Cloning, sequence analysis, and expression of genes encoding xylan-degrading enzymes from the thermophile "Caldocellum saccharolyticum"

A lambda recombinant bacteriophage coding for xylanase and beta-xylosidase activity has been isolated from a genomic library of the extremely thermophilic anaerobe "Caldocellum saccharolyticum." Partial Sau3AI fragments of the lambda recombinant DNA were ligated into pBR322. A recombinant plasmid with an insertion of ca. 7 kilobases of thermophilic DNA expressing both enzymatic activities was isolated. The location of the genes has been established by analyzing deletion derivatives, and the DNA sequence of 6.067 kilobases of the insert has been determined. Five open reading frames (ORFs) were found, one of which (ORF1; Mr 40,455) appears to code for a xylanase (XynA) which also acts on o-nitrophenyl-beta-D-xylopyranoside. Another, ORF5 (Mr 56,365), codes for a beta-xylosidase (XynB). The xynA gene product shows significant homology with the xylanases from the alkalophilic Bacillus sp. strain C125 and Clostridium thermocellum.     

Photoaffinity labelling of nuclear steroid 5 alpha-reductase of rat ventral prostate

In order to get more information on the molecular structure of the rat prostatic 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3:1.22) a systematic photoaffinity labelling study has been performed. To irreversibly freeze the status quo of interaction, either testosterone, the physiological ligand, or diazo-MAPD (21-diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione), a specific 5 alpha-reductase inhibitor, was irradiated with isolated nuclei or with purified nuclear membranes or with solubilized nuclear membrane proteins and checked for optimal labelling conditions. The principal substances covalently labelled were phospholipids and at a minor ratio proteins. Analysis by SDS-PAGE and autoradiofluorography revealed two labelled polypeptides with molecular weights of 20 kDa and 26 kDa. The following evidence indicates that these polypeptides might be derived from the enzyme 5 alpha-reductase: both proteins are labelled only when specific ligands for 5 alpha-reductase are used; binding can be reduced by the addition of an excess of unlabelled ligand; enzyme activity is irreversibly suppressed when irradiated in the presence of these ligands; only subcellular fractions containing 5 alpha-reductase reveal the labelled proteins; in all 5 alpha-reductase containing preparations with increasing specific activity, independent of the polypeptide pattern, the same proteins are labelled.     

Iron overload of the liver by trimethylhexanoylferrocene in rats.

Iron-deficient female Wistar rats were fed a diet, which contained 0.5% trimethylhexanoylferrocene, over a 56-week period. This dietary iron loading resulted in a progressive siderosis and enlargement of the liver with a maximum iron content of 947.0 +/- 148.0 mg (vs. 0.07 +/- 0.04 mg in iron deficiency) and a maximum organ weight of 39.4 +/- 6.6 g (vs. 6.9 +/- 1.4 g in iron-deficient control rats). Up to 43 weeks, whole liver iron rose by increase in iron concentration (max. 28.0 +/- 6.1 mg/g wet weight, w.w.) as well as by enlargement of the organ. Afterwards whole liver iron increased solely by ongoing hepatomegaly. At the commencement of iron loading, stainable iron was almost exclusively stored by hepatocytes equally throughout all areas of the liver lobule. Later, the distribution of iron-loaded hepatocytes became strikingly periportal, and, in addition, Kupffer cells as well as sinus-lining endothelia began to store iron. Animals with a liver iron concentration of more than 10.4 +/- 0.75 mg/g w.w. showed no further increase in ferritin and haemosiderin within hepatocytes. Iron-burdened Kupffer cells/macrophages, however, accumulated permanently, hereby forming intrasinusoidal and portal siderotic nodules and areas. First signs of liver damage such as necrosis of single hepatocytes and mild fibrosis began at a liver iron concentration of 14.7 +/- 1.4 mg/g w.w. With advancement of iron loading, focal necrosis of hepatocytes and iron-burdened macrophages took place, and significant perisinusoidal as well as portal fibrosis developed. Cirrhosis, however, the final stage of impairment in iron overload of the liver in humans, could not be induced in this animal model up to now.     

Multiple origins for phenylketonuria in Europe

Phenylketonuria (PKU), a disorder of amino acid metabolism prevalent among Caucasians and other ethnic groups, is caused primarily by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). PKU is a highly heterogeneous disorder, with more than 60 molecular lesions identified in the PAH gene. The haplotype associations, relative frequencies, and distributions of five prevalent PAH mutations (R158Q, R261Q, IVS10nt546, R408W, and IVS12n1) were established in a comprehensive European sample population and subsequently were examined to determine the potential roles of several genetic mechanisms in explaining the present distribution of the major PKU alleles. Each of these five mutations was strongly associated with only one of the more than 70 chromosomal haplotypes defined by eight RFLPs in or near the PAH gene. These findings suggest that each of these mutations arose through a single founding event that occurred within time periods ranging from several hundred to several thousand years ago. From the significant differences observed in the relative frequencies and distributions of these five alleles throughout Europe, four of these putative founding events could be localized to specific ethnic subgroups. Together, these data suggest that there were multiple, geographically and ethnically distinct origins for PKU within the European population.     

Protein kinase C activity is rate limiting for shedding of the interleukin-6 receptor.

An analysis of the mechanism of generation of the soluble interleukin-6 receptor (IL-6R) has been performed. The membrane-bound receptor is proteolytically cleaved to release a soluble receptor form which retained its ligand binding capacity. Furthermore, the soluble IL-6R is unique in its ability to induce a biological signal in complex with the ligand interleukin-6 (IL-6) on cells which by themselves do not bind IL-6. Shedding of the IL-6R is strongly activated by PMA and can be inhibited by the protein kinase inhibitor staurosporine. The generation of the IL-6R is not dependent on protein synthesis. The inactive PMA analogue 4-alpha-phorbol-12,13-didecanoate fails to induce shedding of the IL-6R. Transfection of a protein kinase C expression plasmid into IL-6R expressing cells leads to enhanced shedding of the receptor. These experiments clearly show that protein kinase C regulates shedding of the IL-6R.     

Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli.

We have designed dimeric antibody fragments that assemble in Escherichia coli. They are based on single-chain FV fragments, with a flexible hinge region from mouse IgG3 and an amphiphilic helix fused to the C-terminus of the antibody fragment. The sequence of the helix was taken either from that of a previously reported four-helix bundle design or from a leucine zipper, optionally extended with a short cysteine-containing peptide. The bivalent fragments associate in vivo, either with covalent linkage or with a monomer-dimer equilibrium, and results from ultracentrifugation sedimentation studies and SDS-PAGE are consistent with dimers. All constructs are able to bind to surface-bound antigen under conditions in which only bivalent but not monovalent antibody fragments bind. The covalent bundle helix construct shows binding characteristics nearly identical to those of the much larger whole mouse antibody, resulting in substantially more stable immunoglobulin-antigen complexes than in the case of monovalent fragments. This modular design of natural and engineered protein domains directly leads to a boost of avidity, and it allows the construction of bispecific antibody fragments in functional form in E. coli.     

Identification of two general diffusion channels in the outer membrane of pea mitochondria.

Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent solubilized mitochondrial membranes of pea seedlings (Pisum sativum). The addition of the detergent-solubilized material to the membranes resulted in a strong increase of the membrane conductance. To identify the proteins responsible for membrane activity the detergent extracts were applied to a hydroxyapatite (HTP) column and the fractions were tested for channel formation. The eluate of the column contained a protein which migrated as a single band with an apparent molecular mass of 30 kDa on SDS-PAGE. This channel was identified as the porin of pea mitochondria since it formed voltage-dependent channels with single-channel conductances of 1.5 and 3.7 nS in 1 M KCl and an estimated effective diameter of about 1.7 nm. Further elution of the column with KCl containing solutions yielded fractions which resulted in the formation of transient channels in lipid bilayer membranes. These channels had a single-channel conductance of 2.2 nS in 1 M KCl and had also the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. Zero-current membrane potential measurements suggested that pea porin was anion-selective in the open state. The selectivity of the second channel was investigated by the measurement of the reversal potential. It was also slightly anion-selective. Its possible role in the metabolism of mitochondria is discussed.