In vivo growth stimulation of collagen gel embedded normal human and mouse primary mammary epithelial cells

A new system for studying growth of normal human mammary epithelial cells in an in vivo environment using athymic nude mice is described. Human mammary epithelial cells dissociated from reduction mammoplasty specimens were embedded within collagen gels and subsequently transplanted subcutaneously into nude mice. Histological sections of recovered collagen gels showed epithelial cells arranged as short tubules with some branching. Proliferation of mammary epithelial cells was quantitated in vivo by 3 days' continuous infusion with 5 bromo-2′-deoxy-uridine followed by immunostaining of sections from recovered gels. Ovarian steroids administered to the host animals, resulting in blood serum levels normally found in the human female, had little or no effect on the proliferation of human mammary epithelial cells. Collagen gel embedded mouse mammary epithelial cells, mouse mammary explants, and host mammary glands all responded similarly to ovarian steroids, suggesting that the unresponsiveness of the human mammary epithelial cells under these conditions was not due to dissociation per se. However, an increased dose of 17β-estradiol or a growth factor combination containing epidermal growth factor, cholera toxin, and cortisol significantly stimulated the proliferation of human outgrowths. The growth factor response was dependent on the location of the cells, with the greatest response seen in the part of the gel proximal to the osmotic pump delivering the growth factors and the effect gradually waning in area more distal to the pump. The effect was especially striking since the mitotic figures could be easily identified and the labeling index was as high as 75%. The host mouse mammary gland also responded to growth factors, resulting in ductal hyperplasia. The proliferative and morphogenetic effects of various agents on normal human mammary epithelial cells embedded in collagen gel can be studied in vivo in nude mice. © 1995 Wiley-Liss, Inc.     

Effects of electrically induced fatigue on the twitch and tetanus of paralyzed soleus muscle in humans

Shields, Richard K., Laura Frey Law, Brenda Reiling, KellySass, and Jason Wilwert. Effects of electrically induced fatigueon the twitch and tetanus of paralyzed soleus muscle in humans.J. Appl. Physiol. 82(5):1499-1507, 1997.We analyzed the twitch and summated torque(tetanus) during repetitive activation and recovery of the human soleusmuscle in individuals with spinal cord injury. Thirteen individualswith complete paralysis (9 chronic, 4 acute) had the tibial nerveactivated every 1,500 ms with a 20-Hz train (7 stimuli) for 300 ms anda single pulse at 1,100 ms. The stimulation protocol lasted 3 min andincluded 120 twitches and 120 tetani. Minimal changes were found forthe acute group. The chronic group showed a significant reduction inthe torque and a significant slowing of the contractile speeds of boththe twitch and tetanus. The decrease in the peak twitch torque was significantly greater than the decrease in the peak tetanus torque early during the fatigue protocol for the chronic group. The twitch time to peak and half relaxation time were prolonged during fatigue, which was associated with improved fusion of the tetanus torque. At theend of the fatigue protocol, the decrease in the peak twitch torque wasnot significantly different from the decrease in the peak tetanustorque. After 5 min of rest, the contractile speeds recovered causingthe tetanus to become unfused, but the tetanus torque became lessdepressed than the twitch torque. The differential responses for thetwitch and the tetanus suggest an interplay between optimal fusioncreated from contractile speed slowing and excitation contractioncoupling compromise. These issues make the optimal design of functionalelectrical stimulation systems a formidable task.

     

Three-dimensional growth and morphogenesis of mouse submandibular epithelial cells in serum-free primary culture

Mouse submandibular epithelial cells can be grown in primary culture using the collagen gel matrix and a chemically defined medium consisting of insulin, transferrin, cholera toxin, and BSA (or FGF). Sustained cell growth leading to a 5–10-fold increase in cell number was observed in less than 2 weeks. In the presence of these additives, clumps of cells proliferate by extending ‘star-like’ projections into the matrix, resulting in three-dimensional outgrowths. The morphology of these outgrowths can be modulated to form a ‘cyst-like’ appearance by deleting BSA and adding cortisol to the basal medium containing insulin, transferrin, cholera toxin and FGF. In brief, a serum-free medium for sustained growth has been devised and a simple manipulation of supplements can modulate the three-dimensional colony morphology in the collagen gel matrix. Finally, the resulting outgrowths can produce epidermal growth factor (EGF) in response to dihydrotestosterone.     

Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

Summary Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture. This investigation was supported by Grants CA05388 and CA09041 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by cancer research funds of the University of California.     

Drought‐induced changes in flow regimes lead to long‐term losses in mussel‐provided ecosystem services

Extreme hydro‐meteorological events such as droughts are becoming more frequent, intense, and persistent. This is particularly true in the south central USA, where rapidly growing urban areas are running out of water and human‐engineered water storage and management are leading to broad‐scale changes in flow regimes. The Kiamichi River in southeastern Oklahoma, USA, has high fish and freshwater mussel biodiversity. However, water from this rural river is desired by multiple urban areas and other entities. Freshwater mussels are large, long‐lived filter feeders that provide important ecosystem services. We ask how observed changes in mussel biomass and community composition resulting from drought‐induced changes in flow regimes might lead to changes in river ecosystem services. We sampled mussel communities in this river over a 20‐year period that included two severe droughts. We then used laboratory‐derived physiological rates and river‐wide estimates of species‐specific mussel biomass to estimate three aggregate ecosystem services provided by mussels over this time period: biofiltration, nutrient recycling (nitrogen and phosphorus), and nutrient storage (nitrogen, phosphorus, and carbon). Mussel populations declined over 60%, and declines were directly linked to drought‐induced changes in flow regimes. All ecosystem services declined over time and mirrored biomass losses. Mussel declines were exacerbated by human water management, which has increased the magnitude and frequency of hydrologic drought in downstream reaches of the river. Freshwater mussels are globally imperiled and declining around the world. Summed across multiple streams and rivers, mussel losses similar to those we document here could have considerable consequences for downstream water quality although lost biofiltration and nutrient retention. While we cannot control the frequency and severity of climatological droughts, water releases from reservoirs could be used to augment stream flows and prevent compounded anthropogenic stressors.     

Facilitative production of an antimicrobial peptide royalisin and its antibody via an artificial oil-body system

Royalisin found in the royal jelly of Apis mellifera is an antimicrobial peptide (AMP). It has a molecular weight of 5.5 kDa, which contains six cysteine residues. In this study, royalisin was overexpressed in Escherichia coli AD494 (DE3) as two oleosin-fusion proteins for preparation of its antibodies and functional purification. The recombinant royalisin, fused with oleosin central hydrophobic domain in both N- and C-termini, was reconstituted with triacylglycerol and phospholipids to form artificial oil bodies (AOBs). The AOBs were then purified to raise the antibodies. These antibodies could recognize both the native and recombinant royalisins, but not oleosin. Another oleosin-intein S-fusion protein was purified by AOBs system, and royalisin was subsequently released from the AOBs through self-splicing of the intein. The recombinant royalisin exhibited high antibacterial activity, which suggested that it was refolded to its functional structure. These results demonstrated that AOBs system is an efficient method to functionally express and purify small AMPs. In addition, it also provides a facile platform for the production of antibodies against small peptides.     

Recombinant Cellulase Accumulation in the Leaves of Mature,Vegetatively Propagated Transgenic Sugarcane

The cost of enzymes that hydrolyse lignocellulosic substrates to fermentable sugars needs to be reduced to make cellulosic ethanol a cost-competitive liquid transport fuel. Sugarcane is a perennial crop and the successful integration of cellulase transgenes into the sugarcane production system requires that transgene expression is stable in the ratoon. Herein, we compared the accumulation of recombinant fungal cellobiohydrolase I (CBH I), fungal cellobiohydrolase II (CBH II), and bacterial endoglucanase (EG) in the leaves of mature, initial transgenic sugarcane plants and their mature ratoon. Mature ratoon events containing equivalent or elevated levels of active CBH I, CBH II, and EG in the leaves were identified. Further, we have demonstrated that recombinant fungal CBH I and CBH II can resist proteolysis during sugarcane leaf senescence, while bacterial EG cannot. These results demonstrate the stability of cellulase enzyme transgene expression in transgenic sugarcane and the utility of sugarcane as a biofactory crop for production of cellulases.     

Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function

Intermediate filaments (IFs) are composed of one or more members of a large family of cytoskeletal proteins, whose expression is cell- and tissue type-specific. Their importance in regulating the physiological properties of cells is becoming widely recognized in functions ranging from cell motility to signal transduction. IF proteins assemble into nanoscale biopolymers with unique strain-hardening properties that are related to their roles in regulating the mechanical integrity of cells. Furthermore, mutations in the genes encoding IF proteins cause a wide range of human diseases. Due to the number of different types of IF proteins, we have limited this short review to cover structure and function topics mainly related to the simpler homopolymeric IF networks composed of vimentin, and specifically for diseases, the related muscle-specific desmin IF networks.     

Functional expression of FIP-gts, a fungal immunomodulatory protein from Ganoderma tsugae in Sf21 insect cells

The mushrooms of diverse Lingzhi species have been traditionally consumed as luxurious functional food supplements in Chinese society. FIP-gts, a fungal immunomodulatory protein found in Song-Shan Lingzhi (Ganodera tsugae) has been proposed to possess therapeutic effects on cancer and autoimmune diseases. To produce active FIP-gts for evaluation of oral administration, a recombinant FIP-gts (rFIP-gts) fused with a 6His-tag at its C-terminus was expressed in Sf21 insect cells by the baculovirus expression system. High yield (about 70%) and purity (about 90%) of rFIP-gts was obtained by one-step nickel-affinity chromatography. The correctness of the harvested rFIP-gts was verified by Western blot and MALDI-MS analyses. Optimal expression of rFIP-gts was observed when the Sf21 cells were infected with multiplicity of infection of 10 for 72 h, and the yield was up to 47.2 microg/3 x 10(6) infected cells. The immunomodulatory activity of the purified rFIP-gts was detected as the induction of interleukin 2 released from murine splenocytes. Compared with the rFIP-gts produced in Escherichia coli cells, the rFIP-gts produced in Sf21 cells possessed evidently higher specific immunomodulatory activity.