PFK inhibition test for cancer detection: clinical applications and mechanisms of PFK inhibition

A newly established cancer marker, the PFK inhibition test, has been further examined for its capacity to detect malignant neoplasms irrespective of the organs in which cancer cells start proliferating. We tested 1,160 sera from cancer patients and compared them with 756 normal sera, using histograms and normal paper for analysis of accumulated frequency. PFK activity through the influence of normal sera showed normal distribution, and cancerous sera shifted to the inhibitory site with an irregular shape. From these analyses, the patients were classified into the following types: normal range: PFK greater than SD (standard deviation of PFK activity in normal sera); suspicious range: SD greater than PFK greater than 2SD, must be given the PFK test again; and dangerous range: PFK less than 2SD, further examination must be carried out to detect cancer. Fifty percent of the sera from all the cancer patients inhibited PFK beyond 2 SD of normal sera. We also analyzed organ-associated PFK distribution, eg, gastric, colorectal, and mammary cancer. In gastric cancer, PFK inhibition was stronger in accordance with how far a particular stage of cancer had progressed. However, 50% of sera from stage I gastric cancer patients was positive beyond the cut-off line of 2 SD. We examined 104 sera from patients diagnosed as benign prostatic hypertrophy and found malignant cells in 10 patients whose sera tended to be positive in PFK inhibition. The PFK inhibitory factor in the body fluids of cancer patients was fractionated by Sephadex G-75 gel filtration and DEAE ion exchange chromatography. The approximate molecular weight of this factor was 13,000 daltons. The factor was resistant to heat and acid (0.1 N HCl and H2SO4) and was sensitive to 0.1 N NaOH and phosphate buffer. Diluted sulfuric acid and ammonium sulfate made an inactive NaOH-treated sample active when lyophilized following dialysis against distilled water. PFK inhibition by cancerous sera was eliminated by fructose-2,6-bisphosphate (the strongest activator of PFK) in a dose-dependent manner. PFK attached to agarose beads was found to be reversible even after being inhibited by cancerous body fluids and ATP water solution. Although PFK is apt to decay in a low pH range, the established procedure did not destroy PFK, but induced a direct inhibition of PFK by ATP through the ATP inhibition site on the PFK molecule. The PFK inhibitor may possibly function as a proton carrier and release protons to activate the ATP inhibition site.(ABSTRACT TRUNCATED AT 400 WORDS)     

An immunohistochemical study of MAP2 and clathrin in gerbil hippocampus after cerebral ischemia

Changes in MAP2 and clathrin immunoreactivity were studied in gerbil hippocampus after transient cerebral ischemia. MAP2 immuno-reactivity decreased significantly by 1 h in the subiculum-CA1 and CA2 areas which correspond to reactive change, while no decrease was observed in CA1 until day 4. Before the initiation of delayed neuronal death, MAP2 immunoreactivity was not changed in CA1. On the other hand clathrin immunoreactivity increased in the pyramidal cell layer of CA1 by 3 h after ischemia and remained high for 2 days. Clathrin immunoreactivity in the pyramidal cell layer of CA1 diminished after delayed neuronal death. The transient change of clathrin was noted especially in CA1 in the period prior to delayed neuronal death. These results imply an abnormal change in clathrin turnover after ischemia, which may participate in the pathogenesis of delayed neuronal death.     

Qualitative abnormalities of choline acetyltransferase in Alzheimer type dementia

The maximum activity of choline acetyltransferase (ChAT), the affinity for choline or acetyl-CoA and the isozyme pattern in the cerebral cortex of 5 cases of Alzheimer type dementia (ATD) and 6 age-matched control subjects were examined post-mortem. Maximum activities of ChAT were estimated in 5 cerebral cortical areas of Brodmann: 4, 7, 10, 17 and 22. A significant reduction in maximum activities of ChAT was found in all cortical areas for the cases of ATD. The affinity for choline or acetyl-CoA was measured in the frontal cortex (Brodmann's areas 4 and 6) and in the temporal cortex (Brodmann's areas 21 and 22). The affinity was significantly decreased in both cortices of demented patients. A significant correlation was observed between maximum activity of ChAT and the affinity for choline or acetyl-CoA. The isozyme pattern obtained by column chromatography on Sephadex G-200 was similar to that obtained by centrifugation on a sucrose density gradient. The isozyme pattern of ATD was different from that of the control subjects. These results suggest qualitative as well as quantitative abnormalities in the ChAT in autopsied brains of ATD.     

Ketamine Suppresses Platelet Aggregation Possibly by Suppressed Inositol Triphosphate Formation and Subsequent Suppression of Cytosolic Calcium Increase

Background: Ketamine has been shown to suppress platelet aggregation, but its mechanisms of action have not been defined. The purpose of the current study is to clarify the effects of ketamine on human platelet aggregation and to elucidate the underlying mechanisms of its action.

Methods: Platelet aggregation was measured using an eight-channel aggregometer, and cytosolic free calcium concentration was measured in Fura-2/AM-loaded platelets using a fluorometer. Inositol 1,4,5-triphosphate (IP3) was measured with use of a commercially available IP3 assay kit. To estimate thromboxane A2 (TXA2) receptor binding affinity and expression, Scatchard analysis was performed using [3H]S145, a specific TXA2 receptor antagonist. TXA2 agonist binding assay was also performed. The membrane-bound guanosine 5'-triphosphatase activity was determined using [[gamma]-32P]guanosine triphosphate by liquid scintillation analyzer.

Results: Ketamine (500 [mu]m) suppressed aggregation induced by adenosine diphosphate (0.5 [mu]m), epinephrine (1 [mu]m), (+)-9,11-epithia-11,12-methano-TXA2 (STA2) (0.5 [mu]m), and thrombin (0.02 U/ml) to 39.1 +/- 30.9, 46.3 +/- 4.3, -2.0 +/- 16.8, and 86.6 +/- 1.4% of zero-control, respectively. Ketamine (250 [mu]m-1 mm) also suppressed thrombin- and STA2-induced cytosolic free calcium concentration increase dose dependently. Although ketamine (2 mm) had no effect on TXA2 receptor expression and its binding affinity, it (1 mm) suppressed intracellular peak IP3 concentrations induced by thrombin and STA2 from 6.60 +/- 1.82 and 4.39 +/- 2.41 to 2.41 +/- 0.98 and 1.90 +/- 0.86 pmol/109 platelets, respectively, and it suppressed guanosine triphosphate hydrolysis induced by thrombin (0.02 units/ml) and STA2 (0.5 [mu]m) to 50.3 +/- 3.2 and 67.5 +/- 5.5%versus zero-control, respectively.     

Spinal Trabecular Bone Loss and Fracture in American and Japanese Women

This study examined trabecular bone mineral density (BMD) in Japanese women with and without spinal fracture, and compared the results to American women with and without fracture. The quantitative computed tomography (QCT) systems used at the University of California, San Francisco (UCSF) and at Nagasaki University were cross-calibrated. Normative BMD was assessed with the K₂HPO₄ liquid phantom in 538 Americans aged 20–85 years, and with the B-MAS200 phantom in 577 Japanese aged 20–83 years. These BMD were adjusted for use with the Image Analysis solid phantom using the result of cross-calibration. The trabecular BMD in 111 postmenopausal American women (55 with fracture), and in 185 postmenopausal Japanese women (67 with fracture) were compared for investigation of the difference in BMD values relative to fracture status. The absolute BMD values in Japanese were lower than those in Americans, and the differences were greater with advancing age. The magnitude of the BMD difference was 8.6, 20.5, 38.1 mg/cm³ in women aged 20–24 years, 40–44 years, 60–64 years, respectively. In premenopausal women, BMD began to decrease at the age of 20 in Japanese, whereas the peak bone mass was maintained until the age of 35 in the American women. In immediate postmenopausal women, BMD significantly decreased in both populations. In later postmenopausal women, BMD significantly decreased with age in the Japanese women but decreased less rapidly in the American women. The aging decrease of BMD was 1.4% and 2.2% per year in the later postmenopausal American and Japanese women, respectively. The fracture threshold is considered to be lower in Japanese women. However, the BMD difference between American and Japanese women with fracture was similar to that without fracture. The Z-scores of fracture subjects versus controls were 2.9 in American and 1.8 in Japanese women. In conclusion, Japanese women were found to have a lower BMD and lower fracture threshold than American women. The significant decrease of spinal trabecular BMD in late postmenopause is potentially responsible for the higher prevalence of spinal fracture in Japanese women. Received: 18 December 1995 / Accepted: 23 September 1996     

Suppressive effect of LD78 on the proliferation of human hemopoietic progenitors.

LD78 is a cDNA newly isolated from human stimulated tonsillar lymphocytes. The expression of LD78 is related to inflammatory responses and its structure has a homology with macrophage inflammatory protein 1-alpha, which is known to have an inhibitory effect on murine CFU-S. Using a colony assay technique, we examined the effects of LD78 on human hemopoietic progenitors. The addition of doses of 100 ng/ml or more of LD78 suppressed the colony formation of KMT-2, a factor-dependent myelomonocytic cell line established from cord blood cells; this suppressive activity was neutralized by the addition of antibody against LD78. The same doses of LD78 suppressed the formation of neutrophil, macrophage, and megakaryocytic colonies which were supported by human interleukin-3 and erythropoietin; however, LD78 did not affect colony formation by either non-phagocytic mononuclear cells or sorted CD34+ cells. The conditioned medium of KMT-2 cells or peripheral blood mononuclear cells cultured with LD78 suppressed colony formation by CD34+ cells. From these findings, it is suggested that LD78 affects phagocytic cells and induces factors that are inhibitory for hemopoiesis. We consider LD78 to be a new cytokine that plays an inhibitory role in hemopoiesis.     

Differential ontogeny of in vitro vascular responses to three categories of calcium channel antagonists in rats.

We examined the ontogeny of relaxation responses to three categories of calcium channel antagonists, represented by verapamil, diltiazem, and nifedipine, for both potential-operated (KCl-mediated) and receptor-operated channels [norepinephrine (NE)-mediated] in rat thoracic aorta. Aortic rings from 2- to 3-d, 1-wk, and 12-wk-old Sprague Dawley rats were mounted in an organ bath, bathed in Krebs' solution, and connected to a force-displacement transducer to measure isometric tension. Endothelium intact vessels at optimal passive force were exposed to a single ED50 of isotonic KCl or NE, equilibrium contraction was measured, then vessels were washed and exposed for 30 min to 1 microM verapamil, 1 microM diltiazem, or 0.1 microM nifedipine, followed by another dose of KCl or NE. Verapamil and diltiazem demonstrated significant (p less than 0.05) age-related increases in effectiveness for blocking KCl-mediated contraction [(% reduction of control contraction +/- SEM) (Verapamil: 2-3 d, 67.7 +/- 4.2; 1 wk, 72.5 +/- 1.8; 12 wk, 89.5 +/- 1.0. Diltiazem: 2-3 d, 64.6 +/- 2.9; 1 wk, 73.5 +/- 3.0; 12 wk, 83.1 +/- 1.8]. Nifedipine was equally effective at all ages: 2-3 d, 85.6 +/- 1.3; 1 wk, 90.0 +/- 1.6; and 12 wk, 91.3 +/- 1.4. Verapamil and diltiazem also showed significant age-related increases in effectiveness for blocking NE-mediated contraction (Verapamil: 2-3 d, 6.2 +/- 3.9; 1 wk, 28.0 +/- 4.8; 12 wk, 44.1 +/- 6.0. Diltiazem: 2-3 d, 8.0 +/- 3.1; 1 wk, 20.5 +/- 3.9; 12 wk, 46.5 +/- 4.8).(ABSTRACT TRUNCATED AT 250 WORDS)     

Discrimination of spinal fracture with various bone mineral measurements

For several different bone mineral measurements and various skeletal sites, we compared capability to discriminate between women in various age decades with and without spinal fracture, and attempted to identify the most effective cutoff level in discrimination of spinal fracture. The subjects were 88 women aged 50–59 years (including 32 with fracture), 95 women aged 60–69 years (including 54 with fracture), and 34 women aged 70–79 years (including 18 with fracture). Spinal trabecular and cortical bone mineral density (BMD) were measured using quantitative computed tomography (CT), and spinal, radial (ultra-distal, 10% distal and 33% distal), and calcaneal BMD were measured by dual X-ray absorptiometry. These BMD values were obtained in each subject on the same day. Three statistical techniques—Student's t-test, the logistic regression analysis, and the receiver operating characteristics (ROC) analysis— were applied and accuracy was calculated using the various cutoff values. The capability to discriminate between women with and those without fracture using these BMD values was different among the three age groups. In women aged 50–59 and 60–69 years, all measurements showed good capabilities for discriminating women with fracture. In women aged 70–79 years, these measurements showed lower capability than in those aged 50–59 and 60–69 years, but among them, the calcaneal and ultradistal radial BMD showed relatively good capability. The 10% and 33% distal radial BMD values were not useful in the detection of the high risk women with fracture. The cutoff BMD values for discrimination of women with fracture varied according to the sites and methods of measurement. For each specific age group, the most suitable measurement methods and the appropriate skeletal sites should be considered, and the effective cutoff values to discriminate those with fracture may differ according to the measurement methods, the skeletal sites examined, and age. Received: 5 February 1996 / Accepted: 18 June 1996