β-Ether cleavage of the lignin model compound 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether and derivatives by Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 4-ethoxy-3-methoxyphenyl-glycerol--guaiacyl ether (V) in low nitrogen, stationary cultures under which conditions the ligninolytic enzyme system is expressed. 4-Ethoxy-3-methoxyphenylglycerol XIII, guaicol and 4-ethoxy-3-methoxybenzyl alcohol (II) were isolated as metabolic products. Exogenously added XIII was rapidly converted to 4-ethoxy-3-methoxybenzyl alcohol indicating that it is an intermediate in the metabolism of V. P. chrysosporium also metabolized 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-3-hydroxypropane VI. The degradation pathway for this dimer also included initial -ether cleavage and -hydroxylation of the diol product 1-(4-ethoxy-3-methoxyphenyl) 2,3 dihydroxypropane (XI) to yield the triol XIII which was cleaved at the , bond to yield 4-ethoxy-3-methoxybenzyl alcohol. Finally P. chrysosporium also cleaved the dimer 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1-hydroxypropane (VIII) at the -ether linkage yielding 1-(4-ethoxy-3-methoxyphenyl) 1,2 dihydroxypropane (IX) which was subsequently cleaved at the , bond to yield II. All of the results indicate that oxidative -ether cleavage is an important initial reaction in the metabolism of -aryl ether lignin substructure dimeric compounds. Metabolities were identified after comparison with chemically synthesized standards by gas liquid chromatography-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography     

Epithelial cell components immunoreact with antiserum thymic factor (FTS) antibodies: Possible association with intermediate-sized filaments

By indirect immunofluorescence microscopy, an antiserum raised in rabbit against serum thymic factor (FTS) was found to decorate the epithelial cells not only in the thymus, but also in the kidney, uterus, urinary bladder, prostatic glands, stomach, ileum, colon, submaxillary glands, trachea, epidermis and epidermal appendages of mouse. The staining ability was completely absorbed with an FTS-binding immunoabsorbent, and affinity-purified anti-FTS IgG showed the same staining patterns as the original antiserum. The staining profiles resembled those described for tissues stained with antiprekeratin and antikeratin antibodies in both distribution in tissue and localization in the epithelial cells. In primary-cultured cells from mouse kidney medullae, the anti-FTS antibodies decorated the cytoplasmic fiber network. The fibers were wavy, bundled together and branched. They were dense in the perinuclear cytoplasm and spread in the cytoplasm toward the cell periphery. This decoration was resistant to colchicine and cytochalasin B, but sensitive to pretreatment with formaldehyde. The organization and shape of the fiber network were similar to those of the networks of intermediate-sized filaments containing cytokeratins, keratins and vimentin. However, the antiserum did not give a precipitin band in immunodiffusion test with prekeratin from bovine muzzle, keratin from human epidermis or 3T3 vimentin. Neither tubulin nor actin formed precipitin bands with the antiserum. These results show that the epithelial cells of various mouse tissues contain FTS or substances close to FTS in chemical structure and suggest that they are associated with the intermediate-sized filaments.     

Identification of mammalian-type transglutaminase in Physarum polycephalum. Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating activity.

Transglutaminase (TGase) catalyses the post-translational modification of proteins by transamidation of available glutamine residues. While several TGase genes of fish and arthropods have been cloned and appear to have similar structures to those of mammals, no homologous gene has been found in lower eukaryotes. We have cloned the acellular slime mold Physarum polycephalum TGase cDNA using RT-PCR with degenerated primers, based on the partial amino acid sequence of the purified enzyme. The cDNA contained a 2565-bp ORF encoding a 855-residue polypeptide. By Northern blotting, an mRNA of approximately 2600 bases was detected. In comparison with primary sequences of mammalian TGases, surprisingly, significant similarity was observed including catalytic triad residues (Cys, His, Asn) and a GTP-binding region. The alignment of sequences and a phylogenetic tree also demonstrated that the structure of P. polycephalum TGase is similar to that of TGases of vertebrates. Furthermore, we observed that the purified TGase had GTP-hydrolysing activity and that GTP inhibited its transamidating activity, as in the case of mammalian tissue-type TGase (TGase 2).     

Enhanced Utilization of Phosphonate and Phosphite by Klebsiella aerogenes

Klebsiella aerogenes ATCC 9621 was able to utilize phosphonates (P_n), including aminoethylphosphonate, ethylphosphonate, methylphosphonate (MP_n), and phosphonoacetate, and inorganic phosphite (P_t) as sole sources of phosphorus (P). The products of the phn gene cluster were absolutely required for P_n breakdown and P_t oxidation to inorganic phosphate (P_i) in this organism. To determine if K. aerogenes ATCC 9621 could be engineered to enhance the utilization of P_n and P_t, a multicopy plasmid, pBI05, which carried the entire phn gene cluster, was introduced into this strain. Despite the increased dosage of the phn genes, K. aerogenes ATCC 9621(pBI05) could utilize only up to 1.1-fold more P_n and P_t than did the control strain with the parent vector alone. These results suggested that P_i, which was generated from P_n and P_t, might limit further utilization of these P compounds. Consequently, to convert the resulting P_i to polyphosphate (polyP), the plasmid pKP28, which carried the K. aerogenes ppk gene (which encodes polyP kinase), was introduced into K. aerogenes ATCC 9621(pBI05). Overexpression of the ppk gene in K. aerogenes ATCC 9621(pBI05, pKP28) resulted in a 2.5-fold increase in P_t utilization over that of the control strain. This recombinant strain also accumulated approximately sixfold more P than did the control strain when the cells were grown with MP_n as a sole source of P.     

Timing and mechanisms of mesodermal and neural determination revealed by secondary embryo formation in Cynops and Xenopus

We examined the timing and mechanisms of mesodermal and neural determination in Cynops , using the secondary embryo induced by transplantation of the prechordal endomesoderm. Two unique approaches were used: one was to observe gastrulation movements induced by the graft, and the other to measure the volumes of formed tissues. Transplanted graft pulled host animal cap cells inside to form a new notochord and other mesoderm of the secondary embryo, showing determination of mesoderm during gastrulation. The graft attained a certain width beneath the host ectoderm and moved near to the animal pole of the host by late gastrula, and a neural plate, which had a similar width to the graft, was formed covering the graft. The volume of neural tissues of the secondary embryo at tail-bud stages was about half that of the normal embryo, while the volumes of notochord were comparable in each case. These data suggest that prechordal endomesoderm, rather than notochord, determines the limit of neural plate in the overlying ectoderm. Similar dorsal grafts were transplanted at early gastrula in Xenopus but did not form well developed secondary embryos, demonstrating that the timing and mechanisms of mesoderm formation in Xenopus are different from those in Cynops .     

Characterization of the Bacillus subtilis CwbA protein which stimulates cell wall lytic amidases

The Bacillus subtilis cell wall binding protein, CwbA, stimulated the cell wall lytic activities of the B. subtilis and B. licheniformis autolysins (CwlA and CwlM, respectively) in addition to that of the major B. subtilis autolysin (CwlB). Even though the substrate for the enzyme reaction was changed from B. subtilis cell wall containing a teichoic acid to Micrococcus luteus cell wall containing a teichuronic acid, the stimulatory effect of CwbA on CwlA activity was observed.     

Sequence analysis and HLA-DR genotyping of a novel HLA-DRw14 allele

Analysis of a Japanese population by oligonucleotide genotyping revealed that one Japanese HLA-DRw14 allele had a DRB1 genotype different from that of the known HLA-DRw14-related alleles, DRB1 ^* 1401 (DRw14-Dw9) and DRB1 ^* 1402 (DRw14-Dw16). The second exon of the DRB1 gene of the novel DRw14 allele (designated DRB1-14c) was amplified enzymatically and sequenced after cloning intto a plasmid vector. The amino acid sequence of the first domain in the DR1 chain encoded in the DRB1-14c allele was more similar to that of the DRB1 ^* 1401 allele (three amino acid substitutions).than to that of the DRB1 ^* 1402 allele (six amino acid substitutions). No polymorphic amino acid residue that could explain the common serologic HLA-DRw14 specificity was identified among the sequences of the three DRw14-related alleles. Sequence-specific oligonucleotides (SSOs) were synthesized on the basis of the DRB1-14c nucleotide sequence and used for genotyping of the Japanese population. These SSOs served as useful probes for identifying the DRB1-14c allele in a wide range of donors.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M33693.     

Electron microscopic demonstration of a common structural motif in human complement factor C3 and rat α1 -inhibitor 3 (murinoglobulin)

Electron microscopy of two homologous giant proteins revealed that complement factor C3 and α_i-inhibitor 3 have a common structural motif of a semicircularly bent string 18–20 nm long with two or three bumps indicating globular domains. C3 had a structure similar to the letter C with a small but distinct hole in the center. α₁-Inhibitor 3 was a more complete ring sometimes ajar at one corner. When the latter was treated with a proteinase, it became slightly flattened and adopted a squarish C-shape.     

Cooperation in a deep helical energy well

In contrast to random coil polymers, polyisocyanates maintain a highly extended helical conformation in solution. This structural characteristic causes unusually large chiral optical properties to arise from copolymerization of tiny proportions of optically active monomer isocyanates with achiral isocyanates or even from stereospecific placement of deuterium in the side chain of poly(n-hexyl isocyanate). These effects can be understood as phenomenologically related to the optical activity amplification properties of vinyl polymers studied by Pino and his co-workers and ascribed to breaking the energetic degeneracy of the otherwise equally populated left- and right-handed helical states of the backbone. Statistical thermodynamic calculations, based on this model, and analogous to those carried out earlier on the vinyl polymers, allow matching the temperature and molecular weight dependence of the optical activity in poly((R)-1-deuterio-1-hexyl isocyanate) to the approximate responsible energy terms.