No description available.

No description available.

The CAPS1 protein was initially discovered as a cytosolic soluble 145kDa protein which was necessary to restore calcium dependent norephinephrine secretion in cracked PC12 cells. Recent findings suggest that CAPS may also play a role in synaptic and dense core vesicle exocytosis as well as in the loading of monoamine neurotransmitters. Recently, studies have implicated CAPS1 in the binding to syntaxin-1. However, no studies have identified the key residues of CAPS1 that facilitate this interaction with syntaxin-1. I show that the binding mode of CAPS1 is independent from that of Munc13 such that CAPS1 requires the full length of syntaxin-1 to bind. Moreover, CAPS1 favors the open conformation of syntaxin-1. Interestingly, the Munc homology (MH) domain of CAPS1 is not critical for this interaction while the C-terminal dense core vesicle binding (DCVB) domain plays an important role. Moreover, truncations to this DCVB domain result in decreased binding to syntaxin-1.

The CAPS1 protein was initially discovered as a cytosolic soluble 145kDa protein which was necessary to restore calcium dependent norephinephrine secretion in cracked PC12 cells. Recent findings suggest that CAPS may also play a role in synaptic and dense core vesicle exocytosis as well as in the loading of monoamine neurotransmitters. Recently, studies have implicated CAPS1 in the binding to syntaxin-1. However, no studies have identified the key residues of CAPS1 that facilitate this interaction with syntaxin-1. I show that the binding mode of CAPS1 is independent from that of Munc13 such that CAPS1 requires the full length of syntaxin-1 to bind. Moreover, CAPS1 favors the open conformation of syntaxin-1. Interestingly, the Munc homology (MH) domain of CAPS1 is not critical for this interaction while the C-terminal dense core vesicle binding (DCVB) domain plays an important role. Moreover, truncations to this DCVB domain result in decreased binding to syntaxin-1.

The eukaryote-associated Pseudoalteromonas tunicata produces two pigments and several other bioactive compounds that are able to inhibit a range of marine organisms including bacteria, protozoa, fungi, algal spores and invertebrate larvae. Early studies suggested that the production of bioactive compounds is correlated with pigmentation in P. tunicata. In one of these studies, a transposon mutagenesis library identified a white mutant, wmpD-, which had been disrupted in a gene encoding a component of the type 11 secretion (T2S) machinery. The T2S system is involved in the transport of different extracellular enzymes in many bacteria. In some cases, the T2S pathway also exports proteins that remain attached to the cells. This thesis aimed to investigate the role of the T2S pathway in the production of the pigments and bioactive compounds in P. tunicata. In order to gain insight into this relationship, two proteomics approaches (2D-PAGE and iTRAQ) were applied to investigate the profile of the secreted proteins (or secretome) in P. tunicata wild-type and the white mutant wmpD-. Proteomic analysis using 2D-PAGE revealed that 23 proteins were differentially expressed between P. tunicata Wt and the mutant wmpD-. The identities of some of these proteins could be correlated with the function of the T2S system in P. tunicata. The role of one of the proteins identified using 2D-PAGE was further investigated through the construction of a gene knockout mutant (hiik mutant). The supernatant activity of the hiik mutant was compared to that of P. tunicata Wt, and it was found that the HiiA protease is required to block the activity of antimicrobial peptides, such as cecropins, produced by eukaryotic hosts in the environment. The second proteomics approach (iTRAQ) used in this thesis, enabled the relative quantitation of a number of proteins in the supernatant of P. tunicata Wt and the white mutant wmpD-. Some proteins with no function to date (hypothetical) were absent in the extracellular fraction of the wmpD- mutant, indicating they may be transported to the extracellular environment via the T2S pathway in P. tunicata. The comparative analysis of the secretome also revealed that TonS-related proteins, involved in iron acquisition, were up-regulated in the wmpD- mutant, possibly to compensate for the lack of TonS-dependant receptors in the outer membrane. Assays for iron binding activity showed that P. tunicata Wt seems to release iron binding compounds (or siderophores) constitutively into the supernatant, in contrast to the white mutant wmpD-, which responds to iron limitation by increasing the production of siderophores. Further outer membrane fractionation studies, indicated that the P. tunicata T2S system is likely to be involved in the transport of TonB-dependant receptors to the outer membrane. The overall results discussed in this thesis indicate that the T2S system has an essential role in the general physiology of P. tunicata, as for iron metabolism, as well as in the in the relationship between this bacterium and eukaryotic hosts in the environment.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1964. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Bibliography: leaves 135-142.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1967. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

No description available.

A radioimmunoassay has been developed for the major metabolite of melatonin, 6-suIphatoxymelatonin (aMT6s). The assay is specific, sensitive and is direct for both urine and plasma samples. Classical validation procedures have been employed and the urinary assay has been compared with an established gas chromatographic/mass spectrometric assay. A number of physiological studies have been carried out. A marked diurnal rhythm in both urinary and plasma aMT6s excretion was found which correlated closely with plasma melatonin values. There were large inter-individual variations in aMT6s excretion but its production was consistent for any one volunteer over a four-day period. No evidence was obtained for the episodic secretion of melatonin or aMT6s when blood samples were taken every thirty seconds for ten minutes. Administration of a peripheral B-blocker to volunteers, resulted in the abolition of the night-time rise in aMT6s excretion. In a seasonal study, a small but highly significant change in the acrophase of the aMT6s rhythm was found during the year with a phase advance in summer compared to winter. Changes in aMT6s excretion with age were also investigated. Total urinary aMT6s excretion was relatively constant in forty children aged 2-20 years. However, when aMT6s excretion was expressed as a function of body weight highly significant age-related changes were observed. In ninety adult volunteers aged 20-80 years there was a significant decline in total 24h aMT6s excretion with age, with significantly lower excretion in elderly subjects. No relationships were found between total 24h aMT6s excretion and body weight, height or pineal calcification. In addition to the above physiological studies, the pharmacokinetics of melatonin and aMT6s were investigated following the oral administration of melatonin to normal volunteers.