Evidence for the location of foreign genes in three different chromosomes in a plant obtained from direct DNA transfer

Tobacco mesophyll protoplasts were treated with plasmids, pCT2 (17.1 kbp) or pCT2T3 (18.3 kbp), which contained a chimeric aminoglycoside phosphotransferase II (APH(3′)II) gene and an intact nopaline synthase gene. Expression of two marker enzymes, APH(3′)II and nopaline synthase, were analyzed in transformed plants. Four out of 16 transformants obtained by pCT2T3 possessed both enzymes. Upon self-pollination, the progeny of one of transformants (T2) segregated to 153∶4 in terms of resistant and susceptible character to kanamycin, suggesting insertion of foreign genes into three independent chromosomes. The kanamycin resistant character in the rest of transformants showed 3∶1 segregation. DNA blot analysis of the T2 transformant and progenies indicated the presence of two marker genes.     

Critical role of a common transcription factor, IRF-1, in the regulation of IFN-beta and IFN-inducible genes.

Interferon regulatory factor 1 (IRF-1) is a protein that binds to cis-elements within the promoter of interferon (IFN)-beta and some IFN-inducible genes. We used a human fibroblast line, GM-637, to generate stable transfectants constitutively expressing IRF-1 mRNA in either the sense or antisense orientation. Upon induction with poly-(I).poly(C) or Newcastle disease virus, cells expressing sense IRF-1 mRNA produced significantly higher levels of IFN-beta mRNA and protein than control cells, whereas cells expressing antisense IRF-1 mRNA produced little or no IFN-beta mRNA and protein. Furthermore, clear differences were seen among the transfectants in the level of expression of two IFN-induced genes (2'-5'-oligoadenylate synthetase and class I HLA). Our data show that IRF-1 is essential for the induced expression of the IFN-beta gene. The results also indicate an important role of IRF-1 in the expression of IFN-inducible genes and suggest a role for IRF-1 in many other cytokine actions.     

Gustatory sensitivities of the hamster's soft palate

The response properties of taste receptors distributed on thesoft palate of the hamster were studied by recording integratedresponses from the greater superficial petrosal (GSP) nerveStimuli were concentration series of sucrose, NaCl, HCl andquinine hydrochloride (QHCl), and several other 0.1 M saltsand 0.5 M sugars. For comparison, integrated responses wererecorded from the chorda tympani (CT) nerve in many of the sameanimals from which recordings were made from the GSP. Responsesin each preparation were scaled relative to the phasic responseto 0.1 M NaCl and were then expressed for each nerve as a proportionof the total response magnitude (TRM)—the sum of all theresponses to the four concentration series. In this way, therelative response of each nerve to all of the stimuli couldbe evaluated. There were significant differences between theGSP and CT nerves in the responses to NaCl, QHCl and sucrose.Both the phasic and tonic responses to sucrose were larger inthe GSP than in the CT, whereas the tonic responses to NaCland QHCl were smaller. The slopes of the concentration-responsefunctions for NaCl, HCl and sucrose were significantly differentbetween the two nerves. The responses to 0.1 M sodium and lithiumsalts were significantly greater in the CT than in the GSP;whereas the 0.5 M sugars elicited responses in the GSP thatwere 2–3 times greater than in the CT nerve. A comparisonof the relative responsiveness to 0.3M sucrose, 0 3 M NaCl,0.01 M QHCl, 0.01 M HCl and distilled water among the GSP, CT,glossopharyngeal (IXth) nerve and superior laryngeal nerve (SLN)indicated that the vast majority of information about sucroseand NaCl is transmitted to the brainstem by the VIIth nerve. ¹Present address: Department of Oral Physiology, Kagoshima UniversityDental School, Kagoshima 890, Japan     

Augmentation of therapeutic effect of adoptive immunotherapy through a synergy between transferred killer cells and host's fresh lymphocytes]

Among several approaches to augment the therapeutic effect of adoptive immunotherapy, we focused the antitumor synergy between transferred killer cells and host's fresh lymphocytes. Immunotherapy models using murine tumors or clinical experiments revealed that preadministration of immunostimulator such as OK-432, followed by chemotherapeutic agents such as cyclophosphamide, can induce host's non-cytotoxic fresh lymphocytes that act synergistically with cultured killer cells against autologous tumor cells. Immuno-chemo-lymphocytotherapy (a sequential treatment with OK-432, chemotherapy and adoptive immunotherapy) is useful to treat the patients with advanced cancer even if the number of transferred lymphocytes is limited.     

Three-dimensional SEM study of arteriovenous anastomoses in the dog's tongue using corrosive resin casts

Arteriovenous anastomoses (AVAs) participate in the regulation of blood flow. Although it has been speculated that AVAs in the dog's tongue play a role in the regulation of the body temperature, no published work is available on the structural characteristics of AVAs in the dog's tongue. The purpose of the present investigation was therefore to determine the frequency of AVAs and their structural characteristics by the fabrication of vascular corrosive resin casts and examination under a scanning electron microscope. This method permitted not only the visualization of the three-dimensional characteristics of AVAs, but also a clear differentiation between arterioles and venules. The total number of AVAs in the mucosal lamina propria of the dorsum of the left tongue half was 2,292. Several essential types such as S-shaped, hook-shaped, straight, bibranching and Y-shaped AVAs were observed, of which the S-shaped and similar types constituted the overwhelming majority; Y-shaped types have never been reported heretofore. This study also revealed that the locations where AVAs were most often distributed were, in descending order of frequency, the tip, the corpus and the root area of the tongue. This high frequency and strategic location of AVAs in the tongue strongly indicate that AVAs of the dog's tongue participate in the thermal regulation.     

Cloning of a complete cDNA encoding human aromatase: immunochemical identification and sequence analysis

A complete cDNA clone encoding a human aromatase was isolated from a human placental cDNA library in lambda gt11. An antibody to the polypeptide specified by the isolated clone was prepared, and Western blot analysis and antibody inhibition experiments of human placental aromatase activity confirmed the identification of the clone as aromatase cDNA. The isolated aromatase cDNA clone of 3030 bp with two unique EcoRI sites contained a 3'-noncoding region of 1397 bp, an open reading frame of 1509 bp encoding 503 amino acid residues, and a 5'-noncoding region of 124 bp. Analysis of the amino acid sequence of aromatase and comparison of aromatase with other forms of cytochrome P-450 indicated that this enzyme is a unique form of the cytochrome P-450 superfamily.     

Suppression of overt diabetes in NOD mice by anti-thymocyte serum or anti-Thy 1, 2 antibody

Effects of anti-thymocyte serum (ATS) and anti-Thy 1, 2 monoclonal antibody on the spontaneously occurring diabetes in NOD mice were examined. Spontaneous diabetes in female mice was markedly suppressed by intravenous injection of rabbit anti-mouse thymocyte serum diluted to 1:4 on three consecutive days during the time period from 70 to 100 days after birth; the cumulative incidence of overt diabetes upto 195 days of age was greatly reduced and the onset of diabetes was delayed. Similar effect was observed with anti-Thy 1, 2 antibody treatment. These findings suggest that T lymphocytes play a role in the production of spontaneous diabetes in this mouse strain.     

Site of alkylation of the major ribonuclease from Aspergillus saitoi with iodoacetate

A base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi (RNase M) was modified by [14C]iodoacetic acid. RNase M was inactivated with concomitant incorporation of about 1 mol equivalent of carboxymethyl group. Carboxymethylated RNase M (CM RNase M) thus obtained was reduced and carboxymethylated (RCM CM RNase M). From tryptic and chymotryptic digests of RCM CM RNase M, two carboxymethylated histidine-containing peptides labeled with radioactivity were isolated. The amino acid sequences of these two peptides were determined to be Thr-Ile-His-Gly-Leu-Trp-Pro-Asp-Asn-Cys-Asp-Gly-Ser-Tyr... and His-Gly-Thr-Cys-Ile-Asn-Thr-Ile-Asp-Pro-Ser-Cys-Tyr-Pro-Asp-Asp-Tyr-Ala. .... The distribution of the radioactivity on the former and latter peptides was 43% and 57%, respectively. The results indicated that two histidine residues are involved in the active site of RNase M, and the modification of either one of the two histidine residues inactivates RNase M. The CD spectrum of carboxymethylated RNase M indicated that some tryptophan residue(s) with a CD band at 287 nm is in the proximity of the active site histidine residues of RNase M.     

Release of endogenous prostaglandins by mild hyperosmotic saline inhibits tetragastrin-stimulated gastric acid secretion in rats

Filling of the gastric lumen of rats with 1.0 M NaCl solution (5 ml) for 10 min under urethane anesthesia caused an increase in the gastric fluid concentrations of prostaglandin (PG) E₂, 13, 14-dihydro-15-keto-PGE₂ and 6-keto-PGF_1α as determined by radioimmunoassay. PGE₂ was the major PG generated. The levels of PGE₂ in the gastric fluid were increased dose-dependently after filling the lumen with 0.3, 0.5, 0.7 or 1.0 M NaCl solutions. The pH of the gastric fluid increased similarly after 0.5 to 1.0 M NaCl solutions. Indomethacin (10 mg/kg, i.p.) suppressed the PGE₂ increase caused by 1.0 M NaCl solution, but did not prevent the increase of the pH of the gastric fluid induced by intragastric 1.0 M NaCl. Infusion of tetragastrin (62.5 μg/kg/hr, i.v., for 10 min) caused a marked increase of acid secretion without modifying intragastic concentration of PGE₂. The acid secretion due to tetragastrin was completely inhibited after intragastric administration of 1.0 M NaCl solution, while indomethacin restored the tetragastrin-induced acid secretion, with prevention of a rise of intragastric PGE₂ levels. These observations suggest that 1.0 M NaCl solutions suppress basal intragastric acid through a mechanism which is independent of prostaglandins. In contrast, the suppression of tetragastrin-induced acid secretion by intragastric 1.0 M NaCl solution appears to be mediated through a release of prostaglandins     

Glycinin A3B4 mRNA. Cloning and sequencing of double-stranded cDNA complementary to a soybean storage protein

The cDNA clones encoding the precursor form of glycinin A3B4 subunit have been identified from a library of soybean cotyledonary cDNA clones in the plasmid pBR322 by a combination of differential colony hybridizations, and then by immunoprecipitation of hybrid-selected translation product with A3-mono-specific antiserum. A recombinant plasmid, designated pGA3B41425, from one of six clones covering codons for the NH2-terminal region of the subunit was sequenced, and the amino acid sequence was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 516 amino acids. Analysis of this cDNA also showed that it contained 1786 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 46 nucleotides, a signal peptide region corresponding to 24 amino acids, an A3 acidic subunit region corresponding to 320 amino acids followed by a B4 basic subunit region corresponding to 172 amino acids, and a 3'-terminal nontranslated region of 192 nucleotides, which contained two characteristic AAUAAA sequences that ended 110 nucleotides and 26 nucleotides from a 3'-terminal poly(A) segment, respectively. Our results confirm that glycinin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs via disulfide bonds. The inferred amino acid sequence of the mature basic subunit, B4, was compared to that of the basic subunit of pea legumin, Leg Beta, which contained 185 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall 42% of the amino acid positions are identical in both proteins. These results led us to conclude that both storage proteins have a common ancestor.