Accelerated development of polyoma tumors and embryonic lethality: different effects of p53 loss on related mouse backgrounds.

Molecular evidence linking polyoma virus to p53 inactivation is thus far lacking, setting this highly oncogenic virus apart from other DNA tumor viruses. As a biological test for interaction, we studied the effects of p53 loss on development of virus-induced tumors. The absence of p53 led to more rapid tumor development on two different mouse backgrounds, indicating synergism between p53 loss and oncogenic pathways controlled directly by the virus. No effects of p53 on tumor type or frequency were noted. Polyoma tumor-derived cells in culture retained p53, and most of these showed induction of p21CIP1/WAF1 in response to DNA damage. These results indicate that p53 functions are not directly and fully impaired by the virus in the intact host. On one mouse background, it was discovered that loss of p53 resulted in complete embryonic lethality prior to 11 days of gestation. This lethality could be rescued by inclusion of gene(s) from a 129/SvJ background.

Wild-derived inbred mice have a novel basis of susceptibility to polyomavirus-induced tumors.

Polyomavirus induces a broad array of tumors when introduced into newborn mice of certain standard inbred strains, notably those bearing the H-2(k) haplotype. Susceptibility in these mice is conferred by an endogenous mouse mammary tumor virus superantigen (Mtv-7 sag) that acts to delete T cells required for polyomavirus-induced tumor immunosurveillance. In the present study we show that mice of two wild-derived inbred strains, PERA/Ei (PE) and CZECH II/Ei (CZ), are highly susceptible to polyomavirus but carry no detectable Mtv sag-related sequences and show no evidence of Vbeta deletion. C57BR/cdJ (BR) mice, which are H-2(k) but lack the endogenous Mtv-7, are highly resistant based on an effective anti-polyomavirus tumor immune response. When crossed with BR, both PE and CZ mice transmit their susceptibility in a dominant fashion, indicating a mechanism(s) that overrides the immune response of BR. Susceptibility in PE and CZ mice is not based on interference with antigen processing or presentation since cytotoxic T cells from BR can efficiently kill F(1)-derived tumor cells in vitro. The expected precursors of polyomavirus-specific cytotoxic T cells are present in both the wild inbred animals and their F(1) progeny. These findings indicate a novel basis of susceptibility that operates independently of endogenous superantigen and prevents the development of tumor immunity.

[Evaluation of screening methods for cholinesterase variants from daily laboratory data].

The genetic variants of the cholinesterase (ChE) are frequently misdiagnosed as a liver dysfunction. We compared three extraction methods for screening of ChE abnormalities. Employing these three methods, total 31 cases were found to be genetic abnormalities from 2985 patients of Hamamatsu University Hospital. We picked up 11 of candidates with low enzyme activities less than 100U/l as group 1 using the first method and effectively detected 9 cases (82%) with genetic abnormalities. The second extraction method was based on the ratio between Albumin (Alb) and ChE and subsequently, 48 of high risk patients were picked up as group 2 and 28 cases (58%) showed genetic abnormalities. Furthermore, all cases of group 1 were contained in the second group. The third method was based on the discrimination function from Alb and total cholesterol (TC) as group 3 and 32 cases were picked up. Fourteen cases (44%) out of them showed the genetic abnormalities using this method, and surprisingly, 13 cases (93%) of them were estimated to be K-variant. Although the three methods showed the different characteristics to extract genetic abnormalities of ChE, the second extraction method could pick up the largest population with genetic abnormalities. Further phenotypic extraction methods should be compared to understand the relationship between phenotype and genotype.

Butyrylcholinesterase genes in individuals with abnormal inhibition numbers and with trace activity: one common mutation and two novel silent genes.

A random population was screened for abnormal dibucaine and fluoride numbers (DN & FN) to find some common mutations in butyrylcholinesterase (BCHE) gene. Of 2375 unrelated individuals, 10 were found to have low DN and FN and were selected for further studies. DNA analysis of these hypocholinesterasemics revealed that seven patients were heterozygous for missense mutation at codon 330 (TTA to ATA; BCHE*330I). The frequency of BCHE*330I mutation was calculated to be at least 0.29% among the Japanese. On the other hand, two novel mutations were found in three families and two individuals including probands whose enzyme activity was very low (silent gene). Polymerase chain reaction and single stranded conformation polymorphism (PCR-SSCP) and restriction fragment length polymorphism (PCR-RFLP) were used for identification of the common and known mutation types such as BCHE*250P (ACT to CCT), BCHE*365R (GGA to CGA), and BCHE*539T (GCA to ACA; K-polymorphism), whereas PCR-SSCP was used in combination with direct DNA sequencing for new mutations like BCHE*446V (TTT to GTT) and BCHE*451X (GAA to TAA).

Genetic mutations of butyrylcholine esterase identified from phenotypic abnormalities in Japan.

We have identified 12 kinds of genetic mutations of butyrylcholine esterase (BCHE) from phenotypic abnormalities, showing that BCHE activities were deficient or diminished in sera. These genetic mutations, detected by PCR-single-strand conformation polymorphism analysis and direct sequencing, consisted of one deletion (BCHE*FS4), nine missense (BCHE*24 M, *1005, *250P, *267R, *330I, *365R, *418S, *515C, *539T), and two nonsense mutations (BCHE*119STOP, *465STOP). All of the individuals deficient in serum BCHE activity were homozygous for silent genes (6 of 6). Fifty-eight percent of the individuals (31 of 53) with slightly reduced serum BCHE activity were heterozygous for silent genes. They also showed a higher frequency (47% as allele frequency) of the K-variant than the general population (17.5%). Finally, we confirmed low serum BCHE activity in 10 of 23 individuals heterozygous for silent genes.

Genetic basis of the silent phenotype of serum butyrylcholinesterase in three compound heterozygotes.

Three Japanese patients showed very low butyrylcholinesterase activity in their sera and appeared to be homozygous for silent genes for butyrylcholinesterase. From DNA analysis, all three patients were compound heterozygotes: GGA(Gly) to CGA(Arg) at codon 365 (G365R) and TTC(Phe) to TCC(Ser) at codon 418 (F418S) in patient 1, G365R and CGT(Arg) to TGT(Cys) at codon 515 (R515C) in patient 2 and ACT(Thr) to CCT(Pro) at codon 250 (T250P) and AGA(Arg) to TGA(Stop) at codon 465 (R465X) in patient 3. The K-variant, GCA(Ala) to ACA(Thr) at codon 539, was also found in patients 1 and 2. Simple identification methods for all the mutations were developed and applied to family analysis and control individuals. The mutant alleles (with silent gene and K-variant) were segregated as predicted by theory in pedigrees of patients 1 and 2. Four of the mutations, F418S, R515C, T250P and R465X, were initially discovered in Japan and genetic heterogeneity among the human population for the butyrylcholinesterase gene was suggested.

Effect of anabolic steroid on the development and treatment of rickets in rats.

The influence of anabolic steroid on the process of production of rickets in vitamin-D deficient animals as well as its influence on healing of rickets with vitamin-D therapy was studied. Long-Evans strains of female rats of two age groups (6 weeks and 4 weeks) were given rachitogenic diet and were divided into five sub-groups-(I) control receiving vitamin-D from the 1st day, (II) rachitic group, (III) receiving vitamin-D from 36th day, (IV) receiving anabolic hormone from the beginning of the experiment and (V) receiving both vitamin-D and anabolic hormone from 36th day of treatment. It was observed grossly as well as histologically and radiographically that: (1) the anabolic steroid minimizes the rachitic changes in the bones of vitamin-D deficient rats, (2) in rachitic animals, a combination therapy of anabolic hormone and vitamin-D results in comparatively greater calcification of osteoid matrix and better healing and remodelling of bone than with vitamin-D alone.