Role of calcium binding by sarcoplasmic reticulum in the contraction and relaxation of uterine smooth muscle

The binding of calcium by isolated sarcoplasmic reticulum from cow uterus was studied. Sarcoplasmic reticulum was prepared by differential centrifugation. Three fractions were obtained: I, sedimented between 2,500–15,000 x g; II at 40,000 x g; and III, at 150,000 x g. Fraction II was further purified on a sucrose density gradient. All three fractions contained considerable amounts of intrinsic calcium, mostly in fraction I. Calcium binding in the presence of ATP¹ and Mg also was greatest in fraction I, followed by fraction II, with less in fraction III. Without ATP no calcium was taken up. 5 and 10 mM sodium azide partially inhibited calcium binding in fraction I, but not in fraction II, suggesting the presence of some mitochondria or mitochondrial fragments in fraction I. Calcium binding in fraction II was completely inhibited by 3 mM salyrgan; this fraction thus appears to be sarcoplasmic reticulum. ATPase activity was found in all three fractions, highest in fraction II. It is computed that calcium binding in fractions I and II, on the basis of a 50% yield of protein, is sufficient to elicit contraction by supplying calcium to the contractile proteins of the smooth muscle cell and to regulate relaxation and contraction.     

Ultrastructure of the parasitic interface ofPucciniastrum,Thekopsora, Naohidemyces,andCalyptospora (Uredinales,Pucciniastraceae) in the dikaryotic stage

The ultrastructure of dikaryotic haustoria of sevenPucciniastrum species,Thekopsora galii, Naohidemyces vaccinii, andCalyptospora goeppertiana was investigated.Pucciniastrum actinidiae, P. agrimoniae, P. pyrolae, andCalyptospora goeppertiana revealed haustoria whose necks were wrapped by a fold of the extrahaustorial matrix. The matrix-fold ofCalyptospora goeppertiana was characteristically shaped.Pucciniastrum circaeae, P. epilobii, P. hikosanense, P. styracinum, Thekopsora galii, andNaohidemyces vaccinii showed typical haustorial necks which were not sheathed by a matrix-fold. Haustorial necks which were wrapped by a fold of the extrahaustorial matrix were designated velopedunculate, and those which were naked gymnopedunculate. The application of haustorial ultrastructure as a character for use in systematics is discussed.Part 112 of the series Studies in Heterobasidiomycetes.     

Agitation,aeration and perfusion modules for cell culture bioreactors

For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode.Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated.Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 m) as well as anchorage dependent cells grown on microcarriers (pore size 75 m) over six weeks to 3 months.Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.