Flavonoids and other phenolic compounds: characterization and interactions with lepidopteran and sawfly larvae

This thesis focuses on flavonoids, a subgroup of phenolic compounds produced by plants, and how they affect the herbivorous larvae of lepidopterans and sawflies. The first part of the literature review examines different techniques to analyze the chemical structures of flavonoids and their concentrations in biological samples. These techniques include, for example, ultraviolet-visible spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy. The second part of the literature review studies how phenolic compounds function in the metabolism of larvae. The harmful oxidation reactions of phenolic compounds in insect guts are also emphasized. In addition to the negative effects, many insect species have evolved the use of phenolic compounds for their own benefit. In the experimental part of the thesis, high concentrations of complex flavonoid oligoglycosides were found in the hemolymph (the circulatory fluid of insects) of birch and pine sawflies. The larvae produced these compounds from simple flavonoid precursors present in the birch leaves and pine needles. Flavonoid glycosides were also found in the cocoon walls of sawflies, which suggested that flavonoids were used in the construction of cocoons. The second part of the experimental work studied the modifications of phenolic compounds in conditions that mimicked the alkaline guts of lepidopteran larvae. It was found that the 24 plant species studied and their individual phenolic compounds had variable capacities to function as oxidative defenses in alkaline conditions. The excrements of lepidopteran and sawfly species were studied to see how different types of phenolics were processed by the larvae. These results suggested that phenolic compounds were oxidized, hydrolyzed, or modified in other ways during their passage through the digestive tract of the larvae.

Regulation of nephron acidification by corticosteroids

The present paper reviews work from our laboratories evaluating the importance of adrenal cortical hormones in acidification by proximal and cortical distal tubules. Proximal acidification was determined by stationary microperfusion, and measurement of bicarbonate reabsorption using luminal pH determination was performed with H+-ion-sensitive microelectrodes. Rats were adrenalectomized (ADX) 48 h before the experiments, and corticosteroids (aldosterone (A), corticosterone (B), and 18-OH corticosterone (18-OH-B)) were injected intramuscularly 100 and 40 min before the experiments. In ADX rats stationary pH increased significantly to 7.03 as compared to sham-operated rats (6.78). Bicarbonate reabsorption decreased from 2.65 ± 0.18 in sham-operated rats to 0.50 ± 0.07 nmol cm-2 s-1 after ADX. The administration of the three hormones stimulated proximal tubule acidification, reaching, however, only 47.2% of the sham values in aldosterone-treated rats. Distal nephron acidification was studied by measuring urine minus blood pCO2 differences (U-B pCO2) in bicarbonate-loaded rats treated as above. This pCO2 difference is used as a measure of the distal nephron ability to secrete H+ ions into an alkaline urine. U-B pCO2 decreased significantly from 39.9 ± 1.26 to 11.9 ± 1.99 mmHg in ADX rats. When corticosteroids were given to ADX rats before the experiment, U-B pCO2 increased significantly, but reached control levels only when aldosterone (two 3-µg doses per rat) plus corticosterone (220 µg) were given together. In order to control for the effect of aldosterone on distal transepithelial potential difference one group of rats was treated with amiloride, which blocks distal sodium channels. Amiloride-treated rats still showed a significant reduction in U-B pCO2 after ADX. Only corticosterone and 18-OH-B but not aldosterone increased U-B pCO2 back to the levels of sham-operated rats. These results show that corticosteroids stimulate renal tubule acidification both in proximal and distal nephrons and provide some clues about the mechanism of action of these steroids

Risk factors for decreased bone density in premenopausal women

Osteoporosis is a major health problem. Little is known about the risk factors in premenopause. Sixty 40-50-year old patients with regular menses were studied cross-sectionally. None of the patients were on drugs known to interfere with bone mass. Patients answered a dietary inquiry and had their bone mineral density (BMD) measured. The Z scores were used for the comparisons. A blood sample was taken for the determination of FSH, SHBG, estradiol, testosterone, calcium and alkaline phosphatase. Calcium and creatinine were measured in 24-h urine. A Z score less than -1 was observed for the lumbar spine of 14 patients (23.3%), and for the femur of 24 patients (40%). Patients with a Z score less than -1 for the lumbar spine were older than patients with a Z score ³-1 (45.7 vs 43.8 years) and presented higher values of alkaline phosphatase (71.1 ± 18.2 vs 57.1 ± 14.3 IU/l). Multiple regression analysis showed that a lower lumbar spine BMD was associated with higher values of alkaline phosphatase, lower calcium ingestion, a smaller body mass index (BMI), less frequent exercising, and older age. The patients with a Z score less than -1 for the femur were shorter than patients with a Z score ³-1 (158.2 vs 161.3 cm). Multiple regression analysis showed that a lower femoral BMD was associated with lower BMI, higher alkaline phosphatase and caffeine intake, and less frequent exercising. A lower than expected BMD was observed in a significant proportion of premenopausal women and was associated with lower calcium intake, relatively lower physical activity and lower BMI. We conclude that the classical risk factors for osteoporosis may be present before ovarian failure, and their effect may be partly independent of estrogen levels.

Purification of bovine pancreatic glucagon as a by-product of insulin production

A process for purifying bovine pancreatic glucagon as a by-product of insulin production is described. The glucagon-containing supernatant from the alkaline crystallization of insulin was precipitated using ammonium sulfate and isoelectric precipitation. The isoelectric precipitate containing glucagon was then purified by ion-exchange chromatography on Q-Sepharose FF, gel filtration on Sephadex G-25 and ion-exchange chromatography on S-Sepharose FF. A pilot scale test was performed with a recovery of 87.6% and a purification factor of 8.78 for the first chromatographic step, a recovery of 75.1% and a purification factor of 3.90 for the second, and a recovery of 76.2% and a purification factor of 2.36 for the last one. The overall yield was 50%, a purification factor of 80.8 was obtained and the fraction containing active glucagon (suitable for pharmaceutical preparations) was 84% pure as analyzed by HPLC

Infrequent p53 gene alterations in ulcerative colitis

The purpose of this study was to determine whether point mutations and loss of the p53 gene take place in ulcerative colitis which is histologically negative for dysplasia. DNA was extracted from 13 frozen rectal or colon biopsies and blood samples. Ulcerative colitis was classified histologically as active (10 cases) and inactive (3 cases). Exons 5-8 were amplified by PCR, treated with exonuclease and shrimp alkaline phosphatase and sequenced by the dideoxy chain termination method with the Sequenase Version 2.0 DNA sequencing kit. PCR products of intron 6 and exon 4 were digested with MspI and AccII, respectively, for RFLP analysis. No p53 gene mutation was detected in these cases. The number of informative patients for loss of heterozygosity (LOH) at the p53 intron 6 was high, 11 out of 12 (92%), whereas no LOH was observed. LOH affecting p53 exon 4 was not detected in lesions from 5 of 12 patients (42%). In ulcerative colitis, tumor progression is similar to that in sporadic colon cancer, and other oncogenes and tumor suppressor genes are likely to be mutated before the p53 gene.

Effects of the partial removal of wood hemicelluloses on the properties of kraft pulp

The objective of this work was to study the effects of partial removal of wood hemicelluloses on the properties of kraft pulp.The work was conducted by extracting hemicelluloses (1) by a softwood chip pretreatment process prior to kraft pulping, (2) by alkaline extraction from bleached birch kraft pulp, and (3) by enzymatic treatment, xylanase treatment in particular, of bleached birch kraft pulp. The qualitative and quantitative changes in fibers and paper properties were evaluated. In addition, the applicability of the extraction concepts and hemicellulose-extracted birch kraft pulp as a raw material in papermaking was evaluated in a pilot-scale papermaking environment. The results showed that each examined hemicellulose extraction method has its characteristic effects on fiber properties, seen as differences in both the physical and chemical nature of the fibers. A prehydrolysis process prior to the kraft pulping process offered reductions in cooking time, bleaching chemical consumption and produced fibers with low hemicellulose content that are more susceptible to mechanically induced damages and dislocations. Softwood chip pretreatment for hemicellulose recovery prior to cooking, whether acidic or alkaline, had an impact on the physical properties of the non-refined and refined pulp. In addition, all the pretreated pulps exhibited slower beating response than the unhydrolyzed reference pulp. Both alkaline extraction and enzymatic (xylanase) treatment of bleached birch kraft pulp fibers indicated very selective hemicellulose removal, particularly xylan removal. Furthermore, these two hemicellulose-extracted birch kraft pulps were utilized in a pilot-scale papermaking environment in order to evaluate the upscalability of the extraction concepts. Investigations made using pilot paper machine trials revealed that some amount of alkalineextracted birch kraft pulp, with a 24.9% reduction in the total amount of xylan, could be used in the papermaking stock as a mixture with non-extracted pulp when producing 75 g/m2 paper. For xylanase-treated fibers there were no reductions in the mechanical properties of the 180 g/m2 paper produced compared to paper made from the control pulp, although there was a 14.2% reduction in the total amount of xylan in the xylanase-treated pulp compared to the control birch kraft pulp. This work emphasized the importance of the hemicellulose extraction method in providing new solutions to create functional fibers and in providing a valuable hemicellulose co-product stream. The hemicellulose removal concept therefore plays an important role in the integrated forest biorefinery scenario, where the target is to the co-production of hemicellulose-extracted pulp and hemicellulose-based chemicals or fuels.

Amifostine (WR-2721), a cytoprotective agent during high-dose cyclophosphamide treatment of non-Hodgkin's lymphomas: a phase II study

Clinical trials indicate that amifostine may confer protection on various normal tissues without attenuating anti-tumor response. When administered prior to chemotherapy or radiotherapy, it may provide a broad spectrum of cytoprotection including against alkylating drugs. The mechanism of protection resides in the metabolism at normal tissue site by membrane-bound alkaline phosphatase. Toxicity of this drug is moderate with hypotension, nausea and vomiting, and hypocalcemia being observed. We report a phase II study using amifostine as a protective drug against high-dose cyclophosphamide (HDCY) (7 g/m2), used to mobilize peripheral blood progenitor cells (PBPC) and to reduce tumor burden. We enrolled 29 patients, 22 (75.9%) affected by aggressive and 7 (24.1%) by indolent non-Hodgkin's lymphoma (NHL), who were submitted to 58 infusions of amifostine and compared them with a historical group (33 patients) affected by aggressive NHL and treated with VACOP-B followed by HDCY. The most important results in favor of amifostine were the reduction of intensity of cardiac, pulmonary and hepatic toxicity, and a significant reduction of frequency and severity of mucositis (P = 0.04). None of the 29 patients died in the protected group, while in the historical group 2/33 patients died because of cardiac or pulmonary toxicity and 2 patients stopped therapy due to toxicity. Amifostine did not prevent the aplastic phase following HDCY. PBPC collection and hematological recovery were adequate in both groups. The number of CFU-GM (colony-forming units-granulocyte/macrophage) colonies and mononuclear cells in the apheresis products was significantly higher in the amifostine group (P = 0.02 and 0.01, respectively). Side effects were mild and easily controlled. We conclude that amifostine protection should be useful in HDCY to protect normal tissues, with acceptable side effects.

Clinical results of the use of mitotane for adrenocortical carcinoma

Mitotane (o,p'-DDD) acts mainly as an inhibitor of intramitochondrial pregnenolone and cortisol synthesis. Its adrenolytic effect depends on metabolic activation due to conversion to o,p'-DDA and o,p'-DDE. The drug has been used for 40 years in the treatment of adrenocortical carcinoma, mainly its regional and metastatic stage, as an adjuvant to surgical resection of the tumor. In the medical literature there are controversial opinions about its efficacy for the treatment of adrenocortical carcinoma. In our experience, mitotane administered immediately after surgery appeared to be much more efficient than when administered later. We have administered this drug in all cases of microscopically confirmed adrenocortical carcinoma, irrespectively of stage at the time of surgery, for fear of a false too optimistic classification. In our series of 82 patients with adrenocortical carcinoma, 59 patients have been treated with mitotane, 32 of them immediately after surgery, and 27 with a delay of 2 to 24 months. Today there are 18 survivors in the group of patients treated with mitotane soon after the operation and only 6 survivors in the group receiving mitotane with a delay. All patients were simultaneously given replacement therapy. Undesired effects of mitotane administration included increased aminotransferase and alkaline phosphatase activity, decreased white cell, platelet or red cell number, and myasthenia. Furthermore, we used mitotane with good results in Cushing's syndrome of non-malignant origin as pre-treatment before surgery or in long-term treatment for patients with poor tolerance of other adrenal inhibitors.

Nutritional status of hemodialysis patients with secondary hyperparathyroidism

The repercussions of secondary hyperparathyroidism on the nutritional status of chronic renal failure patients have not been well established. Therefore, the aim of this study was to compare the nutritional indices of hemodialysis patients with and without secondary hyperparathyroidism. Sixteen hemodialysis patients with serum parathyroid hormone (PTH) levels higher than 420 pg/ml (hyperparathyroidism group) were matched for gender, age and length of dialysis treatment to 16 patients with serum PTH between 64 and 290 pg/ml (control group). The following parameters were assessed: anthropometric indices (body mass index, skinfold thickness, midarm muscle circumference and body fat), 4-day food diaries, protein catabolic rate, biochemical indices (blood urea nitrogen, serum creatinine, albumin, ionized calcium, inorganic phosphorus, serum alkaline phosphatase, PTH, pH and HCO3) and dialysis efficiency. We did not observe differences in the anthropometric indices between the two groups. Only calcium intake was significantly different between groups (307.9 mg/day for the hyperparathyroidism group vs 475.8 mg/day for the control group). Protein catabolic rate tended to be higher in the hyperparathyroidism group compared to the control group (1.3 vs 0.9 g kg-1 day-1; P = 0.08). Except for blood urea nitrogen (86.4 vs 75.7 mg/dl), alkaline phosphatase (175 vs 65 U/l) and PTH (898 vs 155 pg/ml), no other differences were found between groups in the biochemical indices studied. PTH was directly correlated with protein catabolic rate (r = 0.61; P<0.05) and length of dialysis (r = 0.53; P<0.05) only in the hyperparathyroidism group. Considering the indices used, we could not demonstrate the deleterious effect of high PTH levels on the nutritional status of hemodialysis patients. Indirect evidence, however, suggests an action of PTH on protein metabolism.

Sellutehtaan alkalisten valkaisusuodosten käsittely polymeerisilla membraaneilla

Työn tarkoituksena oli tutkia sellutehtaan alkalisen valkaisusuodoksen kierrätystä membraanitekniikkaa käyttäen. Alkalinen valkaisusuodos syntyy sellun valkaisun alkaliuuttovaiheessa ja se on ympäristölle haitallisin sellutehtaan jätevirroista. Suodoksen pH on korkea ja sen lämpötila voi olla suuri, jolloin sopivan membraanin löytäminen on haastavaa. Työssä vertailtiin 4 eri nanosuodatusmembraanin toimivuutta suodatuksessa. NF245, Desal-5 DK, NF270 ja NTR729HG membraaneja käytettiin tutkimuksessa. Suodatukset tehtiin 60 °C lämpötilassa useissa paineissa. Membraanit esikäsiteltiin paineistamalla sekä alkalisella pesulla. Suodatusnäytteistä saadut retentiot ligniinille, TOC:ille sekä johtokyvylle olivat lupaavia. Kaikilla kalvoilla ligniinin retentio oli yli 95 %. NTR729HG kalvoa lukuun ottamatta myös TOC retentiot olivat yli 90%. NTR729HG ja NF270 membraanit kärsivät suodoksen pH:sta sekä lämpötilasta, jolloin niiden retentiot johtokyvylle jäivät alhaisemmiksi, kuin muilla. NTR729HG membraanissa oli merkkejä foulaantumisesta, sekä kalvon hajoamisesta. Muissa membraaneissa oli suodatuksen aikana tapahtunut modifikaatiota.

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Insulin impairs the maturation of chondrocytes in vitro

The precise nature of hormones and growth factors directly responsible for cartilage maturation is still largely unclear. Since longitudinal bone growth occurs through endochondral bone formation, excess or deficiency of most hormones and growth factors strongly influences final adult height. The structure and composition of the cartilaginous extracellular matrix have a critical role in regulating the behavior of growth plate chondrocytes. Therefore, the maintenance of the three-dimensional cell-matrix interaction is necessary to study the influence of individual signaling molecules on chondrogenesis, cartilage maturation and calcification. To investigate the effects of insulin on both proliferation and induction of hypertrophy in chondrocytes in vitro we used high-density micromass cultures of chick embryonic limb mesenchymal cells. Culture medium was supplemented with 1% FCS + 60 ng/ml (0.01 µM) insulin and cultures were harvested at regular time points for later analysis. Proliferating cell nuclear antigen immunoreactivity was widely detected in insulin-treated cultures and persisted until day 21 and [³H]-thymidine uptake was highest on day 14. While apoptosis increased in control cultures as a function of culture time, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-labeled cells were markedly reduced in the presence of insulin. Type II collagen production, alkaline phosphatase activity and cell size were also lower in insulin-treated cultures. Our results indicate that under the influence of 60 ng/ml insulin, chick chondrocytes maintain their proliferative potential but do not become hypertrophic, suggesting that insulin can affect the regulation of chondrocyte maturation and hypertrophy, possibly through an antiapoptotic effect.

Vascular supply of the central nervous system of the land snail Megalobulimus oblongus (Gastropoda, Pulmonata)

The vascularization of the central nervous system of the snail Megalobulimus oblongus was studied by injection of carmine-gelatin solution into the arterial system and using a histochemical technique for the detection of alkaline phosphatase. The central nervous system of M. oblongus is irrigated by the anterior aorta, from which a series of small branches emerge that supply the subesophageal nervous ganglia. In turn, these branches give rise to a series of smaller vessels that irrigate the buccal bulb, the anterior portion of the foot, the cerebral ganglia, the dorsal body gland, and the anterior portion of the reproductive system. No hemolymph vessels were detected within nervous tissue although such vessels were found in the periganglionic connective sheath. This connective sheath contains vascular loops and had a series of overlaps and projections that follow the contour of the nervous ganglia. This arrangement permits a larger area of interaction between the surface of the nervous tissue and the hemolymph and reduces the distance between the deepest portion of a given ganglion and the hemolymph vessels.

Functional cellulose microspheres for pharmaceutical applications

Dissolving cellulose is the first main step in preparing novel cellulosicmaterials. Since cellulosic fibres cannot be easily dissolved in water-based solvents, fibres were pretreated with ethanol-acid solution prior to the dissolution. Solubility and changes on the surface of the fibres were studied with microscopy and capillary viscometry. After the treatment, the cellulose fibres were soluble in alkaline urea-water solvent. The nature of this viscous solution was studied rheologically. Cellulose microspheres were prepared by extruding the alkaline cellulose solution through the needle into an acidic medium. By altering the temperature and acidity of the mediumit was possible to adjust the specific surface area and pore sizes of themicrospheres. A typical skin-core structure was found in all samples. Microspheres were oxidised in order to introduce anionic carboxylic acid groups (AGs). Anionic microspheres are more hydrophilic; their water-uptake increased 25 times after oxidation and they could swell almost to their original state (88%) after drying and shrinking. Swelling was studied in simulated physiological environments, corresponding to stomach acid and intestines (pH 1.2-7.4). Oxidised microspheres were used as a drug carriers. They demonstrated a highmass uniformity, which would enable their use for personalised dosing among different patients, including children. The drug was solidified in microspheres in amorphous form. This enhanced solubility and could be used for more challenging drugs with poor solubility. The pores of themicrospheres also remained open after the drug was loaded and they were dried. Regardless of the swelling, the drug was released at a constant rate in all environments.

Noninvasive classification of liver disease in asymptomatic and oligosymptomatic male alcoholics

The predominant type of liver alteration in asymptomatic or oligosymptomatic chronic male alcoholics (N = 169) admitted to a psychiatric hospital for detoxification was classified by two independent methods: liver palpation and multiple quadratic discriminant analysis (QDA), the latter applied to two parameters reported by the patient (duration of alcoholism and daily amount ingested) and to the data obtained from eight biochemical blood determinations (total bilirubin, alkaline phosphatase, glycemia, potassium, aspartate aminotransferase, albumin, globulin, and sodium). All 11 soft and sensitive, and 13 firm and sensitive livers formed fully concordant groups as determined by QDA. Among the 22 soft and not sensitive livers, 95% were concordant by QDA grouping. Concordance rates were low (55%) in the 73 firm and not sensitive livers, and intermediate (76%) in the 50 not palpable livers. Prediction of the liver palpation characteristics by QDA was 95% correct for the firm and not sensitive livers and moderate for the other groups. On a preliminary basis, the variables considered to be most informative by QDA were the two anamnestic data and bilirubin levels, followed by alkaline phosphatase, glycemia and potassium, and then by aspartate aminotransferase and albumin. We conclude that, when biopsies would be too costly or potentially injurious to the patients to varying extents, clinical data could be considered valid to guide patient care, at least in the three groups (soft, not sensitive; soft, sensitive; firm, sensitive livers) in which the two noninvasive procedures were highly concordant in the present study.