Stress responses to short-term intensified and reduced training in competitive weightlifters.

We sought to identify and evaluate the tolerance to, and consequences of, short-term variations in training load in competitive weightlifters. Seven international-level lifters performed 1 week of initial training followed by 2 weeks of intensified (INT: +100%, 36.5 ± 11.3 × 10(3) kg/week) and 1 week of subsequently reduced (RED: -25%) training within their annual program. After INT, but not RED, 90 min of weightlifting increased mRNA levels of chemokine (C-C motif) ligand 4 (CCL4), chemokine (C-X-C motif) receptor 4 (CXCR4) and cellular stress-associated DNA-damage-inducible transcript 4 (DDIT4) in peripheral blood mononuclear cells by 40-240%. Resting- and weightlifting-induced changes in plasma protein carbonyls, indicative of oxidative stress, but not pro-inflammatory CCL4 concentrations differed between INT and RED. Symptoms of stress (Daily Analysis of Life Demands of Athletes questionnaire) were reported as worse than normal more frequently during INT and RED than initial training. Global (negative) mood state increased during INT and declined during RED. Maximal snatch (-4.3 ± 3.7%) and vertical jump (-7.2 ± 6.5%), but not clean and jerk, were reduced after INT and restored after RED. Chemokine signaling may thus be part of the stress response to intense weightlifting and short-term reductions in training load support recovery from periodic INT training in weightlifters.

Abdominal wall pseudohernia - One secondary to a thoracic extraforaminal disc herniation and other due to thoracic paracentral disc protrusion.

An abdominal wall pseudohernia is a rare clinical entity which consists of an abnormal bulging of the abdominal wall that can resemble a true hernia, but without an associated underlying fascial or muscle defect. It is caused by segmental neuropathy and subsequent denervation of abdominal wall musculature. We present two cases of an abdominal wall pseudohernia. One secondary to a thoracic extraforaminal disc herniation in a 57-year-old male, which, as far as the authors are aware, has not been described previously. The other in a 67 year old male due to right foraminal and paracentral disc protrusion at T9/10.

Informed consent in spinal surgery.

Informed consent is a very important part of surgical treatment. In this paper, we report a number of legal judgements in spinal surgery where there was no criticism of the surgical procedure itself. The fault that was identified was a failure to inform the patient of alternatives to, and material risks of, surgery, or overemphasizing the benefits of surgery. In one case, there was a promise that a specific surgeon was to perform the operation, which did not ensue. All of the faults in these cases were faults purely of the consenting process. In many cases, the surgeon claimed to have explained certain risks to the patient but was unable to provide proof of doing so. We propose a checklist that, if followed, would ensure that the surgeon would take their patients through the relevant matters but also, crucially, would act as strong evidence in any future court proceedings that the appropriate discussions had taken place. Although this article focuses on spinal surgery, the principles and messages are applicable to the whole of orthopaedic surgery. Cite this article: Bone Joint J 2019;101-B:355-360.

Magnetically controlled growing rods in the treatment of early-onset scoliosis: a note of caution.

The MAGnetic Expansion Control (MAGEC) system is used increasingly in the management of early-onset scoliosis. Good results have been published, but there have been recent reports identifying implant failures that may be associated with significant metallosis surrounding the implants. This article aims to present the current knowledge regarding the performance of this implant, and the potential implications and strategies that may be employed to identify and limit any problems. We urge surgeons to apply caution to patient and construct selection; engage in prospective patient registration using a spine registry; ensure close clinical monitoring until growth has ceased; and send all explanted MAGEC rods for independent analysis. The MAGEC system may be a good instrumentation system for the treatment of early-onset scoliosis. However, it is innovative and like all new technology, especially when deployed in a paediatric population, robust systems to assess long-term outcome are required to ensure that patient safety is maintained. Cite this article: Bone Joint J 2017;99-B:708-13.

Cytotoxicity of carcinogenic aromatic amides in normal and xeroderma pigmentosum fibroblasts with different DNA repair capabilities.

The effect of exposure to UV irradiation or to the N-acetoxy-ester derivatives of four carcinogenic aromatic amides, 4-acetylaminobiphenyl (AABP), 2-acetylaminofluorene (AAF), 2-acetylaminophenanthrene, and 4-acetylaminostilbene, on cell survival was compared in strains of cultured human fibroblasts possessing normal rates of excision repair of DNA and in three strains of xeroderma pigmentosum (XP) cells, each differing in its rate of excision repair. The survival of each strain after exposure to UV reflected its capacity to repair DNA. Thus the slope of the survival curve for the XP strain with the poorest capacity for excision repair (XP12BE complementation group A) was 5.8-fold steeper than the exponential portion of the curve for the normally repairing strains; that of XP2BE (complementation group C) was 1.95-fold; and that of XP4BE (a variant capable of a normal rate of dimer excision) was only 1.3-fold steeper. The slope of the survival curves after exposure to each N-acetoxy ester derivative for these same XP strains averaged 6.4, 2.0, and 1.4 times steeper, respectively, than that of the normal strains tested. The excision repair capacity of these lines after exposure to N-acetoxy-AAF (50 muM/ml) was tested with alkaline cesium chloride density gradient centrifugation to detect incorporation of tritiated thymidine into nonreplicated DNA. The normal strains and XP4BE exhibited DNA excision repair by this method, whereas XP patients 2 and 12 did not. The cytotoxic effect of the four parent aromatic amide carcinogens, their N-hydroxy derivatives, as well as the N-acetoxy ester of each of the four N-hydroxy compounds and the N-sulfate ester of N-hydroxy-AAF and N-hydroxy-AABP in the XP2BE strain, was compared with their effect on the normal fibroblasts. The parent amides proved to be noncytotoxic at all doses tested. In contrast, the N-hydroxy derivatives of each aromatic amide were highly cytotoxic, as were the ester compounds. For each active derivative, the slope of the survival curve for XP2BE was 2-2.k times steeper than that of the normally repairing strain.

Should we all go to the PROM? The first two years of the British Spine Registry.

The effective capture of outcome measures in the healthcare setting can be traced back to Florence Nightingale's investigation of the in-patient mortality of soldiers wounded in the Crimean war in the 1850s. Only relatively recently has the formalised collection of outcomes data into Registries been recognised as valuable in itself. With the advent of surgeon league tables and a move towards value based health care, individuals are being driven to collect, store and interpret data. Following the success of the National Joint Registry, the British Association of Spine Surgeons instituted the British Spine Registry. Since its launch in 2012, over 650 users representing the whole surgical team have registered and during this time, more than 27 000 patients have been entered onto the database. There has been significant publicity regarding the collection of outcome measures after surgery, including patient-reported scores. Over 12 000 forms have been directly entered by patients themselves, with many more entered by the surgical teams. Questions abound: who should have access to the data produced by the Registry and how should they use it? How should the results be reported and in what forum?

In vitro processing of proopiomelanocortin by recombinant PC1 (SPC3).

The prohormone convertases, PC1 (SPC3) and PC2, are subtilisin-like serine proteases capable of processing neuropeptide precursors. In cotransfection experiments, other investigators have found that PC1 and PC2 can process POMC to appropriate peptide products. In this study, recombinant rat PC1 was stably expressed in a mouse L-cell line and partially purified. Mouse POMC was cleaved by recombinant PC1 to generate ACTH intermediates, ACTH, ACTH linked to joining peptide, joining peptide, 16-kilodalton N-POMC, N-POMC-(1-74), and beta-lipotropin. Recombinant PC1 was also found to cleave ACTH to ACTH-(1-15) and bovine N-POMC-(1-77) to gamma 3 MSH. The pH optimum of the cleavages was 6.0. We conclude that recombinant PC1 is capable of processing POMC in vitro at all of the paired basic residues, with the exception of Lys-Arg and Lys-Lys in beta-lipotropin and beta-endorphin, respectively. This in vitro study showed a more general specificity of recombinant PC1 for paired and tetrabasic residues of POMC than was previously found in cotransfection experiments. Other cellular regulatory mechanisms probably play a role in limiting the processing of POMC in vivo in the anterior pituitary, where gamma 3 MSH and alpha MSH are not found in significant amounts.

Isolation of two complementary deoxyribonucleic acid clones from a rat insulinoma cell line based on similarities to Kex2 and furin sequences and the specific localization of each transcript to endocrine and neuroendocrine tissues in rats.

We have identified two rat insulinoma cDNAs that code for proteins homologous to the Kex2 dibasic protease of yeast and the mammalian furin gene product. A 5.0-kilobase (kb) cDNA, termed BDP, coding for a 752-amino acid protein and a 2.5-kb cDNA coding for a 636-amino acid protein, which was found to be the rat equivalent of the human insulinoma PC2 protein, were isolated. The proteins encoded by these clones contain a specific N-terminal signal sequence, indicating that both enter the secretory pathway. Neither protein contains a C-terminal transmembrane domain as is found in kex2 and furin, suggesting that the proteins may be soluble. Both proteins contain regions surrounding the active site residues which show amino acid identities to both kex2 (43% for BDP and 41% for RPC2) and furin (57% for BDP and 53% for RPC2). Probes specific for the mRNAs of each protein were used to localize the expression of each protein in endocrine and neuroendocrine tissues.

Processing of prothyrotropin-releasing hormone (Pro-TRH) by bovine intermediate lobe secretory vesicle membrane PC1 and PC2 enzymes.

TRH is synthesized from a larger 26-kilodalton (kDa) prohormone (pro-TRH). Rat pro-TRH contains five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven other cryptic peptides. Each of the five TRH progenitor sequences is flanked by pairs of basic amino acids. We used a bovine intermediate lobe secretory vesicle membrane preparation, which contains the prohormone convertases (PCs) PC1 and PC2, to study the in vitro processing of pro-TRH. Pro-TRH was radiolabeled using [3H]Leu in AtT20 cells transfected with prepro-TRH complementary DNA, and the labeled 26-kDa pro-TRH was isolated from the cell extract by preparative sodium dodecyl sulfate-gel electrophoresis. Incubation of [3H]pro-TRH with the intermediate lobe secretory vesicle membrane preparation was followed by immunoprecipitation with antibodies specific for various regions of the pro-TRH sequence, and the immunoprecipitates were analyzed by sodium dodecyl sulfate-gel electrophoresis. Immunoprecipitation of the reaction mixture with anti-pCC10 antibody (an antibody that recognizes the intact precursor and amino-terminal intermediate products of processing) showed a time-dependent appearance of a 15-kDa and a 6-kDa peptide and, at times, a 3.8-kDa peptide with diminution of the 26-kDa substrate. Immunoprecipitation of the incubate with the C-terminal-directed antibody, pYE17 (an antibody that recognizes the intact precursor and C-terminal intermediate products of processing), showed the generation of 16.5-, 10-, and 5.4-kDa products in a time-dependent manner, with disappearance of the substrate. Western blot analysis demonstrated that the secretory vesicle membrane preparation contains PC1 and PC2. Immunodepletion studies with antiserum specific for PC1 or PC2 demonstrated that PC1 and PC2 can process pro-TRH to these intermediate products. An initial site of cleavage appeared to be either at the 152-153 or the 158-159 pair of basic residues to yield a 15-kDa N-terminal fragment that was then processed to the 6-kDa [TRH-(25-74)] and 3.8-kDa [TRH-(83-112)] forms. The 10-kDa C-terminal peptide generated by this cleavage was then processed to a 5.4-kDa peptide [TRH-(208-255)]. Alternatively, an initial cleavage at the 107-108 or the 112-113 bonds was also observed, yielding a 16.5-kDa C-terminal product that was further processed to the 5.4-kDa peptide. The pH profile for the appearance of both C- and N-terminal products showed a bimodal distribution, with optima at both 5.5 and 7.5. The cleavage of pro-TRH was enhanced by Ca2+ and partially inhibited by Zn2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat mitochondrial coupling factor 6: molecular cloning of a cDNA encoding the imported precursor.

A cDNA clone encoding the precursor to the rat mitochondrial protein coupling factor 6 (F6) has been isolated and sequenced. The deduced amino acid sequence of the rat precursor protein shows 78% and 74% identity with the human and bovine F6 pre-proteins, respectively.

Identification of a 27-kDa enkephalin-containing protein associated with bovine adrenal medullary chromaffin granule membranes by immunoblotting.

An antiserum which recognizes high molecular mass enkephalin-containing proteins was used to compare proenkephalin intermediates in both the soluble and membrane components of bovine adrenal chromaffin granules by immunoblotting. While a range of molecular mass forms were identified in the soluble lysate the major form in the membranes corresponded to a 27-kDa enkephalin-containing protein. Enzymic digestion of bands of 27-kDa material and quantitation of the enkephalin released showed that 22% of this material was membrane-associated. High concentrations of chaotropic agents were required to extract this material from the membranes. Association of hormone and neuropeptide precursors with membrane components may be important for targeting of precursors to secretory granules or correct processing.

Proinsulin-like material in mouse foetal brain cell cultures.

Two main forms of immunoreactive insulin have been identified in cultures of foetal mouse brain using HPLC and gel filtration. The major component which resembled proinsulin was converted by trypsin to the minor form which was similar to authentic pancreatic insulin in chromatographic behaviour. Both components showed immunological properties comparable to insulin and proinsulin including sensitivity of the former to reduction and alkylation.

Differential glycosylation of N-POMC1-77 regulates the production of gamma 3-MSH by purified pro-opiomelanocortin converting enzyme. A possible mechanism for tissue-specific processing.

The amino terminus of bovine pro-opiomelanocortin (N-POMC1-77) is partially processed in the intermediate lobe of the pituitary to N-POMC1-49 and lys-gamma 3-melanotropin. Two pools of N-POMC1-77 were isolated which were differentially glycosylated at threonine45, while N-POMC1-49 isolated from bovine intermediate lobe extracts existed in a non-glycosylated form. This suggested that differential O-linked glycosylation of N-POMC1-77 may regulate cleavage at the Arg49-Lys50 processing site. We tested this hypothesis by incubating N-POMC1-77 glycoforms with purified proopiomelanocortin converting enzyme. Only non-O-glycosylated N-POMC1-77 and O-glycosylated N-POMC1-77 with truncated oligosaccharide sidechains were sensitive to cleavage and generated predominantly lys-gamma 3-melanotropin, identified by high-performance liquid chromatography. These data provide the first functional evidence to support a role for differential O-linked glycosylation in the regulation of the processing of the N-terminus of bovine POMC.

A new intracellular serine protease inhibitor expressed in the rat pituitary gland complexes with granzyme B.

We have cloned a novel serpin (raPIT5a) from a rat pituitary cDNA library which is structurally related to members of the ovalbumin subfamily of serine protease inhibitors. This new cDNA encodes a 374-amino acid protein, designated raPIT5a. raPIT5a was expressed in specific cells in the intermediate and anterior lobes of the pituitary. Recombinant raPIT5a was not secreted suggesting raPIT5a functions to inhibit intracellular proteases. Recombinant raPIT5a formed an SDS-stable complex with human granzyme B, a serine protease which induces apoptosis by activating members of the caspase enzyme family. These data suggest raPIT5a may have a role in regulating granzyme B or related enzymes and apoptosis in the pituitary gland.

Pro-opiomelanocortin and pro-vasopressin converting enzyme in pituitary secretory vesicles.

Peptide hormones are synthesized from larger precursors by cleavages at paired basic residues. We have isolated a pro-hormone converting enzyme from bovine neural and intermediate lobe secretory vesicles that cleaves pro-vasopressin and pro-opiomelanocortin at Lys-Arg residues to yield vasopressin, and adrenocorticotropin/endorphin-related peptides, respectively. The enzyme from both lobes is an aspartyl protease of approximately 70,000 Da, is a glycoprotein and has an optimum pH range of 4.0-5.0. Present within the same secretory vesicles is an aminopeptidase B-like enzyme which is a metalloprotease that is inhibited by Co2+ and Zn2+. This enzyme may play a role in trimming off the N-terminal extended basic residues from peptides liberated by the pro-hormone converting enzyme.

The distribution of lithium and its effects on the distribution and excretion of other ions in the rat.

1. In rats, lithium (ca 1 mEquiv/kg body weight) decreased brain sodium and magnesium, bone sodium and calcium and increased muscle calcium, plasma magnesium, urinary calcium and urine volume.2. Lithium was particularly concentrated in bone.

Distribution of carboxypeptidase H messenger RNA in rat brain using in situ hybridization histochemistry: implications for neuropeptide biosynthesis.

Carboxypeptidase H is an exopeptidase which is highly specific for C-terminal basic amino acids and thought to play a role in neuropeptide biosynthesis. The distribution of carboxypeptidase H mRNA was examined in adult rat brain using in situ hybridization histochemistry. Enzyme transcripts were detected in all major brain areas. Very high levels of carboxypeptidase H mRNA were found in the hippocampus associated with pyramidal cells. Other brain regions showed varied levels of labelling, ranging from high levels in the magnocellular cells of hypothalamic nuclei to very low levels in the striatum. The appearance of enzyme transcripts throughout brain supports a role for this enzyme in the biosynthesis of many neuropeptides. The expression of transcripts in certain ventricular ependymal cells identified them as a new potential peptidergic cell type. The variations in levels of expression of carboxypeptidase H mRNA may reflect differences in peptidergic activity in different neuronal systems.

Effect of lithium in immunodeficiency: improved blood cell formation in mice with decreased hematopoiesis as the result of LP-BM5 MuLV infection.

Lithium salts have been demonstrated to induce the production of hematopoietic cells following administration in vivo and to minimize the reduction of these cells following treatment with either radiation, chemotherapeutic or antiviral drugs. We have previously demonstrated that lithium, when administered in vivo to immunodeficient mice infected with LP-BM5 MuLV (MAIDS) significantly reduced the development of lymphadenopathy, splenomegaly, and the lymphoma associated with late-stage immunodeficiency disease in this model, and increased the survival of these animals compared to virus-infected controls not receiving lithium. We report here the results of in vivo studies in the MAIDS model that determined the effect of lithium on peripheral blood indices and the number of myeloid (CFU-GM), erythroid (BFU-E) and megakaryocyte (CFU-Meg) hematopoietic progenitors from bone marrow and spleen harvested from immunodeficient mice receiving lithium carbonate (1 mM) placed in their drinking water compared to virus-infected controls not receiving lithium. Time-points evaluated were at weeks 1, 5, 9, 13, 17, and 21 postviral infection. Virus-control mice not receiving lithium demonstrated all the signs that are characteristic of MAIDS, i.e., splenomegaly, lymphadenopathy, hypergammaglobulinemia, reduced hematopoiesis, and death. Infected mice receiving lithium demonstrated diminished presence of splenomegaly, lymphadenopathy, hypergammaglobulinemia, no suppression of hematopoiesis nor mortality. Enhanced hematopoiesis was demonstrated by neutrophilia, lymphocytosis, thrombocytosis, and erythrocytosis that was evident by increased myeloid, erythroid, and megakaryocyte progenitor cells cultured from bone marrow and spleen. These studies further demonstrate that lithium influences the disease process in the MAIDS model and restricts the development of hematopoietic suppression that develops in this retroviral animal model of immunodeficiency.

Recovery of cellular activity in cryopreserved human foetal brain tissue.

Since long-term cryopreservation can cause losses in neural tissue viability and function a prerequisite would be the ability to monitor and promote functional recovery in donor tissue intended for neural transplantation. Rapid assessment of cryopreserved tissue's functional status prior to grafting is presently difficult in a clinical setting. A convenient indicator of functional status may be the level of DNA synthesis activity taking place in the tissue. Using immunocytochemical detection of incorporated bromodeox-yuridine we have quantified and compared DNA synthesis activity (expressed as proliferative capacity (PC)) in human foetal mesencephalic, striatal, cortical and cerebellar tissue before and after a 275-376 day storage in liquid nitrogen. There was a post-storage reduction in viability of 48-73% and in PC of 26-59%; the higher the PC before storage the greater the reduction after. Incubation of cryopreserved tissue with fetal calf serum resulted in 2-4-fold higher PC levels than serum-untreated controls and reached 80% of fresh tissue levels in mesencephalic cells after 3-4 h incubation. Assuming that quantification of proliferative activity is a practical indicator of the tissue's functional status, these findings suggest that treatment of the tissue with serum can largely restore the lost function caused by cryopreservation.

Distribution and regulation of the candidate prohormone processing enzymes SPC2 and SPC3 in adult rat brain.

A number of candidate mammalian prohormone processing enzymes related to the yeast Kex2 endoprotease have been cloned and demonstrated to cleave several prohormone precursors at single, pairs and tetra basic amino acid processing sites. We have mapped the distribution of the mRNAs encoding two of these endoproteases in adult rat brain. SPC3 message levels showed a more restricted distribution and generally lower levels than SPC2 transcripts. The highest levels of SPC2 mRNA were found in the pyramidal cells of the hippocampus, several thalamic nuclei, the habenula and selected nuclei in the hypothalamus. SPC3 mRNA was most abundant in dentate gyrus granule cells, the habenula and selected hypothalamic nuclei. In the hypothalamus overlapping and unique distributions of the two transcripts were seen in the paraventricular nucleus with SPC3 mRNA predominantly expressed in lateral magnocellular cells. Both SPC2 and SPC3 mRNA were upregulated in the paraventricular and supraoptic hypothalamic nuclei following chronic salt loading. Combined immunocytochemistry/in situ hybridization histochemistry demonstrated that SPC2 and SPC3 transcripts were both expressed in the vasopressinergic subpopulation of magnocellular neurons in the supraoptic nucleus. SPC3 mRNA, but not SPC2 transcripts, also colocalized with immunoreactive vasopressin-associated neurophysin in the suprachiasmatic nucleus. These results remain consistent with roles for SPC2 and SPC3 in the biosynthesis of neuropeptides and for a specific role for SPC3 in the processing of provasopressin. Increased levels of SPC2 and SPC3 transcripts following a chronic osmotic stimulus suggests these proteases are coregulated with prohormone substrates and may be useful as an indicator of peptidergic activity.