Ontogenetic transition of wound healing pattern in rat skin occurring at the fetal stage

Full-thickness excisional wounds were made in the dorsal skin of rat fetuses at day 16 and day 18 of gestation. A small patch of skin surrounding the open wound was cut out, mounted on a plastic ring and incubated in an organ culture system. In the presence of serum, the open wound in the day-16 fetal skin closed within three days of culture. During the wound-closure process, no new structures were formed in the wound space, and no conspicuous changes were noted in the histological architecture of the surrounding skin during culture, indicating that the wound closure may result from a centripetal movement of the surrounding skin only. In contrast, the size of the open wound in the day-18 fetal skin remained almost unchanged for one week, but a thin acellular network spread over the wound space within one day of culture. The predominant component of the network was cross-linked fibrin, as disclosed by scanning electron microscopy and sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. The network served as a scaffold for the ingrowth of fibroblast-like cells. These stage-dependent differences in fetal wound healing were consistent with an in vivo study showing that the day-16 wound was covered with the surrounding skin itself, whereas the day-18 wound was covered with newly formed epidermis and invaded by inflammatory cells. The present investigation strongly indicates the prenatal occurrence of a fetal-to-adult transition in the wound-healing pattern of rat skin.     

Twelve loci form a continuous linkage map for human chromosome 18

We have constructed a primary genetic map of human chromosome 18 consisting of 11 DNA markers and one serological marker (JK). Two of these loci define highly polymorphic VNTR systems. The markers define a continuous genetic linkage map of 97 cM in males and 205 cM in females; female genetic distances in a panel of 59 three-generation families were consistently about twice those observed in males. The high odds in support of the linear order of the markers on this recombination map, and the extent of coverage of chromosome 18, indicate that this map will permit efficient linkage studies of human genetic diseases that may be segregating on chromosome 18 and will provide anchor points for development of high-resolution maps for this chromosome.     

Detection by DGGE of a new polymorphism closely linked to the adenomatous polyposis coli region

Summary The EF5.44 locus is in close proximity to the chromosome 5 region to which the genetic defect responsible for familial adenomatous polyposis has been mapped. We have devised two oligonucleotides that promote the specific polymerase chain reaction (PCR) amplificiation of a 365-bp sequence in this region. Analysis by denaturing gradient gel electrophoresis of the resulting fragment has unravelled individual differences that could be identified as a single base pair change in aMnlI restriction site. This PCR assayable polymorphism increases the informativeness at this locus, and should be useful in the presymptomatic diagnosis of familial adenomatous polyposis.     

Identification of 57 conventional RFLP and 6 VNTR systems with 32 DNA clones on chromosome 11p15

Fifty-four clones containing human inserts were selected from a cosmid library constructed from a somatic cell hybrid containing chromosome 11p15.3-p15.5 as its only human complement. In 32 of these clones, 63 polymorphic systems were identified with a panel of restriction enzymes: 57 conventional RFLP systems and 6 highly polymorphic VNTR systems. Although we examined the cosmid with only seven enzymes, 18 clones (including 6 VNTRs) were polymorphic with three or more enzymes. The results suggested that DNA sequences on the peritelomeric region of chromosome 11p tend to be highly variable. Because these markers are highly informative, they will be excellent resources for investigations of hereditary diseases and tumor suppressor genes in this region of chromosome 11.     

Establishment of a monoclonal antibody recognizing cyclobutane-type thymine dimers in DNA: a comparative study with 64M-1 antibody specific for (6-4)photoproducts

We obtained a monoclonal antibody (TDM-1) binding to 313-nm UV-irradiated DNA in the presence of acetophenone. The binding of TDM-1 to 254-nm UV-irradiated DNA was not reduced with the subsequent irradiation of 313-nm UV. Furthermore, the treatment of UV-irradiated DNA with photolyase from E. coli and visible light exposure reduced both the antibody binding and the amount of thymine dimers in the DNA. A competitive inhibition assay revealed that the binding of TDM-1 to UV-irradiated DNA was inhibited with photolyase, but not with 64M-1 antibody specific for (6-4)photoproducts. These results suggest that TDM-1 antibody recognizes cyclobutane-type thymine dimers in DNA. Using TDM-1 and 64M-1 antibodies, we differentially measured each type of damage in DNA extracted from UV-irradiated mammalian cells. Repair experiments confirm that thymine dimers are excised from UV-irradiated cellular DNA more slowly than (6-4)photoproducts, and that the excision rates of thymine dimers and (6-4)photoproducts are lower in mouse NIH3T3 cells than in human cells.     

Phosphorylated tau protein is integrated into paired helical filaments in Alzheimer's disease

Antisera to paired helical filaments (PHF) were found to contain a significant amount of tau antibodies specific for a phosphorylated form, but only a negligible amount of those specific for a non-phosphorylated form. Also, the phosphorylated tau-specific antibodies, but not the non-phosphorylated tau-specific ones, labeled neurofibrillary tangles isolated in the presence of sodium dodecyl sulfate (SDS) and stained both tangles and senile plaque neuritis in fixed tissue sections in a very similar way to as the whole antiserum did. Taken together, these results strongly suggest that a major antigenic determinant of PHF is phosphorylated tau itself.     

Predominance of gluconate formation from glucose during germination of Bacillus megaterium QM B1551 spores.

Metabolic pathways of glucose during germination of Bacillus megaterium QM B1551 spores were studied by using specifically labeled glucose and gluconate. The Embden-Meyerhof pathway, the pentose cycle, and the direct oxidation route of glucose to gluconate (the gluconate pathway) were all operative at this stage; among those, gluconate accumulation was most predominant, especially in the early stage. Potassium fluoride, an enolase inhibitor, abolished the catabolism by the Embden-Meyerhof pathway totally without affecting gluconate accumulation. Under these conditions glucose was exclusively oxidized to gluconate. Gluconate thus accumulated could be metabolized further via phosphorylation by gluconate kinase. Remarkable gluconate accumulation was also demonstrated in several other spores requiring alanine as an effective germinant. NADH formed by the direct glucose oxidation may serve as a initial ATP source to phosphorylate glucose in germinating spores.     

Efficient synthesis and secretion of a thermophilic alpha-amylase by protein-producing Bacillus brevis 47 carrying the Bacillus stearothermophilus amylase gene.

Bacillus subtilis and Bacillus brevis 47-5, carrying the Bacillus stearothermophilus alpha-amylase gene on pUB110 (pBAM101), synthesized the same alpha-amylase as the donor strain as determined by the enzyme's thermal stability and NH2-terminal amino acid sequence. Regardless of the host, the 34-amino acid signal peptide of the enzyme was processed at exactly the same site between two alanine residues. B. brevis 47-5(pBAM101) secreted the enzyme most efficiently of the hosts examined, 100, 15, and 5 times more than B. stearothermophilus, Escherichia coli HB101(pH1301), and B. subtilis 1A289(pBAM101), respectively. The efficient secretion of the enzyme in B. brevis 47-5(pBAM101) was suggested to be due to the unique properties of the cell wall of this organism.     

Restriction fragment length polymorphism detected by human salivary amylase cDNA

Summary The plasmid clone which contains human salivary amylase cDNA was used to detect restriction fragment length polymorphisms (RFLPs). After double digestion with Pst 1 and Bam H1, a polymorphism with two alleles was observed. In Japanese, frequencies of these alleles, tentatively called 5.7kb and 6.5kb fragment alleles, are 0.55 and 0.45, respectively.     

Cell-specific properties of red cell and liver ferritin from bullfrog tadpoles probed by phosphorylation in vitro

Cell-specific differences occur in the primary structure of ferritin. For example, red cell and liver ferritin from bullfrog tadpoles differ by 1.5 times in serine content. To determine if cell-specific differences in ferritin primary structure are expressed in the tetraeicosomer, which thus might distinguish the proteins in a functional state, phosphorylation in vitro was employed as a probe using [gamma-32P]ATP and the catalytic subunit from the cAMP-dependent protein kinase of bovine skeletal muscle. Subunits of both proteins in the tetraeicosomers were phosphorylated. Based on tryptic peptide maps, five regions common to both red cell and liver apoferritin were phosphorylated, as confirmed for two peptides by amino acid analyses. [32P]Apoferritin from red cells yielded an additional four 32P-fragments by mapping, at least three of which were unique by amino acid analysis and, in one case, might represent a 32P-Fe complex bound by a fragment of the iron-binding site. One peptide appeared to be unique to liver apoferritin. High concentrations of ATP yielded one additional peptide common to liver and red cell and one red cell-specific peptide in the tryptic peptide maps. The maximum moles of 32P/molecule were 13 +/- 4 and 6 +/- 2, respectively, for red cell and liver apoferritin, which corresponded within experimental error to the number of 32P-tryptic peptides. The level of phosphorylation was, on the average, not more than one site/subunit. Furthermore, above certain levels of phosphorylation, some subunits in the assemblage of 24 appeared to be unavailable as substrates, possibly because of charge repulsion or conformational changes. The possibility that post-translational modifications of ferritin which amplify cell-specific structural features occur in vivo with cytoplasmic components, e.g. protein kinases, is considered in terms of the physiological availability of iron from different iron storage cells and developmental changes in iron storage.