The nuclear matrix is a thermolabile cellular structure.

Heat shock sensitizes cells to ionizing radiation, cells heated in S phase have increased chromosomal aberrations, and both Hsp27 and Hsp70 translocate to the nucleus following heat shock, suggesting that the nucleus is a site of thermal damage. We show that the nuclear matrix is the most thermolabile nuclear component. The thermal denaturation profile of the nuclear matrix of Chinese hamster lung V79 cells, determined by differential scanning calorimetry (DSC), has at least 2 transitions at Tm = 48 degrees C and 55 degrees C with an onset temperature of approximately 40 degrees C. The heat absorbed during these transitions is 1.5 cal/g protein, which is in the range of enthalpies for protein denaturation. There is a sharp increase in 1-anilinonapthalene-8-sulfonic acid (ANS) fluorescence with Tm = 48 degrees C, indicating increased exposure of hydrophobic residues at this transition. The Tm = 48 degrees C transition has a similar Tm to those predicted for the critical targets for heat-induced clonogenic killing (Tm = 46 degrees C) and thermal radiosensitization (Tm = 47 degrees C), suggesting that denaturation of nuclear matrix proteins with Tm = 48 degrees C contribute to these forms of nuclear damage. Following heating at 43 degrees C for 2 hours, Hsc70 binds to isolated nuclear matrices and isolated nuclei, probably because of the increased exposure of hydrophobic domains. In addition, approximately 25% of exogenous citrate synthase also binds, indicating a general increase in aggregation of proteins onto the nuclear matrix. We propose that this is the mechanism for increased association of nuclear proteins with the nuclear matrix observed in nuclei Isolated from heat-shocked cells and is a form of indirect thermal damage.

Platelet ultrastructural and functional studies in myelodysplasia.

We studied the platelets of 8 patients with myelodysplasia aged 49-77 years, using both ultrastructural and functional techniques. Five of the 8 patients were classified as having refractory anaemia, and 3 as refractory anaemia with excess of blasts (RAEB). Electron microscopically, the myelodysplastic patients had in addition to normal ones, platelets containing significantly less alpha-granules. In part of these hypogranular platelets, the dense tubular system was abundant, but in contrast with normal platelets, it was not dispersed between the other organelles, nor did it form a membrane complex with the open cannalicular system. From a functional point of view, collagen-induced shape change was the most frequently disturbed parameter: there was a total loss of collagen-induced shape change in 5 patients. In 2 patients, there was a complete lack of response to collagen in platelet-rich plasma (both shape change and aggregation); in one of them, there was also a total loss of adenosine triphosphate secretion in response to all inducers tested. After 4 years of follow-up, 5 patients had died, of whom 3 were RAEB patients. An initial complete absence of collagen-induced shape change was found in these 5 patients, while in the 3 patients who were still alive at the end of the follow-up period, collagen-induced shape change was normal in 2 and slightly diminished in one.

Interaction of dibucaine with the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

The site of interaction of dibucaine with the Ca(2+)-ATPase of rabbit sarcoplasmic reticulum, an ion-transporting membrane protein, was investigated by determining the effect of dibucaine on the denaturation of the transmembrane domain and the aqueous domain containing, respectively, the high-affinity Ca2+ binding sites and the site of ATP hydrolysis. In the absence of Ca2+, a single irreversible denaturation transition with Tm approximately equal to 49 degrees C is observed for the Ca(2+)-ATPase by differential scanning calorimetry (DSC). In the presence of Ca2+, but not Mg2+, Sr2+, or Ba2+, a new high-temperature transition is observed that has been shown to be due to stabilization of the transmembrane region [Lepock, J. R., Rodahl, A. M., Zhang, C., Heynen, M. L., Waters, B., & Cheng, K. H. (1990) Biochemistry 29, 681-689]. The maximum stabilization corresponds to a shift in Tm of 13.8 degrees C, and Hill analysis indicates that the Ca2+ binding site yielding stabilization has a Kd = 2.5 x 10(-4) M with a cooperativity (n) of 1. Thus, stabilization is due to Ca2+ binding not to the high-affinity sites but to one of the previously observed sites of low or intermediate affinity, which must be located in the transmembrane or stalk subdomains. Dibucaine has little effect on the Tm of the aqueous domain, but it decreases the Tm of the transmembrane domain with Kd approximately equal to 4.1 x 10(-4) M and a cooperativity of approximately 1.6, implying that destabilization is due to the binding of dibucaine to sites of intermediate or moderately high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased thermostability of thermotolerant CHL V79 cells as determined by differential scanning calorimetry.

Heat shock denatures cellular protein and induces both a state of acquired thermotolerance, defined as resistance to a subsequent heat shock, and the synthesis of a category of proteins referred to as heat-shock proteins (HSPs). Thermotolerance may be due to the stabilization of thermolabile proteins that would ordinarily denature during heat shock, either by HSPs or some other factors. We show by differential scanning calorimetry (DSC) that mild heat shock irreversibly denatures a small fraction of Chinese hamster lung V79-WNRE cell protein (i.e., the enthalpy change, which is proportional to denaturation, on scanning to 45 degrees C at 1 degree C/min is approximately 2.3% of the total calorimetric enthalpy). Thermostability, defined by the extent of denaturation during heat shock and determined from DSC scans of whole cells, increases as the V79 cells become thermotolerant. Cellular stabilization appears to be due to an increase in the denaturation temperature of the most thermolabile proteins; there is no increase in the denaturation temperatures of the most thermally resistant proteins, i.e., those denaturing above 65 degrees C. Cellular stabilization is also observed in the presence of glycerol, which is known to increase resistance to heat shock and to stabilize proteins in vitro. A model is presented, based on a direct relationship between the extent of hyperthermic killing and the denaturation or inactivation of a critical target that defines the rate-limiting step in killing, which predicts a transition temperature (Tm) of the critical target for control V79-WNRE cells of 46.0 degrees C and a Tm of 47.3 degrees C for thermotolerant cells. This shift of 1.3 degrees C is consistent with the degree of stabilization detected by DSC.

Thermal denaturation of the Ca2(+)-ATPase of sarcoplasmic reticulum reveals two thermodynamically independent domains.

Inactivation of Ca2+ uptake and ATPase activity of the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum was measured and compared to the thermal denaturation of the enzyme as measured by differential scanning calorimetry (DSC) and fluorescence spectroscopy. Two fluorophores were monitored: intrinsic tryptophan (localized in the transmembrane region) and fluorescein isothiocyanate (FITC)-labeled Lys-515 (located in the nucleotide binding domain). Inactivation, defined as loss of activity, and denaturation, defined as conformational unfolding, were irreversible under the conditions used. Activation energies (EA) and frequency factors (A) for inactivation were obtained for the enzyme in 1 mM EGTA and 1 mM Ca2+. These were transformed to a transition temperature for inactivation, Tm (defined as the temperature of half-inactivation when temperature is scanned upward at 1 degree C/min). All denaturation profiles were fit with an irreversible model to obtain EA and Tm for each transition, and the values of these parameters for denaturation were compared to the values for inactivation. In EGTA, denaturation obeys a single-step model (Tm = 49 degrees C), but a two-step model is required to fit the DSC provile of the enzyme in 1 mM Ca2+. The specific locations of tryptophan and the fluorescein label were used to demonstrate that denaturation in Ca2+ occurs through two distinct thermodynamic domains. Domain I (Tm = 50 degrees C) consists of the nucleotide binding region and most likely the phosphorylation and transduction regions [MacLennan, D. H., Brandl, C. J., Korczak, B., & Green, N. M. (1985) Nature 316, 696-700].(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro and in vivo evaluation of granulocyte labeling with [99mTc]d,1-HM-PAO.

The functional integrity of white blood cells labeled with [99mTc]d,1-HM-PAO containing variable amounts of the ligand or of the 99mTc activity was evaluated by enzymatic tests and by measuring random migration, chemotaxis, phagocytosis, killing, and adhesion. The ultrastructure of labeled cells was studied by electron microscopy. The tracer dose and the HM-PAO concentration did not significantly affect phagocytosis and killing. The results of the other tests remained normal. A maximum labeling efficiency of 80% was reached by incubating the granulocytes for 20 min with 10-20 mCi of [99mTc]d,1-HM-PAO containing 50 micrograms of the ligand in 1 ml of saline. There was only a slow washout of 20% of activity from the labeled cells in 24 hr. The ultrastructure was not influenced by the labeling technique. Proven infection sites of 17 orthopedic patients were clearly visualized. After a short transient lung uptake, there was a clear spleen and moderate liver uptake with early bladder and late prominent colon visualization. Because of the lower cost, favorable radiation dose and more suitable tracer characteristics, this technique is a promising alternative for 111In labeling of white blood cells.

Protein degradation in CHL V79 cells during and after exposure to 43 degrees C.

Cellular protein degradation during and following hyperthermia should be altered due to increased enzymatic activity at elevated temperatures, inhibition of protein synthesis, and denaturation of proteins. We have previously demonstrated by differential scanning calorimetry that approximately 1-2% of total CHL V79 cellular protein denatures during a 10- to 15-min exposure to 43 degrees C (J. E. Lepock et al., J. Cell. Physiol. 137, 14-24 (1988)). Proteolysis was measured during and after exposure to 43 degrees C. The decay curves of the degradation of [3H]Leu-labeled proteins are fit well by a double exponential; however, each component is the sum of the decay curves of a large number of proteins, probably with a distribution of rates of degradation. At 37 degrees C a fast-decaying component (T1/2 congruent to 1.3 h), representing short-term proteins, and a slow-decaying component (T1/2 congruent to 50 h), representing long-term proteins, are observed. At 43 degrees C the rate of degradation of the fast-decaying component is stimulated three- to fivefold (to T1/2 = 0.27-0.45 h). After return to 37 degrees C, the rate of degradation of the slow-decaying component is depressed twofold (to T1/2 = 109-141 h). The period of depression is dose dependent (i.e., time at 43 degrees C) and recovers at approximately the same time as resumption of protein synthesis and growth. Overall stimulation of degradation lasts for approximately 15 min at 43 degrees C and, coupled with an inhibition of synthesis, leads to the loss of at least a small percentage of total cellular protein. It is likely that the initial stimulated degradation is in part due to increased substrate in the form of denatured protein, further supporting the denaturation of proteins during hyperthermia.

Congenital macrothrombocytopenia, leucocyte inclusions, deafness and proteinuria: functional and electron microscopic observations on platelets and megakaryocytes.

We report a patient with a variant of Alport's syndrome: macrothrombocytopenia, leucocyte inclusions, deafness and proteinuria. Ultrastructural studies revealed giant spheroid platelets with a high density of organelles and a disorganized microtubular system. In addition, Fechtner inclusions were observed in neutrophils of the patient and her mother. In platelet rich plasma platelets aggregated normally for the low platelet number, although no shape change was visible. Platelet studies in whole blood using impedance aggregometry gave supernormal aggregation curves; this is not in agreement with the abnormally long bleeding time, showing the limited usefulness of this technique in patients with such large platelets. The megakaryocytes (MK) showed two different distribution patterns of the demarcation membrane system (DMS), which may explain the production of few large platelets. The formation of platelets occurred by fragmentation of the granular zone of the MKs, which seemed to be followed by expulsion of platelets through openings of the peripheral zone. The involvement of cytoskeletal structures in the organization of the DMS and the expulsion of platelets is discussed.

Evaluation of 111In labelled white blood cells by in vitro functional tests and electron microscopy. Comparison of three labelling methods.

We have studied the influence of granulocyte labelling with commercially available 111In-oxine, tropolone (trop) or home made 111In-Mercapto pyridine (Merc) prepared by the method of Thakur (1985) on the cell structure by electron microscopy and on the cell function by enzymatic tests, random migration, chemotaxis, phagocytosis and bactericidal activity. The granulocytes were labelled with 400 microCi 111In-oxine in saline or 111In-trop or Merc in plasma. The effect of the chelating agents with and without addition of the tracer was studied (n = 4) with varying concentrations: 5-10 micrograms/ml oxine, 10-160 micrograms/ml trop and 1-4 micrograms/ml Merc. Chemotaxis and random migration were not affected by 111In-trop and clearly suppressed by 111In-oxine and Merc; the other tests were normal. The cell structure was disturbed by Merc. The labelling efficiency was excellent with oxine (90%), acceptable with trop (30%-80%) and poor with Merc (10%-25%). Without 111In, chemotaxis and random migration were normal up to a concentration of 80 micrograms/ml trop, 8.5 micrograms/ml oxine and 1 microgram/ml Merc. With addition of 111In, chemotaxis and random migration were unaffected up to 80 micrograms/ml by trop and markedly suppressed by Merc and oxine. It is concluded that labelling with 111In-trop assures intact cells.

Role of extracellular calcium in the hyperthermic killing of CHL V79 cells.

Two major questions are addressed by this study: Can an influx of calcium ion sensitize CHL V79 cells to hyperthermia, and, if so, does this occur during heating and does it play a crucial role in cell death? V79 cells are sensitized to hyperthermia by the calcium ionophore A23187 which also induces an influx of calcium at both 37 and 43 degrees C. Sensitization is at least partially dependent on the presence of extracellular calcium. In the absence of A23187, survival is independent of calcium concentration (from 0 to 25 mM) during heating, which differs from the behavior of hepatocytes which are sensitized to hyperthermia by 15 mM CaCl2. Calcium influx, as assayed by uptake of 45Ca measured after washing in LaCl3, is detectable in 3 mM CaCl2 only after 30 min at 45 degrees C, an exposure which reduces reproductive survival to less than 0.1%. Calcium uptake reaches 6 nmol/10(6) cells after 180 min at 45 degrees C. This is not due to a general loss of membrane permeability since there is no trypan blue staining during this time. In 15 mM CaCl2, influx occurs earlier (15 min) but still succeeds the loss of reproductive survival which is less than 1% at this time. Uptake is much higher in 15 mM CaCl2, reaching 10 nmol/10(6) cells by 30 min and 25 nmol/10(6) cells at 180 min, but the temporal pattern of uptake does not correlate with loss of reproductive survival. Thus, although A23187 sensitizes V79 cells to hyperthermia, probably by increased influx of calcium ion, and increased influx occurs during exposure to 45 degrees C, influx is not a crucial early event in the killing of V79 cells. This does not eliminate the possibility of intracellular calcium redistribution during hyperthermia.

Effect of acute lead intoxication on the ultrastructure of rat erythroblasts and reticulocytes. Morphometric analysis and röntgen micro-analysis.

Effect of 17 hr intoxication of lead on the different maturation stages of erythroid cells were studied in rat. Morphometric methods were used to analyse the lead-induced ultrastructural changes in the early, intermediate, late erythroblasts and reticulocytes. It was found that the toxic effect of lead increases with maturation. Energy-dispersive Röntgen micro-analysis showed that fibrillar structures within vesicles and endoplasmic reticulum contained lead.

Autophagy of mitochondria in rat bone marrow erythroid cells. Relation to nuclear extrusion.

Late erythroblasts and reticulocytes from bone marrow of male Wistar rats were studied by electron-microscopic stereology. Late erythroblasts with morphological signs of nuclear extrusion (EN + erythroblasts) and late erythroblasts without these signs (EN-erythroblasts) were analysed separately. The volumes of mitochondria, autophagosomes, autophagocytosed mitochondria, autophagocytosed cytoplasm and degraded material inside autophagosomes were calculated per unit volume of cytoplasm. The results demonstrate that (1) the volume density of mitochondria in the cytoplasm decreases by 34% during maturation from (EN-)- to (EN +)-erythroblasts (P less than 0.001) and by 60% during differentiation from (EN +)-erythroblasts to reticulocytes (P less than 0.001), (2) a fivefold increase in the volume density of autophagosomes in the cytoplasm is noted during maturation from (EN-)- to (EN +)-erythroblasts (P less than 0.01), whereas the value of this parameter remains essentially unchanged during the subsequent differentiation to reticulocytes, (3) no mitochondria are found inside autophagosomes of (EN-)-erythroblasts, whereas mitochondria occupy 26% and 35%, respectively, of the autophagosomal volume in (EN +)-erythroblasts and in reticulocytes. Our results show that autophagocytosis of mitochondria starts at the moment of nuclear extrusion and continues in the bone marrow reticulocytes.

A reliable method with good cell preservation for the demonstration of peroxidase activity in human platelets and megakaryocytes.

We have tried to improve existing methods for demonstration of platelet peroxidase (PPO) in human platelets and megakaryocytes by introducing a fixation of 0.1% glutaraldehyde prior to incubation in the DAB medium. This prefixation with low concentration of glutaraldehyde preserves excellent morphological detail and does not inhibit PPO activity. All 23 platelet-rich plasma samples show PPO reaction product in the dense tubular system after incubation in DAB medium with 0.003% H2O2. When 0.01% H2O2 is used in excessive DAB medium, PPO activity can also be demonstrated in platelets and megakaryocytes of bone-marrow cell suspensions. This method can be used for the identification of megakaryoblasts in acute non-lymphocytic leukemia, myelodysplastic syndromes and in blastic crisis of chronic myeloid leukemia. PPO cytochemistry can be combined with postfixation in a OsO4-ruthenium red mixture. This method reveals alpha-granules, dense bodies, microtubuli, glycogen, mitochondria, dense tubular system and invaginated membrane system in the same platelet and is useful for investigation of platelet ultrastructure.

['Sciatic" artery and vein. Apropos of 3 cases]. / Artère et veine "sciatiques". A propos de 3 observations.

Three cases of sciatic axis are reported: one case, documented by arteriography, of bilateral persistant sciatic artery, which is a rare but well known embryologic abnormality and two cases, documented by varicography, of sciatic vein. Vascular embryology of lower limb is briefly related; the usefulness of arteriography for pulsatile buttock masses and of varicography concerning some leg's varicose veins are developped.

A quantitative ultrastructural study of normal rat erythroblasts and reticulocytes.

Early, intermediate and late erythroblasts and reticulocytes were studied by electron microscopic morphometry. The volumes of mitochondria, Golgi zone and autophagosomes, as well as the surface areas of membranes of rough endoplasmic reticulum (RER) and of mitochondrial cristae and the numbers of ribosomes per unit volume cytoplasm were calculated. the results revealed 3 phases in erythroid maturation: (1) early and intermediate erythroblasts, (2) late erythroblasts, and (4) reticulocytes. The only significant difference between early and intermediate proliferating erythroblasts was a decrease in the surface area of the RER in the latter. After the last mitotic division in late erythroblasts significant reductions occurred in the RER, the Golgi apparatus, in the mitochondria and the number of ribosomes. The numbers of mitochondria and ribosomes were further reduced at the reticulocyte stage (clustered ribosomes more rapidly than single ones). Morphometric analysis showed no evidence of degradation of erythroid mitochondria whilst they are free in the cytoplasm, but there was some evidence of degradation after their uptake into autophagosomes in late erythroblasts and in reticulocytes.

Studies on the mechanism of transferrin iron uptake by rat reticulocytes.

Mechanism of transferrin iron uptake by rat reticulocytes was studied using 59Fe- and 125I-labelled rat transferrin. Whereas more than 80% of the reticulocyte-bound 59Fe was located in the cytoplasmic fraction, only 25-30% of 125I-labelled transferrin was found inside the cells. As shown by the presence of acetylcholine esterase, 10-15% of the cytoplasmic 125I-labelled transferrin might have been derived from the contamination of this fraction by the plasma membrane fragments. Electron microscopic autoradiography indicated 26% of the cell-bound 125I-labelled transferrin to be inside the reticulocytes. Both the electron microscopic and biochemical studies showed that the rat reticulocytes endocytosed their plasma membrane independently of transferrin. Sepharose-linked transferrin was found to be capable of delivering 59Fe to the reticulocytes. Our results suggest that penetration of the cell membrane by transferrin is not necessary for the delivery of iron and that, although it might make a contribution to the cellular iron uptake, internalization of transferrin reflects endocytotic activity of the reticulocyte cell membrane.

Dyserythropoiesis and annulate lamellae.

Cytoplasmic and intranuclear annulate lamellae in the erythroblasts from patients with dyserythropoietic anaemia (megaloblastic anaemia, dysplastic anaemia and erythroleukaemia) are described. Annulate lamellae have mainly been observed in oocytes, in embryonic tissues and in malignant cells. Their occurrence in dyserythropoietic anaemia may be related to the reappearance of fetal characteristics in the erythroblasts and erythrocytes.