Actinobacillus pleuropneumoniae hlyX gene homology with the fnr gene of Escherichia coli.

The hlyX gene from Actinobacillus pleuropneumoniae, which confers a hemolytic phenotype on Escherichia coli, was sequenced, and its role in regulation of gene expression was investigated. No similarity was found between the hlyX sequence and sequences of known hemolysin or cytotoxin genes. However, the hlyX sequence was very similar to that of the fnr gene of Escherichia coli which encodes the global regulatory protein, FNR. Comparison of the deduced amino acid sequence of the hlyX gene product (HlyX) with that of FNR revealed a high degree of well-aligned sequence correlation throughout the polypeptide chain. For example, 23 of 24 amino acids in the DNA-binding region of FNR are identical in the corresponding region of HlyX. Four cysteine residues in the amino-terminal region are also conserved. The promoter region of hlyX is very similar to that of fnr. It has a putative -10 sequence which closely resembles the E. coli -10 consensus sequence. This sequence is overlapped by a potential operator which is very similar to the FNR-binding-site consensus sequence. Functional homology between HlyX and FNR was also demonstrated. Plasmids carrying hlyX complemented the nutritional lesion of an fnr deletion strain of E. coli. These data suggest that HlyX may regulate, rather than mediate, hemolytic activity in E. coli, but the possibility that HlyX is both a regulator of gene expression and a hemolysin cannot be excluded.     

Rotational dynamics of monoclonal anti-dansyl immunoglobulins

The rotational motions of monoclonal mouse anti-dansyl immunoglobulins were studied by nanosecond fluorescence emission anisotropic spectroscopy using a mode-locked argon-ion laser as the pulsed excitation source. Three homogeneous antibodies of the immunoglobulin Gl (IgGl) subclass containing different V regions were prepared. The fluorescence emission maxima of these antibodies (designated as DNS1, DNS2 and DNS3) are at 515, 480 and 500 nm, respectively. Their mean rotational correlation times, 〈φ〉, are 84, 109 and 96 ns, respectively. The binding of protein A or a monoclonal anti-allotype antibody to the F_c unit of DNS1 increased 〈φ〉 to 142 and 150 ns, respectively, whereas reduction of the disulfide bond between the heavy chains decreased 〈φ〉 to 48 ns. These nanosecond measurements show that the rotational motion of the F_ab arms in mouse IgGl is restricted.     

Atherocalcin, a gamma-carboxyglutamic acid containing protein from atherosclerotic plaque.

The specialized calcium binding amino acid, γ-carboxyglutamic acid (Gla) is quantitated in developing atherosclerotic plaque relative to progression of the disease, and a Gla-containing protein isolated from calcified atherosclerotic plaque is partially characterized. Low levels of Gla are found in fatty streak and fibrous plaque lesions, and a marked increase in Gla content occurs in calcified plaque. A unique Gla-containing protein is purified from 0.5M EDTA (pH 8.0) extracts of calcified plaque, named atherocalcin. The protein containing 19 Gla residues/1000 amino acids is 80,000 molecular weight, with a pI of 4.16 – 4.3 and is uniquely different from other known Gla-containing proteins. The implications of this work for the further understanding of the pathogenesis and therapy of atherosclerosis are discussed.     

Lysyl oxidase dependent synthesis of a collagen cross-link containing histidine

To date, collagen appears unique among proteins in that it contains histidine in certain of its cross-links. Synthesis of histidine containing collagen cross-links was studied in vitro with lathyritic L-¹⁴C histidine or L-¹⁴C lysine labelled bone collagen fibrils and purified lysyl oxidase. Synthesis of the tetrafunctional cross-link, dehydrohistidinohydroxymerodesmosine occurred with lysyl oxidase and was inhibited by β-aminopropionitrile. Synthesis began after a lag period of sixteen hours and then proceeded linearly for four days. These data indicate that enzyme dependent synthesis of dehydrohistidinohydroxymerodesmosine occurs in vitro in a biochemically defined system. Biosynthesis in vivo might occur under similar conditions.     

A 25,000 molecular weight protein constituent of human amyloid fibrils related to amyloid protein AA

Amyloid fibrils from a patient with diffuse amyloid disease are dissociated in 6 m guanidine hydrochloride and fractionated by gel chromatography. Two major components are separated on Sepharose 6B. Both proteins are characterized by chromatography, immunodiffusion, discontinuous gel electrophoresis, amino acid tryptic peptide mapping and amino acid sequence analysis. The smaller of the two components is typical of the known protein AA by size (8400 daltons), amino acid composition and a 30-residue N-terminal sequence. The larger of the components (25,000 daltons) undergoes electrophoresis as a single band and appears unaffected by thiol reduction. It differs from protein AA in amino acid content and by its tryptic peptide map, although it contains an N-terminal amino acid sequence identical to protein AA when carried to 20 residues. Treatment of this larger component by mild acid hydrolysis results in the release of the 8400-dalton protein AA. Fractionation after guanidine hydrochloride treatment of this particular amyloid fibril preparation is compared to the fractionation of a typical secondary amyloid preparation that contains only protein AA as the major component. The origin and relationship of the 8,400- and 25,000-dalton protein components is discussed.     

Possibilities of the conjugation process in mycobacteria

Results obtained when studying conjugation in mycobacteria by means of different methods are summarized. The method of conjugation on surface of a solid complete medium was tested with different auxotrophic mutants of different strains ofMycobacterium smegmatis. It was not possible to obtain positive results even by means of the above method. This was probably due to unsuitability of the chosen strains ofMycobacterium smegmatis. Preparation of the donor strain by transfer of the F factor fromEscherichia coli F’ORF 1ade ⁺ lac⁺ pro⁺ toMycobacterium phlei PA adeStm ^r by means of sexduction is described. Frequency of the phenotype PAade ⁺ Stm^r increased in the average by two and a half orders of magnitude with respect to the control, however, a further transfer from cultures of the cellsade ⁺ Stm^r to cells ade could not be demonstrated. Experiments aimed at transferring the R factor from strainsEscherichia coli K-12 toMycobacterium phlei were unsuccessful.