Stimulation of tumor necrosis factor alpha production in human monocytes by inhibitors of protein phosphatase 1 and 2A.

The protein phosphatase 1 and 2A inhibitor, okadaic acid, has been shown to stimulate many cellular functions by increasing the phosphorylation state of phosphoproteins. In human monocytes, okadaic acid by itself stimulates tumor necrosis factor alpha (TNF-alpha) mRNA accumulation and TNF-alpha synthesis. Calyculin A, a more potent inhibitor of phosphatase 1, has similar effects. TNF-alpha mRNA accumulation in okadaic acid-treated monocytes is due to increased TNF-alpha mRNA stability and transcription rate. The increase in TNF-alpha mRNA stability is more remarkable in okadaic acid-treated monocytes than the mRNA stability of other cytokines, such as interleukin 1 alpha (IL-1 alpha), IL-1 beta, and IL-6. Gel retardation studies show the stimulation of AP-1, AP-2, and NF-kappa B binding activities in okadaic acid-stimulated monocytes. This increase may correlate with the increase in TNF-alpha mRNA transcription rate. In addition to the stimulation of TNF-alpha secretion by monocytes, okadaic acid appears to modulate TNF-alpha precursor processing, as indicated by a marked increase in the cell-associated 26-kD precursor. These results suggest that active basal phosphorylation/dephosphorylation occurs in monocytes, and that protein phosphatase 1 or 2A is important in regulating TNF-alpha gene transcription, translation, and posttranslational modification.

Mite-specific immunotherapy using allergen and/or bacterial extracts in atopic patients in Brazil.

OBJECTIVE: This study aimed to evaluate the clinical efficacy and antibody response changes after specific immunotherapy (SIT) using Dermatophagoides pteronyssinus (Dpt) allergens with or without bacterial extracts in Brazilian mite-atopic patients. METHODS: One-hundred patients with allergic rhinitis were selected for a randomized double-blind, placebo-controlled trial and distributed into 4 groups: Dpt (Dpt allergen extract), Dpt+MRB (Dpt allergen plus mixed respiratory bacterial extracts), MRB (MRB extract only) and placebo. Rhinitis symptom and medication scores; skin prick test (SPT) to Dpt extract; and serum immunoglobulin (Ig) E, IgG4, and IgG1 levels to Dpt, Der p 1, and Der p 2 allergens were evaluated before and after a year of treatment. RESULTS: After 1 year, the SPT response was reduced in the Dpt group (P=.03), whereas IgE levels to Der p 2 decreased only in the Dpt (P = .048) and Dpt+MRB (P = .005) groups. IgG4 and IgG1 levels to Dpt and Der p 1 increased in the Dpt group (P < .05), whereas in the Dpt + MRB group the IgG1 level only increased to Dpt (P=.001) and the IgG4 only increased to Der p 1 (P=.049). IgE levels to Dpt decreased only in the MRB (P= .005) and Dpt + MRB (P= .001) groups. Rhinitis symptom and medication scores fell in all groups, including the placebo group (P<.001). CONCLUSIONS: SIT using Dpt extract alone was effective in reducing SPT response and IgE levels to Der p 2 allergen, while bacterial extracts induced decreases in IgE levels to whole Dpt extract. However, only groups receiving Dpt allergen had higher levels of IgG1 and IgG4 to Dpt and Der p 1 after a year of treatment.

Taxis but not private cars are mite allergen reservoirs in Brazil.

Indoor allergens are major causative agents in allergic disease development. Besides homes, public transport vehicles have been considered important mite and pet allergen reservoirs. Our recent studies on allergen exposure in automobiles showed that different allergen levels are found in private cars versus taxis. We quantified group 1 Dermatophagoides spp. (Der 1), Felis domesticus (Fel d 1), and Canis familiaris (Can f 1) allergen levels by ELISA in dust samples from 60 taxi and 60 private car upholstered seats. Mean levels of Der 1 and Fel d 1 were significantly higher in taxis than private cars. A significantly higher percentage of taxis (42%) harboring sensitizing levels of Der 1 compared to private cars (5%) was also found. In spite of the low mean Fel d 1 levels, comparison of the percentage of vehicles with moderate Fel d 1 levels showed a significant difference between taxis and private cars (43% vs. 20%). On the other hand, mean Can f 1 levels were significantly higher in private cars compared to taxis concomitant with a significantly higher percentage of private cars containing moderate Can f 1 levels than taxis (53% vs. 28%). We conclude that upholstered seats from Brazilian taxis but not private cars constitute an important mite allergen reservoir. Thus, additional effective measures for the reduction of allergen exposure in vehicles within the global allergen avoidance strategy should also be routinely accomplished to minimize the induction of sensitization and symptoms in allergic patients.

Differential IgE reactivity to Der p 1 and Der p 2 allergens of Dermatophagoides pteronyssinus in mite-sensitized patients.

Several studies have shown that the presence of IgE antibodies to house dust mites (HDM), particularly Dermatophagoides pteronyssinus (Dpt), is an important risk factor for asthma. Allergen immunotherapy is indicated for patients with IgE antibodies to clinically relevant allergens. The aims of this study were to analyze the levels of specific serum IgE to Der p 1 and Der p 2 allergens in mite-sensitized atopic patients and to compare them with both in vivo (skin prick test) and in vitro (IgE-ELISA) sensitizations to Dpt crude extract. Forty-seven atopic patients with allergic rhinitis with or without intermittent or persistent mild asthma and positive skin prick test (SPT) to Dpt total extract were studied. Thirty age-matched healthy subjects with negative SPT to HDM were included as controls. Levels of total IgE and Dpt-, Der p 1- and Der p 2-specific IgE were measured by ELISAs in SPT-positive atopic patients and SPT-negative control subjects. Among 47 symptomatic atopic patients, 27 (57.4%) were double positive IgE to Der p 1 and Der p 2 allergens, 3 (6.4%) were single positive IgE to Der p 1, 4 (8.5%) were single positive IgE to Der p 2, and 13 (27.6%) were double negative IgE to both allergens. There was a significant correlation between Der p 1- and Der p 2-specific IgE levels, but not between Der p 1- or Der p 2-IgE levels and SPT results. The double negative IgE patients had the smallest skin test reactions although they showed high mean levels of total serum IgE. Therefore, the knowledge of specific IgE levels to Der p 1 and Der p 2 major allergens might support physicians for indication or follow-up in mite-sensitized patients under allergen-specific immunotherapy. These approaches might be important for obtaining improved safety and efficacy of the current clinical practice of allergen immunotherapy.

Responses of IgE, IgG1, and IgG4 to concanavalin A-binding Blomia tropicalis antigens in allergic patients.

Blomia tropicalis (Bt) and Dermatophagoides pteronyssinus (Dp) are the prevalent house dust mites in tropical countries and are associated with allergic diseases. Glycosylated antigens are highly immunogenic and involved in different pathologies. We evaluated the presence of IgE, IgG1, and IgG4 to concanavalin A-binding antigens (Bt-Con-A) isolated from Bt-total extract in sera of allergic and non-allergic subjects. Bt-total and Bt-Con-A extracts were evaluated by SDS-PAGE and ELISA for reacting with IgE, IgG1, and IgG4 in sera of 121 patients with allergic rhinitis and 36 non-allergic individuals. All subjects were skin prick tested with Bt-total extract and inhibition tests were performed for IgE, IgG1, and IgG4 using both extracts (Bt-total and Bt-Con-A). Skin prick test showed that 58% of the patients were sensitized to Bt (Bt+), with 52% reactive to both mites (Bt and Dp) and 6% to Bt only. A broad spectrum of proteins (14-152 kDa) was visualized in Bt-total and components >27 kDa for the Bt-Con-A extract. ELISA showed a similar profile of IgE, IgG1 and IgG4 levels in response to Bt-total and Bt-Con-A extracts in different groups, although Bt+ patients showed a lower IgG4 reactivity to Bt-Con-A extract. Specific IgG1 levels were higher in Bt+ patients than in control subjects, and IgG4 levels showed no significant difference among groups. ELISA inhibition showed a partial IgE and total IgG1 and IgG4 cross-reactivity with Dp extract for Bt-total and Bt-Con-A extracts. We conclude that Con-A-binding components isolated from Bt constitute major allergens and are involved in both allergen sensitization (IgE response) and homeostasis maintenance (IgG1 and IgG4 responses).

Effects of the protein kinase C stimulant bryostatin 1 on the proliferation and colony formation of irradiated human T-lymphocytes.

The protein kinase C stimulant bryostatin 1 (Bryo) was used in examining human peripheral blood T-lymphocyte radiosensitivities in proliferation assays. Bryo was similar to PMA in inducing T-cell proliferation by the CD3, CD28 and CD69 pathways. No difference in radiosensitivities was observed in T-cells stimulated by the three independent surface antigen-mediated activation pathways. CD3 was chosen as the second signal for comparing the potencies of the three different first signals Bryo, phorbol 12-myristate, 13-acetate (PMA), and interleukin 2 (IL-2) in stimulating T-cell proliferation and in maintaining this response after radiation. Though there were radioresponse differences among various individuals, the irradiated lymphocytes consistently showed significantly greater proliferation when treated with Bryo or PMA than with IL-2 (p < 0.05- < 0.005). No difference in proliferative responses was observed in T-cells irradiated between 4 h before and 15 h after the addition of stimulants. Colony forming assays showed higher colony survival for irradiated T-cells stimulated with Bryo than with PMA. These results support the important role of protein kinase C in T-cell radiation responses, and suggest a potential role for Bryo in enhancing T-lymphocyte survival during radiation therapy.

Mite allergen levels and acarologic analysis in house dust samples in Uberaba, Brazil.

Mite allergen exposure has been widely related to sensitization and development of allergic diseases. This study intended to evaluate the degree of allergen exposure in Uberaba, Brazil, through the measurements of Der f 1 and Der p 1 allergen levels associated with the acarologic analysis in house dust samples. A total of 240 dust samples were collected from 60 houses through vacuuming sofas and bedding, during the months of March and July 2000. Indoor temperature and relative humidity were also measured. Mites were counted and identified under light microscopy and allergen levels were measured by two-site monoclonal antibody ELISAs. The major mite family was Pyroglyphidae (39.4%), having D. pteronyssinus as the most frequent species (15.6%), followed by D. farinae (12.3%) and E. maynei (7.9%). The family Glycyphagidae was less commonly found (4.8%), with Blomia tropicalis (4.4%) as its majoritary member. The highest levels of Der f 1 and Der p 1 allergens were found in bedding samples in March (31.7 and 0.9 microg/g of dust, respectively), with Der f 1 levels significantly higher than Der p 1 (p < 0.0001). There was a significant positive correlation between the mite number and allergen levels. These results indicate that Dermatophagoides sp are the most frequent mites in our region followed by E. maynei. Therefore, the knowledge of the local mite fauna would improve the means of investigating the association between allergen exposure and sensitization, allowing the addition of new mite extracts in diagnostic tests.

Spin-induced band modifications of graphene through intercalation of magnetic iron atoms.

Intercalation of magnetic iron atoms through graphene formed on the SiC(0001) surface is found to induce significant changes in the electronic properties of graphene due mainly to the Fe-induced asymmetries in charge as well as spin distribution. From our synchrotron-based photoelectron spectroscopy data together with ab initio calculations, we observe that the Fe-induced charge asymmetry results in the formation of a quasi-free-standing bilayer graphene while the spin asymmetry drives multiple spin-split bands. We find that Fe adatoms are best intercalated upon annealing at 600 °C, exhibiting split linear π-bands, characteristic of a bilayer graphene, but much diffused. Subsequent changes in the C 1s, Si 2p, and Fe 3p core levels are consistently described in terms of Fe-intercalation. Our calculations together with a spin-dependent tight binding model ascribe the diffuse nature of the π-bands to the multiple spin-split bands originated from the spin-injected carbon atoms residing only in the lower graphene layer.

Humoral and cellular immune responses to Blomia tropicalis and concanavalin A-binding fractions in atopic patients.

Blomia tropicalis, Dermatophagoides pteronyssinus and D. farinae are prevalent house dust mites. Concanavalin A-binding components derived from B. tropicalis (Bt-ConA extract) are highly immunogenic in allergic diseases. The aim of the present study was to evaluate the humoral and cellular immune responses to B. tropicalis in mite-sensitized patients. A total of 137 patients with allergic rhinitis with/without asthma and 109 non-atopic subjects were selected and analyzed by the skin prick test, and for total serum IgE and specific IgE levels to both Bt-total and Bt-ConA extracts, their proliferative response and cytokine (IFN-gamma and IL-5) production by peripheral blood mononuclear cells (PBMC) stimulated with both extracts. Skin prick test showed that 70% of the patients were sensitized to Bt (Bt+) and similar levels of specific IgE to Bt-total and Bt-ConA extracts were demonstrable in Bt+ patients. Significant PBMC proliferation was observed in response to Bt-total extract in Bt+, but not in Bt- patients and non-atopic subjects (P < 0.001). Bt-ConA extract induced increased proliferative responses in all patient groups compared to medium alone (P < 0.05), but these responses were significantly decreased in the presence of the mannopyranoside ConA inhibitor (P < 0.05). Significant IFN-gamma production was observed after Bt-ConA stimulation of Bt+ patients (P < 0.05), while Bt-total extract had no effect. IL-5 production was consistently detected in Bt+ patients after allergen-specific stimulation or with no stimulus, indicating that PBMC from allergic patients are prone to produce Th2 profile cytokines, spontaneously or inductively by allergen restimulation. These data showed that ConA-binding components isolated from B. tropicalis may contain relevant antigens that are involved in both humoral and cellular immune responses. However, without an additional purification procedure to eliminate the residual contamination with ConA, its use in immunotherapeutic procedures cannot be recommended.

Mite and pet allergen exposure in hotels in Uberlândia, Midwestern Brazil.

UNLABELLED: Mite allergens are involved in airway sensitization and allergic diseases. We evaluated the exposure to house dust-mite (Der p 1 and Der f 1) and pet (Fel d 1 and Can f 1) allergens in hotels in Uberlândia, Midwestern Brazil. A total of 140 dust samples were collected from bedding (n = 98) and carpet (n = 42) of bedrooms in 20 hotels enrolled for this study. Geometric mean (GM) levels of Der f 1 (11.30 microg/g of dust; 95% CI: 8.34-15.30 microg/g) were significantly higher than Der p 1 (0.15 microg/g of dust; 95% CI: 0.13-0.18 microg/g) in bedding dust samples (P < 0.001), regardless of the hotel classes. Der f 1 levels were significantly higher in bedding (11.30 microg/g of dust; 95% CI: 8.34-15.30 microg/g) than carpet (6.32 microg/g of dust; 95% CI: 4.31-9.26 microg/g) dust samples (P < 0.05). High levels of Der f 1 (>10 microg/g of dust) were found in 58%, 76%, and 69% of dust samples from Simple, Economical, and Tourist/Superior hotels, respectively, while GM levels of Fel d 1 (0.11 microg/g of dust; 95% CI: 0.09-0.14 microg/g) and Can f 1 (0.30 microg/g of dust; 95% CI: 0.20-0.44 microg/g) were relatively low. These results indicate that Der f 1 is the predominant allergen in hotels in Uberlândia, especially in bedding dust samples, regardless of the hotel classes and could represent an important risk factor for exacerbation of allergic symptoms in previously mite-sensitized guests. PRACTICAL IMPLICATIONS: Mites and pets are important sources of indoor allergens. Most people spend the greatest part of their time indoors. Hotels can constitute an important allergen reservoir of the indoor environment and could represent an important risk for exacerbation of allergic symptoms in previously sensitized guests. Thus, hotels should also be included for planning indoor allergen avoidance as part of a global management strategy, especially in patients with respiratory allergy.

IgE, IgG1, and IgG4 antibody responses to Blomia tropicalis in atopic patients.

BACKGROUND: Allergens from house dust mites (HDMs), Dermatophagoides pteronyssinus and Blomia tropicalis are clinically relevant in atopic respiratory diseases in tropical countries. AIMS OF THE STUDY: To evaluate immunoglobulin (Ig)E, IgG1, and IgG4 antibody responses to B. tropicalis in Brazilian atopic patients. METHODS: About 110 patients with allergic rhinitis with/without asthma and 33 control subjects underwent skin prick testing (SPT) with HDM extracts, and their sera were tested for IgE and IgG subclass antibodies to D. pteronyssinus and B. tropicalis by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. RESULTS: Most patients (56%) had positive SPT to B. tropicalis extract (B. tropicalis+ group), although 51% were reactive to both B. tropicalis and D. pteronyssinus and 6% were sensitized to B. tropicalis only. IgE-ELISA detected 43%B. tropicalis positivity with high-specific IgE levels in B. tropicalis+ patients. Specific IgG4 levels were higher in B. tropicalis+ than B. tropicalis- groups and correlated with specific IgE levels. The IgG1 levels to B. tropicalis were higher in patients than controls. The major allergenic B. tropicalis components recognized by B. tropicalis+ patient sera were the 54, 66, and 68 kDa proteins. The IgG4-binding protein profiles closely resembled that of IgE. The IgG1 antibodies recognizing multiple B. tropicalis protein species were detected in sera of all three patient groups. CONCLUSIONS: A large percentage of our allergic patients are B. tropicalis+. They are more frequently sensitized to high-molecular weight (HMW) B. tropicalis components than the major low-molecular weight (11-15 kDa) allergens detected in other studies. The results suggest that HMW B. tropicalis antigenic components are potential candidates for evaluating allergen exposure and sensitization, and for immunotherapy treatment.

Stimulation of interleukin-1 alpha and interleukin-1 beta production in human monocytes by protein phosphatase 1 and 2A inhibitors.

Protein phosphatases 1 and 2A are important in regulating cellular functions by controlling the phosphorylation state of their substrates. In human monocytes, the inhibitors of these phosphatases, okadaic acid and calyculin A, were found to increase the mRNA accumulation and cytokine production of interleukin-1 beta and interleukin-1 alpha. The increased mRNA accumulation was found to be primarily because of the increase in the transcription rate of the interleukin-1 genes. Stimulation of interleukin-1 gene transcription may be caused by the stimulation of transcription factor activities, including those of AP-1, by these protein phosphatase inhibitors. Okadaic acid increased the synthesis of the interleukin-1 beta precursor and mature forms and their secretion. This increased processing and secretion correlated with the stimulation of IL-1 beta convertase mRNA accumulation. The stimulation of interleukin-1 alpha production by okadaic acid was more modest than that of interleukin-1 beta. However, the phosphorylation of the precursor interleukin-1 alpha cytokine was increased. These results show that protein phosphatase 1 and 2A inhibitors exert multiple effects on cytokine production in human monocytes and suggest that these two phosphatases play important roles in regulating interleukin-1 production.

Drug resistance, R plasmids and pigmentation of Serratia marcescens isolated in Taiwan.

The Serratia marcescens isolates used in this study were resistant to ampicillin, tetracycline or cephalothin, streptomycin, tobramycin, kanamycin, carbenicillin, chloramphenicol and gentamicin in descending order. Nalidixic acid was the most effective antibiotic against S. marcescens, followed by amikacin and sulfamethoxazole-trimethoprim. The non-pigmented plasmid-carrying isolates displayed higher resistance to some antimicrobial agents than did the pigmented isolates and plasmid-free white isolates. Nine out of 12 resistant markers were coded by plasmids in S. marcescens. The average number of resistant markers per strain was seven for plasmid-containing white isolates as compared to four for other S. marcescens groups. About 73% of S. marcescens contained plasmids. Thirty eight percent of plasmid-carrying S. marcescens spread their R plasmids to E. coli. Conjugative R plasmids were identified in six out of 17 strains of S. marcescens, which apparently contained a single plasmid.

Inhibition of macrophage phagocytosis by methylation inhibitors. Lack of correlation of protein carboxymethylation and phospholipid methylation with phagocytosis.

Adenosine (Ado), deoxyadenosine (dAdo), and adenine arabinoside (AraA) inhibit the phagocytosis of IgG-coated erythrocytes and zymosan by resident and thioglycollate-elicited macrophages (thio-macrophages) in a dose-dependent and reversible manner. 3-Deazaadenosine (3cAdo) and adenine (Ade) also inhibit the phagocytosis by resident macrophages. Homocysteine thiolactonate (Hcy) potentiates the inhibition by Ado and 3cAdo while erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) potentiates the inhibition by Ado, dAdo and AraA. This inhibition has a very rapid onset and the drugs do not interfere with the binding of IgG-coated erythrocytes to macrophages. The combination of Ado, Hcy and EHNA does not appreciably affect the intracellular level of ATP and S-adenosyl-L-methionine (AdoMet) in thio-macrophages but causes accumulations of Ado and S-adenosyl-L-homocysteine (AdoHcy) up to 135 and 145 nmol/mg of protein, respectively. During phagocytosis reversal, Ado is metabolized within 15 min while AdoHcy decreases log-arithmically with a half-life of 50 min. Carboxymethylation and phospholipid methylation, however, resume about 60-90 min after phagocytosis has recovered, and thus cannot function as transmembrane signals for phagocytosis. Other evidence showing the lack of correlation between phagocytosis and carboxymethylation inhibition include 1) Ado + Hcy inhibit carboxymethylation much better than Ado + EHNA (91 versus 75%) in thio-macrophage, but the two combinations show comparable phagocytosis inhibition potency; 2) Ado + Hcy inhibit carboxymethylation almost as well as Ado + Hcy + EHNA, but the latter is a much more effective drug combination for phagocytosis inhibition; 3) Ade and 3cAdo, although inhibiting resident macrophage phagocytosis as well as Ado + EHNA + Hcy, are much weaker carboxymethylation inhibitors; 4) dAdo and AraA potently inhibit phagocytosis but not carboxymethylation. The difference in the apparent methylation levels is not due to changes in the specific activities of AdoMet, which decrease with a half-life of 88 min. Interestingly, after the initial lag phase of about 90 min after the initiation of inhibition reversal, carboxymethylation and phagocytosis increase in parallel. In a log-log plot of carboxymethylation, phospholipid methylation, or phagocytosis versus the intracellular AdoHcy accumulation, a linear relationship is obtained. It is possible that AdoHcy accumulation is responsible for phagocytosis inhibition but inhibits by a mechanism other than interfering with protein and lipid methylations.

Assessment of skeletal and dental changes by maxillary protraction.

To further evaluate the effect of maxillary protraction on facial growth, cephalometric changes in 129 subjects with conditions diagnosed as skeletal Class III malocclusion and who had been treated with maxillary protraction were compared to 9 male and 12 female subjects with annual cephalometric records from the Yonsei growth study sample. The control subjects had Angle Class I malocclusions with normal overjet and overbite. More maxillary forward displacement and mandibular growth inhibition were observed in the protraction group during treatment, and the difference from the untreated controls was statistically significant. When changes due to treatment according to ages were compared, there was no statistical difference. The direction of maxillary growth was similar in the untreated and protraction groups. Maxillary protraction had a growth-stimulating effect on the maxilla during the treatment period.

Combined treatments of heat, radiation, or cytokines with flavone acetic acid on the growth of cultured endothelial cells.

The antitumour effects of flavone acetic acid (FAA) against a broad spectrum of established experimental tumours has been demonstrated. Damage to the vasculature, which rapidly disrupts blood flow and induces haemorrhagic necrosis, is believed to be a major mechanism contributing to the observed antitumour effects. Despite these established observations, FAA has shown little effect against human tumours. However, other applications of FAA, for examples, for an extended period of treatments or in combination with other antitumour modalities, have not been sufficiently explored. In order to test the direct effects of FAA on vasculature, endothelial cells isolated from human umbilical vein (HUVEC) and bovine pulmonary artery (CPAEC) were used in this study. FAA at the concentrations of 50 to 200 micrograms/ml causes reduction in cell number (from 20 to > 30% of the cells) of HUVEC as measured by MTT assay after 1, 3, and 5 h of treatment at 37 degrees C. FAA did not produce significant effects on similarly treated human squamous cell carcinoma, cell line UM-SCC-2. After 1 h treatment of FAA at 300 micrograms/ml, a large number of HUVECs failed to react with an actin stain, NBD-phallacidin. The growth of HUVECs and CPAEC in the presence of FAA for 1-3 days was progressively reduced. The number of HUVEC treated for 3 days at the concentrations of 100, 200, and 300 micrograms/ml were reduced by 75-86% in comparison with the control culture. The experiments with CPAEC showed similar results. The inhibition of the growth of endothelial cells by FAA was enhanced when it combines with tumour necrosis factor-alpha but not with interleukin-1, interferon-gamma, heat, or radiation. We observed that FAA can initiate both immediate effects and growth inhibition on cultured endothelial cells. These results support the notion that FAA rapidly induces vasculature damage. Furthermore, cytokines such as tumour necrosis factor-alpha can enhance the toxicity of FAA on endothelial cells.

Vascular cambial sucrose metabolism and growth in loblolly pine (Pinus taeda L.) in relation to transplanting stress.

Sucrose synthase (SS) was the dominant enzyme of sucrose metabolism in both stem and root vascular cambial zone tissues of nursery-grown loblolly pine (Pinus taeda L.) seedlings. Acid invertase (AI) and neutral invertase (NT) activities were generally less than 10% of the SS activity in both tissues. In both cambial tissues, seasonal patterns of enzyme activity were observed for SS but not for AI or NI. The seasonal patterns of SS activity in stem and root cambia paralleled the periodic growth of stems and roots. Stems had high SS activity and growth during summer and early fall. Roots had substantial SS activity and growth during summer and fall, but SS activity and growth were even higher in winter. When seedlings were transplanted, about eight months elapsed before stem and root cambia resumed rates of growth and sucrose metabolism similar to those in control nontransplanted seedlings. Two months after transplanting, root SS was at its lowest, whereas AI activity in transplants was 50% higher than in control nontransplanted seedlings. In stems, SS activity decreased in response to transplanting, whereas AI and NI activities did not change appreciably. In loblolly pine tissues, SS was specific for uridylates, whereas the nucleotide triphosphate-dependent phosphofructokinase (NTP-PFK) had similar activity with either UTP or ATP. Except in winter, the NTP-PFK was less active than the pyrophosphate-dependent phosphofructokinase (PPi-PFK) during all seasons. The PPi-dependent PFK activity in nontransplanted seedlings followed similar seasonal and spatial patterns to those of SS activity. In actively growing tissues, such as stem cambial tissues in summer and root cambial tissues in winter, the measured total PFK to SS ratio ranged between 1.5/1 and 3/1. In contrast, in less actively growing tissues or transplanted seedlings, a greater decrease occurred in SS than in PFK activity, hence the ratio rose to as high as 12/1. It was concluded that: (1) SS was the dominant enzyme for sucrose metabolism in root and stem cambial tissues of loblolly pine seedlings; (2) both SS and PPi-PFK in the cambial tissues can be used as biochemical indicators of growth sink strength in stems and roots; and (3) both enzymes can be used as indicators of seedling stress caused by events such as transplanting and winter freezing.

Sucrose metabolism in lima bean seeds.

Developing and germinating lima bean (Phaseolus lunatus var Cangreen) seeds were used for testing the sucrose synthase pathway, to examine the competition for uridine diphosphate (UDP) and pyrophosphate (PPi), and to identify adaptive and maintenance-type enzymes in glycolysis and gluconeogenesis. In developing seeds, sucrose breakdown was dominated by the sucrose synthase pathway; but in the seedling embryos, both the sucrose synthase pathway and acid invertase were active. UDPase activity was low and seemingly insufficient to compete for UDP during sucrose metabolism in seed development or germination. In contrast, both an acid and alkaline pyrophosphatase were active in seed development and germination. The set of adaptive enzymes identified in developing seeds were sucrose synthase, PPi-dependent phosphofructokinase, plus acid and alkaline pyrophosphatase; and, the adaptive enzymes identified in germinating seeds included the same set of enzymes plus acid invertase. The set of maintenance enzymes identified during development, in the dry seed, and during germination were UDP-glucopyrophosphorylase, neutral invertase, ATP and UTP-dependent fructokinase, glucokinase, phosphoglucomutase, ATP and UTP-dependent phosphofructokinase and sucrose-P synthase.

Identification of actively filling sucrose sinks.

Certain actively filling plant sucrose sinks such as a seed, a tuber, or a root can be identified by measuring the uridine diphosphate and pyrophosphate-dependent metabolism of sucrose. Sucrolysis in both active and quiescent sucrose sinks was tested and sucrose synthase was found to be the predominant sucrose breakdown activity. Sucrolysis via invertases was low and secondary in both types of sinks. Sucrose synthase activity dropped markedly, greater than fivefold, in quiescent sinks. The tests are consistent with the hypothesis that the sucrose filling activity, i.e. the sink strength, of these plant sinks can be measured by testing the uridine diphosphate and pyrophosphate-dependent breakdown of sucrose. Measuring the initial reactions of sucrolysis shows much promise for use in agriculture crop and tree improvement research as a biochemical test for sink strength.