Endoplasmic reticulum-associated degradation of the mouse PC1/3-N222D hypomorph and human PCSK1 mutations contributes to obesity.

BACKGROUND: The proprotein convertase 1/3 (PC1/3), encoded by proprotein convertase subtilisin/kexin type 1 (PCSK1), cleaves and hence activates several orexigenic and anorexigenic proproteins. Congenital inactivation of PCSK1 leads to obesity in human but not in mice. However, a mouse model harboring the hypomorphic mutation N222D is obese. It is not clear why the mouse models differ in phenotype. METHODS: Gene expression analysis was performed with pancreatic islets from Pcsk1(N222D/N222D) mice. Subsequently, biosynthesis, maturation, degradation and activity were studied in islets, pituitary, hypothalamus and cell lines. Coimmunoprecipitation of PC1/3-N222D and human PC1/3 variants associated with obesity with the endoplasmic reticulum (ER) chaperone BiP was studied in cell lines. RESULTS: Gene expression analysis of islets of Pcsk1(N222D/N222D) mice showed enrichment of gene sets related to the proteasome and the unfolded protein response. Steady-state levels of PC1/3-N222D and in particular the carboxy-terminally processed form were strongly reduced in islets, pituitary and hypothalamus. However, impairment of substrate cleavage was tissue dependent. Proinsulin processing was drastically reduced, while processing of proopiomelanocortin (POMC) to adrenocorticotropic hormone (ACTH) in pituitary was only mildly impaired. Growth hormone expression and IGF-1 levels were normal, indicating near-normal processing of hypothalamic proGHRH. PC1/3-N222D binds to BiP and is rapidly degraded by the proteasome. Analysis of human PC1/3 obesity-associated mutations showed increased binding to BiP and prolonged intracellular retention for all investigated mutations, in particular for PC1/3-T175M, PC1/3-G226R and PC1/3-G593R. CONCLUSIONS: This study demonstrates that the hypomorphic mutation in Pcsk1(N222D) mice has an effect on catalytic activity in pancreatic islets, pituitary and hypothalamus. Reduced substrate processing activity in Pcsk1(N222D/N222D) mice is due to enhanced degradation in addition to reduced catalytic activity of the mutant. PC1/3-N222D binds to BiP, suggesting impaired folding and reduced stability. Enhanced BiP binding is also observed in several human obesity-associated PC1/3 variants, suggesting a common mechanism.

The histological appearance of the proximal aspect of the dorsal condylar sagittal ridge of the third metacarpal and metatarsal bone in young warmblood horses: normal appearance and correlation with detected radiographic variations.

The objective of this study is to describe the normal histological appearance of the dorsoproximal aspect of the sagittal ridge of the third metacarpal/metatarsal bone in young Warmblood horses, and to compare it to the different radiographic variations (irregular, indentation, lucency, notch) described at this level. A total of 25 metacarpo/metatarsophalangeal joints of 12 Warmblood horses were used. Five samples of each radiographically described group were selected for histological processing. Each category was compared with the normal control group. Each group showed a bone cortex, covered by hyaline cartilage and longitudinally aligned collagen fibres covered by loosely organized connective tissue proximally. The normal and irregular group showed a smooth bone cortex. In the indentation and lucency group, a depression in the cortex was detected. The notch group presented an expansion of the cortex. The collagen fibres and connective tissue were located in the depression in the indentation group whereas the location varied in the lucency and notch group. The radiologic detected differences are translated into detectable histological differences. Further research is warranted to determine whether these variations are developmental or congenital and to evaluate their potential influence on the joint function during hyperextension.

Limitations of inhibitory activities of polyphenols on furin-mediated substrate processing.

Recently, selected polyphenols were reported to exert proprotein convertase (PC) inhibitory activities on in vitro cleavage of a fluorogenic peptide substrate and it was concluded that this anti-protease activity might be responsible for the reported anti-cancer properties of these polyphenols. This prompted investigations to identify PC inhibiting polyphenols that could affect IGF-1R-mediated tumorigenesis since pro-IGF-1R is bioactivated by PCs like furin. Initial screening of polyphenols for their impact on in vitro cleavage of fluorogenic peptide substrate Pyr-RTKR-AMC by human furin (hfurin(573)) indeed revealed varying inhibitory effects. (-)EGCG, chrysin, and quercetin, were subsequently evaluated using uncleaved diphtheria toxin as substrate in vitro. However, none displayed any inhibitory impact on processing. Binding of (-)EGCG to both furin and the diphtheria toxin protein was demonstrated. Subsequently, it was found that for seven polyphenols tested, addition of casein or gamma globulin led to reduction or even annihilation of in vitro Pyr- RTKR-AMC cleavage inhibition. No such effect was seen with the furin inhibitor nona-D-arginine. Western blot studies to investigate possible effects of selected polyphenols on processing in cells of the tumorigenesis-linked proproteins pro-IGF-1R and pro-GPC3 also revealed no inhibitory effects. In conclusion, our results confirm the reported PC inhibitory effects of polyphenols on fluorogenic peptide substrate cleavage in vitro. However, the data show that polyphenolic inhibitory effects on hfurin(573)-mediated in vitro fluorogenic peptide substrate cleavage cannot be extrapolated to similar effects on processing of genuine proproteins, whether in vitro or in cells. This undermines the anti-protease rationale for the reported polyphenolic anti-cancer properties.

Radiographic features of the dorsal condylar sagittal ridge of the third metacarpal and metatarsal bones in young Warmblood stallions.

OBJECTIVES: To describe the radiographic appearance of the dorsoproximal aspect of the sagittal ridge of the third metacarpal/metatarsal bone in Warmblood horses. METHODS: The lateromedial radiographic projections of the metacarpo-/metatarsophalangeal joints performed on horses as a part of stallion selection were used. The dorsal aspect of the distal third metacarpal/metatarsal bone was divided in two areas. The appearance of the bone surface in area I was classified as normal, irregular, notch, indentation and lucency. For area II, the categories were normal, irregular, depression or lucency and flattening of the sagittal ridge. Other abnormalities at the dorsal aspect were also noted. RESULTS: In area I, 51.5% of the ridges appeared normal, 19.3% were irregular, 8.9% had a notch, 8.1% had a lucency, and 12.2% had an indentation. In 1.2% of the horses a fragment was present, and in 1.7% a fragment was suspected. In area II, 90.6% of the metacarpo-/metatarsophalangeal joints were normal, 6.2% were irregular, 2.9% showed a depression or lucency, and the sagittal ridge in 0.2% was flattened. A fragment was present in 0.3%, and suspected in 0.4%. CLINICAL SIGNIFICANCE: Morphological variation is present at the dorsal aspect of the metacarpo-/metatarsophalangeal joint in young Warmblood stallions. These various aspects should be recognised and described in horses presented for prepurchase examination. However, their clinical relevance in the individual horse is unclear and needs further investigation.

A new format of the CATT test for the detection of human African Trypanosomiasis, designed for use in peripheral health facilities.

OBJECTIVES: To test the reproducibility and thermostability of a new format of the Card-Agglutination Test for Trypanosomiasis (CATT) test for Human African Trypanosomiasis (HAT), designed for use at primary health care facility level in endemic countries. METHODS: A population of 4217 from highly endemic villages was screened using the existing format of the CATT test (CATT-R250) on whole blood. All those testing positive (220) and a random sample of negatives (555) were retested in the field with the new format (CATT-D10). Inter-format reproducibility was assessed by calculating kappa. All samples testing positive on whole blood with either method were further evaluated in Belgium by CATT titration of serum by two observers, using both old and new format. CATT-D10 test kits were incubated under four temperature regimens (4, 37, 45 degrees C and fluctuating) with regular assessments of reactivity over 18 months. RESULTS: Inter-format reproducibility of CATT-D10 vs. CATT-R250 on whole blood performed by laboratory technicians in the field was excellent with kappa values of 0.83-0.89. Both inter- and intra-format reproducibility assessed by CATT titration were excellent, with 96.5-100% of all differences observed falling within the limits of +/-1 titration step. After 18 months, reactivity of test kits incubated under all four temperature regimens was still well above the minimum threshold considered acceptable. CONCLUSION: The CATT-D10 is thermostable and can be used interchangeably with the old format of the CATT test. It is highly suitable for use in peripheral health facilities in HAT-endemic countries.

Four cases of omental herniation in cattle.

Four cases in cattle of omental herniation through an acquired omental rent are described. Clinical signs were indistinguishable from other causes of mechanical ileus and exploratory laparotomy was necessary to establish a diagnosis. In all cases a variably sized portion of jejunal loops was obstructed in an omental rent in the deep layer of the greater omentum. In two cases simple reduction was possible, and in one case incision of the hernial ring was necessary before reduction could be performed. Resection was necessary only in a calf, in which the incarceration was complicated by severe abomasal distension and local peritonitis. In all four cases the omental rent was closed by a serosa-serosa suture. Three cases made an uneventful recovery and returned to normal production, but one of these animals died three months postoperatively from an unknown cause. The calf was euthanased two days postoperatively because of persistent ileus.

Dorsoproximal proximal phalanx osteochondral fragmentation in 117 Warmblood horses.

The objective of the present study was to determine clinical and arthroscopic characteristics associated with dorsoproximal proximal phalanx (P1) fragments in Warmblood horses, as well as to examine their histopathological appearance. One hundred sixty-eight fragments were removed from 150 fetlocks of 117 Warmblood horses. Details of signalment and results of clinical examination were collected prior to surgery. After arthroscopic fragment removal and joint evaluation for synovial and/or cartilage abnormalities, the fragments were measured and evaluated histopathologically. The vast majority of the fragments (95.2%) were found medially, without predilection for front or hind limbs. In 10% of the joints, more than one fragment was present. The mean size of the fragments was 6.8 +/- 2.6 mm. Only eight horses presented fetlock-related lameness. Horses of seven years of age and older (OR = 13.32; p = 0.033) and the presence of more than one fragment (OR = 11.12; p = 0.016) were significantly associated with lameness. Arthroscopic evaluation revealed one or more abnormalities in 50.7% of the joints. On histopathology, osteochondral fragments presented as a bony center covered with smooth hyaline cartilage on one side and some fibrous tissue on the other side. No clear histopathological signs were indicating precisely their origin. In Warmblood horses with dorsoproximal P1 fragments, the age (seven years and older) and the presence of more than one fragment in a fetlock significantly increased the risk of lameness. The osteochondral dorsoproximal P1 fragments could be defined as a developmental orthopaedic disease.

Cardiorespiratory effects of enoximone in anaesthetised colic horses.

REASONS FOR PERFORMING STUDY: No studies have been reported on the effects of enoximone in anaesthetised colic horses. OBJECTIVE: To examine whether enoximone improves cardiovascular function and reduces dobutamine requirement in anaesthetised colic horses. METHODS: Forty-eight mature colic horses were enrolled in this prospective, randomised clinical trial. After sedation (xylazine 0.7 mg/kg bwt) and induction (midazolam 0.06 mg/kg bwt, ketamine 2.2 mg/kg bwt), anaesthesia was maintained with isoflurane in oxygen and a lidocaine constant rate infusion (15 mg/kg bwt, 2 mg/kg/h). Horses were ventilated (PaCO2 < 8.00 kPa). If hypotension occurred, dobutamine and/or colloids were administered. Ten minutes after skin incision, horses randomly received an i.v. bolus of enoximone (0.5 mg/kg bwt) or saline. Monitoring included respiratory and arterial blood gases, heart rate (HR), arterial pressure and cardiac index (CI). Systemic vascular resistance (SVR), stroke index (SI) and oxygen delivery index (DO2I) were calculated. For each variable, changes between baseline and T10 within each treatment group and/or colic type (small intestines, large intestines or mixed) were analysed and compared between treatments in a fixed effects model. Differences between treatments until T30 were investigated using a mixed model (a = 0.05). RESULTS: Ten minutes after enoximone treatment, CI (P = 0.0010), HR (P = 0.0033) and DO2I (P = 0.0007) were higher and SVR lower (P = 0.0043) than at baseline. The changes in CI, HR and SVR were significantly different from those after saline treatment. During the first 30 min after enoximone treatment, DO2I (P = 0.0224) and HR (P = 0.0003) were higher than after saline administration. Because the difference in HR between treatments was much clearer in large intestine colic cases, an interaction was detected between treatment and colic type in both analyses (P = 0.0076 and 0.0038, respectively). CONCLUSIONS: Enoximone produced significant, but short lasting, cardiovascular effects in colic horses. POTENTIAL RELEVANCE: Enoximone's cardiovascular effects in colic horses were of shorter duration than in healthy ponies.

Surgical repair of a tibial fracture in a Belgian Landrace pig.

This paper reports the surgical treatment of a tibial fracture in a castrated adult male Belgian Landrace pig of 180 kg. The fracture was repaired using an intramedullary Steinmann pin, combined with cerclage wire and external transfixation. In contrast to other animal species, the fracture repair in the pig was hindered by the short and curved bones, the thick subcutaneous fat layer and the pronounced musculature. Postoperatively, the pig developed an osteomyelitis of the tibia due to pin tract contamination. Despite this complication, the fracture healed acceptably when all fixation material was removed two months after surgery. The infection resolved quickly and a satisfactory clinical result was obtained.

Pustular calicivirus dermatitis on the abdomen of two cats following routine ovariectomy.

An unusual form of calicivirus dermatitis is described in two cats. Two fully vaccinated cats were re-admitted for anorexia and depression following routine ovariectomy. Signs of upper respiratory disease were not present. One cat subsequently showed painful necrosis of the incision wound, the other one developed dyspnoea with pleural effusion and discrete tongue ulcers. Intact pustular lesions confined to the surgically prepared abdomen appeared in both cats, respectively, on days 11 and 9. The histopathological diagnosis was panepidermal pustulosis and necrotizing dermatitis. Positive immunohistochemical staining consistent with feline calicivirus antigen was detected in epithelial cells within pustular lesions. The cats were treated with antibiotics and ketoprofen. The cat with progressive dyspnoea was euthanized. The clinical signs in the other cat rapidly and completely resolved following glucocorticoid therapy. It is hypothesized that the reported cases may represent a distinct calicivirus-induced pustular dermatitis following ovariectomy.

Calcinosis involving multiple paws in a cat with chronic renal failure and in a cat with hyperthyroidism.

Calcinosis of multiple paws is described in two cats. A metastatic pathogenesis was supported by the laboratory findings of hyperphosphataemia and a calcium x phosphorus solubility product > 7 g/L. Hyperparathyroidism could not be confirmed because a valid feline parathyroid hormone assay was not available at the time. One cat was diagnosed with chronic renal failure and presented initially with an irregular nodular calcification on the chin. Dietary and medical management was unsuccessful and ultimately the animal was euthanased. Hyperthyroidism was diagnosed in another cat by laboratory findings and scintigraphic imaging. In addition, the cat had a hyperphosphataemia in the absence of azotaemia. Intravenous administration of radioactive iodine as (131)I was accompanied by reduction and normalization in serum total thyroxine and phosphorus concentrations and resulted in resolution of calcification in the paws.

A twinned monoclinic crystal form of human peroxiredoxin 5 with eight molecules in the asymmetric unit.

The monoclinic crystal form of human peroxiredoxin 5 with eight molecules in the asymmetric unit was obtained under exactly the same conditions as the tetragonal form with one molecule in the asymmetric unit, except that the latter was briefly cryosoaked with halide for derivatization. A merohedral twinning was observed, which is rather unusual in the monoclinic system and only possible with particular unit-cell dimensions. After detwinning the native and a mercury derivative, the structure was solved by the SIR method with the help of the non-crystallographic symmetry. The packing of the monoclinic and tetragonal forms are compared, with special attention to the role of bromide ions in the change of space group after crystallization. The availability of nine (eight monoclinic plus one tetragonal) independent molecules allows an analysis of the mobility. The two Cys residues implicated in the peroxide-reduction mechanism are located in rigid regions but are covered by mobile loops.

Photochemical rearrangement of oxaziridines and nitrones in the hexahydroindole series: a convenient synthetic route to 1-azabicyclo[5.2.0]nonan-2-ones as novel RGD mimetics.

[reaction: see text] Photolysis of oxaziridines a or nitrones b provides a convenient synthetic route to fused bicyclic lactams c adequately substituted on both cycles A and B as scaffolds for mimicking conformationally constrained beta-turn peptides as in the tripeptide RGD signaling motif of fibronectin.

Crystal structure of human peroxiredoxin 5, a novel type of mammalian peroxiredoxin at 1.5 A resolution.

The peroxiredoxins define an emerging family of peroxidases able to reduce hydrogen peroxide and alkyl hydroperoxides with the use of reducing equivalents derived from thiol-containing donor molecules such as thioredoxin, glutathione, trypanothione and AhpF. Peroxiredoxins have been identified in prokaryotes as well as in eukaryotes. Peroxiredoxin 5 (PRDX5) is a novel type of mammalian thioredoxin peroxidase widely expressed in tissues and located cellularly to mitochondria, peroxisomes and cytosol. Functionally, PRDX5 has been implicated in antioxidant protective mechanisms as well as in signal transduction in cells. We report here the 1.5 A resolution crystal structure of human PRDX5 in its reduced form. The crystal structure reveals that PRDX5 presents a thioredoxin-like domain. Interestingly, the crystal structure shows also that PRDX5 does not form a dimer like other mammalian members of the peroxiredoxin family. In the reduced form of PRDX5, Cys47 and Cys151 are distant of 13.8 A although these two cysteine residues are thought to be involved in peroxide reductase activity by forming an intramolecular disulfide intermediate in the oxidized enzyme. These data suggest that the enzyme would necessitate a conformational change to form a disulfide bond between catalytic Cys47 and Cys151 upon oxidation according to proposed peroxide reduction mechanisms. Moreover, the presence of a benzoate ion, a hydroxyl radical scavenger, was noted close to the active-site pocket. The possible role of benzoate in the antioxidant activity of PRDX5 is discussed.

Sleeping sickness resurgence in the DRC: the past decade.

An overview of the evolution of sleeping sickness and control activities in the DRC during the period 1989-1998 is presented. A resurgence was already developing in the mid-1980s and, after a breakdown of active case-finding between 1990 and 1993, annual detection rates attained levels similar to those of the late 1920s. Although a staggering number of 150 591 new cases have been detected during the past decade, the problem is ignored by most of the international community. The major cause for the resurgence appears to be the interruption of active case-finding for a prolonged period of time. Control activities have improved considerably in recent years, but a lot remains to be done and supplementary resources are needed.

Cholic acid derivatives as 1,2,4,5-tetraoxane carriers: structure and antimalarial and antiproliferative activity.

Cholic acid-derived 1,2,4,5-tetraoxanes were synthesized in order to explore the influence of steroid carrier on its antimalarial and antiproliferative activity in vitro. Starting with chiral ketones, cis and trans series of diastereomeric tetraoxanes were obtained, and the cis series was found to be approximately 2 times as active as the trans against Plasmodium falciparum D6 and W2 clones. The same tendency was observed against human melanoma (Fem-X) and human cervix carcinoma (HeLa) cell lines. The amide C(24) termini, for the first time introduced into the carrier molecule of a tetraoxane pharmacophore, significantly enhanced both antimalarial and antiproliferative activity, as compared to the corresponding methyl esters, with cis-bis(N-propylamide) being most efficient against the chloroquine-susceptible D6 clone (IC(50) = 9.29 nM). cis- and trans-bis(N-propylamides) were also screened against PBMC, and PHA-stimulated PBMC, showing a cytotoxicity/antimalarial potency ratio of 1/10 000.

Crystal structure of the EF-hand parvalbumin at atomic resolution (0.91 A) and at low temperature (100 K). Evidence for conformational multistates within the hydrophobic core.

Several crystal structures of parvalbumin (Parv), a typical EF-hand protein, have been reported so far for different species with the best resolution achieving 1.5 A. Using a crystal grown under microgravity conditions, cryotechniques (100 K), and synchrotron radiation, it has now been possible to determine the crystal structure of the fully Ca2+-loaded form of pike (component pI 4.10) Parv.Ca2 at atomic resolution (0.91 A). The availability of such a high quality structure offers the opportunity to contribute to the definition of the validation tools useful for the refinement of protein crystal structures determined to lower resolution. Besides a better definition of most of the elements in the protein three-dimensional structure than in previous studies, the high accuracy thus achieved allows the detection of well-defined alternate conformations, which are observed for 16 residues out of 107 in total. Among them, six occupy an internal position within the hydrophobic core and converge toward two small buried cavities with a total volume of about 60 A3. There is no indication of any water molecule present in these cavities. It is probable that at temperatures of physiological conditions there is a dynamic interconversion between these alternate conformations in an energy-barrier dependent manner. Such motions for which the amplitudes are provided by the present study will be associated with a time-dependent remodeling of the void internal space as part of a slow dynamics regime (millisecond timescales) of the parvalbumin molecule. The relevance of such internal dynamics to function is discussed.