Effects of gonadotropins on in vitro maturation and of electrical stimulation on parthenogenesis of canine oocytes.

The objective of this study was to determine the effects of gonadotropins on in vitro maturation (IVM) and electrical stimulation on the parthenogenesis of canine oocytes. In experiment I, cumulus oocyte complexes were collected from ovaries at a random phase of the oestrus cycle and cultured on maturation medium treated with hCG or eCG for 48 or 72 h. There were no significant differences in the effects on the metaphase II (MII) rate between the hCG and eCG treatment groups over 48 h (5.4% vs 5.5%). The MII rate in the co-treatment group of hCG and eCG for 48 h was higher than in each hormone treated group (15.5%, p < 0.05). In experiment 2, the parthenogenetic effect on oocyte development, at various electrical field strengths (1.0, 1.5, 2.0 kV/cm DC) for 60 or 80 mus with a single DC pulse after IVM on the co-treatment of hCG and eCG, was examined. The rate of pronuclear formation (37.1%) in electrical activation at 1.5 kV/60 mus without cytochalasin B (CB) was higher than that of oocytes activated in the other groups (p < 0.05). However, we did not observe the cleavage stages. Also, CB did not influence parthenogenesis of canine oocytes. The results showed that the pronucleus formation rate, indicative of the parthenogenesis start point, could be increased by electrical stimulation. Therefore, these results can provide important data for the parthenogenesis of canine oocytes and suggest the probability of parthenogenesis in canines.

Parthenogenetic induction of canine oocytes by electrical stimulation and Ca-EDTA.

In this study, we investigated parthenogenetic induction of canine oocytes by electrical stimulation following Ca-EDTA treatment. Oocyte maturation, parthenogenetic development, and cleavage rate in canine after various electrical stimulations (1.5, 1.8, 2.1 kV/cm) for 50 mus with single DC pulse following 1 mM Ca-EDTA treatment were investigated. In oocyte activated electrically at the voltage of 1.5 kV/cm after 1 mM Ca-EDTA treatment, the rate of pronucleus and two-cell was 4.1% and 2.7%, respectively. Although electrical stimulation could parthenogenetically induce immature oocyte to cleavage stage, degeneration rate in all experimental groups was more than 60%. This means that electrical stimulation after Ca-EDTA treatment could cause canine oocytes to be degenerated. However, two-cell in canine oocyte by parthenogenesis was for the first time induced. Therefore, we suggested that electrical stimulation for canine oocytes could induce parthenogenetically early embryonic cleavage. This result can be used as a basic data for parthenogenesis study in canine. Also, to perform more developed embryonic development, further study to parthenogenesis in canine need to be developed.

Prevention of encephalomyocarditis virus-induced diabetes by live recombinant Mycobacterium bovis bacillus Calmette-Guérin in susceptible mice.

The D variant of encephalomyocarditis (EMC-D) virus causes diabetes in susceptible mice by direct cytolysis of pancreatic beta-cells. cDNA covering the major outer capsid protein (VP1) of the EMC-D virus was cloned into Mycobacterium bovis bacillus Calmette-Guerin (BCG). None of the SJL/J mice immunized with live recombinant BCG-VP1 (rBCG-VP1) became diabetic when challenged with the highly diabetogenic EMC-D virus, but the control mice inoculated with normal BCG developed diabetes during the same challenge. VP1-specific antibodies (including neutralizing antibodies) were markedly increased over time and reached the maximum titer at week 10 after a single immunization. The plateau of the titer lasted longer than 4 weeks. Mice and guinea pigs immunized with live rBCG-VP1 showed strong delayed-type hypersensitivity to the VP1 of the EMC-D virus. The preventive immunity still worked effectively 10 months after the primary immunization. At that time, the VP1-specific antibody was almost undetectable in the bloodstream, but a large number of VP1-specific lymphocytes was found in the spleen of the immunized mice. Our results show that live rBCG-VP1 elicits effective humoral and long-lasting cellular immune responses against EMC-D virus infection that results in the prevention of virus-induced diabetes in susceptible mice.

Dietary hematein ameliorates fatty streak lesions in the rabbit by the possible mechanism of reducing VCAM-1 and MCP-1 expression.

Hematein is a compound isolated from Caesalpinia sappan that has been used in oriental medicine as both an analgesic and an anti-inflammatory agent. In this study, we examined the anti-atherogenic potential of hematein using cholesterol-fed New Zealand White (NZW) rabbits. NZW rabbits were divided into a hematein-supplemented (0.05% in diet) group (n=6), a probucol-supplemented (0.25% in diet) group (n=6), and a control group (n=6). After 8 weeks of treatments, the extent of the atherosclerotic lesions was significantly reduced in the hematein-supplemented group and the probucol-supplemented group without changing plasma lipoprotein levels. Hematein and probucol prevented the up-regulation of the vascular cell adhesion molecule-1 (VCAM-1) expression on the descending aorta induced by cholesterol diet. In culture, hematein also significantly inhibited the secretion of soluble VCAM-1 and of monocyte chemotactic protein-1 (MCP-1) respectively induced by tumor necrotic factor alpha (TNF-alpha) and mildly oxidized low density lipoprotein in human umbilical vein endothelial cell (HUVEC) culture. Also, hematein inhibited monocyte adhesion to endothelial cell and the activation of NF-kappaB in HUVECs stimulated with TNF-alpha. The results of the present study suggest that the anti-atherogenic effect of hematein is not related to control of the plasma lipid profile but probably related to the inhibition of VCAM-1 and MCP-1 expression resulting in an amelioration of lesion development in the rabbit.

Sequence and transcription map analysis of early region-1 of porcine adenovirus type-3.

The nucleotide sequence of a region of the genome of porcine adenovirus-3 (PAV-3) between map units 1 and 12.2 was determined. The sequenced region included four major open reading frames, and several transcription control elements. Homology studies, using the deduced amino acid sequences of the open reading frames, revealed genes coding for the E1A, E1B 202R, E1B 474R and pIX proteins. The region was characterized by Northern blot analysis and sequencing of cDNA clones. In PAV-3, the E1A region is located between 1.5 and 3.8 map units. Alternate splice donor sites are used to produce four different types of transcripts from the primary transcript of the E1A region. The E1A proteins of PAV-3 contain a consensus zinc finger motif, which was shown to be the principal transactivation region of human adenovirus-5 (HAV-5) E1A proteins. The PAV-3 E1A proteins also contain a retinoblastoma susceptibility protein (pRb) binding motif, which in HAVs interacts with cellular Rb protein to overcome the pRb mediated transcription repression. The E1B region in PAV-3 maps between 4.0 and 12.2 map units, and shares a polyadenylation signal and polyadenylation sites with the gene coding for pIX. A single major and a number of minor mRNA species are produced from the E1B region. The open reading frame (ORF) analysis of cDNA representing major mRNA produced from the E1B region showed two overlapping ORFs corresponding to 19K and 55K ORFs of HAV-2. In PAV-3, the gene coding for pIX is located between 9.9 and 12.2 map units and codes for a protein of 199 amino acids.

Isolation and phylogeny of new endogenous retroviral sequences belonging to the HERV-F family.

A new human endogenous retroviral family (HERV-F) has been identified from human chromosome 7q31.1-q31.3 that was identical to the XA34 cDNA clone isolated from a human glioma cDNA library with an ERV-9 env probe. We investigated pol gene sequences of the HERV-F family from a human monochromosomal DNA panel and analyzed these with HERV-F. The pol gene sequences of the HERV-F family were detected on chromosomes 3, 6, 7, 10, 11, 14, 19, 20, X, and Y as examined by PCR. Thirty-six pol gene sequences identified from the human chromosomes have a high degree of sequence similarity (80-99%) with that of the HERV-F. Phylogenetic analysis of pol gene sequences distinctively showed four groups, indicating that the HERV-F family could be amplified at least four times after the original integration into the human genome or represent integration events separately during hominid evolution. One clone (HFY-3) on chromosome Y shared 100% sequence identity with a clone (HF19-2) on chromosome 19, and a clone (HF20-6) on chromosome 20 suggests either a recent retrotransposition or a chromosomal translocation. The history of endogenous retroviral sequences may contribute to an understanding of evolutionary change in human genomes.

SINE-R.C2 (a Homo sapiens specific retroposon) is homologous to CDNA from postmortem brain in schizophrenia and to two loci in the Xq21.3/Yp block linked to handedness and psychosis.

We investigated the retroviral/retroposon hypothesis of schizophrenia by generating sequences with PCR primers based on a retroviral sequence recovered by Yee et al. [1998: Schizophr Res 29:92] from a cDNA library from postmortem brain tissue from an individual with psychosis in a genomic region (Xq21.3) that has been tentatively linked to schizophrenia and schizoaffective disorder by Laval et al. [1998: Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:420-427]. Within the block of homology with Yp that was generated by a transposition between the chimpanzee and Homo sapiens we find two sequences, HS307 and HS408, with a high degree of homology to but not identity with the schizophrenic brain cDNA. The closest match of these three sequences is to a family of retroposons, that has evolved from the HERV-K family of endogenous retroviruses, some members of which (e.g., SINE-R.C2) appear to be specific to the human genome. This element has been reported as a cause of Fukuyama-type muscular dystrophy [Kobayashi et al., 1998: Nature 394:388-392]. Such retroposons, as agents of change in the human genome, provide a strategy for investigating pathogenesis. On account of their genomic location in a region that has been subject to change in the course of hominid evolution, and that may have a relationship to psychosis and/or cerebral asymmetry, we conclude that these particular insertions deserve further investigation.

Lymphoid follicles in bone marrow aspirates.

Lymphoid follicles were observed in 260 of 1,450 consecutive bone marrow aspirates (17.9%). As expected, the incidence of lymphoid follicles was less than those reported from autopsies (26.1-62.3%), but was twice as great as those in previous reports based upon study of aspirations (3.3-9.1%). The number of lymphoid follicles is also related to age and sex of the patient: they are rare in childhood and common after the fourth decade of life, particularly in women. Lymphoid follicles are found in higher incidence in bone marrow aspirates with plasma-cytosis and/or lipid granulomas than in those without these reactive changes. This relationship has not been described previously and suggests that the frequent occurrence of lymphoid follicles in the older age group may be a minifestation of a response to chronic immunologic stimulation.

Advances of the past decade in automated hematology.

During the decade of the 1980s, a wealth of information accumulated concerning automation in hematology. Recent technological advances led the way to the development of blood cell analyzers capable of performing a ten-parameter (or greater) complete blood count and five-parameter (or greater) differential leukocyte count on a small amount of whole blood and in an accurate, efficient, and economical way. The authors summarize the available information concerning the data generated by these analyzers, the mechanisms involved in the data generation, and the clinical applications and usefulness or limitations of the so-called new complete blood count parameters and of the automated differential as it compares with the manual differential.

Blood recalcification time. A simple and reliable test to monitor heparin therapy.

The applicability of blood recalcification time as a laboratory test to assess and thereby regulate heparin anticoagulation was evaluated. Normal values for blood recalcification time ranged from 65 to 135 seconds, with a mean and standard deviation of 99 and 12 seconds, respectively. Duplicate determinations showed a mean variation of 5%. Blood recalcification times increased as a linear function of increasing heparin concentrations in vitro. Comparison with whole-blood clotting times revealed reasonably good correlation between the two tests. For whole-blood clotting times of 20 to 45 minutes the corresponding blood recalcification times were 142 to 212 seconds. The therapeutic range is often considered to be twice the normal range (130-270 seconds). In-vivo study revealed the peak blood recalcification time in the first sample collected 30 minutes after heparin injection, and a progressive decline thereafter to the pre-injection level during the subsequent 4 hours. The blood recalcification time is a simple, precise and clinically useful test to monitor heparin therapy.

Reactive plasmacytic lesions of the bone marrow.

A consecutive series of 1,000 bone marrow aspirates was analyzed for percentage of plasma cells, incidence of plasmacytic satellitosis, associated clinical disease states, lymphoid follicles, lipid granulomas, hemosiderin content, and various combinations thereof. Plasmacytosis was a common finding, and tended to parallel the presence of lymphoid follicles, lipid granulomas and plasmacytic satellitosis. The latter is emphasized as a normal phenomenon, may reflect morphologically a physiologic response of the B cell system to antigenic stimulation, and is conspicuously absent in plasmacytic neoplasia. Various secretory forms of plasma cells are illustrated.

Epithelioid granulomas of the bone marrow in non-Hodgkin's lymphoproliferative malignancies.

Ten cases of epithelioid granulomas in the bone marrow of patients with various non-Hodgkin's lymphoproliferative malignancies have been encountered. These included six with non-Hodgkin's lymphoma (three histiocytic and three poorly differentiated lymphocytic types), three with multiple myeloma, and one with acute lymphoblastic leukemia. The bone marrow was not involved by the primary disease in two of the six patients with lymphoma, whereas three with lymphoma showed both granulomatous and lymphomatous lesions in the same marrow specimens, and in one, these lesions were seen in the marrow at different times. The three myeloma patients showed evidence of both myeloma and granulomas in their marrow. In the case of acute lymphoblastic leukemia, the bone marrow showed only granulomas, the leukemic process being in complete remission. Although small numbers of similar cases have been reported before, the authors were unable to find a previous report of acute lymphoblastic leukemia associated with bone marrow granulomas. Although the pathogenesis and the clinical significance of the granulomatous lesion of the bone marrow in non-Hodgkin's lymphoproliferative malignancies are unknown, this lesion should be differentiated from infectious or lipid granulomas as well as from involvement by the primary disease.

Phylogenetic analysis of retroposon family as exemplified on human chromosome 13: further evidence for recent proliferation.

The retroposon SINE-R.C2 was first identified as a human-specific insertion in the complement C2 gene. In our previous study, SINE-R type retroposons, derived from the endogenous retrovirus HERV-K family, have been found to be hominoid specific. In this report on human chromosome 13, we identified eighteen new SINE-R retroposons resembling those we have previously reported on the sex chromosomes and on chromosomes 7 and 17. Phylogenetic analysis using the neighbor-joining method revealed that four SINE-R retroposons (13-16, 21, 23, 25) on chromosome 13 were closely related to the human-specific retroposon SINE-R.C2, with a high degree of sequence homology (95-97%). Such elements differ from the HERV-K10. LTR sequence from which they are derived in being deleted for the promoter region. Therefore while the evidence adds to the case that some classes of SINE-R element have continued to proliferate in hominid and hominoid evolution and may, as in the case of Fukuyama type muscular dystrophy, be a cause of insertional mutagenesis, they are less likely than the HERV-K10 LTR to have a positive effect on host gene activity.

Development of new adherent mutant from human myeloma-derived cell line: in vitro model of anaplastic transformation of myeloma.

Anaplastic myeloma is a rare but distinct, biologically aggressive variant of myeloma which usually results from dedifferentiation or anaplastic transformation of the myeloma cells. The molecular mechanisms that determine the biologic behavior of anaplastic myeloma and effective treatment modalities have not been well known due to lack of in vitro models. In the present study, we have developed an anaplastically transformed mutant from a human myeloma-derived cell line. In the process of long-term culture of the myeloma-derived IM-9 cell line in low serum and nutrient conditions, an adherent mutant line was developed and named IM-9/AD. This mutant cell line displayed several characteristics resembling anaplastic myeloma such as: 1, large cells with large vesicular nucleus and prominent nucleolus, multinuclearity and high mitotic figures; 2, loss of leukocyte-associated antigens; and 3, higher tumorigenecity in scid mice than its parental cell line. This newly developed mutant cell line may serve as a readily available in vitro model to investigate the biology of anaplastic myeloma.

Blood smear examination.

Despite recent advances in the automation of clinical hematology laboratories, a careful microscopic examination of an appropriately prepared and stained blood smear continues to maintain its status as the most informative and useful diagnostic procedure, and offers a simple, reliable means of verifying results generated by automated analyzers. A systematic approach to a comprehensive evaluation of blood cells and related findings is described.

The automated CBC. A current perspective.

CBC traditionally stands for complete blood count. It represents a profile of tests rather than a single test, and over the years it has been given several names, including hemogram, Coulter profile, blood cell profile, and hematology profile. The number and type of tests included in the profile has also changed over time and among laboratories, depending primarily upon capabilities of the automated analyzers used to perform the profile test.

Immunohistochemical evaluation of hematologic malignancies.

This article summarizes the most recent developments and current practice of immunohistochemistry in the diagnosis of hematologic malignancy. Increased availability of monoclonal antibodies applicable in formaldehyde-fixed and paraffin-embedded tissue is discussed as are immunohistochemical definitions for many small cell lymphoma entities. Evaluation is made of the biologic potential of lymphomas and leukemias by the use of antibodies to proliferation antigens, such as Ki-67 and products of tumor suppressor genes (p53).

Structure and function of the bone marrow and hematopoiesis.

Bone marrow, a well-organized tissue located within the bone cavities, is richly innervated and highly vascularized but devoid of lymphatics. Structurally, it consists of two major cellular elements, the stromal cells (reticular cells--fibroblasts, endothelial cells, adipocytes, and so on) and the parenchymal cells (hematopoietic cells). Functionally, it serves as the primary site for hematopoiesis and as a major reticuloendothelial organ involved in immune responses (cellular and humoral) and removal of senescent and abnormal cells and particulate material. An uncommitted pluripotential hematopoietic stem cell, itself a product of the differentiation of mesenchymal cells of the yolk sac and capable of self-replication, undergoes proliferation and differentiation in an orderly manner, generating immature committed progenitors with uni-, bi-, or trilineage specificity. These committed progenitors also multiply and differentiate in a sequential fashion, ultimately producing mature cells that are released into the circulation. Under steady state conditions, the cell death/loss is balanced by cell production by virtue of regulatory mechanisms that apparently involve (1) cell-cell interaction between marrow cells and (2) production of humoral growth and/or inhibitory factors by stromal and parenchymal cells individually or in concert. Some of these regulators of hematopoiesis have been isolated, purified, molecularly cloned, and characterized. The availability of recombinant growth factors has stimulated clinical trials of these factors as therapeutic agents.

Bone marrow examination: techniques and interpretation.

Proper evaluation of the bone marrow requires adequate sampling, appropriate specimen processing, sufficient clinical history, and review of representative blood smears and pertinent laboratory data. Aspiration and needle biopsy should be performed at the same time routinely. Aspirate smears, touch preparations from the biopsy, and sections from the aspirate and biopsy should be processed appropriately and examined in a systematic fashion to perform a comprehensive evaluation considered essential to arrive at or rule out a diagnosis, or as an adjunct in the management of patients, particularly those undergoing chemotherapy and/or radiotherapy.

Fundamentals of bone marrow examination.

Bone marrow evaluation is an important and effective way of diagnosing and evaluating primary hematologic and metastatic neoplasms as well as nonhematologic disorders. Many variations exist for obtaining marrow samples (sites, instruments, techniques); however, the method outlined in this article has proven reliable. Complete evaluation of bone marrow samples should include a brief patient history, pertinent laboratory data, peripheral blood films, bone marrow aspirate smears and sections, and biopsy imprints and sections. Routine examination of the bone marrow as described previously is usually adequate for interpretation. However, application of additional studies using cytochemical, immunocytochemical, immunohistochemical, cytogenetic, and molecular techniques may prove to be of critical importance in the diagnosis of hematologic malignancies.