Tryptic peptides of canine thyroglobulin reactive with sera of patients with canine hypothyroidism caused by autoimmune thyroiditis.

Canine thyroglobulin (cTg) was treated with trypsin at a ratio of trypsin to cTg of 1:100 (w/w). Tryptic peptides of cTg were analysed by Western immunoblotting for their reactivity to serum thyroglobulin autoantibodies (TgAA) from patients with TgAA-positive hypothyroidism and normal individuals. The sera of patients with TgAA-positive hypothyroidism reacted with several peptides: 43, 32.5 and 31 kDa; the sera of normal individuals did not bind these tryptic peptides. Some of the TgAA-positive sera of patients reacted with 25 kDa peptide in addition to three tryptic peptides above. This experiment was the first report about antigenic epitopes of cTg. These small tryptic peptides recognized by TgAA may be related with the induction of TgAA and may be useful as markers for autoimmune thyroid diseases in dog.

Polygalacturonase isolated from the culture of the psychrophilic fungus Sclerotinia borealis.

A polygalacturonase was isolated from the culture medium of Sclerotinia borealis, a psychrophilic fungus that grows on lawn and wheat seedling under the snow in winter and induces the snow mold disease. Pectic acid was a better substrate of this enzyme than pectin when the activity was determined by measuring the reducing sugar produced. However, when the activity was measured by viscosity change, the viscosity of pectin decreased more rapidly than that of pectic acid. The results of viscosity change apparently indicate that the polygalacturonase catalyzes pectin hydrolysis as an endo-type enzyme. Highly methyl-esterified pectin was a poor substrate, as determined by measurements of reducing sugar production and viscosity change. It is suggested from the results that the methoxy group of pectin affects the polygalacturonase reaction. A reaction mechanism was proposed for the polygalacturonase reaction. Molecular mass of this enzyme was 40 kDa and its isoelectric point was pH 7.5. Optimum pH of the enzyme reaction was 4.5 and its optimum temperature was 40-50 degrees C. Thirty percent of the maximum activity was observed at 5 degrees C, but it was only slightly active above 60 degrees C. The activity was preserved for more than 2 years at 5 degrees C and pH 4.5, but it was lost when kept at room temperature overnight or heated at 50 degrees C for 30 min. The amino acid sequence of the N-terminal region of the psychrophilic polygalacturonase of Sclerotinia borealis is compared with those of polygalacturonases of mesophilic fungi. The function of this enzyme against the target plants is discussed with reference to the reaction of polygalacturonases of mesophilic fungi.

Chemical characterization of sialyl oligosaccharides isolated from tammar wallaby (Macropus eugenii) milk.

Two components of the sialyl oligosaccharides were separated from milk of the tammar wallaby (Macropus eugenii) by gel filtration and ion exchange chromatography. Their molecular weights, estimated by gel filtration on HPLC using 5 mM triethylamine-acetate buffer (pH 5.0), were approx. 3,000. Their monosaccharide compositions, determined by GC analysis after methanolysis and by colorimetric assay, were (Glc)1(Gal)9(GlcNAc)2(Neu5Ac)1, and (Glc)1(Gal)8(GlcNAc)2(Neu5Ac)2. Their chemical structures were further elucidated by 1H-NMR and methylation analysis. The results suggest that their approximate structures are: [formula: see text]

[Clinical pharmacology and efficacy of S-1108].

We studied the pharmacokinetics of a new cephem antibiotic, S-1108, in patients with impaired kidney functions. Serum and urinary levels of S-1006 were determined after oral administration of S-1108 at 150 mg to 9 patients with renal dysfunction. In patients with severe renal impairment, high serum levels were maintained over long periods of time. Urinary excretion rates of S-1006 were lower as degrees of kidney failure were severer. S-1108 was administered to treat 27 patients with respiratory tract infections, and its clinical efficacy and safety were evaluated. The clinical efficacies were good in 26 patients, but poor in 1, yielding an efficacy rate of 96.3%. As to adverse reactions; diarrhea was observed in one case. Laboratory tests revealed elevated GOT and GPT in 1, and elevated gamma-GTP in another. These abnormalities, however, were slight and no severe side effects were caused by the drug.

Proteins with molecular masses of 50 and 80 kilodaltons encoded by genes downstream from the fimbrilin gene (fimA) are components associated with fimbriae in the oral anaerobe Porphyromonas gingivalis.

Flanking DNA regions of the fimbrilin gene (designated fimA), which encodes the major subunit protein of Porphyromonas (Bacteroides) gingivalis fimbriae, were cloned in several manners from the P. gingivalis chromosome into Escherichia coli by screening with probes derived from a 2.5-kb SacI DNA fragment previously cloned. A total of 10.4 kb of DNA fragments from the P. gingivalis genome was cloned in the pUC plasmid. Expression of the fimA gene and possible flanking genes in the fragments cloned was examined in a pUC plasmid vector system and in a bacteriophage T7 RNA polymerase-promoter expression vector system. The results show that in the pUC plasmid system, a 45-kDa protein, a product of fimA, was only poorly expressed as a precursor of the fimbrilin protein (FimA) and could be detected from cell extracts in Western blotting (immunoblotting) analysis as a sharp band but not in colony immunoblotting analysis. On the other hand, in the T7 RNA polymerase-promoter system, the product of fimA and products of the possible flanking genes responsible for fimbriation were overproduced as thick bands of the 45-kDa protein and as 63-, 50-, and 80-kDa proteins, respectively, in stained electrophoresis gels. All of the recombinant proteins were insoluble and seemed to be expressed as precursors with leader peptides. The 63-kDa, 45-k*Da (a truncated protein of the 50-kDa protein), and 80-kDa proteins were purified after solubilization with sodium dodecyl sulfate. N-terminal amino acid sequences of the 45-k*Da and 80-kDa proteins were analyzed up to the first 35 residues with a gas-phase sequencer. Monospecific antibodies directed to the recombinant proteins, i.e., the 63-kDa, 45-k*Da, and 80-kDa proteins, were raised in rabbits. By using the antibodies, localization of their matured proteins in P. gingivalis was investigated by Western blotting analysis. Immunoblotting analysis suggests that at least the 50- and 80-kDa proteins, encoded by genes downstream from the fimA gene, are minor components associated with fimbriae.

[Clinical evaluation and pharmacology of DQ-2556 in the patients with compromised renal function].

We studied the pharmacokinetics of a new cephem antibiotic, DQ-2556, in patients with impaired kidney function. The peak concentrations of the compound in the serum were observed irrespective of the degree of kidney failure 5 minutes after its bolus administration of 1.0 g intravenously, and no significant difference was observed in the concentrations among the patients. On the other hand, the decrease in its concentrations in the serum was impeded in proportion to degrees of kidney failure and, in particular, hemodialysis patients showed markedly delayed clearance of the drug from the serum; the half-lives in the serum (beta phase) were prolonged to ca. 6 hours in patients with severe kidney failure (Ccr ca. 20 ml/min) and did so markedly to ca. 17 to 21 hours in patients with hemodialysis as compared with ca. 2.5 hours in patients with slight kidney failure (Ccr ca. 50 ml/min). Urinary excretion rates (0-to-24 hours values) were ca. 70% in patients with slight kidney failure, ca. 60% in patients with moderate kidney failure and ca. 40% in patients with severe kidney failure, showing a tendency toward a decline in relation to increasing degrees of kidney failure. The compound showed a satisfactory dialytic property. The clinical efficacy and safety of DQ-2556 were evaluated upon administering if at daily doses of 0.5 g b.i.d. and 1.0 g b.i.d. for 7 and 14 consecutive days respectively, in patients with lower respiratory tract infections. The clinical efficacies were excellent in 2 patients, good in 11 and poor in 2, yielding a efficacy rate of 86.7%. No side effects were observed, though, a neutrophil sedimentation ratio decreased in a patient, and a down-shift of prothrombin activities was observed in another. These results suggest that DQ-2556 is useful for lower respiratory tract infections, but in patients with kidney failures it is required to seek the most suitable regimen since the excretion rates of the compound decrease as degrees of kidney failure become severer.

[Clinical pharmacology and efficacy of levofloxacin in elderly patients].

We studied a newly developed oral quinolone antimicrobial agent, levofloxacin (LVFX, DR-3355), and obtained the following results. 1. Serum and urine levels of LVFX were determined after oral administration of LVFX 100 mg to 11 elderly patients with various degrees of renal function insufficiencies. The patients were classified according to creatinine clearance (Ccr) values into Group I (n = 1, Ccr greater than or equal to 70 ml/min), Group II (n = 4, 40 less than or equal to Ccr less than 70 ml/min), and Group III (n = 6, Ccr less than 40 ml/min). The peak levels of LVFX did not differ greatly among the 3 groups, but in patients with severely impaired renal functions, serum concentrations decreased more slowly than in those with slightly and moderately impaired renal functions, and high serum levels were maintained over a long period. Urinary excretion of LVFX diminished in relation to degrees of renal failure. 2. LVFX was administered to treat 13 elderly patients with respiratory tract infections. Clinical responses were good in all patients with a high efficacy rate of 100%. Laboratory tests revealed eosinophilia in 1 case. The symptom was mild, however, and no severe side effects due to the drug were observed.

Molecular cloning and expression of a major surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis in Escherichia coli.

A major immunodominant surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis 381 has been purified and its amino-terminal amino acid sequence has been determined. Using oligonucleotide probes corresponding to the sequence, we identified a recombinant plasmid clone carrying a single 4.2-kb BamHI fragment from pUC19 libraries of P. gingivalis. The BamHI fragment transferred to the bacteriophage T7 RNA polymerase/promoter expression vector system produced a slightly larger (77-kDa) protein, a precursor form, immunoreactive to the antibody against the 75-kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 75-kDa protein. Genomic Southern analysis revealed a single copy of the 75-kDa protein gene per genome among all P. gingivalis strains tested, and that no homologous genes are present in other black-pigmented Bacteroides species. These observations suggest that the 75-kDa protein gene may be useful as a specific DNA probe to classify or to detect this organism.

Purification and properties of a 75-kilodalton major protein, an immunodominant surface antigen, from the oral anaerobe Bacteroides gingivalis.

A 75-kilodalton major protein (75K protein) was purified to homogeneity from the cell lysate fraction and the envelope of Bacteroides gingivalis 381. The 75K protein was originally present in the outer membrane or the outermost part of this organism as a large, stable complex with an apparent molecular weight of about 2,000,000. Heating at 80 degrees C and at higher temperatures in the presence of sodium dodecyl sulfate was needed to completely dissociate it to monomers. Amino acid analysis revealed that the 75K protein had about 50% nonpolar amino acids. Various strains of B. gingivalis but not other bacteria, including oral Bacteroides species tested, contained serologically related 75K proteins when tested in Western blotting (immunoblotting) analysis. The abundance and localization of the 75K protein in this organism suggest that it has the potential to participate in the host-parasite interaction in infection. The 75K protein was, indeed, strongly recognized in patients with adult periodontal diseases. Immunoblotting with sera from patients and with rabbit antisera generated by intravenous inoculations of whole B. gingivalis cells revealed that the 75K protein was an immunodominant antigen on the surface of B. gingivalis.

A family case with autosomal-dominantly inherited pituitary dwarfism.

Isolated growth hormone (GH) deficiency (IGHD) is detected in 1/10 of pituitary dwarfism, but there are only a few reports on IGHD as an autosomal-dominant trait. We found one family with autosomal-dominantly inherited IGHD and examined their pituitary functions and GH genomes. Brothers (9.5 year and 11 year) and their mother (37 year) were diagnosed as having IGHD and their grandmother and uncle also seemed to have IGHD. All of their heights were under "mean-4.0 S.D.". Cerebral tomography of brothers and their mother all showed "empty sella", and GH-releasing hormone (GRH) tests showed no responses of GH not only to bolus intravenous injections but also after repeated intramuscular injections of GRH (100 micrograms/day) for 7 days. Although genetic analysis (Southern blotting method) could not detect any mutations in their GH genomes, the IGHD lesion of them seemed to be pituitary in origin.

Two isozymes of chicken muscle acylphosphatase: purification and properties.

Two acylphosphatases, named Ch1 and Ch2, have been purified from chicken skeletal muscle. The molecular weights were determined to be 11,900 and 12,000 for Ch1 and Ch2, respectively, by sedimentation equilibrium. In the amino acid compositions of Ch1 and Ch2, two residues of histidine were contained in Ch2, but none in Ch1, and one residue of cysteine was contained in Ch1, but none in Ch2. There were 11 lysines and 6 arginines in Ch1, whereas there were 6 lysines and 11 arginines in Ch2. In addition, the contents of methionine, serine, glutamic acid and glutamine, and alanine were considerably different between Ch1 and Ch2. There were also differences in the peptide maps and carboxyl-terminal amino acid sequences (-Ser-Thr-Arg-Tyr-COOH for Ch1, and -Phe-Thr-Ile-Arg-Lys-COOH for Ch2). In the double immunodiffusion, Ch2 did not form a precipitin line with the rabbit anti-Ch1 antiserum. These results indicate that Ch1 and Ch2 are different, genetically specified isozymes of acylphosphatase of chicken skeletal muscle. Ch2 is considered to be a new type of acylphosphatase from skeletal muscle.

Fimbriae from the oral anaerobe Bacteroides gingivalis: physical, chemical, and immunological properties.

Circular dichroism spectra indicated the predominance of beta-sheet structure in Bacteroides gingivalis fimbriae regardless of the presence of sodium dodecyl sulfate. By using a computer program, the alpha-helix, beta-sheet, and beta-turn contents and the remainder were estimated to be 0, 55, 18, and 27%, respectively, judging from the circular dichroism spectra of the fimbriae. Heating for 5 min at 100 degrees C in sodium dodecyl sulfate was necessary to denature the fimbriae into their constituent protein (fimbrilin) monomers with a reduced content of beta-sheet structure. The amino-terminal amino acid sequence of the fimbrilin was different from partial or complete amino acid sequences of fimbrilins so far determined from Bacteroides nodosus, which falls into the same nonfermentative species of the genus Bacteroides as B. gingivalis, and from various other bacteria. Fimbrilin monomers had an isoelectric point of 6.0. Examination of antibodies against fimbriae and sodium dodecyl sulfate-denatured fimbrilin by enzyme-linked immunosorbent assay reinforced a previous notion (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) that different sets of antigenic determinants seemed to be exposed on their surfaces.

Antigenic determinants of acylphosphatase from porcine skeletal muscle.

Analysis of the quantitative precipitin reaction of acylphosphatase from porcine skeletal muscle with rabbit antiserum indicated the presence of at least two antigenic determinants on the porcine enzyme molecule. Immunological cross-reactivities of acylphosphatases from equine and rabbit skeletal muscles were examined. In double immunodiffusion with the antiserum, the precipitin lines of the porcine and equine enzymes completely fused, while the rabbit enzyme gave no precipitin line. The reaction between the 125I-labeled porcine enzyme and its antibody was inhibited to the same extent by the porcine and equine enzymes, but not by the rabbit enzyme. The three enzymes were similar in net charge and molecular weight on polyacrylamide gel electrophoreses. No conformational difference among the three enzymes was observed in their circular dichroism spectra. The amino acid composition of the rabbit enzyme differed from those of the porcine and equine enzymes in the contents of Glu, Gly, Lys, and Arg. Differences in the sequence of the rabbit enzyme from that of the porcine enzyme were investigated by comparison of the peptide maps of the tryptic peptides of the two enzymes. Four peptides of the rabbit enzyme were located at different positions from those of the porcine enzyme. Three of the four peptides from both enzymes were sequenced and all the tryptic peptides of both enzymes were characterized by amino acid analysis. The tryptic peptides of rabbit enzyme were tentatively aligned on the basis of their amino acid compositions and sequence homologies, compared with the corresponding peptides of the porcine enzyme. Among five amino acid residues of the porcine enzyme, Arg-4, Asp-28, Arg-31, Glu-56, and Ile-68, which are replaced in the rabbit enzyme, Arg-4 and Asp-28 are considered to be included in the antigenic determinants.

Amino acid sequence of acylphosphatase from porcine skeletal muscle.

The amino acid sequence of acylphosphatase from porcine skeletal muscle was determined. It consists of 98 amino acid residues with N-acetylserine at the amino (N)-terminus: Ac-Ser-Thr-Ala-Arg-Pro-Leu-Lys-Ser-Val-Asp-Tyr-Glu-Val-Phe-Gly -Arg-Val-Gln-Gly-Val-Cys-Phe-Arg-Met-Tyr-Thr-Glu-Asp-Glu-Ala-Arg-Lys-Ile -Gly-Val-Val-Gly-Trp-Val-Lys-Asn-Thr-Ser-Lys-Gly-Thr-Val-Thr-Gly-Gln -Val-Gln-Gly-Pro-Glu-Glu-Lys-Val-Asn-Ser-Met-Lys-Ser-Trp-Leu-Ser-Lys -Ile-Gly-Ser-Pro-Ser-Ser-Arg-Ile-Asp-Arg-Thr-Asn-Phe-Ser-Asn-Glu-Lys- Thr-Ile-Ser-Lys-Leu-Glu-Tyr-Ser-Asn-Phe-Ser-Ile-Arg-Tyr-OH. This sequence has three substitutions of amino acid residues, i.e., Thr/Ala, Ile/Val, and Ile/Val at positions 26, 68, and 96, respectively, from that of horse muscle acylphosphatase, formerly the only mammalian acylphosphatase with known sequence.

Amino acid sequence of acylphosphatase from rabbit skeletal muscle.

The complete amino acid sequence of acylphosphatase from rabbit skeletal muscle has been elucidated by automatic Edman degradation of peptides obtained from staphylococcal protease and trypsin digestions. The enzyme consisted of a single polypeptide chain of 98 amino acid residues, lacking only histidine. Its amino (N)-terminus was blocked by an acetyl group. The presented sequence of rabbit muscle enzyme was compared with those of equine and porcine muscle enzymes. There were four unique replacements, i.e., Arg-4, Asp-28, Arg-31, and Glu-56 in the sequences of both equine and porcine muscle enzymes were replaced by Gly, Gly, Lys, and Asp, respectively, in that of rabbit muscle enzyme. Extensive structural homology was observed among the three enzymes.

Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen.

A lambda gt11 cDNA library containing DNA inserts prepared from human liver mRNA has been screened with an antibody to human alpha 2-thiol proteinase inhibitor that was isolated from fresh plasma. Eighteen positive clones were isolated from one million phage, and each was plaque purified. The cDNA insert of one of these phage was sequenced and shown to code for alpha 2-thiol proteinase inhibitor as identified by a partial amino acid sequence of the light chain of alpha 2-thiol proteinase inhibitor. This cDNA insert contained 1529 base pairs coding for the complete alpha 2-thiol proteinase inhibitor. It included 45 base pairs of 5' noncoding sequence, 1281 base pairs that code for pre alpha 2-thiol proteinase inhibitor, a stop codon, 160 base pairs of 3' noncoding sequence, and 40 base pairs of poly(A) tail. The noncoding sequence on the 3' end contained a potential recognition site (AATAAA) for processing and polyadenylation of precursor messenger RNA. The amino acid sequence of alpha 2-thiol proteinase inhibitor deduced from the cDNA showed a striking similarity (overall homology at 74%) to that of bovine low molecular weight (LMW) kininogen, including two internally repeated sequences and a nonapeptide sequence of bradykinin. These data clearly indicated that alpha 2-thiol proteinase inhibitor and LMW kininogen are identical. This was further supported by immunological cross-reactivity between alpha 2-thiol proteinase inhibitor and LMW kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallization and properties of acylphosphatase from porcine skeletal muscle.

A crystalline acylphosphatase has been obtained from porcine skeletal muscle. The purification procedure consists of extraction with water, phosphocellulose column chromatography, CM-cellulose column chromatography, and crystallization. The enzyme was homogeneous by polyacrylamide gel electrophoresis. A high yield (39%) of the pure enzyme was attained by the use of buffers containing 10 mM 2-mercaptoethanol to prevent dimerization of the enzyme in the purification process. Activity assay in the presence of bovine serum albumin showed a high specific activity of the enzyme (about 7,000 mumol/min/mg at 25 degrees C with benzoyl phosphate as substrate). The molecular weight was determined to be 11,100 by sedimentation equilibrium. The amino acid composition of the enzyme was determined. The amino-terminus of the enzyme was blocked and the carboxyl-terminal residue was tyrosine.

[Basic and clinical studies on the effect of cefmetazole on infection in gyneco-obstetrics].

Cefmetazole (CMZ) was administered to 102 infectious cases in the gynecoobstetric patients, and the basic and clinical studies have been performed. The main findings obtained in the present study are: Amont 78 cases of CMZ administration immediately after the initial infection (A group), 42 remarkably effective (53.8%), 36 effective (46.2%) cass were observed. When other drugs were not effective after the initial infection, CMZ was administered (B group) to 24 cases, and 15 remarkably effective (62.5%), 9 effective (37.5%) cases were observed. overall effectiveness in the A and B groups was 57 remarkably effective (55.9%) and 45 effective (44.1%) cases, which are very excellent clinical effects. Among 102 cases, the pathogenic bacteria were found in 55 cases, and 29 cases out of the 55 (52.7%) showed infections with E. coli and with other bacteria having mixed infections. The effect of CMZ to E. coli as judged by MIC was excellent, providing the excellent clinical results. E. coli and other Gram-negative pathogenic bacteria in the B group showed resistance to ABPC, CEX, CEZ and CET, and after administering CMZ, all cases showed disappearance of these bacteria, but the increase in the resistant bacteria for CEX, CET and CEZ was obviously shown. Subjective and objective adverse effects and clinical laboratory analysis showed no abnormal effect and values due to CMZ, and it was true of the of the case received 140 g in total of CMZ over 35 days, 4 g/day. It may be conducted from the above findings that CMZ is very effective and safe antibiotic agent in the infections in the gynecoobsterics .