Properties of unprimed poly(A)-poly(U) synthesis by Caulobacter crescentus RNA polymerase.

Some properties of unprimed poly(A)-poly(U) synthesis by DNA-dependent RNA polymerase from Caulobacter crescentus were examined. The reaction required ATP and UTP as substrates and manganese as a divalent cation. Rifampicin completely inhibited the reaction at a concentration of 1 micron/ml, and the enzyme catalyzed the polymer synthesis well regardless of the presence of GTP, CTP or both. The chain length of the poly(A)-poly(U) synthesized was about one hundred base pairs, as estimated from a sedimentation velocity and the molar ratio of [3H]AMP to [gamma-32P]ATP incorporated into the poly(A)-poly(U). The reaction was dependent on the square of the enzyme concentration and the enzyme dimers formed complexes with poly(A)-poly(U) during the reaction.

Induction of apoptosis with fusarenon-X in mouse thymocytes.

The effects of fusarenon-X (12,13-epoxytrichothecene; FX) on mouse thymus and T-cell subpopulations were studied. In mice that received three intraperitoneal injections of FX, the thymus showed severe atrophy, the thymic cortex almost completely disappeared, and the total number of thymocytes decreased to 2.2% of that of normal mice. CD4+ CD8+ thymocytes were almost completely depleted by this treatment while CD4+ CD8-, CD4- CD8+ and CD4- CD8- thymocytes were not reduced to such an extent, suggesting that selective damage in CD4+ CD8+ thymocytes was induced by FX. In spleen, CD4+ or CD8+ lymphocytes and CD4- CD8- non-T cells remained unchanged. Next, the mode of damage in thymocytes was investigated by a single injection with FX. The lymphocyte nuclei were fragmented and positive for TUNEL (TdT-mediated dUTP nick-end labeling) staining in the thymic cortex 20 h after FX injection. By electron microscopy, apoptotic lymphocytes with condensed nuclei and stroma cells ingesting many nuclear fragments were frequently observed in the thymic cortex. Internucleosomal DNA fragmentation was apparent in the thymocytes treated with FX both in vivo and in vitro. Thus, we demonstrated that the trichothecene mycotoxin FX is a new cause of apoptosis in CD4+ CD8+ thymocytes of mice besides the other factors that cause similar effects.

Immunological and ultrastructural observations on swine thymic lymphoma.

A case of swine thymic lymphoma is described. A young pig had a mediastinal mass and bone marrow involvement and the microscopic findings showed peripheral blood involvement. The tumour was composed of small to medium sized lymphoid cells which formed rosettes with sheep erythrocytes and were characterized by narrow cytoplasmic bands, nuclear convolution and clustered dense bodies. Such structural and immunological features resembled those of human thymic lymphomas derived from thymic T-cells.

Pathological and immunohistochemical studies of follicular lymphoma in two calves.

Pathological and immunohistochemical studies were performed on two cases of bovine follicular lymphoma with severe leukaemia and antibodies to bovine leukaemia virus. Histologically, numerous neoplastic follicular structures were observed in the lymph nodes and spleen of both cases and in the tonsils and Peyer's patches of one case. The neoplastic cells infiltrated diffusely into the liver, lungs and heart in both cases and into the tonsils, kidneys, abomasum and bone marrow in one case. Intracytoplasmic immunoglobulin was negative in the neoplastic cells. Follicular dendritic reticulum cells were decreased in number in the neoplastic follicles compared with those in normal lymphoid follicles. Follicular lymphoma is rare in cattle. Histological features of the present cases are distinguishable from the adult form of bovine lymphoma and the calf form of lymphoma.

A case of bovine sex cord tumour with annular tubules.

A case of sex cord tumour with annular tubules was found in the right ovary of a 17-month-old Holstein cow. The tumour was characterized by tubules encircling hyaline bodies, which were composed of concentric lamellae of basal lamina. Several irregular tubular structures and a small component of granulosa cell differentiation were seen. Immunohistochemically, the neoplastic cells stained positively for oestradiol or testosterone.

Immunohistochemical studies on ontogeny of bovine lymphoid tissues.

Developing lymphoid tissues of bovine fetuses ranging from 70 to 270 days of fetal age were examined by histological and immunohistochemical procedures. In the peripheral blood, surface membrane immunoglobulin bearing cells (B-lymphocytes) and sheep red blood cell rosette forming cells (T-lymphocytes) had already appeared by 70 days of fetal age. In the lymph nodes intracytoplasmic IgM positive cells appeared at 90 days of fetal age. The cells positive for IgG appeared at 150 days of fetal age and IgA positive cells appeared at 180 days of fetal age. The spleen contained intracytoplasmic immunoglobulin positive cells at almost the same time as those in the lymph nodes. In the ileocecal region, IgM positive cells and IgG positive cells were present at 180 days of fetal age and IgA positive cells were present at 210 days of fetal age. The tonsils contained IgM positive cells and IgG positive cells at 240 days of fetal age. In the thymus, terminal deoxynucleotidyl transferase positive cells appeared at 90 days of fetal age.

Lymphoplasmacytic lymphoma in a stallion.

Lymphoplasmacytic lymphoma found in a 6-year-old Anglo-Arabian stallion was investigated histologically, immunohistochemically and ultrastructurally. The animal showed a large mediastinal mass and generalized lymph node involvement. The neoplastic cells were in various differentiation stages of small lymphocyte, centrocyte, centroblast, immunoblast and plasma cell. Some neoplastic cells showed positive cytoplasmic reactivity for mu and lambda chains. There were well developed rough endoplasmic reticulum (RER) and Golgi complexes in plasmacytoid cells, and slightly developed RER or a few long strands of RER in medium-sized to large lymphoid cells. These findings suggest that this neoplasm is of B-cell origin.

Rapid induction of lymphoid leukosis and ascites by avian leukosis virus from a lymphoid leukosis cell line.

To examine whether a lymphoid leukosis (LL) cell line releases an LL-specific avian leukosis virus (ALV) or not, two viral materials, culture fluid and a concentrated viral material from an LL-cell line, were inoculated into a total of 74 day-old chicks of line 15I in 5 experiments. Spectrum of diseases induced, their incidence and incubation periods to onset were examined. Fifteen chicks were inoculated with the culture fluid and 9 (60%) developed ascites [59-119 days post inoculation (dpi); geometric mean (GM) of dpi, GM: 89.6)], but LL was not induced in any chicks inoculated. Fifty-nine chicks were inoculated with the concentrated viral material and LL was recognized in 13 (22.0%) (27-74 dpi; GM: 48.4), ascites with LL in 11 (18.6%) (34-75 dpi; GM: 41.3), ascites alone in 21 (35.6%) (32-83 dpi; GM: 48.2), erythroblastosis in 2 (3.4%) (70-102 dpi; GM: 84.5), and other diseases in 12 (20.3%) (43-102 dpi; GM: 61.8). LL lesions were frequently observed in the liver, spleen, kidneys, bursa of Fabricius (bursa), bone marrow and gonads. Mild lymphocytic foci in some visceral organs and perivascular cuffing in the central nervous system were observed mainly in several chicks diagnosed as having complication of ascites with LL or other diseases. In addition to these lesions, atrophy of bursa and thymuses was recognized in them. No antibodies against Marek's disease virus (MDV) and reticuloendotheliosis virus were detected in 36 sera taken from the chicks inoculated with the concentrated viral material. Serotype 2 MDV was isolated from the buffy coat of some inoculated chicks. These results suggest that the properties of ALV inoculated and immunosuppression caused by inoculation with high doses of ALV are involved in rapid induction of LL and expression of pathogenicity of serotype 2 MDV released from the LL cell line and included in the viral inoculum. This is the first report describing the rapid induction of LL and ascites in chicks.

Immunohistochemical observations on pneumonic lesions caused by Rhodococcus equi in foals.

An immunohistochemical analysis of Rhodococcus equi-induced pneumonia in 10 foals was performed by biotin-streptavidin system. The detection of R. equi was more sensitive in immuno-stain using anti-R. equi serum than in Gram's stain. This bacteria also reacted to anti-BCG serum. Lysozyme and alpha 1-antitrypsin were detectable in macrophages. A particularly intense staining was observed in association with intracellular bacteria. Though a degree of reaction for alpha 1-antichymotrypsin was very low in comparison with lysozyme and alpha 1-antitrypsin, it was also demonstrated in macrophages ingesting R. equi. These bacteria were almost intact under an electron microscope. Therefore, the surface components of R. equi may play important roles of protection from intracellular enzymes of macrophages. The cells containing intracytoplasmic IgM, IgG or IgA were a few in number and scattered predominantly around the pneumonic lesion. It is considered that the bactericidal activity by immunoglobulins may be weak in comparison with phagocytosis by macrophages.

[Alexia-agraphia of kanji (Japanese morphogram) after left posterior-inferior temporal lesion]. / Alexie-agraphie pour l'écriture kanji après lésion temporale postéro-inférieure gauche.

Several cases of selective alexia with agraphia of kanji have been reported in Japan in this decade. It is well known that the lesion in the posterior inferior temporal lobe of the dominant hemisphere is responsible for this cognitive syndrome. Neuropsychological data in our patient suggest that the postero-inferior region of the temporal lobe of the dominant hemisphere may be the visuo-verbal association area for the analysis of the complex visuo-verbal information. The symptoms caused by the same lesion in western patients might be subangular alexia (alexia without agraphia). Alexia with agraphia of kanji and subangular alexia would appear to be distinct entities, but a dual processing hypothesis of visuo-verbal information and the concept of the visuo-verbal association area might well explain both syndromes.

Effects of acupuncture on peripheral T lymphocyte subpopulation and amounts of cerebral catecholamines in mice.

The aim of this study was to investigate the effects of acupuncture on peripheral lymphocyte subpopulations and cerebral catecholamines. In order to examine the effects of acupuncture, two experiments were performed. Experiment 1: Eighteen female mice (strain; C57BL/6) at the age of 7 weeks were divided three groups, (a) sham operated (control; n=6), (b) ovariectomized (OVX; n=6), and (c) ovariectomized and stimulated by subcutaneous needles on acupuncture point, Shenshu (BL23) at the both sides of the back for 20 days (OVX+Acu; n=6). These animals were sacrificed at 20 days after needle insertion, and the splenic lymphoid cells were examined by two-color flow cytometry, using monoclonal antibodies (mAb) to the cell surface antigens, CD3, CD4, CD8a and NK1.1 (CD56). In the ovariectomized (OVX) group, the peripheral CD4/CD8 ratio was significantly increased and the ratio of natural killer (NK) cells (CD3-NK1.1+; CD3 negative, NK1.1 positive) to T lymphocytes was decreased compared to the sham control group. In the ovariectomized with needle insertion (OVX+Acu) group, the CD4/CD8 ratio was reduced, but the NK cells ratio was not changed compared to the OVX group. Experiment 2: To investigate the acute effects of subcutaneous needle insertion, male C57BL/6 mice (7 weeks old) were used (n=6, each group). The acupuncture points Shen-shu (BL23) on the backs of the male mice were also stimulated by subcutaneous needles for 3 and 7 days. As a result, the CD4/CD8 ratio was significantly decreased at day 3 and day 7, compared to the control group. On the other hand the NK cells ratio and activated T-cells were increased at day 7. The mitogenic activities in the splenic lymphocytes were also increased by acupuncture stimulation at day 3. Catecholamine contents in the hippocampus were measured by high performance liquid chromatography with the electro-chemical detector (ECD-HPLC) method. No significant change was observed in either dopamine contents or norepinephrine; however, dopamine metabolite, homovanillic acid (HVA) and DOPAC (3,4-dihydroxyphenylacetic acid) were increased at day 3. The study suggests that acupuncture has effects on peripheral lymphocyte subpopulations and may modulate mitogenic activity. In addition, acupuncture may stimulate dopamine turnover.

[Hemodynamic effects of STA-MCA anastomosis on patients with occlusion of the main cerebral artery].

PURPOSE: We studied cerebral circulation in patients with occlusion of the main cerebral artery and investigated the efficacy of STA-MCA anastomosis. PATIENTS AND METHODS: Thirty-six patients with occlusion of the main cerebral artery were studied. Twenty-three patients had occlusion of the internal carotid artery and 13 had occlusion of the middle cerebral artery. The mean age was 62 years. Cerebral blood flow (CBF) was measured in all patients and cerebrovascular reactivity (CVR) was examined in 11 patients by xenon enhanced CT. Intraoperatively, cortical arterial pressure and anastomotic flow were measured. RESULTS: There was no perioperative mortality or morbidity. There was no ipsilateral stroke recurrence during the follow-up period averaging 35.1 months. Patency of the anastomosis was verified in 91% of the patients by magnetic resonance angiography. Twenty-three (64%) patients showed decreased CBF before the operation and 57% of these patients showed improvement to the normal range after STA-MCA anastomosis. All of the eight patients with decreased CVR showed improvement after the operation. Anastomotic flow correlated significantly with the cortical arterial pressure. CONCLUSION: STA-MCA anastomosis could improve cerebral circulation of patients with low CBF or low CVR due to occlusion of the main cerebral arterial. It was concluded that STA-MCA anastomosis may contribute to the reduction of stroke recurrence, if perioperative complications are reduced.

Construction of catalase deficient Escherichia coli strains for the production of uricase.

To produce catalase-free uricase preparations, we constructed catalase-deficient strains from Escherichai coli MC1000 and MM294 and used them as recombinant host strains. The parent strains and catalase-deficient strains showed no differences in the growth characteristics by shaking culture in Erlenmeyer flasks. The catalase deficient strain derived from MC1000 transformed with the uricase expression plasmid pUT118 (strain SN0037) had growth characteristics and the uricase productivity comparable to those of the parent host strain MC1000 in fed-batch culture in a jar fermentor and no catalase activity was detected in cell-free extracts. However, the katG disrupted strains from MM294 carrying pUT118 had poor growth and their uricase productivities were low compared to those of the parent strain MM294. Using the strain SN0037, a catalase-free uricase preparation was obtained with fewer purification procedures and the final recovery of uricase activity was improved. The catalase-deficient E. coli host strain will be a suitable host for the production of the uricase, free of catalase activity, in high yield.

Pathological and microbiological studies on calf pneumonia occurring in mass rearing facilities.

Pathological and microbiological studies were conducted on lesions in the lungs of 194 calves from mass rearing facilities. Macroscopically, the lesions were classified into six forms: nonlesion, atelectasis, mild pneumonia, moderate pneumonia, advanced pneumonia, and advanced pneumonia accompanied with abscess. Histopathological examination revealed bronchopneumonia in most of the calves. Lesions more advanced than moderate pneumonia were complicated with desquamation, severe exudation, and necrosis. Bacteriologically, Pasteurella sp. was isolated often in combination with Staphylococcus sp. from about a half of the atelectatic cases. With the development of pneumonic lesions, Pasteurella sp. was isolated at a high frequency in combination with Haemophilus sp., Streptococcus sp., and Corynebacterium sp. Prominent necrosis was more often seen in cases with Pasteurella haemolytica isolated than in cases with only Pasteurella multocida isolated. Mycoplasma sp. and Ureaplasma sp. were isolated from distinctly pneumonic lesions. Advanced pneumonic lesions were observed in many calves over 30 days of age. The importance of environmental and managerial improvement was also emphasized, since calf pneumonia tended to break out in facilities under unsatisfactory conditions in the present work.

Functional interactions of an archaeal sliding clamp with mammalian clamp loader and DNA polymerase delta.

BACKGROUND: By the total genome sequencing of several archaeal organisms, it has been confirmed that many archaeal proteins related to genetic information systems, including DNA replication, transcription and translation, have similar sequences to those of eukaryotes. In eukaryotic DNA replication, proliferating cell nuclear antigen (PCNA) works in clamping DNA polymerases on the DNA template and accomplishes a processive DNA synthesis. Archaea encode PCNA homologues in their genomes and Pyrococcus furiosus PCNA (PfuPCNA) stimulates the DNA synthesizing activities of the DNA polymerases, Pol I and Pol II, in this organism. RESULTS: We have demonstrated that PfuPCNA interacts functionally with calf thymus DNA polymerase delta (Pol delta) and stimulates its activity. Moreover, human replication factor C (RFC) enhances the PfuPCNA-dependent DNA synthesis activity of Pol delta, indicating that human RFC works as the clamp loader for PfuPCNA. These results showed that the three-dimensional structures of archaral PCNA and RFC are actually similar enough to their eukaryotic counterparts to allow a molecular substitution between the two biological domains, albeit at a lower efficiency. CONCLUSIONS: We found that the archaeal molecule interacts functionally with the eukaryotic members in the DNA replication process. This finding supports the idea that studies on the DNA replication mechanism of archaeal organisms will provide many important clues for understanding of the intricate molecular recognition that is inherent to the DNA replication machinery in Eukarya.

Malignant lymphomas of follicular centre cell origin in 14 pigs.

Malignant lymphomas of follicular centre cell (FCC) origin in 14 pigs are described. Seven cases were classified as abdominal type according to the distribution of the neoplastic lesions and the residual cases were of the general type. Most cases were composed of mixed populations of large and medium-sized neoplastic cells and the growing pattern varied from partially follicular to diffuse. The neoplastic cells often showed necrosis, to which macrophages reacted. A part of the neoplastic cells had cytoplasmic immunoglobulin M (CIgM) or cytoplasmic immunoglobulin G (CIgG). The histological features of swine FCC lymphomas are discussed.

Scanning electron microscopy description of a new species of Demodex canis spp.

Between 1997 and 1999, the prevalence of Demodex canis mites was determined in 150 dogs. In two dogs, we found two different species of mites; Demodex canis and another, unidentified, Demodex mite. The unidentified Demodex mite species had several different morphological features. First, it had a short opisthosoma and an obtuse end. In addition, the fourth coxisternal plate was rectangular and there was a band-like segmental plate between the fourth coxisternal plate and opisthosoma. Although all of the morphology and the development of male mites could not be investigated in this study, the location of the opisthosoma and the genital pore clearly differed from Demodex canis, suggesting that this unidentified mite is a new species.

Malignant lymphomas of thymus origin in two sows.

Two cases of swine thymic lymphoma were investigated by electron microscopy and the immunoperoxidase method for terminal deoxynucleotidyl transferase (TdT), and furthermore sheep erythrocyte rosette assay was applied to one case. In case 1, immunoblastoid cells predominated and smaller cells were admixed with them. Many neoplastic cells were TdT-positive and formed rosettes with sheep erythrocytes at 4 degrees C, but about half of them formed rosettes at 37 degrees C. Ultrastructurally the neoplastic cells were characterized by highly irregular nuclei, poorly developed organelles and clustered dense bodies. The neoplasm in case 2 was composed of medium-sized to large cells and the latter predominated. A few of the neoplastic cells stained positively with anti-TdT serum. Ultrastructurally the neoplastic cells were characterized by nuclear convolution, narrow cytoplasmic bands and clustered dense bodies. The morphological features of thymic lymphomas were discussed in comparison with those of other swine lymphomas and lymphomas of other animals. Histological and immunological progression into T-immunoblastoid cells in case 1 was also discussed.

Effects of the amplification of the genes coding for the L-threonine biosynthetic enzymes on the L-threonine production from methanol by a gram-negative obligate methylotroph, Methylobacillus glycogenes.

We constructed recombinant plasmids carrying the genes coding for the L-threonine biosynthetic enzymes, the hom gene, the hom-thrC genes, and the thrB genes, of a gram-negative obligate methylotroph, Methylobacillus glycogenes, and examined the effects of them on the production of L-threonine from methanol. The hom gene, which encodes the homoserine dehydrogenase, and the hom-thrC genes, containing the gene coding for threonine synthase together with the hom gene, were cloned from a wild-type strain, and the thrB gene encoding the desensitized homoserine kinase was cloned from an L-threonine-producing mutant, ATR80. The recombinant plasmids were transferred into ATR80 and its L-isoleucine auxotroph, A513, by conjugation. Amplification of the genes coding for the L-threonine biosynthetic enzymes elevated the activities of the L-threonine biosynthetic enzymes of the transconjugants 10- to 30-fold over those of the strains containing only vectors. The L-threonine production from methanol in test-tube cultivation was increased about 30% and 40% by the amplification of the hom gene and the hom-thrC gene respectively, and it was slightly increased by that of the thrB gene. The effects of gene amplification were confirmed by the cultivation in 5-1 jar fermentors. The best producer, an A513 transconjugant containing the plasmid carrying the hom-thrC genes, produced 16.3 g/l L-threonine for 72 h.