Protein H--a novel IgG binding bacterial protein.

Forty-eight group A streptococcal strains of different M types were screened for binding of human radiolabeled IgG. Three of the strains bound more than 80% of the added radioactivity and one of them, an M protein type 1 strain designated AP1, was selected for further analysis. Attempts were made to solubilize the IgG binding bacterial molecule, and small amounts of an IgG binding protein with a mol. wt of 40 kDa could be solubilized with mutanolysin, a muramolytic agent. The gene encoding this streptococcal protein was cloned and expressed in E. coli, and the E. coli-produced protein was purified in a single step by affinity chromatography on IgG-Sepharose. When tested with IgGs from different species, the molecule was found to bind human IgG almost exclusively. The N-terminal amino acid sequence was determined and showed no homology with previously isolated Ig binding proteins, and the name protein H (as in human IgG) is suggested for this novel Ig binding bacterial protein. Protein H showed preferential affinity for heavy chains and Fc fragments of human IgG, and did not bind Ig light chains. The affinity constant, determined by Scatchard plots, between protein H and human polyclonal IgG was 1.6 x 10(9). No binding was observed between protein H and IgM, IgA, IgD, or IgE. Finally, when tested against several additional proteins and human plasma, protein H only showed weak binding to alpha 2-macroglobulin, a proteinase inhibitor.

Direct expression of urogastrone gene in Escherichia coli.

Human epidermal growth factor (urogastrone; UG) is a 53-amino acid polypeptide hormone. A 192-bp DNA fragment containing the coding sequence for methionyl UG (Met-UG) and the ribosome-binding site (RBS) was chemically synthesized and placed downstream from the promotor for the Escherichia coli outer-membrane lipoprotein gene (lpp) on a plasmid. E. coli cells harboring the plasmid directed the synthesis of Met-UG at 10(2)-10(3) molecules per cell. Next, the coding sequence for Met-UG was inserted in a runaway-replication plasmid and expressed under the control of the lpp promoter and the RBS derived from bacteriophage Mu cII gene. Upon heat induction, the cells harboring the recombinant plasmid synthesized 10(5) molecules of Met-UG per cell.

[Motor adaptation in the Bielschowsky head-tilt test in cases of superior oblique palsy].

To elucidate a motor adaptation phenomenon in the Bielschowsky head-tilt test in cases of superior oblique palsy, a gain of the otolith-ocular reflex was studied. The amplitude of ocular counter-rolling (OCR) of the non-paretic eye was measured with a photographic method, using limbal conjunctival marks as landmarks which were marked with indigo carmine. The average preoperative OCR of the non-paretic eye was 10.49 degrees at 30 degrees of head tilt to both sides, but after corrective surgery in the paretic eye the OCR of the non-paretic eye decreased to 8.43 degrees. To clarify the relation between OCR, duration of palsy and vertical deviation of the Bielschowsky head-tilt test (BHP), which was the difference of vertical deviation measured with the head tilted to the left and right shoulders at an angle of 30 degrees, the BHP/OCR ratio was calculated. We found no relation between BHP and OCR, but the BHP/OCR ratio increased proportionally in cases of long-standing palsy, From these results an increased BHP/OCR ratio could be an adaptive phenomenon caused by secondary innervational changes or muscle contracture to minimizing the contralateral head tilt to maintain binocular single vision.

[Evaluation of preoperative prism adaptation in patients with acquired esotropia].

A prospective study was carried out of the evaluation preoperative prism adaptation in 77 patients with acquired esotropia. Sixty-three patients who showed a stable esodeviation with prisms of 10 prism diopter or less by alternate prism cover test and fusion response to the prisms were classified as prism responders and were randomly classified into two groups. Thirty-one patients of prism responders underwent surgery based on the prism adapted angle. Patients in the other 32 cases were assigned to undergo surgery for their initially measured angle. On the other hand, 14 patients who did not respond to prism adaptation underwent surgery for their preoperative measured angle of deviation. Success rates with deviations of 0 to 10 prism diopters measured 1 year after surgery by the alternative prism cover test at 5 m and with fusion on Bagolini's striated glasses test were highest (26 [84%] of 31 patients) in prism adaptation responders who underwent surgery for prism determined angle and lowest (3 [21%] of 14 patients) in the prism adaptation nonresponder who showed no fusion response to the prisms.

Alignment of protein sequences using the hydrophobic core scores.

Making an alignment of the amino acid sequences is an essential step in the prediction of an unknown protein structure by model building from the known structure of a protein of the same family. To improve the accuracy of the alignments, we introduced the concept of hydrophobic core scores, which restrains putting insertions/deletions in the hydrophobic core regions of the protein. Eight pairs of protein sequences were aligned by this method, and the quality of the alignments were assessed by reference to those obtained by the structural superposition. The introduction of the hydrophobic core scores derived from the knowledge of the tertiary structure of one of each pair resulted in an improvement of the accuracy of the alignments. The quality of the alignment was found to depend on the homology of the protein sequences.

Intraoperative suspension-recession technique for treatment of vertical strabismus in thyroid myopathy.

Ten patients underwent extraocular muscle surgery with an intraoperative suspension-recession technique for diplopia induced by vertical strabismus in thyroid myopathy. The average ratio of the reduction of vertical deviation to the amount of surgery was 1.8 degrees/mm (range, 1.0 degrees/mm-2.8 degrees/mm). Eight (80%) patients had single binocular vision restored in the primary position without prism or compensatory torticollis after surgery. One patient who showed evidence of abnormal thyroid function at surgery developed an overcorrection postoperatively.

The gene sequence and some properties of protein H. A novel IgG-binding protein.

The gene for protein H, a novel bacterial cell wall protein with specific affinity for human IgG Fc, was cloned from a group A Streptococcus and expressed in Escherichia coli. Recombinant E. coli cells produced two forms of a human IgG Fc-binding protein, one with an apparent Mr of 42 kDa in a periplasmic fraction and the other with an apparent Mr of 45 kDa in a mixed fraction of cytoplasms and membranes. Both 42-kDa and 45-kDa protein preparations similarly bound to human IgG1 to IgG4, human IgG Fc, and rabbit IgG, but not to IgG of mouse, rat, bovine, sheep, goat, and human IgA, IgD, IgE, and IgM. The complete nucleotide sequence of the cloned 1.8-kb DNA fragment was determined. An open reading frame encoded a hypothetical protein of 376 amino acid residues (Mr = 42,498). The N-terminal amino acid sequence, consisting of 41 residues, which was removed post-translationally had typical characteristics of Gram-positive bacterial signal peptides. Thus, the mature form of protein H was suggested to consist of 335 residues (Mr = 38,162). There were 3 repeated sequences consisting of 42 residues that were highly homologous to those of protein Arp, an IgA-binding streptococcal cell wall protein, and streptococcal M6 and M24 proteins. The C-terminal amino acid sequence consisting of 93 residues, directly following the repeated sequences, was also highly homologous to that of M6 and M24 proteins. No sequence homology was found between protein H and protein A or protein G, two other IgG-binding bacterial cell wall proteins.

Preoperative prism correction in patients with acquired esotropia.

We performed a prospective study of preoperative prism adaptation in 77 patients with acquired esotropia. Sixty-three of them increased their angle of squint when wearing Fresnel press-on prisms for 5-7 days. After the angle had stabilized to a point that did not exceed the press-on prisms by more than 10 prism D, they were randomly divided into two groups. Thirty-two patients underwent surgery based on the prism-adapted angle. The other 31 patients underwent surgery based on their initially measured angle. Fourteen patients who did not respond to prism correction underwent surgery based on the angle before prism correction. Success rates with deviations between 0 and 10 prism diopters measured 1 year after surgery were highest in those in whom surgery was based on the prism-determined angle and were lowest in the nonresponders, who had no fusion response to the prisms.

Protein H--a surface protein of Streptococcus pyogenes with separate binding sites for IgG and albumin.

Protein H, a molecule expressed at the surface of some strains of Streptococcus pyogenes, has affinity for the constant (IgGFc) region of immunoglobulin (Ig) G. In absorption experiments with human plasma, protein H-sepharose could absorb not only IgG but also albumin from plasma. The affinity constant for the reaction between albumin and protein H was 7.8 x 10(9) M-1, which is higher than the affinity between IgG and protein H (Ka = 1.6 x 10(9) M-1). Fragments of protein H were generated with deletion plasmids and polymerase chain reaction (PCR) technology. Using these fragments in various protein-protein interaction assays, the binding of albumin was mapped to three repeats (C1-C3) in the C-terminal half of protein H. On the albumin molecule, the binding site for protein H was found to overlap the site for protein G, another albumin- and IgGFc-binding bacterial surface protein. Also IgGFc-binding could be mapped with the protein H fragments and the region was found N-terminally of the C repeats. A synthetic peptide (25 amino acid residues long) based on a sequence in this region was shown to inhibit the binding of protein H to immobilized IgG or IgGFc. This sequence was not found in previously described IgGFc-binding proteins. However, two other cell surface proteins of S. pyogenes exhibited highly homologous regions. The results identify IgGFc- and albumin-binding regions of protein H and further define and emphasize the convergent evolution among bacterial surface proteins interacting with human plasma proteins.