Anticoagulants preventing pseudo-thrombocytopenia]

It is well known that some anticoagulants, especially EDTA (ethylene diamine tetraacetic acid), sometimes lead to pseudo-thrombocytopenia due to the aggregation of thrombocytes. We evaluated appropriate anticoagulants that prevent pseudo-thrombocytopenia without affecting other hematological data. In this study, 10 mg EDTA-2K and 1 mg citric acid were added to 1 ml of blood as anticoagulants. Using these anticoagulants (EC method), an aggregation of thrombocytes was clearly inhibited and the platelet counts remained stable in 10 cases with pseudo-thrombocytopenia. This method did not affect the other blood cell counts, the stainability and the morphology of the cells. Since the addition of citric acid to EDTA could prevent the cell volume change induced by the high concentration of EDTA, the micro-hematocrit values were remained unchanged. Hematological data in 20 cases which did not show any pseudo-thrombocytopenia, correlated significantly between the EDTA method and the EC method, and did not change significantly 24 hours after blood sampling. It is concluded that EDTA and citric acid may be a useful combination of anticoagulants for the prevention of pseudo-thrombocytopenia.     

Histological properties and biological significance of pouch in red kangaroo, Macropus rufus

2 embryos, 4 youngs, 4 older youngs and the pouch of 2 mothers of the red kangaroos were examined. The results obtained are as follows: 1. The initial muscle spindles are already observed light microscopically in the vertebral, dorsal neck and forelimb muscles of the newborn baby and a little bit later in the masticatory muscles of the young of 68 mm in craniorump length and 28 g in body weight. 2. In the skin with less hair lining the inner surface of the pouch, abundant apocrine large sweat glands are observed, especially surrounding the basal region of the nipple and in the pleat formation of the skin. 3. The lactiferous mammary gland is enlarged, the lobules being divided by the interlobular muscle fiber tissue and enwrapped by the muscular capsule. The milk is squirted automatically by the muscle fiber contraction from the gland to the nipple, to which the baby attaches itself. 4. The musculature of the pouch wall is developed to form the sphincter muscle in the pouch orifice. The sphincter muscle plays an important role in conditioning the optimum temperature for the naked baby inside the pouch. 5. The apocrine perfume plays an important role in guiding the baby on the journey to the pouch after birth and the apocrine products also in maintaining the optimum humidity of the pouch to accomodate the baby. 6. During the long period of stay in the pouch, the masticatory and locomotive systems and their neuromuscular mechanism related to the herbivorous mastication become fully established and then the young leaves the pouch to feed on the animal's proper diet.     

ABC proteins: key molecules for lipid homeostasis

Forty-nine ABC protein genes exist on human chromosomes. Eukaryotic ABC proteins were originally recognized as drug efflux pumps involved in the multidrug resistance of cancer cells. However, it is now realized that one of their major physiological roles is cellular lipid transport and homeostasis, and their dysfunction is often associated with human diseases. ABCA1 and ABCA7 mediate the apolipoprotein-dependent formation of a high-density lipoprotein–cholesterol complex. ABCA3 is indispensable for pulmonary surfactant secretion. ABCG5 and ABCG8 are involved in the secretion of plant sterols and cholesterol into bile. However, the primary substrates and mechanism of action of these ABC proteins have not been precisely defined. In this review article, we first describe the general structure and functions of eukaryotic ABC proteins. The current model of ABCA1 functionality is then explained based on studies on a topological model, subcellular localization, apoA-I dependence of HDL formation, functional defects of Tangier disease mutants, and ATP hydrolysis of purified ABCA1. ABCA1 is supposed to function as a transporter of lipids as well as a receptor for apoA-I. ABCA3 is likely involved in accumulating phospholipids and cholesterol in lamellar bodies and in generating multivesicular structures.     

Spatial and temporal expression patterns of the cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 during mouse development

The cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 are thought to regulate progression of the cell cycle. We have previously shown that the phenotypes of p27-/- mice are substantially different from those of p57-/- mice, suggesting that spatial and temporal expression patterns of p27Kip1 and p57Kip2 might be distinct. In this study, the roles of p27Kip1 and p57Kip2 in development were examined by characterizing their expression patterns during mouse embryogenesis by immunohistochemical analysis. Whereas certain organs and tissues (brain, lens, ganglion, lung, heart, liver, skin and kidney) expressed both proteins, others expressed only p27Kip1 (thymus, spleen, retina, testis and ovary) or only p57Kip2 (gut, palate, pancreas, cartilage and skeletal muscle). In addition, some organs expressed both p27Kip1 and p57Kip2 but showed mutually exclusive patterns of distribution among tissues. Thus, in the adrenal gland, p57Kip2 was expressed in the cortex but not in the medulla, whereas p27Kip1 was expressed in the medulla but not in the cortex. Whereas the expression of p57Kip2 in most tissues was restricted to embryogenesis, expression of p27Kip1 in many tissues was maintained in adult animals. Double-label immunofluorescence staining with either anti-p27Kip1 or anti-p57Kip2 and anti-BrdU revealed that the expression of p27Kip1 and p57Kip2 was inversely correlated with cell proliferation, suggesting that p27Kip1 and p57Kip2 are expressed exclusively in postmitotic cells. These complex spatial and temporal patterns of expression are consistent with the phenotypes of mice deficient in p27Kip1 or p57Kip2, and they suggest that these proteins might play important roles in tissue development.     

A case of the esophageal candidiasis supposedly caused by rhinenchysis steroid chronic administration before sleep]

In general, steroid is mainly used as anti-inflammatory action in case of allergic diseases. As one of the side effects of inhalation steroid, a report is given below regarding buccal capsule/esophageal candidiasis. The patient came to the hospital with the chief complaint regarding passage dysphagia in the time of deglutition; pharyngitis and esophageal candidiasis were found by endoscopy of upper gastrointestinal tract.The interview after the endoscopy revealed that the patient, a 69-year-old female was diagnosed as chronic perennial allergic rhinitis a few years ago, and had been inhaling rhinenchysis Beclometasone dipropionate (BDP) before sleep every day for the past two years because using this collunarium seemed to mitigate the nasal obstruction and mucus during sleep. The patient did not report this fact before the endocsopy because she did not associate it with her subjective symptom. In this case, it was assumed that nebulized rhinenchysis BDP was accidentally swallowed to the pharynx and esophagus during sleep. As a treatment, rhinenchysis BDP was canceled and instead Azunol mouth washing (gargling/nasal douche) was used. No antifungal agent was used. In two weeks, the patient reported some improvement, and this was confirmed by reexamination of the upper gastrointestinal tract using endoscope in one month and a half. Pharyngitis was improved, and in the digital endoscopic assessment of esophageal candidiasis complicating inhaled steroid therapy the esophageal candidiasis became Grade I (mild grade). As for the later progress, the patient did not report any subjective symptoms such as nasal obstruction and dysphagia. In addition, the inflammation caused by candidiasis and found in the early examination was improved. The patient in this case was under treatment for thrombosis in the vein of lower extremity, but no complications such as diabetes mellitus or immune deficiency syndrome were observed. DISCUSSION: Esophageal candidiasis by chronic administration of inhalation of steroid before sleep for asthmatic patients has been reported. However, there has not been a report of esophageal candidiasis by chronic administration of rhinenchysis steroid before sleep for patients with allergic rhinitis. Similarly, in the case of the use of steroid in the form of collunarium before sleep, steroid stayed in the esophagus via the transendothelial nasal cavity, and that seemed to cause, in the long run, to develop esophageal candidiasis. CONCLUSIONS: One of the implications of the above case is that collunarium can go down, even when it is nebulized in the nasal cavity, to the esophagus via the nasal cavity to buccal capsule. This suggests the necessity for preventative measures in the case of chronic administration of steroid as follows. A. Blowing of the nose just after the use of collunarium B. Daily rinsing (gargling and nasal douche).     

Lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested DNA

The livers of DNase II-deficient mouse embryos contain many macrophages carrying undigested DNA, and the embryos die in utero. Here we report that erythroid precursor cells underwent apoptosis in the livers of DNase II-deficient embryos and that in the liver, interferon-beta mRNA was expressed by the resident macrophages. When the DNase II-deficient mice were crossed with mice deficient in type I interferon receptor, the resultant 'double-mutant' mice were born healthy. The double-mutant embryos expressed interferon-beta mRNA, but the expression of a subset of the interferon-responsive genes dysregulated in DNase II-deficient embryos was restored to normal. These results indicate that the inability to degrade DNA derived from erythroid precursors results in interferon-beta production that induces expression of a specific set of interferon-responsive genes associated with embryonic lethality in DNase II-deficient mice.     

Transgenic rabbits with increased VEGF expression develop hemangiomas in the liver: a new model for Kasabach-Merritt syndrome

Clinical studies have provided ample evidence that high (either systemic or local) levels of vascular endothelial growth factor (VEGF) are associated with several pathophysiological disorders, including hemangiomas. To investigate whether elevated VEGF expression could directly affect these disorders, we created a transgenic (Tg) rabbit model with increased hepatic expression of the human VEGF(165) transgene under the control of the human alpha-antitrypsin promoter. Tg rabbits exhibited marked hepatomegaly, with livers 2.5-fold heavier than those of control rabbits. Histological analysis revealed that the livers of Tg rabbits showed prominent dilation of the sinusoids and formed various-sized blood vessel networks, a feature of diffuse hemangiomas. Immunohistochemical staining revealed that the hepatocytes produced VEGF(165), whereas plasma VEGF(165) was not detected. Furthermore, Tg rabbits suffered from hemolytic anemia, thrombocytopenia and splenomegaly, which was associated with marked extramedullary hematopoiesis. The manifestations of Tg rabbits mimic many of the features of hemangiomatous disorders in humans such as the Kasabach-Merritt syndrome, and therefore this model may be potentially useful for the study of the pathogenesis and complications of hemangiomas as well as the investigation of angiogenesis inhibitors.     

Preparation of ceramic microspheres for in situ radiotherapy of deep-seated cancer

Radiotherapy is one of the most effective treatments for cancers. However, external irradiation provides only small doses to deep-seated cancers, and often causes damage to healthy tissues. It has been reported that 20-30 microm diameter 17Y(2)O(3)-19Al(2)O(3)-64SiO(2) (mol%) glass microspheres are useful for the in situ irradiation of cancers. Yttrium-89 (89Y) in this glass can be neutron bombarded to form the beta-emitter 90Y (half-life=64.1h). When injected in the vicinity of the cancer, such activated glass microspheres can provide a large localized dose of beta-radiation. The Y(2)O(3) content of the glass in the microspheres is limited to only 17 mol%. Chemically durable microspheres with a higher Y(2)O(3) content need to be developed. Phosphorus-31 (31P) with 100% natural abundance can also be activated by neutron bombardment to form the beta-emitter 32P (half-life=14.3d). Chemically durable microspheres containing a high phosphorus content are expected to be more effective for cancer treatment. We prepared pure Y(2)O(3) and YPO(4) microspheres using a high-frequency induction thermal plasma melting technique, and investigated the resulting structure and chemical durability. We successfully prepared smooth, highly spherical polycrystalline Y(2)O(3) and YPO(4) microspheres with diameters in the range 20-30 microm. Both the Y(2)O(3) and YPO(4) microspheres showed high chemical durability in saline solutions buffered at pH=6 and 7. These microspheres are expected to be more effective than the conventional glass microspheres for the in situ radiotherapy of cancer.     

Production of interferon-like substance by mouse spleen cells through contact with BHK cells persistently infected with HVJ

A virus inhibitor, an interferon-like substance, was found in the culture fluid of mouse spleen cells cocultivated with BHK-HVJ cells, the BHK cells persistently infected with HVJ, but not in the medium of cocultivation of mouse spleen cells and normal BHK cells. Neither the culture fluid of spleen cells alone nor that of BHK-HVJ cells alone was shown to contain the virus inhibitor. No virus inhibitory activity could be detected in the culture fluid of mouse spleen cells incubated with either the culture fluid of BHK-HVJ cells contained noninfectious HVJ particles or sonicated BHK-HVJ cell suspension. L cells or mouse liver cells, when cocultivated with BHK-HVJ cells, did not release a virus inhibitor.These findings suggest that mouse lymphoid cells have a capacity to produce a virus inhibitor when cocultivated with BHK-HVJ cells, and that nonlymphoid somatic cells may lack this capacity.Interposition of a Millipore filter between BHK-HVJ monolayer and mouse spleen cells or pretreatment of BHK-HVJ cells with anti-HVJ antiserum resulted in a blockade of virus inhibitor production. These findings suggest that the following sequence is necessary for the mouse spleen cells cocultivated with BHK-HVJ cells to produce the virus inhibitor: first, attachment of the spleen cells to BHK-HVJ cells and, second, recognition by the former of virus antigen(s) present on the surface of the latter.The BHK-HVJ cell membrane, isolated by sucrose density gradient centrifuge, was found to be an active inducer of the virus inhibitor. Moreover, some artificial membranous structures, such as HVJ-erythrocyte complex as well as HVJ spike-erythrocyte complex, exhibited a similar activity.This virus inhibitor induced in the present system appears to have all biologic attributes of interferon and its production might be initiated by membrane-membrane interaction between lymphoid cells and HVJ infected cells.