Alpha-2 adrenergic stimulation triggers Achilles tenocyte hypercellularity: Comparison between two model systems.

The histopathology of tendons with painful tendinopathy is often tendinosis, a fibrosis-like condition of unclear pathogenesis characterized by tissue changes including hypercellularity. The primary tendon cells (tenocytes) have been shown to express adrenoreceptors (mainly alpha-2A) as well as markers of catecholamine production, particularly in tendinosis. It is known that adrenergic stimulation can induce proliferation in other cells. The present study investigated the effects of an exogenously administered alpha-2 adrenergic agonist in an established in vivo Achilles tendinosis model (rabbit) and also in an in vitro human tendon cell culture model. The catecholamine producing enzyme tyrosine hydroxylase and the alpha-2A-adrenoreceptor (α2A AR) were expressed by tenocytes, and alpha-2 adrenergic stimulation had a proliferative effect on these cells, in both models. The proliferation was inhibited by administration of an α2A AR antagonist, and the in vitro model further showed that the proliferative alpha-2A effect was mediated via a mitogenic cell signaling pathway involving phosphorylation of extracellular-signal-regulated kinases 1 and 2. The results indicate that catecholamines produced by tenocytes in tendinosis might contribute to the proliferative nature of the pathology through stimulation of the α2A AR, pointing to a novel target for future therapies. The study furthermore shows that animal models are not necessarily required for all aspects of this research.

Endogenous substance P production in the Achilles tendon increases with loading in an in vivo model of tendinopathy-peptidergic elevation preceding tendinosis-like tissue changes.

OBJECTIVES: To quantify the intratendinous levels of substance P (SP) at different stages of overload in an established model for Achilles tendinopathy (rabbit). Also, to study the distribution of the SP-receptor, the NK-1R, and the source of SP, in the tendon. METHODS: Animals were subjected to the overuse protocol for 1, 3 or 6 weeks. One additional group served as unexercised controls. Immunoassay (EIA), immunohistochemistry (IHC), and in situ hybridisation (ISH) were performed. RESULTS: EIA revealed increased SP-levels in the Achilles tendon of the exercised limb in all the experimental groups as compared to in the controls (statistically significant; p=0.01). A similar trend in the unexercised Achilles tendon was observed but was not statistically significant (p=0.14). IHC and in ISH illustrated reactions of both SP and NK-1R mainly in blood vessel walls, but the receptor was also found on tenocytes. CONCLUSIONS: Achilles tendon SP-levels are elevated already after 1 week of loading. This shows that increased SP-production precedes tendinosis, as tendinosis-like changes occur only after a minimum of 3 weeks of exercise, as shown in a recent study using this model. We propose that central neuronal mechanism may be involved as similar trends were observed in the contralateral Achilles tendon.

Mechanical force modulates scleraxis expression in bioartificial tendons.

Following tendon injury, cartilage, bone and fat metaplasia are often observed, making the optimization of tenocyte differentiation an important clinical goal. In this study we examined the effect of static and cyclic mechanical loading on the expression of genes which play a role in tenocyte differentiation and function, namely scleraxis (Scx) and Type I collagen (Col1a1), and determined the effect of varying mechanical parameters including (1) static vs dynamic load, (2) increasing strain magnitude, (3) inclusion of 10 s rest periods, and (4) increasing cycle number. Cyclic loading resulted in a greater increase of tenocyte gene expression than static loading over 3 weeks in culture. Increasing strain levels potentiated the induction of tenocyte genes. The insertion of a 10 s rest periods further enhanced tenocyte gene expression, as did increasing repetition numbers. These results suggest that mechanical signaling exerts an important influence on the expression of genes which play a role in determining the tendon phenotype. Further work is required to confirm and extend these findings in primary cells such as resident tendon progenitor/stem cells, in order to provide an improved understanding of biology from which optimized rehabilitation programs can be developed.

Physical activity level in Achilles tendinosis is associated with blood levels of pain-related factors: a pilot study.

Physical activity affects the pain symptoms for Achilles tendinosis patients. Brain-derived neurotrophic factor (BDNF), tumor necrosis factor-alpha (TNF-α) and their receptors have been detected in human Achilles tendon. This pilot study aimed to compare serum BDNF and soluble tumor necrosis factor receptor I (sTNFRI) levels in Achilles tendinosis patients and healthy controls and to examine the influence of physical activity, and BMI and gender, on these levels. Physical activity was measured with a validated questionnaire, total physical activity being the parameter analyzed. Physical activity was strongly correlated with BDNF among tendinosis women [Spearman's rho (ρ)=0.90, P<0.01] but not among control women (ρ=-0.08, P=0.83), or among tendinosis and control men. Physical activity was significantly correlated with sTNFRI in the entire tendinosis group and among tendinosis men (ρ=0.65, P=0.01), but not in the entire control group or among control men (ρ=0.04, P=0.91). Thus, the physical activity pattern is related to the TNF and BDNF systems for tendinosis patients but not controls, the relationship being gender dependent. This is new information concerning the relationship between physical activity and Achilles tendinosis, which may be related to pain for the patients. This aspect should be further evaluated using larger patient materials.

The p21(RAS) farnesyltransferase alpha subunit in TGF-beta and activin signaling.

The alpha subunit of p21(RAS) farnesyltransferase (FNTA), which is also shared by geranylgeranyltransferase, was isolated as a specific cytoplasmic interactor of the transforming growth factor-beta (TGF-beta) and activin type I receptors with the use of the yeast two-hybrid system. FNTA interacts specifically with ligand-free TGF-beta type l receptor but is phosphorylated and released upon ligand binding. Furthermore, the release is dependent on the kinase activity of the TGF-beta type II receptor. Thus, the growth inhibitory and differentiative pathways activated by TGF-beta and activin involve novel mechanisms of serine-threonine receptor phosphorylation-dependent release of cytoplasmic interactors and regulation of the activation of small G proteins, such as p21(RAS).

Immunohistochemical evidence of local production of catecholamines in cells of the muscle origins at the lateral and medial humeral epicondyles: of importance for the development of tennis and golfer's elbow?

BACKGROUND: Tennis elbow (TE) is a painful condition affecting the common extensor origin at the lateral humeral epicondyle. Colour Doppler examination has shown increased blood flow at this site and the sensory, and sympathetic innervation patterns have been delineated. However, it is not known whether there is local production of catecholamines and/or acetylcholine in this tissue, which is the case in patellar and Achilles tendinopathies. OBJECTIVE: To investigate the possible presence of local production of catecholamines and acetylcholine in non-neuronal cells (fibroblasts) in connective tissue at the muscle origin at the lateral humeral epicondyle in patients with TE. DESIGN: Immunohistochemical studies were performed on biopsies taken from the extensor origin in patients with TE and in pain-free controls. For reference purpose, biopsies from the flexor origin in patients with golfer's elbow (GE) were also studied. PATIENTS: Seven patients with TE and four patients with GE. Six healthy asymptomatic individuals served as controls. METHOD: Immunohistochemistry, using antibodies detecting synthesising enzymes for catecholamines (tyrosine hydroxylase; TH) and acetylcholine (choline acetyltransferase; ChAT). RESULTS: TH-like immunohistochemical reactions were seen in fibroblasts in four of the seven patients with TE and two of the four patients with GE. No such reactions were detected in controls (0/6). No ChAT reactions were seen in any of the investigated specimens. CONCLUSIONS: There is evidence of local, non-neuronal production of catecholamines, but not acetylcholine, in fibroblasts in the tissue at the muscle origin at the lateral and medial epicondyles in patients with TE and GE, respectively, which might have an influence on blood vessel regulation and pain mechanisms in these conditions.

Transgenic mice expressing beta-galactosidase in mature neurons under neuron-specific enolase promoter control.

To gain insights into transcription factors defining neuronal identity, we generated transgenic mice carrying a 1.8 kb rat neuron-specific enolase (NSE) promoter fragment fused to an E. coli lacZ gene. Four of seven transgenic families expressed transgene RNA in the nervous system but not in most other tissues. Histochemical analysis of adult brain from the two lines with highest lacZ mRNA levels showed neuron-specific, pan-neuronal beta-galactosidase activity. Developmental RNA and histochemical analyses showed parallel onset of transgene and endogenous NSE gene expression in various neuronal cell types, although the magnitude of NSE mRNA accumulation later in development was not matched by the transgene. These results suggest that cis-acting regulatory elements, subject to neuron-specific control, are located within 1.8 kb upstream from the NSE gene.

Putting patients at the center of kidney care transitions: PREPARE NOW, a cluster randomized controlled trial.

Care for patients transitioning from chronic kidney disease to kidney failure often falls short of meeting patients' needs. The PREPARE NOW study is a cluster randomized controlled trial studying the effectiveness of a pragmatic health system intervention, 'Patient Centered Kidney Transition Care,' a multi-component health system intervention designed to improve patients' preparation for kidney failure treatment. Patient-Centered Kidney Transition Care provides a suite of new electronic health information tools (including a disease registry and risk prediction tools) to help providers recognize patients in need of Kidney Transitions Care and focus their attention on patients' values and treatment preferences. Patient-Centered Kidney Transition Care also adds a 'Kidney Transitions Specialist' to the nephrology health care team to facilitate patients' self-management empowerment, shared-decision making, psychosocial support, care navigation, and health care team communication. The PREPARE NOW study is conducted among eight [8] outpatient nephrology clinics at Geisinger, a large integrated health system in rural Pennsylvania. Four randomly selected nephrology clinics employ the Patient Centered Kidney Transitions Care intervention while four clinics employ usual nephrology care. To assess intervention effectiveness, patient reported, biomedical, and health system outcomes are collected annually over a period of 36 months via telephone questionnaires and electronic health records. The PREPARE NOW Study may provide needed evidence on the effectiveness of patient-centered health system interventions to improve nephrology patients' experiences, capabilities, and clinical outcomes, and it will guide the implementation of similar interventions elsewhere. TRIAL REGISTRATION: NCT02722382.

Transcriptional activation of the mouse Mx gene by type I interferon.

Mouse cells of the Mx+ genotype accumulate Mx mRNA in response to type I interferon (IFN). Nuclear runoff experiments show that IFN stringently regulates Mx gene expression at the level of transcription. Mx mRNA synthesis peaks about 3 h after IFN treatment, and within 5 h, Mx mRNA concentration rises from undetectable levels to about 0.1% of polyadenylated RNA.

Structural characterization of a heterogeneous family of rat brain mRNAs.

A large heterogeneous family of RNAs derived from a single rat gene contains members that differ from each other at one or more of three positions. Their 5' ends are nested and transcription can begin at 22 or more sites covering 265 nucleotides. Many of the 5' ends are detectable only in brain RNAs, and even 5' ends common with other tissues appear with different absolute and relative abundances in brain RNA. The central portions of the RNAs are of two forms, differing only by the presence or absence of 17 nucleotides; these forms are probably produced by alternative splicing. Polyadenylation occurs at either of two sites. This complicated family of 88 RNAs encodes two novel putative proteins that differ at their C termini.

Expression of small cytoplasmic transcripts of the rat identifier element in vivo and in cultured cells.

We examined the level of expression of small RNA transcripts hybridizing to a rodent repetitive DNA element, the identifier (ID) sequence, in a variety of cell types in vivo and in cultured mammalian cells. A 160-nucleotide (160n) cytoplasmic poly(A)+ RNA (BC1) appeared in late embryonic and early postnatal rat brain development, was enriched in the cerebral cortex, and appeared to be restricted to neural tissue and the anterior pituitary gland. A 110n RNA (BC2) was specifically enriched in brain, especially the postnatal cortex, but was detectable at low levels in peripheral tissues. A third, related 75n poly(A)- RNA (T3) was found in rat brain and at lower levels in peripheral tissues but was very abundant in the testes. The BC RNAs were found in a variety of rat cell lines, and their level of expression was dependent upon cell culture conditions. A rat ID probe detected BC-like RNAs in mouse brain but not liver and detected a 200n RNA in monkey brain but not liver at lower hybridization stringencies. These RNAs were expressed by mouse and primate cell lines. Thus, tissue-specific expression of small ID-sequence-related transcripts is conserved among mammals, but the tight regulation found in vivo is lost by cells in culture.

The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans.

Cytochrome p450s comprise a superfamily of heme-thiolate proteins named for the spectral absorbance peak of their carbon-monoxide-bound species at 450 nm. Having been found in every class of organism, including Archaea, the p450 superfamily is believed to have originated from an ancestral gene that existed over 3 billion years ago. Repeated gene duplications have subsequently given rise to one of the largest of multigene families. These enzymes are notable both for the diversity of reactions that they catalyze and the range of chemically dissimilar substrates upon which they act. Cytochrome p450s support the oxidative, peroxidative and reductive metabolism of such endogenous and xenobiotic substrates as environmental pollutants, agrochemicals, plant allelochemicals, steroids, prostaglandins and fatty acids. In humans, cytochrome p450s are best know for their central role in phase I drug metabolism where they are of critical importance to two of the most significant problems in clinical pharmacology: drug interactions and interindividual variability in drug metabolism. Recent advances in our understanding of cytochrome p450-mediated drug metabolism have been accelerated as a result of an increasing emphasis on functional genomic approaches to p450 research. While human cytochrome p450 databases have swelled with a flood of new human sequence variants, however, the functional characterization of the corresponding gene products has not kept pace. In response researchers have begun to apply the tools of proteomics as well as homology-based and ab initio modeling to salient questions of cytochrome p450 structure/function. This review examines the latest advances in our understanding of human cytochrome p450s.

Nucleotide sequence and cellular distribution of rat chromogranin B (secretogranin I) mRNA in the neuroendocrine system.

The mRNA of rat secretory-vesicle protein chromogranin B is abundant in brain, adrenal medulla, and anterior pituitary. The primary translation product predicted from the cDNA sequence of this 2,337-nucleotide transcript corresponds to a hydrophilic 655-residue protein preceded by a signal peptide. Both termini of the mature 75-kD protein show extensive similarity to other chromogranins; the more variable internal region is characterized by glutamic acid clusters and numerous pairs of basic residues. In rodent brain, mRNA accumulation starts around embryonic days 13-14 and peaks by postnatal day 20. In situ hybridization in brain sections shows that the mRNA is enriched in the hippocampal formation, the endocrine hypothalamus, the olfactory system, and in anatomically distinct structures in the pons-medulla.

Primary structure of mouse secretogranin III and its absence from deficient mice.

The rat brain- and pituitary-specific 1B1075 mRNA encodes a chromogranin/secretogranin-like protein, called secretogranin III (SgIII), that is a component of intracellular dense core vesicles. In order to study the function of this gene product in a mouse model system, we have isolated the murine homolog of the rat 1B1075 mRNA. This mRNA contains 2163 bp encoding a putative protein of 421 amino acids. Cleavage of the strong putative signal sequence would yield a mature protein of 51 kDa. The sequence of the encoded murine protein preserves the structural features that suggest SgIII is a member of the granin family, and allowed us to recognize and correct errors in our published rat sequence.

Isolation and sequence analysis of cytochrome P450 12B1: the first mitochondrial insect P450 with homology to 1 alpha,25 dihydroxy-D3 24-hydroxylase.

The biosynthesis of steroid hormones is an integral component of insect growth, development and reproduction. Although there is an abundance of biochemical data implicating both microsomal and mitochondrial cytochrome P450s in steroid metabolism, molecular genetic information on mitochondrial P450s is almost entirely limited to vertebrate sequences. In the current study, a degenerate polymerase chain reaction (PCR) primer was targeted to the highly conserved region of P450 genes that encodes the heme-binding decapeptide. Using a 5' rapid amplification of cDNA ends (RACE) approach, seven novel cytochrome P450 genes were isolated from Drosophila acanthoptera, including one sequence (CYP12B1) with high regional homology to vertebrate mitochondrial P450s. Sequence analysis of the conceptual translation of the full length gene, obtained by 5'RACE, revealed an amphipathic NH2-terminus rich in basic and hydrophilic amino acids, a characteristic feature of mitochondrial P450s that distinguishes them from their distantly related microsomal relatives. Phylogenetically, CYP12B1 appears to be most closely related to the mammalian mitochondrial P450s of the CYP24 family that play a critical role in calcium homeostasis through the metabolism of vitamin D3. The potential biological role of CYP12B1 is discussed in the light of what is currently known about arthropod calcium binding proteins and their regulation.

Cloning of a second proopiomelanocortin cDNA from the pituitary of the sturgeon, Acipenser transmontanus.

A recent study on the pituitary of the sturgeon, Acipenser transmontanus, resulted in the cloning of a cDNA that codes for the prohormone, proopiomelanocortin (POMC). This cDNA is designated sturgeon POMC A. Subsequent analysis of the sturgeon pituitary uncovered a second distinct POMC cDNA (sturgeon POMC B). In both sturgeon POMC cDNAs the open reading frame is 795 nucleotides in length. However, the two sturgeon POMC cDNAs differ at 26 amino acid positions in the opening frame. In addition, the 2 forms of POMC differ at 45 nucleotide positions within the open reading frame. The number and types of point mutations are compared in the 2 sturgeons POMC cDNAs, and the origin of the two POMC genes is discussed.

Analyzing the radiation of the proenkephalin gene in tetrapods: cloning of a Bombina orientalis proenkephalin cDNA.

Analyzing the Radiation of the Proenkephalin Gene in Tetrapods: Cloning of a Bombina orientalis Proenkephalin cDNA: A proenkephalin cDNA was cloned from the brain of the anuran amphibian, Bombina orientalis (Family: Discoglossidae). This cDNA is 1358 nucleotides in length, and contains an open reading frame that codes for 251 amino acids. Within the open reading frame there are seven opioid (YGGF) sequences. There were five Met-enkephalin (YGGFM) sequences that are flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended Met-enkephalin sequences: YGGFMRGY and YGGFMRF. No Leu-enkephalin sequences were found in B. orientalis proenkephalin. It was possible to align the amino acid sequences of proenkephalin from several vertebrate taxa (human, Australian lungfish, B. orientalis, Xenopus laevis, Spea multiplicatus) by inserting a minimum of nine gaps. This alignment was then used to analyze the corresponding nucleotides for each proenkephalin sequence using maximum likelihood. This analysis yielded a single tree. In this tree, the Australian lungfish sequence was the outgroup or the tetrapod ingroup. The amphibian sequences form a clade separate from the human sequence. The bootstrap value for the amphibian clade was 100%. Within the amphibian clade the Bombina sequence was the sister group to a clade composed of the X. laevis and S. multiplicatus sequences. The bootstrap value for the X. laevis/S. multiplicatus clade was 94%. Collectively, these data indicate that the sequence of Bombina proenkephalin may be more similar to the proposed ancestral anuran proenkephalin sequence, than either X. laevis or S. multiplicatus proenkephalin.

Organization of proenkephalin in amphibians: cloning of a proenkephalin cDNA from the brain of the anuran amphibian, Spea multiplicatus.

Cloning of a proenkephalin cDNA from the pelobatid anuran amphibian, Spea multiplicatus, provides additional evidence that Leu-enkephalin, although present in the brain of anuran amphibians, is not encoded by the proenkephalin gene. The S. multiplicatus proenkephalin cDNA is 1375 nucleotides in length, and the open reading frame contains the sequences of seven opioid sequences. There are five copies of the Met-enkephalin sequence, as well as an octapeptide opioid sequence (YGGFMRNY) and a heptapeptide opioid sequence (YGGFMRF). In the proenkephalin sequence of S. multiplicatus the penultimate opioid is a Met-enkephalin sequence rather than the Leu-enkephalin present in mammalian sequences. The same order of opioid sequences also is observed for the proenkephalin sequence of the pipid anuran amphibian, Xenopus laevis. Hence, from a phylogenetic standpoint the organization of tetrapod proenkephalin has been remarkably conserved. What remains to be resolved is whether the Leu-enkephalin sequence found in mammalian proenkephalin is an ancestral trait or a derived trait for the tetrapods. Unlike the proenkephalin precursor of X. laevis, all of the opioid sequences in the S. multiplicatus proenkephalin cDNA are flanked by paired-basic amino acid proteolytic cleavage sites. In this regard the proenkephalin sequence for S. multiplicatus is more similar to mammalian proenkephalins than the proenkephalin sequence of X. laevis. However, a comparison of the proenkephalin sequences in human, X. laevis, and S. multiplicatus revealed several conserved features in the evolution of the tetrapod proenkephalin gene. By contrast, a comparison of tetrapod proenkephalin sequences with the partial sequence of a sturgeon proenkephalin cDNA indicates that the position occupied by the penultimate opioid sequence in vertebrate proenkephalins may be a highly variable locus in this gene.

Cloning of a neoteleost (Oreochromis mossambicus) pro-opiomelanocortin (POMC) cDNA reveals a deletion of the gamma-melanotropin region and most of the joining peptide region: implications for POMC processing.

A signature feature of tetrapod pro-opiomelanocortin (POMC) is the presence of three melantropin (MSH) coding regions (alpha-MSH, beta-MSH, gamma-MSH). The MSH duplication events occurred early during the radiation of the jawed vertebrates well over 400 million years ago. However, in at least one order of modern bony fish (subdivision Teleostei; order Salmoniformes; i.e. salmon and trout) the gamma-MSH sequence has been deleted from POMC. To determine whether the gamma-MSH deletion has occurred in other teleost orders, a POMC cDNA was cloned from the pituitary of the neoteleost Oreochromis mossambicus (order Perciformes). In O. mossambicus POMC, the deletion is more extensive and includes the gamma-MSH sequence and most of the joining peptide region. Because the salmoniform and perciform teleosts do not share a direct common ancestor, the gamma-MSH deletion event must have occurred early in the evolution of the neoteleost fishes. The post-translational processing of O. mossambicus POMC occurs despite the fact that the proteolytic recognition sequence, (R/K)-Xn-(R/K) where n can be 0, 2, 4, or 6, a common feature in mammalian neuropeptide and polypeptide hormone precursors, is not present at several cleavage sites in O. mossambicus POMC. These observations would indicate that either the prohormone convertases in teleost fish use distinct recognition sequences or vertebrate prohormone convertases are capable of recognizing a greater number of primary sequence motifs around proteolytic cleavage sites.

Deciphering the origin of Met-enkephalin and Leu-enkephalin in Lobe-finned fish: cloning of australian lungfish proenkephalin.

The previous detection of Met-enkephalin and Leu-enkephalin in the CNS of the Australian lungfish, Neoceratodus forsteri, in a molar ratio comparable to mammals suggested that the lungfish proenkephalin precursor should contain the sequences of both Met-enkephalin and Leu-enkephalin as seen for mammalian proenkephalin. However, the cloning of a full-length proenkephalin cDNA from the CNS of the Australian lungfish indicates that the organization of this precursor is more similar to amphibian proenkephalin than mammalian proenkephalin. The Australian lungfish cDNA is 1284 nucleotides in length and the open reading frame (267 amino acids) contains seven opioid sequences (GenBank #AF232671). There are five copies of the Met-enkephalin sequence flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended forms of Met-enkephalin: YGGFMRSL and YGGFMGY. As seen for amphibians, no Leu-enkephalin sequence was detected in the Australian lungfish proenkephalin cDNA. The fact that Leu-enkephalin has been identified by radioimmunoassay and HPLC analysis in the CNS of the Australian lungfish indicates that a Leu-enkephalin-coding gene, distinct from proenkephalin, must be expressed in lungfish. Potential candidates may include a prodynorphin- or other opioid-like gene. Furthermore, the absence of a Leu-enkephalin sequence in lungfish and amphibian proenkephalin would suggest that the mutations that yielded this opioid sequence in tetrapod proenkephalin occurred at some point in the radiation of the amniote vertebrates.