Trichloroethylene oxidation by the membrane-associated methane monooxygenase in type I,type II and type X methanotrophs

Trichloroethylene (TCE) oxidation was examined in 9 different methanotrophs grown under conditions favoring expression of the membrane associated methane monooxygenase. Depending on the strain, TCE oxidation rates varied from 1 to 677 pmol/min/mg cell protein. Levels of TCE in the reaction mixture were reduced to below 40 nmolar in some strains. Cells incubated in the presence of acetylene, a selective methane monooxygenase inhibitor, did not oxidize TCE.Cultures actively oxidizing TCE were monitored for the presence of the soluble methane monooxygenase (sMMO) and membrane associated enzyme (pMMO). Transmission electron micrographs revealed the cultures always contained the internal membrane systems characteristic of cells expressing the pMMO. Naphthalene oxidation by whole cells, or by the cell free, soluble or membrane fractions was never observed. SDS denaturing gels of the membrane fraction showed the polypeptides associated with the pMMO. Cells exposed to ¹⁴C-acetylene showed one labeled band at 26 kDa, and this protein was observed in the membrane fraction. In the one strain examined by EPR spectroscopy, the membrane fraction of TCE oxidizing cells showed the copper complexes characteristic of the pMMO. Lastly, most of the strains tested showed no hybridization to sMMO gene probes. These findings show that the pMMO is capable of TCE oxidation; although the rates are lower than those observed for the sMMO.     

Molecular defects of type III procollagen in Ehlers-Danlos syndrome type IV

Summary Fibroblasts from most patients with Ehlers-Danlos syndrome (EDS) type IV, a disorder characterized by fragility of skin, blood vessels, and internal organs, secrete reduced amounts of type III procollagen. In 7 of 8 cell strains analyzed, we found evidence of structural defects in half of the type III procollagen chains synthesized, such as deletions or bona fide amino acid substitutions, which cause delayed formation and destabilization of the collagen triple helix and, as a consequence, reduced secretion of the molecule. The data suggest that EDS type IV is often caused by heterozygosity for mutations at the COL3A1 locus, which affect the structure of type III procollagen. The triple-helical region of the molecule, like the homologous region of type I procollagen, appears to be particularly vulnerable.Parts of this work have been presented at the 2nd International Conference on Molecular Biology and Pathology of Matrix, Philadelphia, June 15–18, 1988     

Beyond PrP res type 1/type 2 dichotomy in Creutzfeldt-Jakob disease

Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently subclassified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K (PK) digested abnormal prion protein (PrPres)identified on Western blotting (type 1 or type 2). These biochemically distinct PrPres types have been considered to represent potential distinct prion strains. However, since cases of CJD show co-occurrence of type 1 and type 2 PrPres in the brain, the basis of this classification system and its relationship to agent strain are under discussion. Different brain are as from 41 sCJD and 12 iatrogenic CJD (iCJD) cases were investigated, using Western blotting for PrPres and two other biochemical assays reflecting the behaviour of the disease-associated form of the prion protein (PrPSc) under variable PK digestion conditions. In 30% of cases, both type 1 and type 2 PrPres were identified. Despite this, the other two biochemical assays found that PrPSc from an individual patient demonstrated uniform biochemical properties. Moreover, in sCJD, four distinct biochemical PrPSc subgroups were identified that correlated with the current sCJD clinico-pathological classification. In iCJD, four similar biochemical clusters were observed, but these did not correlate to any particular PRNP 129 polymorphism or western blot PrPres pattern. The identification of four different PrPSc biochemical subgroups in sCJD and iCJD, irrespective of the PRNP polymorphism at codon 129 and the PrPres isoform provides an alternative biochemical definition of PrPSc diversity and new insight in the perception of Human TSE agents variability.     

Evaluation of the pseudocholinesterase activity in hyperlipoproteinemia type II and type IV

Authors have evaluated pseudocholinesterase activity in patients with type IIa and type IV hyperlipoproteinemia. Significative correlation has been found between PCE and C in type IV hyperlipoproteinemia. Authors suggest PCE activity can be proposed as useful biochemical marker of hyperlipoproteinemia.     

nifH sequences and nitrogen fixation in type I and type II methanotrophs

Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N2 as a nitrogen source. However, no sequences are available for nif genes in these strains, and the known nitrogen-fixing methanotrophs are confined mainly to a few genera. The purpose of this work was to assess the nitrogen-fixing capabilities of a variety of methanotroph strains. nifH gene fragments from four type I methanotrophs and seven type II methanotrophs were PCR amplified and sequenced. Nitrogenase activity was confirmed in selected type I and type II strains by acetylene reduction. Activities ranged from 0.4 to 3.3 nmol/min/mg of protein. Sequence analysis shows that the nifH sequences from the type I and type II strains cluster with nifH sequences from other gamma proteobacteria and alpha proteobacteria, respectively. The translated nifH sequences from three Methylomonas strains show high identity (95 to 99%) to several published translated environmental nifH sequences PCR amplified from rice roots and a freshwater lake. The translated nifH sequences from the type II strains show high identity (94 to 99%) to published translated nifH sequences from a variety of environments, including rice roots, a freshwater lake, an oligotrophic ocean, and forest soil. These results provide evidence for nitrogen fixation in a broad range of methanotrophs and suggest that nitrogen-fixing methanotrophs may be widespread and important in the nitrogen cycling of many environments.     

Neurofibromatosis type 1 in pregnancy

The report presents two cases of neurofibromatosis type 1 one previously known and one detected during pregnancy. It describes how the disease was detected and diagnosed, and what was the outcome of pregnancies. This is the first case of prenatal neurofibromatosis type 1 diagnosed in our clinic.     

Identification of cross-linking sites in bovine cartilage type IX collagen reveals an antiparallel type II-type IX molecular relationship and type IX to type IX bonding.

Type IX collagen functions in covalent cross-linkage to type II collagen in cartilage (Eyre, D. R., Apone, S., Wu, J. J., Ericsson, L. H., and Walsh, K. A. (1987) FEBS Lett. 220, 337-341). To understand this molecular relationship better, an analysis of all cross-linking sites labeled by [3H]borohydride was undertaken using the protein prepared from fetal bovine cartilage. Sequence analysis of tryptic peptides containing the 3H-labeled cross-links showed that each of the chains of type IX collagen, alpha 1(IX), alpha 2(IX), and alpha 3(IX), contained a site of cross-linking at the amino terminus of the COL2 triple-helix to which the alpha 1(II)N-telopeptide could bond. The alpha 3(IX)COL2 domain alone also had an attachment site for the alpha 1(II)C-telopeptide. The distance between the alpha 1(II)N-telopeptide and alpha 1(II)C-telopeptide interaction sites, 137 residues, is equal to the length of the hole zone (0.6D) in a type II collagen fibril. This implies an antiparallel type II to type IX cross-linking relationship. Peptide analysis also revealed an unknown amino acid sequence linked to the COL2 cross-linking domains in both the alpha 1(IX) and alpha 3(IX) chains. Using antibodies to this novel peptide, its origin in the collagen alpha 3(IX)NC1 domain was established. In summary, the results confirm extensive covalent cross-linking between type IX and type II collagen molecules and reveal the existence of type IX-type IX bonding. These data provide a molecular basis for the proposed function of type IX collagen as a critical contributor to the mechanical stability and resistance to swelling of the collagen type II fibril framework of cartilage.     

Reduced muscle carnosine content in type 2, but not in type 1 diabetic patients

Carnosine is present in high concentrations in skeletal muscle where it contributes to acid buffering and functions also as a natural protector against oxidative and carbonyl stress. Animal studies have shown an anti-diabetic effect of carnosine supplementation. High carnosinase activity, the carnosine degrading enzyme in serum, is a risk factor for diabetic complications in humans. The aim of the present study was to compare the muscle carnosine concentration in diabetic subjects to the level in non-diabetics. Type 1 and 2 diabetic patients and matched healthy controls (total n=58) were included in the study. Muscle carnosine content was evaluated by proton magnetic resonance spectroscopy (3 Tesla) in soleus and gastrocnemius. Significantly lower carnosine content (-45%) in gastrocnemius muscle, but not in soleus, was shown in type 2 diabetic patients compared with controls. No differences were observed in type 1 diabetic patients. Type II diabetic patients display a reduced muscular carnosine content. A reduction in muscle carnosine concentration may be partially associated with defective mechanisms against oxidative, glycative and carbonyl stress in muscle.     

Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 immunity

The patterns of Ag-induced cytokine coexpression in normal, in vivo-primed CD4 memory T cells has remained controversial because the low frequency at which these cells occur has effectively prevented direct ex vivo measurements. We have overcome this limitation by using two-color cytokine enzyme-linked immunospot assays and computer-assisted image analysis. We found CD4 memory cells that simultaneously expressed IL-2, IL-3, IL-4, IL-5, and IFN-gamma to be rare (0-10%). This cytokine segregation was seen in adjuvant-induced type 1, type 2, and mixed immunity to OVA, in Leishmania infection regardless of the Ag dose used or how long after immunization the assay was performed. The data suggest that type 1 and type 2 immunity in vivo is not mediated by classic Th1 or Th2 cells but by single-cytokine-producing memory cells.     

Autophagic adaptations in diabetic cardiomyopathy differ between type 1 and type 2 diabetes

Little is known about the association between autophagy and diabetic cardiomyopathy. Also unknown are possible distinguishing features of cardiac autophagy in type 1 and type 2 diabetes. In hearts from streptozotocin-induced type 1 diabetic mice, diastolic function was impaired, though autophagic activity was significantly increased, as evidenced by increases in microtubule-associated protein 1 light chain 3/LC3 and LC3-II/-I ratios, SQSTM1/p62 (sequestosome 1) and CTSD (cathepsin D), and by the abundance of autophagic vacuoles and lysosomes detected electron-microscopically. AMP-activated protein kinase (AMPK) was activated and ATP content was reduced in type 1 diabetic hearts. Treatment with chloroquine, an autophagy inhibitor, worsened cardiac performance in type 1 diabetes. In addition, hearts from db/db type 2 diabetic model mice exhibited poorer diastolic function than control hearts from db/+ mice. However, levels of LC3-II, SQSTM1 and phosphorylated MTOR (mechanistic target of rapamycin) were increased, but CTSD was decreased and very few lysosomes were detected ultrastructurally, despite the abundance of autophagic vacuoles. AMPK activity was suppressed and ATP content was reduced in type 2 diabetic hearts. These findings suggest the autophagic process is suppressed at the final digestion step in type 2 diabetic hearts. Resveratrol, an autophagy enhancer, mitigated diastolic dysfunction, while chloroquine had the opposite effects in type 2 diabetic hearts. Autophagy in the heart is enhanced in type 1 diabetes, but is suppressed in type 2 diabetes. This difference provides important insight into the pathophysiology of diabetic cardiomyopathy, which is essential for the development of new treatment strategies.     

FBJ virus-induced osteosarcoma contains type I, type I trimer, type III as well as type V collagens

The FBJ osteosarcoma (a virus-induced osteosarcoma named after its discoverers, Finkel, Biskis, and Jinkins) contains an extensive extracellular matrix. Collagens were extracted by digestion with pepsin in dilute acetic acid from tumors grown in lathyritic mice and fractionated by differential salt precipitation, yielding five fractions. Fraction 1 (precipitated at acidic 0.7 M and neutral 2.0 M NaCl) gave rise mainly to alpha 1(III) chain on phosphocellulose column chromatography. The alpha 1(III) chain was identified by its typical behavior on interrupted electrophoresis and analysis of the CNBr-cleaved peptides. The alpha 1(III) chain of the FBJ tumor had a high content of hydroxylysine and neutral saccharide. Fraction 2 (precipitated at acidic 0.7 M and neutral 4.5 M NaCl) yielded alpha 1(I) and alpha 2(I) chains on the phosphocellulose column from which alpha 1(I) was eluted as a broad peak, conceivably reflecting a high content of hydroxylysine and neutral saccharide. Fraction 4 (precipitated at acidic 1.2 M and neutral 4.5 M NaCl) yielded type V collagen, which also featured an exceptionally high content of neutral saccharide (Yamagata, S., et al. (1982) Biochem. Biophys. Res. Commun. 105, 1208-1214). The proportions of type I, type I trimer, type III, and type V collagens extracted by pepsin digestion from FBJ tumor were calculated to be 33, 29, 26, and 12%, respectively. The FBJ tumor is free from invasion by blood vessels, shows no deposition of calcium, and thus has the appearance of cartilage. But type II collagen, a specific gene product of cartilage, could not be identified in any of the fractions analyzed. Contrary to its appearance, collagen type analyses indicate that FBJ osteosarcoma is literally induced from osteogenic cells.     

In situ radiation-inactivation size of fibroblast membrane-bound acid beta-glucosidase in Gaucher type 1, type 2 and type 3 disease

The radiation-inactivation size of membrane-bound acid beta-glucosidase in cultured skin fibroblasts of four normal individuals, five Gaucher type 1 (non-neuropathic), four Gaucher type 2 (acute neuropathic) and three Gaucher type 3 (sub-acute neuropathic) patients was determined using the radiation-inactivation method. The radiation-inactivation size of the enzyme in the control, Gaucher type 2 and Gaucher type 3 fibroblasts ranged from 94 000 to 128 800, and no statistical significant difference was found in the enzyme size between the normal and Gaucher cells nor among the Gaucher type 2 and type 3 cells. Contrary to the normal, Gaucher type 2 and Gaucher type 3 enzyme, the radiation-inactivation size of membrane-bound acid beta-glucosidase in all of the Gaucher type 1 fibroblasts tested is significantly higher, ranging from 158 400 to 235 300. The size of the control lysosomal enzyme, sphingomyelinase, also determined by the radiation-inactivation method in fibroblasts of normal individuals and patients with the three Gaucher subtypes, was between 70 000 and 74 500 and indistinguishable from each other. Since the molecular weight of acid beta-glucosidase subunit determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was about 60 000 (Pentchev, P.G., Brady, R.O., Hibbert, S.P., Gal, A.E. and Shapiro, C. (1973) J. Biol. Chem. 248, 5256-5261), the above data suggest that: (i) the normal fibroblast enzyme, as well as the Gaucher type 2 and type 3 mutant enzyme, in the membrane-bound form, exists as a dimer; (ii) the underlying biochemical and genetic defect in non-neuropathic (type 1) and neuropathic (type 2 and type 3) Gaucher disease is very different from each other; and (iii) subunit interaction of the mutant enzyme may be present in Gaucher type 1 fibroblasts, resulting in the formation of a higher-molecular-weight aggregate.