BIOCHEMICAL AND PHYSIOLOGICAL ASPECTS OF LEAF DEVELOPMENT IN COCOA (THEOBROMA CACAO)

The dormant phase of the flush cycle of leaf growth in cocoa is known to be correlated with high abscisic acid (ABA) levels in the mature leaves of the new flush (NF) and previous flush (PF) leaves. Defoliation of either the NF leaves or PF leaves of cocoa seedlings reduced the length of the dormant phase of the flush cycle, thus showing that the mature leaves were a source of growth inhibitors which could affect shoot apical activity. The application of ABA to the NF and PF leaves led to an extension of the dormant phase, whereas application of zeatin or gibberellic acid decreased it. The distribution of [¹⁴C]ABA following its application to NF and PF leaves at different stages throughout the growth cycle showed that [¹⁴C] ABA was accumulated by the bud in relatively larger amounts during the final stages of bud dormancy (I-1 and I-2) than in the earlier stage (F-2). The results suggest that internal competition for nutrients may be responsible for the inhibition of growth at the F-2 stage but that ABA translocated from the mature leaves causes the buds to remain dormant during the subsequent stages of I-1 and I-2.     

A novel family of toxin/antitoxin proteins in Bacillus species

The C-terminal regions (CT) of Pfam PF04740 proteins share significant sequence identity with the toxic CdiA-CT effector domains of contact-dependent growth inhibition (CDI) systems. In accord with this homology, we find that several PF04740 CT domains inhibit cell growth when expressed in Escherichia coli. This growth inhibition is specifically blocked by antitoxin proteins encoded downstream of each PF04740 gene. The YobL-CT, YxiD-CT and YqcG-CT domains from Bacillus subtilis 168 have cytotoxic RNase activities, which are neutralized by the binding of cognate YobK, YxxD and YqcF antitoxin proteins, respectively. Our results show that PF04740 proteins represent a new family of toxin/antitoxin pairs that are widely distributed in Gram-positive bacteria.     

Properties of four temperate bacteriophages active on Clostridium perfringens type A.

Four temperature bacteriophages (designated as PF1, PF2, PF3 and PF4) were isolated from lysogenic strains of Clostridium perfringens type A. On the basis of plaque morphology, pH stability, DNase and RNase resistance, buoyant density, one-step growth parameters and electron microscope phage dimensions, it seems that these phages are different and unrelated. Calcium was required for better phage replication. Bacterial strain S107 appears to be the only UV-inducible strain as compared with the other three lysogenic strains. PF2 has a unique pattern of pH stability showing two optima values: one at pH 5 and the second at pH 8-9. Generally, all four phages have a better resistance in acid than in alkaline pH values. The CsC1 equilibrium centrifugation patterns reveal low figures for phage PF1, PF2 and PF3 and show off the fact that PF4 lysates contain two viral particules different with respect to their densities, a property which other determinations failed to demonstrate. Each phage, except PF4, is well characterized by the parameters of the one-step growth cycle.     

Influences of the recombinant artificial cell adhesive proteins on the behavior of human umbilical vein endothelial cells in serum-free culture

To improve the safety of cellular therapy products, it is necessary to establish a serum-free cell culture method that can exclude animal-derived materials in order to avoid contamination with transmissible agents. It would be optimal if the proteins necessary to a serum-free culture could be provided as recombinant proteins. In this study, the influences of recombinant artificial cell adhesive proteins on the behavior of human umbilical vein endothelial cells (HUVECs) in serum-free culture were examined in comparison with the influence of plasma fibronectin (FN). The recombinant proteins used were Pronectin F (PF), Pronectin F PLUS (PFP), Pronectin L (PL), Retronectin (RN), and Attachin (AN). HUVECs adhered more efficiently on PF or PFP than on FN. No cells adhered on PL. Regarding the VEGF or bFGF-induced cell growth, the cells on PF and PFP proliferated at a similar rate to the cells on FN. RN and AN were less effective in supporting cell growth. Since cell adhesion on PF and PFP induced phosphorylation of focal adhesion kinase, they are thought to activate integrin-mediated intracellular signaling. The cells cultured on PF or PFP were able to produce prostaglandin I(2) or tissue-plasminogen activator in response to thrombin. However, thrombin caused detachment of the cells from PF but not from PFP or FN, meaning that the cells were able to adhere more tightly on PFP or FN than on PF. These data indicate that PFP could be applicable as a substitute for plasma FN.     

Interaction of platelet factor 4 with fibroblast growth factor 2 is stabilised by heparan sulphate

The CXC chemokine platelet factor 4 (PF4) appears to inhibit tumour growth through its modulation of the activity of angiogenic growth factors. We investigated the heparan sulphate-dependent mechanism of PF4 inhibition of fibroblast growth factor 2 (FGF-2). The ability of PF4 to bind simultaneously to both FGF-2 and HS was assessed using affinity gel chromatography. Thirty-three to forty-two percent more HS bound to the FGF-2 affinity gel in the presence of PF4 than with HS alone. Protection assays showed that PF4 and FGF-2 bound to adjacent or overlapping sites together covering a 12 kDa stretch of HS. This study suggests that the three components may form a ternary complex. PF4 released at sites of angiogenesis may bind to angiogenic growth factors attached to endothelial cell surface HS to disrupt or prevent them from interacting with their signalling receptors. Manipulation of this mechanism may prove useful for clinical intervention of angiogenesis.     

Autocrine release of TGF-beta by portal fibroblasts regulates cell growth

Portal fibroblasts (PF) are a newly isolated population of fibrogenic cells in the liver postulated to play a significant role in early biliary fibrosis. Because transforming growth factor-beta (TGF)-beta is a key growth factor in fibrosis, we characterized the response of PF to TGF-beta. We demonstrate that PF produce significant amounts of TGF-beta2 and, unlike activated hepatic stellate cells (HSC), express all three TGF-beta receptors and are growth inhibited by TGF-beta1 and TGF-beta2. Fibroblast growth factor (FGF)-2, but not platelet derived growth factor (PDGF), causes PF proliferation. These data suggest a mechanism whereby HSC eclipse PF as the dominant myofibroblast population in biliary fibrosis.     

Suppression by the DNA fragment of the motX promoter region on long flagellar mutants of Vibrio alginolyticus

The axial length of the polar flagellum (Pof) of Vibrio alginolyticus is about 5 microm. We previously isolated mutants that make abnormally long flagella. The swarm sizes of these mutants in a soft agar plate are smaller than that of a wild-type strain. We cloned a DNA fragment into the pMF209 plasmid that restored the swarming ability of the long-Pof strain V10578. The swimming speed and flagellar length of these transformants were almost equal to the wild-type values. The amounts of PF47 flagellin and PF60 sheath-associated protein, which increased in the long-Pof mutants, were retrieved to almost the wild-type level in the transformants. The plasmid pMF209 contained only a 143 bp chromosomal fragment whose sequence is about 80% similar to that of the motX promoter region of V parahaemolyticus. We speculate that this sequence interacts with a regulatory protein that controls Pof expression. The mutation causing the long-Pof phenotype may be in the gene encoding this protein or in the control region of a structural gene that is regulated by this protein.     

Evaluation of various serum and animal protein free media for the production of a veterinary rabies vaccine in BHK-21 cells

We have carried out the adaptation of BHK-21 cells to two serum free (Ex Cell 520 and HyQ PF CHO) and three animal protein free media: Ex Cell 302, HyQ PF CHO MPS and Rencyte BHK. After a direct switch or a gradual adaptation, we have achieved BHK-21 cells growth in the following media: HyQ PF CHO, HyQ PF CHO MPS, Rencyte BHK and Ex Cell 302. The most suitable media for BHK-21 cells growth, with respect to cell density and specific growth rate, were HyQ PF CHO and HyQ PF CHO MPS. Hence we have selected these media to study cell growth and the production of rabies virus. Kinetic studies of cell growth in spinner flasks using the selected media have shown that a maximal cell density of 2x10(6) cells x ml(-1) was reached in both media. For rabies virus production, the viral titer obtained was 1.7x10(6) FFU x ml(-1) in HyQ PF CHO as well as in HyQ PF CHO MPS medium. The optimization of rabies virus production by BHK-21 cells grown in a 2 l bioreactor using the selected media, pointed to the following parameters: culture mode, perfusion rate and multiplicity of infection (MOI), as being the critical factors for achieving a good virus yield. When tested in mice, the activity of the experimental vaccines prepared on HyQ PF CHO MPS medium has shown a protective activity that meets WHO requirements.     

Dopamine affects the stability, hydration, and packing of protofibrils and fibrils of the wild type and variants of alpha-synuclein

Parkinson's disease (PD) is characterized by the presence of cytoplasmic inclusions composed of alpha-synuclein (alpha-syn) in dopaminergic neurons. This suggests a pivotal role of dopamine (DA) on PD development. Here, we show that DA modulates differently the stability of protofibrils (PF) and fibrils (F) composed of wild type or variants of alpha-syn (A30P and A53T) as probed by high hydrostatic pressure (HHP). While in the absence of DA, all alpha-syn PF exhibited identical stability, in its presence, the variant-composed PF acquired a greater stability (DAPFwt < DAPFA30P = DAPFA53T), implying that they would last longer, which could shed light onto why these mutations are so aggressive. When alpha-syn was incubated for long times (18 days) in the presence of DA, we observed the formation of F by electronic microscopy, suggesting that the PF trapped in the presence of DA in short times can evolve into F. The stability of F was also altered by DA. DAFwt was more labile than Fwt, indicating that the former would be more susceptible to breakage. PFA30P and DAPFA30P, when added to mesencephalic and cortical neurons in culture, decreased the number and length of neurites and increased the number of apoptotic cells. Surprisingly, these toxic effects of PFA30P and DAPFA30P were practically abolished with HHP treatment, which was able to break the PF into smaller aggregates, as seen by atomic force microscopy. These results suggest that strategies aimed at breaking and/or clearing these aggregates is promising in alleviating the symptoms of PD.     

Effects of glucose, pH, and dissolved-oxygen tension on Bacillus cereus growth and permeability factor production in batch culture.

The production of a Bacillus cereus enterotoxin, measured as rabbit skin permeability factor (PF), in response to differences in glucose availability, pH, and dissolved oxygen tension was studied in a 1-liter batch fermentor system. Glucose had to be present for toxigenesis to occur. In uncontrolled fermentation an increasing inhibition of PF production and growth occurred as pH dropped occurred below 6.5. Optimum pH for toxigenesis was 7.0 to 7.5, and fermentations maintained at this level yielded 10- to 20-fold more PF than comparable uncontrolled fermentations. PF production was appreciably diminished at or below pH 6.0 and at or above pH 8.5. Peak PF titer was associated with a drop in acid output, and the titrant utilization profile could be used as an indication of this point. Productivity was greatest in the early exponential phase of growth and decreased to zero at the transition phase. Differences in dissolved oxygen tension affected both the maximum productivity early in the fermentation and the rate of its decrease as growth progressed. The optimum dissolved oxygen tension for toxigenesis was 0.002 atm, and the most rapid growth occurred at 0.10 atm. Productivity and growth were reduced under anerobic conditions, whereas a hyperoxic environment severely reduced productivity, but not growth. Postexponential-phase loss of toxic activity coincided with a rapid increase in cellular oxygen demand. Neither was inhibited by the presence of glucose. However, PF loss was completely prevented by stringent oxygen limitation. Extracellular proteolytic activity did not appear to be responsible for the loss of toxic activity.     

Mast cells and pulmonary fibrosis. Identification of a histamine releasing factor in bronchoalveolar lavage fluid.

As elevated bronchoalveolar lavage (BAL) fluid histamine levels are noted in patients with pulmonary fibrosis (PF), we assayed BAL fluid from 16 patients with PF for the presence of a histamine releasing factor (HRF). HRF activity was assayed by measuring release of the preformed mast cell-derived mediators, histamine, or beta-hexosaminidase (beta-hex) from a purified population of IL-3 dependent mouse bone marrow derived mast cells (MBMMC) or human blood basophils. Mean BAL cell free histamine levels in the patients with PF was 1226 +/- 1349 pg/ml, whereas BAL histamine levels in a comparison group of six non-PF patients was 118 +/- 60 pg/ml. HRF was significantly elevated in BAL fluid of patients with PF (mean beta-hex release 24.5 +/- 12.9%; range 6.8 to 52.4%) compared to the non-PF group of patients (mean beta-hex release 7.9 +/- 7.7%; range 1.8 to 20.7%). The PF HRF not only degranulated MBMMC, but also induced the generation of the arachidonic acid metabolite leukotriene C4 from MBMMC (24.6 +/- 4.2 ng leukotriene C4/10(6) MBMMC). The PF HRF did not appear to be a cytokine previously identified in BAL fluid of patients with PF (i.e., platelet derived growth factor or insulin growth factor-1) or a human cytokine able to degranulate human basophils (i.e., IL-1, or granulocyte-macrophage-CSF) as these recombinant human cytokines did not induce MBMMC beta-hex release. Physicochemical characterization of the HRF revealed that it was relatively heat stable, pronase sensitive and on Sephadex G-75 and G-200 column chromatography had an apparent molecular mass of 30 to 50 kDa. The ability of PF BAL to induce beta-hex release from MBMMC was not dependent on IgE as unsensitized or lactic acid treated MBMMC release similar amounts of beta-hex compared to MBMMC sensitized with IgE. Thus, BAL fluid of patients with PF contains an HRF that induces beta-hex release from MBMMC via an IgE-independent mechanism. The presence of the HRF could explain elevated BAL histamine levels in patients with PF.     

Effects of Glucose, pH, and Dissolved-Oxygen Tension on Bacillus cereus Growth and Permeability Factor Production in Batch Culture

The production of a Bacillus cereus enterotoxin, measured as rabbit skin permeability factor (PF), in response to differences in glucose availability, pH, and dissolved oxygen tension was studied in a 1-liter batch fermentor system. Glucose had to be present for toxigenesis to occur. In uncontrolled fermentation an increasing inhibition of PF production and growth occurred as pH dropped occurred below 6.5. Optimum pH for toxigenesis was 7.0 to 7.5, and fermentations maintained at this level yielded 10- to 20-fold more PF than comparable uncontrolled fermentations. PF production was appreciably diminished at or below pH 6.0 and at or above pH 8.5. Peak PF titer was associated with a drop in acid output, and the titrant utilization profile could be used as an indication of this point. Productivity was greatest in the early exponential phase of growth and decreased to zero at the transition phase. Differences in dissolved oxygen tension affected both the maximum productivity early in the fermentation and the rate of its decrease as growth progressed. The optimum dissolved oxygen tension for toxigenesis was 0.002 atm, and the most rapid growth occurred at 0.10 atm. Productivity and growth were reduced under anerobic conditions, whereas a hyperoxic environment severely reduced productivity, but not growth. Postexponential-phase loss of toxic activity coincided with a rapid increase in cellular oxygen demand. Neither was inhibited by the presence of glucose. However, PF loss was completely prevented by stringent oxygen limitation. Extracellular proteolytic activity did not appear to be responsible for the loss of toxic activity.     

残留地膜对番茄幼苗形态和生理特性的影响

采用盆栽试验的方法,研究了土壤中残留地膜对番茄(Lycopersicon esculentum L.)幼苗形态及生理特性的影响.结果表明,残留地膜使番茄幼苗的株高、茎粗、地上部和根系鲜重、根系活力及叶片氮代谢水平等降低;而且根系的IBA含量降低和ABA含量增加,抑制了根系的生长.同时地膜残留量越大,抑制的效果越显著.这...     

Neurite extension by neuroblastoma cells on substratum-bound fibronectin''s cell-binding fragment but not on the heparan sulfate-binding protein, platelet factor-4

Human and rat neuroblastoma cells extend neurites over plasma fibronectin (pFN)-coated substrata. For resolution of which fibronectin binding activities (the cell-binding domain (CBD), the heparan sulfate-binding domains, or a combination of the two) are responsible for neurite outgrowth, CBD was prepared free of heparan sulfate-binding activity as described by Pierschbacher et al. (Cell 26 (1981) 259-267). Neuroblastoma cells attached and extended neurites as stably and as effectively on CBD-coated substrata as on intact pFN, while cytoplasmic spreading was more extensive on pFN-coated substrata. The structures of growth cones on CBD or pFN were virtually identical. On substrata coated with the model heparan sulfate-binding protein, platelet factor 4 (PF4), cells attached and spread somewhat but never extended neurites. When cells were challenged with substrata coated with various ratios of CBD and PF4, PF4 was found to be an effective inhibitor of CBD-mediated neurite extension. Similarly, cells grown on substrata coated at different locations with CBD or PF4 in order to evaluate topographical dependence of growth cone formation extended neurites only onto the CBD-coated region or along the interface between these two proteins, but never onto the PF4 side of cells that bridged the interface. These studies indicate that (a) the CBD activity of pFN, and not its heparan sulfate-binding activity, is the critical determinant in neurite extension of these neural tumor cells from the central nervous system; (b) under some circumstances, heparan sulfate-binding activity can be antagonistic to neurite extension; (c) the chemical nature of the substratum controls the direction of neurite extension; (d) these neuroblastoma cells respond to these binding proteins very differently than fibroblasts or neurons from the peripheral nervous system.     

中华绒螯蟹一龄早熟和二龄成熟家系生长规律的比较研究

中华绒螯蟹(Eridcheir sinensis)1龄性早熟是扣蟹养殖过程中的一个重要问题,尚不清楚1龄性早熟和2龄正常性成熟后代的生长发育规律是否存在差异,本研究通过构建1龄性早熟和正常性成熟中华绒螯蟹家系,综合比较了单养条件下两种家系子一代(以下简称早熟F1和正常F1)在扣蟹和成蟹阶段的生长蜕壳规律、雌蟹腹脐覆盖腹甲宽度比例、成熟后的性腺指数(GSI)和肝胰腺指数(HSI)。结果显示:(1)早熟F1雄体在第1、2次和第7、8次蜕壳后的体重显著大于正常F1雄体(P0.05);而早熟F1雌体在第1~5次和第7次蜕壳后的体重显著大于正常F1雌体(P0.05);(2)第1和2次蜕壳后早熟F1的增重率较高,正常F1在第3~8次蜕壳后的增重率略高于早熟F1,两群体在扣蟹阶段的特定生长率均呈下降趋势,且正常F1高于早熟F1,其中雌雄个体在第3~4次蜕壳后的特定生长率均存在显著差异(P0.05);(3)早熟F1在第1~5次蜕壳间隔较长,而第6~8次蜕壳间隔较短;两种家系在扣蟹养殖阶段蜕壳4~6次,成蟹养殖阶段蜕壳2~4次,其中早熟F1在扣蟹阶段的平均蜕壳次数低于正常F1,而在成蟹阶段的平均蜕壳次数高于正常F1;(4)早熟F1腹脐覆盖腹甲宽度比例一直高于正常F1,但二者无显著差异(P0.05);(5)无论雌体还是雄体,早熟F1和正常F1的性腺指数和肝胰腺指数均无显著差异,单养条件下性腺均可发育成熟(P0.05)。综上,单养条件下,中华绒螯蟹早熟F1和正常F1的生长模式存在显著差异,两者都可以完成生殖蜕壳和性腺发育成熟,这为今后深入研究中华绒螯蟹个体生物学提供了理论依据和参考资料。     

Mediation of pinocytosis in cultured arterial smooth muscle and endothelial cells by platelet-derived growth factor

Pinocytosis was measured in monkey aortic smooth muscle cells (SMC), bovine aortic endothelial cells, and Swiss 3T3 cells in culture as cellular uptake of [U-(14)C]sucrose and horseradish peroxidase (HRP) from the tissue culture medium. Monkey arterial SMC and Swiss 3T3 cells were maintained in a quiescent state of growth at low cells density in medium containing 5 percent monkey plasma-derived serum (PDS). Replacement of PDS with 5 percent monkey whole blood serum (WBS) from the same donor, or addition to PDS of partially purified platelet-derived growth factor(s) (PF), resulted in a marked stimulation of pinocytosis as well as of cellular proliferation. In SMC, enhancement of the rate of pinocytosis occurred 4-6 h after exposure to WBS or PF, and the rate was up to twofold higher than the rate in medium containing PDS. In contrast, [(3)H]thymidine uptake by SMC did not increase until 12-16 h after exposure to PF. In endothelial cells the presence of PF or WBS did not enhance either the rate of pinocytosis or the rate of proliferation over that in PDS. Thus, endothelial cells did not become quiescent at subconfluent densities in PDS but maintained rates of proliferation and pinocytosis that were equivalent to those in WBS. By autoradiography, the fraction of labeled nuclei in SMC cultures 24 h after change of medium increased from 0.061 +/- 0.004 in quiescent cultures to 0.313 +/- 0.028 after exposure to WBS or PF. In contrast, labeling indices of endothelial cells were similar for cultures grown in PDS, WBS, or PF at any single time point after change of medium. These findings suggest that the rate of pinocytosis maybe be coupled in some fashion to growth regulation, which may be mediated in part by specific growth factors, such as that derived from the thrombocyte.     

The Rate of Unequal Crossing Over in the dumpy Gene from Drosophila melanogaster

The PIGSFEAST (PF) exon of the Drosophila dumpy gene is undergoing concerted evolution by the process of unequal crossing over. We have developed a long-range PCR-based assay to amplify the approximately 12 kb long exon which contains variable numbers of 303 or 306 nt long repeats in a tandem array. We applied this procedure to mutation accumulation lines of Drosophila melanogaster established by M. Wayne and L. Higgins. Nine new repeat length variants were found in these lines allowing us to measure the rate of unequal crossing over in the PF exon. The rate, which for several reasons is an underestimate, is 7.05 × 10⁻⁴ exchanges per generation.