Conversion of cholesterol to adreno-cortical hormone intermediates by subcellular fractions of bovine adrenal cortex

Thesis (Ph.D.)--Boston UniversityThe sequence of reactions concerned in the formation of adrenocortical steroids from cholesterol includes the cleavage of the cholesterol side chain between C-22, 23 to form 5 -pregnenolone. This step is accelerated by adrenocorticotrophic hormone (ACTH) administration, whereas the later hydroxylation of progesterone to form corticosterone and cortisol are not. These hydroxylations are known to be dependent upon triphosphopyridine nucleotide (TPNH), but the cofactor requirement for cholesterol side chain cleavage has not been defined. In view of the dominant role proposed fro TPNH by currently held theories of the mechanism of action of ACTH, it is important to know if the only ACTH responsive step in the corticosteroidogenetic sequence requires TPNH as an essential cofactor. The conversion of cholesterol to c-21 steroids was investigated in subcellular fractions of bovine adrenal cortex tissue incubated with cholesterol-4-c^14 and the extent of conversion estimated by separating the radioactive products from unchanged cholesterol-4-c^14 on silicic acid columns. The radioactive products were tentatively identified on the basis of their chromatographic behavior in paper and partition column systems. The subcellular fractions were prepared by conventional differential centrifugation techniques with 0.44M sucrose, with and without ionic additions, for homogenization and isolation media. Nuclei, mitochondrial, microsomal and soluble fractions were prepared from the ionicsucrose media and in the case of pure sucrose homogenates the "fluffy layer". of the mitochondrial pellet was treated as an additional fraction. The mitochondrial fraction from either homogenate required only Mg++ and fumarate for cholesterol conversion activity. Succinate or a-ketoglutarate could replace fumarate and aerobic conditions were necessary. The requirement for citric acid cycle intermediates could be met by the "extemal" TPNH generating system, glucose-6-phosphate (G6P), its dehydrogenase (G6P+DH) and TPN. Acetone dried mitochondrial fractions from pure sucrose homogenates were fully active with fumarate and Mg++, but after restricted dialysis against phosphate buffer, TPN was required in addition to fumarate. The activity could be obtained in a soluble, non-particulate form by extraction of the acetone dried mitochondrial fraction with M/15 phosphate buffer pH 6.8, followed by centrifugation at 105,000g. The products of cholesterol-4-c^l4 conversion by the dialyzed or soluble extract of acetone dried mitochondrial fractions were largely accounted for by ~5 -pregnenolone and progesterone, with the relative amounts of the two intermediates depending upon the endogenous level of DPN in the preparation or the presence of this cofactor in the medium. Distribution studies based on the activity of acetone dried fractions were run in order to avoid problems connected with unequal mixing of tracer and endogenous cholesterol. The results indicated that approximately equal amounts of the total activity were present in the nuclei, mitochondrial and "fluffy layer" fractions. The microsomal and soluble fractions were inactive. The highest activity per mg. protein was found in the "fluffy layer" with the mitochondrial fraction next, a result possibly related to the greater ease of cholesterol-4-C^14 entry into the disorganized structure of the "fluffy layer". Soluble extracts from the acetone dried fractions were active on addition of Mg++, fumarate and TPN, but only the mitochondrial fraction gave acetone powders active in the absence of TPN. The "bound" cofactor in fresh mitochondrial or "fluffy layer" fractions was not available for TPNH generation by G6P and its dehydrogenase and only reacted to a limited extent in acetone dtted preparations. High concentrations of sucrose inhibited cholesterol conversion by particulate preparations, but not the solubilized activity from acetone powders. The effect was possibly due to a reduced entry of cholesterol-4-c^l4, as the specific activities of the products of conversion were reduced, while TPNH generation by either the "internal" or "external" systems was inhibited to an equal extent. The possible relationships of the findings co ACTH action are discussed. [TRUNCATED

Increased Lead Biomarker Levels Are Associated with Changes in Hormonal Response to Stress in Occupationally Exposed Male Participants

Background: Lead (Pb) exposure has been associated with a host of pathological conditions in humans. In rodents Pb exposure has been shown to alter the hypothalamic–pituitary–adrenal (HPA) axis function

A comparison of cholesteryl oleate and 19-iodocholesteryl oleate as substrates for adrenal cholesterol esterase

Radiolabeled 19-iodocholesterol is widely used to obtain images of human adrenals. We have shown in rats and mice that 85-95% of the radiolabel is present in the esterified form. Using rat adrenal cytosolic cholesterol esterase, the kinetic parameters KM and V were determined for commercially available cholesteryl [1-14C]-oleate (I) and [125I]-19-iodocholesteryl oleate (II). The KM and V were found to be 16.2 [mu]M and 602 pmol/min/mg prot. respectively for I compared to 76.2 [mu]M and 37.6 pmol/min/mg prot. for II. Since V/KM is 37.3 ml/min/mg prot. for the normal ester and 0.49 ml/min/mg prot. for the iodinated analog, it appears that the normal substrate is 76 times more specific than II. In addition, direct competition experiments were run in which 67 [mu]M I was used as substrate. The addition of 65 and 325 [mu]M 19-iodocholesteryl oleate caused 20 and 49% inhibition, respectively. On the basis of these studies, it is not surprising that 19-iodocholesterol would accumulate in the adrenals in an esterified form and thus provide an effective agent for adrenal visualization.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23282/1/0000219.pd

Development of a cost-effective ovine antibody-based therapy against SARS-CoV-2 infection and contribution of antibodies specific to the spike subunit proteins.

Antibodies against SARS-CoV-2 are important to generate protective immunity, with convalescent plasma one of the first therapies approved. An alternative source of polyclonal antibodies suitable for upscaling would be more amendable to regulatory approval and widespread use. In this study, sheep were immunised with SARS-CoV-2 whole spike protein or one of the subunit proteins: S1 and S2. Once substantial antibody titres were generated, plasma was collected and samples pooled for each antigen. Non-specific antibodies were removed via affinity-purification to yield candidate products for testing in a hamster model of SARS-CoV-2 infection. Affinity-purified polyclonal antibodies to whole spike, S1 and S2 proteins were evaluated for in vitro for neutralising activity against SARS-CoV-2 Wuhan-like virus (Australia/VIC01/2020) and a recent variant of concern, B.1.1.529 BA.1 (Omicron), antibody-binding, complement fixation and phagocytosis assays were also performed. All antibody preparations demonstrated an effect against SARS-CoV-2 disease in the hamster model of challenge, with those raised against the S2 subunit providing the most promise. A rapid, cost-effective therapy for COVID-19 was developed which provides a source of highly active immunoglobulin specific to SARS-CoV-2 with multi-functional activity

The role of mature mycolic acids in mycobacterial pellicle biofilm formation and interactions with the human complement system

M. tuberculosis cell wall components are essential for host-pathogen interactions and modulating the immune response. M. tuberculosis may persist as pellicle-like biofilms within the peripheries of human cavities, and expectorated bacteria from these biofilms may establish infection in a new host. The complement system is an early cascade of the innate immune response, which plays an important role in M. tuberculosis access to alveolar macrophages. The impact of mycobacterial biofilms on complement activation has not been studied in-depth. This thesis aims to evaluate the role of mycolic acid maturation in pellicle biofilm formation and interactions of the mycobacterial cell envelope with the human complement system. Mycolic acids are a major cell wall component, and their accumulation is a hallmark of pellicle biofilm formation. M. smegmatis, a surrogate organism for M. tuberculosis, was mutated in gene MSMEG4722 (ortholog of M. tuberculosis Rv2509) (ΔMSMEG4722), causing the loss of mature mycolic acids for mycolic acid intermediates (Bhatt et al., 2008). As a result, ΔMSMEG4722 pellicle biofilms could form on the air-liquid interface but could not mature and produced significantly less extracellular material. Biochemical changes in the cell envelope of ΔMSMEG4722 were examined using thin layer chromatography. Changes included alteration to the distribution of glycolipids (TDM, TMM and GMM) within the apolar lipid fractions, occurring in bacteria cultured in Middlebrook 7H9 and as planktonic cells and pellicle biofilms. Within pellicle biofilms, mature free mycolic acids and free mycolic acid intermediates/ pre-mycolates decarboxylated to unsaturated ketones accumulated in the apolar lipid fractions for M. smegmatis mc2 155 and ΔMSMEG4722, respectively. Flow cytometry and ELISA demonstrated increased complement system activation, both on whole bacterial cells and the apolar lipid fractions of ΔMSMEG4722 compared to M. smegmatis mc2 155. Increased complement activator binding was also observed. When examining M. smegmatis mc2 155 pellicle biofilms, comparable complement activation was observed on planktonic cells and pellicle biofilm-derived cells. However, overall activator binding was lower, and there was decreased complement activation by the apolar lipid fractions. ΔMSMEG4722, in contrast, showed higher complement activation by both phenotypes compared to M. smegmatis mc2 155, but activator binding was only increased on biofilm-derived cells and both phenotypes demonstrated decreased complement activation by the outer apolar lipid fraction. The implications of these findings in understanding mycobacterial pellicle biofilm formation and deciphering mycobacterial cell wall component activation of the complement system are discussed