Altered plasma adipokine levels and in vitro adipocyte differentiation in pediatric type 1 diabetes.

CONTEXT: Type 1 diabetes (T1D) is considered a proinflammatory condition. Adipose tissue involvement seems evident because adiponectin levels correlate with disease remission and administration of leptin suppresses the low-grade systemic inflammation in mice with T1D. Whether adipose tissue involvement in T1D already occurs at a young age is yet unknown. OBJECTIVE: The aim was to explore the extent of adipokine alterations in pediatric T1D and gain more insight into the mechanisms underlying the involvement of adipose tissue. DESIGN AND PARTICIPANTS: First, plasma adipokine profiling (24 adipokines) of 20 children with onset T1D, 20 children with long-standing T1D, and 17 healthy controls was performed using a recently developed and validated multiplex immunoassay. Second, the effects of diabetic plasma factors on preadipocyte proliferation and differentiation were studied in vitro. RESULTS: In children with onset and long-standing T1D, plasma adipokine profiling showed increased levels of various adipokines acting at the crossroads of adipose tissue function and inflammation, including CCL2/monocyte chemoattractant protein-1 and the novel adipokines cathepsin S, chemerin, and tissue inhibitor of metalloproteinase-1 (P < 0.05). Furthermore, onset and long-standing diabetic plasma significantly induced preadipocyte proliferation and adipocyte differentiation in vitro (P < 0.05). Two candidate plasma factors, glucose and the saturated fatty acid palmitic acid, did not affect proliferation or adipocyte differentiation in vitro but were found to increase CCL2 (monocyte chemoattractant protein-1) secretion by adipocytes. CONCLUSIONS: The adipogenic effects of diabetic plasma in vitro and the altered adipokine levels in vivo suggest adipose tissue involvement in the low-grade inflammation associated with T1D, already in pediatric patients.

Variants in Apaf-1 segregating with major depression promote apoptosome function.

APAF1, encoding the protein apoptosis protease activating factor 1 (Apaf-1), has recently been established as a chromosome 12 gene conferring predisposition to major depression in humans. The molecular phenotypes of Apaf-1 variants were determined by in vitro reconstruction of the apoptosome complex in which Apaf-1 activates caspase 9 and thus initiates a cascade of proteolytic events leading to apoptotic destruction of the cell. Cellular phenotypes were measured using a yeast heterologous expression assay in which human Apaf-1 and other proteins necessary to constitute a functional apoptotic pathway were overexpressed. Apaf-1 variants encoded by APAF1 alleles that segregate with major depression in families linked to chromosome 12 shared a common gain-of-function phenotype in both assay systems. In contrast, other Apaf-1 variants showed neutral or loss-of-function phenotypes. The depression-associated alleles thus have a common phenotype that is distinct from that of non-associated variants. This result suggests an etiologic role for enhanced apoptosis in major depression.

Driving affinity selection by centrifugal force.

We describe a new approach to affinity selection based on the application of centrifugal force to macromolecules in solution. The method relies on the well known macromolecular hydrodynamic principles of centrifugation. It can be automated and operated in a centralized fashion, or it can be decentralized and used by single researchers or networks of researchers with a minimal additional capital investment. In this method, a centrifugal driving force is used to establish a differential and selective concentration gradient between a therapeutic target and potential ligands in compound libraries. This concentration gradient, in turn, drives the binding of ligands. Once formed, the differential concentration gradient of target macromolecules and ligands is fractionated to capture the self-sorting binding events. Ligand binding is defined by the individual ligand binding constants, so tight binding ligands will essentially distribute identically with the protein target, and weaker binding ligands will not. The level of affinity needed to operationally define tight binding can be adjusted by selecting the initial concentration conditions or centrifugal force. A variety of rapid, commonly available, detection methods can be used to assess binding in the fractionated samples. The method can be broadly applied in drug discovery efforts to examine most types of cell-cell, protein-protein, and protein-small molecule interactions. We describe the application of this method to systems of small molecule interactions with several macromolecules of therapeutic interest.

Study of the in vitro cytotoxic potential of natural and synthetic coumarin derivatives using human normal and neoplastic skin cell lines.

A selection of natural and synthetic coumarin compounds, including the hydroxylated and nitrated derivatives, were assessed for their cytotoxic potential using the microculture 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cellular viability. For the first time this study utilized both human skin malignant melanocytes (SK-MEL-31) and normal human skin fibroblastic cells (HS613.SK), allowing identification of those coumarin derivatives that are selectively toxic. Coumarin was found to exhibit comparatively low toxicity in both cell types, while 7-hydroxycoumarin (7-OHC) and coumarin had similar activity in SK-MEL-31 cells. The entire series of hydroxylated coumarins were considerably more toxic in HS613.SK than in SK-MEL-31 cells. Novel synthetic nitrated coumarins, 6-nitro-7-hydroxycoumarin (6-NO2-7-OHC) and 3,6,8-nitro-7-hydroxycoumarin (3,6,8-NO2-7-OHC), were shown to be significantly more toxic to SK-MEL-31 than HS613.SK cells. In the malignant melanocyte skin cell line (SK-MEL-31) the cytotoxic effects of these nitro-derivatives were shown to be dose and time dependent. Therefore, the cytotoxic potential of coumarins appears to be highly dependent on the nature and position of the functional group. In addition, nitration of 7-OHC produced compounds that were cytotoxic to malignant melanocytes, suggesting that these nitro-derivatives may have a chemotherapeutic role in the future.

Human Daxx regulates Fas-induced apoptosis from nuclear PML oncogenic domains (PODs).

Daxx was first identified as a protein that binds the cytosolic domain of Fas and links this receptor to an apoptosis pathway involving activation of Jun N-terminal kinase (JNK). We show here that cells overexpressing the human homolog of Daxx (hDaxx) display enhanced sensitivity to apoptosis induced by Fas but not by several other cell death stimuli. hDaxx-mediated enhancement of Fas-induced apoptosis was correlated with accelerated activation of caspases but not with JNK induction. Although specifically enhancing Fas function, hDaxx does not bind Fas and instead is found in the nucleus where it localizes to PML oncogenic domains (PODs). Moreover, the hDaxx protein also exhibits the ability to repress transcription. Mutagenesis studies demonstrated a correlation between the localization of hDaxx to PODs and its ability to enhance Fas-induced cell death. Arsenic trioxide (As(2)O(3)), an agent that accentuates POD formation, collaborated synergistically with overexpression of hDaxx to increase cellular sensitivity to Fas-induced apoptosis. Taken together, these findings argue that hDaxx promotes sensitivity to Fas from a nuclear location, probably by modulating the transcription of genes involved in Fas-induced caspase activation and apoptosis.

Compliance to multiple interventions in a high risk population.

PURPOSE: Assess compliance with study medications and examine reasons for noncompliance. Individuals with peripheral arterial disease present the clinician with a unique combination of symptoms and therapeutic needs; the treatment of this population has not been adequately studied. METHODS: The Arterial Disease Multiple Intervention Trial was a randomized double-blind placebo-controlled trial that randomized 468 participants to a combination of antioxidants, niacin and warfarin or matching placebos. Men and women (mean age 65 yrs) with peripheral arterial disease and low-density lipoprotein (LDL) < 190 mg/dl were enrolled and followed for one year. Compliance to the study medications was measured by pill count for each medication. An overall measure of compliance was determined by combining pill counts from all study visits. RESULTS: Mean overall pill counts ranged from 88 to 94% in the eight treatment groups. No statistically significant differences were found in mean pill counts over time or between active and placebo groups. History of coronary artery disease and number of follow-up visits were associated with higher overall pill counts while low compliance during screening was associated with lower counts during follow-up. Participants with an overall mean pill count < 80% had more adverse events compared to those with a higher count. Side effects were reported as the reason for missing pills significantly more often in the active versus placebo niacin group. CONCLUSIONS: Individuals with peripheral arterial disease were able to comply with the complex drug regimen. The ability of this drug combination to reduce cardiovascular events and improve quality of life warrants study.

Molecular genetics of acute promyelocytic leukemia.

The remarkable success of retinoic acid in the treatment of acute promyelocytic leukemias and the subsequent discovery that mutant forms of a retinoid acid receptor (RARalpha) are invariably associated with this disease has generated considerable interest among both clinicians and basic scientists. Studies both in cell culture and in transgenic animals suggest that mutant RARs interfere with normal retinoid-mediated transactivation and granulocytic differentiation. More recently, a pivotal link between transcriptional silencing, the oncogenic functions of RAR mutants, and hormonal responses in APL patients has been established. These studies have greatly advanced our understanding of the molecular changes involved in leukemogenesis, have helped to reveal new aspects of cellular differentiation, and might lead to improved treatment strategies for human leukemias.

Modulation of CREB binding protein function by the promyelocytic (PML) oncoprotein suggests a role for nuclear bodies in hormone signaling.

Disaggregation of the spherical nuclear bodies termed promyelocytic (PML) oncogenic domains (PODs) is a characteristic of acute promyelocytic leukemia. Here, we demonstrate that the cAMP enhancer binding protein (CREB)-binding protein (CBP) associates with PML in vitro and is recruited to the PODs in vivo. Through its association with CBP, wild-type PML dramatically stimulates nuclear receptor transcriptional activity. These results demonstrate that a fraction of CBP is compartmentalized to the POD through its association with PML and thus suggest that PML and other POD-associated proteins may play an unexpectedly broad role in aspects of transcriptional regulation and human disease.

Rationale and design of the Arterial Disease Multiple Intervention Trial (ADMIT) pilot study.

The primary objectives of the pilot study were to: (1) evaluate the feasibility of recruiting patients with peripheral arterial disease (PAD); (2) measure the efficacy and safety of high-density lipoprotein (HDL)-raising treatment, low-density lipoprotein (LDL)-lowering therapy, antioxidant therapy, antithrombotic therapy, and their combinations; and (3) assess adherence to a complex multiple drug regimen. Secondary objectives included measurement of the effect of the interventions on prespecified biochemical markers, maintenance of therapy masking (in particular with niacin), and measurement of the intervention's impact on functional status and on quality of life. To date, no secondary prevention trial has been conducted specifically among patients with PAD. Intermittent claudication affects about 0.5% to 1.0% of persons aged >35 years. There is a striking increase in incidence of PAD with age, particularly among those aged >50 years in both sexes, although men are twice as likely as women to develop PAD. The Arterial Disease Multiple Intervention Trial was a double-blind randomized pilot trial of 468 participants with documented PAD. A 2 x 2 x 2 factorial design was used to evaluate the effect of 3 interventions. The pilot incorporated several major novel design features: first, the use of a simple noninvasive method (measurement of ankle brachial index) to identify a population with either symptomatic or asymptomatic PAD; and second, a lipid modifying strategy to increase HDL with nicotinic acid in the intervention group while lowering LDL levels equally with an hydroxymethylglutaryl-coenzyme A reductase inhibitor as needed in the intervention and control group. Two other arms, the antioxidant arm (consisting of beta-carotene and vitamins E and C) and the antithrombotic arm (using warfarin) were also added. Adherence to therapy was measured by pill count, and success in treatment was measured by the proportion of values in target range for HDL, LDL, and the international normalized ratio.

Domain structure analysis of elongation factor-3 from Saccharomyces cerevisiae by limited proteolysis and differential scanning calorimetry.

Elongation-factor-3 (EF-3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF-3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultracentrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39 degrees C, and a minor transition at 58 degrees C. Ultracentrifugation shows that EF-3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF-3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF-3 with trypsin produced the first cleavage at the N-side of Gln775, generating a 90-kDa N-terminal fragment and a 33-kDa C-terminal fragment. The N-terminal fragment slowly undergoes further digestion generating two major bands, one at approximately 75 kDa and the other at approximately 55 kDa. The latter was unusually resistant to further tryptic digestion. The 33-kDa C-terminal fragment was highly sensitive to tryptic digestion. A 30-min tryptic digest showed that the N-terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C-terminal 40% was readily digested. These results suggest a tight structure of the N-terminus, which may give rise to the 58 degrees C transition, and a loose structure of the C-terminus, giving rise to the 39 degrees C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5-homologous domain (Lys102-Ile368), the N-terminal ATP-binding cassette (Gly463-Lys622), and the aminoacyl-tRNA-synthase homologous domain (Glu820-Gly865). Both the basal and ribosome-stimulated ATPase activities were inactivated by trypsin, but the ribosome-stimulated activity was inactivated faster.

Changes in sample size and length of follow-up to maintain power in the coronary artery bypass graft (CABG) patch trial.

The CABG Patch Trial is testing the hypothesis that prophylactic use of implantable cardiac defibrillators (ICDs) will improve survival in high-risk coronary heart disease patients undergoing CABG surgery. The original design called for 800 patients to be randomized to ICD prophylaxis or to no therapy and followed for 2 to 6.5 years (average, 40 months) to a common termination date. Since the ICD pulse generators used in this trial lasted about 42 months, the original design required ICD replacement in many patients. At its first two meetings in 1993, the Data and Safety Monitoring Board (DSMB) formalized a plan to adjust sample size in October 1994 if the control group mortality rate was lower than expected. In June 1994, an unanticipated and unique event--a subpoena from the Office of the Inspector General (OIG)--made it impossible to replace about half of the ICD generators and threatened to shorten follow-up substantially. If follow-up had been stopped on the date originally planned, but without replacing ICDs, the average follow-up would have fallen from 40 months to about 33 months. Also, in October 1994, the control group mortality rate was found to be somewhat lower than expected. Together, the abbreviated follow-up and lower control group mortality threatened to reduce power substantially. The DSMB reviewed several options for restoring power. Because mortality rates in the first month after CABG surgery were about seven times as high as thereafter and because ICD therapy did not reduce surgical mortality (death during the first 30 days), extending the follow-up benefits power more than does increasing the sample size. However, the limit on extending follow-up was 42 months (the expected battery life of the ICD). Data from the ICD-treated group was not reviewed or considered in making the decision. After reviewing many options for restoring power, the DSMB recommended that the sample size be increased from 800 to 900 patients and that almost all patients be followed for 42 months. This recommendation extended follow-up for 2 years beyond the original termination date planned for the trial and dictated that patients close out after 42 months rather than on a common termination date.

Structure of the E2 DNA-binding domain from human papillomavirus serotype 31 at 2.4 A.

The papillomaviruses are a family of small double-stranded DNA viruses which exclusively infect epithelial cells and stimulate the proliferation of those cells. A key protein within the papillomavirus life-cycle is known as the E2 (Early 2) protein and is responsible for regulating viral transcription from all viral promoters as well as for replication of the papillomavirus genome in tandem with another protein known as E1. The E2 protein itself consists of three functional domains: an N-terminal trans-activation domain, a proline-rich linker, and a C-terminal DNA-binding domain. The first crystal structure of the human papillomavirus, serotype 31 (HPV-31), E2 DNA-binding domain has been determined at 2.4 A resolution. The HPV DNA-binding domain monomer consists of two beta-alpha-beta repeats of approximately equal length and is arranged as to have an anti-parallel beta-sheet flanked by the two alpha-helices. The monomers form the functional in vivo dimer by association of the beta-sheets of each monomer so as to form an eight-stranded anti-parallel beta-barrel at the center of the dimer, with the alpha-helices lining the outside of the barrel. The overall structure of HVP-31 E2 DNA-binding domain is similar to both the bovine papillomavirus E2-binding domain and the Epstein-Barr nuclear antigen-1 DNA-binding domain.

Transcriptional regulation of MDR genes.

The emergence of resistance in a tumor population is most often associated with a disregulation of gene expression, usually at the level of transcription. A major goal in the field of cancer chemotherapy is to define the mechanisms underlying transcriptional regulation of drug resistance genes in an effort to identify targets for therapeutic intervention. Recently, considerable progress has been made in identifying the molecular mechanisms involved in the transcriptional regulation of the P-glycoprotein (Pgp) gene. When overexpressed in tumor cells, Pgp confers resistance to a variety of chemotherapeutic agents; this resistance has been termed MDR (multidrug resistance). Moreover, Pgp is a normal component of a variety of highly differentiated cell types and, as such, is regulated by both internal and external environmental stimuli. In this review, we will discuss the current knowledge regarding the DNA elements and protein factors involved in both constitutive and inducible regulation of Pgp transcription in normal and tumor cells.

NMR-based discovery of lead inhibitors that block DNA binding of the human papillomavirus E2 protein.

The E2 protein is required for the replication of human papillomaviruses (HPVs), which are responsible for anogenital warts and cervical carcinomas. Using an NMR-based screen, we tested compounds for binding to the DNA-binding domain of the HPV-E2 protein. Three classes of compounds were identified which bound to two distinct sites on the protein. Biphenyl and biphenyl ether compounds containing a carboxylic acid bind to a site near the DNA recognition helix and inhibit the binding of E2 to DNA. Benzophenone-containing compounds which lack a carboxylic acid group bind to the beta-barrel formed by the dimer interface and exhibit negligible effects on the binding of E2 to DNA. Structure-activity relationships from the biphenyl and biphenyl ether compounds were combined to produce a compound [5-(3'-(3",5"-dichlorophenoxy)-phenyl)-2,4-pentadienoic acid] with an IC50 value of approximately 10 microM. This compound represents a useful lead for the development of antiviral agents that interfere with HPV replication and further illustrates the usefulness of the SAR by NMR method in the drug discovery process.

Alpha-glutathione s-transferase (alpha-GST) release, an early indicator of carbon tetrachloride hepatotoxicity in the rat.

1. The use of the cytoplasmic enzyme, alpha glutathione s-transferase (alpha-GST) as an early index of carbon tetrachloride (CCl4) toxicity in the rat was investigated and compared with a standard enzyme, marker, aspartate aminotransferase (AST). The hepatotoxic effects of CCl4 in the rat were determined in a time and dose-response study. 2. Following CCl4 exposure, alpha-GST release was shown to be an earlier and more sensitive biomarker of hepatotoxicity than AST. 3. Significant increases in alpha-GST were detected 2 h after CCl4 exposure. Using the enzyme marker AST, this early hepatotoxic injury went undetected. At 6 and 16 h, alpha-GST was also a more sensitive indicator of hepatotoxicity than AST. 4. alpha-GST release was significantly increased at a dose of 5 microliters/kg, the lowest concentration of CCl4 administered and clearly responded in a dose-dependent manner with increasing doses of CCl4. In contrast, release of AST did not reach statistical significance until a dose of 25 microliters/kg. 5. Thus, these findings indicate that alpha-GST is a more sensitive and more accurate reflector of CCl4 induced hepatotoxicity than AST.

Solution structure of the DNA-binding domain of a human papillomavirus E2 protein: evidence for flexible DNA-binding regions.

The three-dimensional structure of the DNA-binding domain of the E2 protein from human papillomavirus-31 was determined by using multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. A total of 1429 NMR-derived distance and dihedral angle restraints were obtained for each of the 83-residue subunits of this symmetric dimer. The average root mean square deviations of 20 structures calculated using a distance geometry-simulated annealing protocol are 0.59 and 0.90 angstroms for the backbone and all heavy atoms, respectively, for residues 2-83. The structure of the human virus protein free in solution consists of an eight-stranded beta-barrel and two pairs of alpha-helices. Although the overall fold of the protein is similar to the crystal structure of the bovine papillomavirus-1 E2 protein when complexed to DNA, several small but interesting differences were observed between these two structures at the subunit interface. In addition, a beta-hairpin that contacts DNA in the crystal structure of the protein-DNA complex is disordered in the NMR structures, and steady-state 1H-15N heteronuclear NOE measurements indicate that this region is highly mobile in the absence of DNA. The recognition helix also appears to be flexible, as evidenced by fast amide exchange rates. This phenomenon has also been observed for a number of other DNA-binding proteins and may constitute a common theme in protein/DNA recognition.