Phenotypic diversification of a cultured tumor line as a function of substratum

We have found that a murine hepatoma displays a considerable phenotypic diversification in culture, which depends upon the substratum utilized, and is manifested by the formation of multicellular structures of differing geometry: Monolayer on glass and plastic, thick multilayer pads on Gelfilm, and spheroids on agar and agarose. These multicellular morphological phenotypes were assayed without disruption to ascertain their antigenicity in vitro and their tumorigenicity in vivo and to obtain quantitative information on the effect of the spatial arrangement of the hepatoma cells upon the ability of each multicellular structure to interact, as a whole, with molecules and cells in its surroundings. The antigenicity of the multicellular structures was determined with calibrated probes and a methodology that measures the total antigenicity, as well as antigenicity per unit of surface area. Antigenicity was found to differ in the following decreasing order: Monolayer on plastic > spheroids on agarose > spheroids on agar > multilayer on Gelfilm. At least part of these antigenic variants arise from different degrees of masking of the structures' surface determinants by a trypsin-sensitive material. The multicellular phenotypes also differed in tumorigenicity. When assayed in syngeneic hosts under comparable conditions, agar-grown spheroids produced the fewest tumors, whereas Gelfilm-grown multilayers produced the most. These two independent sets of data show that the various geometries that a tumor tissue is induced to acquire by the culture substratum are accompanied by a distinctive combination of surface and biological properties.     

Parietal lobe variation in cercopithecid endocasts

In extant primates, the posterior parietal cortex is involved in visuospatial integration, attention, and eye‐hand coordination, which are crucial functions for foraging and feeding behaviors. Paleoneurology studies brain evolution through the analysis of endocasts, that is molds of the inner surface of the braincase. These may preserve imprints of cortical structures, such as sulci, which might be of interest for locating the boundaries of major cortical regions. Old World monkeys (Cercopithecidae) represent an interesting zoological group for evolutionary studies, because of their diverse ecologies and locomotor behaviors. In this study, we quantify parietal lobe variation within the cercopithecid family, in a sample of 30 endocasts including 11 genera and 17 species, by combining landmark‐based and landmark‐free geometric morphometric analyses. More specifically, we quantitatively assess variation of the parietal proportions based on landmarks placed on reliable anatomical references and of parietal lobe surface morphology through deformation‐based methods. The main feature associated with the cercopithecid endocranial variation regards the inverse proportions of parietal and occipital lobes, with colobines, Theropithecus, and Papio displaying relatively larger parietal lobes and smaller occipital lobes compared with cercopithecins. The parietal surface is anteroposteriorly longer and mediolaterally flatter in colobines, while longitudinally shorter but laterally bulging in baboons. Large parietal lobes in colobines and baboons are likely to be independent evolutionary traits, and not necessarily associated with analogous functions or morphogenetic mechanisms.     

Detection of cellular responses to toxicants by dielectrophoresis

The dielectrophoretic (DEP) crossover method has been applied to the detection of cell responses to toxicants. Time and dose responses of the human cultured leukemia (HL-60) line were measured for paraquat, styrene oxide (SO), N-nitroso-N-methylurea (NMU) and puromycin. These toxicants were chosen because of their different predominant mechanisms of action, namely membrane free radical attack, simultaneous membrane and nucleic acid attack, nucleic acid alkylation, and protein synthesis inhibition, respectively. For all treatments, the specific membrane capacitance (C_mem) of the cells decreased while the specific membrane conductance (G_mem) increased in dose- and time-dependent manners. The DEP responses correlated sensitively with alterations in cell surface morphology, especially folds, microvilli, and blebs, observed by scanning electron microscopy. The DEP method was more sensitive to agents that had a direct action on the membrane than to agents for which membrane alterations were secondary. The responses to paraquat and SO, which directly damaged the cell membrane, could be detected 15 min after exposure, while those for puromycin and NMU, which acted on intracellular targets, could be detected after 30 min. The detection times and dose sensitivity results showed that the DEP method is much faster and more sensitive than conventional cell and higher organism viability testing techniques. The feasibility of producing small instruments for toxicity detection and screening based on cellular dielectric responses is discussed.     

Degradation, Recycling, and Shedding of Trypanosoma brucei Variant Surface Glycoprotein

ABSTRACT. Trypanosoma brucei bloodstream forms express a densely packed surface coat consisting of identical variant surface glycoprotein (VSG) molecules. This surface coat is subject to antigenic variation by sequential expression of different VSG genes and thus enables the cells to escape the mammalian host's specific immune response. VSG turnover was investigated and compared with the antigen switching rate. Living cells were radiochemically labeled with either ¹²⁵I-Bolton-Hunter reagent or ³⁵S-methionine, and immunogold-surface labeled for electron microscopy studies. The fate of labeled VSG was studied during subsequent incubation or cultivation of labeled trypanosomes. Our data show that living cells slowly released VSG into the medium with a shedding rate of 2.2 ± 0.6% h⁻¹ (t_1/2= 33 ± 9 h). In contrast, VSG degradation accounted for only 0.3 ± 0.06% h⁻¹ (t_1/2= 237 ± 45 h) and followed the classical lysosomal pathway as judged by electron microscopy. Since VSG uptake by endocytosis was rather high, our data suggest that most of the endocytosed VSG was recycled to the surface membrane. These results indicate that shedding of VSG at a regular turnover rate is sufficient to remove the old VSG coat within one week, and no increase of the VSG turnover rate seems to be necessary during antigenic variation.     

Simultaneous amylase production,raw cassava starch hydrolysis and ethanol production by immobilized Aspergillus awamori and Saccharomyces cerevisiae in a novel alternating liquid phase–air phase system

As a solution to the problems of mass transfer limitation in submerged cultures and scale up of solid-state/liquid-surface cultures, an alternating liquid phase–air phase bioreactor was developed. It consisted of a bioreactor equipped with a siphon system and a reservoir. Aspergillus awamori was immobilized in loofa sponge inside the bioreactor and culture broth was pumped from the reservoir into the bioreactor. Each time the culture broth level reached a critical level, the broth automatically siphoned back into the reservoir. Thus the immobilized cells were alternatingly submerged and exposed to air. The duration of each phase was controlled by the pumping rate and with an on-off timer. During amylase production from soluble starch and raw cassava starch, the optima ratios of the liquid to air phases were 12 h : 12 h and 3 h : 6 h respectively. Saccharomyces cerevisiae IR2 was immobilized in the reservoir and the system was used for simultaneous amylase production, hydrolysis and ethanol production from raw cassava starch. The process was very stable for more than 7 batches with high ethanol yield of 0.46 g-ethanol/g-starch and productivity of 1.73 g-ethanol/L/h. These values are high, the system can be scaled up, and thus it has many potential applications.     

Polyoxoanion-supported organometallic complexes

Synthetic, spectroscopic and x-ray crystallographic research directed at assessing the steric and electronic factors which influence the chemical reactivity of polyoxoanions toward electrophiles is reviewed. Emphasis is placed on studies of hexametalate- and trimetaphosphate-supported organometallic complexes of octahedra d⁶ and square-planar d⁸ transition metals as well as actinide metals. The effects of metal-and-terminal-ligand substitution within the hexametalate cage are demonstrated through protonation and complexation studies.     

Surface properties of sendai virus envelope

Surface properties of Sendai virus envelope membrane have been measured, using both biological and biophysical techniques. Both normal and trypsin-treated virus were studied. SDS gel electrophoresis showed cleavage of the F protein exclusively by trypsin. The major activity change was observed in the hemolysing activity which is an expression of F protein. Hemolysis was reduced to less than 10% of its value for intact virus. ³¹P nuclear magnetic resonance studies of the envelope surface of the native virus showed a highly restricted phospholipid headgroup environment. Interestingly, this restriction was relieved by treatment with trypsin. Thus these data suggest a role of the F protein of Sendai virus in tightly organizing the surface of the viral envelope membrane.