Chemical, physical, and sensory properties of dairy products enriched with conjugated linoleic acid.

Recent studies have illustrated the effects of cis-9,trans-11 conjugated linoleic acid (CLA) on human health. Ruminant-derived meat, milk and dairy products are the predominant sources of cis-9,trans-11 CLA in the human diet. This study evaluated the processing properties, texture, storage characteristics, and organoleptic properties of UHT milk, Caerphilly cheese, and butter produced from a milk enriched to a level of cis-9,trans-11 CLA that has been shown to have biological effects in humans. Forty-nine early-lactation Holstein-British Friesian cows were fed total mixed rations containing 0 (control) or 45 g/kg (on dry matter basis) of a mixture (1:2 wt/wt) of fish oil and sunflower oil during two consecutive 7-d periods to produce a control and CLA-enhanced milk, respectively. Milk produced from cows fed the control and fish and sunflower oil diets contained 0.54 and 4.68 g of total CLA/100 g of fatty acids, respectively. Enrichment of CLA in raw milk from the fish and sunflower oil diet was also accompanied by substantial increases in trans C18:1 levels, lowered C18:0, cis-C18:1, and total saturated fatty acid concentrations, and small increases in n-3 polyunsaturated fatty acid content. The CLA-enriched milk was used for the manufacture of UHT milk, butter, and cheese. Both the CLA-enhanced butter and cheese were less firm than control products. Although the sensory profiles of the CLA-enriched milk, butter, and cheese differed from those of the control products with respect to some attributes, the overall impression and flavor did not differ. In conclusion, it is feasible to produce CLA-enriched dairy products with acceptable storage and sensory characteristics.

Microspectrofluorometry and fluorescence imaging in the study of human cytopathology.

The study of energy pools and dynamics of specific pathways in living cells by microspectrofluorometry and fluorescence imaging produces spectral and topographic images characterizing structural and functional changes associated with cytopathology. Microspectro-fluorometry and fluorescence imaging have been applied, together with organelle morphometry to a number of cells mimicking certain cytopathologies, including melanoma cells, long-term malignant cells, and gene-defective cells. These investigations of cellular pathology indicate that there is a convergence of various physiopathological processes. Cellular states that have similarities include senescence, detoxification, and transformation. While the NAD(P)H metabolic transients have been studied before, our emphasis in this article is on very rapidly scanned fluorescence images related to organelle integration and photoinduced cellular senescence.

Multiprobe fluorescence imaging and microspectrofluorimetry of cell transformation and differentiation: implications in terms of applied biochemistry and biotechnology.

The dichotomy of cellular transformation versus differentiation does not preclude the hypothesis of a unified underlying mechanism that can switch either way as a result of growth factors, cell-membrane receptors, secondary messengers, integrating switch kinases and/or nuclear receptors. Its study for biopharmaceutical and biotechnological applications requires a methodology capable of dealing with such pleiotropy. In the multiprobe-multiparameter approach, one must remain wary of cumulative toxic effects and misinterpretations. 'Smart' instrumentation does not mean 'smart' probes. It turns out that the cell's own endogenous probes, the fluorescent coenzymes, may be akin to 'smart' probes, open to study in situ of many-fold interrelated pathways in cell energetics and dynamics. Resolution at the micro- and even nano-compartment levels is not altogether impossible. Thus an innovative search in terms of what may be called 'intracellular reconnaissance with fluorescent probes and biopharmaceuticals' necessitates recourse to multiple tentative probings along the pleiotropic mechanisms as far in resolution as one can go. Among the characteristic findings using this approach are: (i) morphometric alterations in the mitochondria and melanosomes of melanoma cells treated with azelaic acid; (ii) deregulation of mitochondrial control and extramitochondrial metabolism in similarly treated cells; (iii) considerable acceleration of NAD(P) transient kinetics in atractylate-treated L sarcoma cells; (iv) alterations of mitochondria and Golgi in fusion-deficient myoblasts; (v) tentative recognition of beta-glucosidase deficiency in Gaucher disease cells by the use of fluorescent and fluorogenic lysosomal probes; and (vi) UVA-induced accumulation of Schiff bases (a kind of accelerated photo-aging) in yeast and kidney epithelial cells. Because these studies utilize probing at whatever points along the concerned pathways become accessible, at first glance they may look disconnected. What and where is the connecting thread, for instance, between studying melanoma metabolism, melanosome morphometry, hepatocyte organelle morphogenesis and transformation, myotube organelle morphogenesis and fusion-non-fusion, and lysosomal activity in gene-deficient cells? In the mapping of the regulatory and deregulatory mechanisms involved in the switching of differentiation or transformation, each of the above topics carries an information content towards resolution of the pleiotropic puzzle. The integration of such information with increasing resolution and access to intracellular microdomains may ultimately allow focus on the precise target, the switch from differentiation to transformation or vice versa.

Imaging of cells by autofluorescence: a new tool in the probing of biopharmaceutical effects at the intracellular level.

The success of biopharmaceuticals relies on the ability to have reliable probes to interpret their mechanisms of action in situ at the intracellular level in terms of cell organelles and microcompartments. One of the most effective probes is the endogenous coenzyme NAD(P)H and its fluorescence transients obtained by the microinjection or perfusion of metabolic intermediates and modifiers, in the presence of drugs and inhibitors. The approach in fluorescence microtopography and microspectrofluorimetry is based on the premise that natural cell fluorescence (autofluorescence) holds a decisively greater potential in unravelling intracellular physiopathological processes than extrinsic fluorescence or artificial pseudocolouring. The mounting as a detector of a cooled charge-coupled device camera or alternatively of a non-cooled camera in conjunction with an image intensifier or an investigator (i.e. frame scan accumulator) to enhance sensitivity makes possible the detection of the low-quantum-yield NAD(P)H fluorescence at a level comparable to images previously obtained with high-quantum-yield fluorochromes. The modulation of mitochondrial autofluorescence by rotenone, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and oligomycin, and of cytoplasmic and nuclear autofluorescence by glucose and iodacetamide in CV-1 kidney epithelial cells, Ehrlich-Lettre hypotetraploid CCL77 cells and Saccharomyces cerevisiae, provides examples of the usefulness of fluorescence imaging in the study of biopharmaceuticals. The method goes beyond NAD(P)H to the multiplicity of extrinsic and intrinsic probes already available or in development.

An in situ study of beta-glucosidase activity in normal and Gaucher fibroblasts with fluorogenic probes.

Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post-cleavage increase of fluorescence with fluoresceinyl (bis) beta-glucopyranoside was appreciably above the rather large pre-cleavage emission. In cells incubated overnight with nonylumbelliferyl-beta-glucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta-glucosidase deficiency, is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.

Direct connection between myelinosomes, endoplasmic reticulum and nuclear envelope in mouse hepatocytes grown with the amphiphilic drug, quinacrine.

Mouse hepatocytes grown in 4 microM quinacrine had numerous myelinosomes which were directly connected to expanded cisternae of the rough endoplasmic reticulum (RER). The cisternae of the RER either subtended the electron transparent space of the myelinosome, expanded to form the outer membrane of the myelinosome or penetrated into it. Material of low electron density was frequently seen within the area where the cisternae penetrated into the electron transparent space of the myelinosome. Myelinosomes were also associated with the nuclear envelope in a pattern similar to that of the RER. Quinacrine appears to bind with the phospholipids of the membranes of the endoplasmic reticulum and nuclear envelope and this drug-lipid complex is then moved into myelinosomes effectively removing the drug from the cell.

Bioregulatory mechanisms at the level of cell organelle interactions: microspectrofluorometric in situ studies.

The spatiotemporal analysis of bioregulatory mechanisms at the level of intracellular multienzyme complexes and organelle interactions is made possible by the availability of endogenous and exogenous fluorescence probes, the development of microspectrofluorometers allowing one- and two-dimensional scans of intracellular fluorescence reactions, and the use of micromanipulatory techniques enabling the rapid alteration of metabolic states. Absorbed photons are not only a tool for quantitative evaluation of metabolic processes, they can also trigger alterations of cell membranes and functions as mediated by photosensitizer drugs. In the hierarchy of intracellular organization different levels of complexity are accessible to study, such as the regulation of multienzyme complexes and the interaction of organelle complexes. Typical applications of these methods are the investigation of drug effects (e.g., on melanoma cells), metabolic and structural alterations (e.g., in cystic fibrosis and Gaucher fibroblasts), organelle interactions in cells treated with toxic agents. The implications are relevant to biotechnology for better control of metabolite production and processing, design and testing of new drugs, understanding of drug resistance and better targeting of drugs or probes to selected intracellular sites. In addition, such in vitro methods can contribute to the provision of an alternative to "whole animal experiments" as already achieved in human and mouse fibroblasts, hepatocytes, hepatoma, Swiss 3T3 cells and other cells in culture, especially with regards to an analysis of the action of xenobiotics and drugs in cell physiology and pathology, fluorescence recovery after photobleaching, study of cytoskeleton dynamics and multiparameter probing of organelle activity during in vitro wound repair.

A microspectrofluorometric study of the effect of anthralin, an antipsoriatic drug, on cellular structures and metabolism.

The microspectrofluorometric approach has been used to investigate in single living cells in culture fundamental questions raised by the use of anthralin, a potent antipsoriatic drug. This method allows fluorescence determinations on the intracellular fate of the drug as well as the recognition of structural and metabolic alterations induced by the drug. In the absence of demonstrable adduct formation with DNA, the antipsoriatic, i.e. antiproliferative effect of anthralin, has been attributed to its action at the level of mitochondria or at the level of glucose-6-phosphate dehydrogenase which initiates the pentose phosphate shunt (cf. its prominent role in nucleic acid synthesis). Upon addition of 2.3 to 23 microM anthralin to the L cell culture, the characteristic structure of the anthralin anion fluorescence spectrum is recognized almost immediately in the cytoplasm (much weaker in the nucleus) but disappears within minutes. The vital mitochondrial fluorescence probe dimethylaminostyryl-pyridinium-methyl-iodine reveals striking structural alterations of the mitochondria within 15 min after addition of the drug. At the same time, there is a stimulation of the transient NAD(P)+ reduction observed upon microinjection into the L cell of the Krebs' cycle substrate malate, or the pentose cycle substrate 6-phosphogluconate. Specially, the injection of the latter to anthralin-treated cells suggests that upon release of the mitochondrial control, there is a tremendous disruption of metabolic activity which could have profound consequences on the proliferative activity of the cell. These findings, while they open new possibilities for the intracellular evaluation of therapeutic agents, create also a challenge in understanding the complex and dynamic interrelationships between intracellular organelles and bioenergetic or biosynthetic pathways.

Microspectrofluorometry of fluorescent photoproducts in photosensitized cells. Relationship to cellular quiescence and senescence in culture.

Microspectrofluorometry of L and WI-38 cells reveals chemical/structural changes due to quiescence or senescence, i.e., lipid peroxidation, spontaneous or photosensitized by hematoporphyrin. Cells treated with hematoporphyrin and a lysosomal umbelliferone probe show a fast-rising umbelliferone emission, plus a fluorescent photoproduct. Studies in rapidly growing versus quiescent L, early passage/late passage WI-38 cells, suggest accumulation of fluorescence Schiff bases (i.e., their association with granular regions of cells in stationary phase, spectral properties, fast increase in photosensitized cells) and a possible lysosomal membrane permeabilization in quiescent or senescent cells.

The differential effects of dimethylnitrosamine and ethionine on mitochondrial and extramitochondrial dehydrogenases in single intact cells.

In studying the bioenergetics of living cells, the microfluorometric analysis of coenzyme (NAD(P)H) responses to microinjected respiratory and glycolytic substrates enables, in principle, a search for qualitative/quantitative differences in normal versus carcinogen-treated (short-term, long-term) and malignant cells. Responses are compared in L-cells, same adapted to hypertonic media (i.e. L255, L355) and highly malignant rhabdomyosarcoma (CCL 136) cells. The largest responses to respiratory substrate (malate, isocitrate) and the lowest responses to glycolytic substrate (glucose-6-P) are in the L255, 355 cells which exhibit structural rearrangement and dense packing of mitochondria possibly due to high energy requirement for ion pumping. The converse is observed in the CCl 136 where there is no lack of these organelles, but they could be functionally deficient, as suggested by a predominant response to glucose-6-P compared to malate. In the control L-cell, the malate and glucose-6-P responses are relatively well balanced. Upon addition of dimethylnitrosamine to L-cells, there is an initial acceleration in the rate of glucose-6-P-induced NAD(P) reduction (? NADPH requirement for dimethylnitrosamine metabolization), followed by an upsurge of the malate response. In L355 cells, addition of the carcinogens dimethylnitrosamine or ethionine is followed by a strong reductive response to malate, and minimal response to glucose-6-P. The dramatic intensification of the NAD(P)H response to malate in L355 cells pretreated with an ATP trap (ethionine) or an uncoupler (dinitrophenol) strongly points to a requirement for ATP depletion. Weaker enhancement of NAD(P)H response (preferentially after glucose-6-P) is observed in the CCL 136 upon treatment with ethionine. The findings indicate the need for further study on differences in respiratory/glycolytic pathways and efficiency of ATP cycle in malignant cells exhibiting graded differences of structural/functional specialization.

Metabolic control and compartmentation in single living cells.

Microspectrofluorometry of cell coenzymes (NAD(P)H, flavins) in conjunction with sequential microinjections into the same cell of metabolites and modifiers, reveals aspects of the regulatory mechanisms of transient redox changes of mitochondrial and extramitochondrial nicotinamide adenine dinucleotides. The injection of ADP in the course of an NAD(P)H transient produced by glycolytic (e.g. glucose 6-phosphate, G6P) or mitochondrial (e.g. malate) substrate leads to sharp reoxidation (state III, Chance and Williams, 1955), followed by a spontaneous state III to IV transition, and an ultimate return to original redox steady state. The response to ADP alone is biphasic, i.e. a small oxidation-reduction transient followed by a larger reverse transient. Similarities between responses to injected ATP and ADP suggest possible intracellular interconversions. Sequential injections of glycolytic and Krebs cycle substrates into the same cell, produce a two-step NAD(P) response, possibly revealing the intracellular compartmentation of this coenzyme. A two-step NAD(P)H response to sequentially injected fructose 1,6-diphosphate and G6P indicates the dynamic or even structural compartmentation of glycolytic phosphate esters in separate intracellular pools. The intracellular regulation and compartmentation of bioenergetic pathways and cell-to-cell metabolic inhomogeneities provide the basis on which the quantitative biochemistry of the intact living cell may be reconciled with these in situ findings.

Microspectrofluorometry of carcinogens in living cells.

A microspectrofluorometric approach has been used to follow the changes undergone by the carcinogen benzo(a)pyrene in malignant L cells, inducible Buffalo rat liver (BRL) cells and oncogenic mouse embryo C3H/10 T 1/2, clone 8 (CCL 226) cells. Since it is known that benzo(a)pyrene (BP) is converted metabolically to at least 40 metabolites, including phenols, epoxides, quinones, dihydrodiols, diol epoxides, and water-soluble conjugates, the interpretation of blue- and red-spectral shifts in fluorescence emission observed in BP-treated cells, compared to the original BP emission, undoubtedly presents considerable difficulties, but a certain number of facts clearly emerge. The sequence of blue-red shifts expressive of intracellular interactions and detoxification of the carcinogen is accelerated in the induced BRL compared to non-induced, and it is also generally accelerated in the malignant and inducible lines compared to the oncogenic line. The detection of highly reactive molecules (? of ultimate carcinogens) representing a small fraction of bulk fluorescence, still remains elusive, but two promising approaches are described: the use of phase-specific fluorescence quenchers which enable us to probe for the presence of metabolites in aqueous, hydrophobic or membrane phases of the cell, and the matrix analysis based on plotting of excitation-emission at different wavelengths for resolution of complex spectra. The former approach has enabled some separation or enhancement of red-blue emissions, and the second has helped to differentiate between emission of BP per se and its intracellular conversion products. Finally, observations at nuclear and cytoplasmic sites open the possibility of studying carcinogen interactions at different target sites.

Cell-to-cell communication in rat pancreatic islet monolayer cultures is modulated by agents affecting islet-cell secretory activity.

Single islet cells in monolayer cultures of neonatal rat pancreas were microinjected with the fluorescent dye Lucifer Yellow CH and the cultures were observed by combined phase contrast and fluorescent microscopy. The dye spread from an injected cell directly into neighboring islet cells, and successive microinjections of dye into different cells defined territories comprised of 2-6 communicating cells. The number of communicating cells could be modulated by addition to the cultures of different agents known to affect islet cell secretory activity. Cell communication was significantly increased by a high (16.7 mM) glucose concentration, by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 0.1 mM), and by the calcium ionophore, A23187. The effect of A23187 was transient and dose-dependent. Somatostatin (1 microgram/ml) significantly inhibited cell communication. These results demonstrate that cell-to-cell communication may participate in the regulation of islet cell secretory activity.

Penetration and localization of furocoumarins in single living cells studied by microspectrofluorometry.

The microspectrofluorometric technique has been used to study the penetration and the localization of psoralen, 4,5',8-trimethylpsoralen and 4'-aminomethyltrioxsalen in single living L-cells. The concentration of the different compounds inside the cell reached a plateau in 2 min with psoralen and aminomethyltrioxsalen and in 20 min with trioxsalen. Washing of the cells with culture medium produced only a partial removal of the three furocoumarins, distributed apparently in equivalent amount in the nucleus and cytoplasm.