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Analysis of TpeL secretion in Clostridium perfringensSaadat, Angela P. 11 January 2021 (has links)
Clostridia are a class of gram-positive, anaerobic bacteria best known for their powerful toxins. These bacteria cause many diseases that are difficult to treat and often deadly, including colitis, botulism, tetanus and gas gangrene. These diseases are caused by the secretion of specific toxins, though current treatments do little to nullify these toxins and better therapeutics are urgently needed. The development of such treatments is hindered by our poor understanding of clostridial toxin secretion, which is itself hindered by the innate characteristics of these bacteria that make them difficult to study. Of the pathogenic clostridia, Clostridium perfringens is relatively easy to culture and straddles the line between pathogen and commensal, making it an attractive model organism for studying clostridial toxin secretion. C. perfringens is a bacterium found naturally in soils and in the gastrointestinal tracts of humans and animals that can also cause disease. C. perfringens produces more toxins than any other bacterium, and these toxins generally function as a means to lyse host cells so the bacteria may scavenge their intracellular nutrients. The primary focus of the research in this dissertation is the secretion of the toxin TpeL by a small membrane protein, TpeE. Preceding the study of TpeL secretion were two other projects, which are discussed in Chapters 2 and 3.
Chapter 2 describes an experimental plan to characterize the genes involved in muscle cell adherence as a very basic model to mimic skeletal muscle attachment in gas gangrene. Like many other bacteria, C. perfringens can produce T4P, extracellular filaments that are synthesized, extended and retracted from the cell by the concerted effort of many proteins. Results from initial, proof-of-concept adherence assays are presented and demonstrate that statistical significance was lost when data were compiled. Despite efforts to troubleshoot this, robust test output was not achieved and the project was discontinued November 2016. Chapter 3 describes the experimental plan and initial findings of a project where a link between T4P and virulence was investigated. Such a link had been demonstrated in the T4P model organism Pseudomonas aeruginosa, where PilT, the T4P retraction ATPase, was shown to sense surface attachment and initiate virulence. In C. perfringens, PilT demonstrates a number of characteristics that lead us to think it may also function as a sensor, coordinating host cell attachment and colonization by alternatively associating with PilM and FtsA. We developed an experimental plan to determine if PilT binds both PilM and FtsA by co-immunoprecipitation with live-cell fluorescence imaging. However, we were unable to demonstrate the functionality of a PilT-fluorescent protein fusion with an anti-pilin ELISA assay, nor were we able to detect PilT or FtsA overexpression by immunoblotting, and the project was discontinued in November 2017. In retrospect, these experiments likely failed because of an inactive promoter region in the overexpression plasmid.
Though clostridial diseases require secreted toxins, their secretion mechanisms are largely uncharacterized, and Chapter 4 describes the investigation of a potentially conserved toxin secretion mechanism. TpeL is a recently discovered C. perfringens toxin that is associated with chicken necrotic enteritis, a disease that costs the poultry industry billions of dollars each year. TpeL belongs to a subset of clostridial toxins characterized by their large size and conserved structure, the large clostridial toxins. The gene for tpeL and nearly all other large clostridial toxins lies next to a gene encoding a small membrane protein. Since bacterial genes with a shared function are often found in close proximity, it is suspected that these small proteins share some function with these toxins, and another research group has shown the two large clostridial toxins in C. difficile need this small membrane protein for their secretion. We isolated the small membrane protein and toxin genes tpeE and tpeL from native regulatory elements and overexpressed them heterologously in a different strain of C. perfringens. By immunoblotting, we found rapid TpeL secretion requires TpeE, and secretion was abolished when C-terminal sections of either protein were mutated. By immunoblotting and growth curve analyses, we found that TpeE is maintained at low concentrations and is not lethal in C. perfringens, but was expressed to high levels and was lethal in Escherichia coli. Our results, in conjunction with those from other research groups strongly suggest a conserved secretion mechanism dependent on small, membrane proteins. Our findings further the understanding of toxin secretion, a key step toward novel and effective clostridial disease strategies.
Chapter 5 describes the outcome of an experimental approach where tpeE and tpeL were expressed from two different expression system plasmids. A number of off-target effects materialized with this approach which confounded our experimental results. The predominantly confounding effect was off-target protein secretion, found by immunoblotting to be associated with one of the expression systems. Despite efforts to minimize these effects, it became clear results from this approach would be uninterpretable and the two-plasmid approach for TpeE and TpeL expression was abandoned. A cut-and-paste strategy using the historical, single inducible expression system was implemented in its place.
The exact mechanism for TpeL secretion by the small membrane protein TpeE is unclear. Chapter 6 outlines some hypotheses towards this mechanism and a nascent plan to uncover it. An efficient starting point is to determine if the two proteins are in close enough proximity to one another to interact in vivo. We developed a strategy to determine this by crosslinking and immunoblotting, using the size differential between the proteins to our advantage. Though the results of this study were confounded by an inability of TpeL to solubilize in buffer, the groundwork is laid for future endeavors. / Doctor of Philosophy / Clostridium perfringens is a bacterium found naturally in soils and in the gastrointestinal tracts of humans and animals worldwide. C. perfringens is an important organism to study due to its roles as a decomposer in our ecosystem and its ability to cause a number of diseases. These diseases cause considerable harm to livestock and poultry industries, as well as to human and animal life. Though these diseases vary wildly, they share this in common: they are defined by specific toxins and what defines a harmful lineage of C. perfringens is its ability to produce and secrete these toxins. In fact, this is the common denominator to all clostridial diseases, including the notorious diseases C. difficile-associated colitis, tetanus, botulism, and gas gangrene. Of primary concern in diseases caused by C. perfringens and other clostridia is that effective, novel therapies are grossly lacking. The effects of these powerful toxins can outpace antibiotic therapy and this often leads to extended periods of suffering, even in favorable cases. Of all the pathogenic clostridia, C. perfringens is easy to test in the laboratory and may even be used in place of more dangerous and difficult to work with bacteria. This is useful in developing better treatments and for studying treatment applications for the toxins themselves! Indeed, bacterial toxins have beneficial applications, Botox being a good example, as well as in cancer treatments.
Like many other bacteria, C. perfringens can produce strong, rope-like appendages called pili that are made and extended and retracted from the cell, similar to a lasso, by the concerted effort of many different proteins. These pili confer a number of advantages for bacteria, one being a means for attachment to host cells, an important first step in establishing an infection. With the overarching goal toward sowing future therapeutic developments, Chapter 2 describes an experimental plan to identify and understand the genes for the proteins that allow C. perfringens to attach to muscle cells. Preliminary results are presented for a proof-of-concept method, which was ultimately discontinued November 2016 because reliable, robust results were not obtained.
In addition to host cell attachment, pili have been shown to function a sensor for cell. For example, in another bacterium, pili "sense" a suitable surface for attachment and interpret this signal so the bacterium can attach and "set up shop" by releasing toxins. Based on considerable evidence, we thought C. perfringens might also "sense" a surface with its pili and interpret this signal for attachment and cell growth by means of interactions between three specific proteins. We designed a series of experiments to test this hypothesis, but due to the failure of important, initial studies, this project was discontinued in November 2017.
Even though all clostridial diseases are caused by toxins, which must be secreted outside the bacterium to do harm, how these toxins are secreted is poorly understood. In Chapter 4, we investigate a toxin secretion method where a certain type of toxin is thought to be secreted through a temporary hole formed by many copies of a small, partner. First, we forced C. perfringens bacteria to artificially produce both the small protein and the toxin and found that the toxin needs this small protein to be secreted. We then deleted parts of both the toxin and the small protein and determined which parts of each are essential for this secretion method by linking an absence of secretion in bacteria whose proteins are missing essential parts. Further, we determined that production of this small, partner protein was kept to low levels and was harmless in C. perfringens, but was lethal in a different, unrelated bacterium, Escherichia coli, implying that C. perfringens bacteria have an ability control this hypothetical hole in themselves that E. coli does not. Our results, in conjunction with those from previous groups, suggest a pattern for secretion of this type using these small proteins. This information is a key first step towards developing better therapies for clostridial diseases, since without toxin secretion, clostridial diseases cannot occur.
Chapter 5 describes the surprising outcome of an experimental approach where the toxin and small partner protein are produced in the bacterium by two different mechanisms. We found a number of off-target effects associated with this approach, one of which was the strange facilitation of off-target protein secretion. These off-target effects confused our experimental results and since it was likely that future experiments would also be uninterpretable, we abandoned this approach and used a simpler one instead.
The mechanisms for toxin (TpeL) secretion by its small, partner protein (TpeE) are unclear. A key, initial step towards understanding this mechanism is to determine if the two proteins are in close enough proximity to one another in the bacterium. We developed a strategy to determine if the proteins are close enough together in the cell that takes advantage of the considerable size difference between the two proteins. Presented in this chapter are several initial experiments that can enable this experiment in the future.
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Regulation by glutamate- and adenosine-receptors of dopamine and acetylcholine release from rat striatal slices /Jin, Shaoyu. January 1900 (has links)
Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser.
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The Chlamydia Trachomatis Protein Interaction Network: Insights into the Unique Composition of the Type Three Secretion SystemSpaeth, Kris Edmund 19 November 2008 (has links)
<p>The Gram-negative bacteria Chlamydia trachomatis is a common sexually transmitted pathogen that can cause severe sequelae including cause pelvic inflammatory disease and sterility. This obligate intracellular pathogen effectively manipulates host cellular functions by secreting virulence factors across its membrane bound vacuole. Identifying these virulence components and how they help in establishing an environment conducive for bacterial growth is central to understanding chlamydial pathogenesis. This is experimentally challenging due to a lack of tools to perform molecular genetic studies. In the absence of genetic tools, we developed a yeast model system to identify and characterize chlamydial proteins involved in virulence mechanisms. In this study we describe the identification of twenty-eight proteins potentially involved in modulating host cellular functions and the secretion of virulence factors into the host. Since the delivery of virulence proteins by a type three secretion (T3S) system is a critical step for Chlamydia, we identified the proteins that interacted with the T3S apparatus by yeast two-hybrid analysis. We discovered several novel interactions between and determined that the C. trachomatis T3S apparatus displayed a similar architecture to that of other T3S systems. Furthermore with these approaches we identified networks of proteins that interacted with the secretion apparatus including a novel secretion chaperone protein. We characterized Ct260/Mcsc one of the putative secretion and demonstrated that it represents a novel class 1B secretion chaperone protein. Unlike other known chaperones, Mcsc directly interact with a conserved component of the T3S apparatus cytoplasmic domain, CdsQ. These finding represents a novel mechanism by which the secretion chaperone protein Ct260 may increase the secretion efficiency of its effector cargo and may reveal new facets of secretory cargo recognition by T3S systems.</p> / Dissertation
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Regulation and function of 11β-hydroxysteroid dehydrogenase (11β-HSD1) in pancreatic β-cellsLiu, Xiaoxia January 2011 (has links)
Diabetes Mellitus is characterized by high blood sugar and is caused by resistance to (type 2) or insufficiency of (type 1) the pancreatic β-cell hormone insulin. Most commonly, type 2 diabetes is associated with obesity whereas type 1 diabetes is largely a result of immune-mediated destruction of the β-cell. One rare but significant cause of type 2 diabetes is excess blood glucocorticoid levels (Cushing’s syndrome). High circulating glucocorticoids potently induce metabolic disorders including peripheral insulin resistance in key metabolic tissues (muscle, liver and fat) as well as directly suppressing β-cell function and can precipitate type 2 diabetes. However, in common forms of metabolic syndrome (visceral obesity, type 2 diabetes, increased cardiovascular disease risk) it appears that amplification of local tissue glucocorticoid action by increased levels of the intracellular enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), particularly in adipose tissue, is a key driver of the adverse metabolic phenotype rather than altered circulating glucocorticoid levels. 11β-HSD1 is also elevated in pancreatic islets from obese rodents. This thesis aimed to determine the role of 11β-HSD1 in pancreatic islets (β-cells) under normal conditions and its potential pathogenic role in the development of diabetes. We first determined that 11β-HSD1 acted primarily as a reductase (amplifying glucocorticoid action) in pancreatic islets. We then determined that islet 11β-HSD1 transcription is under the control of the promoters that express in other tissues like liver. Islet 11β-HSD1 is significantly regulated by factors relevant to the diabetic state; high glucose and insulin suppressed whereas fatty acids and TNFα increased 11β-HSD1 activity. To test whether the high islet 11β-HSD1 found in obese rodents was directly diabetogenic, we generated transgenic mice specifically overexpressing β-cell 11β-HSD1 under the mouse insulin promoter (MIP-HSD1 mice) in a mouse strain prone to develop β-cell failure when subjected to diabetic challenge (eg. chronic high fat feeding). Unexpectedly, MIP-HSD1tg/+ mice (expressing ~2 fold elevated 11β-HSD1 activity) exhibited markedly improved β-cell insulin secretory responses, whereas MIP-HSD1tg/tg mice had partially impaired β-cell insulin secretory function in vivo and in vitro. Moreover, MIP-HSD1tg/+ mice completely resisted the mild hyperglycaemia induced by multiple-low doses of the β-cell toxin streptozotocin (40mg/kg i.p. for 5 days) and partially resisted the profound hyperglycaemia induced by a single high dose of streptozotocin (180mg/kg). Notably, MIP-HSD1tg/+ mice exhibited lower macrophage infiltration (MAC-2) and higher T-regulatory cell (Foxp3) infiltration after these challenges with evidence of increased insulin-positive cells and maintenance of normal levels of proliferation-competent β-cells. Overall, MIP-HSD1tg/tg exhibited a partial protection from the streptozotocin challenge. Modestly increased 11β-HSD1 expression in β-cells unexpectedly supports compensatory insulin hypersecretion preventing type 2 diabetes and protects β-cells from inflammatory mediated damage in the setting of type 1 diabetes. Above a protective threshold, elevated β-cell 11β-HSD1 may result in β-cell dysfunction and diabetes. These findings have important implications for the currently advocated therapeutic strategies to inhibit 11β-HSD1 in the context of obesity and diabetes.
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G-protein coupled receptors modulating incretin hormone secretionMoss, Catherine Elizabeth January 2014 (has links)
No description available.
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The Mycobacterium tuberculosis ESX-3 secretion system interactomeNewton-Foot, Mae 03 1900 (has links)
Thesis (MScMedSc (Biomedical Sciences. Human Biology and Human Genetics))--University of Stellenbosch, 2010. / Thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Medical Biochemistry at the Faculty of Health Sciences, University of Stellenbosch. / ENGLISH ABSTRACT: Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease which causes
approximately 2 million deaths each year. Despite extensive research on tuberculosis and M.
tuberculosis, little is understood of the mechanisms of pathogenicity of the organism. The genome of
M. tuberculosis contains five ESAT-6 gene cluster regions, each of which contains genes encoding
proteins involved in the formation of a dedicated protein secretion system. Included in these regions
are genes encoding exported T-cell antigens, serine proteases, ATP-binding proteins and other
membrane-associated proteins. Although it is known that some of these secretion systems are
involved in virulence and phagosomal escape of M. tuberculosis, and that deletion thereof causes
attenuation of the organism, the structure, substrates and functions of the systems are largely
unknown. Understanding the structure of the ESX secretion systems will advance our understanding
of the mechanisms of mycobacterial pathogenicity and provide clues to ways in which to interfere with
these virulence mechanisms.
The ESAT-6 gene cluster region 3, encoding the ESX-3 secretion machinery, is the only ESAT-6 gene
cluster region which is essential for the in vitro growth of M. tuberculosis. It is however not required for
the growth of the saprophytic mycobacterium M. smegmatis. In this study we have identified proteinprotein
interactions within the ESX-3 secretion system, using the Mycobacterial – Protein Fragment
Complementation (M-PFC) mycobacterial two-hybrid system, and created a model of the M.
tuberculosis ESX-3 secretion system. According to this model, the EsxG-EsxH and PE5-PPE4
substrate protein complexes bind to the same components of the ESX-3 secretion machinery and are
secreted via the same mechanism. A knock-out of the ESX-3 secretion system in M. smegmatis was
generated by homologous recombination to allow further research into the functions and properties of
this secretion system. This knock-out was used, together with wild-type M. smegmatis, to investigate
the secretion of the M. tuberculosis EsxH protein by the M. smegmatis ESX-3 secretion system.
The ESX-3 secretion system interactome may serve as a model for the ESX secretion systems and
assist in our understanding of this secretion machinery which is key to the virulence and survival of M.
tuberculosis and other pathogenic mycobacteria. Improved understanding of these mechanisms and
their role in pathogenicity and survival may provide means of interfering with the secretion machinery,
potentially leading to developments in the prevention and treatment of tuberculosis disease. / AFRIKAANSE OPSOMMING: Tuberkulose, wat veroorsaak word deur Mycobacterium tuberculosis, eis jaarliks ongeveer 2 miljoen
lewens. Ten spyte van uitgebreide navorsing oor tuberkulose en M. tuberculosis is min bekend oor die
meganismes van patogenisiteit van díe organisme. Die genoom van M. tuberculosis bevat vyf ESAT-6
geen groep gebiede wat elk proteïene kodeer wat ‘n toegewyde sekresie sisteem vorm. Ingesluit in elk
van díe geen groep gebiede is gene wat T-sel antigene, serien proteases, ATP-bindingsproteïene en
ander membraan-geassosieërde proteïene kodeer. Alhoewel dit bekend is dat sekere van hierdie
sekresie sisteme betrokke is by virulensie en fagosoom-ontsnapping, en dat delesie daarvan die
organisme attenueer, is die struktuur, substrate en funksies van die sisteme grootliks onbekend.
Kennis van die struktuur van die ESX sekresie sisteme sal ons verstaan van die meganismes van
mikobakteriele patogenisiteit verbeter en leidrade verskaf na maniere om in te meng by díe
meganismes van virulensie.
Die ESAT-6 geen groep gebied 3, wat die ESX-3 sekresie sisteem kodeer, is die enigste ESAT-6
geen groep gebied wat noodsaaklik is vir die in vitro groei van M. tuberculosis. Dit is egter nie nodig vir
die groei van die saprofitiese mikobakterium M. smegmatis nie. In hierdie studie het ons proteïenproteïen
interaksies van die ESX-3 sekresie sisteem geïdentifiseer, deur middel van die
Mikobakteriële - Proteïen Fragment Komplementasie (M-PFC) mikobacteriële twee-hibriede stelsel.
Die interaksies is gebruik om ‘n model van die M. tuberculosis ESX-3 sekresie sisteem te skep.
Volgens díe model bind die EsxG-EsxH en PE5-PPE4 substraat proteïen komplekse aan dieselfde
komponente van die ESX-3 sekresie apparaat en word deur dieselfde meganisme uitgevoer. ‘n
uitklopmutant van die ESX-3 sekresie sisteem word deur homoloë rekombinasie in M. smegmatis
gegenereer om verdere ondersoeke na die funksies en eienskappe van hierdie sekresie sisteem in
staat te stel. Hierdie uitklopmutant is tesame met die wilde-tipe M. smegmatis gebruik om die sekresie
van die M. tuberculosis EsxH proteïen deur die M. smegmatis ESX-3 sekresie sisteem te ondersoek.
Die ESX-3 sekresie sisteem interaktoom kan dien as ‘n model vir die ESX sekresie sisteme om te help
om ons kennis van hierdie sekresie apparaat, wat belangrik is vir die virulensie en oorlewing van M.
tuberculosis en ander patogeniese mikobakterieë, te verbeter. Kennis van hierdie meganismes en hul
rol in patogenisiteit en oorlewing mag maniere verskaf om by díe sekresie sisteme in te meng, wat
moontlik kan lei tot ontwikkelings in die voorkoming en behandeling van tuberkulose.
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Development of synthetic signal sequences for heterologous protein secretion from Saccharomyces cerevisiaeKriel, Johan Hendrik 12 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes and
involve the interplay of a multitude of proteins. A unique collection of thermosensitive
secretory mutants allowed scientists to demonstrate that the secretory pathway of the
yeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. All
proteins commence their journey in the endoplasmic reticulum, where they undergo
amino-linked core glycosyl modification. After passage through the Golgi apparatus,
where the remodelling of the glycosyl chains is completed, proteins are transported to
their final destinations, which are either the cell surface, periplasmic space or the
vacuole.
Proteins destined for secretion are usually synthesised with a transient
amino-terminal secretion leader of varying length and hydrophobicity, which plays a
crucial role in the targeting and translocation of their protein cargo. Considerable
effort has been made to elucidate the molecular mechanisms involved in these
processes, especially due to their relevance in a rapidly expanding biotech industry.
The advantages of S. cerevisiae as a host for the expression of recombinant
proteins are well documented. Unfortunately, S. cerevisiae is also subject to a
number of drawbacks, with a relative low product yield being one of the major
disadvantages.
Bearing this in mind, different secretion leaders were compared with the aim of
improving the secretion of the LKA 1 and LKA2 a-amylase enzymes from the
S. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known for
its ability to degrade raw starch and an improved secretion of its amylase enzymes
from S. cerevisiae paves the way for a potential one-step starch utilisation process.
Three sets of constructs were prepared containing the LKA 1 and LKA2 genes
separately under secretory direction of either their native secretion leader, the
S. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1
secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). The
inclusion of a spacer peptide in the latter set of constructs ensured improved Kex2p
proteolytic processing of the leader/protein fusion. Strains expressing the amylase
genes under their native secretion leaders resulted in the highest saccharolytic
activity in the culture medium. In contrast to this, strains utilising the synthetic
secretion leader produced the highest fermentation yield, but had a lower than
expected extracellular activity. We hypothesise that the native amylase leaders may
function as intramolecular chaperones in the folding and processing of their
passenger proteins, thereby increasing processing efficiency and concomitant
enzyme activity. / AFRIKAANSE OPSOMMING: Proteïensekresie en intrasellulêre transport is hoogs gereguleerde prosesse en
betrek die onderlinge wisselwerking van 'n verskeidenheid proteïene. 'n Unieke
versameling van temperatuur-sensitiewe sekresiemutante het wetenskaplikes in staat
gestelom die ooreenkoms tussen die sekresiepad van die gis
Saccharomyces cerevisiae en dié van komplekser eukariote aan te toon. Alle
proteïene begin hul reis in die endoplasmiese retikulum, waartydens hulle ook
amino-gekoppelde kernglikosielveranderings ondergaan. Nadat die proteïene deur
die Golgi-apparaat beweeg het, waar die laaste veranderings aan die
glikosielkettings plaasvind, word hulle na hul finale bestemmings, waaronder die
seloppervlak, die periplasmiese ruimte of die vakuool, vervoer.
Proteïene wat vir sekresie bestem is, word gewoonlik met 'n tydelike,
amino-eindpuntsekresiesein, wat 'n kritiese rol in die teiken en translokasie van hul
proteïenvrag speel, gesintetiseer. Heelwat pogings is in hierdie studie aangewend
om die molekulêre meganismes betrokke by hierdie prosesse te ontrafel, veral as
gevolg van hul toepaslikheid in 'n vinnig groeiende biotegnologiebedryf.
Die voordele van S. cerevisiae as 'n gasheer vir die uitdruk van rekombinante
proteïene is alombekend. S. cerevisiae het egter ook verskeie nadele, waaronder die
relatiewe lae produkopbrengs die belangrikste is.
Teen hierdie agtergrond, is verskillende sekresieseine met mekaar vergelyk met
die doelom die sekresie van die LKA 1 en LKA2 a-amilasegene vanuit die
S. cerevisiae-uitdrukkingsisteem te verbeter. Die gis Lipomyces kononenkoae is
bekend vir sy vermoeë om rou stysel af te breek en 'n verbeterde sekresie van sy
amilasegene vanuit S. cerevisiae baan die weg vir 'n moontlike een-stap
styselgebruiksproses. Drie stelle konstrukte is gemaak wat die LKA 1- en LKA2- gene
onafhanklik onder sekresiebeheer van onderskeidelik hul inheemse sekresiesein, die
S. cerevisiae paringsferomoonsekresiesein (MFa1) of die MFa1-sekresiesein met 'n
sintetiese koppelingspeptied aan die C-eindpunt (EEGEPK), plaas. Die insluiting van
'n koppelingspeptied in die laasgenoemde stel konstrukte verseker verbeterde Kex2p
proteolitiese prosessering van die sein/proteïenfusie. Rasse wat die amilasegene
onder beheer van hul inheemse sekresieseine uitdruk, het die beste saccharolitiese
aktiwiteit in die kultuurmedia getoon. In teenstelling hiermee, het rasse wat van die
sintetiese sekresiesein gebruik maak, die beste fermentasie-opbrengs getoon, maar
met 'n laer as verwagte ekstrasellulêre aktiwiteit. Ons vermoed dat die inheemse
amilaseseine as intramolekulêre begeleiers optree in die vou en prosessering van hul
proteïenpassasiers, wat lei tot verbeterde prosessering en ensiemaktiwiteit.
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Acute effects of dietary fatty acids upon human milk fatty acidsFreer, Cindy A. 15 November 1995 (has links)
Although it is well-established that the fatty acid profile of breast milk will
reflect the dietary fatty acids, the response time with which this occurs is not
known. We hypothesized that fatty acids from a given meal would be transferred
acutely from chylomicrons into breast milk. To test this hypothesis, the
following experiment was performed. Fourteen lactating women drank 700
Calorie breakfast formulas containing six different test fats: 40 grams of cocoa
butter, coconut, safflower or canola oil, 20 grams of menhaden oil or 7 grams of
herring oil. Each fat contained a specific fatty acid whose appearance was
tracked in the milk. After consuming the breakfast formula, subjects collected
mid-feeding milk samples at 0, 6, 10, 14 and 24 hours, and one morning sample
on days two through seven. Fatty acids specifically tracked in milk samples
were: C12:0 (coconut oil), C18:0 (cocoa butter), C18:2n-6 (safflower oil),
C18:3n-3 (canola oil), C22:ln-ll (herring oil), and C20:5n-3 and C22:6n-3
(menhaden oil). There was a significant increase in each of these fatty acids in
human milk (p<0.001). Elevation of these fatty acids was first observed at 6
hours. Maximum increases of these fatty acids occurred 10 h after safflower oil
(177% of baseline), 14 hours after cocoa butter (154%), coconut oil (216%),
canola oil (206%) and menhaden oil (C20:5n-3 [1157%]), and 24 hours after the
herring oil (2621%) and menhaden oil (C22:6n-3 [506%]). Compared to
baseline, these fatty acids were significantly elevated (p<0.05) from 10 to 24
hours. However, after menhaden oil, C20:5n-3 was significantly elevated for 3
days and C22:6n-3 for 2 days. These data support the hypothesis that there is an
acute transfer of dietary fatty acids from chylomicrons into human milk. / Graduation date: 1996
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Glycoprotein hormone expression in the anterior pituitaryAylwin, Simon John Byng January 2001 (has links)
No description available.
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Biochemical and genetic investigation into the mode of action of the anilinopyrimidine fungicides using the cereal pathogen Stagonospora nodurum and the filamentous fungus Aspergillus nidulansHunter-Craig, Alexis C. January 2000 (has links)
No description available.
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