A cell-based PDE4 assay in 1536-well plate format for high-throughput screening

The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,'5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease. Although biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. The authors report the development and validation of a new cell-based PDE4 assay using a constitutively active G-protein-coupled receptor as a driving force for cAMP production and a cyclic nucleotide-gated cation channel as a biosensor in 1536-well plates.     

Lipoprotn lipase content and triglyceride fatty acid uptake in adipose tissue of rats of differing body weights

Increasing body weight appears to alter lipid metabolism in adipose tissue. We have measured the content of lipoprotein lipase and the uptake of chylomicron triglyceride fatty acids in epididymal fat pads of rats of different weights. In order that the results might be expressed in terms of cell numbers, the relationship between the weights of fat pads and the numbers and volumes of fat cells isolated from them was determined. Highly significant correlations were found between fat pad weight and both the number and the volume of the individual adipocytes. In rats weighing from 140 to 350 g, the increase in the size of fat pads was attributable almost equally to increases in cell size and in cell number. Lipoprotein lipase activity was measured in acetone powders of whole fat pads and of isolated fat cell preparations. With both, lipoprotein lipase activity per cell diminished significantly as the weight of fat tissue increased, i.e., larger fat cells contained less enzyme per cell than smaller cells. The uptake of triglyceride fatty acid radioactivity was measured after incubation of fat pads with radiolabeled rat lymph chylomicrons in flasks containing either buffer alone or with added glucose or glucose plus insulin. The addition of glucose and insulin led to a mean increase of 70% in the uptake of radioactivity, but larger adipocytes were stimulated less than smaller cells. This resulted in a significant negative correlation between the weights of fat pads and the uptake of radioactivity. Enlargement of fat cells also led to a diminution in their capacity to esterify fatty acids.     

A genome-wide screen identifies a single β-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins

Background  

Defensins comprise a large family of cationic antimicrobial peptides that are characterized by the presence of a conserved cysteine-rich defensin motif. Based on the spacing pattern of cysteines, these defensins are broadly divided into five groups, namely plant, invertebrate, α-, β-, and θ-defensins, with the last three groups being mostly found in mammalian species. However, the evolutionary relationships among these five groups of defensins remain controversial.     

Physical studies of DNA premelting equilibria in duplexes with and without homo dA.dT tracts: correlations with DNA bending

We have employed a variety of physical methods to study the equilibrium melting and temperature-dependent conformational dynamics of dA.dT tracts in fractionated synthetic DNA polymers and in well-defined fragments of kinetoplast DNA (kDNA). Using circular dichroism (CD), we have detected a temperature-dependent, "premelting" event in poly(dA).poly(dT) which exhibits a midpoint near 37 degrees C. Significantly, we also detect this CD "premelting" behavior in a fragment of kDNA. By contrast, we do not observe this "premelting" behavior in the temperature-dependent CD spectra of poly[d(AT)].poly[d(AT)], poly(dG).poly(dC), poly[d(GC)].poly[d(GC)], or calf thymus DNA. Thus, poly(dA).poly(dT) and kDNA exhibit a common CD-detected "premelting" event which is absent in the other duplex systems studied in this work. Furthermore, we find that the anomalous electrophoretic retardation of the kDNA fragments we have investigated disappears at temperatures above approximately 37 degrees C. We also observe that the rotational dynamics of poly(dA).poly(dT) and kDNA as assessed by singlet depletion anisotropy decay (SDAD) and electric birefringence decay (EBD) also display a discontinuity near 37 degrees C, which is not observed for the other duplex systems studied. Thus, in the aggregate, our static and dynamic measurements suggest that the homo dA.dT sequence element [common to both poly(dA).poly(dT) and kDNA] is capable of a temperature-dependent equilibrium between at least two helical states in a temperature range well below that required to induce global melting of the host duplex. We suggest that this "preglobal" melting event may correspond to the thermally induced "disruption" of "bent" DNA.     

Genotyping single nucleotide polymorphisms by primer extension and high performance liquid chromatography

We have investigated the possibility of genotyping single nucleotide polymorphisms (SNPs) by primer extension and high performance liquid chromatography (HPLC). Using three polymorphisms of current interest to our group (an A/G polymorphism in the proneurotensin gene and A/G and T/C polymorphisms in the 5HT2a receptor gene), we show that robust signal is obtained using this simple analytic method which has the added advantages that sample loading and analysis are essentially automated, analytic time is brief, and no further purification step after primer extension is required. We also show that all stages of the HPLC-primer extension genotyping can be multiplexed which, together with automation, suggests that this system may be suitable for linkage studies based upon emerging SNP maps. Received: 27 April 1998 / Accepted: 26 November 1998     

Conformational selection in trypsin-like proteases

For over four decades, two competing mechanisms of ligand recognition-conformational selection and induced-fit-have dominated our interpretation of protein allostery. Defining the mechanism broadens our understanding of the system and impacts our ability to design effective drugs and new therapeutics. Recent kinetics studies demonstrate that trypsin-like proteases exist in equilibrium between two forms: one fully accessible to substrate (E) and the other with the active site occluded (E*). Analysis of the structural database confirms existence of the E* and E forms and vouches for the allosteric nature of the trypsin fold. Allostery in terms of conformational selection establishes an important paradigm in the protease field and enables protein engineers to expand the repertoire of proteases as therapeutics.     

In vivo operational fascicle lengths of vastus lateralis during sub-maximal and maximal cycling

Instantaneous contractile characteristics of skeletal muscle, during movement tasks, can be determined and related to steady state mechanical properties such as the force–length relationship with the use of ultrasound imaging. A previous investigation into the contractile characteristics of the vastus lateralis (VL) during cycling has shown that fascicles operate on the “weak” descending limb of the force–length relationship, thus not taking advantage of the “strong” plateau region. The purpose of this study was to investigate if VL fascicle lengths change from sub-maximal to maximal cycling conditions, and if maximal cycling results in VL fascicle lengths which operate across the plateau of the force–length relationship. Fifteen healthy male subjects (age 20.9±1.8 yr, wt. 67.0±6.3 kg, ht. 176.7±7.2 cm) were tested to establish the maximal force–length relationship for the VL through ten maximal isometric contractions at various knee angles. Subjects then cycled on an SRM cycle ergometer at cadences of 50 and 80 revolutions per minute at 100 W, 250 W, and maximal effort. Fascicle lengths were determined at crank angles of 0, 90, and 180°. Fascicles operated at or near the plateau of the maximal force–length relationship for maximal cycling, while operating on the descending limb during sub-maximal conditions for both cadences. However, when comparing the fascicle operating range for the sub-maximal cycling conditions to the corresponding sub-maximal force–length relationships, the VL now also operated across the plateau region. We concluded from these results that regardless of cycling effort, the VL operated through the ideal plateau region of the corresponding force–length relationship, hence always working optimally. We hypothesize that this phenomenon is due to the coupling of series elastic compliance and length dependent calcium sensitivity in the VL.     

Sexual Orientation and Functional Pain in U.S. Young Adults: The Mediating Role of Childhood Abuse

Objective

Pain without known pathology, termed “functional pain,” causes much school absenteeism, medication usage, and medical visits. Yet which adolescents are at risk is not well understood. Functional pain has been linked to childhood abuse, and sexual orientation minority youth (gay, lesbian, bisexual, “mostly heterosexual,” and heterosexual with same-sex sexual contact) are more likely to be victims of childhood abuse than heterosexuals, thus may be at greater risk of functional pain.

Methods

We examined sexual orientation differences in past-year prevalence of functional headache, pelvic, and abdominal pain and multiple sites of pain in 9,864 young adults (mean age = 23 years) from a large U.S. cohort. We examined whether childhood abuse accounted for possible increased risk of functional pain in sexual minority youth.

Results

Sexual minority youth, except for gays and lesbians, were at higher risk of functional pelvic and abdominal pain and multiple sites of pain than heterosexuals. Gay and lesbian youth had elevated prevalence only of abdominal pain. Childhood abuse accounted for 14% to 33% of increased experience of multiple sites of pain in minority youth.

Conclusions

Youth who identify as “mostly heterosexual” or bisexual or who identify as heterosexual and have had same-sex partners comprised 18% of our sample. Clinicians should be aware that patients with these orientations are at elevated risk of functional pain and may be in need of treatment for sequelae of childhood abuse. Conventional categorization of sexual orientation as heterosexual or homosexual may fail to distinguish a large number of youth who do not wholly identify with either group and may be at elevated risk of health problems.     

Genetic analysis of lipopolysaccharide core biosynthesis by Escherichia coli K-12: insertion mutagenesis of the rfa locus.

Tn10 insertions were selected on the basis of resistance to the lipopolysaccharide (LPS)-specific bacteriophage U3. The majority of these were located in a 2-kilobase region within the rfa locus, a gene cluster of about 18 kb that contains genes for LPS core biosynthesis. The rfa::Tn10 insertions all exhibited a deep rough phenotype that included hypersensitivity to hydrophobic antibiotics, a reduction in major outer membrane proteins, and production of truncated LPS. These mutations were complemented by a Clarke-Carbon plasmid known to complement rfa mutations of Salmonella typhimurium, and analysis of the insert from this plasmid showed that it contained genes for at least six polypeptides which appear to be arranged in the form of a complex operon. Defects in two of these genes were specifically implicated as the cause of the deep rough phenotype. One of these appeared to be rfaG, which encodes a function required for attachment of the first glucose residue to the heptose region of the core. The other gene did not appear to be directly involved in determination of the sugar composition of the core. We speculate that the product of this gene is involved in the attachment of phosphate or phosphorylethanolamine to the core and that it is the lack of one of these substituents which results in the deep rough phenotype.