Stepwise sarcomere shortening: analysis by high-speed cinemicrography.

Sarcomere shortening in striated muscle appears to follow a regionally synchronized staircase-like time course not anticipated in some cross-bridge models. The visualization method used has been criticized as subject to Bragg diffraction effects. Two independent optical methods were used to visualize a muscle during contraction; agreement between the stepwise behavior observed with the two methods suggests that the phenomenon is genuine.

Translational motion of actin filaments in the presence of heavy meromyosin and MgATP as measured by Doppler broadening of laser light scattering.

Intensity fluctuations of laser light scattering were utilized in order to follow enhancement of translational motion of the actin-heavy meromyosin (HMM) complex in extremely dilute solutions accompanied by the hydrolysis of MgATP. Such enhancement was anticipated on the basis of the idea that active streaming along actin filaments should be associated with their mechanochemical reactivity. Native tropomyosin was added in order to stabilize actin in its filamentous form, thus allowing the reduction of actin concentration below 50 micrograms/ml to enable free movement of neighboring filaments and yet give a reliable signal. Analysis of the data in terms of Doppler broadening led to an approximate evaluation of the average velocity of translation of the mobile filaments. This velocity was found to increase with increasing HMM concentration up to a maximum attained at a molar ratio HMM/actin of 1:2, and then decreased. Total intensity measurements indicate that the mobile scatterer is actually a complex of HMM with an isolated actin filament. HMM subfragment-1 was found to be ineffective. These results suggest that cooperation between the two myosin heads is necessary for efficient induction of active streaming along isolated actin filaments.

High oligomycin concentrations augment 6-keto-PGF1 alpha production in ventricular cardiomyocytes.

Incubation of cultured ventricular cardiomyocytes with high oligomycin concentrations (100 micrograms/ml), either alone or combined with 2-deoxyglucose (20 mM), led to the rapid depletion of cellular ATP. Inositol (poly)phosphate production decreased, and 6-keto PGF1 alpha production was increased. In cells depleted of ATP, either by low oligomycin concentrations or by sodium azide, 6-keto PGF1 alpha was not appreciably increased. There was a 25% rise in the release of fatty acids from the sn-2 position in glycerophospholipids. We suggest that oligomycin at high concentrations causes the release of free arachidonic acid from phospholipids either by non-PIP2-specific PLC and DG lipase or by phospholipase D, phosphatidic acid phosphatase and DG lipase. The effect is unrelated to decreased cellular ATP content.

A study of the dynamic properties of actomyosin systems by quasi-elastic light scattering.

A laser light source and a digital autocorrelator were employed in the study of the molecular dyanmics of acto-heavy meromyosin during the splitting of ATP. Low protein concentrations were used, so that molecular and not gel properties were evident. The addition of Mg2+ to acto-heavy meromyosin solutions in the presence of ATP caused a marked widening of the spectrum at high scattering angles. No such change was observed when chemically inactivated heavy meromyosin was used when actin was cross-linked or when the proteins were in a high ionic strength solution. The data can be interpreted in terms of pronounced change in flexibility of acto-heavy meromyosin induced by active mechanochemical coupling.

Differential aspects in ratio measurements of [Ca(2+)](i) relaxation in cardiomyocyte contraction following various drug treatments.

This study is concerned with the analysis of the time dependency of [Ca(2+)](i), monitored by indo-1-AM, via the ratiometric time response curve R(t) as measured during contractions of spontaneous or electrical stimulated cardiomyocytes (in culture). A mathematical formulation which describes the relaxation phase of R(t) was developed. By fitting formulation to the measured data of R(t), the extraction of characteristic parameters is feasible, which may reflect the factors regulating intracellular Ca concentration. The usefulness of the suggested formulation was examined by monitoring changes induced in those parameters following the exposure of the myocytes to different drugs, among which are: caffeine, ryanodine, thapsigargin db, cyclic AMP, isoprenaline, doxorubicin, and Cl-IB-MECA.

Fluorescence polarisation changes in lymphocyte cytoplasm as a diagnostic test for breast carcinoma.

Lymphocytic cytoplasm from individuals with malignant disease, and from those without, differ in such a way as to be diagnostic both of malignancy generally and of specific types of cancer. Mitogenic stimulation of lymphocytes by phytohaemagglutinin (PHA) and antigenic stimulation by encephalitogenic factor (EF) and certain specific tumour-associated antigens, provokes changes in the structure of the cytoplasmic matrix (SCM) which are detectable upon fluorescence polarisation. The degree of change is quantifiable both by calculating the polarisation ration (PR, polarisation before and after stimulation) and the relative ratio (RRSCM, the ratio between the polarisation obtained after exposure to EF [PEF] and to the polarisation measured after exposure to PHA [PPHA]). A new tumour-associated antigen specific for breast cancer, CaBr, was tested for its diagnostic efficacy in comparison with that of EF, by prospectively testing blood samples from 138 consecutive women with suspicious breast masses. The previously known discriminatory power (sensitivity 60.7% and specificity 90.7%) of the polarisation-derived RRSCM was reconfirmed. However, the RR'SCM (the new ratio using CaBr instead of EF), was significantly more sensitive (77.4%; P < 0.01) and specific (94.4%) than the RRSCM in detecting breast cancers. The polarisation changes in the cytoplasmic matrix after stimulation by CaBr alone suggest the best discriminatory power (sensitivity 90.5% and specificity 94.4%) between cancerous and non-cancerous patients.

Adaptation of the cellscan technique for the SCM test in breast cancer.

The value of the SCM (Structuredness of Cytoplasmic Matrix) cancer test, a procedure based on the detection of differences in lymphocyte activation in the presence and absence of cancer, has remained controversial, with inconsistent results having been reported among investigators. The Cellscan, a high-precision static cytometer system, has been designed to perform the SCM test; the apparatus facilitates the polarisation measurements and can examine cells which have been separated by simpler procedures than were originally described. In this study, using methods and diagnostic criteria adapted for the Cellscan system in a hospital environment, the SCM test correctly classified over 90% (76/80) of patients with breast cancer and differentiated over 90% (72/73) of individuals without cancer.

Fourier analysis of differential light scattering for the quantitation of FSH response associated with structural changes in immortalized granulosa cells.

We have established granulosa cell lines which express constitutively the rat FSH receptors by cotransfection of primary granulosa cells obtained from preovulatory follicles with SV40 DNA, Ha-ras oncogene and a plasmid expressing FSH receptors. These cells respond specifically to ovine and human FSH by cell rounding, intracellular cAMP accumulation, and progesterone secretion in a dose-dependent manner. A new method for the demonstration and quantitation of changes in cell shape-Small Angle Laser Light Scattering (SALLS) analysis-has been utilized for measurement of cell rounding in response to FSH stimulation in these cells. When cells were incubated with increasing doses of either ovine or human FSH, partial rounding of cells was observed at FSH concentrations as low as 24 pM, while complete rounding of cells was observed at a range of 0.24-2.4 nM of FSH. Following aldehyde fixation, hormone-treated cells were examined using the method of SALLS analysis. Histograms obtained by applying SALLS analysis on FSH stimulated GFSHR-17 cells were a reflection of the structural changes induced by the hormone. FSH- and forskolin-incubated cells yielded structured distributions with defined mean size and standard deviations. Moreover, the increase in sharpness of dominant peak in the histogram was correlated with elevated concentration of FSH in a dose dependent manner. In conclusion, cellular response to FSH is correlated with a specific pattern of light scattered in immortalized granulosa cells expressing functional FSH receptors. Therefore, SALLS analysis may serve as a useful tool for in vitro bioassay of the gonadotropic hormone. Moreover, this method may lend itself to in vitro bioassay of any hormone that induces specific morphological changes in target cells.

Active streaming against gravity in glass microcapillaries of solutions containing acto-heavy meromyosin and native tropomyosin.

Solutions containing heavy meromyosin, actin, native tropomyosin, and Mg-ATP exhibited streaming in horizontally placed glass microcapillaries. Up-hill streaming could also be observed when the capillaries were at an inclined position; this served for the clear distinction between active and passive streaming provided surface tension effects were eliminated. The presence of native tropomyosin and actin-activation of the ATPase activity of HMM were essential for the reconstitution of active streaming. The significance of the results for cytoplasmic streaming and muscle contraction is discussed.

Oxygen deprivation and reoxygenation augment prostacyclin synthesis in cultured ventricular myocytes.

Prostacyclin production in cultured cardiomyocytes is not induced by cellular ATP depletion per se, suggesting that the mechanism of ischemic injury is more complex. In the present study we subjected cultured ventricular myocytes to 'simulated ischemia' followed by reoxygenation. A slight increase in 6-keto-PGF(1 alpha) (the stable metabolite of PGI(2)) was found during 'ischemia', which continued to increase markedly during reoxygenation. PGE(2) levels were pronouncedly enhanced during ischemia but decreased during reoxygenation, and TXB(2) levels remained undetectable throughout. These findings reflect a cardiomyocyte response to anoxic injury, suggesting that they act to protect against cardiac injury by producing the potent vasodilators PGI(2) and PGE(2) during ischemia and reoxygenation.

Prelytic stimulation of target and effector cells following conjugation as measured by intracellular fluorescein fluorescence polarization.

The aim of the present study was to detect prelytic intracellular changes induced in target and effector cells following their conjugation at room temperature. Changes in the cytoplasmic matrix were measured by means of intracellular fluorescein fluorescence polarization (IFFP) using the Cellscan apparatus. Both natural killer and lymphocyte activated killer cells were used as effector cells, while K562 and Daudi cell lines were used as targets. The results show that following their conjugation, both the effector and the target cells show significant reductions (>10%) in IFFP values. Changes in IFFP were induced by specific interaction and only between viable cells. No evidence of fluorescein transfer from a stained cell to its nonstained counterpart was found. To the best of our knowledge, this is the first time that effector-target interaction is monitored on an individual cell basis within a population, by means of IFFP measurements. In addition, in order to explain the physical phenomena, measurements of physical parameters which might affect the IFFP, such as changes in osmolality and pH, were performed and discussed. © 1998 Society of Photo-Optical Instrumentation Engineers.

Fluorescence polarization changes in the lymphocytic cytoplasm in the various stages of breast cancer.

Post-surgical pathological staging and ancillary tests determine the initial treatment of breast cancer. Because change in the structuredness of the cytoplasmic matrix (SCM) of peripheral lymphocytes (as assessed by measurement of fluorescein fluorescence polarization, FFP) has already emerged as diagnostic for breast cancer and an aid to staging in other cancers, testing was carried out in 113 pre-surgical patients to see whether such change could contribute to accurate staging of breast cancer. The FFP test was able to distinguish grouped stages of locoregional disease from metastatic disease but was unable to distinguish between any single locoregional stage and the metastatic stage. Technological improvements now underway may create a role for assessment of SCM changes in breast cancer staging.

Potential for anatomic localization in colorectal cancer using the SCM test.

We analysed a series of 81 colorectal cancer cases in which the SCM (structuredness of the cytoplasmic matrix) test had already been performed with a diagnostic sensitivity of 92% and a specificity of 92.6%, yielding positive and negative predictive values of 96.3% and 84.7% respectively. We subdivided this group of 81 patients by anatomic location of the malignancy. Although the resultant subgroups were admittedly small, we noted a tendency for the most prominent changes in observed and calculated polarization parameters to be associated with cecal cancers. This finding was of special interest because the cecum is the most inaccessible site for colonoscopy. Ongoing site-specific surveillance in SCM-tested cases of colorectal cancer is necessary to validate this result.

Rhabdomyolysis due to hereditary torsion dystonia.

Following an acute dystonic crisis, a 6-year-old boy with hereditary torsion dystonia developed rhabdomyolysis. To our knowledge, hereditary torsion dystonia has never been reported as a cause of rhabdomyolysis. Early diagnosis and treatment of rhabdomyolysis should be considered in children with severe dystonia in order to prevent renal failure.

1 kgf/cm2--the isometric tension of muscle contraction: implications to cross-bridge and hydraulic mechanisms.

Attention is drawn to experimental results from many laboratories which indicate that the isometric force (F) in the contraction of striated muscle fibers is linearly proportional to their variable cross-section area (A). Reversible swelling of intact, skinned, or glycinerated fibers can be induced by changes in tonicity, ionic strength or pH. In all cases where careful measurements of F and A are reported, the maximal isometric tension namely, T = F/A, is found around 1 kgf /cm2, even though F and A may change more than threefold for a given fiber at a certain length. These results seem to be independent of the fiber length or temperature. Thus, the isometric tension T in striated muscle does not depend on the number or the rate of the interacting cross-bridges. This result of constant isometric tension, which has so far received little attention, is however, a simple prediction of the hydraulic mechanism which is proposed for muscle contraction. Therefore, the hydraulic model, which is based on the hypothesis of vectorial flux of energetic protons deserves serious consideration.

Immune cytolysis viewed as a stimulatory process of the target.

Humoral and cellular mechanisms of immune cytolysis, as effected by antibody and complement (Ab + C') or by cytolytic T lymphocytes (CTL), have traditionally been considered the end result of early but terminal membrane damage, in turn causing colloid-osmotic lysis of the target cell. A comprehensive theory explaining and relating known prelytic cellular events to subsequent membrane damage is lacking, nor is there a specific picture as to the role and mode of action of Ca2+, which appears to be involved in both complement- and cell-mediated cytolysis (C'MC and CMC, respectively). Recent studies are in support of the view that both Ab + C' and CTL induce a comparable series of prelytic events, in the TC, initiated by membrane depolarization, which in turn bring about voltage-dependent Ca2+ influx or its intracellular release. Persistent elevation of cytosolic Ca2+ can induce massive stimulation of cellular ATPases (actomyosin, Ca2+) and cause exhaustive depletion of ATP. Consequently, Na+-pumping is slowed down and colloid-osmotic lysis ensues. Hence, in our view, membrane damage in immune cytolysis is the result rather than the cause of intracellular events culminating in lysis.

Stepwise shortening: evidence and implications.

The observation that sarcomeres shorten in steps has proved controversial. On the one hand, the phenomenon implies that the contractile process cannot be based on a molecular mechanism that behaves in a random manner: The fact that the steps and pauses characterize the kinetics of large volumes of tissue implies that the elements comprising such volumes must stop and pause synchronously. On the other hand, since current contractile models do not anticipate synchronized behavior, there has been considerable speculation that the phenomenon might not be a genuine feature of contraction, but an instrument-based artifact. We present here a review of observations made with four methods that have been brought to bear on the question. All four show discrete, synchronized contractile behavior. The observation of steps with multiple independent methods implies either that each technique harbors its own " gremlin " that generates spurious steps and pauses of a similar nature, or that the phenomenon is genuine. Finally, some consistent properties of the distribution of step size are considered with respect to possible molecular models.

Carboxyfluorescein as a fluorescent probe for cytoplasmic effects of lymphocyte stimulation.

The application of carboxyfluorescein (CF), as an impermeable fluorescent probe for lymphocyte stimulation with phytohaemagglutinin (PHA), is investigated by following a decrease in the degree of fluorescence polarization. Since CF does not enter the mitochondria, the present results indicate that the measured effect of stimulation occurs in the cytoplasm. The results also reveal that the fluorescence yield of intracellular CF is smaller than that of extracellular CF. Moreover, the degree of fluorescence polarization of intracellular CF is inversely related to its concentration. Following cell disruption, fluorescence intensity increases and polarization decreases. These effects might indicate a weak or reversible association of intracellular CF with cytoplasmic proteins.

Cell activation influences cell staining kinetics.

Stimulation of cells has so far been observed, among other methods, by the decrease of the intracellular fluorescein fluorescence polarization (IFFP). It is shown that the rate constant of leakage of the fluorescent marker out of the cells increases with stimulation much more significantly than the polarization decreases; thus it might provide a more sensitive method to observe cells stimulation. It is also shown that due to negligible leakage of the marker out of the cells shortly after initiation of the staining of the cell suspension, the fluorescein fluorescence polarization (FFP) of the cell suspension, is very close to IFFP.