Orlando J. Martelo

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.

Electron spin resonance (ESR) studies of skeletal protein interactions in human erythrocyte membranes exposed to polyanions and in membranes prepared from inositol hexaphosphate (IHP)-incorporated low-affinity erythrocytes

Previous biophysical investigations, including those from our laboratories, have reported that polyphosphates weaken RBC membrane skeletal protein-protein interactions and decrease hemoglobin affinity for oxygen. We have additionally demonstrated that low-affinity intact RBCs may be produced by inositol hexaphosphate (IHP) incorporation via an osmotic pulse method. In the present electron spin resonance (ESR) study, IHP was shown to cause a concentration-dependent increase in the segmental motion of ghost membrane skeletal proteins, but no alterations in spin-labeled terminal sialic acid. Pyrophosphate and inositol hexasulfate were significantly less effective in altering the physical state of skeletal proteins than was IHP. Additional ESR studies of both the interaction of IHP with membrane skeletal proteins in the presence of hemoglobin and of membranes obtained from osmotic pulse-treated intact cells were performed. The results of all these studies are discussed in terms of previous biophysical investigations of the effects of polyphosphates on membranes and of possible molecular events that occur during the osmotic pulse procedure.

Enzymatic and immunohistochemical evaluation of tyrosine phosphorylation in breast cancer specimens

SummaryUsing 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.

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.

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.

Late Intensification Therapy in Adult Acute Lymphoid Leukemia -Long-Term Follow-Up of the Southeastern Cancer Study Group Experience

One hundred and ninety-two evaluable patients were treated on a multicenter protocol for adult acute lymphoid leukemia to determine in a prospective randomized fashion if late intensification chemotherapy beginning after about six months of treatment would improve remission duration and survival. The complete remission rate was 60%. The median remission duration from beginning of maintenance was 18.7 versus 25.9 months (P = 0.36) for standard maintenance therapy and late intensification, respectively, and the median survival from randomization was 25.8 versus 28.5 months (P = 0.94) respectively. There was a suggestion that the late intensification strategy was helpful with respect to remission duration, and this trend was sustained in long-term follow-up. However, relapse proved to be common during the earlier phases of treatment; thus, insufficient numbers of patients were available at the randomization point to conclusively address the possible value of late intensification. Intensive therapy earlier in the course of treatment should be evaluated, including transplantation in selected patients.

Immunohistochemical classification of acute leukemias using peripheral blood smears.

Immunophenotypic classification of the acute leukemias (AcL) is of well documented value in those of lymphoid or uncertain origin and of increasing importance in those of nonlymphoid origin. Most of these studies have been performed on viable cell suspensions. To study the efficacy of a simpler immunohistochemical approach to the classification of the acute leukemias requiring only peripheral blood smears, 15 AcL (including three CGL-BC) were studied using an immunoalkaline phosphatase method and a panel of anti-lymphoid and anti-myeloid monoclonal antibodies. Routine cytochemistries were also performed (Sudan black, PAS). Using immunohistochemistry, five cases marked as common ALL (four were undifferentiated by cytochemistry, one ALL), eight cases as ANLL (all ANLL by cytochemistry) and two cases marked only with anti-HLA-DR (AUL by cytochemistry). These results show that immunophenotypic analysis of AUL, ALL and ANLL can be successfully performed even when only air dried peripheral blood smears are available.

Lack of inhibition of protein synthesis by double-stranded RNA in a cell-free system prepared from human reticulocytes

Abstract There are two inhibitors of protein synthesis which are related to the activity of interferon. One is a protein kinase which phosphorylates the α subunit of the eucaryotic initiation factor 2 (eIF-2). The other is an enzyme which synthesizes an unusual oligonucleotide that in turn activates a RNA endonuclease. In nucleated cells the synthesis of the inhibitors is induced by interferon but they must be activated in a subsequent lysate by double-stranded RNA (dsRNA). Rabbit reticulocytes, however, contain the inactive forms of the inhibitors in a constitutive manner and require only dsRNA activation. We report here the effect of dsRNA on protein synthesis and the generation of ribosomal eIF-2α kinase and heat-stable (oligonucleotide) inhibitory activity in human reticulocyte lysates. Our findings indicate that human reticulocytes, in contrast to rabbit reticulocytes, do not contain the interferon-related inhibitors of protein synthesis in a constitutive manner. Addition of dsRNA to the human reticulocyte cell-free system does not result in significant inhibition. Furthermore, no generation of ribosomal eIF-2α kinase or heatstable inhibitory activity could be detected. Direct addition of oligonucleotide or eIF-2α kinase (of rabbit origin), however, does result in inhibition of the human system. Thus, the ultimate inhibition mechanisms do appear operative in the human reticulocyte lysates. The differences between the rabbit and human systems may be due to either basic differences in the mechanism of interferon action or simply to variation in the history or maturity of the cells studied.