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The effect of high temperature and high atmospheric CO2 on carbohydrate changes in bell pepper (Capsicum annuum) pollen in relation to its germination.

Pollen viability and germination are known to be sensitive to high temperature (HT). However, the mode by which high temperature impairs pollen functioning is not yet clear. In the present study, we investigated the effect of high temperature on changes occurring in carbohydrate of bell pepper (Capsicum annuum L. cv. Mazurka) pollen in order to find possible relations between these changes and pollen germination under heat stress. When pepper plants were maintained under a moderate HT regime (32/26 degrees C, day/night) for 8 days before flowers have reached anthesis, pollen count at anthesis was similar to that found in plants grown under normal temperatures (NT 28/22 degrees C). However, the in vitro germination, carried out at 25 degrees C, of pollen from HT plants was greatly reduced. This effect matched the marked reduction in the number of seeds per fruit in the HT plants. Maintaining the plants at high air CO2 concentration (800 &mgr;mol mol-1 air) in both temperature treatments did not affect the in vitro germination of pollen from NT plants, but restored germination to near the normal level in pollen from HT plants. Under NT conditions, starch, which was negligible in pollen at meiosis (8 days before anthesis, A-8) started to accumulate at A-4 and continued to accumulate until A-2. From that stage until anthesis, starch was rapidly degraded. On the other hand, sucrose concentration rose from stage A-4 until anthesis. Acid invertase (EC 3.2.1.26) activity rose parallel with the increase of sucrose. In pollen from HT plants, sucrose and starch concentrations were significantly higher at A-1 pollen than in that of NT plants. Under high CO2 conditions, the sucrose concentration in the pollen of HT plants was reduced to levels similar to those in NT pollen. In accordance with the higher sucrose concentration in HT pollen, the acid invertase activity in these pollen grains was lower than in NT pollen. The results suggest that the higher concentrations of sucrose and starch in the pollen grains of HT plants may result from reduction in their metabolism under heat stress. Elevated CO2 concentration, presumably by increasing assimilate availability to the pollen grain, may alleviate the inhibition of sucrose and starch metabolism, thereby increasing their utilization for pollen germination under the HT stress. Acid invertase may have a regulatory role in this system.

Journal Article↗

Pyrimidine nucleotide and nucleic acid synthesis in embryos and megagametophytes of white spruce (Picea glauca) during germination.

Pyrimidine nucleotide synthesis was investigated in isolated germinating zygotic embryos and separated megagametophytes of white spruce by following the metabolic fate of 14C-labelled orotic acid, uridine, and uracil, as well as by measuring the activities of the major enzymes participating in nucleotide synthesis. The rate of nucleic acid synthesis in these tissues was also examined by tracer experiments and autoradiographic studies conducted with labelled thymidine, and by conventional light microscopy. From our results, it emerges that changes in the contribution of the de novo and salvage pathways of pyrimidines play an important role during the initial stages of zygotic embryo germination. Preferential utilization of uridine for nucleic acid synthesis, via the salvage pathway, was observed at the onset of germination, before the restoration of a fully functional de novo pathway. Similar metabolic changes, not observed in the gametophytic tissue, were also documented in somatic embryos previously. These alterations of the overall pyrimidine metabolism may represent a strategy for ensuring the germinating embryos with a large nucleotide pool. Utilization of 14C-thymidine for nucleic acid synthesis increased in both dissected embryos and megagametophytes during germination. Autoradiographic and light microscopic studies indicated that soon after imbibition, DNA synthesis was preferentially initiated along the embryonic axis, especially in the cortical cells. Apical meristem reactivation was a later event, and the root meristem became activated before the shoot meristem. Taken together, these results indicate that precise changes in nucleotide and nucleic acid metabolism occur during the early phases of embryo germination.

Journal Article↗

Soluble antigen can cause enhanced apoptosis of germinal-centre B cells.

Germinal centres are dynamic microenvironments of B-lymphocyte differentiation, which develop in secondary lymphoid tissues during immune responses. Within germinal centres, activated B lymphocytes proliferate and point mutations are rapidly introduced into the genes encoding their immunoglobulin receptors. As a result, new specificities of B cells are created, including those with a heightened capacity to bind the immunizing antigen. Immunoglobulin gene mutation can also lead to reactivity to self antigens. It has been suggested that any newly formed self-reactive B cells are eliminated within the germinal centre in order to avoid autoimmunity. Here we present evidence that antigen-specific, high-affinity, germinal-centre B cells are rapidly killed by apoptosis in situ when they encounter soluble antigen. The effect seems to act directly on the B cells, rather than through helper T cells. Furthermore, the apoptosis is unique to germinal-centre cells, and is only incompletely impeded by constitutive expression of the proto-oncogene bcl-2. This phenomenon may reflect clonal deletion of self-reactive B cells within germinal centres.

Animals↗

Oxalate oxidases and differentiating surface structure in wheat: germins.

Oxalate oxidases (OXOs) have been found to be concentrated in the surface tissues of wheat embryos and grains: germin is concentrated in root and leaf sheaths that surround germinated embryos; pseudogermin (OXO-psi) is concentrated in the epidermis and bracts that 'encircle' mature grains. Most strikingly, the epidermal accumulation of OXO-psi was found to presage the transition of a delicate 'skin', similar to the fragile epidermis of human skin, into the tough shell (the miller's 'beeswing') that is typical of mature wheat grains. A narrow range of oxalate concentration (1--2 mM) in the hydrated tissues of major crop cereals (barley, maize, oat, rice, rye and wheat) contrasted with wide variations in their OXO expression, e.g. cold-tolerant and cold-sensitive varieties of maize have similar oxalate contents but the former was found to contain approx. 20-fold more germin than did the latter. Well-known OXOs in sorghum, a minor cereal, and beet, a dicotyledon, were found to have little antigenic relatedness to the germins, but the beet enzyme did share some of the unique stability properties that are peculiar to the germin-like OXOs that are found only in the major crop cereals. Their concentration in surface structures of domesticated wheat suggests a biochemical role for germin-like OXOs: programmed cell death in surface tissues might be a constitutive as well as an adaptive form of differentiation that helps to produce refractory barriers against tissue invasion by predators. Incidental to the principal investigation, and using an OXO assay (oxalate-dependent release of CO(2)) that did not rely on detecting H(2)O(2), which is often fully degraded in cell extracts, it was found that OXO activity in soluble extracts of wheat was manifested only in standard solution assays if the extract was pretreated in a variety of ways, which included preincubation with pepsin or highly substituted glucuronogalactoarabinoxylans (cell-wall polysaccharides).

Apoptosis↗

The development of ribonuclease and acid phosphatase during germination of Pisum arvense.

1. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic, the time of appearance of the two phases depending on the conditions of growth. 2. Acid phosphatase exhibits a single phase of development. 3. Cycloheximide inhibits development of ribonuclease activity in phase II but not in phase I. 4. (14)C-labelled amino acids are not incorporated into ribonuclease isolated during phase I. 5. The buoyant density of ribonuclease isolated during phase I is not affected by imbibition of the seed in 80% deuterium oxide. 6. Acid phosphatase was isolated from the supernatant fraction of the cotyledons of germinating peas and partially purified. 7. Development of acid phosphatase activity during germination is inhibited by treatment of the seed with cycloheximide or actinomycin D. 8. Partial purification of acid phosphatase from peas germinated in the presence of (14)C-labelled amino acids suggests that the enzyme is radioactively labelled. 9. Germination of peas in the presence of 80% deuterium oxide results in an increase in the buoyant density of acid phosphatase. 10. The results suggest that increase in ribonuclease activity during the first 4 days of germination does not result from synthesis of protein de novo, but that the corresponding increase in acid phosphatase activity does result from synthesis de novo.

Acid Phosphatase↗

Metabolism and the triggering of germination of Bacillus megaterium. Use of L-[3H]alanine and tritiated water to detect metabolism.

L-[2,3-3H]Alanine was used to probe for metabolism of alanine during triggering of germination of spores of Bacillus megaterium KM. No detectable incorporation of label into any compound, including water, was found, indicating that any metabolism involving the alanine germinant must be at a very low rate and also that alanine racemase is absent from spores of this strain. Spores were germinated in 3H2O to find if any of the many metabolic reactions causing irreversible incorporation of 3H into reaction products took place during triggering of germination. No incorporation was detected until 2-3 min after addition of germinants. It is therefore concluded that a wide variety of metabolic routes, including glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and amino acid metabolism are either not involved in the reactions causing the triggering of germination or operate at an extremely low rate during this process.

Alanine↗

Quasi-elastic light scattering studies on dormant and germinating Bacillus subtilis spores.

Spores of Bacillus subtilis in suspension, both dormant and germinating, have been examined by light-scattering methods, both integrated intensity and correlation versions. When intensity of scatter at constant volume was plotted against angle, curves possessing a maximum at about 20 degrees were regularly obtained but without any noticeable features at higher angles. This indicated polydispersity and/or asymmetry among the population. Curves of the intensity correlation function for both dormant and germinating spores at different angles, theta, did not superimpose at the lower angles when plotted appropriately, but did so for theta greater than 35 degrees. This was considered to arise from asymmetry of the spores. By using the high-angle data the apparent diffusion coefficient was determined for both dormant spores and for germinating spores from 1 min after germinant addition. No appreciable difference was observed, from which volume changes greater than 6% during germination could be excluded. The occurrence of germination was confirmed by both absorbance and phase-contrast-microscopy observations.

Alanine↗

Sporulation temperature affects initiation of germination and inactivation by high hydrostatic pressure of Bacillus cereus.

The influence of sporulation temperature (20, 30 and 37 degrees C) on the heat resistance and initiation of germination and inactivation by high pressure on Bacillus cereus ATCC 14579 spores was investigated. Spores sporulated at 37 degrees C were the most heat-resistant. However, spores sporulated at 20 degrees C were more resistant to the initiation of germination and inactivation by high pressure. Spores were more sensitive to pressure at higher treatment temperatures. At 25 degrees C, there was an optimum pressure (250 MPa) for the initiation of germination for the three suspensions; at higher temperatures an increase of pressure up to 690 MPa caused progressively more germination. Resistance to the germinability and inactivation by high pressure of the spore population was distributed heterogeneously. Semilogarithmic curves of the ungerminated and survival fraction of B. cereus spores were concave. The resistant fraction of the spore population was lower at higher treatment temperatures. At 60 degrees C after 30 s of treatment at 690 MPa almost 5 log cycles of the population of B. cereus sporulated at 20 degrees C was germinated, and more than 7 log cycles of the population of B. cereus sporulated at 30 and 37 degrees C. The same treatment inactivated 4, 6 and 7 log cycles of the population of B. cereus sporulated at 20, 30 and 37 degrees C, respectively.

Air Pressure↗

The potential biocontrol agent Pseudomonas antimicrobica inhibits germination of conidia and outgrowth of Botrytis cinerea.

AIMS: Antifungal metabolites of Pseudomonas antimicrobica have previously been shown to inhibit conidial germination of the grey mould pathogen Botrytis cinerea. In this study, metabolites of the bacterium have been tested at different stages of Botrytis germination to determine their effects on germ tube production and extension. METHODS AND RESULTS: Metabolites were added to conidia that had been pre-incubated for either 120 or 255 min. Pseudomonas antimicrobica inhibited B. cinerea conidial germination and caused a significant reduction in germ tube extension, irrespective of the stage of germination. Abnormal germination and a reduction in the frequency of lateral branching of the germ tubes in the presence of the metabolites were also reported, suggesting interference with normal hyphal development. CONCLUSION: The bacterium can inhibit germination of conidia and extension of germ tubes at different stages of Botrytis development. SIGNIFICANCE AND IMPACT OF THE STUDY: The antagonistic activity of the bacterium has promising implications for its use as a biocontrol agent.

Antifungal Agents↗

Examination of the anatomic relationship of the proximal germinal nail matrix to the extensor tendon insertion.

The purpose of this study was to delineate the relationship of the terminal extensor tendon insertion to the proximal limit of the germinal nail matrix. Sixteen fresh-frozen human cadaver fingers without any evidence of trauma (average age, 55 years; 3 males and 1 female) were used for this study. Under x25 magnification the proximal limit of the germinal nail matrix and the terminal bony insertion of the extensor tendon were identified. The distance from the terminal tendon insertion to the germinal nail matrix was ascertained using precision calipers. The average distance from the terminal extensor tendon insertion to the proximal edge of the germinal nail matrix was found to be 1.2 mm. We conclude that the proximal limit of the germinal matrix is extremely close to the terminal extensor tendon bony insertion. When the extensor tendon insertion is visualized during operative exposures of the dorsum of the distal phalanx, care should be taken to avoid damaging the germinal matrix. Conversely, when the nail bed is being completely excised, visualization of the insertion of the extensor tendon will indicate that further proximal dissection is not required.

Adolescent↗

Effect of soaking, germination, and enzyme treatment of whole barley on nutritional value and digestive tract parameters of broiler chickens.

1. An experiment was carried out to determine the effect of soaking at 0 degrees C, soaking at room temperature, germination, or enzyme treatment of whole barley on feeding value and digestive tract parameters of 2- to 4-week old broiler chickens given diets with 700g/kg whole barley. 2. Soaking or germination decreased the soluble and total beta-glucan content (P < 0.05) and, except for soaking at 0 degrees C, the acid extract viscosity of the grain also decreased (P < 0.05). Germination and soaking in the presence of enzymes produced the lowest beta-glucan content and viscosity. 3. Except for soaking in cold water, the soaking, germination and enzyme treatments increased weight gain and decreased food:gain ratio (P < 0.05). Correspondingly, the digestibility of protein, fat, and ash, and the digestible energy content, increased (P < 0.05) after enzyme treatment or germination. 4. Chickens fed on enzyme-treated or germinated barley diets had intestinal contents with a greater proportion of dry matter and lower viscosity than chickens fed on untreated barley (P < 0.05). Consequently, the cages and chickens were cleaner (P < 0.05) and the weight of digestive organs as proportion of live weight was lower. 5. Particle size analysis of excreta revealed that whole barley was efficiently ground by the gizzards of 16-d-old chickens, and very few whole kernels were found.

Animal Feed↗

Specific antibody within lymphoid germinal center cells of mice after primary immunization with horseradish peroxidase: a light and electron microscopic study.

The appearance in mice of specific antibody within newly formed germinal centers in lymph nodes was demonstrated by light and electron microscopy after regional primary antigenic stimulation with horseradish peroxidase (HRP). Lymphoid germinal center cells containing anti-HRP antibody in the perinuclear space and in the cytoplasm were detected from 17 to 26 days after antigenic stimulation. Extracellular anti-HRP antibody within germinal centers, localized between dendritic reticular cells and lymphoid elements, could not be found before the appearance of intracellular antibody. These findings strongly suggest antibody formation by lymphoid germinal center cells. Both antigen and corresponding antibody persisted in intercellular spaces up to 35 days after primary stimulation. The concomitant presence in a given lymph node of germinal centers which are positive or negative with regard to specific antibody provide evidence in favor of monospecificity of individual centers. The mechanisms of antigen-trapping within germinal centers are discussed in the light of the present observations.

Animals↗

Long-oligomer microarray profiling in Neurospora crassa reveals the transcriptional program underlying biochemical and physiological events of conidial germination.

To test the inferences of spotted microarray technology against a biochemically well-studied process, we performed transcriptional profiling of conidial germination in the filamentous fungus, Neurospora crassa. We first constructed a 70 base oligomer microarray that assays 3366 predicted genes. To estimate the relative gene expression levels and changes in gene expression during conidial germination, we analyzed a circuit design of competitive hybridizations throughout a time course using a Bayesian analysis of gene expression level. Remarkable consistency of mRNA profiles with previously published northern data was observed. Genes were hierarchically clustered into groups with respect to their expression profiles over the time course of conidial germination. A functional classification database was employed to characterize the global picture of gene expression. Consensus motif searches identified a putative regulatory component associated with genes involved in ribosomal biogenesis. Our transcriptional profiling data correlate well with biochemical and physiological processes associated with conidial germination and will facilitate functional predictions of novel genes in N.crassa and other filamentous ascomycete species. Furthermore, our dataset on conidial germination allowed comparisons to transcriptional mechanisms associated with germination processes of diverse propagules, such as teliospores of the phytopathogenic fungus Ustilago maydis and spores of the social amoeba Dictyostelium discoideum.

Animals↗

Seed germination of six mature neotropical rain forest species in response to dehydration.

We studied effects of dehydration and rehydration on germination of seeds of six mature tropical rain forest species-Cupania glabra Swartz, Cymbopetalum baillonii Fries, Poulsenia armata (Miq.) Standl., Stemmadenia donnell-smithii (Rose) Woodson, Rheedia edulis Triana & Planch. and an understory palm Chamaedorea alternans H. Wendl.-from Veracruz, México. Before the seeds were sown, their water content was reduced by 0 (control), 30, 54 and 72% of their original water content. Dehydration affects the ability of seeds to rehydrate, as well as the rate and final percentage of germination when seeds are subsequently rehydrated. Seed survival and germination behavior showed three patterns: (1) C. baillonii, P. armata and S. donnell-smithii had greater tolerance to seed dehydration than C. glabra, C. alternans and R. edulis; (2) partial dehydration enhanced germinability of C. glabra and C. baillonii seeds; and (3) partial dehydration of C. alternans and R. edulis seeds resulted in delayed or sporadic germination. A relationship was found between the effects of dehydration on germination and the seasonality of seed production.

Journal Article↗

High expression of methionine aminopeptidase type 2 in germinal center B cells and their neoplastic counterparts.

Methionine aminopeptidase type 2 (MetAP2) is a bifunctional protein that plays critical roles in the regulation of protein synthesis and post-translational processing by (a) protecting the alpha subunit of eukaryotic initiation factor 2 from inhibitory phosphorylation by eukaryotic initiation factor 2 kinases and (b) removing the amino-terminal methionine residue from nascent protein. MetAP2 is also known as the molecular target of the angiogenesis inhibitor TNP-470. In addition, it has been recently suggested that MetAP2 has an antiapoptotic function in mesothelioma. To know the pattern of expression of MetAP2 in normal and neoplastic tissues, we raised two specific rabbit polyclonal Abs and examined the pattern of MetAP2 expression in various normal and pathologic specimens. Unexpectedly, we found a very high and selective expression of MetAP2 in germinal center B cells. In the germinal center, dark zone B cells tended to express more MetAP2 than light zone B cells. When 200 malignant lymphomas of various subtypes were studied, a high level of MetAP2 expression, equivalent to that observed in germinal center B cells, was noted exclusively on B-cell lymphoma subtypes that are currently regarded as the neoplastic counterparts of germinal center B cells. The expression of MetAP2 in diffuse large B-cell lymphomas correlated well with that of BCL6 (p < 0.05) but not with that of either CD10 or BCL2. These data suggest that MetAP2 has specific function(s) in germinal center B cells and that the function is shared by neoplastic counterparts of germinal center B cells.

Aminopeptidases↗

The relationship between conidial germination and esterase activities in Neurospora crassa.

Esterase activity in rapidly germinating Neurospora conidia was several times higher than the esterase activity in conidia which germinate slowly. Starch gel electrophoresis experiments demonstrated the existence of esterase isoenzymes which are specific to the conidia. These isoenzymes completely disappeared during 20 h of conidial germination at 30 degrees C. Electron microscopy showed the successive breakdown of electron-dense compounds in storage bodies during conidial germination. These observations, taken together, indicate that the electron-dense compounds may be hydrolysed by specific esterases to serve as an endogenous energy and material source for germ tube formation. The levels of esterase activity, however, were not always proportional to the time required for conidial germination, indicating the possibility that additional enzyme systems might also be involved in the initial stages of germination.

Acid Phosphatase↗

Properties of the germination inhibitor of Streptomyces viridochromogenes spores.

Germinating spores of Streptomyces viridochromogenes excreted a substance into the surrounding medium which inhibited germination of another sample of the spores. The germination inhibitor (GI) was produced during submerged culture after exponential growth had ceased. The GI was purified 51-fold following extraction from growth liquor with chloroform. It was soluble in alcohol and water and had a molecular weight of less than 1000. The GI blocked growth and respiration of some Gram-positive bacteria and was an inhibitor of the membrane bound, but not solubilized, calcium-dependent ATPase of germinated spores and mycelia of the producing organism. Several sodium-potassium activated ATPases were also inhibited. All four activities (respiration, growth, germination inhibition, ATPase) co-purified during column and thin-layer chromatography. The GI activities released during germination and produced during growth were identical. A role for the GI antibiotic in regulation of dormancy of spores of the producing organism is discussed.

Adenosine Triphosphatases↗

In vivo roles of the germination-specific lytic enzymes of Bacillus subtilis 168.

Germination of endospores of Bacillus subtilis involves the activities of several germination-specific lytic enzymes, including glucosaminidase and lytic transglycosylase. Another non-hydrolytic activity, likely to be due to an epimerase, also occurs. The effect of pH on enzyme activities and the overall germination rate was measured. Optimal germination occurred between pH 7-9; however, optimum glucosaminidase and epimerase activities were noted at pH 5. Conversely, the lytic transglycosylase activity was greatest at pH 8. Treatment of spores (15 min) with heat (90 degrees C) or NaOH (0.25 M) led to impaired cortex hydrolysis/modification, but with <20% loss in viability. Analysis of muropeptides in the germination exudate revealed a reduction of >85% in glucosaminidase and epimerase products, when compared to untreated spores. Conversely, lytic transglycosylase activity was increased by alkali or heat treatment, which was possibly due to increased substrate availability. FB101 (sleB) spores, which lack lytic transglycosylase activity, showed 90-fold greater loss in viability than the wild-type after 1 h at 90 degrees C. Similarly, 97% of FB101 (sleB) spores were unable to form a colony on nutrient agar after 130 min exposure to 0.25 M NaOH at 4 degrees C, whereas the wild-type was unaffected. This demonstrates the crucial role of the lytic transglycosylase in cortex hydrolysis of damaged spores. The respective targets of heat and alkali in spores and their role during germination are discussed.

Amidohydrolases↗