Methods for evaluating the morphological and immunohistochemical properties of human tumor colonies grown in soft agar

Summary Clonogenic assays have been widely adopted for the investigation of hematopoietic and human tumor stem cell biology. Inasmuch as specific, whole colonies need to be analyzed morphologically, we used various methods for fixing and embedding individual colonies in situ that allowed macroscopic, light microscopic (LM), immunofluorescence, and transmission electron microscopic (TEM) evaluation of the intact colony. Melanoma colonies stained with Masson’s Trichrome, hematoxylin and eosin (H&E), periodic acid-Schiff, Best’s carmine, Page-Green method for inclusion bodies, and Snook’s reticulum revealed cellular and extracellular components by LM. Ultrastructural studies revealed specific cellular organelles and extracellular components. Immunofluorescence studies demonstrated cell-surface fibronectin, a high molecular weight, adhesive glycoprotein. Myeloma colonies contained a heterogeneous cell population and produced amyloid fibers that were observed by TEM. Fixation and embedding the colonies in agar for TEM has several advantages over centrifugation methods and other conventional techniques for collecting cells in that (a) an entire specific colony can be studied, (b) there is excellent preservation of the cell and its spatial orientation in the colony, and (c) the extracellular matrix (ECM) of the colony is preserved for immunohistochemical analysis. This work was supported by Grant T32 CA09213, a National Research Service Award from the National Cancer Institute to B. P., by Grants PDT-205 (M. J. C. H.) and PDT-184 (F. M.) from the American Cancer Society, and from the National Cancer Institute (CA-17904).     

Differential uptake and metabolism of sitosterol and cholesterol by Achlya, Pythium, and Phytophthora species.

The relative ability of isolates of Achlya bisexualis and A. ambisexualis and isolates of Pythium and Phytophthora to take up and metabolize sitosterol and cholesterol was studied. Species of Pythium and Phytophthora took up cholesterol and sitosterol efficiently, whereas Achlya species took up booth sterols inefficiently. Species of Pythium and Phytophthora produced a polar metabolite and esters from sitosterol as they did from cholesterol. Achlya species did not produce the polar metabolite from either sterol. In these experiments Achlya species produced esters only from cholesterol; however, their failure to produce esters from sitosterol may have been due to the higher sitosterol than cholesterol concentration.     

Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor.

Modulation of connexin 43 (cx43) in the myometrium of timed pregnant rats was studied using enzyme-linked immunosorbent assay (ELISA), immunocytochemical localization, and immunoblot. These techniques utilized site-specific antibodies directed against a portion of the carboxyl tail of cx43. We found that cx43 is synthesized several days prior to labor but accumulates within the cytoplasm until parturition, when it is rapidly transported to the plasma membrane and assembled into gap junction plaques at the cell surface. These cx43-positive gap junctions begin to disappear from the plasma membrane within hours of delivery of the last pup and are completely absent within 24 hr following delivery. These structures are apparently internalized and degraded within the cytoplasm. ELISA documents a reduction of total cellular cx43 to baseline levels within 5 days following parturition. While the timing of synthesis, cytoplasmic storage, concentration in apparent Golgi vesicles, and transport to and assembly in the plasma membrane are accelerated in three models of preterm labor, the sequence of these events and the correlation of parturition with the formation of gap junctions are identical to those documented in normal labor. These results support the hypothesis that effective labor requires the synthesis and assembly of cx43 into functional gap junctions at the myometrial cell surface.     

Soil Aggregates and Associated Organic Matter under Conventional Tillage,No-Tillage,and Forest Succession after Three Decades

Impacts of land use on soil organic C (SOC) are of interest relative to SOC sequestration and soil sustainability. The role of aggregate stability in SOC storage under contrasting land uses has been of particular interest relative to conventional tillage (CT) and no-till (NT) agriculture. This study compares soil structure and SOC fractions at the 30-yr-old Horseshoe Bend Agroecosystem Experiment (HSB). This research is unique in comparing NT and CT with adjacent land concurrently undergoing forest succession (FS) and in sampling to depths (15–28 cm) previously not studied at HSB. A soil moving experiment (SME) was also undertaken to monitor 1-yr changes in SOC and aggregation. After 30 years, enhanced aggregate stability under NT compared to CT was limited to a depth of 5 cm, while enhanced aggregate stability under FS compared to CT occurred to a depth of 28 cm and FS exceeded NT from 5–28 cm. Increases in SOC concentrations generally followed the increases in stability, except that no differences in SOC concentration were observed from 15–28 cm despite greater aggregate stability. Land use differences in SOC were explained equally by differences in particulate organic carbon (POC) and in silt-clay associated fine C. Enhanced structural stability of the SME soil was observed under FS and was linked to an increase of 1 Mg SOC ha⁻¹ in 0–5 cm, of which 90% could be attributed to a POC increase. The crushing of macroaggregates in the SME soil also induced a 10% reduction in SOC over 1 yr that occurred under all three land uses from 5–15 cm. The majority of this loss was in the fine C fraction. NT and FS ecosystems had greater aggregation and carbon storage at the soil surface but only FS increased aggregation below the surface, although in the absence of increased carbon storage.     

Pseudogene IFN-alpha L: removal of the stop codon in the signal sequence permits expression of active human interferon

Biologically active interferon (10(6)-10(7) units/liter) was produced in Escherichia coli from modified human alpha interferon (IFN-alpha) pseudogene L. IFN-alpha pseudogene L has a stop codon in the signal peptide coding region. The region that contains the stop codon was replaced with the corresponding region of another human IFN-alpha gene, WA, that does not have a stop codon and was previously engineered for expression by fusion to the M13mp11 lac promoter. The interferon L fusion product was induced with IPTG after infecting E. coli JM103 with the M13 bacteriophage that contained the modified human IFN-alpha pseudogene L. Hence, the IFN-alpha L mature interferon coding sequence, which is not identical to any other alpha-interferon gene, has been conserved for active interferon coding information.     

The Preprotein Binding Domain of SecA Displays Intrinsic Rotational Dynamics

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Structure and Energetics of Encapsidated DNA in Bacteriophage HK97 Studied by Scanning Calorimetry and Cryo-electron Microscopy

Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis. We found that, as in other phages, HK97 DNA is organized in coaxially wound nested shells. When DNA-filled capsids (heads) are scanned in buffer containing 1 mM Mg²⁺, DNA melting and capsid denaturation both contribute to the complex thermal profile between 82 °C and 96 °C. In other conditions (absence of Mg²⁺ and lower ionic strength), DNA melting shifts to lower temperatures and the two events are resolved. Heads release their DNA at temperatures well below the onset of DNA melting or capsid denaturation. We suggest that, on heating, the internal pressure increases, causing the DNA to exit—probably via the portal vertex-while the capsid, although largely intact, sustains local damage that leads to an earlier onset of thermal denaturation. Heads differ structurally from empty capsids in the curvature of their protein shell, a change attributable to outwards pressure exerted by the DNA. We propose that this transition is sensed by the portal that is embedded in the capsid wall, whereupon the structure of the portal and its interactions with terminase, the packaging enzyme, are altered, thus signaling that packaging is at or approaching completion.     

Invasion of exotic earthworms into ecosystems inhabited by native earthworms

The most conspicuous biological invasions in terrestrial ecosystems have been by exotic plants, insects and vertebrates. Invasions by exotic earthworms, although not as well studied, may be increasing with global commerce in agriculture, waste management and bioremediation. A number of cases has documented where invasive earthworms have caused significant changes in soil profiles, nutrient and organic matter dynamics, other soil organisms or plant communities. Most of these cases are in areas that have been disturbed (e.g., agricultural systems) or were previously devoid of earthworms (e.g., north of Pleistocene glacial margins). It is not clear that such effects are common in ecosystems inhabited by native earthworms, especially where soils are undisturbed. We explore the idea that indigenous earthworm fauna and/or characteristics of their native habitats may resist invasion by exotic earthworms and thereby reduce the impact of exotic species on soil processes. We review data and case studies from temperate and tropical regions to test this idea. Specifically, we address the following questions: Is disturbance a prerequisite to invasion by exotic earthworms? What are the mechanisms by which exotic earthworms may succeed or fail to invade habitats occupied by native earthworms? Potential mechanisms could include (1) intensity of propagule pressure (how frequently and at what densities have exotic species been introduced and has there been adequate time for proliferation?); (2) degree of habitat matching (once introduced, are exotic species faced with unsuitable habitat conditions, unavailable resources, or unsuited feeding strategies?); and (3) degree of biotic resistance (after introduction into an otherwise suitable habitat, are exotic species exposed to biological barriers such as predation or parasitism, “unfamiliar” microflora, or competition by resident native species?). Once established, do exotic species co-exist with native species, or are the natives eventually excluded? Do exotic species impact soil processes differently in the presence or absence of native species? We conclude that (1) exotic earthworms do invade ecosystems inhabited by indigenous earthworms, even in the absence of obvious disturbance; (2) competitive exclusion of native earthworms by exotic earthworms is not easily demonstrated and, in fact, co-existence of native and exotic species appears to be common, even if transient; and (3) resistance to exotic earthworm invasions, if it occurs, may be more a function of physical and chemical characteristics of a habitat than of biological interactions between native and exotic earthworms.     

Nerve growth factor and its precursor differentially regulate hair cycle progression in mice.

Nerve growth factor (NGF) promotes proliferation via its high affinity receptor (TrkA). Its precursor proNGF promotes apoptosis via the pan-neurotrophin-receptor p75. Recently, we have identified NGF and p75 as important hair growth terminators. However, if proNGF is involved or if NGF can also promote hair growth via TrkA is unclear. By RT-PCR we found that NGF/proNGF mRNA levels peak during early anagen in murine back skin, whereas NGF/proNGF protein levels peak during catagen, indicating high turnover in early anagen and protein accumulation in catagen. By immunohistochemistry, NGF and TrkA are found in the proliferating compartments of the epidermis and hair follicle throughout the cycle. In contrast, strong proNGF is found in the highly differentiated inner root sheath and adjacent to the p75+ regressing epithelial strand in catagen. Commercial 7S NGF, which contains both NGF and proNGF, promotes anagen development in organ-cultured early anagen mouse skin, whereas it promotes catagen development in late anagen skin. Together, our findings suggest an anagen-promoting or anagen-supporting role for NGF/TrkA, and a catagen-promoting role for proNGF/p75 interactions. This has important implications for the future design of specific neurotrophin receptor ligands as novel pharmaceuticals in the modification of tissue remodeling processes such as hair growth or wound healing.