Glycosylation of rat NGF receptor ectodomain in the yeast Saccharomyces cerevisiae

AbstractHere we studied the glycosylation of a mammalian protein, the ectodomain of rat nerve growth factor receptor (NGFRe), in Saccharomyces cerevisiae. NGFRe is secreted to the culture medium of S. cerevisiae if it is fused to a polypeptide (hsp150Δ) carrier. The hsp150Δ-carrier has 95 serine and threonine residues, which were extensively O-glycosylated. In spite of 41 potential sites, NGFe lacked O-glycans, whether fused to the carrier or not. Distortion of the conformation of NGFRe by inhibition of disulfide formation did not promote O-glycosylation, whereas N-glycosylation was enhanced. Thus, the serine and threonine residues of the hsp150Δ-NGFRe fusion protein were highly selectively O-glycosylated

Functional expression of NF1 tumor suppressor protein: association with keratin intermediate filaments during the early development of human epidermis

BACKGROUND: NF1 refers to type 1 neurofibromatosis syndrome, which has been linked with mutations of the large NF1 gene. NF1 tumor suppressor protein, neurofibromin, has been shown to regulate ras: the NF1 protein contains a GTPase activating protein (GAP) related domain which functions as p21rasGAP. Our studies have previously demonstrated that the NF1 protein forms a high affinity association with cytokeratin 14 during the formation of desmosomes and hemidesmosomes in cultured keratinocytes. METHODS: The expression of NF1 protein was studied in developing human epidermis using western transfer analysis, indirect immunofluorescence, confocal laser scanning microscopy, immunoelectron microscopy, and in situ hybridization. RESULTS: The expression of NF1 protein was noted to be highly elevated in the periderm at 8 weeks estimated gestational age (EGA) and in the basal cells at 8–14 weeks EGA. During this period, NF1 protein was associated with cytokeratin filaments terminating to desmosomes and hemidesmosomes. NF1 protein did not display colocalization with α-tubulin or actin of the cytoskeleton, or with adherens junction proteins. CONCLUSIONS: These results depict an early fetal period when the NF1 tumor suppressor is abundantly expressed in epidermis and associated with cytokeratin filaments. This period is characterized by the initiation of differentiation of the basal cells, maturation of the basement membrane zone as well as accentuated formation of selected cellular junctions. NF1 tumor suppressor may function in the regulation of epidermal histogenesis via controlling the organization of the keratin cytoskeleton during the assembly of desmosomes and hemidesmosomes

Localization of general and regulatory proteolysis in Bacillus subtilis cells

Protein degradation mediated by ATP-dependent proteases, such as Hsp100/Clp and related AAA+ proteins, plays an important role in cellular protein homeostasis, protein quality control and the regulation of, e.g. heat shock adaptation and other cellular differentiation processes. ClpCP with its adaptor proteins and other related proteases, such as ClpXP or ClpEP of Bacillus subtilis, are involved in general and regulatory proteolysis. To determine if proteolysis occurs at specific locations in B. subtilis cells, we analysed the subcellular distribution of the Clp system together with adaptor and general and regulatory substrate proteins, under different environmental conditions. We can demonstrate that the ATPase and the proteolytic subunit of the Clp proteases, as well as the adaptor or substrate proteins, form visible foci, representing active protease clusters localized to the polar and to the mid-cell region. These clusters could represent a compartmentalized place for protein degradation positioned at the pole close to where most of the cellular protein biosynthesis and also protein quality control are taking place, thereby spatially separating protein synthesis and degradation

Tight Junction-Related Barrier Contributes to the Electrophysiological Asymmetry across Vocal Fold Epithelium

Electrophysiological homeostasis is indispensable to vocal fold hydration. We investigate tight junction (TJ)-associated components, occludin and ZO-1, and permeability with or without the challenge of a permeability-augmenting agent, histamine. Freshly excised ovine larynges are obtained from a local abattoir. TJ markers are explored via reverse transcriptase polymerase chain reaction (RT-PCR). Paracellular permeabilities are measured in an Ussing system. The gene expression of both TJ markers is detected in native ovine vocal fold epithelium. Luminal histamine treatment significantly decreases transepithelial resistance (TER) (N = 72, p<0.01) and increases penetration of protein tracer (N = 35, p<0.001), respectively, in a time-, and dose-dependent fashion. The present study demonstrates that histamine compromises TJ-related paracellular barrier across vocal fold epithelium. The detection of TJ markers indicates the existence of typical TJ components in non-keratinized, stratified vocal fold epithelium. The responsiveness of paracellular permeabilities to histamine would highlight the functional significance of this TJ-equivalent system to the electrophysiological homeostasis, which, in turn, regulates the vocal fold superficial hydration

Keratinocytes as Depository of Ammonium-Inducible Glutamine Synthetase: Age- and Anatomy-Dependent Distribution in Human and Rat Skin

In inner organs, glutamine contributes to proliferation, detoxification and establishment of a mechanical barrier, i.e., functions essential for skin, as well. However, the age-dependent and regional peculiarities of distribution of glutamine synthetase (GS), an enzyme responsible for generation of glutamine, and factors regulating its enzymatic activity in mammalian skin remain undisclosed. To explore this, GS localization was investigated using immunohistochemistry and double-labeling of young and adult human and rat skin sections as well as skin cells in culture. In human and rat skin GS was almost completely co-localized with astrocyte-specific proteins (e.g. GFAP). While GS staining was pronounced in all layers of the epidermis of young human skin, staining was reduced and more differentiated among different layers with age. In stratum basale and in stratum spinosum GS was co-localized with the adherens junction component ß-catenin. Inhibition of, glycogen synthase kinase 3β in cultured keratinocytes and HaCaT cells, however, did not support a direct role of ß-catenin in regulation of GS. Enzymatic and reverse transcriptase polymerase chain reaction studies revealed an unusual mode of regulation of this enzyme in keratinocytes, i.e., GS activity, but not expression, was enhanced about 8–10 fold when the cells were exposed to ammonium ions. Prominent posttranscriptional up-regulation of GS activity in keratinocytes by ammonium ions in conjunction with widespread distribution of GS immunoreactivity throughout the epidermis allows considering the skin as a large reservoir of latent GS. Such a depository of glutamine-generating enzyme seems essential for continuous renewal of epidermal permeability barrier and during pathological processes accompanied by hyperammonemia

Homeostatic regulation of the endoneurial microenvironment during development, aging and in response to trauma, disease and toxic insult

The endoneurial microenvironment, delimited by the endothelium of endoneurial vessels and a multi-layered ensheathing perineurium, is a specialized milieu intérieur within which axons, associated Schwann cells and other resident cells of peripheral nerves function. The endothelium and perineurium restricts as well as regulates exchange of material between the endoneurial microenvironment and the surrounding extracellular space and thus is more appropriately described as a blood–nerve interface (BNI) rather than a blood–nerve barrier (BNB). Input to and output from the endoneurial microenvironment occurs via blood–nerve exchange and convective endoneurial fluid flow driven by a proximo-distal hydrostatic pressure gradient. The independent regulation of the endothelial and perineurial components of the BNI during development, aging and in response to trauma is consistent with homeostatic regulation of the endoneurial microenvironment. Pathophysiological alterations of the endoneurium in experimental allergic neuritis (EAN), and diabetic and lead neuropathy are considered to be perturbations of endoneurial homeostasis. The interactions of Schwann cells, axons, macrophages, and mast cells via cell–cell and cell–matrix signaling regulate the permeability of this interface. A greater knowledge of the dynamic nature of tight junctions and the factors that induce and/or modulate these key elements of the BNI will increase our understanding of peripheral nerve disorders as well as stimulate the development of therapeutic strategies to treat these disorders

Etiology of Aflatoxin Contamination of Dried Red Chilies and Diversity of Toxic Aspergilli in The United States

Aflatoxins are hepatocarcinogenic mycotoxins produced by fungi within Aspergillus section Flavi. Aflatoxin-producing fungi frequently contaminate crops including maize, cottonseed, groundnuts, tree nuts and spices. Contamination of food and feed with aflatoxins can have severe health consequences, including death of humans and animals. Crops contaminated with aflatoxins may be excluded from markets, and, if in excess of regulatory limits, destroyed; this creates an immense economic burden for growers of crops susceptible to aflatoxin contamination. The research presented in this dissertation focuses on two areas related to aflatoxin contamination of crops. The first one focusses on prevalence of aflatoxin contamination in market-purchased chilies and etiology of contamination. Dried red chili from the genus Capsicum is one of the most extensively consumed spices worldwide to enhance the flavor, color and aroma of different foods. Most chilies are produced in warm regions of the globe that where the climate favors growth and proliferation of filamentous fungi that produce aflatoxins. Appendix A presents a survey of aflatoxin contamination levels in chilies from markets in the United States (US) and Nigeria, two markets differing in regulation enforcement. Unacceptable concentrations of aflatoxins were detected in two percent of samples from US markets and seven percent of Nigerian chilies. Members of Aspergillus section Flavi were recovered from 40% of chilies from the US, although 64% of the samples were contaminated with aflatoxins, indicating sterilization of dried red chilies prior to sale in the US. Both average aflatoxin concentrations and fungal quantities were significantly higher in Nigerian chilies compared to those purchased in the US. Incubation of un-inoculated chilies under conditions conducive for aflatoxin production led to a build-up of lethal concentrations of aflatoxins in Nigerian chilies, indicating higher risk associated with post-purchase practices of consumers in Nigeria compared to that in US. Appendix B presents identification of aflatoxin-producing Aspergilli recovered from chilies from markets in the US and Nigeria. A precise understanding of the etiology of aflatoxin contamination of crops is crucial for development of mitigation strategies. Investigation of Aspergillus communities associated with chilies and the etiology of aflatoxin contamination conducted using aflatoxin-based liquid culture and crop assays as well as multi-locus phylogenetics showed the occurrence of three distinct aflatoxin-producing species in US market chilies. Of these, A. aflatoxiformans is a non-native pathogen and was imported with chilies originating from Nigeria. Five distinct aflatoxin-producers, including a novel lineage, were discovered in Nigerian chilies. Aspergillus aflatoxiformans was identified as the most important etiologic agent of chili contamination in Nigeria based on its overall incidence and aflatoxin producing ability in chilies. The novel lineage has the “S”, small sclerotium (< 400 µm) morphology, and produces high concentrations of B and G aflatoxins in chilies. Several A. flavus L morphotype isolates were recovered from chilies that did not produce detectable concentrations of aflatoxins in vitro and can be a potential resource for development of biological control agents for preventing aflatoxin contamination in chilies. In Appendix C, an in vitro assay has been developed for distinguishing isolates of A. aflatoxiformans and A. minisclerotigenes, aflatoxin-producing species which co-occur in West Africa, and contaminate major crops like maize, groundnuts and chilies with unacceptable concentrations of aflatoxins. These fungi otherwise have largely overlapping morphologies and aflatoxin profiles and are indistinguishable in the absence of molecular tools. The second part of this dissertation focuses on the genetic diversity of fungi with S morphology within Aspergillus section Flavi due to their highly aflatoxigenic potential. Fungal species with S morphology have been implicated as causal agents of crop contamination in both US and Africa. Appendix D describes a novel aflatoxin-producing species Aspergillus texensis from the US. Appendix E presents characterization of S morphology fungi resident in US soils and crops using phylogenetics, aflatoxin profiles and simple sequence repeat markers. Four phylogenetically distinct groups were identified, each of which produced dangerous concentrations of aflatoxins in maize and should be considered when devising management practices for aflatoxin mitigation.Release after 06/24/202

Selvitystyö maatilamittaluokan biokaasulaitoksen perustamisesta Helsingin yliopiston Viikin opetus- ja tutkimustilalle

Tämän opinnäytetyön tavoitteena oli selvittää Helsingin yliopiston Viikin opetus- ja tutkimustilalle perustettavan biokaasulaitoksen toimintaedellytyksiä. Työssä kartoitettiin biokaasuprosessiin sopivia, lähiseudulla helposti saatavilla olevia syötteitä, ja selvitettiin kartoituksen tuloksena valittuun syöteseokseen sopivia biokaasulaitosratkaisuja. Syötteiksi valikoituivat Viikin opetus- ja tutkimustilan maatalousyötteiden lisäksi Vanhankaupunginlahden järviruoko, läheisen ratsastuskeskuksen hevosenlanta sekä lähiseudun pienpanimon olutmäski. Useilta biokaasulaitostoimittajilta saatiin tarjouksia biokaasulaitoksista, jotka perustuivat kolmeen eri biokaasun tuotantoprosessiin: märkä- ja kiintomädätykseen, kuivamädätykseen ja kaksivaiheiseen mädätykseen. Kaikki tarjotut teknologiat soveltuivat valitulle syöteseokselle. Biokaasulaitoksen tuottama biokaasu on taloudellisesti kannattavampaa jalostaa metaaniksi kuin tuottaa siitä sähköä ja lämpöä Viikin opetus- ja tutkimustilan tarpeisiin. Koska kysyntä hiilineutraalille metaanille on suurta, jalostettu metaani voitaisiin hyödyntää joko liikkeenpolttoaineena tai kaasunjakeluverkossa. Investointi- ja kannattavuuslaskelmien perusteella biokaasulaitoksesta ei todennäköisesti tulisi kannattavaa, vaikka investointikustannuksiin saataisiinkin Työ- ja elinkeinoministeriön energiatukea

Kohti onnistumisia hyvinvoivalla mielellä

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