KCl cotransport activity in light versus dense transferrin receptor-positive sickle reticulocytes.

A subset of sickle cells becomes K(+)-depleted and dehydrated before or soon after leaving the bone marrow. These young cells may be identified in blood as transferrin receptor-positive (TfR+) dense reticulocytes. KCl cotransport, which is normally active in young erythroid cells with a maximum at pH 6.8, is a candidate pathway for K+ depletion of sickle reticulocytes. In this investigation, KCl cotransport activity was evaluated in young, TfR+ cells which had become dense in vivo and in age-matched cells which had retained normal hydration. Sickle erythrocytes were first separated into three primary density fractions, with care taken to preserve the in vivo hydration state. After normalization of intracellular hemoglobin concentration with nystatin, the cells were incubated at 37 degrees C for 20 min at pH 6.8 and 7.4. Before and after incubation, each primary fraction was separated into four secondary density fractions. The percentage of TfR+ cells in each secondary fraction was measured and a density distribution for TfR+ cells was determined for each primary fraction before and after incubation. The density shift during incubation was a measure of KCl cotransport. TfR+ cells from the denser primary fractions II and III had significantly more density shift than TfR+ cells from the light fraction I. Although the shifts were larger at low pH, differences between primary fractions were also observed at pH 7.4. These data indicate that the cells which become dense quickly in vivo have more KCl cotransport activity than those which remain light in vivo, and support this pathway as a primary mechanism for dehydration of young sickle cells.

Time-dependent changes in the density and hemoglobin F content of biotin-labeled sickle cells.

Sickle red blood cells (RBC) are subject to a number of important cellular changes and selection pressures. In this study, we validated a biotin RBC label by comparison to the standard 51Cr label, and used it to study changes that occur in sickle cells as they age. Sickle RBC had a much shorter lifespan than normal RBC, but the two labels gave equivalent results for each cell type. A variable number of sickle, but not normal, RBC disappeared from the circulation during the first few hours after reinfusion. The number of biotinylated sickle reticulocytes was decreased by 50% after 24 h and 75% after 48 h, with a gradual decrease in the amount of reticulum per cell. The labeled sickle cells exhibited major density increases during the first 4-6 d after reinfusion, with smaller changes thereafter. A small population of very light, labeled sickle RBC was essentially constant in number after the first few days. Fetal hemoglobin (HbF) content was determined in isolated biotinylated sickle RBC after reinfusion, allowing an estimate of lifespan for RBC containing HbF (F cells) and non-F cells. The lifespan of sickle biotinylated RBC lacking HbF was estimated to be approximately 2 wk, whereas F cells survived 6-8 wk.

Membrane processes associated with the osmotic-pulse incorporation of inositol hexaphosphate.

In previous studies (Biochem. Biophys. Res. Commun. 144, 779-786 (1987); Prog. Clin. Biol. Res. 292, 65-75 (1989)), we showed that inositol hexaphosphate (IHP), when added to erythrocyte membrane ghosts in the range 0.6-2.5 mM, caused a large disruption of skeletal protein-protein interactions as monitored by electron paramagnetic resonance techniques. IHP incorporated into intact cells by an osmotic-pulse method (J. Cell. Physiol. 129, 221-229 (1986)) leads to cells with markedly decreased oxygen affinity. Exposure of the red cells to higher levels of IHP during the osmotic pulse leads to less lysis and more normal cellular indices after healing of the transiently-disrupted membrane (J. Lab. Clin. Med. 113, 58-66 (1989)). In order to determine what effect higher levels of IHP had on skeletal proteins and bilayer lipids of membrane ghosts, spin labeling studies were performed. The main findings were: (a) There was a concentration-dependent alteration in skeletal protein interactions. At concentrations greater than 25 mM IHP, the effectiveness of IHP to disrupt skeletal protein interactions was diminished. (b) No apparent alteration of the motion or order of phospholipids or the lipid water interface of intact cells into which IHP was incorporated occurred, suggesting that higher levels of IHP do not alter the physical state of the lipid bilayer.

Scleromyxedema.

An unusual, nodulocystic form of scleromyxedema (lichen myxedematosus) developed in a 48-year-old man with a six-year history of psoriasis. The scleromyxedema responded to intermittent therapy with melphalan and prednisone. Dermabrasion smoothed and softened the skin and increased the mobility of the perioral skin. Two months after remission of the skin lesions, psoriasis recurred.

Incorporation of inositol hexaphosphate into red blood cells mediated by dimethyl sulfoxide.

Inositol hexaphosphate (IHP) binds to deoxyhemoglobin and markedly decreases the affinity of hemoglobin for oxygen. We introduce here a method for incorporating this polyphosphate into erythrocytes, thus preparing very low affinity cells for use in respiration research. The method uses dimethyl sulfoxide (DMSO) to facilitate entry of IHP. The cells are exposed to a high concentration of DMSO which is rapidly diluted with IHP solution. During this dilution the cells become leaky and IHP enters. The influence of several variables at each step of the process has been investigated and the data support a transient osmotic gradient mechanism for IHP incorporation.

Amino acid requirements of a rat sarcoma as determined by a stem cell assay.

Knowledge of the amino acid requirements of a neoplasm is valuable in determining optimal nutritional support and antineoplastic therapy for the tumor-bearing host. The standard human tumor stem cell assay (HTSCA) was modified by reducing an individual amino acid below the normal plasma concentration of the Fischer 344 rat. All other amino acids were maintained at levels sufficient for normal HTSCA tumor colony growth. Twenty-two amino acids were tested at a mean concentration of 12% (range 3% to 35%) of their normal plasma level. Results indicated that all amino acids except L-glutamine and L-asparagine were present in sufficient quantity for normal tumor growth. Dose-response curves have shown more than 70% inhibition of tumor growth with a glutamine concentration of 50% and an asparagine concentration of 25%. Glutamine and asparagine levels of 4% and 1%, respectively, resulted in 100% inhibition. The data indicate that rat sarcoma stem cells are sensitive to decreased glutamine and asparagine concentrations.

Increased tyrosine protein kinase activity in hairy cell and monocytic leukemias.

Tyrosine protein kinases (TPK) help regulate cellular growth and differentiation. Several proto-oncogenes encode for protein products with associated tyrosine kinase activity. An assay for TPK activity was performed in cell extracts using a synthetic peptide substrate and [32P] adenosine triphosphate (ATP). TPK activity was elevated in K-562 cells, which possess an amplified c-abl oncogene, compared to normal blood mononuclear cells (K-562 = 9.37 +/- 1.72 [mean +/- standard deviation] pmol ATP/10(6) cells/min; normal = 1.14 +/- 0.46, p less than 0.01). TPK activity was measured in peripheral blood mononuclear cells from patients with hairy cell leukemia (HCL), myelomonocytic leukemia (MOL), acute myeloblastic leukemia (AML), and chronic lymphocytic leukemia (CLL). In patients with clinically active disease, elevated TPK activity was measured in mononuclear cells from five HCL patients (range 3.76-24.15) and from seven MOL patients. These elevated levels appeared to parallel disease activity, as low levels of TPK activity were measured in patients with inactive (treated) disease. Low levels of TPK were measured in mononuclear cells from active AML and CLL patients. Elevated TPK levels in patients with HCL and MOL may reflect the overexpression of a proto-oncogene or increased growth factor activity in immature or rapidly dividing leukemic cells. Serial TPK levels in HCL and MOL patients correlated with change in disease activity.

Fractures about the interphalangeal joints in children.

Fracture patterns in 87 interphalangeal joint fractures in children's hands were studied and correlated with unique anatomic features of these joints. The collateral ligaments, which extend beyond the physis into the metaphysis, were found to protect the growth plate in the frontal plane. With laterally directed forces, fractures on the proximal side of the joint occurred, rather than epiphyseal fractures. There was good remodeling of angular deformities in the sagittal plane, but minimal correction in the frontal plane. In those patients treated surgically, angular deformities did not result. Of 24 condylar fractures followed for an average of 55 months, 11 (46%) demonstrated some restriction of joint motion.

Splice variants of tissue factor promote monocyte-endothelial interactions by triggering the expression of cell adhesion molecules via integrin-mediated signaling.

BACKGROUND: TF is highly expressed in cancerous and atherosclerotic lesions. Monocyte recruitment is a hallmark of disease progression in these pathological states. OBJECTIVE: To examine the role of integrin signaling in TF-dependent recruitment of monocytes by endothelial cells. METHODS: The expression of flTF and asTF in cervical cancer and atherosclerotic lesions was examined. Biologic effects of the exposure of primary microvascular endothelial cells (MVEC) to truncated flTF ectodomain (LZ-TF) and recombinant asTF were assessed. RESULTS: flTF and asTF exhibited nearly identical expression patterns in cancer lesions and lipid-rich plaques. Tumor lesions, as well as stromal CD68(+) monocytes/macrophages, expressed both TF forms. Primary MVEC rapidly adhered to asTF and LZ-TF, and this was completely blocked by anti-ß1 integrin antibody. asTF- and LZ-TF-treatment of MVEC promoted adhesion of peripheral blood mononuclear cells (PBMCs) under orbital shear conditions and under laminar flow; asTF-elicited adhesion was more pronounced than that elicited by LZ-TF. Expression profiling and western blotting revealed a broad activation of cell adhesion molecules (CAMs) in MVEC following asTF treatment including E-selectin, ICAM-1 and VCAM-1. In transwell assays, asTF potentiated PMBC migration through MVEC monolayers by ∼3-fold under MCP-1 gradient. CONCLUSIONS: TF splice variants ligate ß1 integrins on MVEC, which induces the expression of CAMs in MVEC and leads to monocyte adhesion and transendothelial migration. asTF appears more potent than flTF in eliciting these effects. Our findings underscore the pathophysiologic significance of non-proteolytic, integrin-mediated signaling by the two naturally occurring TF variants in cancer and atherosclerosis.

Overexpression of Bcl-2 by activated human hepatic stellate cells: resistance to apoptosis as a mechanism of progressive hepatic fibrogenesis in humans.

BACKGROUND AND AIMS: Myofibroblast-like cells, originating from activation of hepatic stellate cells (HSC/MFs), play a key role in liver fibrosis, a potentially reversible process that may rely on induction of HSC/MFs apoptosis. While this possibility has been shown in cultured rat HSC, very limited data are currently available for human HSC/MFs. METHODS: Cultured human HSC/MFs were exposed to several proapoptotic stimuli, including those known to induce apoptosis in rat HSC/MFs, and induction of cell death and related mechanisms were investigated using morphology, molecular biology, and biochemical techniques. RESULTS: In this study we report that fully activated human HSC/MFs did not undergo spontaneous apoptosis and survived to prolonged serum deprivation, Fas activation, or exposure to nerve growth factor, tumour necrosis factor alpha (TNF-alpha), oxidative stress mediators, doxorubicin, and etoposide. Induction of caspase dependent, mitochondria driven apoptosis in HSC/MFs was observed only when protein synthesis or transcription were inhibited. Importantly, the process of HSC activation was accompanied by changes in expression of a set of genes involved in apoptosis control. In particular, activated human HSC/MFs in culture overexpressed Bcl-2. The role of Bcl-2 was crucial as Bcl-2 silenced cells became susceptible to TNF-alpha induced apoptosis. Finally, Bcl-2 was markedly expressed in HSC/MFs present in liver tissue obtained from patients with hepatitis C virus related cirrhosis. CONCLUSIONS: Human activated HSC/MFs are resistant to most proapoptotic stimuli due to Bcl-2 overexpression and this feature may play a key role in the progression of fibrosis in chronic liver diseases.

Modification of the oxygen affinity and intracellular hemoglobin concentration of normal and sickle cells by means of an osmotic pulse.

Important properties of red blood cells (RBCs), including oxygen affinity and intracellular hemoglobin concentration, may be modified by means of an osmotic pulse. The pulse is developed by rapid dilution of a suspension of RBCs to which dimethylsulfoxide has been added. The nature and degree of RBC modification is dependent on the composition of the diluent solution. The purpose of this investigation was to explore the dependence of these changes on diluent composition and to determine the stability of altered cellular properties with in vitro incubation. For both normal control RBCs (A cells) and RBCs from patients with sickle cell anemia (S cells), cells with increased volume and markedly decreased intracellular hemoglobin concentration (65% to 70% of control) or with normal volume and moderately decreased hemoglobin concentration (80% to 85% of control) were prepared. These modifications were accomplished with hemoglobin yields ranging from 60% to 90%, depending on the diluent used, the cell type, and the intensity of treatment. For the same degree of inositol hexaphosphate (IHP) incorporation into A cells, as shown by the shift in oxygen half-saturation pressure (P50), diluent formulations with constant total osmolality but varying IHP concentration gave the same degree of hemoglobin loss per cell. Posttreatment cell size, however, ranged from slightly smaller than control to approximately 20% larger. On incubation, the larger cells returned toward normal size, whereas those with normal posttreatment size remained constant. S cells showed the same general changes with treatment and incubation but with greater variation. The treated S cells exhibited markedly reduced morphologic sickling on deoxygenation.

Inhibition of sickling after reduction of intracellular hemoglobin concentration with an osmotic pulse: characterization of the density and hemoglobin concentration distributions.

Hemoglobin S polymerization is markedly dependent on intracellular hemoglobin concentration. In the studies presented here, sickle RBC were subjected to a transient osmotic stress, which induced a short period of increased membrane permeability and allowed partial efflux of Hb S. Morphological sickling of the resulting hypochromic RBC was inhibited. The response of RBC to this osmotic pulse is influenced by the presence of a polyanion, which in these experiments was either inositol hexaphosphate (IHP, 27 mM or 46 mM) or pyrophosphate (69 mM or 95 mM). The decrease in MCHC, measured manually, ranged from 3.1 +/- 1.7 (1 SD) to 6.3 +/- 2.8 g/dl, depending on the conditions used during modification. Parallel electronic analysis of RBC indices demonstrated a comparable decrease in MCHC which was due to both an increased MCV and a decreased MCH. Since the modified cell population is quite heterogeneous, cells were analyzed using discontinuous stractan gradients and/or a laser-based instrument which measures the hemoglobin concentration (HC) of individual cells. For most treatment conditions, the modified cells have a bimodal HC distribution with one peak centered at about 20 g/dL and the other peak corresponding to the unmodified cells. With the higher concentration of IHP, however, many cells had an intermediate HC. For modified RBC with a bimodal HC distribution (27 mM IHP, 69 mM PP, 95 mM PP), inhibition of morphological sickling was proportional to the change in HC and there were no subpopulations with an increased tendency to undergo sickling. However, the intermediate density cells present when RBCs were treated with the higher concentration of IHP underwent sickling at a higher oxygen partial pressure than control cells.

Activation and partial characterization of a human reticulocyte heme-dependent eIF-Z alpha kinase.

An inhibitor of globin translation initiation that is activated under heme-deficient conditions and whose activity is associated with phosphorylation of the 38,000 dalton (alpha) subunit of the initiation factor eIF-2 has been previously described in rabbit reticulocytes. We describe here an analogous enzyme in human reticulocytes. The human enzyme can be activated in intact reticulocytes under conditions of heme synthesis inhibition or by incubation of lysates in the absence of hemin. Addition of hemin to lysates prevents activation of the eIF-2 alpha kinase. The partially purified kinase is associated with inhibition of globin synthesis in a rabbit cell-free system and phosphorylates the small subunit of highly purified rabbit eIF-2. We conclude that human reticulocytes contain a protein kinase that is analogous to the rabbit HCR and that may play a role in clinical heme deficiency states. l

Asynchronous globin chain synthesis in rabbit reticulocyte lystates induced by hemin-controlled repressor.

To define further the role of hemin-controlled repressor (HCR) in globin synthesis, we studied its effect on the synthesis of individual globin chains in a rabbit reticulocyte lysate cell-free system. In the presence of HCR there was a marked globin chain imbalance, resulting in a lowered alpha/beta ratio. These findings in vitro may have relevance to certain clinical heme deficiency states in which a similar globin chain imbalance has been observed.

The effect of INH-inhibited heme synthesis on globin synthesis.

In the rabbit reticulocyte cell-free system, optimal globin synthesis is dependent upon an adequate level of heme. The effect of heme is mediated by an inhibitor of globin synthesis initiation termed HCR. In addition to marked inhibition of total globin synthesis, HCR results in a decreased alpha/beta globulin synthesis ratio. We describe here the use of INH as a relatively nontoxic inhibitor of heme synthesis in intact rabbit reticulocytes with a resultant inhibition of globin synthesis. In parallel with the inhibition of globin synthesis in reticulocytes, an inhibitor of globin synthesis in the hemin-supplemented cell-free system is generated. No INH-induced alterations in alpha/beta synthesis ratio could be found in "stress" reticulocytes from phenylhydrazine-treated rabbits, but "normal" reticulocytes from untreated rabbits showed a decreased alpha/beta ratio. Inhibition of heme synthesis and the resulting decrease in globin synthesis and intracellular hemoglobin concentration may have application as a potential treatment of homozygous sickle cell disease.

Irreducible palmar dislocation of the proximal interphalangeal joint with bilateral avulsion fractures.

An unusual case of an irreducible anterior fracture dislocation of the proximal interphalangeal joint in a child with open epiphyses is described. Bilateral intraarticular avulsion fractures at the proximal attachment of the collateral ligaments were present, and the phalangeal head was locked through a tear in the central slip.

Deoxygenation-induced cation fluxes in sickle cells. IV. Modulation by external calcium.

Net cation movements were measured in low-density sickle red blood cells (SS RBC) in the presence and absence of oxygen. External Ca2+ (Ca2+o) partially inhibited deoxygenation-induced fluxes of both Na+ and K+. Deoxygenation-induced Na+ influx was reduced by 2 mM Ca2+o to 0.71 +/- 0.04 (SE) of its value in Ca(2+)-free solutions, whereas this ratio was 0.90 +/- 0.05 for K+ efflux (P < 0.01 by paired t-test). Because Ca2+o inhibited Na+ influx more than K+ efflux, net cation loss in deoxygenated SS RBC was higher in the presence of Ca2+o. In separate experiments, Ca2+o reduced deoxygenation-induced Na+ influx to 0.66 +/- 0.03 of its Ca(2+)-free value compared with 0.77 +/- 0.03 for Rb+ influx (P < 0.001), indicating relative selectivity of this effect for Na+ over Rb+. However, this effect is not specific for Ca2+ because other divalent cations also inhibited deoxygenation-induced Na+ and K+ fluxes. Under the conditions of these experiments, no evidence for K+ channel activation was found, indicating that K+ loss measured in deoxygenated SS RBC was mediated by the deoxygenation-induced pathway. These studies show that in the presence of Ca2+o deoxygenation-induced Na+ influx and K+ efflux are unbalanced. This pathway can, therefore, mediate cation loss and contribute directly to cellular dehydration in SS RBC.

Enzymatic and immunohistochemical evaluation of tyrosine phosphorylation in breast cancer specimens.

Using a synthetic peptide substrate, tyrosine protein kinase (TPK) activity was measured in 21 tumors from patients with histologically confirmed breast cancer and in five normal breast tissues from patients undergoing reduction mammoplasty. In 20 of 21 cancer specimens, tumor was available to assess phosphotyrosine (PT) immunohistochemically. Breast cancer specimens possessed significantly more TPK activity than normal breast tissues (Cancer = 43.9 +/- 3.1 pm/mg protein/min, [Mean +/- S.E.M.]; Normal = 3.4 +/- 0.9, p < 0.001). TPK activity was higher in the clinically more aggressive infiltrating ductal cancers compared to the less aggressive intraductal cancers (Infiltrating = 55.9 +/- 5.8; Intraductal = 17.2 +/- 3.4, p < 0.01). TPK activity in tumors with both infiltrating and intraductal histology was intermediate (34.0 +/- 7.2). Significant correlation existed between membrane TPK enzymatic activity and PT expression by immunohistochemistry. There was no relationship between estrogen or progesterone receptor status and TPK activity or PT; however, TPK activity from node negative breast cancer tissue was significantly less than from node positive specimens (p < 0.01). We conclude that breast cancer specimens possess elevated amounts of TPK which correlate with PT expression, and that increased tyrosine phosphorylation appears to correlate with the biologic aggressiveness of the malignant tumor.