Abnormal chemokine-induced responses of immature and mature hematopoietic cells from motheaten mice implicate the protein tyrosine phosphatase SHP-1 in chemokine responses.

Chemokines regulate a number of biological processes, including trafficking of diverse leukocytes and proliferation of myeloid progenitor cells. SHP-1 (Src homology 2 domain tyrosine phosphatase 1), a phosphotyrosine phosphatase, is considered an important regulator of signaling for a number of cytokine receptors. Since specific tyrosine phosphorylation of proteins is important for biological activities induced by chemokines, we examined the role of SHP-1 in functions of chemokines using viable motheaten (me(v)/me(v)) mice that were deficient in SHP-1. Chemotactic responses to stromal call-derived factor 1 (SDF-1), a CXC chemokine, were enhanced with bone marrow myeloid progenitor cells as well as macrophages, T cells, and B cells from me(v)/me(v) versus wild-type (+/+) mice. SDF-1-dependent actin polymerization and activation of mitogen-activated protein kinases were also greater in me(v)/me(v) versus +/+ cells. In contrast, immature subsets of me(v)/me(v) bone marrow myeloid progenitors were resistant to effects of a number of chemokines that suppressed proliferation of +/+ progenitors. These altered chemokine responses did not appear to be due to enhanced expression of CXCR4 or lack of chemokine receptor expression. However, expression of some chemokine receptors (CCR1, CCR2, CCR3, and CXCR2) was significantly enhanced in me(v)/me(v) T cells. Our results implicate SHP-1 involvement in a number of different chemokine-induced biological activities.

Development and characterization of immobilized human organic anion transporter-based liquid chromatographic stationary phase: hOAT1 and hOAT2.

This paper reports the development of liquid chromatographic columns containing immobilized organic anion transporters (hOAT1 and hOAT2). Cellular membrane fragments from MDCK cells expressing hOAT1 and S2 cells expressing hOAT2 were immobilized on the surface of the immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The resulting stationary phases were characterized by frontal affinity chromatography, using the marker ligand [3H]-adefovir for the hOAT1 and [14C]-p-aminohippurate for the hOAT2 in the presence of multiple displacers. The determined binding affinities (Kd) for eight OAT1 ligands and eight OAT2 ligands were correlated with literature values and a statistically significant correlation was obtained for both the hOAT1 and hOAT2 columns: r2=0.688 (p<0.05) and r2=0.9967 (p<0.0001), respectively. The results indicate that the OAT1 and OAT2 have been successfully immobilized with retention of their binding activity. The use of these columns to identify ligands to the respective transporters will be presented.

Principal component analysis uncovers cytomegalovirus-associated NK cell activation in Ph+ leukemia patients treated with dasatinib.

Dasatinib treatment markedly increases the number of large granular lymphocytes (LGLs) in a proportion of Ph+ leukemia patients, which associates with a better prognosis. The lymphocytosis is predominantly observed in cytomegalovirus (CMV)-seropositive patients, yet detectable CMV reactivation exists only in a small fraction of patients. Thus, etiology of the lymphocytosis still remains unclear. Here, we identified NK cells as the dominant LGLs expanding in dasatinib-treated patients, and applied principal component analysis (PCA) to an extensive panel of NK cell markers to explore underlying factors in NK cell activation. PCA displayed phenotypic divergence of NK cells that reflects CMV-associated differentiation and genetic differences, and the divergence was markedly augmented in CMV-seropositive dasatinib-treated patients. Notably, the CMV-associated highly differentiated status of NK cells was already observed at leukemia diagnosis, and was further enhanced after starting dasatinib in virtually all CMV-seropositive patients. Thus, the extensive characterization of NK cells by PCA strongly suggests that CMV is an essential factor in the NK cell activation, which progresses stepwise during leukemia and subsequent dasatinib treatment most likely by subclinical CMV reactivation. This study provides a rationale for the exploitation of CMV-associated NK cell activation for treatment of leukemias.

Effects of Uric Acid on the NO Production of HUVECs and its Restoration by Urate Lowering Agents.

BACKGROUND: Although urate impaired the endothelial function, its underlying mechanism remains unknown. We hypothesized that urate impaired nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) via activation of uric acid transporters (UATs). PURPOSE AND METHOD: In the present study, we studied effects of urate on NO production and eNOS protein expression in HUVEC cells in the presence and absence of urate lowering agents using molecular biological and biochemical assays. RESULTS: HUVECs expressed the 4 kinds of UATs, URATv1, ABCG2, MRP4 and MCT9. Exposure to urate at 7 mg/dl for 24 h significantly reduced production of NO. Pretreatment with benzbromarone, losartan or irbesartan normalized NO production. The same exposure resulted in dephosphorylation of endothelial NO synthase (eNOS) in HUVECs. Again pretreatment with benzbromarone, losartan or irbesartan abolished this effect. CONCLUSION: Urate reduced NO production by impaired phosphorylation of eNOS in HUVEC via activation of UATs, which could be normalized by urate lowering agents.

High levels of Ca(2+)-independent endonuclease activity capable of producing nucleosomal-size DNA fragmentation in non-adherent marrow mononuclear cells from patients with myelodysplastic syndromes and acute myelogenous leukemia.

The endogenous endonucleases capable of producing nucleosomal-size DNA fragmentation are considered candidates of the key enzyme of apoptosis. We examined these activities in the nuclear fraction of non-adherent marrow mononuclear cells (NonAd-MNCs) from patients with myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) using a nuclear autodigestion method. We detected Ca2+/Mg(2+)-dependent endonuclease activity in all samples examined. In contrast, Ca(2+)-independent activity with the ability to produce nucleosomal-size DNA fragmentation was found only in samples from a proportion of patients with MDS (12 of 26 consecutive cases) and all the patients with AML (n = 6), but not in the samples from control group patients (n = 10). This activity was correlated with the percentage of bone marrow (BM) blast cells to some extent. Although the levels of these endogenous endonuclease activities seem not to be correlated directly with the susceptibility of the cells to apoptosis, we postulate that the Ca(2+)-independent endonuclease activity may be associated with apoptosis and/or cell proliferation. Further follow-up study of these patients may be meaningful to clarify the prognostic significance of the Ca(2+)-independent endonuclease activity in patients with MDS.

Marked apoptosis of human myelomonocytic leukemia cell line P39: significance of cellular differentiation.

Myelodysplastic syndrome (MDS)-derived leukemia cell line P39/Tsugane could be induced to apoptosis by a variety of agents including metabolic inhibitors, a calcium ionophore and differentiation-inducing agents. As evaluated by characteristic morphological changes and oligonucleosomal lengths DNA ladder, the levels of apoptosis in P39 cells induced by actinomycin D, or A23187, were far greater than in other myeloid lines examined in this study. When 22-oxa-1 alpha, 25(OH)2D3 (D3), dimethyl sulfoxide (DMSO) and all-trans retinoic acid (RA) were used as differentiation-inducers, varying degrees of apoptosis were seen. D3 induced monocytoid differentiation, but not apoptosis above the control level. On the other hand, RA induced profound apoptosis concomitant with the progressive expression of differentiation markers. Studies on morphology, functions and phenotypes of P39 cells exposed to differentiation inducers suggest that the incidence of apoptosis was not affected by the process of differentiation, but cells in the process of varying degrees of differentiation may die via apoptosis. Moreover, RA-treated P39 cells are unique in the simultaneous occurrence of profound apoptosis and differentiation. We propose that RA-treated P39 differentiation model is ideally suited for the study of MDS.

Apoptosis in myelodysplasia: a paradox or paradigm.

A growing body of evidence indicates that certain diseases are associated with increased apoptosis or inhibition of apoptosis. Among them, the myelodysplastic syndromes (MDS) are unique in that apoptosis is related, in apparently opposite directions, to the various facets of MDS. Ineffective hematopoiesis may be related to excessive intramedullary cell death via apoptosis and leukemic transformation conceivably results from escape from the apoptotic control. Future studies should be directed to define cellular susceptibility to and circumvention from apoptotic control mechanism(s).

Ca2+/Mg(2+)-dependent endonuclease in marrow CD34 positive and erythroid cells in myelodysplasia.

Endonucleases capable of producing internucleosomal DNA cleavage are one of the key enzymes in apoptosis. We examined endonuclease activities contained in nuclei of CD34+ and erythroid cells in the bone marrow (BM) from 12 patients with the myelodysplastic syndromes. The levels of Mg(2+)-dependent and acidic endonucleases showed little changes as compared with those from normal BM. By contrast, the level of Ca2+/Mg(2+)-dependent endonuclease was appreciably higher in MDS erythroid cells than normal counterparts, although the activity varied markedly in CD34+ and erythroid cells. Our results suggested that Ca2+/Mg(2+)-dependent endonuclease is related to ineffective erythropoiesis in MDS.

Intraatrial conduit repair in Scimitar syndrome.

A 26-year-old woman with partial anomalous pulmonary venous drainage into the right atrium (Scimitar syndrome) was successfully operated upon by incorporating an intra-atrial conduit. The single right pulmonary vein present was connected to the right atrial inferior vena cava junction. The atrial septum was extremely deviated to the left, and the left atrium was located entirely on the left of the spine. A small patent foramen ovale was found. The distance between the orifice of the right pulmonary vein and the enlarged atrial septal defect was too great to use an intracardiac patch, so a 14 mm woven Dacron graft, 6 cm long, was interposed between them. The patient is doing well 21 months following the operation.

Ruptured aneurysm of aortic sinus of Valsalva into right ventricle.

We report a patient with ruptured aneurysm of the aortic sinus of Valsalva into the right ventricle, whose heart murmur represented only a diastolic element without aortic regurgitation. Surgery revealed a small opening through myocardium of the ventricular septum without ventricular septal defect. During systole, the opening was constricted and presumably closed with myocardial contraction, and left-to-right shunt might have occurred only in diastole. This might lead to only a diastolic murmur.

Anomalous origin of left coronary artery from pulmonary artery.

An anomalous origin of the left coronary artery from the pulmonary artery in a 26-year-old man was corrected by the ligation of the artery at its anomalous origin, followed by the construction of an aortocoronary bypass with a venous graft. The flow of blood to the myocardium was measured for the purpose of comparing the effect of the bypass method with that of the ligation method. The result suggested that the bypass method provided a greater flow of blood to the myocardium than did ligation alone.

Ruptured aneurysm of aortic sinus of Valsalva into right atrium. Associated atrioventricular block presumable caused by aneurysmal compression of His bundle.

We have seen a case of rupture of an aneurysm of the noncoronary sinus into the right atrium. Surgery revealed an aneurysmal mass the size of the tip of an index finger extending through the inter-atrial septum down to just above the tricuspid valvular ostium. An electrocardiogram showed first-degree atrioventricular block, while the His bundle electrogram demonstrated the presence of disturbances in the intra-atrial as well as His bundle conduction. The disturbance in His bundle conduction was interpreted as being due to compression of the His bundle by the aneurysm.

Common atrium associated with anomalous high insertion of the inferior vena cava.

The case is reported of a 38-year-old housewife with a common atrium associated with anomalous high insertion of the inferior vena cava, absence of the coronary sinus, and drainage of a left hepatic vein into the left side of common atrium. Surgical repair was performed successfully. The procedure was closure of a nearly total atrial septal defect with a Dacron patch. Embroyologically, the development failure of the sinu-atrial fold appears to be the basic causal factor.

Treatment by pacemaker in familial amyloid polyneuropathy.

Amyloid deposits often involve the heart and cause disturbances in conduction and impulse formation in patients with familial amyloid polyneuropathy (FAP). Seven patients with FAP required pacemaker treatment during eight years. The most frequent bradyarrhythmias requiring pacing were sinus node dysfunction with junctional failure. Our seven patients had attacks and symptoms of bradyarrhythmias. A pacemaker relieved symptoms of bradycardias with recurrence of dislocation of electrode, exit block, and relatively high threshold. However, pacing did not improve the ultimate prognosis of FAP, because of progressive inanition of FAP. In our series, pacing should have been started earlier before advanced stage according to the ECG findings as in other diseases with bradycardias.

A new method for quantitative estimation of the degree of DNA fragmentation utilizing agarose gel electrophoresis.

We designed a new method for quantitative analysis of the degree of DNA fragmentation, a characteristic feature of apoptosis. A photograph of an agarose gel electrophoresis of fragmented DNA was incorporated by an optical density scanner or equivalent equipment, and the integrations of middle molecular size area (10,000 to 300 bp), single nucleosomal size area (smaller than 300 bp) and total lane area were calculated. We defined fragmentation rate or (%)FR as the amount of fragmented DNA expressed as the percentage of the total amount of DNA, assigning the coefficients of 1 and 0.5, respectively, to the fragments completely digested into single nucleosomal length and to those partially digested (between 10 k to 300 bp). A standard calibration curve was constructed from triplicate experiments of target nuclei digestion by micrococcal nuclease, which revealed that this method covered a wide range of nuclease activity. We also confirmed that our method was applicable to an autodigestion assay of isolated nuclei which represented endogenous endonuclease activities. This method may be a useful tool for quantitative analysis of endonuclease activities capable of producing nucleosomal-size DNA fragmentation.

Verbal memory disturbances in left temporal lobe epileptics.

The relationship between laterality of paroxysmal discharges and characteristics of disturbances in memory functioning was investigated in temporal lobe epileptics. Subjects consisted of 22 temporal lobe epileptic patients, in whom the foci of the paroxysmal discharges were localized to one side of the temporal regions. The left focus group consisted of 10 patients; the right focus group, 12. Subjects were required to recognize verbal material presented to one hemisphere by means of a tachistoscope. The left focus group alone failed to demonstrate a right visual field superiority in these tasks. It was concluded that the left focus group specifically demonstrate disturbances in verbal memory functioning, particularly when stimuli were presented to the left hemisphere. Paroxysmal discharges seemed to interfere with normal memory functioning in the region where the foci of these discharges were found.

Compensatory function of central dopaminergic system for suppressing psychic seizures--repeated plasma homovanillic acid measurement in refractory temporal lobe epilepsy.

Repeatedly measured plasma homovanillic acid concentration, as a clinically available index of central dopaminergic activity in a patient with temporal lobe epilepsy during a drug-free period, was significantly correlated with seizure frequency in the week immediately following, but not preceding, blood sampling days. This result suggests a compensatory function of the dopaminergic system in suppressing refractory psychic seizures.

Potassium transport and potassium channels in the kidney tubules.

A variety of K+ channels have been identified using electrophysiological techniques including the patch-clamp method. The types of channels include inwardly-rectifying, ATP-dependent, Ca(2+)-dependent, voltage-gated, and so on. Some of them have been cloned by expression and/or PCR cloning techniques, which give us their amino-acid sequences and molecular topology in the cell membrane. Immunohistochemical studies have shown the proteins (channels) to be localized to the luminal and/or basolateral membranes of a certain nephron segment. However, the relationships of these proteins with the ionic channels identified functionally must be examined by their reconstitution in cell-free systems (lipid bilayer membrane) and/or their expression in cells lacking native K+ channels. Much more care should also be taken to avoid artifacts due to channel-inducing factors (CHIF). Structure-function studies at the molecular level will advance our knowledge of the renal K+ channels and provide us with a further understanding of the role of the kidney in K+ homeostasis.

Renal compensation for body water loss during dehydration in neonatal rats.

The present study was designed to investigate whether the limited capacity for concentrating urine in neonatal rats is associated with an immature ability to regulate serum osmolality. During milk deprivation, the percent of reduction in body weight per 10 h (mean +/- SE) was 4.3 +/- 0.2, 3.7 +/- 0.1, 4.8 +/- 0.2, and 6.0 +/- 0.1% in 4-, 7-, 10-, and 14-d-old rats, respectively (n = 23-24, each age). The osmolality of urine increased to 718 +/- 12 (4 d), 741 +/- 28 (7 d), 792 +/- 20 (10 d), and 1,203 +/- 41 mosmol/kg H2O (14 d). Free-water absorption (TcH2O) promptly increased after deprivation of milk: It significantly increased from 2.3 +/- 0.3 (0-4 h) to 3.4 +/- 0.1 (4-7 h) (4 d), from 3.1 +/- 0.3 to 4.1 +/- 0.3 (7 d), from 3. 6 +/- 0.4 to 5.2 +/- 0.3 (10 d), and from 5.0 +/- 0.4 to 7.9 +/- 0.7 microliter/min/100 g (14 d). The raised values were maintained at the later period of dehydration. Thus serum osmolality was unchanged throughout dehydration: 287 +/- 1.0 (7 d) and 292 +/- 0.9 mosmol/kg H2O (14 d). On the other hand, the level of serum sodium concentration slightly but significantly increased (r = 0.61) when the body weight reduction was higher than 5% of the control (14-d-old rats). These results indicate that neonatal rats of 4-14 d control their serum osmolality by reabsorbing free water in the kidney during the 10 or 12 h of milk deprivation.

Roles of vasopressin and hypertonicity in basolateral Na/K/2Cl cotransporter expression in rat kidney inner medullary collecting duct cells.

The basolateral Na/K/2Cl cotransporter mRNA (rNKCC1) increased when the cultured kidney inner medullary collecting duct (IMCD) cells of rats were exposed to vasopressin (10(-8) M) and/or hypertonicity (500 mOsm/kgH2O). However, only hypertonicity was effective in increasing the expression of rNKCC1 protein.