Effects of ionic strength, serum protein and surface charge of membrane movements and vesicle production in heated erythrocytes.

Morphological changes and fragmentation of human erythrocytes heated at various rates through the spectrin inactivation temperature have been examined by cinephotomicroscopy. Most cells heated in 0.20 ionic strength buffered saline developed a wavy disturbance along the cell rim when heated. Vesicles developed from the crests of the growing waves within 0.3 s of the initiation of a wave when the heating rate was 1 degree C/s. At an ionic strength of 0.02, only 48% of the cells developed a wave outline. The average number of waves per cell was half that at 0.2 ionic strength. When the cell surface charge was reduced by neuraminidase treatment, only 12% of the cells fragmented. Bovine serum albumin or homologous plasma also reduced fragmentation. The dependence of the wave growth on ionic strength and surface charge was broadly consistent with theoretical predictions for the growth of a displacement instability on a low interfacial tension interface. Attention has been paid to the importance of bending energy in the development of the wave. Where wave development was suppressed, the morphological changes due to heating appeared to involve membrane internalization in the region of the cell dimple.

Interfacial instability and membrane internalization in human erythrocytes heated in the presence of serum albumin.

The dynamics of morphological change, when human erythrocytes are heated through the spectrin denaturation temperature in the presence of bovine serum albumin, has been studied using differential interference contrast optics and a television video analysis system. Most washed (control) cells developed a wavy disturbance, with an average of 6.6 +/- 0.4 (2 S.E.) waves per cell rim, when heated. The average number of waves per cell rim decreased and the percentage of heated cells showing morphological changes in the dimple region increased with increasing serum albumin concentration, reaching 100% at 1.0 g/l. The change in the dimple region of cells heated in the presence of serum albumin involved the growth of a regular wavy disturbance around the cell dimple rim. The development of the wavy disturbance on the dimple, which resulted in the internalization of membrane, has been examined as an example of an interfacial instability on a biological membrane. Scanning and transmission electron micrographs confirm membrane internalization.

Tetracaine modifies the fragmentation mode of heated human erythrocytes and can induce heated cell fusion.

It is known that human erythrocytes in saline fragment by development of an unstable surface wave on the cell rim when cells are heated through the denaturation temperature of the structural protein, spectrin. Here the influence of tetracaine on the fragmentation process has been recorded and analysed by video microscopy of cells heated in rectangular glass microcapillaries. The number of waves per cell rim decreases with increasing tetracaine concentration until, at 0.5 mM tetracaine, wave growth on the cell rim is suppressed on most cells and the cells internalize membrane at the cell dimple. The rate constant for the change in the number of waves per cell with increasing tetracaine concentration is 9.6 mM-1 at a heating rate of 0.5 K/s. 50% of heated cells internalize membrane at 0.14 mM tetracaine. When cells are heated rapidly in suspension in test tubes the presence of tetracaine reduces the temperature for 50% haemolysis from 66 degrees C for washed control cells to 60.5 degrees C for cells in 2 mMs tetracaine. Cells heat in microcapillaries in tetracaine concentrations of 3 mM and higher begin to swell before the spectrin denaturation temperature is reached. Cell fusion was observed at and above the spectrin denaturation temperature in cells heated in 3 and 4 mM tetracaine. It was also noted that the morphology of erythrocytes maintained in 3.6 mM tetracaine for times up to 30 min at 37 degrees C or 20 degrees C was strongly dependent on temperature and time.

A video image correlation technique for the measurement of electrophoretic mobility.

The electrophoretic movement of blood cells has been examined with a video image correlator using the signal from a 625 line monochrome television camera attached to the optical arrangement. The diffraction pattern of a cell produces a characteristic signal which can be detected and registered in any television scan line to form the data of the first image frame. Under the influence of an electric field, cells moved across the television field of view and after a short interval appeared on different scan lines. The detection and registering process was repeated, and a cross-correlation function between the first and second image frame was computed. This function was stored in an output buffer which was continuously updated as fresh functions were integrated with the existing contents. The mean electrophoretic mobility of a suspension of particles can be calculated from the peak position of the integrated correlation function. Using a triangle deconvolution procedure, the relative contributions of subpopulations of lymphocytes prepared from peripheral blood samples were examined.

The influence of temperature and incubation time on deformability of human erythrocytes.

Human erythrocytes have been heated and stressed in a novel and controlled manner using rectangular microcapillaries. Heated cells attached to the capillary wall were stressed by liquid flow. Under particular conditions of stress, temperature and incubation time the body of the cell could be pulled in the flow, retaining a connection with the glass by means of a narrow process or tether. The tethers appear as: regularly beaded, irregularly beaded or without beads depending upon the incubation conditions. We have outlined the incubation regimes necessary to achieve these different responses in the temperature range 48--55 degrees C. The cells become less deformable as the incubation is continued beyond an optimum time. The behaviour of the tether is compared with that of a viscoelastic liquid. Circular dichroism studies of ghost membranes show that the denaturation of membrane proteins is partially reversible when incubation times are similar to those required to bring about a loss of deformability.

Instability development in heated human erthrocytes.

Heated human erythrocytes gradually lose their form-maintaining structure as the temperature is increased to 50 degrees C and can behave in some respects as a viscous fluid. We have developed a technique for heating and stressing these cells that is novel, simple and quantitatively precise. We have applied this technique to heated human erythrocytes and have measured instability development in cells. We have employed instability growth theory to calculate a value for an effective surface tension which, in contrast to other methods of membrane surface tension measurement sought to minimize the effects of membrane supporting structural elements. The value obtained for the surface tension of the heated erythrocyte membrane was 0.9 . 10(-6) N/m with a range of variation from 0.4 . 10(-6)N/m to 1.4 . 10(-6) N/m. The methods described may be useful for determining fundamental physical parameters such as internal viscosity and interfacial tension in other systems.

Vesicle production of heated and stressed erythrocytes.

Human erythrocytes develop vesicles by budding when heated to temperatures close to the thermal transition for spectrin. Regularly spaced strings of vesicles also develop if cells heated to 51--54 degrees C are pulled into unstable shapes by flow of liquid between cover slips. These strings of vesicles develop when cells which had attached to the glass are restrained in the flow by a long membrane-bound tether which maintains a connection with the attachment site on the glass. Breakup into regularly spaced vesicles suggests the breakup of a liquid-like cylinder by growth of Rayleigh instabilities. The ratio of length:diameter of the fragments of cylinder on which each disturbance grew ranged from 2.2 to 5.4 to 1 with a peak of 3.2, as measured from scanning electron micrographs. The upper limit of the range is slightly less than the ratio for the disturbance most likely to grow if surface tension and viscosity alone controlled the vesicle formation. Similar vesicle formation when the form-maintaining structures were weakened has been reported in other systems.

Determination of radiation-induced damage in lymphocytes using the micronucleus and microgel electrophoresis 'Comet' assays.

DNA damage assays may be useful as rapid predictors of normal tissue radiosensitivity in clinical samples. We measured in vitro radiation-induced (2 Gy) damage to lymphocytes from cancer patients and normal healthy donors using both the micronucleus and microgel electrophoresis (Comet) assays simultaneously. For damage assessment, there was a good correlation (P < 0.001) between the mean comet lengths and the fraction of cells with comets. There was no correlation with initial damage, determined as the proportion of cells within a sample that formed comets, in comparison with the mean frequency of micronuclei per binucleate cell. However, there appeared to be an association between the determination of repair proficiency in the Comet assay and the mean frequency of micronuclei per binucleate cell in lymphocytes from cancer patients.

Grouping and detection of group B streptococci by immunofluorescence.

Forty-nine clinical isolates of group B streptococci (GBS) were correctly grouped from broth culture by the Fluoro-Kit immunofluorescence test. A further 82 beta-haemolytic streptococci of groups A, C, D, F, and G were tested and gave no cross-reactions. The test was simple to perform and gave clear results. The Fluoro-Kit reagents, however, failed to detect GBS in 21 (51%) of 41 smears of rectal or vaginal swabs from pregnant women from which GBS were subsequently grown. Thirty-two (20%) of 159 culture-negative swabs gave positive immunofluorescent reactions.

Nuclear lysate sedimentation measurements of peripheral blood lymphocytes from radiotherapy patients.

When isolated nuclei of human lymphocytes are challenged with 2 M NaCl a histone-free-DNA-protein (HF-DNA) complex is released. In a linear sucrose gradient (pH 8.0) the sedimentation distance of HF-DNA is reduced when immediately isolated from cells irradiated in vitro. At low doses, if irradiated cells are incubated at 37 degrees C the sedimentation behaviour approaches that of unirradiated cells (i.e. repair). In the present study such repair was usually complete within 1 h. The radiation damage to lymphocytes from a healthy donor group and three patient groups consisting of new patients (before radiotherapy), well patients (2 to 6 years post-radiotherapy) and patients with complications attributed to radiotherapy was similar. The lymphocytes from most healthy donors and new patients demonstrated complete repair of radiation damage following an incubation of 1 h at 37 degrees C. However, 2/29 (6.9 per cent) healthy donors and 2/25 (8 per cent) new patients demonstrated poor repair. Of those patients now attending with 'bowel complications' attributed to radiotherapy 7/16 (44 per cent) demonstrated poor repair. In contrast, all those (11/11) described as 'well and complication-free' showed good repair.

The random allocation of patients in clinical trials.

The allocation of patients in controlled clinical trials may be by random selection or by a system of stratification. True matching of the two series can, however, only be achieved by a system of pairing of patients. Such a system is described.

Vitellogenin as a multigene family. Not all Xenopus vitellogenin genes may be in an "expressible" configuration.

DNA-cDNA titrations suggest that the Xenopus genome comprises two albumin genes, whereas there are 12 to 16 vitellogenin-like genes. Both sets of genes are expressed in liver, those coding for albumin constitutively, but vitellogenin only when induced by estrogen in both male and female frogs. Mild micrococcal nuclease digestion of liver nuclei to separate "expressed" from "unexpressed" gene fractions (Tata, J. R., and Baker, B. S. (1978) J. Mol. Biol. 118, 249-272) revealed that the multiple vitellogenin genes are found in two chromosomal states, here termed as "expressible" and "nonexpressible." Both albumin genes were in the chromosomal state associated with expression, but of the 12 to 16 vitellogenin genes, only 4 to 6 are in the potentially expressible state. This distribution pattern is independent of sex and only slightly modified during induction or de-induction of vitellogenesis by estrogenic stimulation or withdrawal.

The effect of intrapartum chemoprophylaxis on the vertical transmission of group B streptococci.

Intrapartum chemoprophylaxis with benzylpenicillin or erythromycin significantly reduced the rate of transmission of group B streptococci (GBS) from mothers colonized during pregnancy to their babies from 45% to 3% (P less than 0.001). None of the babies born to women who were given prophylaxis was colonized with GBS in the first 24 h of life. Six weeks after leaving hospital, however, 23% of the babies in the antibiotic group had become colonized with GBS compared with 44% in the control group. GBS strains resistant and tolerant to both benzyl-penicillin and erythromycin were found in this study. Intrapartum chemoprophylaxis breaks the cycle of GBS transmission at birth and may be useful in preventing early onset GBS disease, but is unlikely to affect late onset infections.

Epidemiology of group B streptococci: one year's experience in an obstetric and special care baby unit.

The epidemiology of group B streptococci (GBS) was studied in an obstetric unit and the related special care baby unit (SCBU). In 1 year 53 (77%) of 69 babies who acquired GBS from their mothers were colonized within 24 h of birth, compared with only 9 (35%) of 38 who acquired GBS from non-maternal sources. While 38 (36%) of 107 GBS colonized babies in the obstetric unit derived the organism from a non-maternal source, the value for the SCBU was only 2 (9%) of 23. In babies rectal and umbilical swabs gave the highest GBS isolation rates. Phage-typing and serotyping suggested that colonized mother baby pairs, rather than staff, were the primary source of hospital acquired GBS. This mode of GBS acquisition did not result in long-term carriage once babies had left hospital. Nosocomial transmission can play an important part in GBS epidemiology, but can be minimized by attention to infection control procedures.