Molecular biological characterization of adenovirus DNA

The agarose gel electrophoresis of DNA, the ethidium bromide fluorescence detection of DNA fragments and restriction endonucleases were discovered at the end of the '60s. The methodological progress enabled institutions equipped with less sophisticated technology to achieve also unique experimental and scientific results in the field of viral DNA research. The team working on virus DNA within the adenovirus research group has constructed several new restriction endonuclease maps of the genomes of human and animal adenoviruses; contributed to the methodology of the determination of specific endonuclease sites, and genome polarity; discovered new restriction endonucleases, adenovirus subtypes, new empty capsid, and genome subpopulations; participated in cooperations leading to novel, although hypothetical approaches in AIDS therapy, taxonomic definition of viruses, and evolutionary origins of adenovirus replication and encapsidation strategy.     

Molecular and phenotypic characterization of genotypic Candida albicans subgroups and comparison with Candida dubliniensis and Candida stellatoidea

There have been increased reports of the isolation of unusual genotypic groups of Candida albicans (groups C and D) based on a well-defined genotypic method; this method uses cellular DNA digested with the EcoRI enzyme and the restriction fragment length polymorphisms (RFLPs) generated by agarose gel electrophoresis. The aim of the present study was to use additional molecular tools to characterize these unusual strains and to compare them with authentic strains of C. dubliniensis, a recently delineated species, and type I C. stellatoidea. The RFLPs of PCR products generated from the intergenic transcribed spacer (ITS) region did not differentiate among C. albicans genotypes A, B, and C and type I C. stellatoidea. However, this method did differentiate the C. albicans genotype D strains, which were identical to C. dubliniensis. The RFLPs generated by HaeIII digestion of the PCR products of the V3 region of the 25S rRNA gene (rDNA) could differentiate the same groups as RFLP analysis of the PCR amplicon of the ITS region. C. albicans genotype B isolates have been shown to have a transposable intron in the 25S rDNA, whereas genotype A isolates do not; C. dubliniensis strains also have an intron that is larger than that in genotype B C. albicans strains but that is in the same location. PCR designed to span this region resulted in a single product for C. albicans genotype A (450 bp), B (840 bp), type 1 C. stellatoidea (840 bp), and C. dubliniensis (1,080 bp), whereas the C. albicans genotype C isolates had two major products (450 and 840 bp). All C. albicans genotype D isolates gave a PCR product identical to that given by C. dubliniensis. These results indicate that those strains previously designated C. albicans genotype D are in fact C. dubliniensis, that no differences were found between type 1 C. stellatoidea and C. albicans genotype B strains, and that the C. albicans genotype C strains appear to have the transposable intron incompletely inserted throughout the ribosomal repeats in their genomes. The results of the antifungal susceptibility testing of 105 of these strains showed that, for fluconazole, strains of C. dubliniensis were significantly more susceptible than strains of each of the C. albicans genotypes (genotypes A, B, and C). The flucytosine susceptibility results indicated that strains of C. albicans genotype A were significantly less susceptible than either C. albicans genotype B or C. albicans genotype C strains. These results indicate that there is a correlation between the Candida groups and antifungal susceptibility.     

Candida glabrata prosthesis infection following pyelonephritis and septicaemia

A 48-year-old woman with no cardiovascular risk factors was admitted to the hospital because of acute dyspnea. At 27-year-old, she developed Hodgkin's disease, that was successfully treated with splenectomy, combined chemotherapy (nitrogen mustard, vincristine, procarbazine, prednisone-MOPP regimen) and radiotherapy (4500 rads). At 43-year-old the lymphoma relapsed and she had further chemotherapy with doxorubicin, bleomycin, vinblastina and dacarbazine. After this treatment, she had an episode of pulmonary edema, attributed to doxorubicin acute cardiotoxicity. She responded to digitalis and diuretics and was discharged with an electrocardiogram (ECG) showing left bundle branch block and a normal echocardiogram. The patient enjoyed good health for several years and 4 months before the present admission the ECG and echocardiogram were unchanged. On this admission there were signs of left ventricular failure with acute pulmonary edema, and a new soft apical murmur (3-4 Levine). The patient required endotracheal intubation and high doses of diuretics, digitalis and vasodilators. The cardiac enzymes were negative, the serial ECGs confirmed left bundle branch block, while the echocardiogram showed moderate to severe mitral regurgitation, akinesia of the interventricular septum and inferior wall with dilation of the left ventricle. A previous silent myocardial infarction was suspected. After recovery, she underwent cardiac catheterization confirming akinesia of the interventricular septum and inferior wall with moderate mitral regurgitation, while coronary angiography showed a critical ostial stenosis of the right coronary artery. In view of a dipyridamole-thallium scan negative for myocardial viability, reperfusion was not attempted. With changes in radiotherapeutic techniques, the incidence of radiation-induced heart disease (pericarditis, myocarditis, conduction abnormalities and, rarely, occlusive coronary artery disease) is declining. Nevertheless, after irradiation of the chest and mediastinum a longterm cardiological follow-up is useful in selecting patients at higher risk of radiation-induced coronary artery disease, who will eventually require coronary angiography and reperfusion intervention.     

Molecular cloning of an Australian isolate of hepatitis C virus

A total of 208 strains of Aeromonas were isolated by monthly sampling from two estuaries (one provided with, and the other devoid of a waste-water treatment system) on the Italian coast of the Adriatic sea between September 1994 and August 1995. Biotyping at the species level allowed the identification of 96 strains (46%) as Aer. caviae, 46 (22%) as Aer. sobria, 33 (16%) as Aer. hydrophila and 25 (12%) as Aer. veronii. Eight strains (4%) were regarded as unnamed aeromonads. Aeromonas caviae was the most prevalent species in water with a high degree of pollution, while Aer. hydrophila strains were more commonly isolated from cleaner water. Aeromonas sobria and Aer. veronii were equally distributed in both estuaries. There was no correlation between temperature and numbers of aeromonads in either estuary. Using a biochemical fingerprinting method, strains were divided into similarity groups (PhP-types) based on their biochemical phenotypes. Several different PhP-types were found in each estuary, yielding a high diversity for these strains. However, some identical PhP-types were also found in both estuaries and at different times of the year, indicating that certain Aeromonas strains can survive more widely varying physico-chemical conditions. The production of toxins capable of causing cytoskeletal-dependent changes in the morphology of Chinese hamster ovary (CHO) cells was detected in 14 strains and appeared to be dependent on the season.     

A clinical isolate of Candida palmioleophila formerly identified as Torulopsis candida

To reassess the impact of renal ultrasonography on the care of children with first febrile urinary tract infection (UTI) we conducted a computer search and review of medical records of (1) all children who were admitted to our hospital with first febrile urinary tract infection and underwent renal ultrasonography during a 25-month period beginning February 1, 1995, (2) all children diagnosed by ultrasound to have hydronephrosis during the same time period. Of a total of 124 patients with UTI, renal ultrasound appeared normal or showed evidence of acute pyelonephritis in 105 (84.7%), and in another nine (7.2%) it showed only minor findings. In 10 children (8.1%) ultrasound showed hydronephrosis and/or hydroureter. In eight of the latter 10, voiding cystourethrography showed vesicoureteral reflux; in one, posterior urethral valves; and in one, who had a unilateral nonobstructed dilatated system, cystography appeared normal. Except for the last patient, who was given prophylactic antibiotics and continued to have urinary tract infections, in no other case did ultrasound alone have any impact on the patient's management. Four children with both abnormal-appearing renal ultrasound and voiding cystourethrography required surgical intervention. One hundred of the 124 children had a voiding cystourethrogram. In 38 children it detected vesicoureteral reflux and, in another two, bladder abnormalities. Thirty-five of those with abnormal-appearing cystogram but without an indication for surgery were given prophylactic antibiotics. During the same 25-month period, 63 children without urinary tract infection were diagnosed by ultrasound with hydronephrosis. In 45 of them (71.4%) the urologic abnormality had already been detected by prenatal ultrasound. Fourteen of these 45 children (31.1%) required surgery, all for congenital anomalies related to obstructive uropathy. We conclude that routine renal ultrasonography in children with first urinary tract infection has negligible influence on their clinical management. This seems to be due to the recent widespread use, in industrialized countries, of maternal-fetal ultrasonography, which already detects a significant number of children with congenital obstructive uropathy prenatally. On the other hand imaging of the lower urinary tract is of high yield and contributes significantly to patient care. Therefore, whereas imaging of the lower urinary tract should continue to be done routinely in children with first urinary tract infection, renal ultrasound may be reserved for more select cases.     

Molecular and biological characterization of a neurovirulent molecular clone of simian immunodeficiency virus

To identify the molecular determinants of neurovirulence, we constructed an infectious simian immunodeficiency virus (SIV) molecular clone, SIV/17E-Fr, that contained the 3' end of a neurovirulent strain of SIV, SIV/17E-Br, derived by in vivo virus passage. SIV/17E-Fr is macrophage tropic in vitro and neurovirulent in macaques. In contrast, a molecular clone, SIV/17E-Cl, that contains the SU and a portion of the TM sequences of SIV/17E-Br is macrophage tropic but not neurovirulent. To identify the amino acids that accounted for the replication differences between SIV/17E-Fr and SIV/17E-Cl in primary macaque cells in vitro, additional infectious molecular clones were constructed. Analysis of these recombinant viruses revealed that changes in the TM portion of the envelope protein were required for the highest level of replication in primary macaque macrophages and brain cells derived from the microvessel endothelium. In addition, a full-length Nef protein is necessary for optimum virus replication in both of these cell types. Finally, viruses expressing a full-length Nef protein in conjunction with the changes in the TM had the highest specific infectivity in a sMAGI assay. Thus, changes in the TM and nef genes between SIV/17E-Cl and SIV/17E-Fr account for replication differences in vitro and correlate with replication in the central nervous system in vivo.     

Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase

We isolated a cDNA clone encoding mouse N-acetylglucosamine-6-O-sulfotransferase based on sequence homology to the previously cloned mouse chondroitin 6-sulfotransferase. The cDNA clone contained an open reading frame that predicts a type II transmembrane protein composed of 483 amino acid residues. The expressed enzyme transferred sulfate to the 6 position of nonreducing GlcNAc in GlcNAcbeta1-3Galbeta1-4GlcNAc. Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc and various glycosaminoglycans did not serve as acceptors. Expression of the cDNA in COS-7 cells resulted in production of a cell-surface antigen, the epitope of which was NeuAcalpha2-3Galbeta1-4(SO4-6)GlcNAc; double transfection with fucosyltransferase IV yielded Galbeta1-4(Fucalpha1-3)(SO4-6)GlcNAc antigen. The sulfotransferase mRNA was strongly expressed in the cerebrum, cerebellum, eye, pancreas, and lung of adult mice. In situ hybridization revealed that the mRNA was localized in high endothelial venules of mesenteric lymph nodes. The sulfotransferase was concluded to be involved in biosynthesis of glycoconjugates bearing the 6-sulfo N-acetyllactosamine structure such as 6-sulfo sialyl Lewis X. The products of the sulfotransferase probably include glycoconjugates with intercellular recognition signals; one candidate of such a glycoconjugate is an L-selectin ligand.     

Molecular biological intervention

Molecular biological intervention including gene therapy is a novel form of molecular medicine that will have an impact on the treatment of many serious diseases. Recent advancement in understanding of molecular mechanism of human disease and development of gene technology allows us practical approaches to molecular biological intervention. In addition to the possibility of correcting inherited genetic disorders, molecular biological intervention is being used to combat acquired disease. In the field of nephrology, the basic study has started on the gene transfer into the kidney in vivo. This review describes some approaches and methods that may be used for molecular biological intervention for glomerulonephritis and the state of our preclinical studies targeting transforming growth factor-beta.     

Inhibition of hyphal growth of azole-resistant strains of Candida albicans by triazole antifungal agents in the presence of lactoferrin-related compounds

The effects of bovine lactoferrin (LF) or the LF-derived antimicrobial peptide lactoferricin B (LFcin B) on the growth of Candida albicans hyphae, including those of three azole-resistant strains, were investigated by a crystal violet staining method. The hyphae of two highly azole-resistant strains were more susceptible to inhibition by LF or LFcin B than the azole-susceptible strains tested. One moderately azole-resistant strain was defective in the formation of hyphae and showed a susceptibility to LF greater than that of the susceptible strains but a susceptibility to LFcin B similar to that of the susceptible strains. The highly azole-resistant strain TIMM3317 showed trailing growth in the presence of fluconazole or itraconazole, while the extent of growth was reduced by the addition of LF or LFcin B at a sub-MIC. Thus, the addition of LF or LFcin B at a sub-MIC resulted in a substantial decrease in the MICs of fluconazole and itraconazole for two highly azole-resistant strains; e.g., the MIC of fluconazole for TIMM3317 was shifted from > 256 to 0.25 micrograms/ml by LF, but the MICs were not decreased for the susceptible strains. The combination effects observed with triazoles and LF-related compounds in the case of the two highly azole-resistant strains were confirmed to be synergistic by the fractional inhibitory concentration index. These results demonstrate that for some azole-resistant C. albicans strains, LF-related compounds combined with triazoles can inhibit the growth of hyphae, an important form of this organism in pathogenesis.     

Molecular characterization of an inwardly rectifying K+ channel from HeLa cells

Retinoid-deficient cultures of airway epithelial cells undergo squamous differentiation. Treatment of such cultures with retinoic acid (RA) leads to restoration of the mucous phenotype. The purpose of our study was to characterize the cellular and molecular changes following RA treatment of retinoid-deficient human tracheobronchial epithelial cell cultures. Of particular interest was to determine when during the conversion of the squamous to the mucous phenotype the mucin genes MUC2, MUC5AC, and MUC5B were expressed. We used cornifin alpha and secreted mucin as markers to monitor the squamous and mucous phenotypes, respectively. Our studies showed that the RA responsiveness of the cultures progressively decreased with protracted retinoid deficiency, requiring higher RA concentrations to restore the mucous phenotype. Within 12 h after the start of RA treatment, cornifin alpha expression decreased, signaling the beginning of a change in cellular phenotype. At 24 h after addition of RA to the cultures, a significant number of mucous cells appeared, and at 72 h mucin was secreted in measurable amounts. Induction of mucin gene expression occurred sequentially: MUC2, MUC5AC, and MUC5B mRNAs were upregulated at 24, 48, and 72 h, respectively. When cultures maintained in 10(-8) M RA were treated with 10(-6) M RA, MUC2 but not MUC5AC and MUC5B mRNA levels were upregulated within 6 h. Our study indicates that MUC2 mRNA is an early marker of mucous differentiation, whereas MUC5AC and MUC5B mRNAs are expressed during more advanced stages of mucous differentiation. Our studies further suggest that each of the mucin genes is regulated by distinct mechanisms.     

A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa

In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria. This isolate, designated So93, did not differ from Mycobacterium tuberculosis in the biochemical tests and in its 16S rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species. This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency. The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease. Both morphotypes had shorter generation times than the M. tuberculosis reference laboratory strain H37Rv and clinical isolates of M. tuberculosis and Mycobacterium bovis. Furthermore, the So93 isolate differed from all M. tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide. This glycolipid had previously been observed only in a smooth isolate of M. tuberculosis obtained in 1969 by Canetti in France. Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M. tuberculosis complex taxa, M. tuberculosis, Mycobacterium africanum, M. bovis, and Mycobacterium microti. The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown. This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M. tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated "M. tuberculosis".     

Molecular characterization of epididymal proteins

Reduction in energy consumption is known to inhibit development of a variety of spontaneous, carcinogen-induced, and hormone-dependent cancers, but the mechanism or mechanisms by which this occurs remain unknown. We hypothesize that energy consumption may modulate development of estrogen-dependent neoplasms by altering the manner in which target cells respond to estrogens. To test this hypothesis, ovariectomized female Fischer 344 rats were fed diets that allowed consumption of different amounts of energy, and the ability of 17beta-estradiol (E2), administered for 10 wk from subcutaneous Silastic implants, to promote development of prolactin-producing pituitary tumors was examined. A 40% restriction of energy consumption virtually abolished the ability of E2 to promote development of pituitary tumors and associated hyperprolactinemia. A 25% restriction of energy consumption appeared to slightly inhibit E2-induced pituitary growth and hyperprolactinemia, but the observed degree of inhibition was not statistically significant. Interestingly, dietary energy restriction did not inhibit induction by E2 of pituitary cell proliferation and lactotroph hyperplasia. Furthermore, E2 treatment inhibited expression of testosterone-repressed prostate message-2 mRNA, a cellular marker of apoptosis, and this inhibitory effect of E2 was blocked by 40% energy restriction. These data suggest that dietary energy restriction virtually abolished E2-induced development of prolactin-producing pituitary tumors, not by blocking the ability of E2 to induce cell proliferation but rather by blocking the ability of E2 to enhance cell survival. This study and the accompanying paper provide the first indication that dietary energy consumption may modulate estrogen action at the level of the target cell.     

Molecular cloning and characterization of an invertebrate homologue of a neuropeptide Y receptor

Neuropeptide Y is an abundant and physiologically important peptide in vertebrates having effects on food intake, sexual behaviour, blood pressure and circadian rhythms. Neuropeptide Y homologues have been found in invertebrates, where they are very likely to play similar, important roles. Although five neuropeptide Y-receptor subtypes have been identified in mammals, none has been reported from invertebrates. Here we describe the cloning of a neuropeptide Y-receptor from the brain of the snail Lymnaea stagnalis. The identity of the receptor was deduced by expressing the neuropeptide Y-receptor-encoding cDNA in Chinese Hamster Ovary cells, which were subsequently challenged with size-fractionated Lymnaea brain extracts. An active peptide, selected on the basis of its ability to induce changes in cAMP levels, was purified to homogeneity, analysed by mass spectrometry and amino acid sequence determination, and turned out to be a Lymnaea homologue of neuropeptide Y.     

Molecular biological aspects of acquired bullous diseases

Bullous diseases of the oral mucosa and skin were originally classified on the basis of clinical and histological criteria. The discovery of autoantibodies in some of these patients and the introduction of molecular biology have resulted in a new understanding of the pathological mechanisms of many of the bullous lesions. In this article, updated topics of the immune-mediated bullous lesions which involve oral mucosa and skin are reviewed. Pemphigus antigens, which are desmosomal-associated proteins and belong to the cadherin superfamily of cell adhesion proteins, have been isolated, and their genes have been cloned. The antigens which react with autoantibodies from patients with bullous pemphigoid, cicatricial pemphigoid, acquired epidermolysis bullosa, and linear IgA disease are all proteins of the hemidesmosome basement membrane complex. Interestingly, most of the antigens also appear to be the target for mutations seen in patients with the inherited type of epidermolysis bullosa in which bullous lesions are a prominent clinical feature.     

Purification and partial characterization of acid phosphatase from Candida lipolytica

Non-specific acid phosphatase from Candida lipolytica cells was purified 111-fold by chromatography on DEAE-cellulose and gel filtration on Sephadex G-100 and Sepharose 4B. The enzyme is a glycoprotein containing 67% neutral sugars. The molecular mass of the highly purified acid phosphatase was found to be approximately 95 kDa by both SDS-PAGE and gel filtration. The pH and temperature optima were 5.8 and 55 degrees C, respectively. The enzyme was stable at pH values between 3.5 and 5.5 and at temperatures up to 60 degrees C. The purified phosphatase had a Km value of 3.64 mM for p-nitrophenyl phosphate and showed broad substrate specificity.