Ectopic expression of the human adenine nucleotide translocase, isoform 3 (ANT-3). Characterization of ligand binding properties.

The adenine nucleotide translocase (ANT) is a key component in maintaining cellular energy homeostasis, and has also been implicated in formation of the mitochondrial permeability transition pore. Human ANT-3 was cloned from a human heart cDNA library and expressed as a histidine-tagged fusion protein in the mitochondria of the Trichoplusia ni. cell line. Overexpression resulted in a concomitant decrease in the endogenous ANT content, allowing for the characterization of binding of known ANT ligands to the human protein. Binding affinities for bongkrekic acid (BKA), ADP, and atractyloside (ATR) were measured in mitochondria from the human ANT-3 expressing cell line, and compared to similar preparations from bovine heart mitochondria by use of a novel radioiodinated derivative of ATR. Binding to ANT-3 by the high affinity inhibitors BKA and ATR, as well as the lower affinity natural ligand ADP, was similar to that measured in bovine heart mitochondria, and to that previously reported for mammalian heart mitochondria. Characterizations such as these of human ANT isoforms may lead to drug development for enhanced mitochondrial function and cellular viability.

Clinical relevance of the primary findings of the MTA: success rates based on severity of ADHD and ODD symptoms at the end of treatment.

OBJECTIVES: To develop a categorical outcome measure related to clinical decisions and to perform secondary analyses to supplement the primary analyses of the NIMH Collaborative Multisite Multimodal Treatment Study of Children With Attention-Deficit/Hyperactivity Disorder (MTA). METHOD: End-of-treatment status was summarized by averaging the parent and teacher ratings of attention-deficit/hyperactivity disorder and oppositional defiant disorder symptoms on the Swanson, Nolan, and Pelham, version IV (SNAP-IV) scale, and low symptom-severity ("Just a Little") on this continuous measure was set as a clinical cutoff to form a categorical outcome measure reflecting successful treatment. Three orthogonal comparisons of the treatment groups (combined treatment [Comb], medication management [MedMgt], behavioral treatment [Beh], and community comparison [CC]) evaluated hypotheses about the MTA medication algorithm ("Comb + MedMgt versus Beh + CC"), multimodality superiority ("Comb versus MedMgt"), and psychosocial substitution ("Beh versus CC"). RESULTS: The summary of SNAP-IV ratings across sources and domains increased the precision of measurement by 30%. The secondary analyses of group differences in success rates (Comb = 68%; MedMgt = 56%; Beh = 34%; CC = 25%) confirmed the large effect of the MTA medication algorithm and a smaller effect of multimodality superiority, which was now statistically significant (p < .05). The psychosocial substitution effect remained negligible and nonsignificant. CONCLUSION: These secondary analyses confirm the primary findings and clarify clinical decisions about the choice between multimodal and unimodal treatment with medication.

Impairment and deportment responses to different methylphenidate doses in children with ADHD: the MTA titration trial.

OBJECTIVE: Results of the NIMH Collaborative Multisite Multimodal Treatment Study of Children With Attention-Deficit/Hyperactivity Disorder (MTA) were analyzed to determine whether a double-blind, placebo-controlled methylphenidate (MPH) titration trial identified the best MPH dose for each child with attention-deficit/hyperactivity disorder (ADHD). METHOD: Children with ADHD assigned to MTA medication treatment groups (n = 289) underwent a controlled 28-day titration protocol that administered different MPH doses (placebo, low, middle, and high) on successive days. RESULTS: A repeated-measures analysis of variance revealed main effects for MPH dose with greater effects on teacher ratings of impairment and deportment (F3 = 100.6, n = 223, p = .0001; effect sizes 0.8-1.3) than on parent ratings of similar endpoints (F3 = 55.61, n = 253, p = .00001; effect sizes 0.4-0.6). Dose did not interact with period, dose order, comorbid diagnosis, site, or treatment group. CONCLUSIONS: The MTA titration protocol validated the efficacy of weekend MPH dosing and established a total daily dose limit of 35 mg of MPH for children weighing less than 25 kg. It replicated previously reported MPH response rates (77%), distribution of best doses (10-50 mg/day) across subjects, effect sizes on impairment and deportment, as well as dose-related adverse events.

A novel mitochondrial DNA-like sequence in the human nuclear genome.

We describe here a nuclear mitochondrial DNA-like sequence (numtDNA) that is nearly identical in sequence to a continuous 5842 bp segment of human mitochondrial DNA (mtDNA) that spans nucleotide positions 3914 to 9755. On the basis of evolutionary divergence among modern primates, this numtDNA molecule appears to represent mtDNA from a hominid ancestor that has been translocated to the nuclear genome during the recent evolution of humans. This numtDNA sequence harbors synonymous and nonsynonymous nucleotide substitutions relative to the authentic human mtDNA sequence, including an array of substitutions that was previously found in the cytochrome c oxidase subunit 1 and 2 genes. These substitutions were previously reported to occur in human mtDNA, but subsequently contended to be present in a nuclear pseudogene sequence. We now demonstrate their exclusive association with this 5842-bp numtDNA, which we have characterized in its entirety. This numtDNA does not appear to be expressed as a mtDNA-encoded mRNA. It is present in nuclear DNA from human blood donors, in human SH-SY5Y and A431 cell lines, and in rho(0) SH-SY5Y and rho(0) A431 cell lines that were depleted of mtDNA. The existence of human numtDNA sequences with great similarities to human mtDNA renders the amplification of pure mtDNA from cellular DNA very difficult, thereby creating the potential for confounding studies of mitochondrial diseases and population genetics.

Evaluation of individual subjects in the analog classroom setting: II. Effects of dose of amphetamine (Adderall).

Multiple dependent variables were graphed for 29 subjects who participated in a double-blind evaluation of 4 doses of Adderall, plus positive (methylphenidate) and placebo control conditions. Five judges ranked the conditions for each subject, and analyses of individual subjects indicated that these rankings were concordant (reliable) across judges. Consensus rankings were assigned to each subject, and an analysis of these ranks showed that the conditions differed significantly. The choice of best conditions were judged to be across 3 doses of Adderall (10, 15, and 20 mg). This confirms the clinical impression of individual differences in optimal dose of stimulant medication. The methodological, graphical, and statistical methods presented in this article provide a systematic, reliable procedure for evaluating relative response of individuals to different doses of stimulant medication.

Laser-enhanced ionization of mercury atoms in an inert atmosphere with avalanche amplification of the signal.

A new method for laser-enhanced ionization detection of mercury atoms in an inert gas atmosphere is described. The method, which is based on the avalanche amplification of the signal resulting from the ionization from a selected Rydberg level reached by a three-step laser excitation of mercury vapor in a simple quartz cell, can be applied to the determination of this element in various matrices by the use of conventional cold atomization techniques. The overall (collisional + photo) ionization efficiency is investigated at different temperatures, and the avalanche amplification effect is reported for Ar and P-10 gases at atmospheric pressure. It is shown that the amplified signal is related to the number of charges produced in the laser-irradiated volume. Under amplifier noise-limited conditions, a detection limit of ∼15 Hg atoms/laser pulse in the interaction region is estimated.

Cloning of a cDNA encoding a novel interleukin-1 receptor related protein (IL 1R-rp2).

We have identified and isolated both the rat and human cDNAs for a novel putative receptor related to the interleukin-1 type 1 receptor. We have named this protein interleukin 1 receptor related protein two (IL 1R-rp2). The rat cDNA for IL1R-rp2 was first identified using oligonucleotides of degenerate sequence in a polymerase chain reaction (PCR) paradigm with rat brain mRNA as the template. The protein encoded by both of these cDNAs are 561 amino acids long and exhibit 42% and 26% overall identity with the interleukin-1 type 1 and type 2 receptors, respectively. RNase protection assays from rat tissues revealed a predominant expression for IL 1R-rp2 in the lung and epididymis with lower levels detected in the testis and cerebral cortex. By in situ hybridization we were able to determine that the expression in rat brain appeared to be non-neuronal and associated with the cerebral vasculature. When expressed transiently in COS-7 cells the receptor was incapable of high affinity binding to either [125I]-recombinant human IL 1 alpha or [125I]-recombinant human IL 1 beta. Together, these data demonstrate the existence of a novel protein that is related to the interleukin-1 receptor but does not bind IL-1 by itself.

Cloning and characterization of an alternatively processed human type II interleukin-1 receptor mRNA.

Two types of interleukin (IL)-1 receptors with three extracellular immunoglobulin-like domains, limited homology (28%), and different pharmacological characteristics termed type I and type II have been cloned from mouse and human cell lines. Both receptors exist in transmembrane and soluble forms; the soluble IL-1 receptor is thought to be post-translationally derived from cleavage of the extracellular portion of the membrane receptors. In preliminary cross-linking studies with radiolabeled IL-1, we found that monkey kidney COS1 cells express a soluble receptor with molecular mass of approximately 55-60 kDa, which is different from previously reported soluble IL-1 receptors. This soluble IL-1 receptor protein from COS1 cells was purified to homogeneity by affinity chromatography using recombinant IL-1beta as the ligand and shown to have an affinity for human 125I-IL-1beta (KD approximately 2-3 nM) comparable to the human type II IL-1 receptor (IL-1RII). The purified protein was microsequenced, and the sequence information was used to design primers to clone the COS1 IL-1RII using reverse transcription-coupled polymerase chain reaction; the DNA comparison with monkey COS1 and human IL-1RII indicate that they are 95% identical at the nucleic acid and amino acid levels. In addition, another cDNA, which represents an alternatively processed mRNA of the IL-1RII gene, was also cloned both from monkey COS1 and human Raji cells and was shown to have approximately 95% sequence identity between these species. While the cDNA of the novel alternatively processed gene has a 5' end identical to the IL-1RII, the 200 base pairs at the 3' end are different and the sequence predicts a soluble IL-1 receptor protein of 296 amino acids. Radioligand binding studies of the alternatively processed IL-1RII mRNA demonstrated kinetic and pharmacological characteristics similar to the known type II IL-1 receptor. COS7 cells (which lack IL-1 receptor) transfected with the transmembrane form of the human IL-1RII cDNA showed 125I-IL-1beta binding in both the membrane fractions and supernatant. In contrast, COS7 cells transfected with the alternatively processed human IL-1RII cDNA showed high affinity 125I-IL-1beta binding (Ki approximately 1.2 nM) predominantly in the supernatant; a very small amount of detectable membrane IL-1 binding activity was also observed presumably due to association of the soluble IL-1 receptor and membrane-integrated proteins. In cross-linking and ligand blot studies, the alternatively processed human IL-1RII cDNA-transfected COS7 cells expressed a soluble IL-1 receptor with molecular masses ranging from 60 to 160 kDa, further indicating the association between this soluble IL-1 receptor and other soluble proteins. In summary, we report the purification and characterization of a soluble IL-1 receptor expressed by COS1 cells and the cloning of an alternatively processed type II IL-1 receptor mRNA from both human and COS1 cells. The alternative splicing of a primary transcript leading to a secreted protein provides a potentially important mechanism by which soluble IL-1RII can be produced.

Peroxisome proliferator-activated receptor response specificities as defined in yeast and mammalian cell transcription assays.

Peroxisome proliferators include a heterogeneous group of xenobiotic agents capable of inducing peroxisome proliferation and hepatocellular carcinomas in rodent model systems. These chemicals appear to mediate their activity through a family of transcription factors known as peroxisome proliferator-activated receptors (PPAR). Recently it has been shown that DNA binding of PPAR is contingent upon heterodimerization with a member of the retinoic acid X (RXR) family of receptors. In this report transcription parameters of a rat PPAR alpha were analyzed using mammalian and yeast cotransfection assays. PPAR activity was observed to be peroxisome proliferator dependent in the mammalian cotransfection assay, and heterodimer dependent but peroxisome proliferator independent in a yeast version of the same assay. Moreover, when the naturally occurring ligand for RXR, 9-cis-retinoic acid (RA), was tested in the same assays, it was observed to generate an RXR-specific response in the yeast cell assay but host cell-specific response in the mammalian cell assay. Finally, the combination of peroxisome proliferator and 9-cis-RA had very little added effect on the yeast cell assay but again produced a cell-specific synergistic response in the mammalian cell assay. These data demonstrate that PPAR transcriptional activity is strongly influenced by the RXR family of receptors, and that peroxisome proliferators may be regulating PPAR mammalian cell activity through a secondary mechanism.

Cloning and characterization of a functionally distinct corticotropin-releasing factor receptor subtype from rat brain.

The present study reports the isolation of a cDNA clone that encodes a second member of the corticotropin-releasing factor (CRF) receptor family, designated as the CRF2 receptor. The cDNA was identified using oligonucleotides of degenerate sequence in a PCR paradigm. A PCR fragment obtained from rat brain was utilized to isolate a full-length cDNA from a rat hypothalamus cDNA library that encoded a 411-amino acid protein with approximately 70% identity to the known CRF1 receptor over the entire coding region. When expressed in mouse Ltk- cells, this receptor stimulates cAMP production in response to CRF and known CRF-like agonists. CRF and the nonmammalian CRF-related peptides sauvagine and urotensin I stimulate adenylate cyclase activity in a dose-dependent manner with a rank order of potency different from that of the CRF1 receptor: sauvagine > urotensin > or = rat/human CRF > ovine CRF. Tissue distribution analysis of the mRNAs by reverse transcriptase-PCR shows CRF2 receptor mRNA is present in rat brain and detectable in lung and heart. In situ hybridization studies indicate specific expression within the brain in the ventromedial nuclei of the hypothalamus, the lateral septum, the amygdala, and entorhinal cortex, but there is unremarkable expression in the pituitary. An additional splice variant of the CRF2 receptor with a different N-terminal domain has been identified by PCR, encoding a putative protein of 431 amino acids. Thus, the data demonstrate the presence of another functional CRF receptor, with significant differences in the pharmacological profile and tissue distribution from the CRF1 receptor, which would predict important functional differences between the two receptors.

Enhanced hematopoietic activity of a human granulocyte/macrophage colony-stimulating factor-interleukin 3 fusion protein.

Granulocyte/macrophage colony-stimulating factor-interleukin 3 (GM-CSF-IL-3) fusion proteins were generated by construction of a plasmid in which the coding regions of human GM-CSF and IL-3 cDNAs were connected by a synthetic linker sequence followed by subsequent expression in yeast. Both GM-CSF-IL-3 and IL-3-GM-CSF fusion proteins were purified to homogeneity and shown to bind to cell-surface receptors through either their GM-CSF or IL-3 domains. The fusion proteins exhibited enhanced receptor affinity, proliferative activity, and hematopoietic colony-stimulating activity compared with either IL-3 and/or GM-CSF alone. This suggests that GM-CSF-IL-3 fusion proteins may hold future promise as therapeutic agents.

Diethylstilbestrol-induced perinatal lethality in the rat. I. Relationship to reduced maternal weight gain.

An analysis was performed to determine the mechanism of depressed maternal weight gain and its effect on perinatal lethality following prenatal exposure to diethylstilbestrol (DES). Pregnant Sprague-Dawley rats were dosed orally by gavage with DES or corn oil (control) during various intervals of gestation. The maternal weight-gain patterns of control and treated dams and the number of live offspring were recorded. The amounts of feed and water intake and feces and urine output in pregnant dams were measured, and metabolic rate and thyroid hormone levels were also determined. DES (at 45 micrograms/kg/day) was embryo- and fetolethal during implantation and parturition, and there was an accompanying decline in maternal weight. Growth of adult males, nonpregnant females, and weanlings of both sexes was also depressed. During pregnancy, the net intake of feed and water was not altered by the drug, but maternal serum thyroxine and metabolic rate were significantly elevated. Reduced metabolic efficiency, then, is the likely mechanism for weight depression. Reduction of maternal weight gain during pregnancy by DES is a diagnostic indicator of fetolethality, but is probably not causally related to it.

Diethylstilbestrol-induced perinatal lethality in the rat. II. Perturbation of parturition.

Evidence is presented suggesting that the fetolethal properties of diethylstilbestrol (DES) are indirect, mediated maternally through a perturbation of the normal mechanisms of parturition. Oral administration of the compound to Sprague-Dawley rats near Day 18 of pregnancy was shown to delay the onset of parturition, prolong labor, and induce dystocia, with a concomitant large increase in perinatal mortality. Exposure during Days 8-16 was without effect, whereas treatment in the Day 18-20 interval resulted in preterm delivery. Inability to initiate labor at term, accompanied by fetal death, also resulted from the administration of hCG on Days 16-18. The relative incidence of stillbirths in DES-exposed pups was markedly decreased by Caesarean delivery. The average weight of the maternal pituitary gland was not affected by treatment, whereas maternal adrenal glands were 30% larger. Maternal blood levels of corticosterone were not significantly elevated, however. The average number of follicles on Day 21 was significantly reduced by DES, and a histological analysis failed to demonstrate a luteotropic effect of the compound. In dams treated on Days 8-18, serum progesterone was reduced by as much as 60%, and total estrogens were 32% lower than in controls. We conclude that DES acts in the rat to depress the preterm levels of steroid hormones, which leads to a failure of uterine contraction accompanied by placental detachment and fetal death.

Human interleukin 7: molecular cloning and growth factor activity on human and murine B-lineage cells.

A cDNA encoding biologically active human interleukin 7 was isolated by hybridization with the homologous murine clone. Nucleotide sequence analysis indicated that this cDNA was capable of encoding a protein of 177 amino acids with a signal sequence of 25 amino acids and a calculated mass of 17.4 kDa for the mature protein. Recombinant human interleukin 7 stimulated the proliferation of murine pre-B cells and was active on cells harvested from human bone marrow that are enriched for B-lineage progenitor cells. Analysis of RNA by blot hybridization demonstrated the presence of two size classes of interleukin 7 mRNA in human splenic and thymic tissue.

Production of recombinant human colony stimulating factors in yeast.

Efficient yeast expression and purification systems for production of recombinant human GM-CSF, IL-3 and G-CSF have been established. Though yeast-derived production of recombinant CSFs (through the use of secretion based system) allows for generation of native molecules which can then be readily separated from fermentation broth, in many instances, natural cDNAs have had to be altered to allow for efficient expression, as well as production of a less heterogeneous product. In the case of CSFs described herein, beneficial mutations (made through site-directed mutagenesis) have included elimination of potential N-linked glycosylation sites, removal of KexII protease recognition sites (notably alterations in dibasic sequences) and elimination of extraneous cysteine residues which might complicate isolation of a homogeneous product due to intermolecular disulfide bonding.

Human interleukin-3 and granulocyte-macrophage colony stimulating factor: site-specific mutagenesis and expression in yeast.

The two human colony stimulating factors, interleukin-3 and granulocyte-macrophage colony stimulating factor, have been molecularly cloned and expressed as secreted proteins in yeast. In both cases, non-glycosylated and glycosylated forms of the molecules were produced. Removal of N-linked glycosylation sites from the genes by site-directed mutagenesis prevented addition of most of the sugar residues, but revealed a low level of residual O-linked glycosylation on a portion of the molecules. No difference in specific biological activity was found between the different forms of the proteins. It was found that a significant proportion of human granulocyte-macrophage colony stimulating factor was degraded by the yeast KEX2 protease that was cleaving after the dibasic sequence Arg-Arg at positions 23-24 of the mature protein. Site-specific mutagenesis was employed to change this sequence to Leu-Arg, and this change resulted in greatly increased expression levels of full length protein and biological activity.

Expression, purification and characterization of recombinant murine granulocyte-macrophage colony-stimulating factor and bovine interleukin-2 from yeast.

Expression and secretion of two lymphokines, murine granulocyte-macrophage colony-stimulating factor (MuGM-CSF) and bovine interleukin-2 (BoIL-2), to levels of 50-60 mg per liter were achieved by placing these cDNAs in a Saccharomyces cerevisiae expression vector that utilized the yeast alcohol dehydrogenase-2 promoter and alpha-factor leader peptide. These lymphokines were purified to homogeneity by direct application of the crude yeast medium to reversed-phase high-performance liquid chromatography. Despite the fact that both lymphokines contain at least one N-glycosylation site and have identical N-terminal residues (Ala-Pro-Thr), recombinant (R) GM-CSF was found to be heterogeneously glycosylated by yeast while RBoIL-2 was secreted without glycosylation. Additionally, approximately 40% of the RGM-CSF was found to be proteolytically cleaved after the second amino acid residue, while RBoIL-2 was found to be intact.

A selection system for unequal crossing-over: plasmid construction and initial studies in Escherichia coli.

Unequal crossing-over is involved in genetic duplication and deletion in such diverse genetic systems as Drosophila, bacteria, and animal viruses. It is proposed to be involved in the form of unequal sister chromatid exchange in gene amplification in cultured animal cells and during carcinogenesis. Studies of the process of unequal crossing-over have been hampered by the lack of genetic systems allowing specific selection for cells that have undergone such unequal crossing-over. We report here on the construction of plasmids designed to provide specific selection of unequal crossing-over. One such plasmid was studied in Escherichia coli. We show that kanamycin resistance is generated, as predicted, by the expected unequal crossover event.