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PubMed1998

Mitogen-activated protein kinase translocates into the germinal vesicle and induces germinal vesicle breakdown in porcine oocytes.

The role of mitogen-activated protein kinase (MAPK) in the meiotic resumption of porcine oocytes was examined. First, using indirect immunofluorescence staining with a specific antibody against rat MAPK, we monitored the dynamics of the subcellular distribution of MAPK during meiosis initiation. We found that the inactive MAPK was already present in immature oocytes arrested at the G2 stage and that this inactive kinase was localized exclusively in the cytoplasm. At the G2/M transition stage, part of the MAPK moved into the germinal vesicle (GV) before germinal vesicle breakdown (GVBD). In addition, immunoblot analysis showed that the nuclear MAPK existed in an active form. To determine whether this active MAPK could induce GVBD, we microinjected active MAPK into immature porcine oocytes. The active MAPK injected into the cytoplasm was quickly inactivated and could not accelerate GVBD. In contrast, MAPK injection into the GV markedly accelerated GVBD. These results show that in porcine oocytes, 1) inactive MAPK localizes in the cytosol of immature GV oocytes, 2) part of the activated MAPK translocates into the GV just before GVBD, and 3) exogenous MAPK maintains its activity level in the GV and induces GVBD, indicating that MAPK mediates the maturation-inducing signal from the cytoplasm into the nucleus and induces meiosis reinitiation.

Animals↗

PubMed1967

Phytochrome and Seed Germination. III. Action of Prolonged Far Red Irradiation on the Germination of Tomato and Cucumber Seeds.

Prolonged irradiation with continuous or intermittent far red prevents the germination of tomato and cucumber seeds. The inhibitory efficiency of intermittent far red decreases with the lengthening of the interval between successive irradiations, and with the increase of temperature. If each far red irradiation is followed by red, germination is restored. Intermittent far red is less inhibitory than intermittent red-far red when red is given immediately before each far red. This effect is more evident when the interval between successive irradiation becomes longer.

Journal Article↗

PubMed1968

Phytochrome and Seed Germination. IV. Action of Light Sources With Different Spectral Energy Distribution on the Germination of Tomato Seeds.

Germination of tomato seeds exposed to a single, saturating irradiation from light sources of different spectral energy distribution seems to be dependent upon the photostationary P(FR)/P(R) ratio established by the irradiation. Germination of tomato seeds exposed to prolonged irradiations from the same light sources does not seem to be controlled solely by the P(FR)/P(R) ratio induced and maintained by the irradiation.

Journal Article↗

PubMed1978

Developmental biochemistry of cotton seed embryogenesis and germination: x. Nitrogen flow from arginine to asparagine in germination.

The enzymic basis for the flow of nitrogen from arginine to asparagine during the first 3 days of germination has been measured in extracts from cotton (Gossypium hirsutum) cotyledons. Evidence that asparagine synthetase regulates asparagine accumulation in germination (for transport to the axis) is presented. Further, evidence that the bulk of the nitrogen passed from one generation to the next in dicots is through an asparagine cycle involving the following sequence asparagine --> arginine --> storage protein --> arginine --> asparagine is discussed.

Journal Article↗

PubMed1969

Protein synthesis during fungal spore germination. IV. Transfer ribonucleic acid from germinated and ungerminated spores.

Transfer ribonucleic acid (tRNA) fractions isolated from germinated and ungerminated spores of Botryodiplodia theobromae and Rhizopus stolonifer had acceptor activity for all 20 amino acids commonly found in protein, when tested with an enzyme fraction from germinated spores. Accordingly, it is unlikely that the absence of tRNA for a particular amino acid limits protein synthesis in fungal spores.

Amino Acids↗

PubMed1975

Lipid metabolism during bacterial growth, sporulation, and germination: differential synthesis of individual branched- and normal-chain fatty acids during spore germination and outgrowth of Bacillus thuringiensis.

The biosynthesis of individual branched- and normal-chain fatty acids during Bacillus thuringiensis spore germination and outgrowth was studied by comparing pulsed and continuous labeling of these fatty acids with [U-14C]acetate. The relative specific activity of each fatty acid varies with time as the cell progresses through outgrowth. However, fatty acid synthesis does occur in two distinct phases. Upon germination, acetate is incorporated only into the iso-isomers i-C13, i-C14, and i-C16; no normal or anteiso synthesis occurs. Subsequent to T30, the full complement of branched- and normal-chain homologues is formed and there is a dramatic enhancement in the overall rate of fatty acid synthesis. Significantly, this rate increase coincides with a marked shift from the synthesis of short-chain to long-chain fatty acids. These findings illustrate a dichotomy in synthesis that may result from initial fatty acid formation by preexisting spore fatty acid biosynthetic enzymes in the absence of de novo protein synthesis. Elucidation of the timing and kinetics of individual fatty acid formation provides a biochemical profile of activities directly related to membrane differentiation and cellular development.

Acetates↗

PubMed1991

Subpopulations of B cells in germinal centers. III. HJ6, a monoclonal antibody, binds globoside and a subpopulation of germinal center B cells.

To identify surface Ag uniquely expressed on human germinal center B cells, we produced a mouse mAb, HJ6. When tonsillar lymphocytes were examined, HJ6 did not label T cells and labeled only about half of PNA+ B cells that were HK23-. HJ6 did not label mononuclear cells from peripheral blood, splenocytes, and any of 29 cell lines including 23 B cell lines. This binding pattern of HJ6 was very similar to that of a mAb named 5B5. It was shown previously that 5B5 bound a glycolipid named CTH (CD77) and its Ag was expressed on HK23- PNA+ tonsillar lymphocytes and Burkitt's lymphoma cell lines. Despite the similarity, HJ6 differed from 5B5: HJ6 did not stain Burkitt's lymphoma cell lines and stained PNA+ tonsillar lymphocytes in the presence of a large concentration of galactose. When its binding to isolated glycolipids was studied, HJ6 was found to bind globoside and Forssman Ag and not to other glycolipids including CTH. When its binding to neutral glycolipids extracted from tonsillar lymphocytes was studied, HJ6 bound only globoside; Forssman Ag was not detected in tonsillar lymphocytes. Taken together, we conclude that globoside is a B cell Ag expressed on a subpopulation of germinal center B cells.

Antibodies, Monoclonal↗

PubMed1982

Changes in germinative cell frequencies in the germinative region of Hymenolepis diminuta during development.

Changes in the frequency of germinative cells were measured daily during prepatent development in five-worm infections of Hymenolepis diminuta as well as at regular intervals during patency in five- and 20-worm infections. Frequency changes of this cell were found to correlate closely to the normal growth pattern of this cestode during prepatent development. Significant changes were also seen to occur during patency. However, the pattern of these changes appeared to be modified as the number of worms per host increased. By 90 days postinfection, cestodes recovered from 20-worm infections could be divided into two populations of distinctly different sizes. Significant differences in germinative cell frequency also occurred between these two populations.

Animals↗

PubMed1995

The Emu-bcl-2 transgene enhances antigen-induced germinal center formation in both BALB/c and SJL mice but causes age-dependent germinal center hyperplasia only in the lymphoma-prone SJL strain.

Emu-bcl-2 transgenic and littermate control BALB/c and SJL mice were immunized in the front footpads with trinitrophenylated Brucella abortus and the germinal center (GC) response in draining brachial lymph nodes was studied by staining with peanut agglutinin peroxidase and methyl green. Although the GCs induced were not larger in transgenic than in control young mice, there was a significant increase in the percentage of B cell follicles exhibiting GCs 7 to 8 days after primary and secondary antigen injections in the transgenic mice of both strains. In addition, glucocorticosteroid injected on day 7 after the primary injection caused a marked decrease in GCs in littermate controls but had no effect in the bcl-2 transgenic SJL mice. Antibody production to B. abortus was only slightly higher in transgenic than in control mice, but anti-TNP immunoglobulin M and G titers were significantly enhanced in the transgenic mice. The bcl-2 transgenic SJL mice, older than 6 months, showed the spontaneous appearance of large numbers of peanut agglutinin-binding GCs that greatly varied in size and were located without regard for the normal lymph node structure or follicle localization. This GC hyperplasia was seen in a large percent of the older transgenic SJL mice and never in similarly aged normal SJL or BALB/c mice with and without the bcl-2 transgene. Frank lymphomatous transformation of peanut agglutinin-binding germinal center-like areas was seen in lymph nodes and Peyer's patches of some of the older bcl-2 transgenic SJL mice. These results suggest that the tendency of SJL mice to develop GC-derived lymphomas synergizes with the presence of the bcl-2 transgene to cause the development of GC hyperplasia.

Aging↗

PubMed2003

Immunobiology of chicken germinal center: II. Accumulation of apoptotic cells within the germinal center.

The germinal center (GC) develops after antigen stimulation and is thought to occur at the site of various immune responses. We observed apoptotic cells within the GC using in situ end labeling (TUNEL), small amount DNA ladder assay, and RT-PCR analysis of Bcl-2 mRNA expression. Apoptosis was detected within GCs at all phases of the GC reaction by both TUNEL and DNA ladder assays. The number of TUNEL(+) nuclei within the GC did not increase over the course of the GC reaction. However, the density of DNA in the ladder assay was higher in later-phase GCs. Bcl-2 mRNA expression was detected within GCs during the early phases of the GC reaction. These results indicate that accumulation of apoptotic cells and rescue from apoptosis occur within chicken GCs. In the present paper, the reasons for the accumulation of apoptotic cells will be discussed.

Animals↗

PubMed2003

Immunobiology of chicken germinal center: I. Changes in surface Ig class expression in the chicken splenic germinal center after antigenic stimulation.

The germinal center (GC) develops after antigenic stimulation and is thought to occur at the site of various immune responses. We separated a single GC from chicken spleen after antigenic stimulation. Flow cytometric analysis of the cells derived from a single GC and RT-PCR analysis of Ig mRNA expression in GC was performed. Direct evidence indicates that: (1) there was a considerable difference in the cell population of each GC, (2) the ratio of CD3(+) cells in a GC remains constant at 10-20%, (3) the highest proportion of sIgY(+) cells in a GC occurs 1 week after the time of highest proportion of sIgM(+) cells, and (4) RT-PCR analysis was used to detect IgY mRNA expression in a GC. The continuous existence of CD3(+) cells, the alterations in sIgM(+) and sIgY(+) cell ratios, and the expression of IgY mRNA strongly suggest that Ig class switching occurs in the GC during an immune response.

Animals↗

PubMed2003

Epstein-Barr virus-infected B cells expanding in germinal centers of infectious mononucleosis patients do not participate in the germinal center reaction.

To assess the impact of the germinal center (GC) reaction on viral spread in Epstein-Barr virus (EBV) infection, we isolated EBV(+) GC B cells from the tonsils of two infectious mononucleosis patients, sequenced their rearranged V genes, and determined expression of the EBV latency genes EBV nuclear antigen 2 and latent membrane protein 1. Most EBV(+) GC B cells belonged to clones of cells harboring somatically mutated V gene rearrangements. Ongoing somatic hypermutation, the hallmark of the GC reaction, was seen only in uninfected GC B cell clones, not in EBV(+) B cell clones. Thus, in infectious mononucleosis, GC and/or memory B cells are directly infected by EBV and expand without somatic hypermutation, whereas the GC passage of EBV-infected naive B cells does not contribute detectably to the generation of infected memory B cells, the main reservoir of EBV during persistence. Most, if not all, EBV-infected cells in GCs exhibited an unusual EBV gene expression pattern in that they were positive for EBV nuclear antigen 2 but negative for latent membrane protein 1. Although the three main types of EBV-associated B cell lymphomas (Burkitt's, Hodgkin's, and posttransplant lymphomas) presumably are derived from GC B cells, EBV(+) GC B cells resembling these EBV(+) GC B cell lymphomas in terms of EBV gene expression and somatic hypermutation pattern could not be identified.

Adolescent↗

PubMed2005

Expression and prognostic implications of cell cycle regulatory molecules, p16, p21, p27, p14 and p53 in germinal centre and non-germinal centre B-like diffuse large B-cell lymphomas.

AIMS: To evaluate the different expression patterns and the prognostic significance of cell cycle regulatory molecules in diffuse large B-cell lymphomas (DLBCLs) of germinal centre (GC) and non-GC phenotypes. METHODS AND RESULTS: Tissue microarray slides composed of 126 extranodal and 88 nodal DLBCLs were immunostained for p16, p21, p27, p14 and p53. DLBCLs were classified into GC and non-GC phenotype according to the immunohistochemical expression of bcl-6, CD10, and MUM1. Aberrant expression of p53 was more frequent in the GC phenotype in nodal cases (P = 0.038), and the loss of p16, p21 and p14 expression was significantly more common in the non-GC phenotype (P = 0.004, P = 0.001, P < 0.001). Concurrent disruptions of the p16-Rb and p14-p53 pathways as represented by the immunoprofile of p16/p14/p53 (-/-/+) were associated with a poor prognosis in the GC phenotype [mean survival 31 months in the p16/p14/p53 (-/-/+) group versus 62 months in the other groups, P =0.0485]. CONCLUSIONS: The expression and prognostic implications of cell cycle regulatory molecules differ between GC and non-GC phenotypes in DLBCLs. The immunoprofile of p16/p14/p53 (-/-/+) within the GC phenotype of DLBCLs can be defined as a poor prognostic subgroup.

Adolescent↗

PubMed2001

Cutting edge: cellular Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein protects germinal center B cells from apoptosis during germinal center reactions.

During germinal center (GC) reactions, follicular dendritic cells are believed to select memory B lymphocytes by switching off apoptosis in the successfully binding B cells. The cellular signals involved in this process are largely unknown. Here, we show that GC B lymphocytes have a long isoform of the cellular homologue of the viral Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein (cFLIP(L)), which is capable of inhibiting death receptor-induced caspase activation. In isolated GC B cells, cFLIP(L) decays rapidly even without Fas ligation, and this results in activation of caspase activity and apoptosis. Contact with follicular dendritic cells prevents cFLIP(L) degradation and blocks all signs of apoptosis, even in the presence of anti-Fas ABS: cFLIP(L) expression is sustained by CD40 ligation as well, suggesting that at least at some stage of the GC reaction activated T cells may help selected B cells to leave the follicular dendritic cell network without becoming apoptotic.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Cellular and molecular factors that regulate the differentiation and apoptosis of germinal center B cells. Anti-Ig down-regulates Fas expression of CD40 ligand-stimulated germinal center B cells and inhibits Fas-mediated apoptosis.

To investigate the molecular and cellular mechanisms underlying the selection, differentiation, and apoptosis of germinal center (GC) B cells, we have established a culture system containing a follicular dendritic cell (FDC) line, HK. The mAb, 3C8, which is specific to HK cells and recognizes dendritic network in the GC, was developed and provided additional evidence that HK cells are related to FDC by sharing a unique surface Ag. The roles for CD40 ligand (CD40L) and T cell-derived cytokines in the differentiation of GC B cells were investigated in our culture system. We show that there are two distinct stages of GC B cell differentiation. In the early stage, GC B cells undergo spontaneous apoptosis unless they are stimulated by CD40L. In the secondary stage, IL-10 directs GC B cell differentiation toward the generation of plasma cells, while the absence of IL-10 stimulation leads to the generation of memory B cells. The major function of CD40L was found in the enhancement of cell recovery and the augmentation of memory B cell generation. Although GC B cells are Fas+, GC B cells are at first resistant to, but then become sensitive to, anti-Fas killing after 24 h in culture with CD40L, which coincides with the gradual increase in Fas expression on GC B cells. Furthermore, anti-Ig down-regulated Fas expression on CD40L-stimulated GC B cells, suggesting that Ag receptor engagement down-regulates Fas expression and prevents Fas-mediated apoptosis of GC B cells. Our data imply that GC T cells have an important role in the differentiation and apoptosis of GC B cells. GC T cells expressing both CD40L and Fas ligand have a dual function on GC B cells, helper or killer, depending on the status of target B cells. In the early stage, GC T cells stimulate the extensive proliferation of GC B cells, ensuring a large repertoire of B cells for selection. In the later stage, GC T cells kill B cells via Fas-Fas ligand interactions unless GC B cells are positively selected by Ags present on FDC.

ADP-ribosyl Cyclase↗

PubMed1981

Germinal centres and the B-cell system. V. Presence of germinal centre-precursor cells among lymphocytes of the thoracic duct in the rat.

Thoracic duct lymphocytes (TDL) were studied with respect to their capacity to give rise to germinal centres (GC) and to form primary antibody in an adoptive transfer system of the rat. Challenge with sheep erythrocytes (SRBC) 24h after lethal irradiation (900 rads) and syngeneic TDL reconstitution (10(8)) lead to conspicuous GC activity already 7 days after transfer. In contrast, using syngeneic bone marrow (BM) in the adoptive transfer system, no GC formation was observed over the period studied (14 days after reconstitution). Reconstitution experiments using in vivo-separated T-TDL (1-5% s-Ig+) and B-TDL (greater than 90% s-Ig+) subpopulations, either separately or in combination, indicated that GC originate from B-TDL but require T-TDL for induction.

Animals↗

PubMed1984

Homing of germinal-center cells into germinal centers of lymph node via afferent lymphatics. An autoradiographic study in rabbits.

Affinity of lymphoid cells for the microenvironment of germinal centers (GC), as detectable in transfer experiments by rapid homing in spleen GC from the blood, is a capacity expressed by only a subset of lymphoid cells, in particular by those constituting a GC. However, when introduced into the blood stream, these cells do not home into GC of lymph nodes and gut-associated lymphoid tissues. To investigate further this homing inability for high endothelial venule (HEV)-containing lymphoid tissues, GC cells isolated from donor rabbit appendix were labeled in vitro with 3H-leucine and injected into an afferent lymph vessel of recipient popliteal lymph nodes. Draining lymph nodes were removed 15 min to 24 h after cell administration and prepared for radioautography. For reference, the migration of cells isolated from Peyer's patches and thoracic duct lymph was also studied. By use of appendix GC cells, large numbers of labeled cells were found to migrate into GCs of the outer cortex centripetally, i.e., from the subcapsular sinus through the lymphocyte corona into the GC proper. The same was observed for cells from Peyer's patches, although in smaller numbers. Thoracic duct lymphocytes were only localized in the lymphocyte corona and the deep cortex. Thus, appendix GC cells and a subpopulation of cells from Peyer's patches can reach lymph node GC, but only when administered intralymphatically.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Similarity of spontaneous germinal and in vitro somatic cell mutation rates in humans: implications for carcinogenesis and for the role of exogenous factors in "spontaneous" germinal mutagenesis.

The rate of spontaneous mutation resulting in electrophoretic variants per cell generation in a human lymphoblastoid cell line, on the basis of experiments described in this paper, is found to be 7.2 x 10(-8) per locus. A review of similar data on electrophoretic variants resulting from spontaneous mutation in the human germ line leads to an estimate of 3.3 x 10(-8) per locus per cell generation. It is argued that the similarity of these two estimates, despite an average cell generation time of 18.5 hr for the cultured somatic cells but about 26 days in the germ line, suggests that spontaneous mutation involving nucleotide substitutions is much more dependent on cell generation than on time. This finding permits the inference that environmental (exogenous) variables make a relatively small contribution to the rate of this type of human germinal spontaneous mutation. While in vitro somatic-cell mutation rates, such as derived in this study, provide a basis for modeling the contribution of nucleotide substitutions in multihit/clonal theories of carcinogenesis, it is also argued that the complex of events involved in carcinogenesis, including chromosomal rearrangements and mitotic recombination, could have very different individual probabilities. Estimates for the rates of these other types of mutation are needed to provide a better understanding of the manner in which multiple mutations accumulate in malignant cells.

Alleles↗