A Screening Method for Adrenoleukodystrophy Using a Dried Blood Spot on Filter Paper

The authors have devised a new, simple method for the diagnosis of adrenoleukodystrophy (ALD) using a dried blood spot sample. Fatty acid from the dried blood spot was extracted and methylated with HCl-methanol. Fatty acid methyl esters were analyzed by gas chromatography-mass spectrometry. Fatty acid composition of the blood spot from five patients with ALD and from five healthy controls was determined from the mass chromatogram of the m/z 143 ion, [(CH₂) ₆ COOCH₃]±. The ratios of tetracosanoic acid to docosanoic acid (C24:0/C22:0) and of hexacosanoic acid to docosanoic acid (C26:0/C22:0) were significantly greater in ALD patients than in controls. The C24:0/C22:0 ratios of ALD patients and of controls were 2.08 ± 0.33 and 1.47 ± 0.16 (mean ± SD), respectively. The C26:0/C22:0 ratios of ALD patients and of controls were 0.31 ± 0.024 and 0.092 ± 0.041, respectively. The fatty acid composition of the dried blood spot did not change at room temperature within a week. Since the specimens can be sent by mail, this method could be applied to the screening of other “peroxisomal” diseases, such as neonatal ALD and Zellweger syndrome, as well.     

In vivo mechanical condition plays an important role for appearance of cartilage tissue in ES cell transplanted joint

The objective of this study was to evaluate the effects of the mechanical environment on the formation of cartilage tissue in transplanted embryonic stem (ES) cells. Full‐thickness osteochondral defects were created on the patella groove of SD rats, and ES cells (CCE ES cells obtained from 129/Sv/Ev mice and Green ES FM260 ES cells obtained from 129SV [D3] ‐ Tg [NCAG‐EGFP] CZ—001–FM260Osb mice) were transplanted into the defects embedded in collagen gel. The animals were randomly divided into either the joint‐free group (JF group) or the joint‐immobilized group (JI group) for 3 weeks after a week postoperatively. The results showed that cartilage‐like tissue formed in the defects of the JF group whereas large teratomatous masses developed in the defects of the JI group. Some parts of the cartilage‐like tissue and the teratomatous masses were positively stained with immunostain for GFP when the Green ES FM260 ES cells were transplanted. It is suggested that the environment plays an important role for ES cells in the process of repairing cartilage tissue in vivo. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:10–17, 2008     

Immunohistochemical analysis of CYP2A13 in various types of human lung cancers

Human CYP2A13, which is expressed in the respiratory tract, is the most efficient enzyme for the metabolic activation of tobacco‐specific nitrosamines such as 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK). The relevance of CYP2A13 in carcinogenicity and toxicity in the respiratory tract has been suggested, but the expression of CYP2A13 protein in lung cancer tissues remains to be determined. We first prepared a mouse monoclonal antibody against human CYP2A13. The antibody showed no cross reactivity with the other CYP isoforms including CYP2A6. Using the specific antibody, we performed immunohistochemical analysis for human lung carcinomas. In adenocarcinomas (n = 15), all specimens were positive for the staining and five samples showed strong staining. In squamous cell carcinomas (n = 15) and large cell carcinomas (n = 15), each 14 samples were positive for the staining and two and three samples showed strong staining, respectively. In small cell carcinoma samples (n = 15), eight samples were negative for the staining and five samples showed weak or moderate staining. In conclusion, we first found that the expression of CYP2A13 was markedly increased in non‐small cell lung carcinomas. The high expression might be associated with the tumor development and progression in non‐small cell lung carcinomas. (Cancer Sci 2010; 101: 1024–1028)     

A case of idiopathic portal hypertension associated with systemic lupus erythematosus

A 29-year-old male who was diagnosed as having systemic lupus erythematosus (SLE) with hemolytic anemia and renal dysfunction at the age of 18 was diagnosed as having idiopathic portal hypertension (IPH) by angiography and liver biopsy. Improvement of thrombocytopenia and proteinuria, and transient increase of complement was observed after transabdominal devascularization with splenectomy (Hassab’s operation). The present case suggests that one of the aspects of the pathogenesis of IPH may be related with autoimmune mechanisms seen in patients with SLE.     

Hypertension in obese children: fasting serum insulin levels are closely correlated with blood pressure

The relationship between blood pressure and anthropometric or metabolic factors was studied in 324 obese children aged 9.5 +/- 1.8 years (mean +/- standard deviation). Obese children had a significantly higher blood pressure than non-obese children (systolic blood pressure: 121 +/- 14 mmHg in obese children vs 112 +/- 11 mmHg in non-obese children, P less than 0.001; diastolic blood pressure: 72 +/- 9 mmHg in obese children vs 66 +/- 7 mmHg in non-obese children, P less than 0.001). When the obese children were divided into hypertensive and normotensive groups, there was a significant difference in fasting serum insulin levels between the two groups (19.3 +/- 9.3 microU/ml in the hypertensive group vs 13.0 +/- 6.1 microU/ml in the normotensive group), and a close correlation between fasting serum insulin levels and systolic blood pressure was demonstrated (r = 0.63, P less than 0.001). However, there was no significant correlation between blood pressure and the degree of obesity itself or the waist-to-hip ratio in the obese children. There was no significant correlation between blood pressure and fasting plasma glucose, serum total cholesterol, or triglycerides levels in the obese children. Moreover, the correlation between fasting insulin levels and blood pressure was shown to be independent of the degree of obesity or waist-to-hip ratio and age by multiple regression analysis. These results indicate that hyperinsulinemia itself may play an important role in the pathogenesis of hypertension in obese children.     

Phosphorylation of the Mr 170,000 to 180,000 glycoprotein specific to multidrug-resistant tumor cells: effects of verapamil, trifluoperazine, and phorbol esters

An overexpression of plasma membrane glycoprotein with a relative molecular mass (Mr) of 170,000-180,000 is consistently found in different multidrug-resistant human and animal cell lines, although the functional role of the protein in multidrug resistance is not fully understood. It has been reported previously that the Mr 170,000-180,000 glycoprotein is involved, directly or indirectly, in the drug transport mechanism and the proliferation of multidrug-resistant tumor cells. In an attempt to clarify further the function of the Mr 170,000-180,000 glycoprotein, we have studied the phosphorylation state of the protein in intact K562/ADM cells and found that: the protein is phosphorylated in the basal state; verapamil and trifluoperazine, which inhibit the active drug efflux and restore drug sensitivity in resistant cells, caused an increase in the phosphorylation of the Mr 170,000-180,000 glycoprotein; 4 beta-phorbol 12 beta-myristate 13 alpha-acetate and 1-oleoyl 2-acetylglycerol enhanced phosphorylation of the protein; the protein was phosphorylated at serine residues; tryptic phosphopeptide mapping of the Mr 170,000-180,000 glycoprotein showed that 4 beta-phorbol 12 beta-myristate 13 alpha-acetate treatment induced an increase in phosphorylation at different sites of the protein from those induced by verapamil or trifluoperazine treatment, suggesting that the protein is phosphorylated by an array of complex regulation mechanisms. Phosphorylation of the Mr 170,000-180,000 glycoprotein might play a role in the regulation of processes affecting cellular function in multidrug resistance.     

A new enzymatic assay of urinary guanidinoacetic acid.

We describe a new enzymatic determination of urinary guanidinoacetic acid (GAA) with guanidinoacetate kinase (ATP: guanidinoacetate N-phosphotransferase, EC 2.7.3.1), which does not require a blank to correct for endogenous constituents (ADP and pyruvate). In the first step, pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) and lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) were used to eliminate endogenous constituents (ADP and pyruvate) in the presence of phosphoenolpyruvate and NADH. In the second step, urinary GAA was phosphorylated in the presence of ATP by guanidinoacetate kinase to form phosphoguanidinoacetate and ADP. The resultant ADP was sequentially measured at 340 nm in a coupled reaction catalyzed by pyruvate kinase and lactate dehydrogenase. The standard curve was linear up to 20 mg/dl for standard solutions of GAA. Analytical recovery of GAA added to normal urines ranged from 97.0 to 103.2% (mean 100.7%). The within-run and between-run studies gave CV values of less than or equal to 3.6% and less than or equal to 4.8%, respectively. No significant interference by endogenous urinary compounds were observed with the proposed method under this study. The results obtained by the present method correlated well with those obtained by a high-performance liquid chromatographic method. This method is accurate and simple, and less time-consuming than those previously reported. We determined the concentrations of GAA in 24-h urine samples by the proposed method, and observed that the urinary excretion of GAA decreased markedly in patients with renal failure.     

Depletion of bone-marrow cells due to deficiencies of trace elements.

Male rats were divided into five diet-dependent groups: the Control group; two kinds of Zn-deficient diet groups with different amounts of Mn and Mg (the CLEA and OBS group); the Pair-fed group, where the body weight gain of the rats was restricted; and the OBS + Zn group. In those groups where the diets were deficient in Zn, Mn, and Mg, bone-marrow cells were suppressed and the peripheral reticulocyte count was decreased, but plasma corticosterone increased significantly. The OBS + Zn group demonstrated an improvement in the reticulocyte count and serum corticosterone level.