In vitro carcinogenesis of mammary epithelial cells byN-nitroso-N-methylurea using a collagen gel matrix culture

Summary Carcinogenesis is a lengthy process which eventually culminates in the transformed phenotype, cancer. However, much remains to be defined about the process of transformation. In vivo models for the study of the carcinogenic process present limitations because it is not possible to detect the premalignant stages in the animals. An in vitro model, on the other hand, facilitates the study of the carcinogenic process because it enables one to dissect out the crucial events required for carcinogenesis to occur. As carcinogenesis is believed to be a multistep process; initiation, promotion, and progression, a multistep, in vitro system has been devised in our laboratory to mimic each of these stages. We have previously shown the formation of “microtumors” in collagen gels, induced by 7,12-dimethylbenz(a)anthracene. In the present study the direct acting water soluble, mammary carcinogen,N-nitroso-N-methylurea (NMU) was used for tumorigenesis of mammary epithelial cells in culture. Mammary epithelial cells from virgin Sprague-Dawley rats were propagated and exposed to single or multiple doses of NMU while growing as a monolayer in glass petri dishes (initiation). Initiated cells were then plated into a collagen gel matrix culture. Prolonged growth in the collagen gels afforded for the progression of the transformed cells into discernable microtumors in the three-dimensional matrix of the collagen. The morphology of these “tumors” was determined by histologic sections of the gels. Fewer, if any, such structures existed in the untreated gels.     

Enrichment of adeno-associated virus serotype 5 full capsids by anion exchange chromatography with dual salt elution gradients

Adeno-associated virus-based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or “empty” capsids, represent a product-related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno-associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3-fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%.     

Circumscription of Fulbrightiella gen. nov. and Sherwoodiella gen. nov., Two Novel Genera in the Calotrichaceae (Nostocales,Cyanobacteria)

Three novel strains in Calotrichaceae from tropical habitats were isolated and characterized with regard to their morphology, phylogenetic placement, and secondary structures of conserved domains in the 16S-23S internal transcribed spacer (ITS). The strains fell into two clades formerly identified as Calothrix from freshwater and brackish habitats. Based on both morphology and ecology, they differed from the type species of Calothrix, C. confervicola, which is marine, has wide trichomes with short cells, and narrows abruptly to a hyaline hair. The first clade grouped species with heteropolar filaments widened at the base and narrowed gradually toward the apex but not ending in a hair, with basal heterocytes that are formed in series as the apically placed heterocytes senesce; this clade is being named Fulbrightiella gen. nov., with two named species, F. bharadwajae sp. nov. and F. oahuensis sp. nov. The second clade was comprised of a single species with isopolar trichomes that are untapering as hormogonia, but which widen midfilament and taper toward both ends following growth. These trichomes develop pairs of heterocyte mid-filament, causing fragmentation into heteropolar trichomes with basal heterocytes and ends that taper, but not to a hair. This clade consists of a single species at present, Sherwoodiella mauiensis. With this action, four clades in the Calotrichaceae have been named: Macrochaete, Dulcicalothrix, Fulbrightiella, and Sherwoodiella. Calothrix sensu stricto is truly marine, morphologically distinct, and unsequenced; finding and sequencing the generitype for Calothrix remains as the most important and unfinished task in the revision of the Calotrichaceae.     

Habitat selection and space use overlap between feral horses,pronghorn, and greater sage-grouse in cold arid steppe

Populations of feral horses (Equus ferus caballus) in the western United States have increased during the past decade, consequently affecting co-occurring wildlife habitat. Feral horses may influence 2 native wildlife species, greater sage-grouse (Centrocercus urophasianus; sage-grouse) and pronghorn (Antilocapra americana) through mechanisms of habitat alteration and competition. Wyoming, USA, contains the largest populations of pronghorn and sage-grouse of any state and also has the highest degree of range overlap between feral horses and these species. Consequently, the effects that horses may have on pronghorn and sage-grouse populations in Wyoming have implications at local, state, and population-wide levels. Managers need information concerning habitat selection and space use overlap among these species to develop appropriate management strategies; yet this information is absent for most feral horse management areas. To address this knowledge need, we attached global positioning system (GPS) transmitters to horses, pronghorn, and sage-grouse within the greater Bureau of Land Management–Adobe Town Herd Management Area in southern Wyoming and northern Colorado, USA, between 2017 and 2021 to evaluate habitat selection and space use of all species during 3 biologically relevant seasons: spring (Apr–Jun; sage-grouse breeding, nesting, and early-brood rearing; pronghorn late gestation and early parturition), summer (Jul–Oct; sage-grouse summer and late-brood rearing; pronghorn late parturition and breeding), and winter (Nov–Mar; non-breeding season). Feral horses selected flatter slopes and shorter mean shrub height across all seasons and were closer to water in spring and summer. Pronghorn habitat selection was similar to horses, but they also avoided oil and gas well pads year-round. During spring, sage-grouse selected greater herbaceous cover, flatter slopes, and areas farther from well pads. In summer, sage-grouse selected greater mean shrub height, flatter slopes, and were closer to water. In winter, sage-grouse selected flatter slopes and areas with greater vegetation production during the preceding summer. Our results indicate strong year-round overlap in space use between horses and pronghorn, whereas overlap between horses and sage-grouse is greatest during the summer in this region. Consequently, managers should recognize the potential for horses to influence habitat quality of pronghorn and sage-grouse in the region.     

FACTORS AFFECTING RED SPRUCE REGENERATION IN DECLINING AREAS OF CAMELS HUMP MOUNTAIN,VERMONT

Scarcity of red spruce (Picea rubens Sarg.) seedlings in declining spruce-fir forests of Camels Hump mountain, Vermont, prompted a study on some contributing factors involved in failure of spruce regeneration. Cones were shorter than those from low elevation red spruce trees from unaffected sites. Seed number in cones collected in declining areas of Camels Hump was low as were seed sizes and weights. Seed germination was at control levels only in good seed years. Capacity of seeds to form seedlings was reduced relative to that of controls, although seedling growth was normal. Coniferous litter contains presumed allelopathic substances leachable by contemporary precipitations that affect seed germination and seedling root development in red spruce, but not in balsam fir. Shield fern contains leachable substances that reduce seed germination and seedling root development in red spruce, but not balsam fir. Roots of red spruce germlings have lower capacity to penetrate through the increased forest duff depths of declining forests than do balsam fir roots. It is anticipated that substantial reproduction of red spruce will not occur in declining montane conifer forests under present conditions.     

THE ULTRASTRUCTURE OF SAHNIA POLLEN (PENTOXYLALES)

The micromorphology and fine structure of in situ pentoxylalean pollen are described from the holotype of Sahnia laxiphora Drinnan and Chambers 1985 collected from the Lower Cretaceous (Valanginian-Aptian) of Victoria, southeastern Australia. Pollen grains are ovoid, monosulcate, and relatively small, averaging 26 μm in length. Exine ornamentation is psilate. The sporoderm is two-parted with the sexine staining lightly throughout and approximately six times the thickness of the more darkly staining nexine. The exine over the sulcus is typically strongly invaginated, and may or may not include an extremely thin sexine layer. The outer part of the sexine is homogeneous, while the inner part is composed of relatively large granules separated by irregular lacunae of various sizes; lacunae are most pronounced at the sexinenexine interface. Faint lamellae characterize the nexine in both apertural and nonapertural regions. Granular orbicules are often associated with the exine surfaces and also occur appressed to pollen sac walls along with lamellated tapetal membranes. Sporoderm ultrastructure is compared to that of nonsaccate pollen of other groups, and particularly to pollen of Bennettitales, Gnetales, angiosperms, and similar plants, to which the Pentoxylales have been thought to be closely related. Although Sahnia laxiphora pollen is not identical to that of any of these taxa, the strongest similarity is with pollen of Bennettitales.     

THE ECOLOGICAL IMPACT OF ALLELOPATHY IN AILANTHUS ALTISSIMA (SIMAROUBACEAE)

Compounds inhibitory to the growth of neighboring plant species were found in significant concentrations in the leaves and stems of young Ailanthus altissima ramets. The surrounding soil also contained appreciable concentrations of similarly acting toxins. Individuals of neighboring plant species have either incorporated active portions of inhibitory compounds or responded to Ailanthus by producing growth-inhibiting substances. Under greenhouse conditions, individuals of neighboring plant species previously unexposed to Ailanthus in the field were found to be more susceptible to the Ailanthus toxins than individuals previously exposed. Moreover, seeds produced by unexposed populations were also more susceptible to Ailanthus toxins than seeds produced by previously exposed populations. These differences demonstrated that the allelochemicals of Ailanthus altissima exhibited a measurable impact upon neighboring plant species. Since the progeny of these populations displayed a differential response to Ailanthus toxin, this phenotypic difference between the two populations may have a heritable basis.     

Notes on TYachoma, Tüberolabium and Parapteroceras (Orchidaceae)

The genus Trachoma is considered congeneric with Tüberolabium. All necessary transfers to Tüberolabium are made. Four new combinations are made in the recently established Parapteroceras. A key to the species of Parapteroceras and a key to Parapteroceras and allied genera are provided.     

Regulation of collagenase production by steroids in uterine smooth muscle cells: An enzymatic and immunologic study

An enzyme-linked immunosorbent assay (ELISA) has been developed for rat collagenase. The assay is capable of measuring the enzyme from a variety of rat cell sources at concentrations of 10–;50 ng/ml, approximately 500–;1,000-fold more sensitive than radiolabelled collagen fibril assay systems. The assay is specific to collagenase from the rat: enzymes from human, tadpole, mouse, and bacterial sources failed to cross-react significantly with rat enzyme. The assay is reproducible and accurate, and is capable of detecting enzyme in the presence of serum or tissue inhibitors. Using the ELISA, we have examined the effect of a variety of hormones on the production of collagenase by rat myometrial smooth muscle cells in culture. Of all the reproductive hormones examined, only progesterone and its synthetic derivative medroxyprogesterone acetate were capable of inhibiting the production of the enzyme by these cells. The maximally effective concentration of progesterone was 1 x 10^?6M, and that of medroxyprogesterone acetate was 1 x 10^?7M. The effect of the steroid was selective: no effect on cell proliferation or on general protein synthesis was observed. In addition to the progestational steroids, the glucocorticoids were also capable of inhibiting the production of collagenase by the cells at similar nominal concentrations. However, the myometrial cells were found actively to metabolize progesterone but not hydrocortisone in culture. Thus, the effective inhibitory concentration of progesterone was approximately ten-fold lower than that of hydrocortisone. The results of this study support the concept that progesterone plays a major role in preventing the production of collagenase in the rat uterus.