bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical, Endocrine, and Molecular Genetic Findings in Patients with 17β-Hydroxysteroid Dehydrogenase Deficiency
Authors: Twesten Wiebke , Holterhus Paul-Martin , Sippell Wolfgang G. , Morlot Michel , Schumacher Heinz , Schenk Bernd , Hiort Olaf
Published: April 2003

Mutations in the 17β-hydroxysteroid dehydrogenase (17β-HSD) type 3 gene are associated with the clinical findings of 17β-HSD deficiency. We investigated 5 patients of German descent with 46,XY karyotype and predominantly female phenotype. Androstenedione (A) and testosterone (T) levels in serum were determined before and after stimulation with human chorionic gonadotropin. DNA analysis of the whole coding region of the 17β-HSD type 3 gene was performed by PCR, single-strand conformation analysis, and direct sequencing. In all patients we found highly variable A and T levels before and after stimulation. However, the A-to-T ratio was abnormal in all cases suggestive of 17β-HSD deficiency. Molecular genetic analysis revealed mutations in all patients. We conclude that A and T levels may be highly variable in patients with 17β-HSD deficiency. Molecular genetic analysis of the 17β-HSD gene may support the diagnosis of this disorder.
Role of Growth Factors in the Development of Diabetic Complications
Authors: Chiarelli F. , Santilli F. , Mohn A.
Published: April 2003

The structural changes characterising diabetic microangiopathy, which may be referred to as ’abnormal growth’ and ’impaired regeneration’, strongly suggest a role for a number of aberrantly expressed growth factors, possibly acting in combination, in the development of these complications. This initial speculation has been supported by the detection of increased concentrations of several growth factors in the target tissues of diabetic long-term complications, and by enhanced expression of these growth factors consequent to the activation of the biochemical pathways linking hyperglycaemia to microvascular changes: the polyol pathway; non-enzymatic glycation of proteins; vasoactive hormones; oxidative stress, and hyperglycaemic pseudohypoxia. As to nephropathy, insulin-like growth factor I (IGF-I) seems to be implicated in the earlier stages of the disease, while transforming growth factor β (TGFβ) is involved both in the early and later stages, being responsible, at least in part, for extracellular matrix (ECM) accumulation. Vascular endothelial growth factor (VEGF) plays a pivotal role both in non-proliferative and proliferative retinopathy. Finally, deficiency of several neurotrophic factors, namely nerve growth factor (NGF) and IGF-I has been related to the degeneration or impaired regeneration occurring in diabetic neuropathy. Knowledge of the involvement of growth factors in diabetic microangiopathy opens the way to new therapeutic interventions aimed at blocking the deleterious actions of several growth factors.
Idiopathic Growth Hormone Deficiency: A Vanishing Diagnosis?
Authors: Pfäffle Roland , Blankenstein Oliver , Wüller Stephan , Heimann Konrad , Heimann G.
Published: April 2003

Some non-organic causes for growth hormone (GH) deficiency (GHD) can be attributed to genetic defects within the hypothalamo-pituitary axis. Using modern molecular biology techniques micromutations within the GH and GH-releasing hormone receptor genes have been detected as a rare cause of isolated GHD. Combined pituitary hormone deficiencies (CPHD), on the other hand, are associated with defects that manifest during the organogenesis of the anterior pituitary gland. In recent years an increasing number of patients with CPHD has been reported, showing mutations within pituitary transcription factors Pit-1, Prop-1 and HesX1. Such defects can be observed with different frequencies in patients. Some disorders, such as CPHD due to Pit-1 mutations, display a hormonal phenotype that seems more or less invariable. In most other forms of genetic CPHD both the combination and severity of anterior pituitary hormone deficiencies vary considerably. Ongoing research concentrates on factors involved in the differentiation and proliferation of cells that belong to the hypothalamo-pituitary growth axis. As not every possible candidate turns out to be a frequent cause of GHD or CPHD in humans, it will be many more years before the term ‘idiopathic’ becomes a vanishing attribute to the clinical diagnosis of pituitary insufficiency.
Is There Growth Hormone Deficiency in Prader-Willi Syndrome?
Authors: Eiholzer Urs , Bachmann Sara , l’Allemand Dagmar
Published: April 2003

Prader-Labhart-Willi syndrome (PWS) is the most frequent form of syndromal obesity. Its main features are associated with hypothalamic dysfunction, which has not yet been comprehensively described. The aim of this review is to present arguments to define the presence of genuine growth hormone (GH) deficiency (GHD) in these patients. Decreasing growth velocity despite the onset of obesity, reduced lean body mass in the presence of adiposity, small hands and feet, relatively low insulin-like growth factor-I and low insulin levels, as well as the dramatic effect of GH treatment on growth, support the presence of hypothalamic GHD in PWS. Even though it might be difficult to ultimately prove GHD in PWS because of the obesity-induced counterregulation, the hormonal situation differs from that in simple obesity. The effects of long-term therapies with GH on body composition in these patients are summarized. GH therapy dramatically changes the phenotype of PWS in childhood: height and weight become normal and there is a sustained impact on the net loss of body fat. We conclude that GHD may account for several features of PWS.
Macrophage Inhibition of Lymphocyte and Tumor Cell Growth Is Mediated by 25–Hydroxycholesterol in the Cell Membrane
Authors: Kato Hiroshi , Horino Atsuko , Taneichi Maiko , Fukuchi Naoyuki , Eto Yuzuru , Ushijima Hiroshi , Komuro Katsutoshi , Uchida Tetsuya
Published: April 2003

We have previously reported that a lipid molecule in the membrane fraction of cloned macrophage hybridomas inhibited the growth of lymphocytes and several tumor cell lines. In this study, the inhibitory lipid molecule in the membrane fraction of macrophages was analyzed by thin–layer chromatography and identified as 25–hydroxycholesterol, a family of oxysterols. This conclusion was confirmed by analysis using gas chromatography–mass spectrometry. In addition, both 25–hydroxycholesterol and the lipid molecule recovered from macrophage cell membrane induced apoptosis of the murine T cell lymphoma, BW–5147. These results suggest that an oxysterol expressed in the macrophage cell membrane may participate in the regulation of cell growth through cell contact.
Inverse Association between Skin Response to Aeroallergens and Schistosoma mansoni Infection
Authors: Araujo Maria Ilma , Lopes Antônio Alberto , Medeiros Manoel , Cruz Álvaro A. , Sousa-Atta Luiza , Solé Dirceu , Carvalho Edgar M.
Published: April 2003

Background: Helminthic infections and allergic disease are highly prevalent in many areas of the world. It is known that IgE antibodies are involved in the pathogenesis of both helminthiasis and atopy. However, the consequences of the presence of helminthic infections in atopic patients are still not completely understood. Methods: Subjects infected by Schistosoma mansoni with more than 200 eggs/g of feces (n = 42) and uninfected subjects (n = 133) were selected from an endemic area of schistosomiasis. The history of allergy and results of the immediate hypersensitivity prick tests with inhalant allergen extracts were registered. Total IgE and IgE specific to S. mansoni and aeroallergens were measured in serum by ELISA. Results: The proportion of individuals with a positive skin test to allergens was higher in the uninfected group (24.3%) than in the group with more than 200 eggs/g of feces (4.8%). The odds of atopy (defined as a positive test for at least one of the antigens) were 5 times higher (odds ratio = 7.0; 95% confidence interval = 1.6–31.1%; p = 0.01) in the uninfected group, after taking into account the potential influence of gender and age. While there was a tendency for higher total and S. mansoni-specific IgE levels in infected patients, an opposite trend, that is higher aeroallergen-specific IgE, was observed in uninfected subjects. Conclusions: There was a strong and statistically significant inverse association between the immediate skin test response to common aeroallergens and infection by S. mansoni. The results indicate that immediate hypersensitivity reactions may be suppressed in S. mansoni-infected individuals.
Serum Obtained from Rats after Partial Hepatectomy Enhances Growth of Cultured Colon Carcinoma Cells
Authors: de Jong Koert P. , Brouwers Mark A.M. , van Veen Marco L. , Brinker Marja , de Vries Elisabeth G.E. , Daemen Toos , Scherphof Gerrit L. , Slooff Maarten J.H.
Published: April 2003

Tumour-bearing rats were randomized to a 70% partial hepatectomy or a sham operation. At days 1, 3 or 14, portal and systemic serum was obtained and colon carcinoma cells were cultured in the presence of 5, 10, 20 or 50% serum. Proliferation and epidermal growth factor receptor (EGFr) expression was measured in tumour cells. Proliferation was 25–40% higher in tumour cells cultured with portal serum after hepatectomy than after sham operation when using serum obtained at day 3, but not days 1 and 14 after operation. In cultures with serum obtained at day 14 after operation CC 531 cells showed a 30% higher proliferation rate with systemic hepatectomy serum than CC 531 cells with sham systemic serum. These effects were not mediated by a change in EGFr mRNA and protein levels as the used colon carcinoma cells did not reveal EGFr activity by any of the three detection methods used.
Invasive Potential and Substrate Dependence of Attachment in the Dunning R-3327 Rat Prostate Adenocarcinoma Model
Authors: Donald Carlton D. , Montgomery David E. , Emmett Nerimiah , Cooke III David B.
Published: April 2003

Cancer cell attachment to and invasion of the extracellular matrix has been associated with the metastatic potential of cell lines of the Dunning R-3327 rat prostatic adenocarcinoma model. We investigated the cell-matrix interactions of prostate tumor cells by comparing the invasive ability through reconstructed extracellular matrix and attachment upon EHS NATRIX (natural extracellular matrix), fibronectin, laminin, and collagen Type IV. We observed a correlation between metastatic potential and substrate dependence of attachment in prostate cancer cells. Nonmetastatic AT-1 cells possessed a higher adhesive potential to extracellular matrix components than the highly metastatic cells (ML, MLL and AT-3). It was also found that the invasive potential of the three highly metastatic cell lines was significantly higher than that of the nonmetastatic cell line. Here, it is reported that the ability to traverse a matrigel matrix correlates with their metastatic potential. These observations suggest that the extracellular matrix components are highly involved in influencing prostate cancer cell activities. In addition, we investigated the effects of two differentiation agents, retinoic acid (RA) and difluoromethylornithine (DFMO), on the adhesive and invasive profiles of the tumor cells. After treatment with both agents, adhesion was increased to levels not different from nonmetastatic cells. Furthermore, the ability of highly metastatic cells to traverse a matrigel barrier was significantly reduced after treatment with both differentiation agents. These results suggest that RA and DFMO are capable in reversing the metastatic potential of prostate cancer cells in vitro and may give a possible insight into their role as potential therapeutic agents in vivo.
A Model System to Quantitate Tumor Burden in Locoregional Lymph Nodes during Cancer Spread
Authors: Moore Anna , Sergeyev Nikolay , Bredow Sebastian , Weissleder Ralph
Published: April 2003

In order to quantitate the metastatic burden of secondary tumor deposits in locoregional lymph nodes, we produced a green fluorescent protein (GFP)-expressing murine cell line (B16F1) metastatic to lymph nodes in immunocompetent mice. When implanted into the hindleg of mice, all animals developed paraaortic lymph node metastases. Fluorescence microscopy, immunohistochemistry, and RT-PCR confirmed the presence of metastatic cells in lymph nodes. Tumoral deposits occurred preferentially in marginal sinuses and spread superficially to the subcortical area. Western blot analysis showed that the local tumor burden could be quantitated in individual lymph nodes. This model system should be useful for quantitating metastatic invasion particularly of micrometastases and aid in the development of lymphotropic drugs to detect and/or treat lymph node metastases in advanced cancers.
Establishment of High and Low Metastasis Cell Lines Derived from a Human Tongue Squamous Cell Carcinoma
Authors: Nakayama Shuko , Sasaki Akira , Mese Hiroshi , Alcalde Rafael E. , Matsumura Tomohiro
Published: April 2003

Malignant tumors are composed of cells with different phenotypic properties and only certain cell subpopulations present metastatic potential. The establishment of cell lines with high or low metastatic potential is necessary to investigate the molecular mechanisms of the metastatic process. However, human oral squamous cell carcinoma (SCC) cell lines that are suitable for the above investigation are scarce. High and low metastatic cells were obtained from a primary lesion of a patient with tongue carcinoma who had not received any therapy. Two distinct cell lines were selected, UM1 with a scattered growth pattern and loose cell-cell adhesion, and UM2 with a colony-formed growth pattern and firm cell-cell adhesion. The expression of E-cadherin in UM1 was clearly lower than that in UM2. UM1 exhibited a higher motility, invasive and metastatic activity than UM2 in vivo and in vitro. A low invasive and a metastatic oral SCC cell line, useful to investigate invasion and metastasis mechanisms, have been established.
Expression of the Human Papillomavirus Type 11 E5A Protein from the E1∧E4,E5 Transcript
Authors: Brown Darron R. , McClowry Tracy L. , Sidner Richard A. , Fife Kenneth H. , Bryan Janine T.
Published: April 2003

The abundant human papillomavirus type 11 (HPV 11) E1∧E4,E5 transcript potentially encodes the E1∧E4,E5a and E5b proteins. It is not known if either of the E5 proteins are expressed from this transcript. For HPV 16, E5 is a single open reading frame (ORF), and the E5 protein is expressed from an unspliced E2,E5 transcript but not from the spliced E1∧E4,E5 transcript. This study was undertaken to determine if the HPV 11 E5a protein is expressed from the E1∧E4,E5 transcript. To detect E5a expression in eukaryotic cells, the green fluorescent protein (GFP) gene was fused to the 3′ end of the E5a gene in the pEGFP-N1 vector. Several recombinant plasmid constructs were made to determine if E5a translation is influenced by upstream sequences present in the E1∧E4,E5 transcript. COS-7 cells were transfected with each construct, and flow cytometry was performed after 24 h of growth. The amount of E5a-GFP expressed from each construct was determined by the mean fluorescence of 2,000 transfected cells. Although the E5a-GFP fusion was expressed by all but one construct, the quantity of expressed E5a-GFP varied considerably. The most abundant expression was detected in cells transfected with the E1∧E4,E5a construct that lacked the 5′ noncoding sequence between nucleotides (nts) 714 and 831 that is present in the authentic transcript. Other constructs expressed E5a-GFP in variable amounts, suggesting that sequences between nt 714 and the start of the E5a ORF affect expression of the E5a protein. An E2,E5a construct was made to compare the HPV 11 E5a expression to that of HPV 16. In contrast to HPV 16, no E5a-GFP was expressed from the HPV 11 E2,E5a construct. E1∧E4 protein was detected by immunofluorescence in COS-7 cells transfected with a construct that expressed E1∧E4 as a T7-epitope-tagged protein, and E5a as a GFP fusion. We conclude that the abundant HPV 11 E1∧E4,E5 transcript is a functional message that can support both E1∧E4 and E5a expression in eukaryotic cells.
Inhibition of HIV-1 Replication by Chimeric Phosphorothioate Oligodeoxynucleotides Applied in Free Solution
Authors: Lund Ole Søgaard , Hansen John-Erik Stig
Published: April 2003

Oligodeoxynucleotides (ODNs) containing a variable number of 3′ and 5′ terminal phosphorothioate linkages were applied in free solution to cells infected by HIV-1. ODNs of 28 nt length were applied at up to 5 µM concentration. The ODNs were found to inhibit HIV-1 infection in a dose dependent manner, which correlated with the number of modified linkages (4, 8 and 12, respectively). A target sequence in the HIV-1 rev mRNA, previously reported as sensitive to antisense inhibition by full length phosphorothioate ODNs, only revealed non-sequence dependent inhibition of HIV-1, when tested by these modified chimers.
Coinfection of Spodoptera exigua and Spodoptera frugiperda Cell Lines with the Nuclear Polyhedrosis Viruses of Autographa californica andSpodoptera exigua
Authors: Yanase Tohru , Yasunaga Chisa , Hara Toshio , Kawarabata Takeshi
Published: April 2003

The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) has a broad host range among Lepidoptera. In contrast, the Spodoptera exigua MNPV (SeMNPV) can replicate efficiently only in S. exigua larvae or S. exigua-derived cell lines. In this study, we examined the coinfection of S. exigua Se301 and Spodoptera frugiperda IPLB-SF21AEII (Sf21) cell lines with SeMNPV and AcMNPV recombinant (Ac360-501β-gal) which was constructed for expression of β-galactosidase under control of the polyhedrin promoter. Coinfection led to the restriction as the level of late gene expression, nonoccluded virus production, and DNA replication of Ac360-501β-gal in both Se301 and Sf21 cell lines. In contrast, Ac360-501β-gal supported the SeMNPV replication in Sf21 cells. Occurrence of recombinants, between Ac360-501β-gal and SeMNPV, with expanded host range was not observed in coinfected Sf21 cells. This suggests that Ac360-501β-gal supports the SeMNPV replication through trans-activation.
Hepatitis A Virus Proteins
Authors: Totsuka Atsuko , Moritsugu Yasuo
Published: April 2003

The RNA genome of hepatitis A virus (HAV) shares common characteristics of the picornavirus family. However, the nucleotide or amino acid sequences are distantly related with other members of the family. Like other picornaviruses, HAV proteins are cleaved from a large polyprotein (PO), but the processing and some products are quite different. The 3C protein is the sole processing enzyme, and the primary cleavage takes place at the 2A/2B site. Several VP1-2A sites are proposed. In some strains, the intermediate VP1-2A polypeptides are assembled in the virion. The VP4 is very small and not detected in the mature virion. Some mutations in 2B, 2C and 3A proteins are identified to enhance viral replication or to induce cytopathogenic effects in the viruses adapted to cell cultures.
Truncated Hepatitis B Virus RNA in Human Hepatocellular Carcinoma: Its Representation in Patients with Advancing Age
Authors: Kairat Astrid , Beerheide Walter , Zhou Ge , Tang Zhao-You , Edler Lutz , Schröder Claus H.
Published: April 2003

RNA from tissue samples of 46 HBsAg seropositive hepatocellular carcinoma (HCC) patients was analysed by an RT/PCR assay which discriminates full-length hepatitis B virus (HBV) RNA polyadenylated at the unique viral poly(A) signal governing replication from truncated HBV RNA polyadenylated at a cryptic poly(A) signal. In the tumor the apparent coexistence was less frequent than in the peritumor while the predominance of one of the two RNAs was more frequent. The mean age of patients with a predominance of truncated RNA in the tumor was 9 years above those patients with a predominance of full length RNA (p < 0.05). An inverse relationship existed between the presence of truncated RNA and the presence of RNA carrying core gene sequences. The results of this study establish truncated RNA as a frequent marker of the chronic infection but leave it open whether it is found preferentially in patients developing HCC or generally in chronically infected individuals.
Intracellular Replication of Choleraphage φ92
Authors: Barman Subrata , Majumdar Sabita
Published: April 2003

The broad host range choleraphage φ92 contains a linear double-stranded DNA molecule of 68 kilobase (kb) pairs with 3′ overhang cohesive ends. Following infection with phage φ92, the host macromolecular synthesis is shut off within the first 5 min, and synthesis of phage-specific DNA is detectable after 7 min of infection. At early times during infection, phage DNA is replicated in circular form and the synthesis is membrane associated. The closed circular form serves as the precursor for the synthesis of the mature phage DNA which is eventually packaged into the phage head. Pulse labeling of ultraviolet-irradiated infected cells at different times during infection has allowed identification of about 30 phage-specific proteins of which 6 are structural proteins. These proteins appear during the infection cycle in two distinct phases, early and late. Eighteen early and 12 late proteins have been identified.
Co-Infection of Acyclovir-Resistant and Acyclovir-Sensitive Herpes simplex Type 2 Virus Strains in BS-C-1 Cells
Authors: Keywan Kazem , Katz Ehud
Published: April 2003

Co-infection of BS-C-1 cells by the herpes simplex virus type 2 (HSV-2) Curtis strain with its acyclovir (ACV)-resistant mutant (HSV-2-ACVR), resulted in a severalfold increase in virus yield, as compared to a single infection. On the other hand, when two viruses (G strain and HSV-2-ACVR) belonging to different strains of HSV-2 were involved, their growth was significantly inhibited; the decrease in the titer of the second virus to infect the cells was greater when its infection took place at later times following infection by the first. This inhibition was not due to the shut-off of host cell protein synthesis caused by the first virus, since an HSV-2 mutant which is unable to inhibit the protein synthesis of the host cell was still capable of efficiently inhibiting the growth of the superinfecting virus.
Close Association between High Serum ALT and More Rapid Recurrence of Hepatocellular Carcinoma in Hepatectomized Patients with HCV-Associated Liver Cirrhosis and Hepatocellular Carcinoma
Authors: Tarao Kazuo , Rino Yasushi , Takemiya Shoji , Tamai Setsuo , Ohkawa Shinichi , Sugimasa Yukio , Miyakawa Kaoru , Morinaga Soichiro , Yoshida Muneki , Shibuya Akitaka , Kokubu Shigehiro , Kakita Akira , Endo Osamu
Published: April 2003

We investigated whether or not a high serum alanine aminotransferase (ALT) level is associated with a more rapid recurrence of hepatocellular carcinoma (HCC) in hepatectomized patients with hepatitis C virus (HCV)-associated liver cirrhosis (LC) (HCV-LC) and HCC. Thirty-three hepatectomized patients with HCV-LC and HCC of a single nodule who had no histologic evidence of portal or hepatic vein invasion and who had been followed up for more than 3 years were included in the study. They were subdivided into two groups according to their serum ALT levels, ALT being a well-known marker of inflammatory necrosis in the liver. Seventeen patients whose serum ALT levels showed several peaks or plateaus above 80 international units (IU) were designated as the high ALT group, and 16 patients whose serum ALT levels showed a sustained low level below 80 IU until the first recurrence were designated as the low ALT group, and the interval between hepatectomy and the first recurrence was observed. In the high ALT group, HCC recurred within 3 years in 70.6% of the patients. In contrast, it recurred in only 18.8% of the low ALT group within the same period (p < 0.05). There was a significant difference (p = 0.0201) between the two groups in the cumulative nonrecurrence rate. The mean interval in recurrent patients between hepatectomy and the first recurrence in the high ALT group (23.6 ± 2.8 months; mean ± SE) was significantly (p < 0.02) shorter than that in the low ALT group (49.3 ± 9.7 months). The expected interval between hepatectomy and recurrence was as short as 2.8 ± 0.5 years (mean ± SE) in the high ALT group, compared with 5.8 ± 0.7 years in the low ALT group (p < 0.05). These results showed that the recurrence of HCC was accelerated in the high ALT group, suggesting that suppression of the rise in ALT level after hepatectomy by treatment with anti-inflammatory drugs may prolong the interval until recurrence by about 2 years in hepatectomized patients with HCC and HCV-LC.
Hepatitis A Virus Replication: An Intermediate in the Uncoating Process
Authors: Bishop Naomi E.
Published: April 2003

Dense, RNase-sensitive, VP2-containing, non-infectious hepatitis A virus (HAV) particles were found to be formed at early times after the infection of cultured cells. These particles formed with kinetics mirroring those reported for HAV uncoating. The kinetics of the formation of dense HAV particles corresponded to a decrease in detectable, mature input virions, as detected by RNA dot blot hybridization of CsCl density gradient fractions. The dense HAV particles did not appear to have altered sedimentation coefficients, and as the fate of small capsid protein VP4 is not yet known, these particles cannot yet be termed ‘A particles’ or ‘infectosomes’, as have the uncoating intermediates in some picornavirus-cell systems.
An Arthrogenic Alphavirus Induces Monocyte Chemoattractant Protein-1 and Interleukin-8
Authors: Mateo Luis , La Linn May , McColl Sean R. , Cross Simone , Gardner Joy , Suhrbier Andreas
Published: April 2003

Cytokines and chemokines play important roles in both autoimmune and infectious arthritides. Here we describe the cytokines and chemokines induced by Ross River (RR) virus infection of synovial fibroblasts and macrophages in vitro. RR virus is the aetiological agent of epidemic polyarthritis (EPA), a principally acute and chronic rheumatic disease affecting up to 7,000 Australians annually. Infected fibroblasts increased expression of mRNA coding for monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), and granulocyte-macrophage colony-stimulating factor. MCP-1, IL-8, macrophage inflammatory protein-2, and to a lesser extent interferon γ-induced protein-10 mRNA were upregulated in infected macrophages. Expression of MCP-1 is consistent with the predominantly monocytic effusion found in EPA synovia.
Influence of Viral Load and Alanine Aminotransferase on Viral Genetic Heterogeneity in Patients with Chronic Hepatitis C Virus Infection
Authors: Bozdayi A. Mithat , Uzunalimoglu Özden , Aslan Nuray , Bozkaya Hakan , Türkyilmaz Ahmet Resat , Kayhan Burhan , Sahin Tulay , Sivri Bulent , Uygun Ahmet , Altiok Aysin , Çetinkaya Hulya , Karayalçin Selim , Yurdaydin Cihan
Published: April 2003

Background/Aim: Hepatitis C virus (HCV) populations in vivo consist of genetically different heterogeneous mixtures defined as ‘quasispecies’, which vary in the hypervariable region 1 (HVR1) mostly. To further address the role of quasispecies diversity in hepatitis C infection, this study aimed to evaluate the influence of ALT, viral load and genotypes on quasispecies heterogeneity in patients with HCV infection. Methods: Thirty-six chronic hepatitis C patients with high levels of alanine aminotransferase (ALT) were studied. None of them received any antiviral therapy. HCV RNA serum levels, genotype and genetic heterogeneity were determined by branched-chain DNA assay, restriction fragment length patterns and RT-PCR single-strand conformational polymorphism analysis of HVR1, respectively. Results: Twenty-eight patients had genotype 1b (28/36; 78%), 6 patients had genotype 1a (6/36; 17%), 1 patient was 2a (1/36; 3%) and genotype could not be determined in 1 patient. The patients were categorized into two groups according to the number of bands representing the dominant strains in the circulation: group A with 2 bands having 1 strain (14/36 patients; 39%) and group B with more than 2 bands indicating more than 1 strain (22/36 patients; 61%). The serum viremia and ALT levels for these groups were 11 ± 8.8 and 5.3 ± 4.6 mEq/ml (p < 0.05), and 79 ± 20, and 127 ± 80 IU/l (p < 0.05), respectively. Conclusion: The results of this study suggest that hepatitis C patients having 1 dominant strain in the circulation may show a relatively weaker immune response resulting in lower ALT and higher viremia levels, whereas patients with high degrees of virus quasispecies diversity have higher ALT levels and a more active immune response causing the selection of new genome variants and depressing viral replication partly.
Human T Lymphotropic Virus I in Indonesia
Authors: Tanggo Y. , Gultom S.P. , Simanjuntak T. , Sibuea W.H. , Matsuzaki H. , Yamaguchi K.
Published: April 2003

Human T lymphotropic virus I (HTLV-I) is a retrovirus associated with adult T cell leukemia/lymphoma, tropical spastic paraparesis and possibly with other diseases. The actual incidence of HTLV-I carriers in Asia and Africa is difficult to assess and values of 0–37% have been reported. A study in Papua New Guinea by Yamaguchi et al. [6]found 1.8% positive for antibody to HTLV-I and this area is geographically close to the eastern part of Indonesia. A collaborative study was carried out to establish the seroprevalence of HTLV-I in Indonesia using the same method as that used standardly by Yamaguchi et al. in Japan. In a first step we measured antibody to HTLV-I in 657 subjects including 127 healthy persons around Jakarta, 451 patients with various nonhematological diseases and 79 blood donors. The majority of these healthy persons and patients came from various tribes of west Indonesia. All samples were initially screened for HTLV-I by the aggutination test. Serum that caused agglutination of the final dilution of 1:16 or more and/or showed positivity on ELISA was subjected to immunofluorescence testing using HTLV-I-producing MT-2 cells and Western blot analysis. Of the 657 samples, only 1 sample (0.15%) was found to be positive by the agglutination test, but this sample was negative when subjected to WB. From these result we concluded that there was very low seroprevalence of HTLV-I in the Jakarta area, which was also reflected in the western part of Indonesia. Routine examination of blood donors for HTLV-I in this area may not be necessary.
Attachment and Penetration Properties of Bovine Herpesvirus 1 Recombinants Expressing Pseudorabies Virus Glycoproteins gC and gB
Authors: Nakamichi Kazuo , Otsuka Haruki
Published: April 2003

Objective: Bovine herpesvirus 1 (BHV-1) has a relatively narrow host cell range in vitro and in vivo when compared to pseudorabies virus (PrV). The aim of this study was to elucidate whether homologous glycoproteins gC and gB from PrV can function in a heterologous BHV-1 background and whether the expression of these PrV glycoproteins influences the in vitro host cell specificity of BHV-1. Methods: We constructed BHV-1 recombinants in which PrV gC and gB were expressed either individually or in combination, and examined their attachment and penetration properties in permissive Madin-Darby bovine kidney (MDBK), semipermissive hamster lung (HmLu-1) and nonpermissive murine embryo fibroblast (A31) cells. Results: Two BHV-1 recombinants which expressed PrV gC exhibited remarkable competence in virus attachment to cells. The expression of PrV gB improved the virus attachment only a little but penetration, especially into HmLu-1 and A31 cells, was enhanced. Conclusions: These results indicate that PrV gC and gB can function in a BHV-1 environment and facilitate virus attachment and penetration by BHV-1.
Full or Near Full Length Nucleotide Sequences of TT Virus Variants (Types SANBAN and YONBAN) and the TT Virus-Like Mini Virus
Authors: Takahashi Kazuaki , Hijikata Minako , Samokhvalov Evgenyi I. , Mishiro Shunji
Published: April 2003

TT virus (TTV) is a common virus and consists of many genotypes and variants. In addition, there exists a virus which both differs greatly from and retains a considerable resemblance to TTV, such as the TTV-like mini virus (TLMV) as we reported previously. Here we report the near full length genomic sequences of 4 isolates of a new variant of TTV (designated YONBAN) along with the full length sequences of 2 isolates of the TTV-SANBAN lineage and 7 isolates of the TLMV species derived from human sera. The TTV-YONBAN sequences showed only about 50% identity at the nucleotide level to those of the prototype TTV (TA278) and to SANBAN, and even less to TLMV. Moreover, the ORF1 of YONBAN lacked the ATG initiation codon which is shared by all the TTV and TLMV isolates so far identified in humans; instead, YONBAN had a Kozak’s rule-compatible ACG codon as the candidate initiation site for the ORF1 translation. Nevertheless, the overall genetic structure and the conserved amino acid motifs within the ORF1 and the ORF2 were well shared among the prototype TTV strains, the SANBAN and YONBAN variants, and TLMV. The most conserved nucleotide sequence was found in the noncoding region just upstream from the ORF2, allowing construction of a phylogenetic tree which implied that the TTV genotypes and variants, the TLMV, and chicken anemia virus could be coclassified under a superfamily for which we proposed the name of ‘Paracircoviridae’ in our previous report.
Persistent Infection Mechanism of GB Virus C/Hepatitis G Virus Differs from That of Hepatitis C Virus
Authors: Orii Koji , Tanaka Eiji , Rokuhara Akinori , Maruyama Atsushi , Ichijo Tetsuya , Yoshizawa Kaname , Kiyosawa Kendo
Published: April 2003

Objective: Changes in the deduced amino acid sequence of the envelope 2 (E2) region of the GB virus C/hepatitis G virus (GBV-C/HGV) were analyzed to investigate whether or not the region contributes to persistent infection with the virus. Methods: Eight patients with acute hepatitis C and 1 patient with acute hepatitis of unknown etiology were included in the study. GBV-C/HGV RNA was detected in 6 patients, including the patient with hepatitis of unknown origin. The nucleotide sequence of the E2 region of hepatitis C virus (HCV) and GBV-C/HGV was determined by direct sequencing of polymerase chain reaction products in 5 patients with HCV infection and in 6 patients with GBV-C/HGV infection twice during the period of early infection and several months or years later in each patient. Results: The mean substitution rate of the deduced amino acid sequence in the E2 region was over 100 times lower (p < 0.001) in GBV-C/HGV (0.01 ± 0.04/month/100 sites) than in HCV (2.4 ± 1.7/month/100 sites). The amino acid sequence of the loop domain of GBV-C/HGV-E2 did not change in any of the 6 patients. On the other hand, the sequence of the hypervariable region of HCV-E2 changed remarkably (5.9 ± 4.3/month/100 sites). No amino acid substitution in the loop domain was observed in 7 additional patients who showed persistent GBV-C/HGV viremia for more than 2 years. Conclusion: These results indicate that changes in the amino acid sequence of the E2 region are not involved in the mechanism of persistent GBV-C/HGV infection.
Contribution of Individual Structural Components in Determining the Zero-Stress State in Small Arteries
Authors: Zeller Peter J. , Skalak Thomas C.
Published: April 2003

Arterial-wall stress distributions are important determinants of arterial function and are determined by the mechanical properties of the vessel wall, the physiological loading conditions, and the zero stress state of the artery. In this study, the relationship between this zero-stress state, the contractile state of the vessel, and the extracellular matrix components was investigated. Rat saphenous arteries were excised, and cross-sectional slices were cut. Wall thickness, intimal circumference, medial to adventitial border circumference, and the outer adventitial circumference were measured. Each slice was cut once longitudinally to relieve the residual stresses, and after a period of 30 min, vessel dimensions were again measured, and the angle to which the arterial section opened, a measure of the residual stress present in the intact artery, was recorded in this new zero-stress state. Control ‘opening angle’ was 112 ± 10° (mean ± SD, n = 8) as compared to 138 ± 6° (n = 18) and 127 ± 6° (n = 11) for slices placed in 10–4 M adenosine at 25 and 37°C, respectively. Fluorescein isothiocyanate dextran was also injected intravenously in 8 animals to measure the homeostatic lumen diameter and wall thickness using intravital microscopy. Comparison of homeostatic strains calculated from these in situ dimensions to residual strains indicated that a given opening angle produced a radially uniform circumferential strain only for a specific level of tone. These results suggest that smooth muscle tone can acutely modify residual strain, possibly through interconnections with matrix components. Furthermore, selective elimination of matrix components by post-treatment of zero-stress slices with elastase or collagenase in 6 animals each increased the opening angle by 53 and 70%, respectively, suggesting that the extracellular matrix structure may regulate arterial strains in the long term.
Role of Intracellular Calcium for Noradrenaline-Induced Depolarization in Rat Mesenteric Small Arteries
Authors: Nilsson Holger , Videbæk Lars M. , Toma Catalin , Mulvany Michael J.
Published: April 2003

We have investigated the effect of intracellular calcium levels for membrane potential during noradrenaline application in isolated small arteries. Rat mesenteric small arteries were mounted for isometric tension measurement. Smooth muscle membrane potentials were measured by conventional intracellular electrodes, and intracellular calcium concentration was measured using Fura-2 fluorescence. Under control conditions, noradrenaline caused contraction and depolarization from –55.5 to –29.3 mV. In intact arteries, depleting intracellular calcium stores with thapsigargin caused smooth muscle hyperpolarization and inhibited contraction to noradrenaline. In de-endothelialized vessels, thapsigargin still depleted calcium stores, but did not affect either the depolarization or contraction caused by noradrenaline. In noradrenaline-activated vessels, inhibition of calcium influx by amlodipine caused tension and calcium levels to fall to near-baseline levels, but membrane potential returned by only 55%. Treatment with a combination of thapsigargin, D-600 and BAPTA-AM inhibited the tension and calcium responses to noradrenaline, but the membrane potential response was reduced by only 34%. Acute reduction of extracellular chloride concentration caused similar, small depolarization at rest and during noradrenaline exposure. It is concluded that an elevation of intracellular calcium concentration is not essential for noradrenaline depolarization, although part of the depolarization is associated with the raised intracellular calcium level.
Abnormal Deposition of Extracellular Matrix Proteins by Cultured Smooth Muscle Cells from Human Varicose Veins
Authors: Sansilvestri-Morel Patricia , Nonotte Isabelle , Fournet-Bourguignon Marie-Pierre , Rupin Alain , Fabiani Jean-Noël , Verbeuren Tony J. , Vanhoutte Paul M.
Published: April 2003

The aim of the present study was to verify whether the modifications of the extracellular matrix, described in varicose veins, are also present in cultures of smooth muscle cells from human varicose veins. The accumulation of collagen type III and fibronectin was determined by immunofluorescence in cultures of smooth muscle cells at passage 2–3 during the proliferation phase. After 5 days of culture, the immunostaining of both collagen type III and fibronectin was weaker in cells from varicose than in those of control veins while the expression of collagen type III and fibronectin messenger ribonucleic acids was not significantly different. Collagen type I and III synthesis were quantified by tritiated proline incorporation in control and varicose cell layers at postconfluence. Collagen type I deposition was similar in both types of cell layers while collagen type III was decreased in cell layers from varicose veins. Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) were also quantified by enzyme immunoassays in supernatants from smooth muscle cell cultures at postconfluence. No significant difference was observed in the synthesis of any of the MMPs (–1, –2 and –9) or their inhibitors (–1 and –2) tested. These data illustrate that smooth muscle cells cultured from varicose veins deposit less collagen type III and fibronectin than control cells despite comparable levels of mRNAs for these proteins suggesting dysregulation of posttranslational steps in the synthesis of both proteins by smooth muscle cells from varicose veins.
Inhibition of Vascular Smooth Muscle Cell Migration by Intact Endothelium Is Nitric Oxide-Mediated: Interference by Oxidised Low Density Lipoproteins
Authors: Gorog Peter , Kovacs Iren B.
Published: April 2003

Migration of vascular smooth muscle cells (SMCs) leading to neointimal hyperplasia is an early and cardinal feature of atherogenesis. Migration of rat aortic SMCs from an upper chamber towards a lower one has been studied in a microchemotaxis (Boyden) chamber. Spontaneous migration of SMCs was practically prevented by the presence of endothelium in the lower chamber and was reduced if endothelial cells were substituted with endothelial cell-conditioned medium. Endothelial cells which had been treated with either the inhibitor of protein synthesis cycloheximide or nitric oxide synthesis NG-nitro-L-arginine showed no inhibitory effect on SMC migration. Addition of a nitric oxide donor S-nitroso-N-acetylpenicillamine to cell-free medium in the lower chamber prevented SMC migration. Addition of native LDL to endothelial cells had no effect on SMC migration, while (UV light) oxidised LDL completely abolished the inhibitory effect of endothelial cells on SMC migration. It is concluded that via nitric oxide, endothelium exerts a powerful inhibitory effect on SMC migration. This effect of intact endothelium is completely abolished by oxidised LDL applied in a concentration, which is relevant to those measured in plasma of patients with severe coronary artery disease. It is suggested that oxidised LDL may contribute to the pathogenesis of atherogenesis by stimulating migration of SMCs from media to the intima via abolishing the physiological inhibitory effect of normal endothelium.
Bioassay of an Endothelium-Derived Hyperpolarizing Factor from Bovine Coronary Arteries: Role of a Cytochrome P450 Metabolite
Authors: Gebremedhin Debebe , Harder David R. , Pratt Phillip F. , Campbell William B.
Published: April 2003

An endothelium-derived hyperpolarizing factor (EDHF) mediates a part of the vasodilatory action of bradykinin. A bioassay method was developed to investigate the properties of EDHF on bovine coronary arterial smooth muscle cells. Cannulated bovine coronary arteries with an intact endothelium that were treated with indomethacin and NG-nitro-L-arginine methyl ester served as the EDHF donor. The effect of the donor vessel perfusate was examined on a 240 pS single-channel calcium (Ca2+)-activated potassium (K+) current (KCa) and resting membrane potential in recipient coronary arterial smooth muscle cells. The open state probability (NPo) of the channel averaged 0.011 ± 0.003 during basal perfusate flow. After stimulation of the donor vessels with bradykinin (10–10–10–6 M), the perfusate induced a 1.2- to 5-fold increase in the NPo (n = 7, p < 0.001). This increase in channel activity was attenuated by either removing the endothelium of the donor arterial segment or upon inhibition of cytochrome P450 in the donor arterial segment with the combination of 17-octadecynoic acid and miconazole. The resting cell membrane averaged –60 ± 2 mV, and hyperpolarized to –69 ± 1.5 mV (n = 6, p < 0.05) in response to the perfusate following stimulation of the donor vessel with bradykinin. Addition of 14,15-epoxyeicosatrienoic acid mimicked the effects of the perfusate and increased the NPo of the KCa channel from 0.01 ± 0.001 to 0.05 ± 0.001. These findings suggest that bradykinin stimulates the release of a transferable endothelial factor that activates KCa channels and hyperpolarizes coronary arterial smooth muscle cell membranes. These findings support the hypothesis that coronary arteries release an EDHF which is a cytochrome P450 metabolite of arachidonic acid.
Urokinase Plasminogen Activator Colocalizes with CD25+ Cells in Atherosclerotic Vessels
Authors: Falkenberg Mårten , Giglio Daniel , Björnheden Tom , Nygren Håkan , Risberg Bo
Published: April 2003

Proteolytic activity in vascular tissue is necessary for cellular migration, remodelling of extracellular matrix and the development of atherosclerotic lesions. Inflammatory cells, mainly macrophages, are numerous in atherosclerotic plaques and may synthesize and secrete proteolytic enzymes. The principal activator of plasminogen in tissues is urokinase plasminogen activator (u-PA). To determine if an activated phenotype of inflammatory cells colocalizes with local expression of u-PA in atherosclerotic vessels, vascular biopsies from 15 patients with peripheral atherosclerotic disease were analyzed by immunohistochemistry on consecutive sections. Anti-CD68 antibodies were used as markers for macrophages and were positive in 14/15 specimens. Anti-CD25 (interleukin-2 receptor-α) antibodies were used to identify inflammatory cells with an activated phenotype and were positive in 9/14 CD68+ specimens. The same 9 specimens were positive for u-PA. A positive reaction for u-PA was found only in specimens with CD25+ cells. Specimens with positive reactions for all three antibodies were further analyzed with computer-assisted image analysis. The colocalization with u-PA was higher for CD25 compared to CD68 in all specimens. Mean percentage of the u-PA-positive area in regions positive for cellular markers was 52% (SEM 6%) for CD25 and 19% (SEM 5%) for CD68 (p < 0.01). The results indicated that the activation of macrophages in atherosclerotic vessels may modulate local proteolysis and be of importance in plaque development and stability.
Segmental Differences in Geometric, Elastic and Contractile Characteristics of Small Intramural Coronary Arteries of the Rat
Authors: Szekeres Mária , Nádasy György L. , Dézsi László , Orosz Miklós , Tökés Annamária , Monos Emil
Published: April 2003

The depedence of elastic and contractile properties on the caliber of small intramural coronary arteries was investigated in the rat in vitro. Different segments of the left anterior descending coronary artery branching system were prepared for microarteriography. The segments were cannulated at both ends, immersed in oxygenated normal Krebs Ringer (nKR) solution. Intraluminal pressure was changed at a rate of about 0.5 mm Hg/s between 0 and 150 mm Hg in repeated cycles. The outer diameter was continuously measured with microangiometry. Pressure-diameter curves were recorded after preconditioning pressure cycles in nKR, with PGF2α in the bath (7.5 × 10–6 M), and in maximal relaxation with papaverine (2.8 × 10–4 M). Biomechanical parameters were computed for vessels grouped according to their calibers (inner diameters: 50–150, 150–250, 250–350, >350 µm). Distensibility and contractility decreased with increasing caliber of the vessels, while the elastic modulus increased. Spontaneous tone was (at 100 mm Hg in mechanically preconditioned vessels) 18.8 ± 4.5, 8.4 ± 4.4, 9.7 ± 3.7 and 8.3 ± 3.8% in the four groups, respectively. PGF2α contraction was maximal around the 300-µm caliber. Our study is the first direct demonstration that intramural small coronary arteries exhibit characteristic variability in their elastic and contractile properties as a function of their caliber. Such differences may be important in segmentally specific control processes of the coronary microcirculation.