Molecular Diagnosis of a Case of Hb Phnom Penh [α117(GH5)Phe-I1e-α118(H1)Thr (α1)]

We report the first case of Hb Phnom Penh where a molecular study was done on the patient's sample. The result confirmed the predicted DNA sequence change involved in the mutation, which was delineated by another group in 1998 using amino acid analysis.     

Anti-angiogenic effects of SN38 (active metabolite of irinotecan): inhibition of hypoxia-inducible factor 1 alpha (HIF-1α)/vascular endothelial growth factor (VEGF) expression of glioma and growth of endothelial cells

Purpose Inhibition of angiogenesis is an important new treatment modality for malignancies, including gliomas. Vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 (HIF-1) have been investigated as potent mediators of tumor angiogenesis. We investigated whether four major chemotherapeutic agents (ACNU, cisplatin, etoposide, and SN38) showed an angiosuppressive effect in vitro.Method The effects of ACNU, cisplatin, etoposide, and SN38 for endothelial cells were assessed by cell growth inhibition assay (WST-8 assay) and vessel formation assay (angiogenesis kit). The inhibitory effects of the HIF-1 and VEGF expression of glioma cells after SN38 treatment were assessed by real-time RT-PCR, Western blot, and ELISA.Results SN38, but not other chemotherapeutic agents, selectively inhibited endothelial cell proliferation and three-dimensional tube formations at the 0.01 M. Furthermore, SN38 significantly decreased the HIF-1 and VEGF expression of glioma cells in a dose- and time-dependent manner under normoxic and hypoxic conditions. SN38 has dual angiosuppressive actions, including both the inhibition of endothelial proliferation and tube formation, and the inhibition of the angiogenic cascade in glioma cells.Conclusion SN38 is an attractive agent as both a direct and indirect angiogenesis inhibitor and provides the anti-glioma agents with an angiosuppressive function. This study was supported by a Grant-in-Aid for Scientific Research from Japanese Ministry of Education, Science, and Culture and by a grant provided by the Tsukuba Advanced Research Alliance     

Expression of p21~(WAF1) and p53 and polymorphism of p21~(WAF1) gene in gastric carcinoma

AIM:To investigate the relationship between expressionof p21~(WAF1) and p53 gene,and to evaluate the deletion andpolymorphism of p21~(WAF1) gene in gastric carcinoma (GC).METHODS:Expression of p21 and p53 proteins,anddeletion and polymorphism of p21 gene in GC wereexamined by streptavidin-peroxidase conjugated method(SP) and polymerase chain reaction-restriction fragmentlength polymorphism (PCR-RFLP) respectively.RESULTS:The expression of p21 and p53 was found in100% (20/20) and 0% (0/20) of normal gastric mucosae(NGM),92.5% (37/40) and 15.0% (6/40) of dysplasia (DP)and 39.8% (43/108) and 56.5% (61/108) of GC,respectively.The positive rate of p21 in GC was lower than that in NGMand DP (P<0.05),while the positive rate of p53 in GC washigher than that in NGM end DP (P<0.05).p21 and p53were significantly expressed in 63.3% (19/30) and 36.7%(11/30),35.0% (14/40) and 77.5% (31/40),26.7% (4/15)and 80.0% (12/15),30.8% (4/13) and 30.8% (4/13),and20.0% (2/10) and 30.0%,(3/10) of well-differentiated,poorly-differentiated,undifferentiated carcinomas,mucoidcarcinomas and signet ring cell carcinomas.The expressionof p21 in well-differentiated carcinomas was significantlyhigher than that in poorly-differentiated,un-differentiated,mucoid carcinomas and signet ring cell carcinomas (P<0.05).Contrarily,The expression of p53 was increased from well-differentiated to poorly-differentiated and un-differentiatedcarcinomas (P<0.05).The expression of p21 and p53 inpaired primary and metastatic GC (35.3% and 70.6%) wasdifferent from non-metastatic GC (62.5% and 42.5%)markedly (P<0.05).The expression of p21 in invasivesuperficial muscle (60.0%) was higher than that in invasivedeep muscle or total layer (35.2%) (P<0.05) and was higherin TNM stages Ⅰ (60.0%) and Ⅱ (56.2%) than in stages Ⅲ(27.9%) and Ⅳ (22.2%) (P<0.05),whereas the expressionof p53 did not correlate to invasion depth or TNM staging(P>0.05).The exoression patterns of p53 /p21-,and ofp53-/p21 were found in 5.0% and 82.5% of DP.Therewas a significant correlation between expression of p21and p53 (P<0.05).But there was no significant correlationbetween expression of both in GC (P>0.05).There was nodeletion in exon 2 of p21 gene in 30 cases of GC and 45cases of non-GC,but polymorphism of p21 gene at exon 2was found in 26.7% (8/30) of GC and 8.9% (4/45) of non- GC,a significant difference was found between GC andnon-GC (P<0.05).There was no significant relationbetween p21 expression of polymorphism (37.5%,3/8)and non-polymorphism (45.5%,10/22) in GC (P>0.05).CONCLUSION:The loss of p21 protein and abnormalexpression of p53 are related to carcinogenesis,differentiationand metastasis of GC.The expression of p21 is related toinvasion and clinical staging in GC intimately.Theexpression of p21 protein depends on p53 protein largelyin NGM and DP,but not in GC.No deletion of p21 gene inexon 2 can be found in GC.The polymorphism of p21 genemight be involved in gastric carcinogenesis.There is nosignificant association between polymorphism of p21 geneand expression of p21 protein.     

Wilms’ tumor gene 1 (WT1) expression in subtypes of acute lymphoblastic leukemia (ALL) of adults and impact on clinical outcome

Wilms’ tumor gene 1 (WT1) is gaining increasing attention as a therapeutic target molecule due to its common expression in acute leukemias and its involvement in cell proliferation. Here, we reported on WT1 messenger RNA expression levels at diagnosis in a series of 238 adult acute lymphoblastic leukemia (ALL) samples of various subtypes and clinical outcome. WT1 expression was found in 219 out of 238 ALL samples (92%). Compared to a cohort of acute myeloid leukemia patients, the median WT1 expression level in ALL was significantly lower with large variations among different ALL subgroups. Specifically, WT1 expression levels were low in mature B-ALL and highest in ALL cases with co-expression of myeloid markers, making it a useful therapeutic target molecule in adult ALL with the exception of mature B-ALL. Cox regression analysis, considering ALL phenotype as well as molecular-cytogenetic subsets, revealed no independent prognostic role of WT1 expression level for disease-free and overall survival.     

Genomic studies of the Lucké tumor herpesvirus (RaHV-1)

Ranid herpesvirus 1 (RaHV-1) is the etiological agent of the Lucké renal adenocarcinoma of the North American leopard frog Rana pipiens. Construction of cosmid libraries containing RaHV-1 DNA inserts allowed the derivation of a BamHI map for the viral genome. Summation of fragment sizes indicates that the genome is 217 kbp in size, a value in accordance with the most recent published estimate (220 kbp) obtained by field-inversion gel electrophoresis. The DNA sequence of the 39,757-bp insert in 1 cosmid (cos54) was determined and was predicted to contain 21 complete and 3 partial genes. In all, 12 genes have distant counterparts in a fish herpesvirus (ictalurid herpesvirus 1) and are present in 2 blocks, 1 of which is relatively inverted. This indicates that RaHV-1 belongs to the fish virus lineage of the herpesvirus family rather than to the lineage populated by mammalian and avian viruses. The remainding 12 genes in cos54 lack counterparts in any other herpesvirus. One of these encodes a putative DNA (cytosine-5) methyltransferase. This raises the possibility that biological processes induced in the host by RaHV-1 might involve methylation of cellular DNA by the viral enzyme. Received: 1 December 1998 / Accepted: 27 January 1999     

Continuous stimulation of human glucagon-like peptide-1 (7–36) amide in a mouse model (NOD) delays onset of autoimmune type 1 diabetes

Aims/hypothesis We examined the effect of glucagon-like peptide-1 (GLP-1) on the development of diabetes and islet morphology in NOD mice by administering GLP-1 to prediabetic mice. Methods Eight-week-old female NOD mice were infused subcutaneously with human GLP-1 via a mini-osmotic pump for 4 or 8 weeks. In mice treated with GLP-1 for 4 weeks, blood glucose levels and body weight were measured. An intraperitoneal glucose tolerance test (IPGTT) and evaluation of insulitis score were also performed. Beta cell area, proliferation, apoptosis, neogenesis from ducts and subcellular localisation of forkhead box O1 (FOXO1) were examined by histomorphometrical, BrdU-labelling, TUNEL, insulin/cytokeratin and FOXO1/insulin double-immunostaining methods, respectively. Results Mice treated with human GLP-1 for 4 weeks had lower blood glucose levels until 2 weeks after completion of treatment, showing improved IPGTT data and insulitis score. This effect continued even after cessation of the treatment. In addition to the increase of beta cell neogenesis, BrdU labelling index was elevated (0.24 vs 0.13%, p < 0.001), while apoptosis was suppressed by 54.2% (p < 0.001) in beta cells. Beta cell area was increased in parallel with the translocation of FOXO1 from the nucleus to the cytoplasm. The onset of diabetes was delayed in mice treated with GLP-1 for 4 weeks, while mice treated with GLP-1 for 8 weeks did not develop diabetes by age 21 weeks compared with a 60% diabetes incidence in control mice at this age. Conclusions/interpretation Continuous infusion of human GLP-1 to prediabetic NOD mice not only induces beta cell proliferation and neogenesis, but also suppresses beta cell apoptosis and delays the onset of type 1 diabetes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users. J. Zhang and Y. Tokui contributed equally to this study.     

Diagnosis and management of patients with α1-antitrypsin (A1AT) deficiency

Alpha(1)-antitrypsin (A1AT) deficiency is an autosomal codominant disease that can cause chronic liver disease, cirrhosis, and hepatocellular carcinoma in children and adults and increases risk for emphysema in adults. The development of symptomatic disease varies; some patients have life-threatening symptoms in childhood, whereas others remain asymptomatic and healthy into old age. As a result of this variability, patients present across multiple disciplines, including pediatrics, adult medicine, hepatology, genetics, and pulmonology. This can give physicians the mistaken impression that the condition is less common than it actually is and can lead to fragmented care that omits critical interventions commonly performed by other specialists. We sought to present a rational approach for hepatologists to manage adult patients with A1AT deficiency.     

Increased levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in tracheal aspirates of newborns with pneumonia

Summary Pneumonia is one of the major sites of infection in ventilated newborns. We investigated whether the cytokines IL-1 and TNF- are detectable in tracheal aspirates of newborns with pneumonia as a diagnostic marker. All 12 infants with pneumonia had elevated levels of IL-1 (range 30–300 pg/ml) and TNF- (range 60–680 pg/ml), whereas control infants (n=21; respiratory distress syndrome, very low birth weight or infants intubated preoperatively) had no detectable levels of IL-1 or TNF-.In vitro investigations with mononuclear cells of umbilical cord blood were performed to rule out that exogenously added surfactant influences IL-1 and TNF- production. It is concluded that IL-1 and TNF- are important and specific mediators of neonatal pneumonia which may be of diagnostic importance.

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Erhöhte Spiegel von TNF- und IL-1 im Trachealsekret von Neugeborenen mit Pneumonie

Zusammenfassung Die Pneumonie ist eine der Hauptlokalisationen von Infektionen bei Neugeborenen. Wir untersuchten, inwieweit die Zytokine IL-1 und TNF- im Trachealsekret von Neugeborenen mit Pneumonie als diagnostischer Marker nachgewiesen werden können. Alle 12 Kinder mit Pneumonie hatten erhöhte Spiegel für IL-1 (30–300 pg/ml) und TNF- (60–680 pg/ml), während die Kontrollen (n=21; Atemnotsyndrom, sehr untergewichtige Neugeborene, präoperativ intubierte Kinder) keine nachweisbaren Spiegel für IL-1 und TNF- hatten. Um auszuschließen, daß exogen appliziertes Surfactant die IL-1 und TNF--Produktion beeinflußt, wurdenIn vitro-Untersuchungen mit mononukleären Zellen von Nabelschnurblut durchgeführt. Wir schließen aus den Ergebnissen, daß IL-1 und TNF- wichtige und spezifische Mediatoren der Neugeborenenpneumonie sind, die von diagnostischer Bedeutung sein können.

     

Association of 86 bp variable number of tandem repeat (VNTR) polymorphism of interleukin-1 receptor antagonist (IL1RN) with susceptibility and clinical activity in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic, systemic disease of unknown etiology. Several studies have reported a variable number of tandem repeat (VNTR) 86 bp (rs2234663) in the intron 2 of IL1RN gene with RA risk. The present study was designed to determine the frequencies of this polymorphism in patients with RA and control subjects (CS) and its association with RA in a western Mexican population. An analytical cross-sectional study was performed, in which 350 patients with RA and 307 CS were included. The identification of IL1RN VNTR polymorphism was carried out by polymerase chain reaction (PCR), and genotypes were associated with clinical variables (DAS28 and CRP). The presence of A1/A2 genotype was associated with RA risk (p = 0.03, OR = 1.45, 95% CI = 1.02–2.05). Also, results indicate that the presence of heterozygote genotypes which include A2 was associated with RA risk (p = 0.01, OR = 1.5, 95% CI = 1.07–2.11). Patients carrier of A2/A2 genotype have a higher score of DAS28 (5.64 [4.49–6.70]). A-/A- has higher level of CRP (2.30 [0.62–9.10]) in comparison with A2/A- (1.06 [0.37–2.82]). A1/A2 genotype was associated with susceptibility to RA in a western Mexican population. The presence of the A2/A2 genotype in RA is associated with increased disease activity.     

Neuroprotection and repair of 3′-blocking DNA ends by glaikit (gkt) encoding Drosophila tyrosyl-DNA phosphodiesterase 1 (TDP1)

Tyrosyl-DNA phosphodiesterase (TDP1) is a phylogenetically conserved enzyme critical for the removal of blocking lesions at the 3′ ends of DNA or RNA. This study analyzes the Drosophila TDP1 gene ortholog glaikit (gkt) and its possible role(s) in the repair of endogenous DNA lesions and neuroprotection. To do so, we studied a homozygous PiggyBac insertion (c03958) that disrupts the 5′ UTR of gkt. Protein extracts of c03958 flies were defective in hydrolyzing 3′-DNA–tyrosyl residues, demonstrating that gkt is the Drosophila TDP1. Although the mutant is generally healthy and fertile, females exhibit reduced lifespan and diminished climbing ability. This phenotype was rescued by neuronal expression of TDP1. In addition, when c03958 larvae were exposed to bleomycin, an agent that produces oxidative DNA damage, or topoisomerase I-targeted drugs (camptothecin and a noncamptothecin indenoisoquinoline derivative, LMP-776), survivors displayed rough eye patches, which were rescued by neuronal expression of TDP1. Our study establishes that gkt is the Drosophila TDP1 gene, and that it is critical for neuroprotection, normal longevity, and repair of damaged DNA.DNA repair is indispensable to maintaining genomic integrity against the various endogenous and exogenous agents and enzymes that react with DNA, including reactive oxygen species (1–3), base-damaging agents (4, 5), and chain-terminating nucleosides (6), which yield nonligatable DNA ends. In addition, topoisomerase I (Top1) (7–9) is capable of generating strand breaks with protein-blocked 3′ ends. To ligate and extend the ends of broken DNA, the modified 3′ end must be removed. Tyrosyl-DNA phosphodiesterase (TDP1), first identified in yeast, catalyzes the hydrolysis of 3′-phosphotyrosyl, 3′-phosphoglycolate, and 3′-nucleoside bonds (8, 10–14). The irreversible Top1–DNA complexes and 3′-blocking nucleoside lesions are cleaved by TDP1, leaving 3′-terminal phosphates, which are further processed by polynucleotide kinase phosphatase before ligation and/or extension by polymerases.Deleterious TDP1 mutants in yeast, mice, and humans are viable but have decreased capacity to repair oxidative and topoisomerase-induced damage (15–17). In humans, a homozygous mutation in TDP1 (A1478G) causes spinocerebellar ataxia with axonal neuropathy (SCAN1) (18). Cells from SCAN1 patients contain enhanced levels of Top1 cleavage complexes (Top1cc) and have defective repair of Top1cc (19, 20) and oxidative damage (21). In mice, TDP1 was recently shown to play a critical role in removing endogenous Top1cc and preventing neurodevelopmental defects (22).A Drosophila TDP1 ortholog, encoded by the glaikit gene (gtk), has been reported, but its functions in DNA repair have not been studied because the knockout flies were found to be nonviable, with defects in epithelial polarity formation and neuronal development (23). This finding was surprising because TDP1 is not essential for viability in other eukaryotes, including yeast (10, 11), chicken and human cells (4, 24), and mice (16, 25). Here we use an insertional mutant (Exelixis line c03958) to elucidate the role of TDP1 in flies and its critical activity for the repair of 3′-blocked DNA termini and neuroprotection.     

Effects of methylprednisolone (MPS) on induction of apoptosis and expression of interleukin-1 B converting enzyme (ICE) in the kidneys of MRL/Ipr mice.

Objective: To explore the effects of methylprednisone on induction of apotosis and expression of interleukin-1 B converting enzyme in the kidneys. Methods: We investigated the effects of MPS on the     

Neuropsychological profile of adult patients with Niemann–Pick C1 (NPC1) mutations

Niemann-Pick type C disease is a fatal neurovisceral disorder linked to dysregulation in cholesterol processing. A medication for this disease is currently being tested in clinical trials. However, there is a lack of information on neuropsychological testing parameters for this disease. One aim of this pilot study was to evaluate a test battery that could be used to assess cognitive deficits in different stages of the disease. A second aim was to determine whether specific functional deficits are associated with certain disease stages. Eight men and two women (19-40 years of age) harbouring mutations in the gene coding for the cholesterol trafficking protein NPC1 were put through the same test battery independently of their disease stage. The external staging criterion was based on a five-step clinical scale. Trail Making tests A & B and verbal fluency were sensitive indicators at early stages of NPC. Corsi Block-Tapping, Mini Mental Status, Find Similarities and Clock Drawing showed abnormal results in patients with advanced disease. The Grooved Pegboard, Trail Making and Mosaic tests were unsuitable in advanced disease due to impaired fine motor skills. We observed that visuospatial working memory was less affected by the neurodegenerative process than verbal working memory. The series of tests used here could be supplemented by the severe impairment battery and Raven matrices tests for patients with advanced disease.     

α-Lipoic acid regulates lipid metabolism through induction of sirtuin 1 (SIRT1) and activation of AMP-activated protein kinase

Aims/hypothesis  

Sirtuin 1 (SIRT1) is a longevity-associated protein, which regulates energy metabolism and lifespan in response to nutrient deprivation. It has been proposed to be a therapeutic target for obesity and metabolic syndrome. We investigated whether α-lipoic acid (ALA) exerts a lipid-lowering effect through regulation of SIRT1 activation and production in C₂C₁₂ myotubes.     

Influence of the carboxylesterase inhibitor bis-p-nitrophenylphosphate on the rates of hydrolysis of various α-esters of 1-(N-methyl-N-nitrosamino)-methanol in vitro and in vivo and on the acute toxicity and carcinogenicity of 1-(N-methyl-N-nitrosamino)-methylacetate

Summary The effect of an in vivo treatment with the carboxylesterase inhibitor bis-p-nitrophenylphosphate (BNPP) on the hydrolysis of 1-(N-methyl-N-nitrosamino)-methylacetate (NNMA), 1-(N-methyl-N-nitrosamino)-methylbutyrate (NNMB), 1-(N-methyl-N-nitrosamino)-methylbenzoate (NNMBz) and 1-(N-methyl-N-nitrosamino)-methylpivaloate (NMMP) in rat tissue homogenates was studied. The rates of hydrolysis were specific for each compound and different in every organ tested; the extent of inhibition of the hydrolysis by BNPP was also substrate and organ specific. In some cases no inhibition at all was observed. The rate of elimination of NNMA, NNMB, and NNMP from blood was not influenced by BNPP pretreatment. The LD₅₀ of NNMA after i.v. application showed a rise of 85% with a BNPP pretreatment. BNPP also influenced the carcinogenicity of NNMA, whereby the total carcinogenic potency was not altered, but the organotropism had changed slightly.Abbreviations NNMA 1-(N-methyl-N-nitrosamino)-methylacetate - NNMB 1-(N-methyl-N-nitrosamino)-methylbutyrate - NNMBz 1-(N-methyl-N-nitrosamino)-methylbenzoate - NNMP 1-(N-methyl-N-nitrosamino)-methylpivaloate - BNPP bis-p-nitrophenylphosphate - DSF disulfiram Dedicated to Professor Dietrich Schmähl on the occasion of his 60th birthday