Effect of different collagenases on the isolation of viable hepatocytes from rat liver

The isolation of viable hepatocytes from rat liver was found to depend on the source of collagenase (EC 3.4.24.3) more than any other single factor examined. Collagenase purified from Achromobacter iophagus/Bacillus polymyxa (collagenase/dispase) gave reproducibly high viability without the use of complex perfusion protocols.     

Effect of different carbon sources on community composition of bacterial enrichments from soil

Soil is a highly heterogeneous matrix, which can contain thousands of different bacterial species per gram. Only a small component of this diversity (maybe <1%) is commonly captured using standard isolation techniques, although indications are that a larger proportion of the soil community is in fact culturable. Better isolation techniques yielding greater bacterial diversity would be of benefit for understanding the metabolic activity and capability of many soil microorganisms. We studied the response of soil bacterial communities to carbon source enrichment in small matrices by means of terminal restriction fragment length polymorphism (TRFLP) analysis. The community composition of replicate enrichments from soil displayed high variability, likely attributable to soil heterogeneity. An analysis of TRFLP data indicated that enrichment on structurally similar carbon sources selected for similar bacterial communities. The same analysis indicated that communities first enriched on glucose or benzoate and subsequently transferred into medium containing an alternate carbon source retained a distinct community signature induced by the carbon source used in the primary enrichment. Enrichment on leucine presented a selective challenge that was able to override the imprint left by primary enrichment on acetate. In a time series experiment community change was most rapid 18 hours after inoculation, corresponding to exponential growth. Community composition did not stabilize even 4 days after secondary enrichment. Four different soil types were enriched on four different carbon sources. TRFLP analysis indicated that in three out of four cases communities enriched on the same carbon source were more similar regardless of which soil type was used. Conversely, the garden soil samples yielded similar enrichment communities regardless of the enrichment carbon source. Our results indicate that in order to maximize the diversity of bacteria recovered from the environment, multiple enrichments should be performed using a chemically diverse set of carbon sources.     

Cytokine effect on ex vivo expansion of haemopoietic stem cells from different human sources

Human pluripotential stem cells (PSC) are currently the target for transplantation attempts and genetic manipulation. We have therefore investigated the frequency and the expansion potential of PSC’s in different types of blood samples. CD 34⁺ cells were thus obtained from human bone marrow (BM), as well as from peripheral blood (PB) and cord blood (CB) samples. After immuno-magnetic separation the highest yields of CD 34⁺ cells were from BM (1.08–2.25%) and CB (0.42–1.32%) while PB samples gave much lower values. Suspension cultures of PSC’s from the three sources were then set up, in the presence of combinations of haemopoietic growth factors. A remarkable amplification of the nucleated cell pool was observed reaching a maximum between 10 and 15 days of culture; earliest and maximum expansion (up to 220-fold) was achieved when Erythropoietin (Epo) was added to the culture medium, but this resulted in reduction of colony-forming cells and differentiation into erythroid progenitors. Clonogenic tests for BFU-E’s derived colonies showed a peak value at 5 days of liquid culture. Further studies are advisable to establish the best cytokine combination for a valuableex vivo expansion, coupled with preservation of stem cell properties.     

Pancreatic islets isolation using different protocols with in situ flushing and intraductal collagenase injection

Human islet transplantation seems to be a very promising clinical procedure for patients with type I diabetes mellitus. The aim of our study was to investigate the influence of in situ intravascular flushing with University of Wisconsin (UW) solution and intraductal collagenase injection at the time of pancreas procurement on the isolated islets and exocrine tissue injury. Our experiments indicated that in situ perfusion with the UW solution has a beneficial effect on pancreatic islets and intraductal distention results in an increase in the concentration of pancreatic enzymes released into the cold preservation solution during ischemic conditions. Cold ischemia reduced islet yield, but pancreas perfusion with the UW solution showed better ischemic tolerance of isolated islets during glucose static incubation. We conclude that intravascular pancreas flushing has a crucial effect on recovery and yield of pancreatic islets and protects against exocrine tissue injury.     

Copper-chelatin: isolation from various eucaryotic sources.

Bovine factor V was treated with highly purified neuraminidase to yield asialo factor V. Different preparations of factor V were found to exhibit heterogeneity with respect to the content of sialic acid, which ranged from 5% to 12%. The activity of asialo factor V in accelerating blood coagulation was found to be significantly greater than native factor V; thus, sialic acid is not essential for activity but may modulate its function. Asialo-factor V was 3.8 times more stable as calculated from the thermal decay constant at 37 °C. when compared to unmodified factor V.The increase in activity of factor V by thrombin was biphasic after removal of all sialic acid residues. The binding of asialo-factor V to phosphatidyl ethanolamine, as, characterized both by kinetics and by sucrose density gradient ultracentrifugation, resulted in an inactive complex. Double immunodiffusion shows that the sialic acid component of factor V is not necessary for its antigenicity.     

The origins of blood: induction of hematopoietic stem cells from different sources

Ex vivo hematopoiesis from embryonic sources offers exciting promises in basic research and medicine. In this issue of Cell Stem Cell, Ledran et al. (2008) describe human embryonic stem cell (hESC)-derived hematopoiesis, while Taoudi et al. (2008) define the origin of definitive hematopoietic stem cells (HSCs) from the mouse aorta-gonad-mesonephros (AGM) region.     

Effect of divalent metal ions on collagenase from Clostridium histolyticum.

The collagenase from Clostridium histolyticum (EC 3.4.24.3) degrades type IV collagen with Km 32 nM, indicating a high affinity for this substrate. Ferrous and ferric ions can inhibit Clostridium collagenase. Inhibition by Fe++ was of the mixed, non-competitive type, with Ki 90 microM. The inhibitory effect of Fe++ may be due to Zn++ displacement from the intrinsic functional center of this metalloprotease, since in the presence of excess amounts of Zn++ enzyme activity is retained. This inhibitory effect of Fe++ may be common for all types of collagenases, since this ion can also inhibit type IV collagenase purified from Walker 256 carcinoma, with IC50 80 microM. Cu++ can only partially inhibit Clostridium collagenase, while other divalent metal ions such as Cd++, Co++, Hg++, Mg++, Ni++ or Zn++ are devoid of any inhibitory effect on the enzyme.     

Comparison of direct and indirect radiation effects on osteoclast formation from progenitor cells derived from different hemopoietic sources

Hemopoietic stem and progenitor cells from different sources differ in radiosensitivity. Recently, we have demonstrated that the multinucleated cell responsible for bone resorption and marrow cavity formation, the osteoclast, is in fact of hemopoietic lineage. In this investigation we have studied the radiosensitivity of osteoclast formation from two different hemopoietic tissues: fetal liver and adult bone marrow. Development of osteoclasts from hemopoietic progenitors was induced by coculture of hemopoietic cell populations with fetal mouse long bones depleted of their own osteoclast precursor pool. During culture, osteoclasts developed from the exogenous cell population and invaded the calcified hypertrophic cartilage of the long bone model, thereby giving rise to the formation of a primitive marrow cavity. To analyze the radiosensitivity of osteoclast formation, either the hemopoietic cells or the bone rudiments were irradiated before coculture. Fetal liver cells were found to be less radiosensitive than bone marrow cells. The D0, Dq values and extrapolation numbers were 1.69 Gy, 5.30 Gy, and 24.40 for fetal liver cells and 1.01 Gy, 1.85 Gy, and 6.02 for bone marrow cells. Irradiation of the (pre)osteoclast-free long bone rudiments instead of the hemopoietic sources resulted in a significant inhibition of osteoclast formation at doses of 4 Gy or more. This indirect effect appeared to be more prominent in the cocultures with fetal than with adult hemopoietic cells. Furthermore, radiation doses of 8.0-10.0 Gy indirectly affected the appearance of other cell types (e.g., granulocytes) in the newly formed but underdeveloped marrow cavity. The results indicate that osteoclast progenitors from different hemopoietic sources exhibit a distinct sensitivity to ionizing irradiation. Radiation injury to long bone rudiments disturbs the osteoclast-forming capacity as well as the hemopoietic microenvironment.     

Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells.

Perlecan, a major heparan sulfate proteoglycan of vascularized tissues, was immunopurified from media conditioned by human endothelial cells of both arterial and venous origin. The heparan sulfate moiety of perlecan from cultured arterial cells differed in amount and/or composition from that produced by a transformed cell line of venous origin. Both forms of perlecan bound basic fibroblast growth factor with Kd approximately 70 nM. In ELISA experiments, perlecan and its protein core bound to various extracellular matrix components in a manner that was strongly influenced by the format of the assay. Human vascular smooth muscle cells and human endothelial cells adhered to perlecan-coated surfaces, and both cell types adhered better to the venous cell-derived than to the arterial cell-derived perlecan. Removal of the heparan sulfate chains abolished this difference and increased the ability of both types of perlecan to adhere vascular cells. Denaturation of perlecan and its protein core also rendered each of them more adhesive, indicating the presence of conformation-independent adhesion determinants in the polypeptide sequence. Their location was investigated using recombinant perlecan domains. Overall, our results represent the first demonstration of human perlecan acting as an adhesive molecule for human vascular cells and suggest that it may play a role in vascular wound healing.     

Structural comparison of phosphorylases from different sources

All of the -glucan phosphorylases so far purified from diverse origins have similar molecular and catalytic properties, whereas they differ in regulatory properties and glucan specificities. The activity of the rabbit muscle enzyme is regulated by phosphorylation-dephosphorylation and activated by AMP. On the other hand, the potato and Escherichia coli enzymes exist only in the active form, and are unaffected by the nucleotide. To elucidate the structural bases for these differences, we have determined the complete amino acid sequence of potato phosphorylase and compared it with those of the rabbit muscle and E. coli enzymes. The monomer of the potato enzyme contains 916 amino acids with a molecular weight of 103,916. About one-fourth of the amino-terminal threonine is blocked by an acetyl group. Sequence comparison among these enzymes reveals the presence of a characteristic 78-residue insertion in the middle of the polypeptide chain of the potato enzyme. Except for the large inserted portion, 51 and 40% of the amino acids in the potato enzyme are identical with the rabbit muscle and E. coli enzymes, respectively. The regions relevant to the regulation of the activity are completely different among the three enzymes, whereas those involved in the catalytic reaction are well conserved. The potato enzyme sequence is consistent with the tertiary structure of the rabbit muscle enzyme. The 78-residue insertion is located at the junction of the amino- and carboxyl-terminal domains on the molecular surface near the glycogen-storage site. This insertion could account for the substrate discrimination of the potato enzyme. The molecular evolution of phosphorylase is discussed based on the structural comparison among the three enzymes.     

Effect of shear stress on migration and integrin expression in macaque trophoblast cells

During fetal development, trophoblast cells enter endometrial capillaries, migrate within the uterine vasculature, and eventually reside within spiral arteries of the uterus. This invasive activity is accompanied by upregulation of trophoblast beta1 integrin expression. Fluid mechanical shear stress regulates migration and expression of adhesion molecules in vascular endothelial cells, but nothing is known about the effects of shear stress on trophoblast cells. We tested the hypothesis that shear stress regulates the motility and beta1 integrin expression of trophoblast cells. Early gestation macaque trophoblast cells were cultured in 1 x 1-mm square cross-section capillary tubes within which the flow field was determined using three-dimensional computational fluid dynamic simulations. Trophoblast cells in the capillary tubes were exposed to a steady shear stress of 7.5, 15, or 30 dyn/cm2 for up to 24 h. In the absence of flow, trophoblast cells were highly dynamic with constant nondirectional positional shifts but with no net cell migration. Exposure of the cells to shear stress within 24-72 h of cell plating significantly increased the level of this activity and led to net cell migration in the direction of flow. Shear stress also increased the expression and altered the topography of beta1 integrin. These results suggest that shear stress regulates trophoblast motility and beta1 integrin expression in vitro.     

Effect of different carbon sources on lipase production by Candida rugosa

Different carbon sources affecting growth and lipase production in Candida rugosa were studied by using batch cultures on defined medium. Carbohydrates and acids non-related to fats did not induce lipase production. The highest yields of enzyme were obtained with lipids or fatty acids as carbon sources. Tween 80 stimulated lipase biosynthesis and secretion outside the cell. Combinations of two types of substrates, carbohydrates and fatty acids, did not improve lipase production, and in some cases, their consumption was produced in a sequential pattern. Glucose presented a repressing effect on lipase production. Moreover, glucose was found to be effective in stimulating lipase secretion by cells with a high level of cell-bound lipase activity because of their previous growth in oleic acid.