Mixed-function oxidase activity in enriched populations of ciliated cells freshly isolated from rabbit tracheas

A method is described for the preparation of enriched populations of ciliated cells from rabbit tracheas. Following protease digestion of tracheal lumen tissue, cells were subjected to centrifugal elutriation. This produced two cell fractions of interest: an 8 µm diameter fraction believed to be composed largely of basal cells, and a 15 µm diameter fraction containing a mixture of ciliated cells and Clara cells. Further treatment of the 15 µm cells with a dextran/polyethylene glycol/phosphate buffer system resulted in separation of a highly enriched ciliated cell fraction (84.3 ± 2.7% ciliated cells with 6.5 ± 1.5% Clara cells) from a fraction containing both ciliated cells (42.0 ± 2.1%) and Clara cells (27.0 ± 3.5%). The yield of cells in the enriched ciliated cell fraction was 0.68 ± 0.09 × 10⁶ cells/ trachea. Analysis of mixed-function oxidase activity in tracheal cells showed 7-ethoxycoumarin deethylase and coumarin hydroxylase activities to be present in the 8 µm cells as well as in ciliated cells and Clara cells. Enzyme activities measured in the ciliated cells (152 ± 66 pmol/ min/ mg protein or 51.2 ± 20.5 pmol/ min/ 10⁶ cells for 7-ethoxycoumarin deethylase and 31.7 ± 15.4 pmol/ min/ mg protein or 10.5 ± 4.8 pmol/ min/ 10⁶ cells for coumarin hydroxylase) were not attributable to contamination with Clara cells.Abbreviations CD cell digest - DNase deoxyribonuclease I - E-1 first elutriator fraction - E-2 second elutriator fraction - E-3 third elutriator fraction - 7-Ec 7-ethoxycoumarin - FCS fetal calf serum - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid - HpBS HEPES-buffered salt solution - NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - NBT nitro blue tetrazolium - PEG Carbowax polyethylene glycol 6000     

Electroporation-mediated transient gene expression in intact cells of sweetpotato

Summary Transient expression of the β-glucuronidase (GUS) gene has been studied in leaf-derived embryogenic callus of sweetpotatoIpomoea batatas L. (Lam.) by electroporation. The influence of several factors including electric field strength, buffer composition, time course of transientGUS gene expression, DNA concentration, enzyme, and polyethylene glycol (PEG) treatment was examined onGUS gene expression (number of blue spots). MaximumGUS gene expression (an average of 90 blue spots/fifty mg fresh weight callus tissue) was observed after 48 h when callus pieces were preincubated with electroporation (EPR) buffer for 1 h, followed by electroporation with a single electric pulse of 500 V/cm discharged from a 960-μF capacitor in the presence of 20 μg DNA/ml and 8.3 μl NaCl (3M). Changing the electroporation buffer conductivity (by varying the buffer composition with low-high salt concentrations), had only slight effect on the number of blue spots. Similarly, the time course study ofGUS gene expression revealed that GUS activity could be detected 12 h after electroporation with a maximum activity after 72 h (112 blue spots). Increasing the amount of DNA from 5 to 50 μg/ml in the EPR buffer had a slight effect on the expression frequency (from 20–110 blue spots, and 112 blue spots with 20 μg/ml). The number of blue spots was increased by enzymatic wounding of callus pieces for 10 min and by addition of 200 μl PEG 4000 (15%) before electroporation. These results suggest that intact cell electroporation can be used for producing transgenic sweetpotato tissue.     

Metabolism of arachidonic acid by guinea pig Clara cells

A method for the isolation of non-ciliated bronchiolar epithelial (Clara) cells from the guinea pig is described. Following digestion of the lung tissue with Type XXIV protease, the isolated lung cells showed a viability greater than 90 % and contained 3 % of Clara cells. Several cell populations were then separated on the basis of size using 2 centrifugal elutriations. The macrophages and endothelial cells were removed from the Clara cells enriched fractions by differential adherence on Petri dishes. The Clara cell-rich suspension was then further purified by centrifugation on Percoll non-continuous density gradients consisting of 48-52-55 % Percoll solution. The lower interface and the pellet of the non-continuous gradient consisted of approximately 80 % Clara cells. Identification of isolated Clara cells was confirmed by light microscopic observations after nitroblue tetrazolium staining and by ultrastructural characteristic features as observed by electron microscopy. The metabolism of arachidonic acid into prostaglandins and TxB₂ by purified Clara cells was examined by enzyme immunoassay (EIA) and leukotriene formation was investigated by reverse phase high performance liquid chromatography (RP-HPLC). Enriched guinea pig Clara cells incubated with arachidonic acid released TxB₂, PGE₂ and 6-keto PGF_1α, but did not produce leukotrienes. These cells could however transform exogenous leukotriene A₄ into leukotriene B₄. These results suggest that guinea pig Clara cells possess the enzymes of the cyclooxygenase pathway required for TxB₂, PGE₂ and 6-keto-PGF_1α synthesis. Clara cells do not possess the 5-lipoxygenase enzyme but show some leukotriene A₄ hydrolase activity since they can produce leukotriene B₄ upon incubation with leukotriene A₄.     

One-step isolation of neutrophils using an elutriator

Summary To simplify the isolation of neutrophils, we developed a one-step procedure using elutriation. The perfusate (0.2% gelatin and 0.1% glucose in phosphate buffered saline) was pumped through an elutriator rotor at 4 ml/min (25° C) with the rotor speed at 2370 rpm. Twenty milliliters of anticoagulated porcine venous blood were mixed with 60 ml of perfusate and loaded into the elutriator chamber. The flow rate was increased by 2 ml/min increments and 100-ml fractions of effluent were collected at each increment. Concentrations of neutrophils and mononuclear cells were measured in each fraction, and the percentage of total neutrophils or mononuclear cells was plotted against flow rate. The optimal yield (46%) and purity (95.1%) of neutrophils (n=8) was obtained in pooled fractions at flow rates greater than 20 ml/min. Neutrophils in this preparation were round, the granules were intact, and the nuclei were lobulated. In addition, the cells produced superoxide in the presence of phorbol myristate acetate and phagocytosed zymosan particles. These characteristics were similar to those of porcine neutrophils prepared by a conventional sedimentation method. The yield (43%) and purity (94%) of human neutrophils isolated using the elutriator method was similar to that for porcine cells. This one-step method provides a moderate yield of pure neutrophils that have retained their morphology and function. This work was supported by the Canadian Heart Foundation.     

Metabolism of arachidonic acid by guinea pig Clara cells.

A method for the isolation of non-ciliated bronchiolar epithelial (Clara) cells from the guinea pig is described. Following digestion of the lung tissue with Type XXIV protease, the isolated lung cells showed a viability greater than 90% and contained 3% of Clara cells. Several cell populations were then separated on the basis of size using 2 centrifugal elutriations. The macrophages and endothelial cells were removed from the Clara cells enriched fractions by differential adherence on Petri dishes. The Clara cell-rich suspension was then further purified by centrifugation on Percoll non-continuous density gradients consisting of 48-52-55% Percoll solution. The lower interface and the pellet of the non-continuous gradient consisted of approximately 80% Clara cells. Identification of isolated Clara cells was confirmed by light microscopic observations after nitroblue tetrazolium staining and by ultrastructural characteristic features as observed by electron microscopy. The metabolism of arachidonic acid into prostaglandins and TxB2 by purified Clara cells was examined by enzyme immunoassay (EIA) and leukotriene formation was investigated by reverse phase high performance liquid chromatography (RP-HPLC). Enriched guinea pig Clara cells incubated with arachidonic acid released TxB2, PGE2 and 6-keto PGF1 alpha, but did not produce leukotrienes. These cells could however transform exogenous leukotriene A4 into leukotriene B4. These results suggest that guinea pig Clara cells possess the enzymes of the cyclooxygenase pathway required for TxB2, PGE2 and 6-keto-PGF1 alpha synthesis. Clara cells do not possess the 5-lipoxygenase enzyme but show some leukotriene A4 hydrolase activity since they can produce leukotriene B4 upon incubation with leukotriene A4.     

Characterization of the separation properties of the beckman elutriator system

The role of fluid flow in the elutriation process was visualized by pumping dye solution through the Beckman JE-6 elutriator rotor. Three major fluid flow disturbances were observed in the separation chambers, namely; jet-streaming, ripple flow, and whirl flow. In order to evaluate the effects of these non-ideal fluid flow patterns on the separation of homogeneous populations of particles or cells, 12–35 μm diameter latex spheres and 9L rat brain tumor cells were fractionated with the Beckman elutriator system. The elutriator system was evaluated on the basis of: (1) recovery, (2) elution loss during loading, (3) homogeneity of the size distributions, and (4) the relationship of the median volume of eluted particles or cells to the rotor speed and the collection fluid velocity. Both a conventional collection method (two 40-mL fractions at each collection rotor speed) and a long collection method (10–15 40-mL fractions at several collection rotor speeds) were compared to determine if collection procedures could compensate for some of the difficulties caused by the non-ideal fluid flow patterns. Although more than 90% of the particles or cells were always recovered, about 5% eluted during the loading procedure. Neither collection method altered this phenomenon. The collected populations, but this was accompanied by a reduction in cell yield. The median particle or cell volume of each fraction agreed with that expected under ideal fluid flow conditions except at high and low rotor speeds when the conventional collection method was used.     

Characterisation of lectin binding patterns of mouse bronchiolar and rat alveolar epithelial cells in culture

Lung epithelial cell differentiation pathways remain unclear. This is due in part to the plasticity of these cells and the lack of markers which accurately reflect their differentiation status. The aim of this study was to determine if lectin binding properties are useful determinants of functional differentiation status in vitro. Mouse Clara cells were cultured for 5 days. During this time, no alteration in differentiation was evident by electron microscopy. No significant alteration in binding reactivity of Bauhinia purpurea (BPA), Maclura pomifera (MPA), Concanavalin A, Wheat germ or Helix pomatia lectins occurred in cultures compared with Clara cells in mouse lung tissue. In contrast, nitrotetrazolium blue reductase activity and CC10 expression declined in culture. Rat type II cells were cultured for 8 days. Between days 0 and 4, the number of type II cells identified by electron microscopy was constant at 70–80%, decreasing to 8% by day 6. In contrast, by day 4, only 42% cells retained alkaline phosphatase activity. BPA and MPA reactivity was altered at day 0 and day 4 respectively, compared with cells in situ. Therefore, the reactivity of lectins analysed here does not reflect functional differentiation status of cultured mouse Clara cells. However, BPA and MPA reactivity may be a sensitive indicator of alterations in rat type II cell differentiation in vitro.     

Poly(ethylene glycol) modification of β-glucuronidase-antibody conjugates for solid-tumor therapy by targeted activation of glucuronide prodrugs

 Methoxypoly(ethylene glycol) (PEG) modification of Escherichia coliβ-glucuronidase (βG) was examined as a method to improve the stability and pharmacokinetics of antibody-βG conjugates for the targeted activation of glucuronide prodrugs at tumor cells. Introduction of 3 PEG molecules did not affect βG activity whereas higher degrees of PEG modification produced progressively greater loss of enzymatic activity. The enzyme was found to be stable in serum regardless of PEG modification. PEG-modified βG was coupled via a thioether bond to mAb RH1, an IgG_2a antibody that binds to the surface of AS-30D hepatoma cells, to produce conjugates with 3 (RH1-βG-3PEG), 5.2 (RH1-βG-5PEG) or 9.8 (RH1-βG-10PEG) PEG molecules per βG with retention of 75%, 45% and 40% of the combined antigen-binding and enzymatic activity of the unmodified conjugate RH1-βG. In contrast to the rapid serum clearance of RH1-βG observed in mice, the PEG-modified conjugates displayed extended serum half-lives. RH1-βG-3PEG and RH1-βG-5PEG also exhibited reduced spleen uptake and greater tumor accumulation than RH1-βG. BHAMG, the glucuronide prodrug of p-hydroxyaniline mustard (pHAM), was relatively nontoxic in vivo. Injection of 6 mg/kg or 12 mg/kg pHAM i.v. depressed white blood cell numbers by 46% and 71% whereas 80 mg/kg BHAMG reduced these levels by 22%. Although the tumor/blood ratio of RH1-βG-5PEG was adversely affected by slow clearance from serum, combined therapy of small solid hepatoma tumors with this conjugate, followed 4 and 5 days later with i.v. injections of BHAMG, cured all of seven mice with severe combined immunodeficiency. Combined treatment with a control antibody-βG conjugate and BHAMG delayed tumor growth and cured two of six mice while treatment with pHAM or BHAMG alone was ineffective. Received: 27 February 1997 / Accepted: 6 May 1997     

DIFFERENTIAL ENRICHMENT OF CELLS FROM EMBRYONIC RAT CEREBRA BY CENTRIFUGAL ELUTRIATION

—Centrifugal elutriation was used to obtain different populations of cells dissociated from 16-day-old rat embryo cerebra. The cell populations recovered were viable and could be maintained for several weeks in vitro. Sterile conditions were maintained throughout a preparation. Rat pups were removed by Caesarean section, the cerebra dissected and the cells dissociated by brief exposure to trypsin (0.125%, 6 min). An equivalent volume of elutriation medium (Dulbecco's medium containing 1% fetal calf serum, sodium bicarbonate, penicillin and streptomycin, EDTA, and deoxyribonuclease) was added to the trypsin-cell suspension, the dissociated cells pelleted, resuspended in elutriation medium and counted. Up to 4 x 10⁸ cells were injected into the previously sterilized elutriator. Seven fractions were usually recovered from a preparation. The first fraction contained primarily red blood cells and cell debris, which could not be maintained in vitro. Upon culture, fraction 2 consisted of predominantly non-neuronal cells, while fractions 3–6 contained neuronal and non-neuronal cells. The morphological characteristics of the neurons differed in these fractions. Fraction 7 contained cells that had reaggregated during the elutriation procedure and exhibited a variety of cell types in vitro.     

A method for isolating large numbers of viable disaggregated cells from various human tissues for cell culture establishment

Summary A method is described for the isolation of large numbers of viable disaggregated cells from human tissues. This method combined the mechanical action of a Stomacher Model 80 Lab Blender, 0.1 mg/ml trypsin or 0.5 mg/ml collagenase, and 0.1 mM [ethylene bis(oxyethylenenitrolo)]-tetraacetic acid (EGTA). Tissue (0.2 to 1.0 g) obtained from human fetal intestine, kidney, liver, lung, and skin were separately minced into approximately 1-mm³ pieces. The pieces were placed in a sterile bag containing 60 ml of calcium- magnesium-free phosphate buffered saline, the appropriate enzyme (0.1 mg/ml trypsin or 0.5 mg/ml collagenase) plus 0.1 mM EGTA, and 0.1% methylcellulose. The bag was then placed into the blender and mixed at a low speed for 3 to 20 min at room temperature. After a single cell suspension was observed by phase contrast microscopy, 10 ml of bovine calf serum was added to the cell suspension to inactivate the proteolytic enzymes. At this time 130 ml of cold Hanks' balanced salts solution containing 5% bovine calf serum was added and the entire cell suspension passed through a tissue sieve (100 mesh, 140 μm) and the cells collected by centrifugation. These cells were then resuspended into the appropriate culture medium. In comparison to other methods for establishment of cell cultures from human tissues, the method described requires shorter incubation times with relatively low concentrations of proteolytic enzymes, and yields two- to three-fold greater number of cells per tissue with 86 to 93% viability. Also, depending on the cell type, 50 to 75% of the isolated cells attached to the culture vessel within 24 h. Variation of the time and concentration of digestive enzymes can be used to select different cell types for culture. This work was supported by research grants from the National Institute of Environmental Health Sciences, Bethesda, MD (ES3101) and the United States Environmental Protection Agency, Washington, D. C. (R810146).     

Stimulation of the side population fraction of ATDC5 chondroprogenitors by hypoxia

The influence of oxygen tension on the side population (SP) fraction sorted from ATDC5 chondroprogenitor cells was investigated. ATDC5 cells cultured in normoxia (20%) or hypoxia (1% oxygen) were compared. The SP fraction was significantly higher in the cells cultured in hypoxia. The gene expression of 3 ABC transporters, abcb1a/b (mdr1a/b) and abcg2 (bcrp1) was quantified by RT-PCR. SP cells were characterized by the higher expression of abcb1a. The expression levels of abcb1b and abcg2 were higher than abcb1a. However, there was no significant difference between SP and non-SP fractions in the expression of abcb1b and abcg2. The telomeric repeat amplification protocol assay showed that SP cells tended to show lower telomerase activity than non-SP cells. Chondrogenic properties of ATDC5 cells derived from SP or non-SP were assessed by micromass culture. There were not significant differences between SP and non-SP derived cells in Alcian blue staining and sox9, Aggrecan, Col2a1 and SZP mRNA expression. The results demonstrate that the SP fraction was stimulated by hypoxia and chondrogenic properties of SP and non-SP fraction of ATDC5 cells were similar.     

Enzymatic activity and thermal stability of PEG-α-chymotrypsin conjugates

α-Chymotrypsin was chemically modified with methoxypoly(ethylene glycol) (PEG) of different molecular weights (700, 2,000, and 5,000 Da) and the amount of polymer attached to the enzyme was varied systematically from 1 to 9 PEG molecules per enzyme molecule. Upon PEG conjugation, enzyme catalytic turnover (k _cat) decreased by 50% and substrate affinity was lowered as evidenced by an increase in the K _M from 0.05 to 0.19 mM. These effects were dependent on the amount of PEG bound to the enzyme but were independent of the PEG size. In contrast, stabilization toward thermal inactivation depended on the PEG molecular weight with conjugates with the larger PEGs being more stable.     

Evaluation of encapsulation stress and the effect of additives on viability and photosynthetic activity of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 encapsulated in silica gel

Stresses imposed on the cyanobacterium Synechocystis sp. PCC 6803 by various compounds present during silica sol–gel encapsulation, including salt, ethanol (EtOH), polyethylene glycol (PEG), glycerol, and glycine betaine, were investigated. Viability of encapsulated cells and photosynthetic activity of cells stressed by immediate (2 min) and 24-h exposure to the five stress-inducing compounds were monitored by pulse amplitude modulated fluorometry. Cells of Synechocystis sp. PCC 6803 readily survive encapsulation in both alkoxide-derived gels and gels from aqueous precursors and can remain active at least 8 weeks with slight degradation in PSII efficiency. Post-encapsulation survival was improved in gels containing no additive when compared with gels containing PEG or glycerol. Glycerol was shown to have a detrimental effect on Synechocystis sp. PCC 6803, reducing ϕPSII and F _v′/F _m′ by as much as 75%, possibly a result of disrupting excitation transfer between the phycobilisomes and photosystems. PEG was similarly deleterious, dramatically reducing the ability to carry out a state transition and adequately manage excitation energy distribution. EtOH stress also hindered state transitions, although less severely than PEG, and the cells were able to recover nearly all photosynthetic efficiency within 24 h after an initial drop. Betaine did not interfere with state transitions but did reduce quantum yield and photochemical quenching. Finally, Synechocystis sp. PCC 6803 was shown to recover from salt stress.     

Abnormal expression of Muc5b in Cftr-null mice and in mammary tumors of MMTV-ras mice

Gel-forming mucins are large, high molecular weight, and heavily O-glycosylated proteins that are responsible for the rheological properties of mucus gel. Among them, the mucin MUC5B has been implicated in breast cancer and cystic fibrosis. We obtained a new polyclonal serum, named CP1, which was isolated from a rabbit immunized with a mouse Muc5b peptide. The immunoprofile of Muc5b was determined on paraffin-embedded and frozen mouse tissue sections and showed a similar expression pattern in mouse to that in the human. The “nonmammary” mucin Muc5b was detected in all mammary tumors analyzed from MMTV-ras mice, suggesting that the CP1 antibody is a valuable tool for investigating the involvement of this mucin in mammary cancer. We also found that uninfected Cftr ^−/− mice harbored more Clara cells, which were Muc5b-positive, than did their wild-type control littermates. The number of Muc5b-positive cells increased in Cftr ^−/− mice infected experimentally with Pseudomonas aeruginosa, and the mice developed mucus plugs in their bronchi and bronchioles with a high frequency of Muc5b content (87%, Cohen’s kappa = 0.82; p < 0.0001). These findings suggest that mice genetically deficient in the Cftr gene are predisposed to develop mucus plugs and that MUC5B may provide a valuable target for decreasing mucus viscosity in cystic fibrosis.     

Metabolism of 2-acetylaminofluorene by clara cells,type II cells and alveolar macrophages isolated from rabbit lung,and use of a new chamber incubation mutagenicity test system

Clara cells, alveolar type II cells and pulmonary alveolar macrophages (PAM) were isolated in high yield from rabbit lung. The purity of the cell fractions was 80–90%, 98% and above 99%, respectively. Cytochrome P-450 total content was determined in microsomes from freshly prepared cells. The Clara cells contained significantly more cytochrome P-450 than was found in whole lung microsomes. Furthermore, the cytochrome content of the Clara cells was 2 -fold higher than in the type II cells and 4 -fold higher than in the macrophages. 2-aminofluorene (AF) was the major metabolite in all preparations when intact cells were incubated with 2-acetylaminofuorene (AAF). The PAMs produced AF in the highest rates, while the Clara cells showed the largest rates of cytochrome P-450-dependent, ring hydroxylation of AAF. Mutagenic activation of AAF by isolated lung cells was assayed with a chamber-incubation method. The Clara cells were far more active than the type II cells in this respect, while the macrophages were inactive.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethyl sulfoxide - NBT nitro blue tetrazolium - 7-OH-AAF 7-hydroxy-AAF - 9-OH-AAF 9-hydroxy-AAF     

Simultaneous production and partition of chitinase during growth of Serratia marcescens in an aqueous two-phase system

Summary Partition and production of the extracellular chitinase from Serratia marcescens were studied in PEG/dextran aqueous two-phase systems. The enzyme partitions into the bottom phase and the cells segregate into the top phase. The best system is 2% (w/v) PEG 20000 and 5% (w/v) dextran T500. The cell growth and enzyme production kinetics are similar in the aqueous two-phase system and in the polymer-free reference system. However, the maximum enzyme concentration in the former system is 1.5 times that in the latter one.     

Water status and growth of loblolly pine (Pinus taeda L.) callus

Water status of Pinus taeda L. callus supported on Murashige and Skoog (MS) liquid medium was characterized over an 8 week period using thermocouple psychrometry. Medium with 30 gl⁻¹ sucrose was used to produce a high water potential (Ψ_w) of −0.4 MPa (H), and the same medium was used to create a moderate Ψ_w of −0.7 MPa (M) by the addition of 10% polyethylene glycol (PEG, w/v, MW=8000). Calli were produced from cotyledon explants on H medium for 2 weeks and then transferred to either M or H medium. Callus absorption of PEG accounted for 40% of the callus dry weight and less than 7% of the callus fresh weight. Callus dry weight (without the PEG fraction) on M medium was 40% of that observed on H medium. Fresh weight on M medium was only 15% of that observed on H medium. The Ψ_w of both H and M media remained constant throughout the culture period. On H medium, callus Ψ_w and osmotic potential (Ψ_s) both increased 0.05 MPa/week with the callus Ψ_w approaching that of the external medium. On M medium, callus Ψ_w and Ψ_s both decreased more than 0.1 MPa/week with the callus Ψ_w decreasing greatly below that of the external medium. The latter was attributed to a rapidly produced osmotic shock induced upon callus transfer and/or PEG which caused less callus hydration and resulted in reduced growth. Callus turgor potential (Ψ_p) was estimated to be +0.02 to +0.09 MPa and turgor was maintained as callus Ψ_w increased or decreased. After 8 weeks, cell volumes from callus on M medium were 50 to 60% less than on H medium, suggesting that reduced cell volumes were related to turgor maintenance.     

Glucans in the Cell Walls Regenerated from Vinca rosea Protoplasts

Protoplasts prepared from suspension-cultured Vinca rosea cellswere cultured for 5 days. The cell walls regenerated from theprotoplasts were mainly composed of glucans having 1,3- and1,4-linkages. To investigate the molecular species, these glucanswere separated into four fractions: EDTA (50 mM, pH 4.5)-soluble(fraction E), KOH (24%)- soluble but not precipitatable by neutralizationwith acetic acid (fraction K-S), KOH (24%)-soluble and precipitatableby neutralization with acetic acid (fraction K-P), and KOH (24%)-insoluble(fraction C). By means of sugar composition analysis, methylationanalysis, periodate oxidation and enzymatic digestion, the molecularspecies of the glucans contained in the regenerated cell wallswere deduced to be ß-1,4-glucan (cellulose) and ß-1,3-glucan.Fraction C was mainly composed of ß-1,4-glucan; ß-1,3-glucanwas mainly recovered in fraction K-P. The ß-l,3-glucanwas soluble in dilute alkali solution, but was only slightlysoluble in water. The ß-1,3-glucan had an essentiallyunbranched structure, and its weight average molecular weightestimated by gel permeation chromatography was 4.5–5.0x 10⁴. ¹ Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabe, Tsukuba, Ibaraki305, Japan (Received May 21, 1981; Accepted October 13, 1981)     

Cytochrome P-450 monooxygenase,epoxide hydrolase and flavin monooxygenase activities in clara cells and alveolar type II cells isolated from rabbit

The activities of several enzymes which metabolize xenobiotics were measured and compared in freshly isolated rabbit Clara cells (50–70% purity) and alveolar type II cells (80–95% purity) or microsomal preparations from the isolated cell fractions. The presence of 1 mM nicotinamide in protease and cell isolation buffers increased significantly 7-ethoxycoumarin (7-EC) deethylase and epoxide hydrolase activities in the isolated Clara and type II cells. Isolated Clara cell fractions metabolized 7-EC to umbelliferone at a rate of 241 ± 27 pmoles/mg prot/min (mean ± S.E., N=5), while the 7-EC deethylation rate in type II cells was 111 ± 15 pmoles/mg prot/min. Coumarin hydroxylation activity, however, was more than ten times greater in the Clara cells than in the type II cells on a per mg cellular protein basis. N-oxidation of N,N-dimethylaniline, catalyzed by a flavin monooxygenase, was about 2 times as great in microsomes of Clara cells as in microsomes of type II cells. Epoxide hydrolase activity with benzo(a)pyrene 4,5-oxide as substrate was about 10 times higher in Clara cells than in type II cells. Because of the greater cellular, structural and functional heterogeneity in lung, differential distribution of enzymes responsible for xenobiotic metabolism in this tissue may contribute to cell selective chemical toxicity and carcinogenesis.Abbreviations 7-EC 7-ethoxycoumarin - DMA N,N-dimethylaniline     

Isolation of polyhydroxyalkanoate from hydrolyzed cells of <Emphasis Type="Italic">Bacillus flexus</Emphasis> using aqueous two-phase system containing polyethylene glycol and phosphate

Main objective of present work was to isolate polyhydroxyalkanoate (PHA) from cell lysate of Bacillus flexus by aqueous-aqueous two-phase system (ATPS). Selected ATPS having polyethylene glycol (12%, w/v) and potassium phosphate (9.7%, pH 8.0) containing cell lysate obtained by sonication or hypochlorite treatment of B. flexus biomass (1 g%, dry weight), was held at 28°C for 30 min, which partitioned PHA into top PEG phase and residual cell materials into bottom phase. For enzymatic cell hydrolysis, Microbispora sp. culture filtrate having protease (3 U/mL) was mixed with B. flexus biomass and ATPS, incubated at 37°C for 2 h prior to phase separation. PHA recovered by centrifugation was 19∼51% of cell dry weight, depending on the mode of cell disruption. Protease was recovered along with PHA in the PEG phase and showed 7 fold increase in activity. PHA was characterized by GC, FTIR, and ¹H NMR. Results indicated that ATPS can be used for the isolation of PHA from hydrolyzed bacterial cells and purified protease can be recovered as a byproduct, in a single defined experiment. Results have indicated that ATPS can be successfully employed as a non-organic solvent method for the isolation of PHA.