The Use of PTI-Marker Genes to Identify Novel Compounds that Establish Induced Resistance in Rice.

Compounds that establish induced resistance (IR) in plants are promising alternatives for the pesticides that are progressively being banned worldwide. Screening platforms to identify IR-establishing compounds have been developed, but none were specifically designed for monocot plants. Here, we propose the use of an RT-qPCR screening platform, based on conserved immunity marker genes of rice as proxy for IR induction. Central regulators of biotic stress responses of rice were identified with a weighted gene co-expression network analysis (WGCNA), using more than 350 microarray datasets of rice under various sorts of biotic stress. Candidate genes were narrowed down to six immunity marker genes, based on consistent association with pattern-triggered immunity (PTI), both in rice plants as in rice cell suspension cultures (RCSCs). By monitoring the expression of these genes in RCSCs upon treatment with candidate IR-inducing compounds, we showed that our marker genes can predict IR induction in rice. Diproline, a novel IR-establishing compound for monocots that was detected with these marker genes, was shown to induce rice resistance against root-knot nematodes, without fitness costs. Gene expression profiling of the here-described PTI-marker genes can be executed on fully-grown plants or in RCSCs, providing a novel and versatile tool to predict IR induction.

The Toxic Effects of Aflatoxin B1 and Aflatoxin M1 on Kidney through Regulating L-Proline and Downstream Apoptosis.

The toxic effects and potential mechanisms of aflatoxin B1 (AFB1), aflatoxin M1 (AFM1), and AFB1+AFM1 in the kidney were studied and compared in HEK 293 cells model and CD-1 mice model. The 35-day subacute toxicity mice model was constructed, biochemical indicators and kidney pathological staining were detected, kidney metabonomics detection was performed, and the metabolites were analyzed, and then the related toxicity mechanism was validated. Results showed that AFB1 (0.5 mg/kg), AFM1 (3.5 mg/kg), and AFB1 (0.5 mg/kg)+AFM1 (3.5 mg/kg) activated oxidative stress and caused renal damage. The relative concentration of the metabolite L-proline was found to be lower in aflatoxins treatment groups when compared with the control (P < 0.05). Moreover, with the treatment of aflatoxins, proline dehydrogenase (PRODH) and proapoptotic factors (Bax, Caspase-3) were upregulated, while the inhibitor of apoptosis Bcl-2 was downregulated, at both the mRNA and the protein levels, comparing with the control (P < 0.05). In addition, the combined effect of AFB1 and AFM1 was validated, for the toxicity of the combination was stronger than the other two groups. In conclusion, AFB1 and AFM1 caused kidney toxicity by activating oxidative stress through altering expression of PRODH and L-proline levels, which then induced downstream apoptosis.

Inhibitory effects of low decibel infrasound on the cardiac fibroblasts and the involved mechanism.

INTRODUCTION: Infrasound is a mechanical vibration wave with frequency between 0.0001 and 20 Hz. It has been established that infrasound of 120 dB or stronger is dangerous to humans. However, the biological effects of low decibel infrasound are largely unknown. The purpose of this study was to investigate the effects of low decibel infrasound on the cardiac fibroblasts. MATERIALS AND METHODS: The cardiac fibroblasts were isolated and cultured from Sprague-Dawley rats. The cultured cells were assigned into the following four groups: control group, angiotensin II (Ang II) group, infrasound group, and Ang II+infrasound group. The cell proliferation and collagen synthesis rates were evaluated by means of [3H]-thymidine and [3H]-proline incorporation, respectively. The levels of TGF-ß were determined by enzyme-linked immunosorbent assay. Moreover, RNAi approaches were used for the analysis of the biological functions of miR-29a, and the phosphorylation status of Smad3 was detected using western blotting analysis. RESULTS: The results showed that low decibel infrasound significantly alleviated Ang II-induced enhancement of cell proliferation and collagen synthesis. DISCUSSION: Compared with the control, Ang II markedly decreased the expression of miR-29a levels and increased the secretion of TGF-ß and phosphorylation of Smad3, which was partly reversed by the treatment with low decibel infrasound. Importantly, knockdown of miR-29a diminished the effects of infrasound on the cardiac fibroblasts. In conclusion, low decibel infrasound inhibits Ang II-stimulated cardiac fibroblasts via miR-29a targeting TGF-ß/Smad3 signaling.

Silicon alleviates salt and drought stress of Glycyrrhiza uralensis seedling by altering antioxidant metabolism and osmotic adjustment.

This study was conducted to determine effect and mechanism of exogenous silicon (Si) on salt and drought tolerance of Glycyrrhiza uralensis seedling by focusing on the pathways of antioxidant defense and osmotic adjustment. Seedling growth, lipid peroxidation, antioxidant metabolism, osmolytes concentration and Si content of G. uralensis seedlings were analyzed under control, salt and drought stress [100 mM NaCl with 0, 10 and 20% of PEG-6000 (Polyethylene glycol-6000)] with or without 1 mM Si. Si addition markedly affected the G. uralensis growth in a combined dose of NaCl and PEG dependent manner. In brief, Si addition improved germination rate, germination index, seedling vitality index and biomass under control and NaCl; Si also increased radicle length under control, NaCl and NaCl-10% PEG, decreased radicle length, seedling vitality index and germination parameters under NaCl-20% PEG. The salt and drought stress-induced-oxidative stress was modulated by Si application. Generally, Si application increased catalase (CAT) activity under control and NaCl-10% PEG, ascorbate peroxidase (APX) activity under all treatments and glutathione (GSH) content under salt combined drought stress as compared with non-Si treatments, which resisted to the increase of superoxide radicals and hydrogen peroxide caused by salt and drought stress and further decreased membrane permeability and malondialdehyde (MDA) concentration. Si application also increased proline concentration under NaCl and NaCl-20% PEG, but decreased it under NaCl-10% PEG, indicating proline play an important role in G. uralensis seedling response to osmotic stress. In conclusion, Si could ameliorate adverse effects of salt and drought stress on G. uralensis likely by reducing oxidative stress and osmotic stress, and the oxidative stress was regulated through enhancing of antioxidants (mainly CAT, APX and GSH) and osmotic stress was regulated by proline.

The effects of cadmium on the biochemical and physiological parameters of Eruca sativa.

In this study, Eruca sativa (Rocket) seedlings were treated with different cadmium (Cd) concentrations (0, 150, 300 and 450 µg ∙ g-1). The effects of Cd on lipid peroxidation, enzymatic (APx, CAT, GPX, SOD) and non-enzymatic antioxidants (total ascorbate, dehydroascorbate, ascorbate, non-protein thiol), fresh and dry masses, water content were determined. Also, Cd content of the leaves and the roots were analysed. The highest cadmium accumulation of leaves was at 450 µg ∙ g-1 Cd treatment and the accumulation was 2.62 times greater than those in the roots. The translocation factor was 3.89 at 300 µg ∙ g-1 Cd treatment. Cd treatments caused decreases of fresh, dry mass and water content of leaves and roots. Malondialdehyde content, which is an index of lipid peroxidation, was increased in proportion with the increase in Cd. While there was not change in the activity of GPX according to control, a decrease in activities of SOD, CAT and APX were observed with the increase of cadmium concentration. Although a significant increase in the amounts of non-protein thiol groups and proline were observed in 450 µg ∙ g-1 Cd treated plants, Cd did not lead to a significant change in AsA, DHA and total AsA contents. According to the results of the research, E. sativa may be a Cd hyperaccumulator plant and we suggest that the plant may be a candidate plant for remediation of Cd-contaminated soil.

Analysis of salt-induced physiological and proline changes in 46 switchgrass (Panicum virgatum) lines indicates multiple response modes.

Switchgrass (Panicum virgatum) is targeted as a biofuel feedstock species that may be grown on marginal lands including those with saline soils. Our study investigated salt stress responses in 46 switchgrass lines from the lowland and upland ecotypes by assessing physiological phenotypes and proline concentrations. Lowland switchgrass lines demonstrated less severe responses to salt stress than most upland switchgrass lines, but a number of upland lines performed as well as lowland individuals. Photosynthetic rate (Pn), the most important physiological trait measured, was reduced by salt treatment in all lines. Tolerant lines showed ∼50% reduction in Pn under salt stress, and sensitive lines exhibited ∼90% reduction in Pn after salt stress. Proline analysis showed the largest amount of variation under salt stress with some lines exhibiting minor increases in proline, but some salt-sensitive lines demonstrated more than 5000-fold increase in proline concentration in response to salt treatment. Clustering of salt-stress phenotypic responses revealed five groups of switchgrass. Lowland lines were present in two of the phenotypic clusters, but upland lines were found in all five of the phenotypic clusters. These results suggest that there are multiple modes of salt response in switchgrass including two distinct modes of salt tolerance.

Role of reactive oxygen species and proline cycle in anthraquinone accumulation in Rubia tinctorum cell suspension cultures subjected to methyl jasmonate elicitation.

Elicitors are compounds or factors capable of triggering a defense response in plants. This kind of response involves signal transduction pathways, second messengers and events such as Reactive Oxygen Species (ROS) generation, proline accumulation and secondary metabolite production. Anthraquinone (AQs) biosynthesis in Rubia tinctorum L. involves different metabolic routes, including shikimate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADP+ generated by this cycle could act as a cofactor of the first enzymes of the PPP. The end-product of this pathway is erithrose-4-phosphate, which becomes the substrate of the shikimate pathway. The aim of this work was to study the effect of methyl jasmonate (MeJ), a well-known endogenous elicitor, on the PPP, the proline cycle and AQs production in R. tinctorum cell suspension cultures, and to elucidate the role of ROS in MeJ elicitation. Treatment with MeJ resulted in AQs as well as proline accumulation, which was mimicked by the treatment with a H2O2-generating system. Both MeJ-induced effects were abolished in the presence of diphenyliodonium (DPI), a NADPH oxidase inhibitor (main source of ROS). Treatment with the elicitor failed to induce PPP; therefore, this route did not turn out to be limiting the carbon flux to the shikimate pathway.

Trehalose inhibits inflammatory cytokine production by protecting IkappaB-alpha reduction in mouse peritoneal macrophages.

OBJECTIVE: The aim of this study was to examine whether trehalose, a disaccharide, could inhibit Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS)-enhanced production of inflammatory cytokines in mouse peritoneal macrophages. DESIGN: Mouse peritoneal macrophages were treated with trehalose and stimulated with P. gingivalis LPS. Interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) levels in the culture supernatant were measured by ELISA. The mRNA levels of the cytokines in macrophages were analysed by semi-quantitative RT-PCR. DNA and protein synthesis were measured by incorporation of [(3)H] thymidine or [(14)C] praline into mouse peritoneal macrophages. IkappaB-alpha reductions were assessed by western blot. RESULTS: Treatment with trehalose suppressed LPS-induced IL-1beta and TNF-alpha production and downregulated transcription of these cytokines. Furthermore, trehalose inhibited LPS-induced reduction of IkappaB-alpha. In addition, we also observed expression of the trehalose receptor (T1R3) in mouse peritoneal macrophages. CONCLUSION: These results may suggest that trehalose inhibits LPS-induced production of IL-1beta and TNF-alpha in mouse peritoneal macrophages by inhibiting degradation of IkappaB-alphavia the trehalose receptor T1R3.

Effects of selenium on wheat seedlings under drought stress.

The paper reports the effects of selenium (Se) supply on growth and some physiological traits of wheat (Triticum aestivum L. cv Shijiazhuang NO. 8) seedlings exposed to drought stress. The growth and physiological responses of seedlings were different depending on the Se concentration. The higher (3.0 mg Se kg(-1)) and lower amount used (0.5 mg Se kg(-1)) did not significantly affect on biomass accumulation. Treatments with 1.0 and 2.0 mg Se kg(-1) promoted biomass accumulation of wheat seedlings. Treatments at 1.0, 2.0, and 3.0 mg Se kg(-1) significantly increased root activity, proline content, peroxidase (POD), and catalase (CAT) activities, carotenoids (Car) content, chlorophyll content, and reduced malondialdehyde (MDA) content of wheat seedlings. Lower Se treatment did not significantly effect on chlorophyll content and MDA content, although it also increased some antioxidant index (proline and Car content, POD and CAT activities) in wheat seedlings. These results suggest that optimal Se supply is favorable for growth of wheat seedlings during drought condition.

Hyperprolinemia is a risk factor for schizoaffective disorder.

DNA sequence variations within the 22q11 DiGeorge chromosomal region are likely to confer susceptibility to psychotic disorders. In a previous report, we identified several heterozygous alterations, including a complete deletion, of the proline dehydrogenase (PRODH) gene, which were associated with moderate hyperprolinemia in a subset of DSM III schizophrenic patients. Our objective was (i) to determine whether hyperprolinemia is associated with increased susceptibility for any of three psychiatric conditions (schizophrenia, schizoaffective disorder and bipolar disorder) and (ii) to establish a correlation between hyperprolinemia and PRODH genotypes. We have conducted a case-control study including 114 control subjects, 188 patients with schizophrenia, 63 with schizoaffective disorder and 69 with bipolar disorder. We report that, taking into account a confounding effect due to valproate treatment, hyperprolinemia is a risk factor for DSM IIIR schizoaffective disorder (P=0.02, Odds ratio=4.6, 95% confidence interval 1.3-16.3). We did not detect 22q11 interstitial deletions associated with the DiGeorge syndrome among the 320 patients of our sample and we found no association between common PRODH polymorphisms and any of the psychotic disorders. In contrast, we found that five rare PRODH alterations (including a complete PRODH deletion and four missense substitutions) were associated with hyperprolinemia. In several cases, two variations were present simultaneously, either in cis or trans in the same subject. A total of 11 from 30 hyperprolinemic subjects bore at least one genetic variation associated with hyperprolinemia. This study demonstrates that moderate hyperprolinemia is an intermediate phenotype associated with certain forms of psychosis.

Stress reactions in Vitis vinifera L. following soil application of the herbicide flumioxazin.

In order to evaluate the stress effects of flumioxazin (fmx) on grapevine, a non-target plant (Vitis vinifera L.), physiological parameters such as carbohydrate content, water status or nitrogenous metabolites were investigated on fruiting cuttings and plants grown in vineyard. In the leaves of cuttings, the soil-applied herbicide induced stress manifestations including a decrease of the dry weight percentage and the soluble carbohydrate content during the first week after treatment. Thereafter, a decrease of the osmotic potential was observed, as well as a decrease of total protein content and a parallel accumulation of free amino acids, including proline. Altogether, these results suggest that soil-applied fmx induced a stress in grapevines, leading to leaf proteolysis. However, this stress was partially recovered 3 weeks after herbicide application, suggesting that the cuttings were capable to adapt to the fmx exposure. In the vineyard, the flumioxazin effects were still significant 5 months after the treatment, particularly in the CH cv. They included a decrease of the leaf dry weight percentage and soluble carbohydrate content, as well as an increase of the osmotic potential. The decrease of leaf soluble carbohydrates may have dramatic consequences for the berry growth and the reserve constitution. Moreover, treated plants were characterized by a decrease of the free amino acid content and an accumulation of ammonium, while the protein level did not significantly increase, suggesting a degradation of amino acids. The alteration of carbon and nitrogen status after herbicide treatment may affect the grapevine vigour in a long term.

Pin1 acts catalytically to promote a conformational change in Cdc25.

Pin1 is an essential protein that can peptidyl-prolyl-isomerize small phosphopeptides. It has been suggested that Pin1 regulates entry into mitosis by catalyzing the cis/trans-isomerization of prolines on critical protein substrates in response to phosphorylation. We show that Pin1 catalytically generates a conformational change on the mitotic phosphatase Cdc25, as assayed by limited protease digestion, differential reactivity to a phosphoserine-proline-directed monoclonal antibody (MPM-2), and by changes in Cdc25 enzymatic activity. Pin1 catalytically modifies the conformation of Cdc25 at stoichiometries less than 0.0005, and mutants of Pin1 in the prolyl isomerase domain are not active. We suggest that, although difficult to detect, phosphorylation-dependent conformational changes mediated by prolyl isomerization may play an important regulatory role in the cell cycle.

The mechanism for anthracycline-induced inhibition of collagen biosynthesis.

One of the recognized side effects accompanying anti-neoplastic anthracyclines administration is poor wound healing resulting from impairment of collagen biosynthesis. However, the precise mechanism of anthracyclines-induced inhibition of collagen synthesis has not been established. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for anthracyclines-induced inhibition of synthesis of this protein. Prolidase [EC 3.4.13.9] cleaves imidodipeptides containing C-terminal proline, providing large amount of proline for collagen synthesis. Therefore, we compared the effect of daunorubicin and doxorubicin on prolidase activity and collagen biosynthesis in confluent cultured human skin fibroblasts. We have found that daunorubicin and doxorubicin coordinately induced the inhibition of prolidase activity (IC(50)=0.3 and 10 microM, respectively) and collagen biosynthesis (IC(50)=1 and 15 microM, respectively) in cultured human skin fibroblasts. The inhibitory effect of daunorubicin or doxorubicin on prolidase activity and collagen biosynthesis was not due to anti-proliferative activity of these drugs as shown by cell viability tetrazoline test. The decrease in prolidase activity due to the treatment of confluent cells with the anthracyclines was not accompanied by any difference in the amount of enzyme protein recovered from these cells as shown by Western immunoblot analysis. It may be suggested that the inhibition is a post-translational event. Since prolidase is metalloprotease, requiring manganese for catalytic activity, and anthracyclines are known as chelators of divalent cations, we considered that the chelating ability of anthracyclines might be an underlying mechanism for the anthracyclines-induced inhibition of prolidase activity. In order to determine the ability of daunorubicin or doxorubicin to form complexes with manganese (II), potentiometric method was employed based on the measurement of protonation constant by pH-metric titrated assay. We have found that both anthracyclines form stable complexes with manganese (II). The composition of the daunorubicin-Mn(II) complex was calculated as 3:1 while that of doxorubicin-Mn(II) complex was 2:1. The constant stability value for the investigated complexes were calculated as beta(av)=(1.74+/-0.01)x10(23) for daunorubicin, and beta(av)=(1.99+/-0.025)x10(11) for doxorubicin. The higher ability of daunorubicin vs. doxorubicin to chelate manganese and inhibit prolidase activity may explain the potential mechanism for its greater potency to inhibit collagen biosynthesis.

[Experimental aortic aneurysm]. / Eksperymentalny tetniak aorty.

Experimental aortic aneurysm may be evoked in animals by application of chemical compounds disturbing biosynthesis, posttranslating modifications and degradation of elastin and collagen, local damage of the aortic wall by chemical, thermal and mechanical factors and by increased blood pressure. Administration of protease inhibitors, anti-inflammatory drugs, agents enhancing synthesis and formation of elastic and collagen fibers and hypotensive drugs prevent formation enlargement and rupture of aneurysm.

Effects of TGFbeta2 on collagen synthesis in cultured normal and wounded fetal mouse palates.

OBJECTIVE: It has been demonstrated in a number of models that fetal wounds heal with little or no scar. Since collagen is an integral part of the extracellular matrix in adult scar formation, we studied the synthesis and localization of collagen in an in vitro mouse palate model for fetal wound healing. METHODS: Palates, dissected from fetal mice at 15, 16, and 17 days of gestation and from newborn mice, were cultured in medium containing serum (for 8 hours); this was followed by culture in serum-free medium (for 12 hours). One-half of the samples from each age group were wounded in the midline. All samples were placed in serum-free medium containing 20 microCi/mL 3H-proline for 8 hours. In addition, palates from 15-day gestation and from newborn mice were also incubated with transforming growth factor TGF-beta2 (10 ng/mL). Palates were washed with saline, homogenized, and radioactivity was counted. Proline uptake was calculated for each sample as counts per milligram of protein and was subjected to statistical analysis (three-way analysis of variance). Samples of the homogenate were subjected to sodium dodecyl sulfate-gel electrophoresis and Western blotting in order to determine the types of collagen that were synthesized. Immunohistochemical localization of collagen types I, III, and VI was carried out on paraffin-embedded samples from each group. RESULTS: There were no significant differences in proline uptake between wounded mouse palates and nonwounded mouse palates at any age, and there was no histological evidence of regeneration of the palate at the site of the wound. Proline uptake was significantly greater in untreated wounded palates at 15 days' gestation than it was in newborns. After treatment with TGF-beta2, proline uptake was significantly greater in both wounded and nonwounded palates in the newborn group and had no effect on collagen synthesis in palates from 15-day gestation animals. Collagen types I and III were localized in histological specimens using immunohistochemistry and on nitrocellulose using Western blotting. No type VI collagen was demonstrated by Western blotting, but it was localized around blood vessels and on basement membranes using immunohistochemistry. CONCLUSION: Treatment with TGF-beta2 significantly increased collagen synthesis, as assessed by 3H-proline uptake, in cultured palates from newborn mice as compared with palates from untreated newborn mice and from both treated and untreated palates of 15-day gestation mice. These data suggest a differential response to TGF-beta2 by mouse palates as a function of fetal development.

Effects of butyrate and propionate on the adhesion, growth, cell cycle kinetics, and protein synthesis of cultured human gingival fibroblasts.

BACKGROUND: Various periodontal and root canal pathogens, such as the Bacteroides species, can produce significant amounts of short chain fatty acids (SCFA). The roles of SCFA in the pathogenesis of periodontal disease are still not fully understood. METHODS: We therefore investigated 2 main SCFA, butyrate and propionate, on the functional behavior of cultured human gingival fibroblasts (GF) such as cell growth, protein synthesis, cell adhesion capacity, and cell cycle progression. RESULTS: Butyrate and propionate inhibited the growth of healthy (HGF) and inflamed gingival fibroblasts (IGF) in a dose dependent manner. At concentrations of 4, 8, and 16 mM, butyrate suppressed the cell growth by 11 to 58%, 16 to 60%, and 50 to 71%, respectively. The response of cultured gingival fibroblasts to SCFA showed individual differences. Morphologically, GF became larger and more flattened in appearance following exposure to butyrate (>8 mM) and propionate (>24 mM) for 5 days. Inhibitory effects of butyrate (>2 mM) and propionate (>8 mM) on the growth of GF were due possibly to their inhibition of cell-cycle progression. At concentrations of 2 and 8 mM, butyrate led to G0/G1 arrest. Elevation of the exposure concentration to 8 to 24 mM further result in G2/M phase arrest of GF. On the other hand, propionate, at concentrations ranging from 4 to 24 mM, led to G0/G1 arrest. Butyrate (>2 mM) inhibited the proline-rich protein synthesis of GF. At concentrations of 4, 8, 16, and 24 mM, butyrate inhibited the protein synthesis of HGF-1 by 42%, 43%, 51%, and 54%, respectively. In all strains of cultured GF, the suppressive effect of propionate is less than that of butyrate. At concentration range of 4 to 24 mM, propionate suppressed the protein synthesis of HGF-1 by 23 to 43%. However, both butyrate and propionate (4 to 48 mM) exerted little effects on the adhesion of GF to type I collagen within 3 hours of incubation. CONCLUSIONS: These results suggested that SCFA released by pathogenic microorganisms can contribute to the gingival tissue dysfunction and breakdown through their actions on specific biological functions of GF.

Isolation and characterization of two different cDNAs of delta1-pyrroline-5-carboxylate synthase in alfalfa, transcriptionally induced upon salt stress.

Two different cDNA clones, MsP5CS-1 and MsP5CS-2, encoding delta1 -pyrroline-5-carboxylate synthase (P5CS). the first enzyme of the proline biosynthetic pathway, were isolated from a lambdaZap-cDNA library constructed from salt stressed Medicago sativa roots. MsP5CS-1 (2.6 kb) has an open reading frame of 717 amino acids, as well as a non-spliced intron at a position corresponding to the evolutionary fusion point of the bacterial proA and proB genes. MsP5CS-2 (1.25 kb) is a partial clone. The clones share 65% identity in nucleotide sequences, 74% homology in deduced amino acid sequences, and both show a high similarity to Vigna aconitifolia and Arabidopsis thaliana P5CS cDNA clones. Southern blot analysis confirmed the presence of two different P5CS genes. The effect of salinity on the transcription of MsP5CS-1 and MsP5CS-2 in roots was studied, using northern blot analysis and a RT-PCR approach. A rapid increase in the steady-state transcript level of both genes in roots was observed by RT-PCR upon exposure of hydroponically grown 6-day old seedlings to 90 mM NaCl, suggesting that both are salt-inducible genes, yet a higher response was observed for MsP5CS-2.

Salivary secretions: narcolepsy and central nervous system stimulants.

In an ongoing attempt to develop a model to study the influence of various diseases and drugs on saliva, we studied persons with narcolepsy treated with central nervous system stimulants. The aim was to study the secretion of salivary proteins in narcolepsy in the presence and absence of central nervous system stimulants. For this purpose, two proteins synthesized in acinar cells, acidic proline-rich proteins (PRPs) and statherin, were selected. Persons with narcolepsy treated with central nervous system stimulants only were included, n = 12, ages 14 to 68, seven females. Citric-acid-stimulated parotid and submandibular/sublingual saliva samples were collected from these persons during medication, after a drug-free period of one week, at least two weeks after the drug had been re-introduced, and from a matched healthy control group. PRP and statherin concentrations were determined by means of reversed-phase and anion exchange chromatography, respectively. Both concentration and output of statherins and PRPs were increased in persons with narcolepsy receiving central nervous system stimulants compared with healthy control individuals. When the drug was withdrawn, salivary flow rates were not influenced. In contrast, withdrawal of the drug led to a significantly decreased secretion of PRPs and statherin. The reduced protein secretion may reflect decreased adrenergic activation in narcolepsy, to be reversed by treatment with central nervous system stimulants. It can be concluded that measurements of both salivary fluid and salivary proteins may be necessary for an overall evaluation of the effects of a given drug or disease on salivary secretion.

Prevention of bleomycin-induced pulmonary fibrosis after adenovirus-mediated transfer of the bacterial bleomycin resistance gene.

A serious limitation in the use of the DNA-cleaving, antitumoral-antibiotic, bleomycin during chemotherapy is pulmonary toxicity. Lung injury induced by bleomycin is characterized by an increased deposition of interstitial extracellular matrix proteins in the alveolar wall that compromises respiratory function. Several drugs have been tested in animal models to prevent the pulmonary toxicity of bleomycin, but have not led to a useful clinical treatment because of their adverse effects on other tissues. We have shown that transgenic mice expressing Streptoalloteichus hindustanus (Sh) ble bleomycin resistance protein in pulmonary epithelial cells in the lungs are protected against bleomycin-induced toxicity in lungs. In the present study, we used intranasal administration by adenovirus-mediated gene transfer of the bleomycin resistance Sh ble gene to mouse lung for prevention of bleomycin-induced pulmonary fibrosis. We constructed recombinant adenoviruses Ad.CMVble and Ad.RSVble harboring the bleomycin resistance Sh ble gene under the control of the cytomegalovirus early promoter and the Rous sarcoma virus early promoter, respectively. Transgene expression was detected in epithelia of conducting airways and alveolar septa by immunostaining with a rabbit polyclonal antibody directed against the bleomycin resistance protein and persisted for the duration of drug treatment; i.e., up to 17 d. No toxic effect was seen in adenovirus-treated mice. Pretreatment of mice with Ad.CMVble or Ad.RSVble completely prevented collagen deposition 42-133 d after bleomycin treatment, as measured by lung OH-proline content. Histologic studies indicated that there was little or no lung injury in the adenovirus/bleomycin-treated mice compared with the bleomycin-treated mice. These observations may lead to new approaches for the prevention of bleomycin-induced pulmonary fibrosis.

Effect of transamniotic administration of epidermal growth factor on fetal rabbit small intestinal nutrient transport and disaccharidase development.

As fetal swallowing is documented in utero, supplementation of the ingested amniotic fluid with nutrients or hormones has been postulated as a potential prenatal treatment for intrauterine growth retardation (IUGR). To study the effect of epidermal growth factor (EGF) on the developing fetal small intestine, 12 pregnant rabbits underwent operation on day 24 of a normal 31-day gestation. Bilateral ovarian end fetuses underwent catheterization of their respective amniotic cavities with attachment to a miniosmotic pump. Study fetuses received recombinant human EGF at approximately 300 micrograms/kg/d for 1 week; controls received carrier solution only at an equivalent rate. On gestational day 31, fetuses were delivered by cesarean section and somatic measurements were recorded. The small intestine was harvested and proximal, middle, and distal regions were analyzed for lactase and maltase enzyme activity. Additionally, the uptake of radiolabeled glucose and proline was measured by a standard everted mucosal sleeve technique for each segment. Results were analyzed by Student's paired t test and reported as mean +/- SEM. Nine fetal pairs survived (75%). Small intestinal (SI) length was increased in EGF fetuses (54.8 +/- 1.9 cm) versus control (50.4 +/- 2.7 cm) (P = .02). Lactase activity, reported as UE/g protein, was significantly increased in the proximal segments in the EGF-infused fetuses; maltase was significantly increased in both the proximal and middle segments (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)