Casbene synthase is commonly found in Euphorbiaceae species and is likely to be the precursor to the majority of complex diterpenes found in this family.
A large number of diterpenes have been ...isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways. Other than the enzymes involved in gibberellin biosynthesis, the only diterpene synthase isolated to date from the Euphorbiaceae has been casbene synthase, responsible for biosynthesis of a macrocyclic diterpene in the castor bean (
Ricinus communis). Here, we have selected five Euphorbiaceae species in which to investigate terpene biosynthesis and report on the distribution of diterpene synthases within this family. We have discovered genes encoding putative casbene synthases in all of our selected Euphorbiaceae species and have demonstrated high-level casbene production through expression of four of these genes in a metabolically engineered strain of
Saccharomyces cerevisiae. The only other diterpene synthase found among the five plants was a neocembrene synthase from
R. communis (this being the first report of a neocembrene synthase gene). Based on the prevalence of casbene synthases, the lack of other candidates, and the structure of the casbene skeleton, we consider it likely that casbene is the precursor to a large number of Euphorbiaceae diterpenes. Casbene production levels of 31
mg/L were achieved in
S. cerevisiae and we discuss strategies to further increase production by maximizing flux through the mevalonate pathway.
Environmental DNA analysis has revolutionized the way we study rare, invasive, and endangered taxa. However, if eDNA testing is to become an increasingly reliable tool, high detection sensitivity is ...crucial. Current eDNA sampling methods, like filtration and precipitation, can only process small volumes of water per sample. If only a few samples are collected, eDNA from the target organism might be missed, leading to false‐negative results. We developed an eDNA collection method for lentic systems that improved detection sensitivity while keeping the total number of samples low. Unlike filtration and precipitation, which mainly target extracellular DNA, this method specifically targets eDNA in larger particle sizes and is not limited to processing small volumes of water. A 64‐micrometer mesh tow net was used to process >3,000 L of water per eDNA sample. We compared the tow net method to a common collection method, a 0.45 μm cellulose nitrate filter that processes about 1 L of water per eDNA sample. Paired tow and filter samples were collected at 37 locations and tested for two taxa: an aquatic plant, Northern watermilfoil (Myriophyllum sibiricum), and aquatic mollusks, including Helisoma anceps, using Kompetitive Allele Specific PCR (KASP) assays. We detected M. sibiricum significantly more frequently in tow samples than filter samples. Mollusks were detected in all eDNA samples (tow nets and filters), but when eDNA samples were diluted 25‐fold to mimic a low target concentration scenario, mollusk DNA was detected significantly more frequently in tow samples than filter samples. This high‐volume eDNA sampling method, using a tow net to process thousands of liters of water, can improve detection sensitivity for multiple taxa, making it a useful tool for researchers and managers.
Current eDNA sampling methods, like filtration and precipitation, process relatively small volumes of water per sample. If only a few samples are collected, eDNA from the target organism might be missed leading to false‐negative results. Tow nets, a high‐volume eDNA collection tool for lentic systems, process thousands of liters of water per eDNA sample and significantly improve detection sensitivity over cellulose nitrate filters for an aquatic plant and mollusk.
Adeno-associated virus (AAV) vectors are currently being used in several clinical gene-therapy trials (see the NIH OBA Human Gene Transfer Clinical Trials Database); however, little is known about ...the chromosomal effects of vector integration. Here we report that integrated vector proviruses are associated with chromosomal deletions and other rearrangements and are frequently located on chromosome 19 (although not at the wildtype AAV integration site).
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The BB (BioBreeding) rat is one of the best models of spontaneous autoimmune diabetes and is used to study non-MHC loci contributing to Type 1 diabetes. Type 1 diabetes in the diabetes-prone BB ...(BBDP) rat is polygenic, dependent upon mutations at several loci. Iddm1, on chromosome 4, is responsible for a lymphopenia (lyp) phenotype and is essential to diabetes. In this study, we report the positional cloning of the Iddm1/lyp locus. We show that lymphopenia is due to a frameshift deletion in a novel member (Ian5) of the Immune-Associated Nucleotide (IAN)-related gene family, resulting in truncation of a significant portion of the protein. This mutation was absent in 37 other inbred rat strains that are nonlymphopenic and nondiabetic. The IAN gene family, lying within a tight cluster on rat chromosome 4, mouse chromosome 6, and human chromosome 7, is poorly characterized. Some members of the family have been shown to be expressed in mature T cells and switched on during thymic T-cell development, suggesting that Ian5 may be a key factor in T-cell development. The lymphopenia mutation may thus be useful not only to elucidate Type 1 diabetes, but also in the function of the Ian gene family as a whole.
The
Gimap gene family has been shown to be integral to T cell survival and development. A frameshift mutation in
Gimap5, one of seven members of the
Gimap family, results in lymphopenia and is a ...prerequisite for spontaneous type 1 diabetes (T1D) in the BioBreeding (BB) rat. While not contributing to lymphopenia, the
Gimap family members proximal to
Gimap5, encompassed within the
Iddm39 quantitative trait locus (QTL), have been implicated in T1D. We hypothesized that expression of the
Gimap family members within the
Iddm39 QTL, during thymocyte development as well as in peripheral T and B cells contribute to T1D.
Cell sorted subpopulations were analyzed by quantitative real time (qRT) PCR.
Gimap4 expression was reduced in DR.
lyp/lyp
rat double negative, double positive and CD8 single positive (SP) thymocytes while expression of
Gimap8,
Gimap6, and
Gimap7 was reduced only in CD8 SP thymocytes. Interestingly, expression of the entire
Gimap gene family was reduced in DR.
lyp/lyp
rat peripheral T cells compared to non-lymphopenic, non-diabetic DR.
+/+
rats. With the exception of
Gimap6, the
Gimap family genes were not expressed in B cells from spleen and mesenteric lymph node (MLN). Expression of
Gimap9 was only detected in hematopoietic cells of
non B cell lineage such as macrophage, dendritic or NK cells.
These results suggest that lack of the Gimap5 protein in the DR.
lyp/lyp
congenic rat was associated with impaired expression of the entire family of
Gimap genes and may regulate T cell homeostasis in the peripheral lymphoid organs.
Biobreeding‐diabetes prone (BB‐DP) rats spontaneously develop organ‐specific autoimmunity and are severely lymphopenic and particularly deficient in ART2+ regulatory T cells. A special breed, the ...so‐called BB‐diabetic‐resistant (DR) rats, are not lymphopenic and do not develop organ‐specific autoimmunity. The genetic difference between both strains is the lymphopenia (lyp) gene. Intrathymic tolerance mechanisms are important to prevent autoimmunity, and next to thymus epithelial cells, thymus APC play a prominent part in this tolerance. We here embarked on a study to detect defects in thymus APC of the BB‐DP rat and isolated thymus APC using a protocol based on the low‐density and nonadherent character of the cells. We used BB‐DP, BB‐DR, wild‐type F344, and F344 rats congenic for the lyp gene‐containing region. The isolated thymus, nonadherent, low‐density cells appeared to be predominantly ED2+ branched cortical macrophages and not OX62+ thymus medullary and cortico‐medullary dendritic cells. Functionally, these ED2+ macrophages were excellent stimulators of T cell proliferation, but it is more important that they rescued double‐positive thymocytes from apoptosis. The isolated thymus ED2+ macrophages of the BB‐DP and the F344.lyp/lyp rat exhibited a reduced T cell stimulatory capacity as compared with such cells of nonlymphopenic rats. They had a strongly diminished capability of rescuing thymocytes from apoptosis (also of ART2+ T cells) and showed a reduced Ian5 expression (as lyp/lyp thymocytes do). Our experiments strongly suggest that branched cortical macrophages play a role in positive selection of T cells in the thymus and point to defects in these cells in BB‐DP rats.
Context: The expression of adipogenic genes in sc adipose tissue has been reported to be lower among patients with HIV-associated lipoatrophy than HIV-uninfected controls. It is unclear whether this ...is a result or cause of lipoatrophy.
Objective: The objective of the study was to investigate the temporal relationships among changes in adipogenic gene expression in sc adipose tissue and changes in body fat distribution and metabolic complications in HIV-infected subjects on antiretroviral therapy.
Design: This was a prospective longitudinal study.
Setting: The study was conducted at HIV clinics in Seattle, Washington.
Participants: The study population included 31 HIV-infected and 12 control subjects.
Interventions: Subjects were followed up for 12 months after they initiated or modified their existing antiretroviral regimen.
Main Outcome Measures: Changes in body composition, plasma lipids, insulin sensitivity, and gene expression in sc abdominal and thigh adipose tissue.
Results: Subjects who developed lipoatrophy (n = 10) had elevated fasting triglycerides 3.16 (sd 2.79) mmol/liter and reduced insulin sensitivity as measured by frequently sampled iv glucose tolerance test 1.89 (sd 1.27) × 10−4 min−1/μU·ml after 12 months, whereas those without lipoatrophy (n = 21) did not show any metabolic complications triglycerides 1.32 (sd 0.58) mmol/liter, P = 0.01 vs. lipoatrophy; insulin sensitivity 3.52 (sd 1.91) × 10−4 min−1/μU·ml, P = 0.01 vs. lipoatrophy. In subjects developing lipoatrophy, the expression of genes involved in adipocyte differentiation, lipid uptake, and local cortisol production in thigh adipose tissue was significantly reduced already at the 2-month visit, several months before any loss of extremity fat mass was evident.
Conclusions: In HIV-infected subjects, lipoatrophy is associated with elevated fasting triglycerides and insulin resistance and might be caused by a direct or indirect effect of antiretroviral drugs on sc adipocyte differentiation.
Waterfowl are natural reservoirs for zoonotic pathogens, and abundant resident (nonmigratory) Canada Geese (Branta canadensis) in urban and suburban environments pose the potential for transmission ...of Campylobacter through human contact with fecal deposits and contaminated water. In June 2008 and July 2009, we collected 318 fecal samples from resident Canada Geese at 21 locations in and around Greensboro, North Carolina, to test for Campylobacter. All campylobacter species detected were C. jejuni isolates, and prevalences in 2008 and 2009 were 5.0% and 16.0%, respectively. Prevalence of C. jejuni–positive sampling sites was 21% (3/14) and 40% (6/15) in 2008 and 2009, respectively. All C. jejuni isolates were susceptible to a panel of six antimicrobial agents (tetracycline, streptomycin, erythromycin, kanamycin, nalidixic acid, and ciprofloxacin). We used pulsed-field gel electrophoresis and fla-typing to identify several strain types among these isolates. Multilocus sequence typing of representative isolates revealed six sequence types, of which two (ST-3708 and ST-4368) were new, two (ST-702 and ST-4080) had been detected previously among C. jejuni from geese, and two (ST-991 and ST-4071) were first reported in C. jejuni from an environmental water source and a human illness, respectively. These results indicate a diverse population of antibiotic-susceptible C. jejuni in resident Canada Geese in and around Greensboro, North Carolina, and suggest a need for additional assessment of the public health risk associated with resident Canada Geese in urban and suburban areas.
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