Protection of cryopreserved Onchocerca microfilariae (nematoda) from dilution shock by the use of serum

This paper describes the use of newborn calf serum during the cooling and warming/dilution phases of the cryopreservation of Onchocerca gutturosa microfilariae using an interrupted slow cool to ?196 °C in the presence of 5% (v/v) methanol. Serum proved detrimental at concentrations above 20% (v/v) in the cooling medium unless it was also present in high concentrations, 60% (v/v) in the warming/ dilution medium.Damage to the organisms occurred predominantly during the thawing/dilution phase of cryopreservation and not the cooling phase and could be reduced greatly by the presence of high serum concentrations when thawing. This indicates that the major protective action of serum is that of reducing dilution shock—shock produced by a rapid influx of water and/or the effects of high solute concentrations established during cooling.     

Collagen modulates cell shape and cytoskeleton of embryonic corneal and fibroma fibroblasts: Distribution of actin, α-actinin,and myosin

In the embryo, fibroblasts migrating through extracellular matrices (ECM) are generally elongate in shape, exhibiting a leading pseudopodium with filopodial extensions, and a trailing cell process. Little is known about the mechanism of movement of embryonic cells in ECM, for studies of fibroblast locomotion in the past have been largely confined to observations of flattened cells grown on planar substrata. We confirm here that embryonic avian corneal fibroblasts migrating within hydrated collagen gels in vitro have the bipolar morphology of fibroblasts in vivo, and we show for the first time that highly flattened gerbil fibroma fibroblasts, grown as cell lines on planar substrata, can also respond to hydrated collagen gels by becoming elongate in shape. We demonstrate that the collagen-mediated change in cell shape is accompanied by dramatic rearrangement of the actin, α-actinin, and myosin components of the cytoskeleton. By immunofluorescence, the stress fibers of the flattened corneal fibroblasts grown on glass are seen to stain with antiactin, anti-α-actinin, and antimyosin, as has been reported for fibroma and other fibroblasts grown on glass. Stress fibers, adhesion plaques, and ruffles do not develop when the corneal or fibroma fibroblast is grown in ECM; these features seem to be a response to strong attachment of the cell underside to a planar substratum. When the fibroblasts are grown in ECM, antimyosin staining is distributed diffusely through the cytoplasm. Antiactin and anti-α-actinin stain the microfilamentous cell cortex strongly. We suggest that locomotion of the fibroblast in ECM is accompanied by adhesion of the cell to the collagen fibrils and may involve an interaction of the myosin-rich cytosol with the actin-rich filamentous cell cortex. Interestingly, the numerous filopodia that characterize the tips of motile pseudopodia of cells in ECM are very rich in actin and α-actinin, but seem to lack myosin; if filopodia use myosin to move, the interaction must be at a distance. Soluble collagen does not convert flattened fibroblasts on planar substrata to bipolar cells. Thus, the effect of collagen on the fibroblast cytoskeleton seems to depend on the presence of collagen fibrils in a gel surrounding the cell.     

The MAS-encoded processing protease of yeast mitochondria. Interaction of the purified enzyme with signal peptides and a purified precursor protein

The matrix of yeast mitochondria contains a chelator-sensitive protease that removes matrix-targeting signals from most precursor proteins transported into this compartment. The enzyme consists of two nonidentical subunits that are encoded by the nuclear genes MAS1 and MAS2. With the aid of these cloned genes, we have now overexpressed the active holoenzyme in yeast, purified it in milligram amounts, and studied its biochemical and physical properties. Atomic absorption analysis shows that the purified enzyme lacks significant amounts of zinc, manganese, or cobalt; if none of these metal ions is added during the assay, the enzyme is catalytically inactive but can still cleave substoichiometric amounts of substrate. The amino-terminal sequences of the two mature subunits were determined; comparison with the deduced amino acid sequences of the corresponding precursors revealed that the MAS1 and MAS2 subunits are synthesized with prepeptides composed of 19 and 13 residues, respectively, which have similar sequences. The enzyme is inhibited competitively by chemically synthesized matrix-targeting peptides; the degree of inhibition correlates with the peptides' targeting efficacy. Matrix-targeting peptides containing the cleavage site of the corresponding authentic precursor protein are cleaved correctly by the purified enzyme. A purified artificial precursor protein bound to the holoenzyme can be photocross-linked to the MAS2 subunit.     

Homeobox gene expression in the intestinal epithelium of adult mice.

Using a polymerase chain reaction-based strategy, we have detected the expression of nine different homeobox genes in adult mouse intestine. Included among these are the recently described intestine-specific Cdx-1 gene and a new, related gene, Cdx-2. Southern blot experiments show that Cdx-2 is present in a single copy in the mouse genome. Of several adult mouse tissues assayed, intestine was the only one that contained detectable levels of Cdx-2 mRNA. Expression of all nine homeobox genes in different regions of the intestine was quantitated by RNase protection analysis, which revealed a unique expression profile for each gene. These observations suggest that homeobox gene expression may play an important role in cellular differentiation in the adult intestine.     

Identification and primary structure of calmodulin binding domains in the phosphorylase kinase holoenzyme

Fast twitch skeletal muscle phosphorylase kinase was isolated and incubated with a radioactive, bifunctional, photoactivable, and cleavable cross-linker conjugated to calmodulin. Incubation of the holoenzyme only resulted in the labeling of the alpha-subunit in the presence of Ca2+. After cleavage with CNBr (and subdigestion with Asp-N protease), a sequence was identified (residues 1069-1087) in the alpha-subunit which had the predominant basic character and the propensity to form an amphiphilic helix like other calmodulin binding domains. If cross-linked calmodulin was incubated with the isolated subunits of phosphorylase kinase, radioactivity was recovered in seven CNBr peptides: three came from the alpha-subunits, one of them corresponding to the sequence labeled in the holoenzyme. Three came from the beta-subunit, and one came from the gamma-subunit. The latter contained the two adjacent calmodulin binding domains recently identified in the gamma-subunit (Dasgupta, M., Honeycutt, T., and Blumenthal, D. K. (1988) J. Biol. Chem. 264, 17156-17163).     

Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type

Insulin stimulates glucose transport in adipocytes via the rapid redistribution of the GLUT1 and GLUT4 glucose transporters from intracellular membrane compartments to the cell surface. Insulin sensitivity is dependent on the proper intracellular trafficking of the glucose transporters in the basal state. The bulk of insulin-sensitive transport in adipocytes appears to be due to the translocation of GLUT4, which is more efficiently sequestered inside the cell and is present in much greater abundance than GLUT1. The cell type and isoform specificity of GLUT4 intracellular targeting were investigated by examining the subcellular distribution of GLUT1 and GLUT4 in cell types that are refractory to the effect of insulin on glucose transport. Rat GLUT4 was expressed in 3T3-L1 fibroblasts and HepG2 hepatoma cells by DNA-mediated transfection. Transfected 3T3-L1 fibroblasts over-expressing human GLUT1 exhibited increased glucose transport, and laser confocal immunofluorescent imaging of GLUT1 in these cells indicated that the protein was concentrated in the plasma membrane. In contrast, 3T3-L1 fibroblasts expressing GLUT4 exhibited no increase in transport activity, and confocal imaging demonstrated that this protein was targeted almost exclusively to cytoplasmic compartments. 3T3-L1 fibroblasts expressing GLUT4 were unresponsive to insulin with respect to transport activity, and no change was observed in the subcellular distribution of the protein after insulin administration. Immunogold labeling of frozen ultrathin sections revealed that GLUT4 was concentrated in tubulo-vesicular elements of the trans-Golgi reticulum in these cells. Sucrose density gradient analysis of 3T3-L1 homogenates was consistent with the presence of GLUT1 and GLUT4 in discrete cytoplasmic compartments. Immunogold labeling of frozen thin sections of HepG2 cells indicated that endogenous GLUT1 was heavily concentrated in the plasma membrane. Sucrose density gradient analysis of homogenates of HepG2 cells expressing rat GLUT4 suggested that GLUT4 is targeted to an intracellular location in these cells. The density of the putative GLUT4-containing cytoplasmic membrane vesicles was very similar in HepG2 cells, 3T3-L1 fibroblasts, 3T3-L1 adipocytes, and rat adipocytes. These data indicate that the intracellular trafficking of GLUT4 is isoform specific. Additionally, these observations support the notion that GLUT4 is targeted to its proper intracellular locale even in cell types that do not exhibit insulin-responsive glucose transport, and suggest that the machinery that regulates the intracellular targeting of GLUT4 is distinct from the factors that regulate insulin-dependent recruitment to the cell surface.     

The effects of temperature, pH and growth rate on secondary metabolism in Streptomyces thermoviolaceus grown in a chemostat.

Streptomyces thermoviolaceus was grown in a chemostat under conditions of glutamate limitation. The effects of growth rate on production of the antibiotic granaticin, extracellular protein and protease activity as components of secondary metabolism were studied at 37, 45 and 50 degrees C. The amount of each secondary metabolite synthesized was highly dependent on growth rate and temperature. Granaticin yields were highest at growth rates of 0.1 to 0.15 h-1 at 37 degrees C, 0.175 h-1 at 45 degrees C and 0.045 h-1 at 50 degrees C. Protease activity of culture supernatants responded to low nutrient concentration and/or low growth rate. Measurements of extracellular protein revealed complex changes in amount which were dependent on growth rate and temperature. At 45 degrees C and a growth rate of 0.15 h-1, biomass yield was highest between pH 5.5 to 6.5 whereas granaticin synthesis was low at pH 5.5 and rose to highest values at between pH 6.5 and 7.5.     

Production and characterization of osteoclast-selective monoclonal antibodies that distinguish between multinucleated cells derived from different human tissues

Osteoclastoma-derived giant cells were used to produce 11 mouse monoclonal antibodies (MAb) reactive against human osteoclasts on undecalcified sections of adult human bone. All exhibited unique reactivities across a wide range of human tissues. Three in particular demonstrated distinctive reactivities; C35 was highly selective for bone osteoclasts, C27 showed selective reactivity for osteoclasts, tissue macrophages and blood-borne monocytes, and C22 showed selective membrane staining of osteoclasts. Consequently, C22 was used to coat Dynabeads to affinity-purify viable human osteoclasts from osteoclastoma-derived cell suspensions. Immunocytochemical staining of inflammatory osteoarthritic synovium/granulation tissue demonstrated positivity in the majority of giant cells with MAb C22 and C27. In contrast, C35 reacted with only very occasional giant cells. Furthermore, multinucleated cells formed in long-term human bone marrow cultures demonstrated similar selective staining. C27 stained all giant cells and the majority of mononuclear cells. C22 detected only a small proportion of giant cells. In contrast to its staining on bone osteoclasts, C22 demonstrated granular cytoplasmic staining in cultured giant cells. C35 stained no cells at all in these cultures. These MAb can therefore distinguish between giant cells of various origins and authentic mature osteoclasts. Alternatively, they can recognize antigens expressed at different stages of osteoclast differentiation and therefore provide an excellent tool for the study of the human osteoclast lineage.     

A review of the salt sensitivity of the Australian freshwater biota

In Victoria, Australia, both dryland salinity and salinity in irrigation regions are serious agricultural problems. One option to control the latter is to pump groundwater to maintain it below the surface. However, this leaves a saline wastewater for disposal, probably into local streams or wetlands. This review of the salt sensitivity of the biota of Australian streams and wetlands gives information of interest to those responsible for developing controls on these discharges. The review addresses the lethal and sub-lethal effects of salinity on microbes (mainly bacteria), macrophytes and micro-algae, riparian vegetation, invertebrates, fish, amphibians, reptiles, mammals, and birds. Data suggest that direct adverse biological effects are likely to occur in Australian river, stream and wetland ecosystems if salinity is increased to around 1 000 mg L⁻¹. The review highlights a general lack of data on the sensitivity of freshwater plants and animals to salinity increases.     

Phylogenetic analysis and evolution of RNase P RNA in proteobacteria.

The secondary structures of the eubacterial RNase P RNAs are being elucidated by a phylogenetic comparative approach. Sequences of genes encoding RNase P RNA from each of the recognized subgroups (alpha, beta, gamma, and delta) of the proteobacteria have now been determined. These sequences allow the refinement, to nearly the base pair level, of the phylogenetic model for RNase P RNA secondary structure. Evolutionary change among the RNase P RNAs was found to occur primarily in four discrete structural domains that are peripheral to a highly conserved core structure. The new sequences were used to examine critically the proposed similarity (C. Guerrier-Takada, N. Lumelsky, and S. Altman, Science 246:1578-1584, 1989) between a portion of RNase P RNA and the "exit site" of the 23S rRNA of Escherichia coli. Phylogenetic comparisons indicate that these sequences are not homologous and that any similarity in the structures is, at best, tenuous.