Electron microscopy of thin-sectioned three-dimensional crystals of SecA protein from Escherichia coli: structure in projection at 40 A resolution.

SecA is a single-chain, membrane-associated polypeptide (102 kDa) which functions as an essential component of the protein export machinery of Escherichia coli. SecA has been crystallized from ammonium sulfate as small, three-dimensional bipyramidal crystals (0.1 x 0.1 x 0.05 mm). These crystals did not demonstrate detectable diffraction of X-rays from rotating anode sources. For study by electron microscopy, individual crystals were cross-linked in glutaraldehyde and OsO4 solutions, dehydrated, embedded in epoxy resin, and sectioned normal to crystallographic axial directions inferred from the external morphology of the crystals. Fourier transformation of processed images of untilted thin sections stained with uranyl acetate and lead citrate show reflections extending to 31 A resolution. Diffraction data and reconstructed images of the projected density of the unit cell contents indicate that the bipyramidal SecA crystals belong to orthorhombic space group C222(1) with unit cell dimensions a = 414 A, b = 381 A, and c = 243 A. Filtered images and density maps of mutually orthogonal projections of the unit cell contents are consistent with a three-dimensional model in which the asymmetric unit contains eight SecA monomers. The large unit cell dimensions and packing of protein monomers suggest that SecA is crystallizing as an oligomer of either dimers or tetramers.     

Comparison of cold enrichment and U.S. Department of Agriculture methods for isolating Listeria monocytogenes from naturally contaminated foods. The Listeria Study Group.

We compared the cold enrichment (CE) and U.S. Department of Agriculture (USDA) methods for isolating Listeria monocytogenes by examining 402 food samples. The food samples were collected from refrigerators of listeriosis patients as part of a multistate active surveillance project to determine the role of foods in sporadic listeriosis in the United States. L. monocytogenes was isolated from 51 food samples (13%). The USDA method was significantly better (P less than 0.001) than the CE method. The isolation efficiencies of the USDA and CE methods were 96 and 59%, respectively. Quantitation of L. monocytogenes in the food samples revealed that many food samples containing less than 0.3 CFU/g were negative as determined by the CE method but positive as determined by the USDA method.     

Influence of myc overexpression on the phenotypic properties of Chinese hamster lung cells resistant to antitumor agents

In the Chinese hamster lung fibroblast cell line DC-3F, the development of resistance to different drugs, through several mechanisms like MDR expression or alteration of the DNA topoisomerase II activity, has been shown to be associated with a decreased tumorigenicity. Multiple studies have shown that the myc oncogene, in cooperation with ras, plays a major role in the oncogenic transformation of fibroblasts. As an approach to a better understanding of the relationship between the different phenotypic traits, we analyzed the expression of myc and ras oncogenes in the drug-sensitive DC-3F cells and in variants resistant to 9-hydroxy-ellipticine (9-OH-E) (DNA topoisomerase II alteration) or to actinomycin D (AD) (multidrug (MDR) expression). Southern and Northern blot analyses revealed about a 10-fold amplification and a 20-fold overexpression of the c-myc gene in the DC-3F cells as compared to the normal lung fibroblasts. Both amplification and overexpression are markedly decreased in the two resistant variants. ras gene copy number and expression were found to be identical in all cell types. In order to analyze the contribution of the decreased myc expression on the different phenotypic traits, the DC-3F/9-OH-E cells were transfected with the plasmid pSV-c-myc, and six clones expressing high amounts of the transfected myc were isolated and characterized. Morphological and caryological alterations, as well as an increased cloning efficiency in soft agar, indicated that the myc gene product was made in these cells. However, the tumorigenicity of the sensitive parental cells was not restored, thus showing that the decreased myc expression alone does not account for the loss of tumorigenicity in the resistant cells. 9-OH-E resistance was not modified in the transfected cells, while the cross-resistance of these cells to MDR-sensitive drugs, such as vincristine, actinomycin D, and taxol, was reversed roughly in proportion of the expression of the transfected myc.     

Five cases of cyclical Cushing's syndrome.

Reported cases of cyclical Cushing''s syndrome are rare. Of 14 successive patients with Cushing''s syndrome nine collected sequential urine samples for the estimation of cortisol:creatinine ratio. Five had cyclical Cushing''s syndrome while two had considerable variation in urinary cortisol excretion without a cyclical pattern being established. Two of the five patients with a cyclical syndrome had paradoxical responses to dexamethasone. In only one patient with a cyclical pattern did the cortisol:creatinine ratio fall after treatment with bromocriptine or cyproheptadine, or both. The high incidence of the cyclical form of Cushing''s syndrome has important clinical implications. A high index of suspicion of the syndrome is required in patients with symptoms or signs of Cushing''s syndrome but with normal cortisol values, in patients with fluctuating cortisol values, and in patients with anomalous responses to dexamethasone. Because of possible variations in steroidogenesis the results of drug studies in Cushing''s syndrome must be interpreted cautiously.     

Survival ofRhizobium trifolii in soil following inoculation of arrowleaf clover

Summary Arrowleaf clover (Trifolium vesiculosum Savi) is an annual forage legume that is sown in south-eastern USA when temperature and moisture conditions may not be suitable for survival of inoculant rhizobia. Survival of two antibiotic-resistant strains of rhizobia on seed or quartz particles was evaluated under controlled conditions in the laboratory. At 30°C and 75% r.h., lime coating of inoculated seed, soil pH (4.2 and 6.7), and surface or subsurface sowing of inoculated seed did not significantly (P = 0.05) affect the survival of either rhizobial strain. The use of gum arabic as an inoculant adhesive resulted in better rhizobial survival than sucrose or water, but even with gum arabic treatment the population of strain 162Y10 declined fromc. 10,000/seed to less than ten/seed in six days. Survival of strain 162Y15 appeared better andc. 200 rhizobia/seed survived for six days. When inoculated seed were incubated at 45°C, soil pH, method of sowing, lime coating of the seed, and r.h. (75% and 100%) did not influence rhizobial survival. The gum arabic adhesive enhanced survival over the use of sucrose but survival was still poor. No viable rhizobia were detected after two days of incubation. High r.h. (100%) and 30°C favoured survival and growth of both rhizobial strains when inoculated onto quartz particles placed on, or mixed into, soil at pH 4.2. Coating inoculated quartz particles with lime increased the growth of rhizobia under these conditions, but sucrose and gum arabic were equally effective as inoculant adhesives.

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Resumen Supervivencia de Rhizobium trifoliien el suelo después de la inoculación de trébol El trébol en punta de flecha arrowleaf (Trifolium vesiculosum Savi) es una leguminosa de forraje anual que se siembra en el S.E. de EUA en un periodo en el cual las condiciones de humedad y temperatura no suelen ser las adecuadas para la supervivencia de inóculo deRhizobium. La supervivencia en semillas o partículas de cuarzo de dos cepas deRhizobium resistentes a antibiôticos se evaluó bajo condiciones controladas en laboratorio. A 30°C y 75% de humedad relativa, ni el recubrimiento con cal de la semilla inoculada, ni la profundidad de la siembra (en superficie o no) ni el pH del suelo (4.2 y 6.7) alteraron significativamente la supervivencia de las dos cepas estudiadas. El uso de goma arábiga como adhesivo para el inóculo comparado con el uso de sucrosa o agua mejoró la supervivencia delRhizobium. Sin embargo incluso con el tratamiento con goma arábiga la población de la cepa 162Y10 declinó desde 10000/semilla hasta valores inferiores a 10/semilla en seis días. La cepa 162Y15 pareció tener mejor supervivencia y 200Rhizobium por semilla sobrevivieron a los seis días. Cuando las semillas inoculadas se incubaron a 45°C, el pH del suelo, el encalado de la semilla y la humedad relativa (75 y 100%) no influenciaron la supervivencia deRhizobium. El uso de goma arábiga mejoró relativamente la supervivencia aunque esta fue pobre de todos modos. No se detectaronRhizobium viables después de un periodo de incubación de dos días. Una elevada humedad relativa (100%) y una temperatura de 30°C favorecieron la supervivencia de ambas cepas cuando estas se inocularon en particulas de cuarzo, las cuales se situaron o, alternativamente, se mezclaron con suelo a pH 4.2. El recubrimiento con cal de las particulas de cuarzo inoculadas favoreció el crecimiento deRhizobium annque tanto sucrosa como goma arábiga fueron igualmente efectivos como adhesivos para el inóculo.

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Résumé Survie dans le sol de Rhizobium trifoliiaprès inoculation de trèfle sagittaire Le trèfle sagittaire (Trifolium vesiculosum Savi) est une légumineuse annuelle fourragère cultivée dans le Sud-Est des USA à une période de l'année où les conditions de température et d'humidité peuvent ne pas convenir à la survie de rhizobiums inoculés. La survie de deux souches de rhizobiums résistants aux antibiotiques sur des graines ou sur des particules de quartz a été evaluóe au laboratoire, dans des conditions contrôlées. A 30°C et r.h. 75%, l'enrobage dans la chaux des graines inoculées, le pH du sol (4, 2 et 6, 7) et le semis, soit en surface, soit recouvert, ne modifient pas significativement (P = 0,05) la survie des deux souches de rhizobiums. L'emploi de gomme arabique comme adhésif de l'inoculum assure une meilleure survie des rhizobiums que le saccharose ou l'eau. Mais, même avec la gomme arabique, la population de la souche 162Y10 décroit en six jours de 10,000 à moins de dix cellules par graine. La survie de la souche 162Y15 semble meilleure: 200 cellules survivantes par graine au bout de six jours. Lorsque les graines inoculées sont incubées à 45°C, le pH du sol, la méthode de semis, l'enrobage de la graine dans la chaux et le r.h. (75 et 100%) n'ont pas d'influence sur la survie des rhizobiums. La gomme arabique améliore la survie mieux que le saccharose, mais trés faiblement. Après deux jours d'incubation, on ne trouve plus de rhizobiums viables. Un r.h. élevé (100%) et une température de 30°C améliorent la survie et la croissance des deux souches de rhizobiums lorsque celles-ci sont inoculées sur des particules de quartz ou mélangées au sol à pH 4, 2. L'enrobage par la chaux des particules de quartz inoculées accroit la survie des rhizobiums dans ces conditions, mais le saccharose et la gomme arabique sont aussi efficaces somme agents d'adhésion de l'inoculum.

     

Circadian Activity Rhythm of the House Fly Continues after Optic Tract Severance and Lobectomy

Under constant conditions, locomotor activity in about 50% of 63 adult Musca domestica continued to be rhythmic after bilateral severance of optic tracts or bilateral lobectomy. Apparently, the optic lobes of Musca do not contain the oscillator for rhythmic control of locomotor activity as has been proposed for other insects. In 20% of the individuals, several circadian components of activity rhythms were found after operation indicating a role of the optic lobes in the coupling of oscillators. The remaining 30% of the flies with severed optic tracts appeared to be arrhythmic. Most of these flies had vacuolized tissue in the central brain. However, disruption of rhythmicity did not correlate with a common pattern of degeneration. Therefore no conclusions can be drawn as to the localization of the circadian control of locomotor activity in the brain. Flies showing an arrhythmic activity pattern could still be synchronized by LD cycles. Activity did not occur solely during the light period as is the case in controls; but was phase delayed by about 6 hr towards the dark period. Since all flies with severed optic tracts could be synchronized by LD cycles, Musca domestica must possess extraocular photoreceptors.     

Differences in proliferation rate between CADASIL and control vascular smooth muscle cells are related to increased TGFβ expression

Cerebral autosomal‐dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial fatal progressive degenerative disorder. One of the pathological hallmarks of CADASIL is a dramatic reduction of vascular smooth muscle cells (VSMCs) in cerebral arteries. Using VSMCs from the vasculature of the human umbilical cord, placenta and cerebrum of CADASIL patients, we found that CADASIL VSMCs had a lower proliferation rate compared to control VSMCs. Exposure of control VSMCs and endothelial cells (ECs) to media derived from CADASIL VSMCs lowered the proliferation rate of all cells examined. By quantitative RT‐PCR analysis, we observed increased Transforming growth factor‐β (TGFβ) gene expression in CADASIL VSMCs. Adding TGFβ‐neutralizing antibody restored the proliferation rate of CADASIL VSMCs. We assessed proliferation differences in the presence or absence of TGFβ‐neutralizing antibody in ECs co‐cultured with VSMCs. ECs co‐cultured with CADASIL VSMCs exhibited a lower proliferation rate than those co‐cultured with control VSMCs, and neutralization of TGFβ normalized the proliferation rate of ECs co‐cultured with CADASIL VSMCs. We suggest that increased TGFβ expression in CADASIL VSMCs is involved in the reduced VSMC proliferation in CADASIL and may play a role in situ in altered proliferation of neighbouring cells in the vasculature.     

High-throughput marker assays for <Emphasis Type="Italic">FaRPc2</Emphasis>-mediated resistance to Phytophthora crown rot in octoploid strawberry

Phytophthora crown rot (PhCR) caused by Phytophthora cactorum is a destructive disease of the allo-octoploid cultivated strawberry (Fragaria ×ananassa Duch). Many major strawberry cultivars grown worldwide are susceptible to PhCR. Resistance is conferred by the recently-discovered FaRPc2 locus, but high-throughput markers are not yet available for marker-assisted breeding. In the current study, we developed DNA markers for two haplotypes at the FaRPc2 locus associated with resistance, H2 and H3. Marker validation and marker-assisted selection were performed in University of Florida (UF) breeding population. Seven single nucleotide polymorphism-based high resolution melting (HRM) markers linked to H2 and four HRM markers for H3 were developed. One HRM marker, RPCHRM3 linked to H3, was converted to a Kompetitive Allele Specific PCR (KASP) marker. To further examine the utility of the markers, they were screened in University of California Davis cultivars with known phenotypes as well as in 20 diverse accessions with phenotypes that are reported in the literature and that are preserved at the USDA-ARS National Clonal Germplasm Repository, in Corvallis, Oregon. The most informative markers for FaRPc2 resistance are being implemented in the UF strawberry breeding program to improve PhCR resistance.     

Are ME/CFS Patient Organizations “Militant”?

Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) is a contested illness category. This paper investigates the common claim that patients with ME/CFS—and by extension, ME/CFS patient organizations (POs)—exhibit “militant” social and political tendencies. The paper opens with a history of the protracted scientific disagreement over ME/CFS. We observe that ME/CFS POs, medical doctors, and medical researchers exhibit clear differences in opinion over how to conceptualize this illness. However, we identify a common trope in the discourse over ME/CFS: the claim of “militant” patient activism. Scrutinizing this charge, we find no compelling evidence that the vast majority of patients with ME/CFS, or the POs representing them, have adopted any such militant political policies or behaviours. Instead, we observe key strategic similarities between ME/CFS POs in the United Kingdom and the AIDs activist organizations of the mid-1980s in the United States which sought to engage scientists using the platform of public activism and via scientific publications. Finally, we explore the contours of disagreement between POs and the medical community by drawing on the concept of epistemic injustice. We find that widespread negative stereotyping of patients and the marginalization and exclusion of patient voices by medical authorities provides a better explanation for expressions of frustration among patients with ME/CFS.