Clinical analysis of HLA-DR-negative non-M3 AML

The type of leukemia was defined as HLA-DR(-) non-M3-AML, when HLA antigens were detected by flow cytometry at an incidence of < 20% of the blast population excluding M3-AML. Out of 109 patients with de novo acute myeloid leukemia, 8 patients had HLA-DR(-) non-AML-M3. According to the French-American-British criteria, 7 patients could be subdivided into 3 patients with M1, 4 patients with M2 and 1 patient with M4. The morphological features of bone marrow aspiration demonstrated no dysplasia and peroxidase stain positivity was noted in over 86% of the blast cells in all patients, the blast cells with fine granularity in 7 patients. The cytogenetic analysis revealed a normal karyotype. There was no expression marker of the blast antigens except CD13, CD14, CD33, CD34 and CD56. All of 7 patients who underwent induction therapy attained complete remission. Overall survival and disease-free survival showed no significant differences between the HLA-DR(-) non- M3-AML group and the HLA-DR(+) AML group.     

MR features of the substantia innominata and therapeutic implications in dementias

We measured the thickness of the substantia innominata using magnetic resonance imaging in 122 patients with Alzheimer's disease (AD), 31 patients with dementia with Lewy bodies (DLB) and 34 patients with vascular dementia (VaD), and examined the correlates of cognitive response to donepezil. Although all dementia groups showed significant atrophy of the substantia innominata compared to 28 age-matched controls, atrophy was greater in the DLB group, but less in the VaD group than the AD group. Mini-Mental State Examination score changes at 12 weeks after donepezil administration inversely and significantly correlated with the thickness of the substantia innominata in patients with AD (n=103, r=-0.43, p<0.0001) and in patients with DLB (n=24, r=-0.57, p<0.01), but not in patients with VaD (n=12, r=-0.22, p>0.1). There may be some differences in cholinergic impairment among AD, DLB and VaD, reflecting cholinergic neuropathology. Clinical response to cholinergic therapy may be partly attributable to damaged cholinergic neurons in AD and DLB, but not in VaD, suggesting differences in the therapeutic implication of cholinergic system degeneration.     

Sequential compound exocytosis of large dense-core vesicles in PC12 cells studied with TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis

We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of ∼7 s even though exocytosis was induced with large increases in cytosolic Ca²⁺ concentration by uncaging of a caged-Ca²⁺ compound. We also found that 97% of exocytic LVs remained poised at the plasma membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a polar membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.     

Effects of Maharishi Amrit Kalash 5 as an Ayurvedic herbal food supplement on immune functions in aged mice

Background  

Maharishi Amrit Kalash (MAK) 5, one of the Ayurvedic food supplements, belongs to a group of substances known as Rasayana. MAK5 and other Rasayanas are believed to enhance the body's resistance to infections and disease, and enhance longevity. In this study, we determined the effects of administration of MAK5, one of the Ayurvedic food supplements on immune functions in young and old mice.     

Plasma biopterin levels and tetrahydrobiopterin responsiveness

Tetrahydrobiopterin (BH4) responsive hyperphenylalaninemia (HPA) with a mutant phenylalanine hydroxylase (PAH) gene was found during neonatal screening for PKU. This study determined blood BH4 and phenylalanine in two patients with hyperphenylalaninemia following oral load with BH4 10 mg/kg. Our patients underwent neonatal screening for PKU, had normal biopterin metabolism and their PAH mutations were determined. Peak plasma biopterin levels in Case 1, which were reached at between 2 and 4h after loading, were 612, 297, and 178 nmol/L at age 30 days, 55 days, and 19 months, respectively, and the maximum phenylalanine decreasing rates, which were found at 24h, were 54, 16, and 4%, respectively. In Case 2, peak plasma biopterin levels were 747 and 327 nmol/L at age 20 and 55 days, respectively, and the maximum phenylalanine decreasing rates were 39 and 32%, respectively. In the BH4 loading test, the peaks of BH4 in both patients lowered ( approximately 50%), on the same dose schedule of BH4, as patients got older.     

Identification of a novel adhesion molecule involved in the virulence of Legionella pneumophila

Legionella pneumophila is an intracellular bacterium, and its successful parasitism in host cells involves two reciprocal phases: transmission and intracellular replication. In this study, we sought genes that are involved in virulence by screening a genomic DNA library of an L. pneumophila strain, 80-045, with convalescent-phase sera of Legionnaires' disease patients. Three antigens that reacted exclusively with the convalescent-phase sera were isolated. One of them, which shared homology with an integrin analogue of Saccharomyces cerevisiae, was named L. pneumophila adhesion molecule homologous with integrin analogue of S. cerevisiae (LaiA). The laiA gene product was involved in L. pneumophila adhesion to and invasion of the human lung alveolar epithelial cell line A549 during in vitro coculture. However, its presence did not affect multiplication of L. pneumophila within a U937 human macrophage cell line. Furthermore, after intranasal infection of A/J mice, the laiA mutant was eliminated from lungs and caused reduced mortality compared to the wild isolate. Thus, we conclude that the laiA gene encodes a virulence factor that is involved in transmission of L. pneumophila 80-045 and may play a role in Legionnaires' disease in humans.     

Spatiotemporal changes of coxsackievirus and adenovirus receptor in rat hearts during postnatal development and in cultured cardiomyocytes of neonatal rat

Coxsackievirus B is the most common cause of viral myocarditis and is particularly virulent in neonates and children. Adenovirus is also a leading cause of the disease. The determinant of tropism for both viruses is considered to be the expression of coxsackievirus and adenovirus receptor (CAR) in target organs. However, developmental change and physiological localization of CAR in the heart are unknown. We examined expression levels of CAR in rat hearts by quantitative real-time polymerase chain reaction and Western blot analysis and found that CAR decreased gradually during postnatal development, although CAR was detectable, even in adults. Immunohistochemistry revealed CAR on the whole surface of cardiomyocytes in immature rat hearts. In contrast, CAR was detected predominantly on intercalated disks in the adult heart and was accumulated especially at the contact point between the cultured cardiomyocytes, even though they were prepared from the neonatal rat heart. In conclusion, CAR was expressed abundantly on the whole surface of cardiomyocytes in immature rat hearts. Both the expression level and the localization of CAR are possible determinants of the susceptibility to viral myocarditis of neonates and children.     

Activity of synthetic enzymes of tetrahydrobiopterin in the human placenta

Tetrahydrobiopterin (BH4) is an essential co-factor for nitric oxide (NO) synthase (NOS) and regulates the production of NO, or endothelium-derived relaxation factor. Although NOS is highly expressed in the placenta and NO plays a critical role in the regulation of feto-placental circulation, the mechanism maintaining the level of BH4 is not known. To investigate the de novo synthesis of BH4 in the human placenta, the activity of guanosine triphosphate cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SR) in the chorionic tissue during the first, second and third trimester of pregnancy was analyzed. GTPCH activity was the lowest of the three enzymes and became negligible after the second trimester. There was no significant change in PTPS activity throughout pregnancy. Although SR activity decreased significantly after the second trimester, the levels remained abundant throughout pregnancy. These results showed that GTPCH is a rate-limiting enzyme and the total activity of the de novo synthesis of BH4 is negligible in the mature placenta after the second trimester when fetal growth is accelerated. The present study suggests that the level of BH4 in the placenta depends principally on the system other than de novo synthesis. The salvage pathway is considered the most potent system, which is formed by the transfer of the substrates from the fetus and their enzymatic conversion to BH4 in the placenta.     

Close association of aquaporin-2 internalization with caveolin-1

Aquaporin 2 (AQP2) is a membrane water channel protein that traffics between the intracellular membrane compartment and the plasma membrane in a vasopressin-dependent manner in the renal collecting duct cell to control the amount of water reabsorption. We examined the relation between AQP2 internalization from the plasma membrane and caveolin-1, which is a major protein in membrane microdomain caveolae, in Mardin-Darby canine kidney cells expressing human AQP2 (MDCK-hAQP2 cells). Double-immunofluorescence microscopy showed that AQP2 is colocalized with caveolin-1 in the apical plasma membrane by stimulating the intracellular signaling cascade of vasopressin with forskolin. After washing forskolin, both AQP2 and caveolin-1 were internalized to early endosomes and then separately went back to their individual compartments, which are subapical compartments and the apical membrane, respectively.Double-immunogold electron microscopy in ultrathin cryosections confirmed the colocalization of AQP2 with caveolin-1 at caveolar structures on the apical plasma membrane of forskolin-treated cells and the colocalization within the same intracellular vesicles after washing forskolin. A co-immunoprecipitation experiment showed the close interaction between AQP2 and caveolin-1 in forskolin-treated cells and in cells after washing forskolin. These results suggest that a caveolin-1-dependent and possibly caveolar-dependent pathway is a candidate for AQP2 internalization in MDCK cells.     

Primary somatosensory evoked magnetic fields elicited by sacral surface electrical stimulation

To explore the brain response to sacral surface therapeutic electrical stimulation (SSTES) for the treatment of refractory urinary incontinence and frequent micturition, evoked magnetic fields were measured in six healthy males. Electrical stimuli were applied between bilateral surface electrodes over the second through fourth posterior sacral foramens with intensity just below the pain threshold. Somatosensory evoked magnetic fields (SEFs) for the bilateral median (MN) and posterior tibial nerves (PTN) were also measured for the comparison. Sources of the early SEF peaks were superimposed on individual magnetic resonance images. The first peak latency for sacral stimuli, M30, occurred at 30.2+/-0.8 ms (mean+/-standard deviation, N=6), with shorter latency than those for PTN stimulus (39.3+/-1.4 ms, N=12) and longer latency than those for MN stimulus (21.0+/-0.9 ms, N=12). The second peak latency for sacral stimuli, M50, occurred at 47.2+/-2.9 ms (N=6). Both M30 and M50 peaks showed a single dipole pattern over the vertex in the isofield maps. The equivalent current dipoles of M30 and M50 were both estimated near the medial end of the central sulcus with approximately posterior current direction. These results suggest that the sacral M30 and M50 are responses from the primary somatosensory cortex. The relatively long time lag between the onset and peak of M30 suggests that SSTES directly affects both the cauda equina and cutaneous nerve of the sacral surface.