Gas chromatography/mass spectrometry characterization of corticosteroid metabolism in human immortalized keratinocytes

To continue our studies on the cutaneous expression of a proopiomelanocortin/corticotropin-releasing hormone system, we investigated whether this is accompanied by adrenal-type enzymatic activity. Immortalized cultured human keratinocytes were incubated with radiolabeled corticosteroids. Analysis by thin-layer chromatography showed rapid transformation of both progesterone and deoxycorticosterone; one of the progesterone metabolites migrated at the same rate as deoxycorticosterone. Gas chromatography/mass spectrometry further identified as major species of deoxycorticosterone metabolites 3beta,6alpha,21-trihydroxy-5alpha-pregnan-20-one, 3alpha,6alpha,21-trihydroxy-5alpha-pregnan-20-one, and 3alpha5alpha- and 3beta5alpha-tetrahydrodeoxycorticosterone. Minor metabolites were 3alpha,21-dihydroxy-5-pregnen-20- one (3alphaDelta5-21-OHpregnenolone), 3beta,21-dihydroxy-5-pregnen-20-one (3betaDelta5-21-OHpregnenolone), 3alpha,21-dihydroxy-4-pregnen-20-one (3alphaDelta4-21-OHpregnenolone), 6-hydroxy-dihydrodeoxycorticosterone, and two 5-dihydrodeoxycorticosterone species. Thus, in addition to sex steroids keratinocytes also actively metabolize corticosteroids along similar enzymatic pathways. The surprising detection of 3alphaDelta5-21-OHpregnenolone and 3 betaDelta5-21-OHpregnenolone, indicating Delta4-ketosteroids to Delta5-hydroxysteroids conversion, provides strong evidence for the occurrence, at least in human keratinocytes, of isomerase activity that allows the reaction to proceed in reverse of its usual direction. As skin expresses 3alpha/beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerases, cutaneous reactions catalyzed by these enzymes must be reversible. In conclusion, besides elements of the corticotropin-releasing hormone/proopiomelanocortin system human keratinocytes show high levels of corticosteroid metabolizing activity. Moreover, the wide array of steroid products generated from a single substrate indicates serial progressive conversion involving 5alpha-reductase, 6alpha-hydroxylase, 3alpha/beta-hydroxysteroid dehydrogenase, and reverse Delta4minus signDelta5 isomerase enzymes. As distinct from the adrenal cortex, production of A, B, Aldo, 18OHdeoxycorticosterone, or F in keratinocytes was absent or below limits of detectability.     

Prevalence of fragile X syndrome in males and females in Indonesia

AIM: To investigate the prevalence of fragile X syndrome (FXS) in intellectually disabled male and female Indonesians. METHODS: This research is an extension of a previously reported study on the identification of chromosomal aberrations in a large cohort of 527 Indonesians with intellectual disability (ID). In this previous study, 87 patients had a chromosomal abnormality, five of whom expressed fragile sites on Xq27.3. Since FXS cannot always be identified by cytogenetic analysis, molecular testing of the fragile X mental retardation 1 CGG repeat was performed in 440 samples. The testing was also conducted in the five previously identified samples to confirm the abnormality. In total, a molecular study was conducted in 445 samples (162 females and 283 males). RESULTS: In the cohort of Indonesian ID population, the prevalence of FXS is 9/527 (1.7%). The prevalence in males and females is 1.5% (5/329) and 2% (4/198), respectively. Segregation analysis in the families and X-inactivation studies were performed. We performed the first comprehensive genetic survey of a representative sample of male and female ID individuals from institutions and special schools in Indonesia. Our findings show that a comprehensive study of FXS can be performed in a developing country like Indonesia where diagnostic facilities are limited. CONCLUSION: The prevalence of FXS is equal in females and males in our study, which suggests that the prevalence of FXS in females could be underestimated.     

Relation between regional and global systolic function in patients with ischemic cardiomyopathy after β-Blocker therapy or revascularization

Background

To assess the relationship between improved regional and global myocardial function in patients with ischemic cardiomyopathy in response to β-blocker therapy or revascularization.

Materials and methods

Cardiovascular Magnetic Resonance (CMR) was performed in 32 patients with ischemic cardiomyopathy before and 8 ± 2 months after therapy. Patients were assigned clinically to β-blocker therapy (n = 20) or revascularization (n = 12). CMR at baseline was performed to assess regional and global LV function at rest and under low-dose dobutamine. Wall thickening was analyzed in dysfunctional, adjacent, and remote segments. Follow-up CMR included rest function evaluation.

Results

Augmentation of wall thickening during dobutamine at baseline was similar in dysfunctional, adjacent and remote segments in both patient groups. Therefore, baseline characteristics were similar for both patient groups. In both patient groups resting LV ejection fraction and end-systolic volume improved significantly (p < 0.05) at follow-up. Stepwise multivariate analysis revealed that improvement in global LV ejection fraction in the β-blocker treated patients was significantly related to improved function of remote myocardium (p < 0.05), whereas in the revascularized patients improved function in dysfunctional and adjacent segments was more pronounced (p < 0.05).

Conclusion

In patients with chronic ischemic LV dysfunction, β-Blocker therapy or revascularization resulted in a similar improvement of global systolic LV function. However, after β-blocker therapy, improved global systolic function was mainly related to improved contraction of remote myocardium, whereas after revascularization the dysfunctional and adjacent regions contributed predominantly to the improved global systolic function.     

Interaction of 16beta-hydroxydehydroepiandrosterone with renal mineralocorticoid receptors.

A previously unidentified mineralocorticoid, 16beta-hydroxydehydroepiandrosterone (16beta OH-DHEA) has recently been isolated from urine extracts of patients with low-renin hypertension and reported to possess one-fortieth the mineralocorticoid potency of aldosterone by bioassay. However, using a radioreceptor assay, we demonstrate the affinity of 16beta-OH-DHEA for renal 3H-aldosterone receptors to be only 0.011 per cent that of unlabeled aldosterone. 16beta-OH-DHEA therefore would not be expected to possess significant mineralocorticoid properties unless its mechanism of action involves binding to a unique class of renal receptors. Until these discrepancies between bioassayable mineralocorticoid activity and affinity for mineralocorticoid receptors can be resolved, the role of 16beta-OH-DHEA in the pathogenesis of low-renin hypertension remains speculative.     

Multi-unit ribozyme-mediated cleavage of bcr-abl mRNA in myeloid leukemias

Chronic myelogenous leukemia is characterized by the Philadelphia chromosome, which at the molecular level results from the fusion of the bcr gene on chromosome 22 and the abl gene on chromosome 9. The bcr-abl fusion gene encodes a novel tyrosine kinase with transforming activity. In this study, we have synthesized a multi-unti ribozyme that targets bcr-abl mRNA. In vitro ribozyme cleavage reactions show increased cleavage efficiency of this multi-unit ribozyme compared with single or double ribozymes. The multiunit ribozyme was then transfected into murine myeloblasts transformed with the bcr-abl gene (32D cells). Ribozyme transfection was accomplished either by liposomes or using follic acid-polylysine as a carrier. Multi-unit ribozyme transfection reduced the level of bcr-abl mRNA 3 logs when transfected via folate receptor-mediated uptake into transformed 32D cells. These results suggest that a multi-unit ribozyme could be an effective therapeutic agent for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia.     

Contrast medium-induced adverse reactions: economic outcome

Because the cost of managing an expected greater number of adverse reactions when high-osmolality contrast media (HOM) are used could offset the higher material cost of low-osmolality contrast media (LOM), a prospective study was done of 795 inpatients undergoing any of four procedures involving intravascular injection of HOM: cardiac catheterization, peripheral angiography, head computed tomography (CT), or body CT. The resources used in managing HOM-induced adverse reactions were measured, and the costs of these resources were estimated. Four hundred five patients (51%) had adverse reactions. Reactions were grouped into three classes according to their severity. Class 1 (mild) reactions occurred in 358 patients (45%), class 2 (moderate) reactions occurred in 44 patients (6%), and class 3 (severe) reactions occurred in three patients (0.4%). Ninety-nine patients (12%) consumed resources as a result of an adverse reaction. The average cost of these resources per patient undergoing examination was $1.07 to the radiology department, $5.83 to the hospital, and $12.93 to a charge-paying insurer. Mean (+/- standard deviation) cost to the hospital for managing class 1, class 2, and class 3 reactions were $2.52 +/- $5.33, $24 +/- $54, and $910 +/- $749, respectively. By comparison, the difference in material cost of HOM versus LOM ranged from $93 for body CT to $179 for cardiac catheterization. Even if LOM were to induce no adverse reactions, the increased material cost associated with universal substitution of LOM for HOM would be greater than the expected cost of managing adverse reactions when HOM are used.