(NZW x BXSB)F1 mouse. A new animal model of idiopathic thrombocytopenic purpura.

A decrease in thrombocyte count was observed in (NZW x BXSB)F1 (W/B F1) mice at the age of greater than 5 mo, whereas megakaryocyte counts were found to increase in such mice. FACS analyses revealed the presence of both platelet-associated antibodies (PAA) and circulating antiplatelet antibodies. There is a correlation between the presence of these antibodies and the degree of thrombocytopenia. The transplantation of normal bone marrow cells from BALB/c nu/nu mice to W/B F1 mice was found to have preventative and curative effects on thrombocytopenia; the mice showed normal platelet counts and no evidence of circulating antiplatelet antibodies. These results indicate that thrombocytopenia in W/B F1 mice is due to the presence of antibodies to platelets. We therefore think that W/B F1 mice serve as a useful animal model of idiopathic thrombocytopenic purpura (ITP) not only for elucidating the mechanism of the development of antiplatelet antibodies, but also for characterizing autoantibodies to platelets.

Incidence of precancerous foci of mammary glands and growth rate of transplantable mammary cancers in sialoadenectomized mice.

Sialoadenectomies performed on 8-week-old female SHN and GR mice markedly reduced the numbers of precancerous and cancerous lesions in their mammary glands that had been mildly hypoplastic; the mice were necropsied when they were 30 weeks old. The success rate of the mammary cancer transplantation to isogenic male SHN or C3H mice was lower in the sialoadenectomized animals, and growth of the grafted tumors was delayed after gland removal. Some tumor development resumed in the hosts that received mouse epidermal growth factor after surgery. Therefore, we believe this growth factor may play a role in the multistage process of mouse mammary carcinogenesis.

Prophylaxis of spontaneous mammary tumorigenesis by temporal inhibition of prolactin secretion in rats at young ages.

The effects of temporal prolactin suppression during the limited periods of early age on spontaneous mammary tumorigenesis at advanced ages were examined in rats. Daily s.c. injections of 0.5 mg 2-bromo-alpha-ergocryptine mesylate, a potent suppressor of pituitary prolactin secretion, into virgin rats for 7 weeks beginning at 4 weeks of age resulted in almost complete prevention of mammary tumor appearance by 20 months of age. The incidence of tumors in this group (Group 1) was 3.3% (1 of 30), significantly smaller than that of the control [Group 2, 47.6% (10 of 21)]. Treatment given between 11 and 18 weeks of age inhibited mammary tumor incidence to a lesser extent [Group 3, 20% (6 of 30)], although tumor incidence was still significantly smaller than that in the corresponding control [Group 4, 41.2% (7 of 17)]. Serum prolactin level was decreased significantly by 2-bromo-alpha-ergocryptine mesylate; the levels on the evening of proestrus in the last week of injection were 18 +/- 3 (S.E.), 326 +/- 37, 15 +/- 2, and 460 +/- 55 ng/ml in Groups 1, 2, 3, and 4, respectively. Body weight change and pattern of estrous cycles were not affected by the treatment.

Necrotizing effect of ethanedimethanesulfonate on spontaneously occurring Leydig cell tumors in old F344 rats.

Thirty 18-month-old male F344/DuCrj rats were divided into the following groups: 10 untreated controls; eight vehicle-injected controls; and 12 ethanedimethanesulfonate (EDS)-injected rats. Untreated controls were killed immediately to check for testicular tumor incidence. In rats of the test group, a 75-mg/kg dose of EDS dissolved in dimethyl sulfoxide:water (1:3) was injected i.p. At intervals of 1, 2, 3, and 10 days after injection, two vehicle-injected control rats and three EDS-injected rats were sacrificed, and the testes were fixed by vascular perfusion. The midsagittal sections of all the fixed testes were examined to determine the incidence of macroscopic Leydig cell tumors, and some tumor tissues of the injection-treated groups were also investigated ultrastructurally. In 28 of 30 animals, a total of 78 Leydig cell tumors could be distinguished. Extensive and severe necrotic alterations accompanying fresh, multiple hemorrhages in early stages and reparative changes in later stages could be observed in a total of 78% of the 32 tumors examined from the EDS-injected group. The tumor cells exhibited ultrastructurally degenerative changes such as chromatin condensation and cytoplasmic vacuolation from 1 day after EDS injection. Therefore, EDS may be a necrotic agent for rat Leydig cell tumor.

Intervention of T-cells in transportation of mouse mammary tumor virus (milk factor) to mammary gland cells in vivo.

Using BALB/c nu/nu, BALB/c nu/nufC3H (BALB/c nu/nu mice raised by C3H/HeN foster mother), BALB/c thymus-engrafted BALB/c nu/nufC3H, BALB/c nu/+, and BALB/c nu/+fC3H mice, we examined what kinds of cells are carriers of blood-borne mouse mammary tumor virus (B-MMTV). A radioimmunoassay and an immunoperoxidase assay revealed the presence of MMTV-gp52 antigen in the mammary glands of all BALB/c nu/+fC3H and BALB/c thymus-engrafted BALB/c nu/nufC3H mice (more than 60 days old) but only of some BALB/c nu/nufC3H mice (more than 120 days old): those that possessed a significant number of functional T-cells. BALB/c nu/+ mice did not show the antigen expression at any age. Transfer experiments of cells or plasma from young (less than 12 weeks) BALB/c nu/nufC3H to BALB/c +/+ virgins revealed that cells besides T-cells can also become carriers of B-MMTV. This was confirmed by Southern blotting analyses; exogenous provirus DNA sequences were found in B-cells as well as T-cells of BALB/c nu/+fC3H mice. However, when young BALB/c nu/nu mice were inoculated with BALB/c nu/nufC3H blood, they did not show the MMTV-gp52 antigen expression. Transfer experiments of purified T-cells, B-cells, natural killer cells, and macrophages from BALB/c fC3H mice to BALB/c nu/nu mice revealed that only T-cells have the ability to transfer viral activity to the mammary glands. These results suggest that B-MMTV is carried from the gastrointestinal tract to the mammary glands by lymphoid cells such as T-cells and B-cells, then transferred to the mammary gland cells by the T-cells.

Reactivity of rat pial arterioles and venules to adenosine and carbon dioxide: with detailed description of the closed cranial window technique in rats.

This study describes a closed cranial window technique that allows the observation and measurement of rat pial arterioles and venules in situ. The resolving power of this system is 1-2 microns. Using this sensitive technique, we characterized the responses to 7% carbon dioxide inhalation and adenosine in arterioles (10-70 microns) and venules (15-100 microns). During carbon dioxide inhalation, larger arterioles (greater than 40 microns) dilated more than smaller arterioles (less than 20 microns). There was limited vasoreactivity of pial venules during CO2 inhalation. Dilation of arterioles was initially observed with an adenosine concentration of 10(-8) M. Almost a twofold increase in diameter was noted at 10(-3) M. In contrast to the effect of CO2 inhalation, the degree of dilation with topical application of adenosine was not size dependent. Pial venules did not respond to adenosine. The technique for observation of pial vessels using the closed cranial window and for measurement of vessel diameter by video camera system microscopy is a powerful tool for studying in vivo the cerebral circulation in the rat.

Moderate hypothermia reduces hypotensive, but not hypercapnic vasodilation of pial arterioles in rats.

Two types of acid-base strategies are available for the blood gas management of patients during hypothermia: alpha-stat and pH-stat management. However, the more suitable strategy for therapeutic hypothermia is unclear. We studied the effects of hypothermia (30 degrees C) and acid-base management on reactivity to hypercapnia and hypotension in rat pial arterioles, using a closed cranial window. The baseline diameter during hypothermia decreased in the alpha-stat (PaCO2 was maintained at 35 mm Hg when measured at 37 degrees C, n = 8), but not in the pH-stat (PaCO2 was maintained at 35 mm Hg when corrected to the animal's actual temperature, n = 7). Vasodilation induced by hypotension was significantly reduced in hypothermic groups compared with the normothermic group (n = 7), whereas responses to hypercapnia were preserved. Moreover, hypotensive vasodilation was more attenuated in the pH-stat, than the alpha-stat, management. These findings show that moderate hypothermia and acid-base management alter cerebrovascular autoregulation.

Ultracytochemical observations on intracytoplasmic lipids by enzymic digestive methods: histochemical trials.

The penetration of Rhizopus arrhizus lipase into ethionine fatty livers was examined histochemically. A free-floating, 30 microns thick Vibratome section of the livers perfused with aldehydes was shown to be suitable for ultracytochemical analysis by the lipase digestive method. Accessibility of phospholipids in the outer membrane of erythrocytes to Naja naja phospholipase A2 was observed under a light microscope, and intact red cells were used for the ultracytochemical analysis by the phospholipase digestive method. Ultracytochemical pictures of cytoplasmic lipids in these materials subjected to the enzymatic digestive method are presented.

Establishment of DDD/1-Mtv-2/Mtv-2, nu/nu and DDD/1-Mtv-2/Mtv-2, nu/+ mice: preliminary characterization in relation to MTV antigen expression and mammary tumorigenesis.

To investigate the effects of the T-cell deprivation on viral mammary tumorigenesis, two double congenic mouse strains of the DDD genetic background, DDD/1-Mtv-2/Mtv-2, nu/nu and DDD/1-Mtv-2/Mtv-2, nu/+, were produced by the cross between DDD/1-Mtv-2/Mtv-2 (DDD-Mtv-2) and DDD/1-nu/nu mice, followed by repeated intercross breedings. Expression of the mouse mammary tumor virus (MTV)-gp52 antigen was demonstrated in the mammary glands of mice from 14 days on, in both -nu/nu and -nu/+ females. Mammary gland development was comparable in both strains, but, the incidence of mammary cancer was lower in the T-cell-deprived mice.

Neoplastic response to dmba powder in mammary and interscapular tissues in wistar furth and copenhagen female rats.

To investigate genetic factors in local tumorigenesis, a dusting of 1 mg of 7,12-dimethylbenz(a)-anthracene (DMBA) powder was directly applied to exposed mammary tissue in 15 Wistar/Furth (WF), 20 Copenhagen (Cop), 19 (WF x Cop) F1, 16 backcross (BC) (F1 x WF) and 19 BC (F1 x Cop) females, at the age of 30 days. By 28 weeks after dusting, gross tumors were induced in 15 WF, 17 Cop, 12 (WF x Cop) F1, 11 BC (F1 x WF) and 14 BC (F1 x Cop) rats. Depending on their neoplastic response, carcinomatous response was 93% in WF, 5% in Cop, 16% in (WF x Cop) F1, 31% in BC (F1 x WF) and 0% in BC (Fl x Cop) rats, and sarcomatous response was 20% in WF, 85% in Cop, 58% in (WF x Cop) F1, 44% in BC (F1 x WF) and 73% in BC (F1 x Cop) rats. When dusting was performed on the interscapular fat tissue of 21 WF and 22 Cop females at 38 days of age, sarcomas were found in 67% of WF and 91% of Cop rats. Therefore, susceptibility and supressor gene(s) are suspected in mammary epithelial cells of the WF and Cop, respectively, for carcinogenesis, while co-dominant allele for sarcomagenesis exists in the Cop and WF mammary stromal cells.

Strain differences in neoplastic response to dmba-induced uterine vascular tumors in mice.

We examined strain differences in neoplastic response to DMBA-induced uterine vascular tumors in mice. Female BALB/c, C57BL, C3H, SWR/J, GRS/A and DDD/1-Mtv-2 mice were treated with a single intraperitoneal injection (ip) of 1 mg of 7,12-dimethylbenz(a)anthracene (DMBA) at 4 weeks of age, and observed until they were 32 weeks of age. The BALB/c, C57BL, C3H strains showed a high incidence of uterine vascular tumors (87%, 93%, and 90%, respectively), while in contrast, the SWR/J, GRS/A, and DDD/1-Mtv-2 strains showed a low incidence (17%, 11% and 15%, respectively); these differences were striking. The mice of American origin (BALB/c, C57BL and C3H) appeared to be susceptible to DMBA, while those of European origin (SWR/J, GRS/A and DDD/1-Mtv-2) were resistant to the induction of uterine vascular tumors.

Keratin phenotypes in Basal-cell carcinomas of the skin.

Immunohistochemical profiles of 23 cases of basal cell carcinoma (BCC) were examined and compared with adjacent normal skin using three keratin antibodies, 34betaB4, 312C8-1 and E3, which recognize keratin 1, 14 and 17, respectively. In normal skin, 34betaB4 was expressed in suprabasal cells of the epidermis, sebaceous duct cells and the outer root sheaths of hair follicles at the level of sebaceous duct insertion. 312C8-1 was seen in basal cells of the epidermis, the outer root sheaths of hair follicles, and the peripheral cells of sebaceous glands. E3 was detected in the outer root sheaths of hair follicles, and the peripheral cells of sebaceous glands, while it was absent in the epidermis. In BCC, 312C8-1 and E3 were homogeneously found in all 23 cases, and 34betaB4 was sporadically expressed in 3. Therefore. the results suggest that the keratin expression of BCC resembles that of the pilosebaceous apparatus.

Immunohistochemical evaluation of mam-3 and mam-6 antigens in squamous-cell carcinoma of the oral cavity.

MAM-3 and MAM-6 antigens of human milk fat globule membrane designated as human polymorphic epithelial mucin (PEM) were detected immunohistochemically in a total of 86 specimens from 39 cases of oral squamous cell carcinoma (SCC) and 6 cases of normal oral mucosa. The two antigens were identified by MoAbs 67D11 and 115G3 (MAM-3 antigen) and by MoAbs 115D8 and 115F5 (MAM-6 antigen) in paraffin sections. MoAb 115G3 staining was found in well keratinized foci; whereas, MoAb 115D8 and 115F5 staining was very low in keratinized tumor cells. The cytochemical distribution of the antigens was classified into 3 types; (i) cell membrane positive type, (ii) whole cell positive type and (iii) antigen negative type. Reduction and disappearance of the two antigens were correlated to the mode of tumor invasion and suggested that glycocalyx complex in cell membrane properties was changed for malignant transformation.

Esophageal carcinoma in house musk shrews, Suncus murinus (Insectivora), induced by N-methyl-N'-nitro-N-nitrosoguanidine.

Female 6-week-old shrews were given a solution of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at a concentration of 50 micrograms/ml or 100 micrograms/ml in the drinking water. All 11 shrews receiving 100 micrograms/ml MNNG died 8-13 days after the beginning of carcinogen administration and 6 of the 20 shrews receiving 50 micrograms/ml MNNG died after 10-54 days. When animals were between 43 and 54 weeks of age, multiple esophageal lesions were evoked in all 14 that had received 50 micrograms/ml MNNG for 30 weeks. All shrews developed a protruding, ulcerative, or superficial type of squamous-cell carcinoma of the esophagus, accompanied by papillomas. Local invasion was seen in squamous-cell carcinoma but no distant metastasis was noted. None of the 5 control shrews developed any esophageal abnormality. No gastric adenocarcinoma, intestinal sarcoma, or other tumors were induced with MNNG. It can be concluded that MNNG has a carcinogenic effect on shrew esophageal epithelium.

3-Ketosteroid-delta1-dehydrogenase of Rhodococcus rhodochrous: sequencing of the genomic DNA and hyperexpression, purification, and characterization of the recombinant enzyme.

The gene encoding 3-ketosteroid-Delta1-dehydrogenase from Rhodococcus rhodochrous was cloned and sequenced. The gene (ksdD) consists of 1,536 nucleotides and encodes an enzyme protein of 511 amino acid residues. The amino terminal methionine residue was deleted in the mature protein. The amino acids involved in the flavin binding site are conserved in the dehydrogenase sequence. The deduced amino acid sequence is highly homologous to that from Arthrobacter simplex but less so to that from Pseudomonas testosteroni. Upstream of the gene was located a heat shock protein gene, dnaJ, and downstream, a gene of a hypothetical protein. The enzyme gene was ligated with an expression vector to construct a plasmid pDEX-3 and introduced into Escherichia coli cells. The transformed cells hyperexpressed the 3-ketosteroid-Delta1-dehydrogenase as an active and soluble protein at more than 30 times the level of R. rhodochrous cells. Purification of the recombinant 3-ketosteroid-Delta1-dehydrogenase from the E. coli cells by a simplified procedure yielded about 13 mg of enzyme protein/liter of the bacterial culture. The purified recombinant dehydrogenase exhibited identical molecular and catalytic properties to the R. rhodochrous enzyme.

Essential tyrosine residues in 3-ketosteroid-delta(1)-dehydrogenase from Rhodococcus rhodochrous.

Tetranitromethane treatment of 3-ketosteroid-Delta(1)-dehydrogenase of Rhodococcus rhodochrous caused loss of the catalytic activity in a time- and concentration-dependent manner. Peptides (P-81) and (PN-83) were isolated from tryptic digests of the native and tetranitromethane-treated enzyme proteins, respectively. PN-83 was the nitrated form of P-81. The amino acid sequence was GGAPLIDYLESDDDLEFMVYPWPDYFGK (positions 97-124 of the dehydrogenase sequence). PN-83 showed a low yield of PTH-Tyr of position 116, i.e. less than 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine. This indicated that tetranitromethane modifies Y-116 under the experimental conditions used. Mutation of Y-104, Y-116, and Y-121 to smaller amino acid residues, Phe, Ser, or Ala, significantly changed the catalytic activity of the dehydrogenase. All of the mutants contained FAD and exhibited the same spectrophotometric properties as those of the wild type enzyme. The K(m) values for 4-androstene-3,17-dione of the Y-104, Y-116, and Y-121 mutants changed to large values. The most drastic change was observed for Y116A. The K(d) values for 1,4-androstadiene-3,17-dione of the Y116 mutants changed to 1.5-2.6-fold larger values than that of the recombinant enzyme. The Y-121 mutant enzymes exhibited catalytic activities like those of the recombinant enzyme, but the catalytic efficiencies of Y121F and Y121A drastically decreased to 0. 014-0.054% of that of the recombinant enzyme. The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104 act on binding of the substrate steroid.

Steroid monooxygenase of Rhodococcus rhodochrous: sequencing of the genomic DNA, and hyperexpression, purification, and characterization of the recombinant enzyme.

Steroid monooxygenase of Rhodococcus rhodochrous is a Baeyer-Villigerase catalyzing the insertion of an oxygen atom between the C(17)- and C(20)-carbons of progesterone to produce testosterone acetate. The 5.1-kbp-long BamHI DNA fragment containing the steroid monooxygenase gene, smo, was cloned from the chromosomal DNA and sequenced. The smo gene is 1,650 nucleotides long, starts with a TTG codon, and ends with a TGA codon. The deduced amino acid sequence indicates that the enzyme protein consist of 549 amino acid residues with a molecular mass of 60,133. Thus, the molecular mass of the holoenzyme is 60,919. The amino acid sequence is highly homologous (41.2% identity) to that of cyclohexanone monooxygenase of Acinetobacter sp. In the upstream of the smo gene, the genes of heat shock proteins, dnaK, grpE, and dnaJ, located on the complementary strand, and the DNA-inserts of pSMO and pD1, which contains the ksdD gene, were joined at the BamHI site of the dnaJ gene. The smo gene was modified at the initiation codon to ATG and ligated with an expression vector to construct a plasmid, pSMO-EX, and introduced into Escherichia coli cells. The transformed cells hyperexpressed the steroid monooxygenase as an active and soluble protein at more than 40 times the level in R. rhodochrous cells. Purification of the recombinant monooxygenase from the E. coli cells by simplified procedures yielded about 2.3 mg of enzyme protein/g wet cells. The purified recombinant steroid monooxygenase exhibited indistinguishable molecular and catalytic properties from those of the R. rhodochrous enzyme.

Coexisting primary early gastric plasmacytoma and sarcoidosis with hypercalcaemia.

We report on a 61-year-old woman with coexisting early stage primary gastric plasmacytoma and sarcoidosis with hypercalcaemia. Laboratory data on admission showed hypercalcaemia, with 12.8 mg/dl, parathyroid hormone-related peptide (PTHrP) 1.2 pmol/l, C-PTHrP 69.5 pmol/l, and 1,25-dihydroxyvitamin D3 46.7 pg/ml. Neoplastic plasma cells proliferated in the propria mucosa of the stomach, showed a monoclonal immunoglobulin of cytoplasmic IgA (lambda light chain) and were positive for leucocyte common antigen and epithelial membrane antigen on paraffin section prepared from a stomach biopsy specimen. Russel bodies were present, as were crystals. Abundant sarcoid granulomas were observed in many of the regional lymph nodes around the stomach and in the dermis of a skin nodule. The patient underwent subtotal gastrectomy with administration of antimyeloma chemotherapy. We suggest that the hypercalcaemia in this patient was due to PTHrP production by neoplastic plasma cells.

Accumulation of 125I-factor XI in atheroma of rabbit with hereditary hyperlipidemia (WHHL-rabbit).

We have studied the turnover and accumulation of rabbit factor XI (F.XI) in atherosclerotic lesion in Watanabe-hereditable hyperlipidemic rabbit (WHHL rabbit) to reveal the participation of blood coagulation in atherosclerotic lesion. Rabbit F.XI was iodinated and administered intravenously to WHHL rabbits and Japanese white rabbits. The turnover of 125I-rabbit F.XI was significantly faster in WHHL rabbits (T1/2 = 2.84 +/- 0.44 days) than in normal rabbits (T1/2 = 4.44 +/- 0.42 days). The thoracic aorta of WHHL rabbit was strongly labelled with 125I-rabbit F.XI, in sections obtained after 5 days by en-face autoradiography, whereas no radioactivity was detected in normal aorta. By an immunohistochemical study of WHHL rabbit aorta, we confirmed that many F.XI- and fibrin-related compounds existed in the atheroma, whereas albumin did not in these area. These results suggest that the activation of F.XI proceeds on the atherosclerotic lesions of WHHL rabbits.

Lectin-binding patterns in transplantable mouse mammary tumors and their metastases.

Lectin binding was assessed in a transplantable pregnancy-dependent mouse mammary tumor line (TPDMT-4), its autonomous sublines (T4-0196 and T4-01165) and their artificial metastases (lung colonies), using the avidin-biotin-peroxidase technique. Soybean agglutinin (SBA) and peanut agglitinin (PNA) bound to the luminal surfaces of TPDMT-4 tumor cells, while dolicos biflorus agglutinin (DBA) showed no binding. In T4-0196 and T4-01165 tumors as well as their lung metastases, SBA and PNA binding was mixed and both positive and negative cells were detected, indicating that these lectins were not associated with the metastatic phenotype. Although the T4-0196 and T4-01165 sublines had a mixture of DBA-positive and DBA-negative cells, all the metastatic T4-0196 subclones contained only DBA-positive cells and all the metastatic T4-01165 subclones had DBA-negative cells. Thus DBA-positive, and DBA-negative subclones had respectively metastasized to the lungs from these autonomous sublines, implying that the carbohydrate moieties detected by DBA were not associated with metastatic potential but that the lung metastases were clonal in origin.