Molecular profiling of a 6-hydroxydopamine model of Parkinson's disease.

Convection enhanced delivery of 6-hydroxydopamine (6-OHDA) to the rat striatum results in a model of Parkinson's disease. An important feature of this unilateral model is the progressive loss of dopaminergic (DA) neurons over the course of several weeks. To improve the understanding of this model, gene expression changes in the substantia nigra, which contains the DA neuron cell bodies, and the striatum, which contains the DA neuron synaptic terminals, were examined using DNA microarrays. Samples were collected and behavior was analyzed from vehicle and toxin treated animals at 3 days, 1 week, 2 weeks and 4 weeks following 6-OHDA treatment. Tissue DA content was determined and samples from animals which exhibited a substantial depletion of striatal DA were included in the subsequent gene expression analysis. The results of the gene expression analysis indicated that 6-OHDA elicits a vigorous inflammatory response, comprised of several distinct pathways, in the striatum at the earliest time point tested. In contrast, relatively few gene expression changes were observed in the SN at the 3-day time point. In both tissues examined there was evidence for a vigorous inflammatory response at the 1- and 2-week time points, which was substantially diminished by the 4-week time point. Inflammation plays a prominent role in the 6-OHDA model of Parkinson's disease.

Library preparation for next generation sequencing: A review of automation strategies.

Next generation sequencing is in the process of evolving from a technology used for research purposes to one which is applied in clinical diagnostics. Recently introduced high throughput and benchtop instruments offer fully automated sequencing runs at a lower cost per base and faster assay times. In turn, the complex and cumbersome library preparation, starting with isolated nucleic acids and resulting in amplified and barcoded DNA with sequencing adapters, has been identified as a significant bottleneck. Library preparation protocols usually consist of a multistep process and require costly reagents and substantial hands-on-time. Considerable emphasis will need to be placed on standardisation to ensure robustness and reproducibility. This review presents an overview of the current state of automation of library preparation for next generation sequencing. Major challenges associated with library preparation are outlined and different automation strategies are classified according to their functional principle. Pipetting workstations allow high-throughput processing yet offer limited flexibility, whereas microfluidic solutions offer great potential due to miniaturisation and decreased investment costs. For the emerging field of single cell transcriptomics for example, microfluidics enable singularisation of tens of thousands of cells in nanolitre droplets and barcoding of the RNA to assign each nucleic acid sequence to its cell of origin. Finally, two applications, the characterisation of bacterial pathogens and the sequencing within human immunogenetics, are outlined and benefits of automation are discussed.

A gradient of sequence divergence in the human adult alpha-globin duplication units.

The nucleotide sequences of the two 5'-homology blocks of human alpha-globin gene duplication units were determined. The sequence difference between the two blocks is essentially zero in the 5' portions, and increases gradually toward the 3' ends until it reaches a value of 18 percent. This gradient of sequence divergence is similar to the distribution of the frequencies of gene conversion along several loci in Ascobolus and yeast. Hot spots for initiation of gene correction processes appear to exist near the 5' ends of the human alpha-globin duplication units. The data provide the physical evidence for polar gene correction process in a mammalian genome.

Review on pneumatic operations in centrifugal microfluidics.

Centrifugal microfluidics allows for miniaturization, automation and parallelization of laboratory workflows. The fact that centrifugal forces are always directed radially outwards has been considered a main drawback for the implementation of complex workflows leading to the requirement of additional actuation forces for pumping, valving and switching. In this work, we review and discuss the combination of centrifugal with pneumatic forces which enables transport of even complex liquids in any direction on centrifugal systems, provides actuation for valving and switching, offers alternatives for mixing and enables accurate and precise metering and aliquoting. In addition, pneumatics can be employed for timing to carry out any of the above listed unit operations in a sequential and cascaded manner. Firstly, different methods to generate pneumatic pressures are discussed. Then, unit operations and applications that employ pneumatics are reviewed. Finally, a tutorial section discusses two examples to provide insight into the design process. The first tutorial explains a comparatively simple implementation of a pneumatic siphon valve and provides a workflow to derive optimum design parameters. The second tutorial discusses cascaded pneumatic operations consisting of temperature change rate actuated valving and subsequent pneumatic pumping. In conclusion, combining pneumatic actuation with centrifugal microfluidics allows for the design of robust fluidic networks with simple fluidic structures that are implemented in a monolithic fashion. No coatings are required and the overall demands on manufacturing are comparatively low. We see the combination of centrifugal forces with pneumatic actuation as a key enabling technology to facilitate compact and robust automation of biochemical analysis.

Production of recombinant human tektin 1, 2, and 4 and in vitro assembly of human tektin 1.

Proteins predicted to be composed of large stretches of coiled-coil structure have often proven difficult to crystallize for structural determination. We have successfully applied EPR spectroscopic techniques to the study of the structure and assembly of full-length human vimentin assembled into native 11 nm filaments, in physiologic solution, circumventing the limitations of crystallizing shorter peptide sequences. Tektins are a small family of highly alpha helical filamentous proteins found in the doublet microtubules of cilia and related structures. Tektins exhibit several similarities to intermediate filaments (IFs): moderate molecular weight, highly alpha helical, hypothesized to be coiled-coil, and homo- and heteromeric assembly into long smooth filaments. In this report, we show the application of IF research methodologies to the study of tektin structure and assembly. To begin in vitro studies, expression constructs for human tektins 1, 2, and 4 were synthesized. Recombinant tektins were produced in E. coli and purified by chromatography. Preparations of tektin 1 successfully formed filaments. The recombinant human tektin 1 was used to produce antibodies which recognized an antigen in mouse testes, most likely present in sperm flagella. Finally, we report the creation of seven mutants to analyze predictions of coiled-coil structure in the rod 1A domain of tektin 1. Although this region is predicted to be coiled-coil, our EPR analysis does not reflect the parallel, in register, coiled-coil structure as demonstrated in vimentin and kinesin. These results document that tektin can be successfully expressed and assembled in vitro, and that SDSL EPR techniques can be used for structural analysis.

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage.

BACKGROUND: Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes. RESULTS: A single tenascin gene was identified in the genome of C. intestinalis that encodes a polypeptide with domain features common to all vertebrate tenascins. Both pufferfish genomes encode five tenascin genes: two tenascin-C paralogs, a tenascin-R with domain organization identical to mammalian and avian tenascin-R, a small tenascin-X with previously undescribed GK repeats, and a tenascin-W. Four tenascin genes corresponding to tenascin-C, tenascin-R, tenascin-X and tenascin-W were also identified in the X. tropicalis genome. Multiple sequence alignment reveals that differences in the size of tenascin-W from various vertebrate classes can be explained by duplications of specific fibronectin type III domains. The duplicated domains are encoded on single exons and contain putative integrin-binding motifs. A phylogenetic tree based on the predicted amino acid sequences of the fibrinogen-related domains demonstrates that tenascin-C and tenascin-R are the most closely related vertebrate tenascins, with the most conserved repeat and domain organization. Taking all lines of evidence together, the data show that the tenascins referred to as tenascin-Y and tenascin-N are actually members of the tenascin-X and tenascin-W gene families, respectively. CONCLUSION: The presence of a tenascin gene in urochordates but not other invertebrate phyla suggests that tenascins may be specific to chordates. Later genomic duplication events led to the appearance of four family members in vertebrates: tenascin-C, tenascin-R, tenascin-W and tenascin-X.

Cloning and pharmacological characterization of a rabbit bradykinin B1 receptor.

A rabbit B1 bradykinin receptor cDNA was isolated from a rabbit aorta smooth muscle cell library. The 1223 bp cDNA clone encodes a protein of 352 amino acids which is 78% identical to the human bradykinin B1(3) receptor protein. Heterologous expression of the rabbit B1 receptor cDNA in COS-7 cells imparts a high affinity specific binding for 3H-labeled [des-Arg10,Leu9]kallidin. Scatchard analysis indicates that the receptor binds the radiolabeled ligand with a Kd of 0.5 nM. The ability of kallidin (Lys-bradykinin) and bradykinin analogues to compete with binding of 3H-labeled [des-Arg10,Leu9]kallidin was determined and defined a rank order of potency: [des-Arg10,Leu9]kallidin = [des-Arg10]kallidin > [des- Arg9]bradykinin = kallidin >> bradykinin. This receptor exhibits the classical B1 pharmacological property of preferentially binding to kinin analogues which lack the C-terminal arginine. In addition, the affinities for [des-Arg10]kallidin and [des-Arg10,Leu9]kallidin are 100-fold higher than those for the corresponding bradykinin analogues [des-Arg9]bradykinin and [des-Arg9,Leu8]bradykinin which lack the N-terminal lysine. This pharmacological profile is characteristic of the B1 receptor subtype.

Functional STAT 1 and 3 signaling by the leptin receptor (OB-R); reduced expression of the rat fatty leptin receptor in transfected cells.

The leptin receptor (OB-R) bears homology to members of the class I cytokine receptor family. We demonstrate that leptin binding to OB-R stimulates formation of STAT-1 and STAT-3 complexes, thereby defining transcriptional motifs for genes that are under leptin control. Transfected fa OB-R bound leptin with equal affinity to that of wild type OB-R. fa OB-R abundance was about 7 fold reduced compared to control cells. Surprisingly, the low level of fa OB-R is fully capable of activating the STAT signal transduction pathway. We discuss plausible explanations for the obese phenotype in Zucker fatty rats.

Nucleotide sequence of the bovine vimentin-encoding cDNA.

Using reverse transcription-polymerase chain reaction (RT-PCR), we have amplified vimentin (Vim)-encoding sequences from both total and polyadenylated bovine lens RNA. The amplified products were cloned and the nucleotide sequences determined. The amino-acid sequence of bovine vimentin shows 97.2, 96.8, 96.3 and 84.9% homology with reported aa sequences of human, mouse, hamster and chicken Vim, respectively.

Cloning of a novel member of the low-density lipoprotein receptor family.

A gene encoding a novel transmembrane protein was identified by DNA sequence analysis within the insulin-dependent diabetes mellitus (IDDM) locus IDDM4 on chromosome 11q13. Based on its chromosomal position, this gene is a candidate for conferring susceptibility to diabetes. The gene, termed low-density lipoprotein receptor related protein 5 (LRP5), encodes a protein of 1615 amino acids that contains conserved modules which are characteristic of the low-density lipoprotein (LDL) receptor family. These modules include a putative signal peptide for protein export, four epidermal growth factor (EGF) repeats with associated spacer domains, three LDL-receptor (LDLR) repeats, a single transmembrane spanning domain, and a cytoplasmic domain. The encoded protein has a unique organization of EGF and LDLR repeats; therefore, LRP5 likely represents a new category of the LDLR family. Both human and mouse LRP5 cDNAs have been isolated and the encoded mature proteins are 95% identical, indicating a high degree of evolutionary conservation.

Effects of the bradykinin B1 receptor antagonist des-Arg9[Leu8]bradykinin and genetic disruption of the B2 receptor on nociception in rats and mice.

The contributions of B1 and B2 bradykinin receptors to acute and chronic inflammatory hyperalgesia were examined using the peptide B1 receptor antagonist des-Arg9[Leu8]bradykinin and transgenic Bk2r-/- mice. In normal rats and mice, des-Arg9[Leu8]bradykinin (30 nmol/kg i.v. or s.c.) inhibited carrageenan-induced hyperalgesia and the late phase nociceptive response to formalin. The active dose range was narrow, suggesting partial agonist activity of this peptide. In rats with monoarthritis, des-Arg9[Leu8]bradykinin (up to 30 nmol/kg i.v.) failed to reduce the number of vocalisations elicited by gentle flexion and extension of the inflamed limb; however, hyperalgesia was exacerbated by administration of the B1 receptor agonist des-[Arg9]bradykinin (100 nmol/kg i.v.), consistent with other evidence for local induction of B1 receptors during adjuvant-induced arthritis. The nociceptive response to intraplantar injection of bradykinin (10 nmol) and hyperalgesia induced by carrageenan (0.6 mg) were absent in Bk2r-/- mice, indicating that stimulation of B2 receptors is an essential step in the initiation of some nociceptive and inflammatory reactions. However, the nociceptive response to formalin (2.5% intraplantar), including inhibition of the late phase by des-Arg9[Leu8]bradykinin (0.3 nmol), and induction of thermal hyperalgesia by Freund's adjuvant (0.1%) appeared intact in Bk2r-/- mice. These findings support other evidence for an involvement of B1 receptors in inflammatory hyperalgesia and suggest that B1 receptor antagonists may be clinically useful as anti-inflammatory and analgesic drugs.

Protein phosphorylation in the bacterial chemotaxis system.

Bacterial chemotaxis involves the detection of changes in concentration of specific chemicals in the environment of the cell as a function of time. This process is mediated by a series of cell surface receptors that interact with and activate intracellular protein phosphorylation. Five cytoplasmic proteins essential for chemotaxis have been shown to be involved in a coupled system of protein phosphorylation. Ligand binding to cell surface receptors affects the rate of autophosphorylation of the CheA protein. In the absence of an attractant bound to receptor and in the presence of the CheW protein, the rate of CheA autophosphorylation is markedly increased. Phosphorylated CheA can transfer phosphate to the CheY or CheB proteins; phosphorylation of these "effector" proteins may increase their activity. The CheY protein is thought to regulate flagellar rotation and thus control swimming behavior. The CheB protein modifies the cell surface receptor and thus regulates receptor function. Finally, another chemotaxis protein, CheZ, acts to specifically dephosphorylate CheY-phosphate. This system shows marked similarity to the 2-component sensor-regulator systems found to control specific gene expression in a variety of bacteria.

Expression of B1 and B2 bradykinin receptor mRNA and their functional roles in sympathetic ganglia and sensory dorsal root ganglia neurones from wild-type and B2 receptor knockout mice.

Bradykinin has been implicated in nociception and inflammation. To examine the relative significance of B1 and B2 bradykinin receptor subtypes in sympathetic and sensory ganglia, the electrophysiological effects of bradykinin analogues and the expression of receptor subtype mRNA were examined in wild-type and "B2 knockout" mice from which the B2 receptor gene had been deleted. In wild-type mice the B2 receptor agonist bradykinin depolarized superior cervical ganglia (SCG) and activated inward currents in dorsal root ganglia (DRG) neurones. Responses to the B1 receptor agonist, [des-Arg10]-kallidin, were seen only in SCG that had been pre-treated with interleukins and the peptidase inhibitor captopril, but not in DRG neurones. The up-regulation of responses to [des-Arg10]-kallidin and substance P were blocked by indomethacin and, thus, were dependent upon cyclo-oxygenase activity. The effects of bradykinin were abolished in SCG and DRG's from B2 knockout mice and this was correlated with the absence of B2 receptor mRNA in ganglia from these animals. However, despite the presence of B1 receptor mRNA in interleukin treated SCG from B2 knockout mice, no depolarizing effects of the B1 receptor agonist [des-Arg10]-kallidin were observed. The successful elimination of bradykinin responses and B2 mRNA in sympathetic and sensory ganglia from B2 knockout mice, confirms that B2 receptors are the predominant functional bradykinin receptor subtype in these tissues and that B1 receptor mRNA is expressed in both sympathetic and sensory ganglia from these animals.

Expression of the type I diabetes-associated gene LRP5 in macrophages, vitamin A system cells, and the Islets of Langerhans suggests multiple potential roles in diabetes.

LRP5 is a novel member of the low-density lipoprotein receptor family that is genetically associated with Type 1 diabetes. As a start to defining the normal function of LRP5 and to generate testable hypotheses of its potential role in Type 1 diabetes pathogenesis, we carried out an extensive expression analysis of this gene at the mRNA and protein levels in normal human, monkey, and mouse, as well as in non-obese diabetic (NOD) mice at several stages of diabetes development. In all species, expression of LRP5 was found in four functionally important cell types: the distributed mononuclear phagocyte system, the islets of Langerhans, vitamin A-metabolizing cells, and CNS neurons. Given the critical role of macrophages in the onset and progression of islet cell destruction in Type 1 diabetes and the hypothesized role of retinoids as modifiers of diabetes progression, these findings suggest that LRP5 may confer Type 1 diabetes risk by altering the normal functioning of one or more of these regulatory systems. Specifically, given that the LRP5 polymorphisms associated with diabetes are in the promoter region of the gene, alterations in LRP5 expression may be responsible for diabetes susceptibility and therefore may be potential targets for therapeutic intervention. (J Histochem Cytochem 48:1357-1368, 2000)

Development- and differentiation-dependent reorganization of intermediate filaments in fiber cells.

PURPOSE: To define the remodeling of lens fiber cell intermediate filaments (IF) that occurs with both development and differentiation. METHODS: Prenatal and postnatal mice were probed for the IF proteins phakosin, filensin, and vimentin, using light microscope immunocytochemical methodology. RESULTS: The pattern of vimentin accumulation in elongating fiber cells changed with development. Early in development vimentin first emerged predominantly as focal accumulations in the basal region of both epithelial and primary fiber cells. A light diffuse cytoplasmic staining was also noted. Later in embryonic development, and through maturity, vimentin in fiber cells was predominantly associated with the plasma membrane with no anterior-posterior polarity. Phakosin and filensin were first detected in the very latest stages of primary fiber elongation and continued to accumulate well after cells had completed elongation. Initially, these proteins accumulated in the anterior half of the fiber cells and were cytoplasmic in distribution. After P13, the pattern of initial distribution in differentiating fiber cells changed to a predominantly plasma membrane localization. Neither beaded filament protein showed focal basal accumulations. In mature lenses, all three proteins ultimately disappeared from the nuclear fiber cells. CONCLUSIONS: Beaded filament protein accumulation lags significantly behind both primary and secondary fiber cell elongation, suggesting a functional role subsequent to elongation. The subcellular distribution of vimentin and the beaded filament proteins showed marked differences within the cell, with differentiation, and with development. The differences in time of initial synthesis and in distribution of these IF proteins may bear on hypotheses about the role of IFs in fiber cell elongation and in structural-functional polarity of the fiber cell.

Protection of a restriction enzyme from heat inactivation by [alpha]-crystallin.

PURPOSE: To determine whether the chaperone activity of human alpha-crystallin can protect a restriction enzyme from heat inactivation. METHODS: The restriction enzyme Nde I was heated in the presence or absence of purified bovine alpha-crystallin. Following heat treatment, the enzymatic activity of the heat treated samples was assayed by cleavage of plasmid DNA. The extent of digestion was monitored by agarose gel electrophoresis and visualization of DNA fragments by ethidium bromide staining. RESULTS: Heating of Nde I in the absence of alpha-crystallin resulted in inactivation. However, Nde I heated in the presence of alpha-crystallin remained active. Furthermore, an increased amount of alpha-crystallin provided a longer period of thermal protection. CONCLUSIONS: The chaperone activity and thermo-protective effect of alpha-crystallin extend to protection of enzymatic activity, not merely the protection from thermally induced aggregation/denaturation. In addition, inclusion of alpha-crystallin during some enzymatic reactions may be beneficial.

Lack of DNase I mRNA sequences in murine lenses.

Clarity of the mammalian lens is due in part to the complete lack of internal organelles, including nuclei, within the lens fiber cells that compose the bulk of the lens. Experimental evidence shows that as the differentiation of lens fiber cells progresses, nuclei and nuclear DNA are actively degraded. Prior characterization of chick lens development suggests that DNase I could be involved in lens DNA degredation. However, recent data suggest that DNase I is unlikely to be the nuclease responsible for DNA degredation in the differentiating lens. In this report, we find that in the murine lens, mRNA for DNase I is undetectable by northern blotting or PCR. We conclude that mRNA for DNase I is either not present or present in very low levels in murine lens. Our results are consistent with the hypothesis that DNase I is not involved in lens DNA degredation.

Differential expression of mRNA for leptin receptor isoforms in the rat brain.

Leptin plays an important role in the control of food intake and energy metabolism by interacting with its receptor (OB-R) in the brain. Several alternatively spliced isoforms of OB-R have been identified. To study the expression patterns and the potential biological function of these OB-Rs in the brain, the distribution of mRNA encoding OB-R isoforms was examined by in situ hybridization. In agreement with previous studies, strong signals for OB-R mRNA were detected in the hypothalamus, thalamus and choroid plexus. In addition, intense signals were observed in several other brain areas including piriform cortex, granule cell layer of the cerebellum and substantia nigra. With isoform-specific probes, a differential expression pattern of OB-Rs was revealed: OB-Ra and OB-Rb, but not OB-Rc and OB-Rf, are abundantly expressed in the hypothalamus, whereas OB-Ra, OB-Rc and OB-Rf, but not OB-Rb, are significantly expressed in the choroid plexus. The preferential expression of OB-Rb in the hypothalamus is in support of its role in mediating the satiety effect of leptin. The co-expression of OB-Ra with OB-Rb in the hypothalamus may suggest a possible interaction between the two isoforms. Finally, the detection of OB-R mRNA in a number of other brain regions may indicate the involvement of leptin in additional as yet undefined physiological functions.

Bradykinin B1 receptors in rabbit aorta smooth muscle cells in culture.

Kinin B1 receptors on rabbit aorta smooth muscle cells in culture were investigated. [3H]Des-Arg10-kallidin labeled a single site in cells at early passage with an equilibrium dissociation constant of 258 pM and a maximal binding density of approximately 680 sites/cell. Treatment of the same cells for 18 h with epidermal growth factor increased the binding density over 6-fold without affecting the ligand's affinity. At latter passages, the density of binding sites was found to increase and the growth factor had a much less pronounced effect. The rank order of potencies for agonist inhibition of binding (des-Arg10-kallidin > des-Arg9-BK = kallidin > bradykinin) was consistent with the specific labeling of a B1 receptor. Also, [3H]des-Arg10-kallidin binding was potently inhibited by the B1 receptor antagonist des-Arg9[Leu8]bradykinin but not by the B2 receptor antagonist Hoe 140. The agonists were found to stimulate phosphoinositide hydrolysis in the smooth muscle cells with an order of potencies that reflected their binding assay activities. Des-Arg9[Leu8] BK blocked the des-Arg10-kallidin response with a potency consistent with its known B1 receptor activity while Hoe 140 was inactive. These results demonstrate the presence of inducible B1 receptors on rabbit aorta smooth muscle cells in culture that couple to phospholipase C activation. These cells should be useful in future studies of the mechanisms and factors involved in the regulation of expression of the B1 receptor.