Matrix metalloproteinase activity in equine synovial fluid: influence of age, osteoarthritis, and osteochondrosis

OBJECTIVE: To investigate the influence of age, osteoarthritis (OA), and osteochondrosis (OC) on the matrix metalloproteinase (MMP) activity in the synovial fluid (SF) of equine joints. METHODS: SF was collected from normal and osteoarthritic metacarpophalangeal joints (normal: 14 adult, 28 juvenile; OA: 22 adult). And from normal and osteochondrotic tarsocrural joints (5 months: 11 normal, 8 OC; 11 months: 7 normal, 6 OC). Subsequently, overall MMP activity was measured. RESULTS: The level of active MMPs was almost twofold higher in SF from juvenile horses (age up to 11 months) than in SF from mature animals (4-30 years; p < 0.001). In juvenile horses MMP activity was higher in 5 month old foals than in 11 month old foals (p < 0.01). In adult horses MMP activity was independent of age. In OA joints the activity was nearly twice as high as in normal joints (p < 0.001). In OC joints MMP activity was not significantly different from normal, age matched, control joints. CONCLUSIONS: MMP activity in SF from normal adult joints is not related to age. In juvenile joints MMP activity is significantly higher than activity in joints from adult animals. It is hypothesised that the gradual decrease in MMP activity with increasing age reflects the declining metabolic activity resulting from ceasing growth and the accompanying decrease in cartilage remodelling. The increased MMP activity in osteoarthritis joints most likely reflects matrix destruction. In osteochondrosis MMP mediated matrix degradation appears not to be different from normal joints.     

Plasma glucose and insulin responses to orally administered simple and complex carbohydrates

We have studied the effects of glucose, sucrose, and various starches on postprandial plasma glucose and insulin responses in 19 subjects. All carbohydrate loads were calculated to contain 50 gm. of glucose, and the response to each carbohydrate was tested twice: when given alone in a drink or when given in combination with other nutrients as a meal. The data demonstrate: (1) Glucose and sucrose elicited similar plasma glucose response curves, but sucrose elicited a somewhat greater (20 per cent) plasma insulin response. (2) Raw starch ingestion resulted in a 44 per cent lower glucose response and a 35-65 per cent lower insulin response than did either glucose or sucrose ingestion. (3) When carbohydrate was given as a meal the plasma glucose responses were 40-60 per cent lower than when the same carbohydrate was given as a drink, while the insulin responses were generally similar, and (4) when different cooked starches were compared, the plasma glucose and insulin responses to rice were significantly lower (50 per cent) than to potato. In conclusion, the size of the carbohydrate molecule appears to influence the postprandial glucose and insulin responses such that more complex carbohydrates (starches) elicit lower responses. This effect may be related to differences in digestion rather than to differences in absorption.     

ECT in Parkinson's disease. Changes in motor symptoms, monoamine metabolites and neuropeptides

Electroconvulsive therapy (ECT) was given to 16 non-depressed, non-demented patients with advanced Parkinson's disease (PD). In all the patients an antiparkinsonian effect was seen, lasting for 18 months in one patient, 3-5 months in seven patients, and a few days to four weeks in eight patients. After ECT the levels of homovanillic acid and neuropeptide Y in cerebrospinal fluid (CSF) were significantly increased. The eight patients with long lasting motor improvement after ECT had significantly lower CSF-3-methoxy-4-hydroxyphenylglycol compared to the group with short lasting improvement. Five patients developed transitory mental confusion after ECT. In these patients, and in no others, a high albumin-ratio was found already before ECT was given - an indication of blood CSF barrier damage. Our results suggest that ECT is valuable in patients with drug refractory PD or PD with intolerance to antiparkinsonian drugs.     

Prevalence of rheumatic fever in children from a public high school in Belo Horizonte

Genetic studies have suggested homogeneity between the Baltic-type and Mediterranean-type progressive myoclonic epilepsy. Magnetoencephalography was applied to elucidate the mechanism underlying the giant evoked responses in cortical reflex myoclonus. A new concept of negative myoclonus mediated by cortical reflex mechanism was proposed. Cortical myoclonus was demonstrated in various neurodegenerative or metabolic disorders, such as presenile or senile dementia, olivopontocerebellar atrophy, and myoclonus epilepsy associated with ragged-red fibres. Myoclonus in corticobasal degeneration is especially noteworthy because it has clinical and electrophysiological features of cortical reflex myoclonus, but its latency is shorter compared with the conventional cortical reflex myoclonus. Clinical features of 'palatal myoclonus' were reported by the name of 'palatal tremor'.     

Cloning, sequencing and functional analysis of a truncated cDNA encoding red deer prolactin receptor: an alternative tyrosine residue mediates beta-casein promoter activation

The purpose of these studies was to compare directly the percutaneous absorption,excretion and metabolism of all-trans-retinoyl beta-glucuronide (RAG), a nontoxic retinoid, with all-trans-retinoic acid (RA) in the rat. Previously, it was demonstrated that topical treatment of human acne with either RAG or RA in cream resulted in a significant reduction of lesions. Whereas 0.1% RA showed adverse effects, concentrations of RAG up to 2.4% did not cause any adverse reactions. In the present studies, radiolabeled RAG or RA, dispersed in a water-based cream, was applied to the shaved dorsal skin of vitamin A-sufficient rats. Both RAG and RA were absorbed from the skin in a similar way. In both cases, radioactivity peaked in the plasma within 2-4 h and within the liver at 4-12 h. During a 7-day period, the overall excretion of radioactivity derived from RA and RAG in the feces and urine were similar, e.g. 17 and 12%, respectively. it is concluded that: (1) the transport, metabolism and excretion of topically applied radioactive RA and RAG are similar, although not identical, in the rat and (2) the toxic skin manifestations induced by RA but not by RAG cannot be attributed to major differences in their overall absorption, metabolism and excretion.     

Protein kinase C and mouse sciatic nerve regeneration

We have studied the role of protein kinase C (PKC) in peripheral nerve regeneration by using the cultured adult mouse sciatic nerve, which displays regrowth of sensory axons under serum-free conditions. By the use of immunohistochemistry we show that one of the isoforms of PKC, PKC beta, is present in the nerve cell bodies of normal nerves and is upregulated after injury. In spite of this, the specific PKC inhibitor chelerythrine at 5 microM, a concentration well above its IC50 value for PKC, failed to reduce the outgrowth distance of new axons. This was not due to impermeability of the drug, since the same concentration caused a clear reduction of the injury-induced proliferation of Schwann cells in the crush region. Likewise, HA-1004, an inhibitor of cyclic nucleotide-dependent protein kinases, also lacked effect on outgrowth when used on its own, even at very high concentrations (100 microM). In contrast, outgrowth was significantly reduced when 5 microM chelerythrine and 5 microM HA-1004 were used in combination. In conclusion, the present results suggest that PKC-activity is important but not indispensable for the regeneration process. Successful completion of the latter could be achieved by several, perhaps redundant, phosphorylation systems.     

A yeast protein related to a mammalian Ras-binding protein, Vps9p, is required for localization of vacuolar proteins

In the yeast Saccharomyces cerevisiae, mutations in vacuolar protein sorting (VPS) genes result in secretion of proteins normally localized to the vacuole. Characterization of the VPS pathway has provided considerable insight into mechanisms of protein sorting and vesicle-mediated intracellular transport. We have cloned VPS9 by complementation of the vacuolar protein sorting defect of vps9 cells, characterized its gene product, and investigated its role in vacuolar protein sorting. Cells with a vps9 disruption exhibit severe vacuolar protein sorting defects and a temperature-sensitive growth defect at 38 degrees C. Electron microscopic examination of delta vps9 cells revealed the appearance of novel reticular membrane structures as well as an accumulation of 40- to 50-nm-diameter vesicles, suggesting that Vps9p may be required for the consumption of transport vesicles containing vacuolar protein precursors. A temperature-conditional allele of vps9 was constructed and used to investigate the function of Vps9p. Immediately upon shifting of temperature-conditional vps9 cells to the nonpermissive temperature, newly synthesized carboxypeptidase Y was secreted, indicating that Vps9p function is directly required in the VPS pathway. Antibodies raised against Vps9p immunoprecipitate a rare 52-kDa protein that fractionates with cytosolic proteins following cell lysis and centrifugation. Analysis of the VPS9 DNA sequence predicts that Vps9p is related to human proteins that bind Ras and negatively regulate Ras-mediated signaling. We term the related regions of Vps9p and these Ras-binding proteins a GTPase binding homology domain and suggest that it defines a family of proteins that bind monomeric GTPases. Vps9p may bind and serve as an effector of a rab GTPase, like Vps2lp, required for vacuolar protein sorting.     

Detergent binding in trigonal crystals of OmpF porin from Escherichia coli

The structure of the detergent, ocytyl hydroxyethylsufoxide (C8(HE)SO), bound to the OmpF porin from E coli (in the trigonal crystal form) has been determined by neutron crystallography. Due to a dynamic exchange of detergent molecules with their environment they are not ordered on an atomic scale. The structure reported here is therefore at a resolution of approximately 16 A. The X-ray crystallographically determined structure of the protein provides a starting point for the neutron analysis in which the detergent is visualized primarily thanks to its high contrast against D2O. The structure shows the detergent to be located mainly in two areas. It forms toroidal annuli around each OmpF trimer, these annuli fusing to form a detergent belt surrounding a solvent filled column traversing the crystal. Those areas of the protein to which the detergent binds are formed almost exclusively of hydrophobic residues and form a band about 30 A high around the trimer. Its upper and lower bounds are defined by two bands of aromatic residues, tyrosines pointing away from the detergent belt and interacting with the polar headgroups while phenylalanines point inwards. This strongly suggests that the same areas define, in vivo, the location at which protein interacts with lipid. The hydrophobic moiety of detergent is also found mediating the hydrophobic protein-protein interactions at the interface between two trimers on the crystallographic two-fold axis.     

Expressed protein ligation: a general method for protein engineering

A protein semisynthesis method-expressed protein ligation-is described that involves the chemoselective addition of a peptide to a recombinant protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant protein with an N-terminal cysteine containing synthetic peptide, near quantitative chemical ligation of the peptide to the protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intramolecular phosphotyrosine-Src homology 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiologically relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed protein ligation is a simple and powerful new method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size.