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PubMed1995

Human cell mutants with very low mitochondrial DNA copy number (rho d).

Mutants of human Namalwa cells are described, denoted rho d (mtDNA-depleted), which contain a very low mtDNA copy number. Three isolates, denoted FV02, FV03 and FV05, each selected on the basis of their pyruvate-dependent phenotype, were shown to have severely reduced mitochondrial respiratory functions consistent with results of histochemical and cytochrome analysis. Analyses of mtDNA by the polymerase chain reaction (PCR) and Southern blotting indicated that FV02, FV03 and FV05 each contained a full complement of mtDNA sequences, 16.6 kb in length, in circular form but at a greatly reduced level compared with that of parent rho + cells. Quantitative PCR tests showed first, that the parent Namalwa rho + cells contain about 1800 molecules of mtDNA and second, that cells of each of FV02, FV03 and FV05 contain between 100- and 1000-fold less mtDNA than rho + cells. Subclones of each of FV02, FV03 and FV05 all contained detectable mtDNA, thus eliminating the possibility that these isolates are mixtures of rho o (devoid of mtDNA) and rho + cells. Each of the rho d cell lines FV02, FV03 and FV05 had a more rapid growth rate than the corresponding rho o cells but less than that of the parent Namalwa rho + cells. The experimentally induced rho d cells described here may represent a useful model for human diseases in which severe depletion of cellular mtDNA levels in tissues is encountered.

Cell Division↗
PubMed1993

Stereochemistry of a multistep/bipartite methyl transfer reaction: thymidylate synthase.

Atomic structures of thymidylate synthase (TS) reveal key steps in a multi-step reaction and show quantitatively how conformation change is involved in mediating the methyl transfer reaction catalyzed by TS. Numerous alterations in TS produced by mutation, screened by complementation, and further characterized can be understood in terms of the structure and profound structure change required during the TS reaction.

Chemical Phenomena↗
PubMed1976

The relationship between pregnancy, HCS and B lymphocytes.

The quantitative distribution of the lymphocyte subpopulations (B, T and null) and the serum concentrations of Human Chorionic Somatomammotropin (HCS), immunoglobulins IgG, IgM and IgA, complement component C4 and C1 inactivator, were estimated in venous blood samples from 32 women at various stages of pregnancy and compared with a control series of 7 non-pregnant normal women. A significant decline in the B cell percentage during pregnancy was seen. The fall in the percentage of the B lymphocytes was found to be concomitant with the rise in HCS concentration. No significant changes in the other parameters studied were present.

B-Lymphocytes↗
PubMed1974

Assessment of phagocytic and antimicrobial activity of human granulocytes.

A microassay for assessing two functions of polymorphonuclear leukocytes in the presence or absence of complement is presented. Requiring only minute amounts of blood (0.1 ml) and a minimum of laboratory equipment (microscope, incubator, and centrifuge), it allows the quantitation of phagocytosis and intracellular killing of microorganisms by leukocytes. To demonstrate the value of this assay, the phagocytic and microbicidal activity of leukocytes from healthy subjects and patients against Candida albicans was investigated. Apart from individual cases, no differences in the phagocytic activity between groups of healthy subjects and patients with candida vaginitis or different types of cancer could be found. However, the killing capacity of the leukocytes from women with recurrent candida vaginitis was reduced. The leukocytes of one patient showing a very low killing capacity lacked myeloperoxidase. Also, low values of killing were seen with leukocytes from three patients suffering from osteosarcoma, chronic lymphatic leukemia, or Hodgkins disease.

Journal Article↗
PubMed1973

Radiobacteriolysis: a new technique using chromium-51 for assaying anti-Vibrio cholerae antibodies.

A new method for detecting and quantitating antibodies against Vibrio cholerae is described. The reaction involves the release of radiochromium from prelabeled vibrios in the presence of specific antibody and complement. The entire assay can be completed within 5 hr. The method is highly reproducible, immunologically specific, temperature- and complement-dependent, and significantly more sensitive than other methods currently used for titration of anti-Vibrio cholerae antibodies. The technique is also potentially applicable to titration of antibodies against other gram-negative bacteria.

Aeromonas↗
PubMed1985

Mass and molecular composition of vesicular stomatitis virus: a scanning transmission electron microscopy analysis.

Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the remaining virion mass is contributed by lipids in the viral envelope, we obtained a value of 56.1 MDa for its lipid content. In addition, four different electron microscopy procedures were applied to determine the nucleocapsid length, which we conclude to be 3.5 to 3.7 micron. The nucleocapsid comprises a strand of repeating units which have a center-to-center spacing of 3.3 nm as measured along the middle of the strand. We show that these repeating units represent monomers of N protein, each of which is associated with 9 +/- 1 bases of single-stranded RNA. From scanning transmission electron microscopy images of negatively stained nucleocapsids, we inferred that N protein has a wedge-shaped, bilobed structure with dimensions of approximately 9.0 nm (length), approximately 5.0 nm (depth), and approximately 3.3 nm (width, at the midpoint of its long axis). In the coiled configuration of the in situ nucleocapsid, the long axis of N protein is directed radially, and its depth corresponds to the pitch of the nucleocapsid helix.

Capsid↗
PubMed1996

vif-negative human immunodeficiency virus type 1 persistently replicates in primary macrophages, producing attenuated progeny virus.

The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzyme-linked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif-negative HIV-1 made in MDM revealed that the virus had reduced p24 content compared with wild-type HIV-1. Cell-free MDM-derived vif mutant HIV-1 was infectious in macrophages as determined by the synthesis and maintenance of full-length viral DNA and by the produc- tion of particle-associated viral RNA, but its infectivity was approximately 2,500-fold lower than that of wild-type virus whose titer was determined in parallel by measurement of the viral DNA burden. MDM infected with MDM-derived vif-negative HIV-1 were able to transmit the virus to uninfected MDM by cocultivation, confirming the infectiousness of this virus. We conclude that mutations in vif significantly reduce but do not eliminate the capacity of HIV-1 to replicate and produce infectious progeny virus in primary human macrophages.

Base Sequence↗
PubMed2006

Hereditary angioedema associated with heterozygous factor V Leiden mutation in a patient with Purpura fulminans.

BACKGROUND: Hereditary angioedema (HAE) is an autosomal dominant, quantitative or functional defect of the C1 esterase inhibitor. The main role of the C1 esterase inhibitor is to regulate the activation of the complement system, the contact phase of the intrinsic coagulation system. On the other hand, factor V Leiden is the most common cause of primary and recurrent venous thromboembolism and displays a strong interaction with oral contraceptives. Here we report the case of a patient with HAE associated with the factor V Leiden mutation who had purpura fulminans when put on an oral contraceptive therapy. CASE: A 42-year-old woman presented to the emergency room with a history of livid skin changes on her legs during a flight. On physical examination, the vital signs were normal, but there were edemas in her legs. The livid skin changes tended to increase, eventually leading to skin necrosis on the next day. She had a history of a similar episode two months before and experienced both episodes of necrotic skin lesions just after she had been put on a hormone replacement therapy due to irregular menstruations. She also had a history of recurrent angioedema attacks since her childhood, triggered by stress and physical stimulants such as pressure and heat. Subsequent investigations revealed heterozygous factor V mutation, activated protein C resistance and reduced C1 inhibitor levels. CONCLUSION: Thrombosis is a multifactorial disease. The coexistence of multiple hereditary and acquired factors eases its occurrence. Women with HAE should be screened for the factor V Leiden mutation before pregnancy or the start of a hormone replacement therapy.

Adult↗
PubMed2001

[Epidemiology and laboratory diagnostics of legionellae].

The severe pulmonary disease caused by the inhalation of the different Legionella species is called Legionella pneumonia, while the name of the pulmonary disease caused by the most common Legionella (L. pneumophila) is Legionnaires' disease. Another type of disease caused by legionellae is Pontiac fever with influenza-like symptoms. Legionella spp. are facultative intracellular parasites. They survive within both monocytes in the human organism and amebae in the environment. To prevent and control the occurrence of legionelloses, legionellae should be surveyed and detected in the environmental (water pipes, air-conditioning systems, cooling towers, respiratory equipments, etc.) and clinical (blood, bronchoalveolar lavage, sputum, abscess, etc.) samples. Laboratory diagnosis is complicated by the limitations of the available assays. Thus, it is proposed that the microbiological laboratory diagnosis should be based on the simultaneous application of at least three methods (culturing [on BCYE medium], followed by biochemical assays, serology, molecular biologic methods, such as polymerase chain reaction [PCR], direct demonstration [immunofluorescence microscopy], antigen determination are the most important ones) and on the simultaneous demonstration from three different samples (e.g. lower respiratory tract secretions, sputum, urine, blood culture, serum, moreover, water samples from all potential infectious sources, sediment of hot water tanks, as well as swab samples of faucets and shower heads). The advantage of PCR is that is gives reliable results in one day, in contrast to conventional culturing. However, its sensitivity can not be improved by increasing the sample volume, and neither can it give quantitative results nor can it produce strains for epidemiologic studies, contrary to the method of culturing. It is concluded that PCR and culturing do complement, but do not substitute each other.

Antigens, Bacterial↗
PubMed2003

Going global: protein expression profiling using shotgun mass spectrometry.

Protein expression profiling, the science of monitoring global sets of proteins produced by any given cell type, tissue or organism, has been invigorated by the introduction of proteomic technologies capable of characterizing large numbers of proteins. This review summarizes recent advances in mass spectrometry-based techniques for high-throughput protein identification and quantitation that are fueling rapid growth in the field. Key publications applying state-of-the-art 'shotgun' methods for investigating the entire protein complement of whole organelles, cells and tissues are highlighted. An overview of current proteomic challenges, particularly in the area of data analysis, and the long-term prospects of protein profiling strategies in basic biomedical research, therapeutics development and clinical discovery is also provided.

Animals↗
PubMed1976

Quantitation of the spatial distribution of 'prespore vacuoles' in pseudoplasmodia of Dictyostelium discoideum.

The axial distribution of an organelle, the prespore vacuole (PV), previously reported absent from the prestalk region, was determined in pseudoplasmodia of varying sizes, under differing conditions of photostimulation of migration. The distribution of these organelles, determined quantitatively by electron microscopy of sections from known axial locations, was found to have a spatial pattern which varied with pseudoplasmodial size. The total complement of these organelles appeared constant for any size of pseudoplasmodium under similar conditions of illumination. Increased illumination decreased the total number of the organelles. The spatial distribution of PV varies with total cell number, and the size of the region with no PV bears no relationship to the proportion of the cell mass which would form stalk cells. Similarly, the number of cells containing PV bears no fixed relationship to the number of cells which will form spores. On these grounds, the reported role of PV, that of directing or reflecting spore differentiation, appears unlikely.

Cell Differentiation↗
PubMed1989

Brucella abortus-specific immunoglobulin isotypes in serum and vaginal mucus from heifers vaccinated with Brucella abortus salt-extractable proteins and challenge exposed with virulent Brucella organisms.

Serum and vaginal Brucella-specific immunoglobulin isotypes (IgG1, IgG2, IgM, and IgA), obtained from 62 crossbred beef heifers vaccinated with Brucella abortus salt-extractable proteins and subsequently challenge exposed with B abortus S2308, were studied. Brucella-specific IgG antibodies and Brucella-specific immunoglobulin isotypes were quantitated by a fluorometric immunoassay. Serum and vaginal immunoglobulin responses were evaluated as a method of distinguishing infected from noninfected heifers. Rivanol precipitation, complement-fixation, buffered-antigen brucellosis tests and an ELISA were performed on sera. For immunoglobulin isotypes, vaccinated heifers had mean antibody responses higher than baseline mean antibody responses for at least 31 weeks after vaccination. After challenge exposure, significant differences (P greater than 0.05) were not detected between mean antibody responses of vaccinated and nonvaccinated heifers. Vaginal Brucella-specific antibody responses did not correlate with protection from disease. Vaginal Brucella-specific IgM was detected only at the time of abortion. Vaginal IgA appeared specific for identification of virulent B abortus infection. All serotests appeared adequate in distinguishing baseline titers from titers of heifers that had aborted and were considered bacteriologic culture-positive. Results of serotests neither consistently distinguished vaccinates from challenge-exposed cattle nor distinguished heifers that were challenge exposed, had aborted, and were considered bacteriologic culture-positive adequately from heifers that were challenge-exposed, had not aborted, and were considered bacteriologic culture-negative. Brucella-specific IgA appeared to be the most effective in distinguishing vaccinated heifers from challenge- exposed heifers and heifers that were challenge exposed and had aborted, from heifers that were challenge exposed and had not aborted. Brucella-specific serum IgA was detected up to 13 weeks after abortion.

Animals↗
PubMed1987

[Comparative immunofluorescence study of actinic keratosis and chronic discoid lupus erythematosus].

Regarding systemic (SLE) and chronic discoid lupus erythematosus (CDLE), the diagnostic value of the lupus band test ist generally accepted. In the literature, however, there are but few obligatory criteria concerning the definition of a positive lupus band. In order to illustrate the influence of sunlight on the evolution of junctional deposits of immunoglobulins, we examplarily studied actinic keratosis (AK) as a chronic light-dependent dermatosis. The junctional deposits in AK were qualitatively and quantitatively compared with the lupus band typical for CDLE. In CDLE we mostly found more distinct band-like junctional deposits of immunoglobulins and complements. Light-dependent, non-specific junctional patterns of immunofluorescence similar to LE, therefore, require clear morphological criteria of immunohistology.

Aged↗
PubMed1985

[Respiratory manifestations in hereditary angioneurotic edema].

Hereditary angioneurotic oedema is an autosomal dominant state associated with a quantitative, and sometimes purely functional, deficiency of C1 esterase inhibitor (C1 INH). The clinical manifestations may begin during adulthood or childhood; they are periodical and of varying severity. Beside oedema of the skin and digestive disorders, respiratory disorders are bound to attract attention. They consist of laryngeal oedema, which may end in lethal asphyxia if tracheotomy is not performed, or, exceptionally, of pulmonary oedema requiring assisted ventilation, as in the case reported here. The diagnosis, suspected in the presence of a decrease in CH50 and C4, is confirmed by a quantitative assay of C1 INH, which is low, and/or by the Fong and Good's functional tests. The physiopathological mechanisms are complex. They involve complement activation through the classical route, and activation of the coagulation system contact phase. Patients with severe attacks now benefit from treatment with preparations of C1 INH in high concentrations. The best treatment, however, is prophylactic, using testosterone derivatives, danazol and stanozolol, which can be prescribed for long periods taking into account their usually moderate side-effects.

Acute Disease↗