Joint study on the teratogenic sensitivity of the Pika (Ochotona rufescens rufescens) to selected drugs

A collaborative study was conducted to investigate the teratological susceptibility of the Pika (Ochotona rufescens rufescens) to selected teratogenic chemicals: cyclophosphamide, 6-mercaptopurine, 5-fluorouracil, 6-aminonicotinamide, actinomycin D, ethylurethan, ampicillin, tetracycline, thalidomide, diphenylhydantoin, hypervitaminosis A, aspirin, dexamethasone, betamethasone and bredinin. Some of the chemicals were shown to be teratogenic in the Pika, but this animal was generally more resistant to their teratogenicity than the rabbit and rodents. In the Pika, thalidomide did not induce any typical limb defects, which have been produced in the rabbit. Pikas reproduce well and appear to have no substantial disadvantages as an animal species for teratological studies. Thus, the Pika may be useful as a new non-rodent species for teratological testings.     

UV endonuclease-mediated enhancement of UV survival in Micrococcus luteus: evidence revealed by deficiency in the Uvr homolog.

Unlike its phage T4 counterpart (also known as endonuclease V), Micrococcus luteus UV endonuclease (pyrimidine dimer DNA glycosylase/apurinic-apyrimidinic endonuclease) has suffered from lack of genetic evidence to implicate it in the promotion of UV survival of the cell, i.e., mutants with its deficiency are no more UV-sensitive than the wild type. On the assumption that the contribution of UV endonuclease is obscured by the presence of a homolog of Escherichia coli UvrABC endonuclease, which has recently been identified in this bacterium, survival studies were carried out in its absence. With 254-nm UV irradiation, which generates not only pyrimidine dimers but also 6-4 photoproducts as lethal lesions, a double mutant defective in both UV endonuclease and the Uvr homolog was shown to be more sensitive than a single mutant defective only in the latter, with a dose reduction factor of approximately 2 at the survival level of 37%. Furthermore, molecular photosensitization, which produces only pyrimidine dimers, revealed an even greater difference in sensitivity, the dose reduction factor being about 3.4. These results indicate that the contribution to cell survival of UV endonuclease, an enzyme specific for pyrimidine dimers, is manifest if the backup by the Uvr homolog is absent.     

The origin of O6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells transfected with human DNA

Transfection of Chinese hamster ovary (CHO) cells with human DNA has been shown in several laboratories to produce clones which stably express the DNA-repair protein, O6-methylguanine-DNA methyltransferase (MGMT), that is lacking in the parent cell lines (Mex- phenotype). We have investigated the genetic origin of the MGMT in a number of such MGMT-positive (Mex+) clones by using human MGMT cDNA and anti-human MGMT antibodies as probes. None of the five independently isolated Mex+ lines has human MGMT gene sequences. Immunoblot analysis confirmed the absence of the human protein in the extracts of these cells. The MGMT mRNA in the lines that express low levels of MGMT (0.6-1.4 x 10(4) molecules/cell) is of the same size (1.1 kb) as that present in hamster liver. One cell line, GC-1, with a much higher level of MGMT (4 x 10(4) molecules/cell) has two MGMT mRNAs, a major species of 1.3 kb and a minor species of 1.8 kb. It has also two MGMT polypeptides (32 and 28 kDa), both of which are larger than the 25 kDa MGMT present in hamster liver and other Mex+ transfectants. These results indicate that the MGMT in all Mex+ CHO cell clones is encoded by the endogenous gene. While spontaneous activation of the MGMT gene cannot be ruled out in the Mex+ cell clones, the intervention of human DNA sequences may be responsible for activation of the endogenous gene in the GC-1 line.     

Insulin-like growth factor-I stimulates endothelin-3 secretion from rat anterior pituitary cells in primary culture

Since we found relatively high concentrations of immunoreactive (ir-) ET-3 in the rat pituitary gland (190 pg/g tissue), we have investigated the possible ET-3 secretion from the primary culture of anterior pituitary cells and the effects of various growth factors on the ET-3 secretion. The ir-ET-3 was detected in the incubation medium within 2 h, and 24 h of culture attained the concentrations of 1.15 +/- 0.26 pg/well/6 x 10(5) cells. The ir-ET-3 secretion was stimulated by insulin, insulin like growth factor-II (IGF-II), and most effectively by insulin like growth factor-I (IGF-I) in a dose- and time-dependent manner, whereas the production of ir-ET-1 and ir-big ET-1 was slightly inhibited by IGF-I and IGF-II. In reverse-phase HPLC, the ir-ET-3 released into the culture media showed identical retention time with authentic ET-3. Although ir-ET-1 and ir-big ET-1 secretion was stimulated by transforming growth factor-beta (TGF-beta), ir-ET-3 secretion was inhibited. These results indicate that the anterior pituitary cells secrete ET-3 and the secretion is stimulated by IGF-I.     

Calcium metabolism, serum thyroid-stimulating hormone, prolactin, and growth hormone in spontaneously hypercholesterolemic rats

We have shown previously that spontaneously hypercholesterolemic (SHC) rats exhibit abnormal bone metabolism with advanced bone resorption, which develops with age. In this study, we measured serum levels of growth hormone, thyroid-stimulating hormone, and prolactin in addition to several parameters of calcium metabolism and renal function in young (6-week) and old (24-week) SHC rats and compared these with age-matched Sprague-Dawley rats. In young SHC rats, urinary excretion of hydroxyproline and serum levels of calcium were significantly elevated and excretion of protein into urine and urea nitrogen in the serum were normal, suggesting that calcium metabolism was abnormal without kidney dysfunction at this age. Serum growth hormone and thyroid-stimulating hormone levels were markedly higher (20- to 30-fold and 4- to 5-fold, respectively) in young and old SHC rats, whereas serum prolactin levels were similar. A high level of serum thyroid-stimulating hormone was associated with elevated levels of thyroxine and triiodothyronine in young SHC rats, but not old ones. These results demonstrate that the rat exhibits abnormalities in endocrine function as well as calcium metabolism preceding the occurrence of renal dysfunction.     

Albutensin A,an ileum-contracting peptide derived from serum albumin,acts through both receptors for complements C3a and C5a

Summary Albutensin A is an ileum-contracting peptide derived from serum albumin. The sequences of bovine, human and porcine albutensin A are ALKAWSVAR, AFKAWAVAR, and AFKAWSLAR, respectively. These albutensin A homologs all exhibited biphasic ileal contractions in the longitudinal strips of guinea pig ileum. The order of potency in the contraction was porcine>bovine>human homologs. The ileal contraction profiles were similar to those of oryzatensin and casoxin C, agonist peptides for complement C3a receptors derived from rice albumin and bovine κ-casein, respectively. All three homologs of albutensin A have homology with the COOH-terminal sequences of complements C3a and C5a, which are essential for their activities; porcine albutensin A showed the highest homology. Indeed, porcine albutensin A was confirmed to act through both C3a and C5a receptors by a radioreceptor assay and cross-desensitization in the ileal contraction. In addition, bovine and human homologs also showed affinity for both receptors. This study suggests that a bioactive peptide acting through both C3a and C5a receptors is released by the proteolytic cleavage of serum proteins other than complement components.     

Identification of the gene variations in human CD22

 CD22, a member of the immunoglobulin superfamily, is a B-cell transmembrane glycoprotein that acts as an accessory-signaling component of the B-cell antigen receptor (BCR). Recent evidence indicating the role of CD22 as a negative regulator of BCR signal transduction prompted us to test the possibility that genetic variations of human CD22 may be associated with autoimmune diseases. In this study, variation screening of the entire CD22 coding region was performed, and possible association with rheumatic diseases was tested, using the genomic DNA from 207 healthy Japanese individuals, 68 patients with systemic lupus erythematosus (SLE), and 119 patients with rheumatoid arthritis (RA). Through the variation screening, seven non-synonymous and four synonymous substitutions were identified. In addition, single base substitutions were found in two introns flanking exon-intron junctions. Among these variations, Q152E substitution within the second extracellular domain was observed with a marginally higher frequency in the patients with SLE (3/68, 4.4%) than that in healthy individuals (1/207, 0.5%) (P=0.048. SLE vs healthy individuals), although this difference was no longer significant after correction for the number of comparisons (Pc=0.62). No significant association was observed between any of the variations and RA. These findings indicate that a number of genetic variants are present in CD22, and suggest that CD22 could be considered a candidate for the susceptibility genes to autoimmune diseases. Received: 14 July 1998 / Revised: 7 September 1998     

Substrate-induced Nuclear Export and Peripheral Compartmentalization of Hepatic Glucokinase Correlates with Glycogen Deposition

Hepatic glucokinase (GK) is acutely regulated by binding to its nuclear-anchored regulatory protein (GKRP). Although GK release by GKRP is tightly coupled to the rate of glycogen synthesis, the nature of this association is obscure. To gain insight into this coupling mechanism under physiological stimulating conditions in primary rat hepatocytes, we analyzed the subcellular distribution of GK and GKRP with immunofluorescence, and glycogen deposition with glycogen cytochemical fluorescence, using confocal microscopyand quantitative image analysis. Following stimulation, a fraction of the GK signal translocated from the nucleus to the cytoplasm. The reduction in the nuclear to cytoplasmic ratio of GK, an index of nuclear export, correlated with a >50% increase in glycogen cytochemical fluorescence over a 60min stimulation period. Furthermore, glycogen accumulation was initially deposited in a peripheral pattern in hepatocytes similar to that of GK. These data suggest that a compartmentalization exists of both active GK and the initial sites of glycogen deposition at the hepatocyte surface.