Simultaneous detection of beta-galactosidase activity and surface antigen expression in viable haematopoietic cells

The quantitation of intracellular beta-galactosidase activity has been described for viable cells. By using the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside (FDG) in conjunction with flow cytometry, the proportion of positive cells as well as the level of expression can be determined. In this paper we describe beta-galactosidase expression in lymphoid and myeloid cells from transgenic mice that widely express beta-galactosidase from an inserted lacZ transgene. Both foetal and adult haematopoietic tissues are able to express beta-galactosidase. The intracellular fluorescence reflecting beta-galactosidase activity can be readily combined with fluorescently labelled antibodies against cell surface antigens. Thus, beta-galactosidase can be used as a marker in transplantation experiments to study the development of lymphoid and myeloid precursor cells.     

In situ histochemical detection of beta-galactosidase activity in lung: assessment of X-Gal reagent in distinguishing lacZ gene expression and endogenous beta-galactosidase activity

Bacterial lacZ is one of the most commonly used reporter genes for assessing gene transfer to lung. However, lung contains endogenous beta-galactosidase (beta-Gal), which can confound estimation of exogenous lacZ expression by histochemical techniques (i.e., X-Gal) for in situ demonstration of enzyme activity. We investigated several parameters of the X-Gal reaction, including time and temperature of X-Gal exposure as well as lung tissue processing and fixation techniques, and found that none of these could be used to distinguish between endogenous and exogenous beta-Gal activities. The mammalian and bacterial beta-Gal enzymes, however, have pH optima in the acidic and neutral ranges, respectively. Exposing whole lung, lung minces, or mounted frozen sections of lung to X-Gal at mildly alkaline pH (pH 8.0-8.5), minimized detection of endogenous activity in lungs from a variety of species while preserving that resulting from bacterial enzyme activity in a transgenic mouse expressing lacZ. This technique was also useful in distinguishing endogenous activity from that resulting from adenovirus-mediated lacZ gene transfer to diploid lung fibroblasts in primary culture. An appropriate buffer that maintains the desired pH throughout the duration of X-Gal exposure must be used.     

Alcohol dehydrogenase (ADH) in yeast cells. I. Cytoplasmic, mitochondrial and nuclear ADH in Saccharomyces carlsbergensis and Kluyveromyces fragilis

When grown in a medium containing lactat, Saccharomyces carlsbergensis produces 5 times more ground-plasmatic ADH than Kluyveromyces fragilis. Upon gelectrophoresis, K. fragilis exhibits 7 bands while S. carlsbergensis shows only one. In a polyacrylamid gradient, the ADH's of both strains yield one band, the position of which corresponds to a molecular weight of about 160 000 D. In cell homogenates and mitochondrial fractions of S. carlsbergensis treated with ultrasound, an ADH is detected which exhibits 3 subbands and a molecular weight greater than 1 Megadalton. This ADH does not occur in K. fragilis. Mitochondrial fractions from K. fragilis contain an ADH, the electrophoretical mobility of which is identical to that of the ADH of the groundplasma. Nuclei of S. carlsbergensis also possess ADH whereas those of K. fragilis probably do not.     

Verapamil inhibition of enzymatic product efflux leads to improved detection of beta-galactosidase activity in lacZ-transfected cells

BACKGROUND: We studied the usefulness of nuclear DNA patterns and argyrophylic nucleolar organizer regions (AgNORs) for evaluating the malignant potential of colorectal cancers, which is increasingly being regarded as important in predicting patients' prognosis and for their appropriate postoperative management. METHODS: We measured these two factors in curatively resected specimens of 91 colorectal cancer cases, which were followed up for 1,549 +/- 788 days postoperatively. Ploidy pattern was either diploid or aneuploid, and AgNORs score was either low (LS) or high (HS). Thus, we classified our cases into Group I (diploid, LS). Group II (aneuploid, LS), Group III (diploid, HS), and Group IV (aneuploid, HS). Postoperative survival curves in the cases belonging to these groups were analyzed. RESULTS: Survival rates in Groups I and II were significantly higher than those in Group IV. Correlation between subgroups and clinicopathological factors such as average age, histologic type, depth of invasion, and histologic stage were observed. Incidence of lymph node metastasis at the time of operation and that of postoperative recurrence were higher in group IV than that in groups I and II. CONCLUSIONS: Measurement of DNA ploidy patterns and AgNORs score were found to be useful in evaluating malignant potential of colorectal cancers.     

Biodegradation of benzene, toluene, ethylbenzene, and o-xylene by a coculture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a fibrous-bed bioreactor

A fibrous-bed bioreactor containing the coculture of Pseudomonas putida and P. fluorescens immobilized in a fibrous matrix was developed to degrade benzene (B), toluene (T), ethylbenzene (E), and o-xylene (X) in synthetic waste streams. The kinetics of BTEX biodegradation by immobilized cells adapted in the fibrous-bed bioreactor and free cells grown in serum bottles were studied. In general, the BTEX biodegradation rate increased with increasing substrate concentration and then decreased after reaching a maximum, showing substrate-inhibition kinetics. However, for immobilized cells, the degradation rate was much higher than that of free cells. Compared to free cells, immobilized cells in the bioreactor tolerated higher concentrations (> 1000 mg l-1) of benzene and toluene, and gave at least 16-fold higher degradation rates for benzene, ethylbenzene, and o-xylene, and a 9-fold higher degradation rate for toluene. Complete and simultaneous degradation of BTEX mixture was achieved in the bioreactor under hypoxic conditions. Cells in the bioreactor were relatively insensitive to benzene toxicity; this insensitivity was attributed to adaptation of the cells in the bioreactor. Compared to the original seeding culture, the adapted cells from the fibrous-bed bioreactor had higher specific growth rate, benzene degradation rate, and cell yield when the benzene concentration was higher than 100 mg l-1. Cells in the fibrous bed had a long, slim morphology, which is different from the normal short-rod shape found for suspended cells in solution.     

Monocyte-derived dendritic cells: development of a cellular processor for clinical applications

Since dendritic cells (DCs) are the most professional antigen-presenting cells, (Schuler et al., 1997), increasing interest in their use in clinical approaches has been observed. (Nestle et al., 1998; Murphy G. et al., 1996). We have developed an ex vivo standardized process for the generation of dendritic-like cells (MAC-DCs) from human blood circulating monocytes. Human monocytes can differentiate into very different functional cells according to the conditions of culture, media and cytokines used. In the present study, we demonstrate that both pure monocytes and mononuclear cells differentiate into DCs when they are grown in defined medium AIM-V in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus IL13 and in approved biocompatible non-adherent bags. Quality and functional controls of the immature DCs obtained rely on bacterial sterility, viability, morphology and recovery. The MAC-DCs also present an immature DC phenotype with a low expression of CD14 and CD64, and high expression of MHC-I, MHC-II and CD40. They also express B7 costimulatory molecules (CD80, CD86), CD83, and CD1a molecules. They induce strong allogenic T-cell proliferation (mixed lymphocyte reaction as well as proliferation of autologous memory T lymphocytes when incubated in the presence of recall antigens (tuberculosis, Candida albicans, and tetanus toxoid). They also show an increase in phagocytic uptake of yeast, tumour cells and debris. The global closed system which, under reproducible good medical practice (GMP) conditions, enables the production of dendritic cells of clinical quality, has been optimized ("Vac Cell Processor"). It contains all bags, connections, media, reagents, washing solutions, control antibodies, standard operating procedures, data management, traceability and help in the form of dedicated software.     

Mitogenic activity of purified capsular polysaccharide A from Bacteroides fragilis: differential stimulatory effect on mouse and rat lymphocytes in vitro

Bacteroides fragilis, a Gram-negative colonic bacterium, induces the formation of abscesses associated with intra-abdominal sepsis in humans. The singular ability of this organism to modulate abscess formation in experimental rodent models resides in the structurally distinct and ionically charged capsular polysaccharides A (PS A) and B (PS B). The regulation of abscess formation in animals is dependent on T lymphocytes. However, the manner in which PS A interacts with T cells remains unknown. We therefore tested the T cell stimulatory capacity of purified PS A on mouse and rat lymphocytes in cellular proliferation assays and found that the PS A molecule possesses mitogenic characteristics distinguishable from those of the polyclonal B cell activator LPS, the T cell mitogen Con A, and staphylococcal enterotoxin A superantigen. Further, PS A stimulated proliferation of normal mouse and rat lymphocytes differentially. Mouse B cells responded to PS A in a fashion that did not require exogenous APC function, while rat T lymphocyte responses to PS A required APC function derived from autologous or xenogenic feeder cells. Cellular depletion experiments showed that the CD4+ subset of rat spleen cells was the primary responder cell type to PS A in vitro. The differential stimulatory effects of PS A on mouse and rat lymphocytes may reflect its ability to stimulate different lymphocyte subsets in vivo through the activities of receptor/counter-receptor pairs present on responder lymphocytes and cognate APC.     

Expression profile of senescence-associated beta-galactosidase and activation of telomerase in human ovarian surface epithelial cells undergoing immortalization

Senescence is a specific physiological stage of cells characterized by long population doubling time. It accounts for the inability of normal somatic cells to undergo indefinite cell division. As the number of population doublings increase, cell cycle regulatory mechanisms come into play and signal cells to exit the cell cycle and become senescent. Senescence has been implicated in the aging process and may function as a tumor suppressor mechanism in human cells. The ability to measure the degree of cellular senescence is important in understanding the biological processes regulating cell aging and immortalization. Senescent cells exhibit an enzyme termed senescence-associated histochemical staining. Cells immortalized by viral oncogenes often enter a stage of crisis at the early phase of immortalization. The cells at crisis have a long population doubling time. Cells at the crisis stage resemble senescent cells and the expression of SA- beta-Gal may be used to monitor the process of immortalization. In this study the expression profile of SA-beta-Gal was examined in human ovarian surface epithelial cells (HOSE 6-3) undergoing immortalization by the human papilloma viral oncogene E6 and E7 (HPV E6 and E7). Our results showed a low percentage (12.0%) of HOSE 6-3 cells expressing SA-beta-Gal activity at the pre-crisis stage. The percentage of HOSE 6-3 cells expressing SA-beta-Gal activity was highest (39.2%) at the crisis stage. When HOSE 6-3 cells achieved immortalized status there was a sharp decrease in cells (1. 3%) expressing SA-beta-Gal activity. In addition, an inverse relationship between the expression of SA-beta-Gal activity and telomerase activity was noted in cells undergoing immortalization. The results confirm that the SA-beta-Gal enzyme is a good marker for monitoring the population of cells undergoing senescence at different stages of immortalization and that telomerase activation is a characteristic feature of post-crisis cells.     

Expression and purification of a truncated macrophage colony stimulating factor in Kluyveromyces lactis

A truncated human macrophage colony stimulating factor (M-CSF) cDNA encoding amino acid residues from 3 to 149 of the native M-CSF was obtained by using polymerase chain reaction. When inserted into plasmid pCXJ1 and psPHO5 and introduced into Kluyveromyces lactis, it directs the the secretory expression of the biologically active dimeric form of M-CSF. Through a four-step purification protocol, i.e. ammonium sulfate salting out, DEAE-cellulose column chromatography, hydrophobic chromatography on phenyl-sepharose and Mono Q fast protein liquid chromatography, the recombinant truncated M-CSF was purified to homogenerity and show its apparent molecular mass at 21KDa on reduced SDS-PAGE, with a specific activity of 1.21 x 10(7) units/mg protein.     

Peripheral blood stem cells in acute myeloid leukemia: biology and clinical applications

Fifteen persons with profound mental retardation were divided into two groups. One group was identified with chronic training needs by habilitative staff and the other group served as a control. In an attempt to identify a reinforcer, each participant received a preference assessment and a simple, low-effort treatment procedure. In Experiment 1, only individuals who approached at least one stimulus on 80% or more of the preference assessment trials ("high preference") showed reinforcement effects in treatment. However, three individuals showing high preference failed to show treatment effects. All persons identified with chronic training needs failed to show reinforcement effects. Experiment 2 analyzed characteristics of the two groups and found significant differences in overall movement and response latency. Limitations of the current reinforcement technology were apparent for identifying reinforcers in the group with chronic training problems. Research is suggested for evaluating training alternatives for people with profound multiple disabilities who move very little or who respond with very long latencies.     

Phospholipase D activity in L1210 cells: a model for oleate-activated phospholipase D in intact mammalian cells

The effect of 2-aminobicyclo[2.2.1]heptan-2-carboxylic acid (BCH), an L-leucine nonmetabolizable analogue and an allosteric activator of glutamate dehydrogenase, on glucose and glutamine synthesis was studied in rabbit renal tubules incubated with alanine, aspartate or proline in the presence of glycerol and octanoate, i.e. under conditions of efficient glucose formation. With alanine+glycerol+octanoate the addition of BCH resulted in a stimulation of alanine and glycerol consumption, accompanied by an increased glucose, lactate and glutamine synthesis. In contrast, when alanine was substituted by either aspartate or proline, BCH altered neither glucose formation nor glutamine and glutamate synthesis, while an accelerated glycerol utilization was accompanied by a small increase in lactate production. In view of the BCH-induced changes in intracellular metabolite levels the acceleration of gluconeogenesis by BCH in the presence of alanine+glycerol+octanoate is probably due to (i) increased uptake of alanine via alanine aminotransferase, (ii) stimulation of phosphoenolpyruvate carboxykinase, a key-enzyme of gluconeogenesis, (iii) rise of glucose-6-phosphatase activity, as well as (iv) activation of the malate-aspartate shuttle resulting in an augmented glycerol utilization for lactate and glucose synthesis.     

Proteolytic release of membrane proteins: studies on a membrane-protein-solubilizing activity in CHO cells

Diverse membrane proteins are solubilized by a specific proteolytic cleavage in the stalk sequence adjacent to the membrane anchor, with release of the extracellular domain. Examples are the amyloid precursor protein, membrane-bound growth factors and angiotensin-converting enzyme (ACE). The identities and characteristics of the responsible proteases remain elusive. We have studied this process in Chinese hamster ovary (CHO) cells stably expressing wild-type ACE (WT-ACE) or juxtamembrane (stalk) deletion or chimaera mutants. Determination of the C termini (i.e. the cleavage sites) of released, soluble wild-type and mutant ACE by MALDI-TOF mass spectrometry indicated that the membrane-protein-solubilizing protease (MPSP) in CHO cells is not constrained by a particular cleavage site motif or by a specific distance from the membrane, but instead may position itself with respect to the putative proximal, folded extracellular domain adjacent to the stalk. Nevertheless, kinetic analyses of release rates indicated that a minimum distance from the membrane must be preserved. Interestingly, soluble full-length (anchor-plus) WT-ACE incubated with fractions of, or intact, CHO cells was not cleaved. In all cases, release was stimulated by a media change or by the addition of phorbol ester, with rate enhancements of 5- and 50-fold, respectively, for WT-ACE. The phorbol ester effect was abolished by staurosporine, a protein kinase C (PKC) inhibitor. We propose that the CHO cell MPSP that solubilizes ACE: (1) only cleaves proteins embedded in a membrane; (2) requires an accessible stalk and cleaves at a minimum distance from both the membrane and proximal extracellular domain; (3) positions itself primarily with respect to the proximal extracellular domain and (4) is regulated in part by a PKC-dependent mechanism.     

Application of yeasts in gene expression studies: a comparison of Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactis -- a review

The performance of a supervised k-nearest neighbor (kNN) classifier and a semisupervised fuzzy c-means (SFCM) clustering segmentation method are evaluated for reproducible measurement of the volumes of normal brain tissues and cerebrospinal fluid. The stability of the two segmentation methods is evaluated for (a) operator selection of training data, (b) reproducibility during repeat imaging sessions to determine any variations in the sensor performance over time, (c) variations in the measured volumes between different subjects, and (d) variability with different imaging parameters. The variations were found to be dependent on the type of measured tissue and the operator performing the segmentations. The variability during repeat imaging sessions for the SFCM method was < 3%. The absolute volumes of the brain matter and cerebrospinal fluid between subjects varied quite large, ranging from 9% to 13%. The intraobserver and interobserver reproducibility for SFCM were < 4% for the soft tissues and 6% for cerebrospinal fluid. The corresponding results for the kNN segmentation method were higher compared to the SFCM method.     

Comparison of the in vitro bactericidal activity of human serum and leukocytes against bacteroides fragilis and Fusobacterium mortiferum in aerobic and anaerobic environments

In aerobic and anaerobic in vitro environments, Fusobacterium mortiferum was killed by human leukocytes or serum alone or in combination; clinical isolates of Bacteroides fragilis were killed only by leukocytes in the presence of serum.     

Gene transfer: a review of methods and applications

Gene transfer is a potentially powerful tool for the treatment of a wide variety of diseases. The transfer of these genes is achieved by utilizing a variety of vectors, including retroviral, adenoviral, adeno-associated virus (AAV) and a number of non-viral mechanisms. Numerous studies have successfully demonstrated transduction of genes into target cells with a variety of vectors, and have provided 'proof-in-principle' that gene transfer can result in prolonged in vivo expression of transduced genes, albeit at low quantities. Furthermore, gene marking studies in acute myeloblastic leukemia (AML), chronic myeloid leukemia (CML) and neuroblastoma have elegantly demonstrated that gene-marked tumor cells contribute to relapse following autologous transplantation. However none of the studies examining the therapeutic benefit of gene therapy has definitively demonstrated a clinically meaningful benefit. Nonetheless, the results of studies involving gene transfer for severe combined immunodeficiency (SCID), chronic granulomatous disease (CGD), melanoma and lung cancer highlight the potential benefit of this strategy. This review will discuss mechanisms of achieving gene transfer into target cells. It will examine some of the pre-clinical and clinical results to date and will discuss some of the potential uses of gene transfer for therapeutic purposes.     

Transient steel quality under non-isothermal conditions in a multi-strand billet caster tundish: part II. Effect of a flow-control device

A mathematical model was built to simultaneously analyse the effects of non-isothermal conditions and flow-control device on steel quality in a real steelmaking tundish. Liquid steel was used as the operating liquid with a step-input of 23° in a full-scale delta-shaped multi-strand billet caster tundish fitted with a standard impact pad (SIP). The changes in flow pattern and temperature fields of liquid steel in the tundish under isothermal, step-up and step-down conditions were thoroughly studied. Similar to the case of a bare tundish, buoyancy effects were seen to dominate at regions away from the ladle shroud. The presence of SIP modified the flow patterns and resulted into markedly different values of RRI as compared to bare tundish. Finally, the calculated results were qualitatively compared to results obtained from a real steelmaking tundish.     

High sensitivity detection of HPV-16 in SiHa and CaSki cells utilizing FISH enhanced by TSA

Detection of integrated human papillomavirus type 16 (HPV-16) DNA in SiHa and CaSki cells was used as a model system to demonstrate sensitivity and resolution of a well defined target. Using 293- to 1987-base polymerase chain reaction (PCR)-synthesized probes to the E6 and E7 open reading frames of HPV-16, several fluorescent in situ hybridization (FISH) detection methods, enhanced with tyramide signal amplification (TSA), were compared. The synthetic probes were biotin labeled by a nick translation method and the hybridized probes were detected by various fluorescent TSA methods using cyanine 3 tyramide, biotinyl tyramide and a biotin TSA Plus reagent. High sensitivity detection in SiHa cells was demonstrated using a 619-base probe to detect two single copies of integrated HPV-16 DNA. In CaSki cells, which contain up to 600 copies of HPV-16 DNA, a 293-base probe was used for detection. The results of these comparisons show that with refinement of TSA methods and reagents, increasing levels of high sensitivity detection can be achieved and that these methods allow subnuclear localization as well.