No presente trabalho, foram padronizados os radionuclídeos emissores beta puros 32P e 90Sr 90Y. O primeiro foi medido nos sistemas comerciais de cintilação líquida TRICARB 2100 e HIDEX 300SL, que utilizam, respectivamente, dois e três tubos fotomultiplicadores. A análise dos dados adquiridos pelo primeiro e segundo sistemas foi realizada pela aplicação dos métodos CIEMAT/NIST e TDCR, respectivamente. Para padronização da solução de 32P, foi também utilizado o sistema 4πβ empregando-se a técnica de autoabsorção. A solução de 90Sr 90Y foi padronizada no sistema de cintilação líquida, HIDEX 300SL, pelo método TDCR, e pela técnica do traçador, no sistema de coincidência 4πβ-γ o qual é composto por um contador proporcional à gás fluente, acoplado a dois cristais cintiladores de NaI(Tl). A taxa de desintegração foi determinada pela técnica de extrapolação, por meio de absorvedores externos. Para aplicação da técnica do traçador, foi utilizada uma solução de 60Co, emissor beta-gama previamente padronizado no sistema de coincidências. Foi realizada, também, uma simulação dos processos de detecção neste sistema por meio do programa ESQUEMA, que, pela simulação de Monte Carlo, prediz a curva de extrapolação da eficiência. Os resultados obtidos para o 32P nos sistemas utilizados apresentaram um bom acordo, dentro das incertezas experimentais, bem como os obtidos para o 90Sr 90Y, em sistema de cintilação e sistema de coincidência, apresentando bom acordo, dentro das incertezas experimentais. O resultado obtido pela técnica de Monte Carlo apresentou boa concordância com o valor obtido com o sistema de coincidência. Os resultados indicam a qualidade e boa precisão dos sistemas de detecção utilizados neste trabalho, quando empregados para fins metrológicos. / In the present work, pure beta emitters radionuclides 32P and 90Sr - 90Y were standardized. The first one was measured in commercial liquid scintillation systems TRICARB 2100 and HIDEX 300SL, which use, respectively, two and three photomultiplier tubes. The analysis of the data acquired by the first and second systems was performed using the CIEMAT / NIST and TDCR methods, respectively. For standardization of the 32P solution, the 4πβ system was also used, using the self-absorption technique. The 90Sr - 90Y solution was standardized in the liquid scintillation system, HIDEX 300SL, by the TDCR method, and by the tracer technique, in the coincidence system 4πβ-γ which is composed by a proportional counter to the flowing gas, coupled to two NaI(Tl) scintillation crystals. The rate of disintegration was determined by the extrapolation technique, by means of external absorbers. For the application of the tracer technique, a 60Co solution, a beta-gamma emitter previously standardized in the coincidence system, was used. It was also performed a simulation of the detection processes in this system through the program ESQUEMA, which, by Monte Carlo simulation, predicts the efficiency extrapolation curve. The results obtained for the 32P in the systems used presented a good agreement, within the experimental uncertainties, as well as those obtained for the 90Sr - 90Y, in scintillation system and coincidence system, showing good agreement, within the experimental uncertainties. The result obtained by the Monte Carlo technique showed good agreement with the value obtained with the coincidence system. The results indicate the quality and good accuracy of the detection systems used in this work, when used for metrological purposes.

Atualmente existe um considerável interesse no processamento de materiais com estrutura ultrafina de grãos. Estes materiais podem ser obtidos por deformação plástica severa via extrusão por canal angular (ECAE). Durante ECAE, uma barra lubrificada é pressionada através de uma matriz rígida consistindo de dois canais de mesma seção transversal, os quais se interceptam a um ângulo ?. Cisalhamento simples é o mecanismo de deformação predominante e ocorre paralelamente ao plano de intersecção entre os dois canais. Este trabalho enfoca a estabilidade térmica e sua influência sobre as propriedades magnéticas de ferro puro severamente deformado por ECAE. Uma barra de ferro puro foi deformada em temperatura ambiente por múltiplos passes de ECAE (8 passes), usando uma matriz com ângulo de intersecção ??= 90º, resultando em uma deformação equivalente _N = 9,2. Esta barra foi girada de 90o depois de cada passe de extrusão. Amostras da barra deformada foram recozidas em várias temperaturas entre 100 e 800°C, variando-se o tempo de recozimento entre 1 e 120 min. Uma segunda barra de ferro puro foi deformada por um único passe de ECAE, com um ângulo ??= 120o, resultando em uma deformação equivalente _N = 0,67. Amostras retiradas desta barra foram recozidas em várias temperaturas entre 300 e 800°C por 15 min. Em uma condição correspondente à segunda barra, uma terceira foi deformada sendo o passe de ECAE interrompido. A caracterização microestrutural foi efetuada utilizando-se microscopias ótica e eletrônica de varredura, microdureza Vickers e textura via difração de raios X. Curvas de magnetização em função do campo magnético aplicado até cerca de 9 T foram obtidas para amostras representativas da barra deformada por múltiplos passes de ECAE. Foram determinados os intervalos de temperatura de recozimento em que ocorrem a recuperação e a recristalização para esta barra e para a barra deformada por um único passe de ECAE. Com relação à barra do ensaio interrompido, várias seções do plano normal à direção de extrusão da barra foram analisadas a fim de se investigar a evolução da textura durante extrusão em canal angular. Os resultados foram comparados com aqueles reportados para outros materiais deformados via ECAE com estrutura ccc e também com as texturas previstas pelo Modelo Visco-plástico Autoconsistente (do inglês VPSC model). / There is considerable current interest in fabricating ultrafine-grained materials. Such materials can be obtained by severe plastic deformation via equal-channel angular extrusion (ECAE). During ECAE, a lubricated billet is pressed through a rigid die consisting of two channels of the same cross section intersecting each other at an angle ?. Simple shear is the predominant deformation mechanism and occurs parallel to the intersecting plane of the channels. This work focuses on thermal stability and its influence on the magnetic properties of severely deformed pure iron via ECAE. A billet of pure iron was deformed at room temperature by multiple ECAE passes (8 passes), using a die angle ??= 90o, to a total equivalent strain of _N = 9.2. The billet was rotated by 90o after each extrusion pass. Samples of the deformed billet were annealed at several temperatures between 100 and 800oC, varying the annealing time from 1 to 120 min. A second billet of pure iron was deformed using 1-pass ECAE, with ??= 120o, with an equivalent strain of _N = 0.67. Samples of this billet were annealed at several temperatures between 300 and 800°C for 15 min. Corresponding to second condition, a third billet was deformed by interrupting the ECAE pass. Microstructural characterization was performed using optical and scanning electron microscopies, Vickers microhardness, and texture measurements via X-ray diffraction. Magnetization curves as a function of applied magnetic field up to 9 T were obtained for representative samples of the billet deformed by multiple ECAE passes. The annealing temperature ranges corresponding to recovery and recrystallization for this billet and 1-pass ECAE billet were determined. Regarding the interrupted 1-pass ECAE billet, several sections normal to the extrusion direction were analyzed in order to investigate the texture evolution during equal channel angular extrusion. The obtained results were compared to those ones found in other deformed bcc materials via ECAE and also by using the visco-plastic self-consistent (VPSC) model to predict the final texture.

The effect of erythromycin on the 50S ribosomal subunit during cell growth has been extensively investigated. Sucrose density gradient analysis of ribosomes formed in the presence and absence of the drug revealed a 50S specific assembly defect is partially responsible for erythromycin's inhibitory effects on wild type cells. Examination of two erythromycin-resistant mutants of E. coli (N281 and N282) revealed that mutant N281 (L22 mutant) but not N282 (L4 mutant) was assembly defective in the presence of the drug, although only at much higher drug concentrations (300 ug/ml vs. 75 ug/ml for wild type cells). The altered genes from each mutant have been isolated and sequenced. The L22 mutant was found to contain a 9 bp deletion which eliminated codons 82-84, and the sequence Met-Lys-Arg from the protein. The L4 mutant had an A to G transition mutation in codon 63 resulting in a Lys to Glu change in the protein. Complementation of each mutant by their respective wild type genes resulted in an increased sensitivity to the drug in the partial diploid strains. Two other macrolide antibiotics (oleandomycin and spiramycin) were also examined but revealed no apparent assembly effect on wild type cells. The MLS antibiotics also appeared to be unable to effect assembly. However, the erythromycin derivative azithromycin showed a similar effect on assembly to that of the parent compound although clarithromycin (another erythromycin derivative) did not. These results suggest erythromycin and azithromycin effect assembly through ribosomal protein L4 and to a lesser extent through protein L22.

Tryptase, a mast cell-specific serine protease with trypsin-like specificity, has been identified in a mouse mast cell line (ABFTL-6) based on it's enzymatic activity, inhibition properties, and cross-reactivity to a human mast cell tryptase antibody. The effects of fibroblast-conditioned medium and sodium butyrate on ABFTL-6 mast cell differentiation and tryptase expression have been examined. ABFTL-6 mouse mast cells undergo phenotypic changes upon culturing in media supplemented with fibroblast-conditioned media at 50% or 1 mM sodium butyrate. The induced cells increased in size, had larger and more metachromatic cytoplasmic granules, and increased their total cellular protein about four-fold. Tryptase activity increased 13- and 6-fold upon fibroblast-conditioned media and butyrate induction, respectively. However, tryptase antigen levels increased dramatically from 2.3 $\mu$g/10$\sp6$ uninduced cells to 125 (54-fold) and 75 (33-fold) $\mu$g/10$\sp6$ cells induced with fibroblast-conditioned media or butyrate, respectively. A cDNA library was constructed in $\lambda$gt10 from ABFTL-6 cell poly(A)$\sp+$ RNA, and screened with dog mast cell tryptase and rat mast cell chymase cDNAs. Clones encoding two distinct tryptases (mouse tryptases I and II), a chymase (mouse chymase I) and a novel carboxyl terminal chymase (mouse chymase II) were isolated and sequenced. Mouse tryptases I and II have 75% and 70% sequence identity at the nucleotide and amino acid levels, respectively. The deduced amino acid sequence for the mature active enzyme for each mouse tryptase contains 245 residues and all the characteristics of a serine protease. Asp is found in the substrate binding pockets, consistent with a trypsin-like specificity for Arg-X and Lys-X bonds. It is predicted that tryptases are synthesized with prepropeptides, requiring signal peptidase processing and removal of a three amino acid propeptide for activation. Mouse chymase I consists of a 226 amino acid catalytic portion and a 21 amino acid preprosequence. An Asn occurs in the substrate binding pocket, a feature that has not been observed in any other serine protease.

Extraction of the lipids of Legionella pneumophila yields phases unlike those produced from other Gram-negative bacteria. A viscous interface forms between the aqueous (wash) and organic phases. More than half of the hydroxylated fatty acids were found distributed between the aqueous phase and the interfacial material, fractions in which such constituents have not been reported in other Gram-negative species. It was further observed that after the material from the aqueous/interfacial phase was dissolved in methanol or chloroform/methanol (2:1 (V/V)), the addition of acetone would create a white, flocculent precipitate. Analyses showed that the supernatant contained fatty acids that were nonhydroxylated and the precipitate contained both nonhydroxylated and hydroxylated fatty acids. The acetone precipitate could be further purified by column chromatography. Material was eluted from a silicic acid column with sequential additions of chloroform, acetone, and methanol. It was found that the methanol fraction contained the majority of the hydroxylated fatty acid containing material. An improved method for extracting LPS-like material from Legionella pneumophila is presented. This study suggests that LPS-like material can be obtained from L. pneumophila in higher yield (6.4% of total cell weight), of higher purity (as indicated by SDS-PAGE), and by a simpler method than those previously reported. SDS-PAGE profiles of purified (acetone precipitation and column chromatographic separation) LPS-like material extracted with chloroform/methanol (2:1 (V/V)) from L. pneumophila are identical to the previously reported profiles for G. pneumophila LPS. The chemical analyses of the LPS-like material can only account for approximately one-half the isolated material weight. This is suggestive of a moiety that is as of yet undetectable by the means employed to characterize the LPS.

The hypothesis that antioxidant vitamins (ascorbate and tocopherols) along with urate protect blood plasma lipids from oxidation was tested. Dietary fat is also an important factor influencing plasma lipid peroxidation. The purpose of this study was to investigate the role of plasma antioxidants and dietary fat on low density lipoprotein (LDL) and plasma lipid oxidation. In the first part of this study, we compared the ability of urate and ascorbate to protect human LDL from in vitro oxidation. LDL oxidation was initiated by 15 mM of a water soluble azo-initiator in the presence or absence of ascorbate or urate. The rate of lipid hydroperoxide (LOOH) formation was increased after the LDL tocopherols were totally consumed, i.e., after the lag phase. Urate (50 $\mu$M) was more effective than ascorbate (50 $\mu$M) in extending the lag phase. Moreover, urate was consumed more slowly than ascorbate under identical oxidation conditions. The combination af 25 $\mu$M ascorbate and 25 $\mu$M urate was more effective in extending the lag phase than ascorbate alone but less effective than urate alone. An empirical mathematical model was developed to describe the oxidation kinetics of LDL tocopherols. In the second part of this study, we studied the role of dietary fat and dietary $\alpha$-tocopherol ($\alpha$-toc) levels on rat plasma oxidation. The fatty acid composition of plasma was found to be modulated by the type of dietary fat. Neither dietary fat nor $\alpha$-toc influenced the plasma levels of water soluble antioxidants (ascorbate, urate and sulfhydryl content). Rat plasma was oxidized either by a water soluble azo-initiator (25 mM) or a lipid soluble azo-initiator (10 mM). In both cases, the rate of LOOH formation in plasma from rats fed butter oil diets was markedly suppressed compared to the plasma from rats fed corn oil diets. When oxidation was initiated by a lipid soluble azo-initiator, plasma from rats fed $\alpha$-toc supplemented diets showed higher LOOH levels than plasma from rats fed $\alpha$-toc deficient diets. Surprisingly, when oxidation was initiated by water soluble azo-initiator, tocopherol appeared to act as a pro-oxidant. The results suggest that urate may be more significant than ascorbate in delaying the consumption of tocopherols in human LDL and that low dietary PUFAs levels are more important in preventing the in vitro oxidation of plasma lipids than high dietary levels of $\alpha$-tocopherol.

A temperature-sensitive mutant of Escherichia coli affected in the alpha subunit of RNA polymerase has been investigated. Gene mapping and complementation experiments placed the mutation to temperature-sensitivity within the alpha operon at 72 min on the bacterial chromosome. The rate of RNA synthesis in vivo and the accumulation of ribosomal RNA were significantly reduced in the mutant at 44$\sp\circ$C. The thermostability at 44$\sp\circ$C of the purified holoenzyme from mutant cells was about 20% of that of the normal enzyme. Assays with T7 DNA as a template showed that the fraction of active enzyme competent for transcription was reduced as a function of assay temperature but that initiation and elongation were not significantly affected by the alpha mutation. A major effect on the fidelity of transcription was observed with the mutant enzyme, with misincorporation on two different templates stimulated about four fold at 37$\sp\circ$C. The role of the alpha dimer in the structure and function of RNA polymerase is discussed. In addition during the course of this study a new procedure for the purification of E. coli RNA polymerase was developed. This method is rapid, convenient, and useful for the preparation of enzyme from 1-5 grams of cells in two days. The ease and speed of this method allowed the rapid characterization of the mutant enzyme. This system should also find application for the purification of small quantities of other bacterial RNA polymerases that share the general chromatographic properties of E. coli RNA polymerase.

Glycerophospholipids of mammalian cells exist as chemically diverse structures with various fatty acids at the sn-1 and sn-2 positions. Arachidonic acid, a polyunsaturated fatty acid, which may be converted to biologically active eicosanoids such as prostaglandins, thromboxanes, and leukotrienes, is found predominantly in the sn-2 position of glycerophospholipids. The purpose of this study was to examine, at the level of the individual molecular species, the incorporation of arachidonate into phospholipids and its release from phospholipids during stimulation. In this way, the specificity of the enzymes controlling arachidonate metabolism could be examined in order to clarify the processes that control the metabolism of this precursor of potent biological mediators. An investigation of the deacylation-reacylation mechanisms for the incorporation of arachidonic acid into the cellular phospholipids revealed that both the CoA-independent transacylation and CoA-dependent acylation mechanisms are active in the P388D1 macrophages. The CoA-independent transacylase preferentially acylated the alkyllysoglycero-phosphatidylcholine substrate with the polyunsaturates, arachidonate, and docosahexaenoate. The CoA-dependent pathways exhibited less selectively and acylated the alkyl-substrate with more saturated fatty acids. Supplementation of the P388D1 macrophages with the n-3 marine oil fatty acids, eicosapentaenoate and docosahexaenoate resulted in the enrichment of the cellular phospholipids with these polyunsaturates at the expense of arachidonate-containing molecular species. Using methodology, which permits the measure of both mass and specific radioactivity changes in the molecular species of phospholipids, it was determined that the arachidonate-containing species are preferentially degraded during stimulation with the calcium ionophore, A23187. Stimulation with calcium ionophore results in the activation of a calcium dependent phospholipase specific for the arachidonate-containing species. Together, these results demonstrate that the incorporation and release of arachidonic acid is regulated by enzymes that bear distinct substrate specificities. The specificities of these enzymes can be directly related to the trafficking of arachidonate and its various esterified forms in cell phospholipids.

Enoyl-acyl carrier protein (ACP) reductase from chloroplast nonaggregated fatty acid synthetase (FAS) of Euglena gracilis variety bacillaris was purified to a single band on a denaturing polyacrylamide gel. The enzyme was partially characterized with respect to substrate specificity, reduced nucleotide requirement, and the effect of ACP and Ca$\sp{++}$ on enzyme activity. Antibodies against the purified protein were raised in hens and isolated from eggs. ACP was purified from Euglena in yields of about 1mg/100g (wet weight) of cells. Antibodies were raised against the purified protein. ACP antibodies inhibited the Euglena chloroplast FAS using Euglena or E. coli ACP as a substrate. Comparisons with other ACPs included the following items: biological activity, pI, behavior in size exclusion media, and amino acid sequence of the N-terminal portion of the molecule. ACPs from E. coli and Euglena have been shown to interact with melittin, a cationic peptide from bee venom. E. coli ACP is a small (Mr, 8847), acidic, Ca$\sp{++}$-binding protein which possesses some characteristics resembling those of regulatory Ca$\sp{++}$-binding proteins including interaction with melittin. Melittin inhibited activity of the nonaggregated FAS from Euglena using either E. coli or Euglena ACP as a substrate. The peptide also inhibited activity of the aggregated FAS from Euglena. Antibodies against melittin were raised. Anti-melittin inhibited activity of both the nonaggregated and aggregated FAS enzyme systems from Euglena relative to nonimmune antibody. Investigation of inhibition of the nonaggregated FAS enzyme system demonstrated that acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and keto-acyl-ACP synthetase activities were inhibited to different degrees by anti-melittin antibodies, while keto-acyl-ACP reductase and enoyl-ACP reductase enzyme activities were not inhibited.

To explore the possibility that 1-O-alkyl-sn-glycerol (alkylglycerol) may serve a regulatory role in the control of cell proliferation or PKC activity, we examined the ability of alkylglycerol to influence PKC activity and subcellular distribution as well as the ability of alkylglycerol to effect cell proliferation. MDCK cells grown to confluence show a loss of PKC activity associated with the membrane, as reported in fibroblasts. Preconfluent cultures of MDCK cells have a high level of PKC activity associated with the membrane. However, treatment of preconfluent cultures with alkylglycerol causes a reduction of PKC activity. A similar inhibition was observed with alkylglycerol when cells were treated with TPA, an activator of PKC. To confirm that alkylglycerol was exerting an effect directly on PKC, alkylglycerol was shown to inhibit PKC activity in vitro in a dose dependent manner. Since PKC exists as a family of closely related isozymes, we have determined the effects of growth arrest and alkylglycerol treatment on PKC $\rm\alpha,\ \epsilon,\ and\ \zeta$ (expressed in MDCK cells). The active forms of PKC $\alpha$ and $\epsilon$ are lost early in the growth of MDCK cells during the endogenous accumulation of alkylglycerol and synthetic alkylglycerol inhibits the membrane form of PKC $\alpha$ and $\epsilon.$ However, alkylglycerol inhibits the TPA induced translocation of PKC $\alpha$ but not $\epsilon$ suggesting a differential inhibition among these isoforms. Neither TPA or alkylglycerol had any effects on the distribution of PKC $\zeta.$ To examine the effect of alkylglycerol on cell proliferation, Swiss 3T3 cells were used. GLC analysis shows that 3T3 cells accumulate alkylglycerol in a similar manner as MDCK cells. Since this accumulation occurs just prior to cell growth arrest, the effects of alkylglycerol on preconfluent cells was observed. Preconfluent cultures of 3T3 cells were treated with alkylglycerol on day 1 of growth. After 8 days of culture, the treated group showed a slower growth rate and saturation density. Furthermore, after these cells were reseeded in the absence of alkylglycerol, the original growth rate and saturation density returned. Thus alkylglycerol induces a decrease in cell proliferation without causing any detrimental effects. Similarly, alkylglycerol was found to inhibit the induction of mitogenesis by TPA (a PKC dependent pathway) and these effects were shown not to be stereospecific. To further investigate the effect of alkylglycerol on cell proliferation, the content of the monoglycerides in ras-transformed cells was analyzed. These cells have lost contact dependent growth arrest indicating a disruption of cell growth regulation. We observed a massive increase in the content of alkylglycerol during the culture of ras transformed cells. This increase is 3 fold higher than MDCK or 3T3 cells. This raises the possibility that alkylglycerol may be the end result of an increased number of cell-cell contacts. We have observed an increase in the accumulation of alkylglycerol in normal and ras-transformed cells. This accumulation is accompanied by a decrease in PKC activity and alkylglycerol was shown to be a potent in vitro inhibitor of PKC. Similarly, alkylglycerol was shown to inhibit PKC $\alpha$ under stimulation by TPA. Alkylgylcerol is a inhibitor of the TPA induced induction of mitogenesis and slows the growth rate of proliferating cultures of 3T3 cells. These results indicate that the endogenous ether-linked glycerolipid, alkylglycerol, is a regulator of cell proliferation through its inhibitory effects on protein kinase C. (Abstract shortened by UMI.)