Substitution of the human beta-spectrin promoter for the human agamma-globin promoter prevents silencing of a linked human beta-globin gene in transgenic mice

During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human beta-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Agamma-globin gene linked to a 576-bp fragment containing the human beta-spectrin promoter. In these mice, the beta-spectrin Agamma-globin (betasp/Agamma) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, betasp/Agamma-, psibeta-, delta-, and beta-globin genes showed no developmental switching and expressed both human gamma- and beta-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Agamma-globin gene promoter showed developmental switching and expressed Agamma-globin mRNA in yolk sac and fetal liver erythroid cells and beta-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the gamma-globin promoter with the beta-spectrin promoter allows the expression of the beta-globin gene. We conclude that the gamma-globin promoter is necessary and sufficient to suppress the expression of the beta-globin gene in yolk sac erythroid cells.     

Presence of immunocytes and sulfidopeptide leukotrienes in the inflamed guinea pig distal colon

This study examined whether the immunocyte recruitment associated with a mild inflammatory state induced by acetic acid would produce detectable sulfidopeptide leukotriene (LT) levels from colonic tissues or in dialysates. Histological examination and measurements of peroxidase activities of inflamed tissues indicated edema, hyperplasia and neutrophil infiltration. Significant elevated LTB4 and prostaglandin E2(PGE2) levels were found but only slight elevations in sulfidopeptide LTs occurred. A slight elevation in eosinophil peroxidase indicated that eosinophil infiltration also occurred. The increase in sulfidopeptide LT levels appeared insufficient by itself to alter secretory responses in the distal colon. However, combined with other immunocyte products such as PGs, the sulfidopeptide LTs may influence the symptomology of inflammatory bowel disease.     

WD-40 repeat region regulates Apaf-1 self-association and procaspase-9 activation

The casp9 protein plays a critical role in apoptosis induced by a variety of death stimuli. A regulator of apoptosis, Apaf-1, binds to and activates pro-casp9 in the presence of cytochrome c and dATP, a requirement that is bypassed by deletion of the WD-40 repeats located in the C-terminal half of Apaf-1. In this report, we used constitutively active Apaf-1 mutant lacking the WD-40 repeat region to study the mechanism and regulation of pro-casp9 activation. Mutational analysis revealed that only a small portion of the CED-4 homologous region (residues 456-559) could be deleted without destroying the ability of Apaf-1-(1-559) to activate pro-casp9. Apaf-1 can self-associate to form oligomers. Disruption of Apaf-1 self-association by deletion (Delta109-559) or mutation of the P-loop region (K149R) abrogated Apaf-1-mediated pro-casp9 activation. Forced oligomerization of the caspase recruitment domain of Apaf-1 was sufficient for pro-casp9 activation. Dimerization of chimeric Fpk-pro-casp9 protein with the dimerizer drug FK1012 induced pro-casp9 processing and apoptosis in cells. Significantly, the C-terminal region containing WD-40 repeats interacted with its N-terminal CED-4 homologous region, as determined by immunoprecipitation experiments. Importantly, expression of the WD-40 repeat region inhibited Apaf-1 self-association and proteolytic activation of pro-casp9. These studies provide a mechanism by which Apaf-1 promotes autoactivation of pro-casp9 through Apaf-1 self-association, a process that is negatively regulated by the WD-40 repeats.     

Diagnosis of fetal renal anomalies

Urinary tract abnormalities are fairly common in the perinatal period. Ultrasonography is an effective diagnostic tool for identifying fetal urinary tract anomalies and can be helpful in establishing prognosis and guiding treatment decisions. The sonographer is encouraged to characterize and localize the fetal urinary tract abnormality when identified. In doing so, the correct diagnosis can be selected from the differential possibilities, providing vital clinical information needed to develop a care plan for both the pregnant woman and her fetus.     

Identification of an opioid kappa receptor subtype-selective N-substituent for (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine

A three-component library of compounds was prepared in parallel using multiple simultaneous solution-phase synthetic methodology. The compounds were biased toward opioid receptor antagonist activity by incorporating (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (a potent, nonselective opioid pure antagonist) as one of the monomers. The other two monomers, which included N-substituted or unsubstituted Boc-protected amino acids and a range of substituted aryl carboxylic acids, were selected to add chemical diversity. Screening of these compounds in competitive binding experiments with the kappa opioid receptor selective ligand [3H]U69,593 led to the discovery of a novel kappa opioid receptor selective ligand, N-?(2'S)-[3-(4-hydroxyphenyl)propanamido]-3'-methylbutyl?-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (8, RTI-5989-29). Additional structure-activity relationship studies suggested that 8 possesses lipophilic and hydrogen-bonding sites that are important to its opioid receptor potency and selectivity. These sites appear to exist predominantly within the kappa receptor since the selectivity arises from a 530-fold loss of affinity of 8 for the mu receptor and an 18-fold increase in affinity for the kappa receptor relative to the mu-selective ligand, (+)-N-[trans-4-phenyl-2-butenyl]-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (5a). The degree of selectivity observed in the radioligand binding experiments was not observed in the functional assay. According to its ability to inhibit agonist stimulated binding of [35S]GTPgammaS at all three opioid receptors, compound 8 behaves as a mu/kappa opioid receptor pure antagonist with negligible affinity for the delta receptor.     

Regional 5-hydroxyindoleacetic acid production in humans

Veno-arterial plasma concentration differences and regional organ plasma flows were used to quantify the relative amounts of 5-hydroxyindoleacetic acid (5-HIAA) contributed by various sites into the peripheral circulation. Positive venoarterial concentration gradients were found in the hepatosplanchnic, forearm, cardiac and jugular vessels in the healthy subjects. The renal circulation was determined to be the principal site of 5-HIAA clearance, extracting 18 +/- 2 nmol/min. The gut was the greatest contributor to the total 5-HIAA plasma pool with the relative contributions of the various organs being as follows: hepatosplanchnic organs 58%, skeletal muscle 26%, brain 6% and the heart 3%. The source of 5-HIAA stemming from these regional beds remains unknown, it may derive from serotonin taken up by and deaminated in ubiquitous endothelial cells, enterochromaffin cells of the gut, peripheral serotonergic nerves, serotonin turnover in platelets or perhaps the metabolism of serotonin taken up by sympathetic nerves. To test the latter hypothesis we examined 23 patients with chronic congestive heart failure and 9 patients with pure autonomic failure to investigate the possible effects of sympathetic nervous system overactivity and underactivity on peripheral 5-HIAA production and plasma 5-HIAA concentration. The resting arterial plasma 5-HIAA concentration in the heart failure patients was increased three-fold. This elevated plasma 5-HIAA concentration was attributable to an increased rate of whole body 5-HIAA production. The arterial 5-HIAA plasma concentration in the autonomic failure patients was paradoxically elevated, being 70% greater than that of the healthy subjects. The increased 5-HIAA plasma concentration in these patients was accounted for by a reduction in 5-HIAA plasma clearance. In all subjects studied there was a weak relationship only between total body norepinephrine spillover to plasma and the arterial 5-HIAA plasma concentration. We found that in healthy subjects the overflow of 5-HIAA into the hepatic vein was significantly related to the underlying degree of sympathetic activity. It can be concluded that 5-HIAA is produced at a number of sites throughout the body with the arterial plasma concentration being dependent on both the level of production and plasma clearance. By far the majority of 5-HIAA in plasma is derived from the gut with only minimal contribution from the brain.     

Fryns syndrome: prenatal diagnosis and pathologic correlation

From 1988 to 1995, 96 patients (161 feet) underwent a modified Mitchell distal metatarsal osteotomy performed for mild-to-moderate hallux valgus. On AP x-rays of the standing foot, the average intermetatarsal angle was corrected from 15 degrees to 9 degrees, and the first metatarsophalangeal angles were corrected from an average of 41 degrees to 15 degrees. Criteria for evaluation of clinical results included relief of pain, appearance of foot, and shoe wear. After an average follow-up of 38 months, the overall satisfaction rate was 92.5%. Complications included 13 pin tract infections, two delayed unions, and two correction losses. The most common late sequela was transfer metatarsalgia of the lesser toes, which occurred in 20 feet (12.4%), leading to some dissatisfaction. The Mitchell osteotomy can be used on cases with less than 20 degrees of intermetatarsal angle, offering a stable construct with easy postoperative care.