Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens.

A culture from human stool for diagnosis of Campylobacter-based intestinal illness takes several days, a wait that taxes the fortitude of the physician and the patient. A culture is also prone to false negative results from random loss of viability during specimen handling, overgrowth of other fecal flora, and poor growth of several pathogenic Campylobacter species on traditional media. These problems can confound clinical decisions on patient treatment and have limited the field from answering fundamental questions on Campylobacter growth and infections. We describe a procedure that estimates the lower limit of bacterial numbers that can be detected by a culture and a method for quantifying survival of C. jejuni in media used for transport of this fragile organism. Knowing this information, it becomes possible to set clinically relevant detection thresholds for diagnostic tests and address unstudied issues of whether non-symptomatic colonization is prevalent, if co-infection with other enteric pathogens is common, or if bacterial load correlates with symptoms or serious sequelae. The study also included testing of 1,552 prospectively collected patient diarrheal fecal specimens that were initially classified by conventional culture and further tested by a new enzyme immunoassay. Positive and discrepant specimens were then screened by four molecular methods to assign true-positive or true-negative status. The 5 non-culture methods showed complete agreement on all 48 positive and discrepant specimens, while the culture mis-identified 14 (28%). The specimens that were incorrectly identified by culture included 13 false negative and 1 false positive sample. This basic protocol can be used with multiple Campylobacter spp. and will allow the numbers of Campylobacter bacteria that produce symptoms of gastroenteritis in humans to be determined and for prevalence rates to be updated.

Campylobacter culture fails to correctly detect Campylobacter in 30% of positive patient stool specimens compared to non-cultural methods.

Campylobacter diagnosis is hampered because many laboratories continue to use traditional stool culture, which is slow and suffers false-negative results. This large multi-site study used a composite reference method consisting of a new FDA-cleared immunoassay and four molecular techniques to compare to culture. Prospectively collected patient fecal specimens (1552) were first preliminarily categorized as positive or negative by traditional culture. All specimens were also tested by EIA, and any EIA-positive or culture-discrepant results were further characterized by 16S rRNA qPCR, eight species-specific PCR assays, bidirectional sequencing, and an FDA-cleared multiplex PCR panel. The five non-culture methods showed complete agreement on all positive and discrepant specimens which were then assigned as true-positive or true-negative specimens. Among 47 true-positive specimens, culture incorrectly identified 13 (28%) as negative, and 1 true-negative specimen as positive, for a sensitivity of 72.3%. Unexpectedly, among the true-positive specimens, 4 (8%) were the pathogenic species C. upsaliensis. Culture had a 30% false result rate compared to immunoassay and molecular methods. More accurate results lead to better diagnosis and treatment of suspected campylobacteriosis.

Transplantation of BDNF-secreting mesenchymal stem cells provides neuroprotection in chronically hypertensive rat eyes.

PURPOSE: To evaluate the ability of mesenchymal stem cells (MSCs) engineered to produce and secrete brain-derived neurotrophic factor (BDNF) to protect retinal function and structure after intravitreal transplantation in a rat model of chronic ocular hypertension (COH). METHODS: COH was induced by laser cauterization of trabecular meshwork and episcleral veins in rat eyes. COH eyes received an intravitreal transplant of MSCs engineered to express BDNF and green fluorescent protein (BDNF-MSCs) or just GFP (GFP-MSCs). Computerized pupillometry and electroretinography (ERG) were performed to assess optic nerve and retinal function. Quantification of optic nerve damage was performed by counting retinal ganglion cells (RGCs) and evaluating optic nerve cross-sections. RESULTS: After transplantation into COH eyes, BDNF-MSCs preserved significantly more retina and optic nerve function than GFP-MSC-treated eyes when pupil light reflex (PLR) and ERG function were evaluated. PLR analysis showed significantly better function (P = 0.03) in BDNF-MSC-treated eyes (operated/control ratio = 63.00% ± 11.39%) than GFP-MSC-treated eyes (operated/control ratio = 31.81% ± 9.63%) at 42 days after surgery. The BDNF-MSC-transplanted eyes also displayed a greater level of RGC preservation than eyes that received the GFP-MSCs only (RGC cell counts: BDNF-MSC-treated COH eyes, 112.2 ± 19.39 cells/section; GFP-MSC-treated COH eyes, 52.21 ± 11.54 cells/section; P = 0.01). CONCLUSIONS: The authors have demonstrated that lentiviral-transduced BDNF-producing MSCs can survive in eyes with chronic hypertension and can provide retina and optic nerve functional and structural protection. Transplantation of BDNF-producing stem cells may be a viable treatment strategy for glaucoma.

H-ras resides on clathrin-independent ARF6 vesicles that harbor little RAF-1, but not on clathrin-dependent endosomes.

Internalization of H-Ras from the cell surface onto endomembranes through vesicular endocytic pathways may play a significant role(s) in regulating the outcome of Ras signaling. However, the identity of Ras-associated subcellular vesicles and the means by which Ras localize to these internal sites remain elusive. In this study, we show that H-Ras is absent from endosomes initially derived from a clathrin-dependent endocytic pathway. Instead, both oncogenic H-Ras-61L and wild type H-Ras (basal or EGF-stimulated) bind Arf6-associated clathrin-independent endosomes and vesicles of the endosomal-recycling center (ERC). K-Ras4B-12V can also be internalized via Arf6 endosomes, and the C-terminal tails of both H-Ras and K-Ras4B are sufficient to mediate localization of GFP chimeras to Arf6-associated vesicles. Interestingly, little Raf-1 was found on these Arf6-associated endosomes even when active H-Ras was present. Instead, endogenous Raf-1 distributed primarily on EEA1-containing vesicles, suggesting that this H-Ras effector, although accessible for H-Ras interaction on the plasma membrane, appears to separate from its regulator during early stages of endocytosis. The discrete and dynamic distribution of Ras pathway components with spatio-temporal complexity may contribute to the specificity of Ras:effector interaction.

Characterization of the retinal proteome during rod photoreceptor genesis.

BACKGROUND: The process of rod photoreceptor genesis, cell fate determination and differentiation is complex and multi-factorial. Previous studies have defined a model of photoreceptor differentiation that relies on intrinsic changes within the presumptive photoreceptor cells as well as changes in surrounding tissue that are extrinsic to the cell. We have used a proteomics approach to identify proteins that are dynamically expressed in the mouse retina during rod genesis and differentiation. FINDINGS: A series of six developmental ages from E13 to P5 were used to define changes in retinal protein expression during rod photoreceptor genesis and early differentiation. Retinal proteins were separated by isoelectric focus point and molecular weight. Gels were analyzed for changes in protein spot intensity across developmental time. Protein spots that peaked in expression at E17, P0 and P5 were picked from gels for identification. There were 239 spots that were picked for identification based on their dynamic expression during the developmental period of maximal rod photoreceptor genesis and differentiation. Of the 239 spots, 60 of them were reliably identified and represented a single protein. Ten proteins were represented by multiple spots, suggesting they were post-translationally modified. Of the 42 unique dynamically expressed proteins identified, 16 had been previously reported to be associated with the developing retina. CONCLUSIONS: Our results represent the first proteomics study of the developing mouse retina that includes prenatal development. We identified 26 dynamically expressed proteins in the developing mouse retina whose expression had not been previously associated with retinal development.

Effects of Echinacea extracts on macrophage antiviral activities.

Type I interferons are a class of cytokines synthesized by leukocytes such as macrophages that limit viral replication. We hypothesized that one mechanism whereby Echinacea spp. extracts may enhance immunity is through modulating interferon-associated macrophage pathways. We used herpes simplex viral infection in the murine macrophage cell line RAW264.7 and monitored virus-induced cell death, interferon secretion, and two intracellular proteins that indicate activation of interferon pathways. Cells were incubated with control media or extracts from four different species (E. angustifolia, E. purpurea, E. tennesseensis, E. pallida). Cells incubated with extracts prior to infection showed very modest enhancement of viability, and no increase in the secretion of interferons alpha or beta as compared to control cells. Virus-infected macrophages treated with extracts from E. purpurea showed a small (<2-fold) induction of guanylate binding protein (GBP) production, but no effect of extracts from other species was observed. In virus-infected cells, all the extracts increased the amount of inducible nitric oxide synthase (iNOS) protein, and this effect varied by type of extraction preparation. Together, these results suggest that any potential antiviral activities of Echinacea spp. extracts are likely not mediated through large inductions of Type I interferon, but may involve iNOS.

H-Ras does not need COP I- or COP II-dependent vesicular transport to reach the plasma membrane.

Although vesicular transport of the H-Ras protein from the Golgi to the plasma membrane is well known, additional trafficking steps, both to and from the plasma membrane, have also been described. Notably, both vesicular and nonvesicular transport mechanisms have been proposed. The initial trafficking of H-Ras to the plasma membrane was therefore examined in more detail. In untreated cells, H-Ras appeared at the plasma membrane more rapidly than a protein carried by the conventional exocytic pathway, and no H-Ras was visible on Golgi membranes in >80% of the cells. H-Ras was still able to reach the plasma membrane when COP II-directed transport was disrupted by two different mutant forms of Sar1, when COP I-mediated vesicular traffic from the endoplasmic reticulum to the Golgi was inhibited with brefeldin A, or when microtubules were disrupted by nocodazole. Although some H-Ras was present in the secretory pathway, protein that reached the membranes of the endoplasmic reticulum-Golgi intermediate compartment was unable to move further in the presence of nocodozale. These results identify an alternative mechanism for H-Ras trafficking that circumvents conventional COPI-, COPII-, and microtubule-dependent vesicular transport. Thus, H-Ras has two simultaneous but distinct means of transport and need not depend on vesicular trafficking for its delivery to the plasma membrane.

Transforming activity of the Rho family GTPase, Wrch-1, a Wnt-regulated Cdc42 homolog, is dependent on a novel carboxyl-terminal palmitoylation motif.

Wrch-1 is a Rho family GTPase that shares strong sequence and functional similarity with Cdc42. Like Cdc42, Wrch-1 can promote anchorage-independent growth transformation. We determined that activated Wrch-1 also promoted anchorage-dependent growth transformation of NIH 3T3 fibroblasts. Wrch-1 contains a distinct carboxyl-terminal extension not found in Cdc42, suggesting potential differences in subcellular location and function. Consistent with this, we found that Wrch-1 associated extensively with plasma membrane and endosomes, rather than with cytosol and perinuclear membranes like Cdc42. Like Cdc42, Wrch-1 terminates in a CAAX tetrapeptide (where C is cysteine, A is aliphatic amino acid, and X is any amino acid) motif (CCFV), suggesting that Wrch-1 may be prenylated similarly to Cdc42. Most surprisingly, unlike Cdc42, Wrch-1 did not incorporate isoprenoid moieties, and Wrch-1 membrane localization was not altered by inhibitors of protein prenylation. Instead, we showed that Wrch-1 is modified by the fatty acid palmitate, and pharmacologic inhibition of protein palmitoylation caused mislocalization of Wrch-1. Most interestingly, mutation of the second cysteine of the CCFV motif (CCFV > CSFV), but not the first, abrogated both Wrch-1 membrane localization and transformation. These results suggest that Wrch-1 membrane association, subcellular localization, and biological activity are mediated by a novel membrane-targeting mechanism distinct from that of Cdc42 and other isoprenylated Rho family GTPases.

Glucose uptake in PC12 cells: GLUT3 vesicle trafficking and fusion as revealed with a novel GLUT3-GFP fusion protein.

The distribution of glucose transporters at the cell surface has a major impact on cellular glucose uptake. In muscle cells and adipocytes, this distribution is under the control of insulin; however, neuronal glucose uptake is not acutely regulated by insulin. Factors that affect the translocation of the neuronal glucose transporter isoform GLUT3 vesicles to and their fusion with the plasma membrane are not well understood. We report that GLUT3 in PC12 cells colocalizes with SNARE complex proteins SNAP-25 and syntaxin 1, suggesting that fusion of GLUT3-containing vesicles with the plasma membrane is mediated by these proteins. In addition, it seems that GLUT3 vesicle fusion is regulated, as depolarization increases GLUT3 insertion into the plasma membrane. To study the dynamics of GLUT3 vesicle trafficking, we have created a GLUT3-GFP fusion protein that is easily expressed in PC12 cells. Trafficking of GLUT3-GFP seems normal, as 1). its distribution is similar to endogenous GLUT3, 2). GLUT3-GFP containing vesicles fuse with the plasma membrane evidenced by labeling of the fusion protein with an antibody directed against the exofacial epitope of GLUT3, and 3). glucose uptake is similar to PC12 cells not transfected with GLUT3 fusion protein. These studies are the first to examine GLUT3 trafficking and fusion in PC12 cells, and establish a model system to study regulation of the neuronal glucose transporter.

Distinct rates of palmitate turnover on membrane-bound cellular and oncogenic H-ras.

H-Ras displays dynamic cycles of GTP binding and palmitate turnover. GTP binding is clearly coupled to activation, but whether the palmitoylated COOH terminus participates in signaling, especially when constrained by membrane tethering, is unknown. As a way to compare COOH termini of membrane-bound, lipid-modified H-Ras, palmitate removal rates were measured for various forms of H-Ras in NIH 3T3 cells. Depalmitoylation occurred slowly (t(1/2) approximately 2.4 h) in cellular (H-RasWT) or dominant negative (H-Ras17N) forms and more rapidly (t(1/2) approximately 1 h) in oncogenic H-Ras61L or H-RasR12,T59. Combining this data with GTP binding measurements, the palmitate half-life of H-Ras in the fully GTP-bound state was estimated to be less than 10 min. Slow palmitate removal from cellular H-Ras was not explained by sequestration in caveolae, as neither cellular nor oncogenic H-Ras showed alignment with caveolin by immunofluorescence. Conversely, although it had faster palmitate removal, oncogenic H-Ras was located in the same fractions as H-RasWT on four types of density gradients, and remained fully membrane-bound. Thus the different rates of deacylation occurred even though oncogenic and cellular H-Ras appeared to be in similar locations. Instead, these results suggest that acylprotein thioesterases access oncogenic H-Ras more easily because the conformation of its COOH terminus against the membrane is altered. This previously undetected difference could help produce distinctive effector interactions and signaling of oncogenic H-Ras.