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The ability to discriminate between stereoisomers of a-tocopherol was studied in five patients with abetalipoproteinemia (ABL) because an impairment in secretion of apolipoprotein B-containing lipoproteins might impede the normally enhanced plasma transport of RRR-a-tocopherol. An oral dose containing 3.7 g of each 2R,4′R,8′R-α-[5-C2H3]tocopheryl acetate (d3RRR-α-tocopheryl acetate) and 2RS,4′RS,8′RS-α-[5,7-(C2H3),]tocopheryl acetate (d6all rac-α-tocopheryl acetate) was administered, then the labeled and unlabeled a-tocopherol contents of plasma and red blood cells from multiple blood samples obtained at selected times up to 72 h following the dose were quantitated. ABL plasma contained about 1%-10% of the d3-RRR-a-tocopherol concentrations of normal subjects given only 150 mg of each isotope. Three of the patients discriminated between forms of a-tocopherol with ratios of RRR-/allrac-α-tocopherol ≥ 1.8, similar to normals. These data suggest that the hepatic tocopherol binding protein is present and functional in ABL patients. Although two of the patients did not discriminate between stereoisomers of a-tocopherol, it is likely that this resulted from nearly a complete block in very low density lipoprotein (VLDL) secretion. Thus, the ability of ABL patients to absorb and transport orally administered vitamin E is markedly impaired and variable among patients.

Toxicity preventing activity (TxPA) is a recently identified substance in serum which counteracts the toxic effect of very low density lipoproteins upon endothelial cells in vitro. In two clinical studies, TxPA was low in individuals with angiographically demonstrable coronary artery disease. An atherogenic index which combines TxPA with lipoprotein cholesterol values classifies individuals with coronary artery disease with an accuracy of greater than 93%. TxPA precipitates with 0.15 M trichloroacetic acid and above 3 M (NH4)2SO4. Activity is present in Cohn fractions IV4 and V and is stabilized by antioxidants. TxPA co-lutes with the albumin peak on gel filtration chromatography and as a subcomponent of albumin on ion-exchange chromatography. Isoelectric focusing resolves albumin into two major peaks with pI values of 4.8 and 5.6. The TxPA is identified as the pI 5.6 albumin peak.

This report demonstrates the utilization of a new serum factor, Toxicity Preventing Activity (TxPA) in the diagnosis of coronary disease prone individuals. Our laboratory has recently identified TxPA, which offsets the toxicity of very low density lipoproteins (VLDL) upon arterial cells in vitro. In the present study, we measured TxPA activity and serum lipoprotein levels in 73 individuals undergoing coronary angiography. Serum from control subjects demonstrated 270% more TxPA than aged matched individuals with angiographically demonstrable coronary disease (CHD). When TxPA was combined with serum lipoprotein values, a new atherogenic index was generated which further distinguished these individuals with CHD from non-angiographed controls. These results demonstrate that TxPA is a new protective factor in coronary artery disease, and that the new atherogenic index provides for the first time an accurate classification of individuals with coronary artery disease.

Twenty-one 8-14 kg adult male stumptailed macaques, Macaca arctoides, were fed a standard laboratory diet and divided into 3 groups. The high-dose group and low-dose group were exposed to cigarette smoke at the human equivalent of 3 packs and 1 pack per day, respectively, 7 days per week, for 3-5 years. Eight animals served as cage and sham controls. Peak blood carboxyhemoglobin (COHb) levels measured immediately after smoking showed levels of 0.5 ± 0.1%, 3.6 ± 1.0%, and 5.7 ± 2.8% for. sham controls, low, and high dose smokers, respectively. Hemoglobin and hematocrit values were 2-7% higher (N.S. to P < 0.05) for smoking groups, presumably as a consequence of chronically elevated COHb levels. No significant differences were seen in total plasma cholesterol and lipoprotein cholesterol concentration measured at four intervals over a period of one year. We conclude from these data that, while fed a low fat diet, chronic cigarette smoke inhalation fails to alter plasma lipoprotein levels in this animal model.

Many endospore-forming bacteria cause diseases such as anthrax and food poisoning. Spores however also contribute to various agricultural and industrial processes. Spores possess extreme resistance properties, notably to chemical, et and dry heat, desiccation, and UV damage. For pathogenic spore formers, this poses an issue as spores are resistant to most decontamination methods currently in use. This work focuses on characterizing proteins thought to contribute to spore stability and efficient spore germination. Understanding how spores can remain stable for long periods of dormancy and against various insults and rapidly initiate germination could allow for the development of techniques that induce germination early and rapidly, promoting inexpensive decontamination. Physiological studies found that a family of spore-associated lipoproteins is needed for efficient spore germination and influences membrane fluidity in dormant spores. All the members of the lipoprotein family serve the same function, as each can fulfill the role of another. In vivo cross-linking was used to characterize protein-protein interactions found on the inner spore membrane. Glutaraldehyde crosslinking revealed that the four lipoproteins appear to interact. Bacterial two-hybrid analysis on individual protein domains further suggests the lipoproteins seem to interact through their predicted ring-building motif within their otherwise uncharacterized domains. Additionally, the absence of the spore lytic enzyme SleB seems to alter the crosslinking pattern of the lipoproteins, suggesting either it's interacting or helping facilitate lipoprotein interactions. Fluorescence microscopy reveals an unequal spatial distribution of the lipoproteins on the spore membrane, which seems to be supported by preliminary super-resolution microscopy studies. Further work aiming to characterize the entire inner spore membrane interactome is currently being conducted. The presented research used many methods and built many collaborations with the goal of providing insight to spore dormancy and efficient spore germination with an additional goal of understanding inner spore membrane protein behavior and how it leads to the highly resistant properties native to bacterial endospores. / Doctor of Philosophy / Certain species of Gram-positive bacteria can form a dormant cell called a bacterial endospore. Endospores, or spores, are highly resistant to insults such as noxious chemicals, wet and dry heat, and UV irradiation. These resistance properties make spores immune to standard sanitation methods, and result from various anatomical structures innate to the spore. In medicine, this poses a problem as spore forming bacteria can be causative agents of diseases such as food poisoning, anthrax, infant botulism, hospital-acquired diarrhea, and others. In agriculture and other industries, spore forming bacteria can be used as insecticides or fungicides, and have a long shelf life, making them ideal for long term storage. Research in the following document aims to answer questions relating to how spores transition from dormant spore to a typical cell. Understanding of these processes can inform novel decontamination techniques, better more stable spore-based products, and subversion of disease, depending on which process/ structure in the spore is altered.

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No description available.

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Most of the lipid present in lactating mammary gland cytosol was associated with a high molecular weight aggregate isolated from cytosol by gel exclusion chromatography or by density gradient centrifugation. The major polypeptide constituent of this lipoprotein aggregate was the monomer of fatty acid synthase (FAS). The major milk lipid globule proteins, butyrophilin (8u) and xanthine oxidase (XO), as well as the small GTP-binding protein ARF, also were present. This lipoprotein complex was abundant in cytosol from lactating but not from involuting mammary glands. HPTLC analysis of lipids extracted from the low density FAS (LDFAS) complex demonstrated the presence of the five major milk phospholipids as well as triacylglycerols, cholesterol, unesterified fatty acids, and diacylglycerols. ³²P-labeled phospholipids present in cytosol could be transferred to microlipid droplets (MLD) and endoplasmic reticulum (ER), in vitro, and could be precipitated along with FAS, and other polypeptide constituents of the LDFAS complex. Complexed FAS could be separated from noncorrlplexed FAS by density gradient centrifugation, native PAGE, and gel exclusion chromatography. A large amount of phospholipid consistently was retained with the complexed form of FAS. These results suggest that FAS migrates to a low density fraction by virtue of its association to other proteins and lipids. FAS was found to be associated with ER, intracellular lipid droplets, and the milk lipid globule membrane (MLGM). A similar complex to LDFAS was isolated from ER from liver and mammary gland homogenates following incubation in buffer containing ATP. Polypeptide constituents of this complex had similar electrophoretic patterns to LDFAS, but behaved differently from LDFAS constituents when fractionated with the detergent TX-114. While most of the polypeptides in LDFAS partitioned equally into the detergent and aqueous phases, a constituent with an approximate molecular weight of 70 kDa was enriched in the detergent phase. For the ER-derived FAS complex, most of the polypeptides remained in the aqueous phase but the detergent phase also was enriched with a polypeptide similar in size to the LDFAS detergent enriched constituent. Western blot analysis failed to detect Bu in the ER-derived complex. However, protein disulfide isomerase (PDI) was detected in this complex as well as a polypeptide with approximate molecular weight 50 kDa that cross-reacted with PDI antibody. Extraction of lipids from this ER-derived complex demonstrated the presence of large quantities of unesterified fatty acids, with relatively low amounts of complex lipids. In studies using ¹²⁵I labeled LDFAS, labeled polypeptides were shown to associate with ER and intracellular lipid droplets and their dissociation was stimulated by ATP. Immunocytochemistry using antibody to rat liver FAS revealed distribution of FAS at localized regions of the cytoplasmic surface of rough endoplasmic reticulum and on surfaces of intracellular lipid droplets. Electron micrographs of the LDFAS complex showed a homogeneous morphology of granular, symmetrical particles ranging in size from 40 nm to 170 nm in diameter. These particles resembled low density lipoprotein (LDL) in morphology. From the available data, the following model was proposed for a possible involvement of FAS in lipid droplet secretion in the mammary gland. During active lipogenesis, FAS is targeted to ER membrane by association with a signal or targeting peptide(s) in the cytosol. The signal peptide then binds to selected regions of ER where signal receptors reside. Binding of FAS may initiate synthesis and accumulation of triacylglycerol between ER membrane bilayers. Upon the achievement of a "critical mass", the lipid core may be released into the cytoplasm in an ATP-dependent manner, surrounded by the membrane components that provided the hydrophobic pocket for lipid accumulation. Butyrophilin and the 70 kDa detergent-extractable constituent released from the ER and present in LDFAS are possible sources of such a function. Polypeptides from the cytosolic leaflet of the ER, and proteins peripherally associated with the leaflet then would comprise the polypeptide constituents of the lipid particle. / Ph. D.