Production of a protein A--lymphotoxin hybrid protein for cancer-targeted therapy.

A hybrid protein between staphylococcal protein A and human lymphotoxin, ALT, was produced in Escherichia coli by expression of a recombinant plasmid containing the respective genes. IgG-binding activity of ALT was confirmed by Western blotting analysis and by affinity purification with IgG-Sepharose column chromatography. The purified ALT had cytotoxicity on mouse L-929 cells and its specific activity was approximately 3.5-5.0 X 10(6) U mg-1. ALT was partially degraded by a protease including in the E. coli lysate or trypsin and was converted to lymphotoxin which lacks the NH2-terminal 19 residues but possesses higher cytotoxic activity than ALT.     

Identification of Mutations in the Pigment Cell-Specific Gene Located at the Brown Locus in Mouse

The pigment cell-specific gene, located at the brown (b)-locus in mouse, encodes the protein that determines the type of melanin synthesized. This protein is known as tyrosinase-related protein, but here we tentatively term it b-locus protein to avoid confusions with the related sequence cross-hybridizing to the tyrosinase gene. In order to identify the mutation at the b-locus, we have cloned and characterized the b-locus protein gene of BALB/c mouse (b/b, c/c). The gene is about 18 kb long and organized into 8 exons and 7 introns. Sequence analysis of the b-locus protein gene reveals four base changes within the protein-coding regions: two missense mutations and two silent mutations. Two missense mutations result in the Cys to Tyr substitution at position 86 (codon 110) and the Arg to His substitution at position 302 (codon 326) of a b-locus protein molecule. Using allele-specific amplification, we confirmed that these missense mutations are actually present in the genomic DNA of two b-mutant strains examined, BALB/c and DBA/2 (b/b, C/C) mice, suggesting that these mutations are specific for the mutant mice at the b-locus. Moreover, we are able to show that the b-locus protein containing Tyr 86 is not reactive with the anti-b-locus protein monoclonal antibody, TMH-1, in transient expression assays.     

Enterobacter kobei sp. nov., a new species of the family Enterobacteriaceae resembling Enterobacter cloacae

The name Enterobacter kobei sp. nov. is proposed for a group of organisms referred to as NIH Group 21 at the National Institute of Health, Tokyo. The members of this species are Gram-negative, motile rods conforming to the definition of the family Enterobacteriaceae. The DNA relatedness of 23 strains of NIH Group 21 to the representative proposed as the type strain of this species averaged 82% at 70°C, whereas the relatedness to other species within the family Enterobacteriaceae was less than 42%. Because the phenotypic resemblance to Enterobacter cloacae is very close and the DNA relatedness (12–42%) is closer to species of the genus Enterobacter than to other species of the family, the members of NIH Group 21 were placed in the genus Enterobacter. Close phenotypic and genetic relationships were also found between NIH Group 21 and a member of a group of organisms referred to as Enteric Group 69 at the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA. It is suggested that the latter could be regarded as a subspecific rank of E. kobei, though this is subject to study of further strains. The majority of strains of E. kobei were isolated from clinical specimens. A culture of the type strain (NIH 1485-79) has been deposited in the Japan Collection of Microorganisms as JCM 8580. Received: 22 March 1996 / Accepted: 19 April 1996     

Phosphorylation of a Protein (pp56) is Related to the Regeneration of Rice Cultured Suspension Cells

Short-term cultured suspension cells of rice (Oryza sativa L.)are capable of regeneration, but not in long-term culture. Forclarification of the mechanism of regeneration, protein phosphorylationin short-term and long-term cultured suspension cells was comparedby two dimensional- polyacrylamide gel electrophoresis. A 56kDa protein having an isoelectric point of 4.5 was phosphorylatedin vitro in short-term cultured suspension cells, but was notphosphorylated after regeneration. This protein in longtermcultured suspension cells remained phosphorylated after transferto the regeneration medium. However, using an antibody raisedagainst this protein from short-term cultured suspension cells,it was always detected in long-term and short-term culturedsuspension cells after transfer to the regeneration medium.The partial amino acid sequence of the HPLC-purified proteinshowed homology to a calcium-binding protein from maize. Thephosphorylation of the 56 kDa protein (pp56) appears to be associatedwith the regeneration of cultured rice cells. (Received December 11, 1995; Accepted June 3, 1996)     

Thermodynamic response of soft biological tissues to pulsed infrared-laser irradiation.

The physical mechanisms that achieve tissue removal through the delivery of short pulses of high-intensity infrared laser radiation, in a process known as laser ablation, remain obscure. The thermodynamic response of biological tissue to pulsed infrared laser irradiation was investigated by measuring and analyzing the stress transients generated by Q-sw Er:YSGG (lambda = 2.79 microns) and TEA CO2 (lambda = 10.6 microns) laser irradiation of porcine dermis using thin-film piezoelectric transducers. For radiant exposures that do not produce material removal, the stress transients are consistent with thermal expansion of the tissue samples. The temporal structure of the stress transients generated at the threshold radiant exposure for ablation indicates that the onset of material removal is delayed with respect to irradiation. Once material removal is achieved, the magnitude of the peak compressive stress and its variation with radiant exposure are consistent with a model that considers this process as an explosive event occurring after the laser pulse. This mechanism is different from ArF- and KrF-excimer laser ablation where absorption of ultraviolet radiation by the collagenous tissue matrix leads to tissue decomposition during irradiation and results in material removal via rapid surface vaporization. It appears that under the conditions examined in this study, explosive boiling of tissue water is the process that mediates the ablation event. This study provides evidence that the dynamics and mechanism of tissue ablation processes can be altered by targeting tissue water rather than the tissue structural matrix.     

Effects of estradiol and testosterone on the synthesis,expression and degradation of androgen receptor in human uterine endometrial fibroblasts

The mechanism of known receptor-mediated androgen effects on the endometrial stroma was studied in endometrial fibroblasts derived from human uterus. 17-Estradiol (E) induced the expressions of androgen receptor (AR) mRNA, and predominantly increased the level of testosterone-binding sites (TBS) in uterine endometrial fibroblasts. The effect on the level of dihydrotestosterone-binding sites (DHTBS) was similar but smaller. This result suggests that the AR mRNA expressed might encode TBS, but probably not DHTBS. The TBS level increased by estrogen was down-regulated by testosterone (T) + E, but the AR mRNA expression increased by E was not down-regulated by E + T in the fibroblasts. Although the synthesis rate of AR was slightly increased (p<0.05) by E alone or E + T, the degradation rate of AR was significantly accelerated (p<0.05) by E + T in the fibroblasts. This result suggests that T might stimulate the metabolic rate of TBS, but does not inhibit the synthesis rate of AR mRNA to TBS in endometrial fibroblasts.     

Keratinocyte growth factor

Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, albeit from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal-epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr-2 gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and studies of KGF regulation by sex sterorid hormones reinforced the idea that KGF acts predominantly on epithelial cells to elicit a variety of responses including proliferation, migration and morphogenesis.     

A novel oncogene, ost, encodes a guanine nucleotide exchange factor that potentially links Rho and Rac signaling pathways.

Transfection of NIH3T3 cells with an osteosarcoma expression cDNA library led to the appearance of foci of morphologically transformed cells which were found to harbor a novel oncogene, ost. The ost product was activated by truncation of the N-terminal domain of the ost proto-oncogene and was highly tumorigenic in nude mouse assays. The proto-ost cDNA, isolated subsequently, encodes a predicted protein of 100 kDa containing DH (Db1 homology) and PH (pleckstrin homology) domains. Ost is mainly phosphorylated on serine and localized in the cytoplasm. Purified Ost protein catalyzed guanine nucleotide exchange on RhoA and Cdc42 among the Rho and Ras family members tested, indicating that Ost can activate these small GTP-binding proteins. Ost did not detectably associate with RhoA or Cdc42, but interacted specifically with the GTP-bound form of Rac1, suggesting that Ost can function as an effector of Rac1. These results suggest that Ost is a critical regulatory component which links pathways that signal through Rac1, RhoA and Cdc42. Of the tissues examined, expression of ost was the highest in brain and could be localized to neurons and alpha-tanycytes, suggesting that Ost may participate in axonal transport in these specialized cells.