Effect of substitution of glycine for arginine at position 146 of the A1 subunit on biological activity of Escherichia coli heat-labile enterotoxin.

The ADP-ribosyltransferase activity of polypeptide A1 of cholera toxin and that of Escherichia coli heat-labile enterotoxin (LT) are primarily responsible for the toxic activities of these toxins. Since the amino acid sequences of the two A1 polypeptides are very similar, their functional mechanisms are considered to be the same. Arg-146 of polypeptide A1 is thought to be involved in the active site, because this amino acid of cholera toxin has been identified as the site of self-ADP-ribosylation. However, the exact role of Arg-146 and the significance of self-ADP-ribosylation in toxicity remain unclear. We substituted Arg-146 of polypeptide A1 of LT with Gly by oligonucleotide-directed mutagenesis and examined the biological property of the resultant mutant LT. The substitution changed the mobility of subunit A on sodium dodecyl sulfate-polyacrylamide gel but did not reduce the vascular permeability activity of LT. This result indicates that Arg-146 is not absolutely required for toxic activity and that LT can express its toxic activity without self-ADP-ribosylation at Arg-146.     

Establishment of a new hereditary-cataract mouse (CSM) strain derived from SENCAR mouse

A mouse representing a new hereditary cataract strain was found in a mouse colony and a new line was established strain CSM. These mice were investigated genetically, histologically and biochemically. The results suggested that this cataract was apparently inherited through two recessive autosomal genes. Histologically the denucleation process of lens fibers was abnormal and small vacuoles appeared in the equatorial region of the lens cortex at 12 days. Biochemically, insoluble protein and sodium increased in the lens with age.     

The development of newborn C 57 BL/KsJ-dbm mice produced from eggs fertilized in vitro

The purpose of this study was to examine the development of newly born C57BL/KsJ-dbm mice produced from eggs fertilized in vitro. The embryos derived from fertilization in vitro (which was performed by using db/db eggs and adrenalectomized db/db (Adrex) spermatozoa,) were transferred to the oviduct of MRL/MpJ pseudopregnant recipients 30 hr after insemination. 376 of these embryos yielded 65 young. Weight gain and urine glucose, plasma glucose and insulin levels were measured in these young as well as in Adrex males. The young produced by fertilization in vitro showed hyperglycemia, hyperinsulinemia and obesity. The physiological abnormalities in these young were similar to those in db/db young produced by natural mating between heterozygote (db/+) males and females. Adrex males did not show hyperglycemia but did show hyperinsulinemia. These results indicate that in vitro fertilization and embryo transfer is an effective means of producing fetuses or newborns with an overt genotype in genetically diabetic obese (db) mice.     

IL-4, but not IL-5, can act synergistically with B cell activating factor (BCAF) to induce proliferation of resting B cells

B cell activating factor (BCAF) was initially identified in the supernatant of the murine T helper cell clone 52-3 (52-3 SN) because of its ability to promote activation and proliferation of resting B cells in the absence of any other costimulus. In this paper, we show that 52-3 T helper cells also secrete IL-4 and IL-5 and we have analyzed the influence of these two lymphokines on B cell proliferation induced by BCAF-containing 52-3 SN. Using the neutralizing anti-IL-4 monoclonal antibody 11B11, we observed partial inhibition of B cell proliferation. 52-3 SN free of IL-4 prepared using an immunoabsorbent column was still able to induce significant B cell proliferation. Although recombinant IL-4 alone does not induce B cell proliferation, it increased the proliferation induced by IL-4-free 52-3 SN. Kinetic studies showed that IL-4 is required at the start of B cell cultures in order to exert optimal synergistic effects. In contrast, anti-IL-5 monoclonal antibody NC17 did not affect the B cell proliferative activity of 52-3 SN whether or not IL-4 was present. When 52-3 SN was tested on dextran-sulfate-activated B cells, IL-5 and BCAF activities were detected but only the IL-5 activity was neutralized by monoclonal antibody NC17. These results demonstrate that (i) BCAF-containing SN can induce proliferation of resting B cells independently of IL-4 and IL-5, and (ii) IL-4, but not IL-5, can act synergistically with BCAF to induce B cell proliferation.     

Preparation and properties of the water-soluble chlorophyll-bovine serum albumin complexes

By mixing chlorophyll (Chl) a or b with a dense bovine serum albumin solution, the water-soluble Chl-bovine serum albumin complexes were prepared. These complexes, eluted near the void volume on a gel filtration, were separated well from unreacted bovine serum albumin, indicating an aggregation of such molecules in the complexes. Preparation of chlorophyllide (Chlide) a- or Chlide b-bovine serum albumin complex was unsuccessful, while the phytol-, and β-carotene-bovine serum albumin complexes could be obtained. Chls in the Chl-bovine serum albumin complexes had the following characteristics. (i) Main absorption peak of Chl a or b in the red region occurred at 675 nm or 652 nm, respectively. The Chl a-bovine serum albumin complex having absorption peak at 740 nm was also prepared. As compared with the stabilities of Chl a and b in Triton X-100. (ii) Both Chls in the bovine serum albumin-complexes were stable against oxidative stresses, such as photobleaching, Fenton reagent, peroxidase-H₂O₂ system. But (iii) they were easily hydrolyzed by chlorophyllase. These properties of Chls in the bovine serum albumin-complexes were similar to those of Chls in the isolated light-harvesting Chl a/b protein complex. A possible localization of Chls within the bovine serum albumin complexes was suggested that the porphyrin moiety of Chl was buried in bovine serum albumin; however, the hydrophilic edge of porphyrin ring, adjacent to the phytol group, occurred in the hydrophilic region of a bovine serum albumin molecule.     

Genetic studies on the muscle protein turnover rate of coturnix quail

The validation of the urinary excretion of N-methylhistidine (N-MH) by quail as an index of the muscle protein turnover rate was tested using the criterion of the rate of recovery of radioactivity in urine following an intraperitoneal dose of l-[3-¹⁴C]methylhistidine. A genetic study on muscle protein turnover in quail was conducted using three genetically diverse lines (LL, large body size; SS, small body size; RR, random-bred control line) selected for body size. When l-[3-¹⁴C]methylhistidine was administered to 20-week-old male and female coturnix quail by direct intraperitoneal injection, approximately 90% of the l-[3-¹⁴C]methylhistidine was recovered by 96 hr postinjection. Recoveries were low in the egg and muscle. These results show that N-MH released from myofibrillar protein is not reutilized and the excretion of N-MH is a satisfactory index of muscle protein breakdown. In all lines, the amount of urinary N-MH excretion and fractional synthesis (K_s) and degradation (K_d) rates at the high growing period were higher than those at the low growing period. The K_s and K_d are significantly different among selected lines at both 3 and 6 weeks of age. At 3 weeks of age, the fractional rate of synthesis of the LL line (13.2%/day) was higher than that of the RR line (11.5%/day), whereas the SS (8.1%/day) was lower than that of the RR line (11.5%/day). The fractional rates of degradation of both the LL line (4.1%/day) and the SS line (5.6%/day) were lower than that of the RR line (7.0%/day) at 3 weeks of age. From these results, it was recognized that selection for body size gave rise to the changes in the muscle protein turnover rate.     

Structure-activity relationships of nitro and methyl-nitro derivatives of indoline, indole, indazole and benzimidazole in Salmonella typhimurium

The mutagenic activities of eleven nitro derivatives and eleven N-methyl-nitro derivatives of indoline, indole, indazole and benzimidazole were investigated in Salmonella TA98 and TA100. The presence of a nitro group at C4 or C7 resulted in only weakly or nonmutagenic compounds, while a nitro group at C2, C5 or C6 usually resulted in measurable mutagenic activity in the non-N-methylated compounds. Methylation of a ring nitrogen usually reduced the mutagenic activity of these nitroheterocyclics except 2-nitro-benzimidazole, which resulted in a better than 300-fold increase in mutagenic activity. A proposed mechanism for the increased mutagenic activity obtained by methylation of imidazole nitrogens may provide insights into the reasons for the potent mutagenicities observed for several similarly methylated cooked-food mutagens.     

Molecular cloning and sequence analysis of full-length cDNA for rabbit liver NADPH-cytochrome P-450 reductase mRNA

The nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2,269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2,037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76,583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleotides of poly(A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85% homology to that of rat reductase (Porter, T.D. & Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U.S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91% identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system.