for cartilage repair tissue were found. No statistical differences were found between the 2 later follow-up groups of reparative cartilage either for T1 (Gd) or DeltaR1. CONCLUSION: The high correlation between T1 (Gd) and DeltaR1 and the comparable conclusions reached utilizing metric implies that T1 mapping before intravenous administration of MR contrast agent is not necessary for the evaluation of repair tissue. This will help to reduce costs, inconvenience for the patients, simplifies the examination procedure, and makes dGEMRIC more attractive for follow-up of patients after cartilage repair surgeries.

Randomized trial of daily versus weekly administration of 2-chlorodeoxyadenosine in patients with hairy cell leukemia: a multicenter phase III trial (SAKK 32/98)

Daily administration of 2-chlorodeoxyadenosine (Cladribine, CDA) is a standard treatment for hairy cell leukemia, but may cause severe neutropenia and neutropenic fever. This trial compared toxicity and efficacy of weekly versus daily CDA administration. One hundred patients were randomized to receive standard (CDA 0.14 mg/kg/day day 1-5 [Arm A]) or experimental treatment (CDA 0.14 mg/kg/day once weekly for 5 weeks [Arm B]). The primary endpoint was average leukocyte count within 6 weeks from randomization. Secondary endpoints included response rates, other acute hematotoxicity, acute infection rate, hospital admission, remission duration, event-free, and overall survival. There was no significant difference in average leukocyte count. Response rate (complete + partial remission) at week 10 was 78% (95% confidence interval (CI) 64-88%) in Arm A and 68% (95% CI 54-80%) in Arm B (p = 0.13). Best response rates during follow-up were identical (86%) in both arms. No significant difference was found in the rate of grade 3+4 leukocytopenia (94%vs. 84%), grade 3+4 neutropenia (90%vs. 80%), acute infection (44%vs. 40%), hospitalization (38%vs. 34%), and erythrocyte support (22%vs. 30%) within 10 weeks. Overall, these findings indicate that there are no apparent advantages in toxicity and efficacy by giving CDA weekly rather than daily.

Use of concatamers to study GABAA receptor architecture and function: application to delta-subunit-containing receptors and possible pitfalls

Many membrane proteins, including the GABA(A) [GABA (gamma-aminobutyric acid) type A] receptors, are oligomers often built from different subunits. As an example, the major adult isoform of the GABA(A) receptor is a pentamer built from three different subunits. Theoretically, co-expression of three subunits may result in many different receptor pentamers. Subunit concatenation allows us to pre-define the relative arrangement of the subunits. This method may thus be used to study receptor architecture, but also the nature of binding sites. Indeed, it made possible the discovery of a novel benzodiazepine site. We use here subunit concatenation to study delta-subunit-containing GABA(A) receptors. We provide evidence for the formation of different functional subunit arrangements in recombinant alpha(1)beta(3)delta and alpha(6)beta(3)delta receptors. As with all valuable techniques, subunit concatenation has also some pitfalls. Most of these can be avoided by carefully titrating and minimizing the length of the linker sequences joining the two linked subunits and avoiding inclusion of the signal sequence of all but the N-terminal subunit of a multi-subunit construct. Maybe the most common error found in the literature is that low expression can be overcome by simply overloading the expression system with genetic information. As some concatenated constructs result by themselves in a low level of expression, this erroneous assembly leading to receptor function may be promoted by overloading the expression system and leads to wrong conclusions.

Structure of alpha6 beta3 delta GABA(A) receptors and their lack of ethanol sensitivity

Delta (delta) subunit containing GABA(A) receptors are expressed extra-synaptically and mediate tonic inhibition. In cerebellar granule cells, they often form a receptor together with alpha(6) subunits. We were interested to determine the architecture of these receptors. We predefined the subunit arrangement of 24 different GABA(A) receptor pentamers by subunit concatenation. These receptors (composed of alpha(6), beta(3) and delta subunits) were expressed in Xenopus oocytes and their electrophysiological properties analyzed. Currents elicited in response to GABA were determined in presence and absence of 3alpha, 21-dihydroxy-5alpha-pregnan-20-one and to 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol. alpha(6)-beta(3)-alpha(6)/delta receptors showed a substantial response to GABA alone. Three receptors, beta(3)-alpha(6)-delta/alpha(6)-beta(3), alpha(6)-beta(3)-alpha(6)/beta(3)-delta and beta(3)-delta-beta(3)/alpha(6)-beta(3), were only uncovered in the combined presence of the neurosteroid 3alpha, 21-dihydroxy-5alpha-pregnan-20-one with GABA. All four receptors were activated by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol. None of the functional receptors was modulated by physiological concentrations (up to 30 mM) of ethanol. GABA concentration response curves indicated that the delta subunit can contribute to the formation of an agonist site. We conclude from the investigated receptors that the delta subunit can assume multiple positions in a receptor pentamer composed of alpha(6), beta(3) and delta subunits.

Unanticipated structural and functional properties of delta-subunit-containing GABAA receptors

GABA(A) receptors mediate inhibitory neurotransmission in the mammalian brain via synaptic and extrasynaptic receptors. The delta (delta)-subunit-containing receptors are expressed exclusively extra-synaptically and mediate tonic inhibition. In the present study, we were interested in determining the architecture of receptors containing the delta-subunit. To investigate this, we predefined the subunit arrangement by concatenation. We prepared five dual and three triple concatenated subunit constructs. These concatenated dual and triple constructs were used to predefine nine different GABA(A) receptor pentamers. These pentamers composed of alpha(1)-, beta(3)-, and delta-subunits were expressed in Xenopus oocytes and maximal currents elicited in response to 1 mm GABA were determined in the presence and absence of THDOC (3alpha, 21-dihydroxy-5alpha-pregnane-20-one). beta(3)-alpha(1)-delta/alpha(1)-beta(3) and beta(3)-alpha(1)-delta/beta(3)-alpha(1) resulted in the expression of large currents in response to GABA. Interestingly, the presence of the neurosteroid THDOC uncovered alpha(1)-beta(3)-alpha(1)/beta(3)-delta receptors, additionally. The functional receptors were characterized in detail using the agonist GABA, THDOC, Zn(2+), and ethanol and their properties were compared with those of non-concatenated alpha(1)beta(3) and alpha(1)beta(3)delta receptors. Each concatenated receptor isoform displayed a specific set of properties, but none of them responded to 30 mm ethanol. We conclude from the investigated receptors that delta can assume multiple positions in the receptor pentamer. The GABA dose-response properties of alpha(1)-beta(3)-alpha(1)/beta(3)-delta and beta(3)-alpha(1)-delta/alpha(1)-beta(3) match most closely the properties of non-concatenated alpha(1)beta(3)delta receptors. Furthermore, we show that the delta-subunit can contribute to the formation of an agonist site in alpha(1)-beta(3)-alpha(1)/beta(3)-delta receptors.