A micro-Raman spectroscopy approach was used for the direct in situ characterization of lipid bodies in the water-conducting branch xylem of an African resurrection plant and three deciduous European tree species. Because of average diameters of at least 1 microm, the lipid bodies of all investigated species proved to be easily accessible by this technique. All vesicle-forming xylem lipids were identified as fatty acid esters, which may correspond to phospholipids. Whereas in the resurrection plant saturated lipids were dominant, the lipid bodies of the European trees consisted of highly unsaturated fatty acids. A comparison of the spectra of lipid droplets of lime obtained in situ and from isolated xylem sap revealed slightly different signatures. This finding suggests that micro-Raman spectroscopy may be used to detect modifications of the chemical composition of biological substances as a result of the extraction mode.

Biophysical Phenomena↗

PubMedDate not supplied

Arginine vasopressin and adrenocorticotropin secretion in response to psychosocial stress is attenuated by ethanol in sons of alcohol-dependent fathers.

Familial risk and environmental stress promote the development of alcohol dependence. We investigated whether a positive family history of alcoholism affects the neuroendocrine response to a standardized laboratory stress test in healthy subjects without alcohol use disorders. Twenty-four high-risk subjects with a paternal history of alcoholism (PHA) and 16 family history negative (FHN) controls were evaluated. Psychosocial stress was induced by having subjects deliver a 5-min speech and mental arithmetics in front of an audience on separate days, after drinking either placebo or ethanol (0.6 g/kg) in a randomized sequence. Adrenocorticotropin (ACTH) was measured in 10 plasma samples covering up to 75 min after the stress test. Plasma arginine vasopressin (AVP) was determined before the stressor, at the time of maximum ACTH secretion, and at 75 min after stress onset. The stress test induced a phasic increase in ACTH secretion. At the time of maximum ACTH, AVP was significantly increased in relation to baseline. Compared to placebo, alcohol administration significantly attenuated maximum ACTH concentration in PHA but not FHN subjects, and decreased AVP measured in the same samples in PHA but not FHN subjects. We conclude that activation of the hypothalamic-pituitary-adrenal system by psychosocial stress is accompanied by an increase in peripheral plasma AVP levels. Secretion of both ACTH and AVP suggest that alcohol attenuates the stress response selectively in PHA but not FHN subjects. This might imply some short-term positive alcohol effect in sons of alcoholics, but also constitute a mechanism by which their risk to develop alcohol use disorders is increased.

Adolescent↗

PubMedDate not supplied

Beneficial effects of human serum albumin on stability and functionality of alginate microcapsules fabricated in different ways.

A key engineering challenge in designing microcapsules made from biocompatible alginate is maintaining adequate exchange of nutrients and oxygen between the entrapped cells and the environment, while simultaneously avoiding swelling and subsequent failure of the microcapsule. Approval for the use of alginate in pharmaceutical and/or biomedical applications also strictly requires that the components of the microcapsule material must meet the safety criteria of the ASTM and FDA. Incorporation of foetal calf serum (FCS) into the microcapsules for stabilization is not in accordance with the guidelines affirmed by these organizations. FCS should be substituted by microcapsule-stabilizing additives that are medically approved. In this communication, it is shown that 10% FCS can be replaced by 1% human serum albumin (i.e. by an agent for which medical approval is granted) without compromising effects on long-term in vitro stability. Furthermore, it is demonstrated that human serum albumin (HSA) significantly enhances cell survival and, particularly, insulin secretion of encapsulated rat islets over a time period of 3 weeks when incubated in culture medium. Thus, HSA-stabilized microcapsules made from UHV(Lam) alginate are apparently a promising system for immunoisolation of cells, particularly when alginate is cross-linked by injection of BaCl(2) crystals into the alginate droplets. Slight adjustments of the alginate concentration can tailor the microcapsule permeability to the released therapeutic factor.

Alginates↗

PubMedDate not supplied

Fractions from commercial collagenase preparations: use in enzymic isolation of the islets of Langerhans from porcine pancreas.

Transplantation of isolated islets of Langerhans is an intriguing possibility for the treatment of diabetes mellitus. The isolation of islets from pancreata requires the specific dissociation of the tissue. Commercial collagenases from Clostridium histolyticum are widely used for this purpose. Unfortunately, the effectiveness of these commercial enzymes is not predictable and differs considerably between suppliers and even from lot to lot. This is due mainly to differences in their specific collagenase activity and to the presence of other lytic enzymes, as well as to other contaminants. Free flow zone electrophoresis (FFZE) was used to separate the effective protein components from undesired compounds and to prepare a digestive enzyme mixture with controlled composition of lytic activities. Fractionation of crude collagenases by FFZE resulted in partially purified protein fractions that were enriched for collagenase and tryptic activities, and contained only trace amounts of neutral protease. These preparations proved to be highly effective in an in vitro assay for the libration of viable islets from porcine pancreas. To scale up the production of these collagenases with defined enzyme composition, we fractionated two different lots of a commercial collagenase from C. histolyticum (one lot effective in islet isolation, the other not) by using fast protein liquid chromatography (FPLC) on hydroxyapatite. Again, high efficacy of islet release from pancreatic tissue was correlated to high specific tryptic and collagenase activities and low levels of neutral protease. The chromatographic protocol developed in this study converted a non-effective collagenase lot into a preparation that allowed successful islet isolation.

Animals↗

PubMedDate not supplied

Preparation of human erythrocyte ghosts in isotonic solution: haemoglobin content and polypeptide composition.

Human red blood cell ghosts were prepared by dielectric breakdown of the cell membrane and subsequent electrical haemolysis in isotonic and isoionic solutions. Almost complete removal of haemoglobin (to about 0.1%) could be achieved by subjecting the erythrocytes suspended in isotonic solution to three consecutive electric field pulses of 16 kV/cm in the presence of 10 mM EDTA, followed by dilution with EDTA-free isoionic medium. The cup-shaped ghost cells were electrically homogeneous. The polypeptide composition of the electrically prepared ghost cells, as analyzed by polyacrylamide electrophoresis in 1% SDS, revealed a pattern similar to ghost cells prepared by osmotic haemolysis. However, the band 6, corresponding to glyceraldehyde-3-phosphate-dehydrogenase protomer, was absent from the electrically prepared ghost cells. Also the band 8, which is eluted at low ionic strength, appeared to be very prominent in the electrical preparations.

Cell Fractionation↗

PubMedDate not supplied

Membrane fusion and deformation of red blood cells by electric fields.

Human red blood cells suspended in a slightly hypotonic solution of low electric conductivity were exposed to an inhomogeneous and alternating electric field (sine wave, 30 V peak-to-peak value, electrode distance 120 microns, 0.5 to MHz). Due to the dielectrophoretic effect the cells align parallel to the field lines under the formation of pearl chains. At high voltages (10 V amplitude) membrane fusion is observed between the adhered red blood cells in the pearl chains, whereby the chains become attached to the electrodes. In contrast to the pearl chains observed at voltages of up to 5 V amplitude the resulting fused and uniform aggregates which exhibit no recognisable individual cells under the light microscope, remain stable, even after the alternating electric field has been switched off or after haemolysis in response to osmotic shock. The fused aggregates are highly elastic. If the field strength of the applied alternating electric field is further increased they are stretched in the direction of the opposite electrode. Frequently, bridges are formed between the two electrodes. The uniform bridges remain stable for some time even in the absence of an electric field. The possibility of cell fusion and its initiation by electrical breakdown of the cell membranes are discussed.

Cell Aggregation↗

PubMedDate not supplied

Immobilization of human red blood cells.

Human red blood cells were immobilized in an alginate network which was cross-linked with Ca2+ ions. The immobilized cells were stored for longer than 5 weeks at 4 degrees C in an isotonic buffered NaCl solution containing glucose, inosine, adenine, and guanosine for energy supply. The immobilized cells were released from the alginate network by dissolving the matrix with citrate. Both the immobilized and released cells retain their biconcave shape over the storage period. Measurements of the released cell population in a hydrodynamically focussing Coulter Counter demonstrated that the mean size of the size distribution, the breakdown voltage, and the internal conductivity have not changed in contrast to control measurements on red blood cells stored conventionally in suspension indicating that immobilization preserves cellular functions.

Alginates↗

PubMedDate not supplied

Rotation of cells in an alternating electric field: the occurrence of a resonance frequency.

Cells suspended in a low-conducting medium were exposed to an alternating electric field whose frequency was altered between 1 kHz and 2 MHz. A resonance frequency was observed at which all suspended cells rotated about an axis normal to the field lines (when the electric field strength was larger than a threshold value of about 400 V/cm). This resonance frequency varied from species to species of cells (mesophyll protoplasts of Avena sativa = 20-40 kHz, human erythrocytes and ghost cells = 80-100 kHz, yeast cells = 140-180 kHz, Friend cells = 30-40 kHz, at room temperature). The resonance frequency of cell rotation was observed only under specific experimental conditions which excluded interference by reversible electrical breakdown of cell membranes and by gravitational forces. Glutardialdehyde fixed and heated cells exhibited no rotation in the frequency and field range investigated. The phenomenon of rotation is discussed in terms of dipole orientation within the membrane.

Animals↗