CAR2 displays unique ligand binding and RXRalpha heterodimerization characteristics.

The constitutive androstane receptor (CAR; NR1I3) regulates the expression of genes involved in xenobiotic metabolism. Alternative splicing of the human CAR gene yields an array of mRNAs that encode structurally diverse proteins. One form of CAR, termed CAR2, contains an additional four amino acids (SPTV) that are predicted to reshape the ligand-binding pocket. The current studies show a marked, ligand-independent, CAR2-mediated transactivation of reporters containing optimal DR-3, DR-4, and DR-5 response elements, and reporters derived from the natural CYP2B6 and CYP3A4 gene promoters. Overexpression of the RXRalpha ligand binding domain was critical for achieving these effects. CAR2 interaction with SRC-1 was similarly dependent on the coexpression of RXRalpha. Mutagenesis of Ser233 (SPTV) to an alanine residue yielded a receptor possessing higher constitutive activity. Alternatively, mutating Ser233 to an aspartate residue drastically reduced the transactivation capacity of CAR2. The respective abilities of these mutagenized forms of CAR2 to transactivate a DR-4 x 3 reporter element correlated with their ability to interact with RxRalpha and to recruit SRC-1 in a ligand-regulated manner. Together, these results demonstrate a robust RXRalpha-dependent recruitment of coactivators and transactivation by CAR2. In addition, CAR2 displays novel dose responses to clotrimazole and androstanol compared with the reference form of the receptor while at the same time retaining the ability to bind CITCO. This result supports a hypothesis whereby the four-amino-acid insertion in CAR2 structurally modifies its ligand binding pocket, suggesting that CAR2 is regulated by a set of ligands distinct from those governing the activity of reference CAR.     

Localization of Apaf1 gene expression in the early development of the mouse by means of in situ reverse transcriptase-polymerase chain reaction.

Apoptosis is an essential ubiquitous process that controls the duration of the life span of cells, thus playing a crucial role in morphogenetic, histogenetic, and phylogenetic developmental processes. Apaf1 (apoptosis protease activating factor 1) is one of the central mediators of the intrinsic apoptotic pathway and a part of the apoptosome, which activates procaspase-3 and promotes cell death. Gene knockout of Apaf1 in mice leads to late embryonic lethality with malformations such as the persistence of interdigital webs and hyperplasia of brain and retina. Therefore, Apaf1 is generally believed to play a crucial role in developmental apoptosis and have a widespread expression. However, its pattern of expression in early development remains unknown. To specify whether Apaf1 indeed plays this key role, we investigated the pattern of gene expression for Apaf1 in mouse embryos on day 7, 9, and 12 of development. Our results show, that gene expression for Apaf1 first occurs within the embryo between day 7 and 9 of development, becoming more widespread toward day 12 and then includes structures, such as yolk sac, mesenchyme, cartilage, heart anlage, otic vesicle, peridermis, and anlagen of the spinal ganglia and vertebral bodies. Our results also show that gene expression for Apaf1 is not ubiquitous in early mouse development. This finding indicates that cell death processes are independent of or less dependent on Apaf1 during this time. Of interest, an active gene expression for Apaf1 is also present in organ anlagen such as heart or intestine, in which no obvious phenotype is seen after Apaf1 deletion. This finding suggests a possible role for Apaf1 in such anlagen as a putative alternative compensatory pathway, which could be switched on in the case of defects in the mediators that are normally involved in such organs.     

Does antenatal identification of small-for-gestational age fetuses significantly improve their outcome?

OBJECTIVES: Most obstetric clinics have a program for the identification of small-for-gestational age (SGA) fetuses because of the increased risk of fetal complications that they present. We have a structured model for the identification and follow-up of SGA pregnancies. We aimed to determine whether the recognition of SGA antepartum improves fetal outcome. METHODS: All pregnancies at Malm? University Hospital from 1990 to 1998 (n = 26 968) were reviewed. SGA fetuses identified prior to delivery (n = 681) were compared with those not identified (n = 573). Also, all pregnancies with SGA fetuses were compared with those appropriate-for-gestational age (AGA) (n = 24 585). The risk of serious fetal complications (hypoxic encephalopathy grade 2 or 3, intracranial hemorrhage, Apgar score <4 at 5 min, neonatal convulsions, umbilical pH <7.0, cerebral palsy, mental retardation, stillbirth, intrapartum or infant death) was assessed with cross-tabulation and logistic regression analysis, adjusted for gestational age and degree of SGA. RESULTS: When compared with SGA fetuses identified before delivery (54%), SGA fetuses not identified before delivery were characterized by a four-fold increased risk of adverse fetal outcome (odds ratio, 4.1; 95% CI, 2.5-6.8). Similarly, compared with AGA fetuses, SGA fetuses were associated with a four-fold increased risk of serious fetal complications. CONCLUSIONS: A structured antenatal surveillance program for fetuses identified as SGA results in a lower risk of adverse fetal outcome, compared with cases of SGA fetuses not identified antepartum.     

Preferential liver gene expression with polypropylenimine dendrimers.

Previously, the lower generation (DAB 8-generation 2 and DAB 16-generation 3) polypropylenimine dendrimers have been shown to be effective gene delivery systems in vitro. In the current work, we sought to: (a) test the effect of the strength of the carrier, DNA electrostatic interaction on gene transfer and (b) to study the in vivo gene transfer activity of these low molecular weight (<1687 Da) non-amphiphilic plain and quaternary ammonium gene carriers. Towards this aim, methyl quaternary ammonium derivatives of DAB 4 (generation 1), DAB 8, DAB 16 and DAB 32 (generation 4) were synthesised to give Q4, Q8, Q16 and Q32, respectively. Quaternisation of DAB 8 proved to be critical in improving DNA binding, as evidenced by data from the ethidium bromide exclusion assay and dendrimer-DNA colloidal stability data. This improved colloidal stability had a major effect on vector tolerability, as Q8-DNA formulations were well tolerated on intravenous injection while a similar DAB 8-DNA dose was lethally toxic by the same route. Quaternisation also improved the in vitro cell biocompatibility of DAB 16-DNA and DAB 32-DNA dendrimer complexes by about 4-fold but not that of the lower generation DAB 4-DNA and DAB 8-DNA formulations. In contrast to previous reports with non-viral gene delivery systems, the intravenous administration of DAB 16-DNA and Q8-DNA formulations resulted in liver targeted gene expression as opposed to the lung targeted gene expression obtained with the control polymer-Exgen 500 [linear poly(ethylenimine)] and a lung avoidance hypothesis is postulated. We conclude that the polypropylenimine dendrimers are promising gene delivery systems which may be used to target the liver and avoid the lung and also that molecular modifications conferring colloidal stability on gene delivery formulations have a profound effect on their tolerability on intravenous administration.     

Histological changes over time around the site of sustained release naltrexone-poly(DL-lactide) implants in humans.

In order to assess the histological tissue changes over time around the site of implant, tissue biopsies were taken at 1 to 38 months post-implant from 54 (34 male) consenting human subjects who had received the Australian subcutaneous naltrexone-poly(DL-lactide) implant for heroin dependence. The implant consists of multiple tablets containing compressed naltrexone-poly[trans-3,6-dimethyl-1,4-dioxane-2,5-dione] (DL-lactide) loaded microspheres. Assessment of tissue samples by pathologists showed an early phase (up to 12 months post-implant) of inflammation, foreign body reaction, and fibrosis. This subsided gradually over the next 12 months until tissue returned to normal by 25+ months. Sufficient evidence was not available to conclude that the poly(DL-lactide) implant matrix was totally biodegradable within the study period. While implant material was not identified in most of the latter biopsies, its presence was noted in one biopsy at 26 months post-implant. Nevertheless the study results did demonstrate the implant's biocompatibility by the lack of inflammation, foreign body reaction, and fibrosis detected by 25+ months. It seems highly probable that surgical technique rather than the implant itself was associated with the additional finding of fat necrosis. Moderate fat necrosis was observed as a common feature of biopsies carried out during the first 6 months following implant. It subsided to mild levels over the next 18 months, and was notably absent by 25+ months. The results of the study indicated that the Australian naltrexone-poly(DL-lactide) implant is well tolerated and may have a role for use in the management of medical conditions such as heroin dependence.     

Contribution of autologous blood transfusion in cardiac surgery in the adult

The use of autologous blood transfusion in cardiac surgery is still controversial. This study was prospectively designed to evaluate the haemodynamic and haematological benefits of this method, with special attention to its impact on reducing bank blood requirements. Between November 1983 and October 1984, 160 patients underwent cardiac surgery with extracorporeal circulation and were randomly assigned to two groups: group I (81 patients) was the control group and group II (79 patients) received autologous transfusion following extracorporeal circulation. Blood was withdrawn immediately after the induction of anaesthesia via a jugular catheter and stored in CPD solution at room temperature. The volume of blood removed was replaced with gelatin solutions; after bypass, blood was returned to the patient. There was no difference in systolic, diastolic or mean blood pressures between the two groups. Right atrial pressure and heart rate were not statistically different in both groups. Myocardial perfusion and myocardial oxygen consumption remained unchanged in group II compared with group I. Complete haematological evaluation was carried out before and during bypass, and thereafter daily for the first twelve days of the postoperative period. There was no significative difference between the two groups in platelet counts, fibrinogen levels, prothrombin and partial thromboplastin times. During extracorporeal circulation, mean haematocrit was 22.9 +/- 0.4% in group II and 25.3 +/- 0.5% in group I (p less than 10(-3)). The mean haematocrit time course was similar in both groups during the postoperative period and returned to preoperative value at discharge.(ABSTRACT TRUNCATED AT 250 WORDS)