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Approximately 10-15% of field-grown eastern white pine (Pinus strobus L.) within a nursery plantation expressed foliar characteristics similar to that induced by oxidant pollution. Sensitive genotypes (based on foliar characteristics), had a 50% reduction in needle growth, severe needle tip burn, mottling, and early needle shed during a high O₃, drought-type growing season (1988) compared to a low O₃, non-drought growing season (1989). Tolerant genotypes showed little difference in needle growth or visible injury during the two growing season. Seasonal needle ascorbate concentrations were similar during the two years however, needle glutathione (GSH) content has not. Total GSH content was two-to three-fold greater in both genotypes during the summer of 1989 compared to 1988. Cloned, tolerant trees also had 23% more total GSH when exposed to forced ambient air compared to charcoal-filtered air in open-top chambers. Cloned sensitive trees had similar GSH concentrations when exposed to different chamber treatments. One-year-old needles always had lower ratios of ascorbate/ dehydroascorbate, ascorbate/α-tocopherol and GSH/GSSG than current year needles. One-year-old needles from the tolerant tree also maintained a higher glutathione reductase (GR) activity than the sensitive tree during the late summer. Needles of eastern white pine had two isoforms of GR (GRA and GRB). GRA and GRB accounted for 17% and 83% of the GR recovered, respectively. GRA and GRB had different physical and kinetic properties. Antibody produced from GRR was reactive with both native and denatured GRB, but was cross-reactive with only native GRA. Tolerant and sensitive clones exposed to control (< 0.025 ppm) or high (4.5 ppm∙hr total dose) O₃ for O to 72 hr, showed no increase in GR activity. Only in the high-O₃-treated trees did the amount of GR protein increase. Needles from the sensitive clone contained 14, 62, and 464 ng GR mgP⁻¹ and needles from the tolerant clone contained 21, 138, and 2800 ng GR mgP⁻¹ after 0, 24 and 72 hr O₃ exposure, respectively. The results of this dissertation indicate that differential foliar characteristics in eastern white pine may be correlated with GSH turnover and its regulation by GR during periods of high oxidant stress. Ph. D.

Manipulation of dietary zinc produced a different body zinc status in male Japanese quail during 3 weeks of treating period. After intramuscular injection with 8μmol/100g body weight of estradiol-17β, Japanese quail were sacrificed and vitellogenin (phosphoprotein) production was assessed in these birds by analyzing plasma for protein-bound phosphorus concentrations (PBP). Plasma PBP concentrations of estrogen-injected male quails increased from undetectable values for control birds to O.66mg/ml for high-zinc birds and O.42mg/ml for low-zinc birds. About 36% loss of vitellogenin synthesis was associated with the consumption of the zinc-deficient diet. Plasma zinc concentrations also increased on the estrogen injection because of the zinc-binding property of vitellogenin. The extra zinc ions in the plasma were considered to be come from the body zinc pools. Liver as well as other major tissues was believed to act as a reservoir of zinc for egg development but it was not observed that liver was the major source of the extra zinc in the plasma. Master of Science

The biosynthesis of cellulolytic enzymes by resting cells of T. reesei QM 9414 incubated in 17 mM potassium phosphate buffer, pH 6.0, and Q-β-D-glucopyranosy1-(172)α-D-glucopyranose was investigated. A maximum of 200 mi1liunits aryl-β-D-g1ucosidase, 9000 mi1liunits endo-l,4-β-D-glucanaseand 200 milliunits Avicelase per milliliter of culture supernate was produced after 24 hours of incubation; at that time, extracellular protein production reached a maximum of 0.5 mg/ml. Optimum enzyme yields were obtained with 3-4 mg dry weight cells/ml, and 1 nIDi 0-β-D-glucopyranosyl-(1->2)α-D-glucopyranose. Inclusion of metals, e.g., zinc, cobalt, manganous and ferrous ions enhanced enzyme production when glutamic acid was present, in which case aryl-β-D-glucosidase, endoglucanase and Avicelase activity were enhanced by 20, 100 and 40 percent, respectively. Under the same conditions asparagine enhanced only ary1-β-D-glucosidase activity. Some of the enzymic components of the cellulase system produced by T. reesei under these conditions were purified by ion exchange chromatography on DEAE-Sephadex A-50. Two cellobiohydrolases were isolated in pure form. One of these was identical to the previously isolated ce1lobiohydrolase D on the basis of amino acid composition carbohydrate content, electrophoretic mobility and ultraviolet spectrum. Similar data for the other cellobiohydrolase suggest that it represents an enzyme not previously identified. An endoglucanase was also isolated which, on the basis of its amino acid composition and electrophoretic mobility, appears similar to the previously identified Endoglucanase IV. More precise characterization of these enzymes is currently under investigation. Master of Science

Tris-soluble protein and total nitrogen of tall and dwarf tomato (Lycopersicon esculentum Mill.) seedlings were determined by the method of Lowry et al. and a modified micro-Kjeldahl direct Nesslerization technique. The tall tomato 'Fireball' differs from the dwarf 'Md. 412-4' in several ways: (1) the tall has more total nitrogen and protein; (2) it exhibits a significant increase in protein during the initial 48-hours of seeds imbibition; (3) it has a higher amount of protein in the roots; (4) the rate of decline of total nitrogen is faster than the dwarf, and (5) the tall tomato is more efficient in the utilization of plant nitrogen and protein. The greater growth of the tall resulted in a more rapid decline in nitrogen. However, in 10-18 day-old seedlings, growth was still rapid while nitrogen and protein did not change appreciably. The tall was more efficient and utilized a lesser amount of nitrogen and protein in the production of a comparable amount of fresh weight. It is suggested that the roots have considerable influence(s) on the growth of the shoot and that the decline in nitrogen and protein is a resultant of growth. It is indicated that the differences in growth may be more dependent on the manner in which nitrogen and protein are utilized rather than their absolute content. Master of Science

A subcutaneous injection of sodium selenite (30 umol/kg body weight) in 10â day old rats produced a cataract within 72 hours. Lens opacification was preceded by a 15% decrease in ATP content. Lens ATP did not fully recover to control concentrations by 11 days post-injection. A moderate correlation existed between lens weight and total ATP content in control lenses

The effect of various growth conditions on the type of superoxide dismutase (SGD) formed anaerobically in three Bacteriodes species was studied. B. fragilis, B. distasonis, and B. thetaiotaomicron were grown in ironâ restricted media with or without manganese supplementation. Iron availability was decreased by treatment of the media with chelex-100, a metal-chelating resin, and addition of desferrioxamine mesylate (desferal, Ciba-Geigy), an iron chelator. Mn-containing (MnSOD) and Fe-containing superoxide dismutase (EeSOD) activities in cell extracts were differentiated by inhibition with azide and inactivation by H₂0₂. The amount of Mn-containing superoxide dismutase was estimated by the fraction of azide- and H₂0₂, -resistant activity. Cells grown in untreated media contained approximately 90% FeSOD and 10% MnSOD. Cells grown in Fe-restricted media supplemented with graded amounts of manganese synthesized a progressively larger fraction of MnSOD. Hemin, added to the Fe-restricted media, did not serve as an iron source for FeSOD formation. Superoxide dismutase specific activities varied (3-6 U/mg) in each extract but not as a function of manganese concentration. Master of Science

Studies of the binding of coenzyme analogs to yeast 6-phosphogluconate dehydrogenase indicate that NADP binding to the enzyme results from selective interactions between regions of the coenzyme binding site and portions of the NADP molecule. These studies suggested the existence of coenzyme binding site regions which selectively interact with the adenosine, 2'-phosphate, and pyrophosphate moieties of NADP. The importance of the 2'-phosphate to coenzyme binding was indicated by enhanced binding of adenosine derivatives possessing this moiety when compared to adenosine derivatives not phosphorylated at this position. The better binding of the 2'-phosphorylated derivatives became more pronounced with increasing resemblance of the derivative to the NADP molecule, and NAD was not inhibitory up to 70 roM. These results substantiate the concept that interaction of the enzyme with the d2'-phosphate is a key factor in the specificity of yeast 6-phosphogluconate dehydrogenase for NADP. Structural analogs of the pyridinium portion of the NADP molecule, Nl-alkylnicotinamide chlorides, did not inhibit yeast 6-phosphogluconate dehydrogenase at concentrations normally required for selective interactions with dehydrogenases; however, enzyme activity was decreased at micellar concentrations of Nl-dodecylnicotinamide chloride. Investigations of the role and environment of the essential sulfhydryl group of this enzyme were also performed. N-alkylmaleimides (N-methyl - N-hexyl, inclusive) were shown to inactivate the enzyme, but without a chainlength effect. In the presence of 6-phosphogluconate, the enzyme was protected from N-ethylmaleimide inactivation and this protection was enhanced by the addition of NADPH or AADP. Master of Science

Several new pterins have been discovered in halophilic and thermoacidophilic archaebacteria. Two of these were identified in the extreme halophiles and were thus called halopterins. One of these halopterins is produced by Halobacterium salinarium, Halobacterium halobium, and Halococcus morrhuae and is called phosphohalopterin-1. It was given this name because it was the first halopterin discovered and it has four monophosphate esters per dimeric pterin. The proposed structure of phosphohalopterin-1 is as follows. [see document for diagram of chemical structure] The other halopterin, which is produced by Halobacterium marismortui, Halobacterium volcanii, and Halobacterial strain GN-1, is called sulfohalopterin-2 because it has two sulfate esters per dimeric pterin and it was isolated and recognized after the isolation of phosphohalopterin-1. The proposed structure of sulfohalopterin-2 is as follows. [see document for diagram of chemical structure] As shown above, both pterins are dimers with an ether linkage connecting the polyol side chains. Both of the halopterins are negatively charged because of the phosphate and sulfate esters on the side chains. In addition to the halopterins, a positively charged pterin has been isolated from Sulfolobus solfataricus. This pterin is very special since no positively charged unconjugated pterin had ever been found in nature before. This pterin is named solfapterin after the species name of the bacteria from which it was obtained. The structure of this pterin is still unknown but the preliminary data indicate that it is an unconjugated pterin with a polyol containing an amine on the side chain. Another positively charged pterin which is different from solfapterin was found in Thermoplasma. All of the above pterins are different from any previously described pterins and thus represent new pterins in the archaebacterial kingdom. The discovery of these new pterins is important both to pterin biochemistry and to archaebacterial taxonomy. These discoveries also open up a new field, that is, the exploration of the function of these new pterins in norunethanogenic archaebacteria. Ph. D.

Alkylation of lens proteins with iodoacetamide during homogenization of tissue (50 millimolar excess) immediately followed by gel-permeation chromatography yielded a crystallin population devoid of βH-crystallin. This result occurred in lens homogenates from both young (100 g) and older (400 g) male rats. BetaH-crystallin was not converted to insoluble protein with alkylation. Each crystallin fraction reacted with radioactive iodoacetamide in proportion to sulfhydryl content; at a ratio of 1 mg iodoacetamide/mg protein total free-sulfhydryl of the crystallins had reacted after 1 hr at pH 8, 25°C. Alkylated α-, βL-' and y-crystallin fractions demonstrated no altered chromatographic behavior on Sephacryl S-200; only alkylated βH-crystallin was altered so that it co-chromatographed with control or alkylated βL-crystallin. Master of Science

Procedures are described for the routine analysis of amines and other compounds produced in bacterial cultures. Whole cultures were grown in six mls of chopped meat-glucose medium, acidified, and extracted with ethyl ether or chloroform to obtain organic acids, alcohols, ketones, aldehydes, and other neutral compounds. The residual broth was then made basic and extracted with chloroform to obtain the amines. For chromatographic analyses, alcohols and amines were converted to trifluoroacetyl derivatives. Organic acids were converted to their methyl esters. Analyses were made by a temperature- programmed gas chromatograph equipped with flame ionization detectors. Sixty-two strains of clostridia, representing 13 species, were analyzed for soluble products in whole cultures. Many amines were found and total amine concentrations exceed 0.5% (weight) in cultures of several species examined. Neutral products were found to be major products of several species. Alcohols both short and long chain were detected and were valuable in differentiation of some groups of clostridia. The 13 species were grouped as follows: on the basis of fatty acids and neutral products, 7 groups; on the basis of amine products, 10 groups; on the basis of alcohol and neutral products, 12 groups; and on the basis of amine, alcohol, and neutral products, 13 groups. Ph. D.