Molecular QTAIM Topology Is Sensitive to Relativistic Corrections.

The topology of the molecular electron density of benzene dithiol gold cluster complex Au4 -S-C6 H4 -S'-Au'4 changed when relativistic corrections were made and the structure was close to a minimum of the Born-Oppenheimer energy surface. Specifically, new bond paths between hydrogen atoms on the benzene ring and gold atoms appeared, indicating that there is a favorable interaction between these atoms at the relativistic level. This is consistent with the observation that gold becomes a better electron acceptor when relativistic corrections are applied. In addition to relativistic effects, here, we establish the sensitivity of molecular topology to basis sets and convergence thresholds for geometry optimization.

Detection of Mycobacterium bovis lymphocyte stimulating antigens in culture filtrates of a recombinant Mycobacterium smegmatis cosmid library.

Culture filtrates derived from a Mycobacterium bovis cosmid library in Mycobacterium smegmatis were screened for bovine lymphocyte stimulatory antigens using peripheral blood mononuclear cells (PBMC) from cattle vaccinated with a low dose of Mycobacterium bovis BCG. Lymphocyte proliferation and interferon-gamma (IFN-gamma) production were used as cellular response markers for antigen recognition. In the primary screen, approximately 28% of all culture filtrates (CF) stimulated responses by PBMC from at least two out of four vaccinated cattle. In one of these CF, the M. bovis Ag85-B antigen was detected by Western-blot analysis. Despite heterogeneous lymphocyte responses of the animals, twenty-four of the culture filtrates stimulated lymphocyte proliferation and IFN-gamma production from at least six out of eight vaccinated animals in a secondary screen. Analysis of the cosmid DNA associated with these positive CF demonstrated that several contained homologous DNA sequences. It appears that the lymphocyte screening has detected M. bovis antigens that are immuno-dominant in cattle vaccinated with M. bovis BCG.

Daily supplementation with aged garlic extract, but not raw garlic, protects low density lipoprotein against in vitro oxidation.

The oxidation of low density lipoprotein (LDL) is believed to be an important process in the development and progression of atherosclerosis. In this study, human subjects were supplemented daily with one of: 6 g raw garlic; 2.4 g aged garlic extract (AGE); or 0.8 g DL-alpha-tocopherol acetate for 7 days to determine the effect on the susceptibility of LDL particles to Cu2+-mediated oxidation. LDL isolated from subjects given either alpha-tocopherol or AGE, but not raw garlic, was significantly more resistant to oxidation than LDL isolated from subjects receiving no supplements. These results suggest that if antioxidants are proven to be antiatherogenic, AGE may be useful in preventing atherosclerotic disease.

Antigen-specific lymphocytes enhance nitric oxide production in Mycobacterium bovis BCG-infected bovine macrophages.

Nitric oxide (NO) production was evaluated in macrophages isolated from Mycobacterium bovis bacille Calmette-Guérin (BCG)-immunized, and control non-immunized, cattle. Incubation of M. bovis BCG-infected macrophages with recombinant bovine IFN-gamma led to increased nitrite levels in culture supernatants. It was also demonstrated that NO production by autologous M. bovis BCG-infected macrophages increased in a linear relationship with the number of antigen-specific lymphocytes added to cultures. The elevated NO levels were also associated with increased IFN-gamma secretion. Treatment of cultures with the NO inhibitor, N-monomethyl L-arginine (L-NMMA), reduced the levels of NO without affecting the metabolic activity of internalized M. bovis BCG. Our results suggest that synthesis of NO may constitute an integral part of the cell-mediated antigen-specific response against M. bovis BCG. However, although the presence of lymphocytes does partially inhibit multiplication of M. bovis BCG in macrophages, it appears that the activity of NO, or the levels produced in monocyte-derived macrophages, may be insufficient to influence the growth of the intracellular mycobacteria.

Cellular responses and Mycobacterium bovis BCG growth inhibition by bovine lymphocytes.

Cellular responses of a group of cattle immunized subcutaneously with a low dose of Mycobacterium bovis bacille Calmette-Guerin vaccine (BCG) were measured in vitro and compared with nonimmunized control animals. PBMC taken from immunized animals proliferated and produced IFN-gamma in the presence of M. bovis BCG culture filtrate proteins. The addition of PBMC from immunized animals to M. bovis BCG-infected autologous macrophages also resulted in secretion of IFN-gamma. In contrast, the responses of PBMC from control animals were comparatively low over the period of study. In experiments to study the interaction of non-adherent lymphocytes with infected macrophages, M. bovis BCG growth was inhibited in cultures containing autologous PBMC from immunized and non-immunized control animals. The degree of inhibition was related to lymphocyte concentration but did not correlate with IFN-gamma production. Treatment of macrophages with recombinant IFN-gamma prior to, or postinfection did not alter the intracellular growth kinetics of mycobacteria. It appears, therefore, that although M. bovis BCG immunization of cattle stimulates the generation of a T cell-mediated immune response to M. bovis BCG, the cattle may already possess a high level of innate resistance to M. bovis BCG that requires the presence of lymphocytes.

Identification of bovine T cell stimulatory antigens using a cosmid library of Mycobacterium bovis in Mycobacterium smegmatis.

Culture filtrates derived from a Mycobacterium bovis cosmid library in Mycobacterium smegmatis were screened for T cell antigens. Recognition and reactivity were measured by the levels of lymphocyte proliferation and the levels of gamma interferon (IFN-gamma) produced when the culture filtrates were incubated with peripheral blood mononuclear cells (PBMC) taken from cattle immunised with M. bovis BCG. The screening system was optimised to distinguish between M. bovis secreted antigens and normal M. smegmatis secreted proteins. From ten culture filtrates screened, two were identified that induced lymphocyte proliferation and IFN-gamma production. Analysis of the DNA inserts from the recombinant cosmids suggest that they may code for different proteins. The results demonstrate that screening recombinant M. smegmatis culture filtrates can be used to identify M. bovis T cell antigens that are recognised by immunised cattle. These antigens may be important for the development of vaccines with protective ability against bovine tuberculosis.

Responses of bovine T cells to fractionated lysate and culture filtrate proteins of Mycobacterium bovis BCG.

Bacterial cell lysates and culture filtrate proteins of Mycobacterium bovis BCG were each separated in a two-dimensional system that yields soluble protein fractions immediately available for probing with T cells. The fractions were used in lymphocyte proliferation assays using blood lymphocytes from cattle immunized with either viable or gamma-irradiated BCG. Cattle immunized with either form of BCG responded similarly to fractionated lysate proteins. Cattle immunized with viable BCG responded to culture filtrate proteins that were not recognized by cattle immunized with dead BCG. Marked heterogeneity of the responses to the culture filtrate proteins was seen.

Antibody detection ELISAS for malaria diagnosis.

Parasite extracts of Plasmodium falciparum and P. chabaudi and three synthetic peptides from the P. falciparum MSA2 merozoite antigen were tested for suitability as antigens in an antibody detection ELISA using sera from malaria patients in Brisbane. The P. chabaudi extract was superior to P. falciparum extract for detecting P. vivax cases, while for P. falciparum cases the two parasite extracts were equivalent. Single peptide antigens were generally less sensitive than parasite extracts; however, peptides G3 and G7 were more sensitive than parasite extracts in detecting first attacks of P. vivax. Examination of isotype specific responses demonstrated that this may be explained by higher IgG responses to these peptides in first than in subsequent P. vivax attacks. Because of the differing antibody specificities in primary and secondary P. falciparum and P. vivax cases, the best sensitivity was achieved by using the combined results of assays with three antigens: P. chabaudi, peptide G3 and peptide G7. The combined sensitivity was 77.1% for P. falciparum and 88.6% for P. vivax acute cases with 91.1% specificity.

Design of DNA intercalators to overcome topoisomerase II-mediated multidrug resistance.

Murine P388 (P) leukemia cell lines resistant to amsacrine (P/AMSA), dactinomycin (P/DACT), and doxorubicin (P/DOX) were compared with the parental strain in their sensitivity to a number of derivatives of amsacrine. The P/DACT cell line, which shows the characteristics of a transport-mediated multidrug-resistant cell line, was cross-resistant to vincristine, doxorubicin, etoposide, and a number of acridine-substituted amsacrine derivatives, but was sensitive in vitro and in vivo to amsacrine and its analog CI-921. The P/DOX cell line was cross-resistant to amsacrine but showed a similar pattern of cross-resistance to that of P/DACT in its in vitro response to amsacrine derivatives. In contrast, the P/AMSA line was substantially cross-resistant (from 27- to 146-fold) to all acridine-substituted amsacrine derivatives. However, when the substituents on the anilino side chain of amsacrine were changed, the in vitro cross-resistance of the P/AMSA line could be substantially reduced and even overcome. Derivatives with low cross-resistance ratios were tested in vivo against the P/AMSA leukemia and, in contrast to amsacrine and CI-921, were found to be active. Since the target enzyme for amsacrine action, topoisomerase II, is thought to be structurally modified in the P/AMSA line as well as in some other multidrug-resistant lines, these results suggest the feasibility of tailoring topoisomerase II-directed drugs specifically for the altered enzymes in resistant cells. New drug design approaches are therefore available for overcoming two major types of multidrug resistance.

Comparison of the effects of flavone acetic acid, fostriecin, homoharringtonine and tumour necrosis factor alpha on colon 38 tumours in mice.

Advanced subcutaneous Colon 38 tumours in mice were used for the assessment of activity of a number of anticancer drugs. Activity was measured by histological examination of tumours 24 h after a single dose of the drug and in some cases by tumour growth delay. Agents thought to exert their cytotoxic effect by damaging DNA, including Adriamycin, amsacrine and its analogue CI-921, cyclophosphamide, 5-fluorouracil and methotrexate produced no gross histological changes after 24 h, even though some delayed the growth of subcutaneous tumours. In contrast, flavone acetic acid, fostriecin and homoharringtonine caused extensive necrosis of tumours after 24 h, and each delayed the growth of advanced subcutaneous tumours by at least 10 days when administered as a single dose. The histological effects of flavone acetic acid and fostriecin were indistinguishable from those of recombinant human tumour necrosis factor alpha. It is proposed that histological assay of advanced tumours may provide a useful adjunct to existing methods in screening for antitumour agents with novel mechanisms of action.

Effect of flavone acetic acid on Lewis lung carcinoma: evidence for an indirect effect.

Flavone-8-acetic acid (FAA), a new antitumor agent currently undergoing clinical trial, fails to inhibit the growth of early stage Lewis lung (LL) tumors growing in the lung. However, the growth of advanced subcutaneous tumors, arising from inoculation of either the original in vivo LL line or a tissue culture-adapted cell line (LLTC) derived from the LL line was delayed significantly by FAA treatment. Comparison, by clonogenic survival assays, of the cytotoxic effect of FAA on LLTC cells demonstrated that most cell killing occurred between 2 and 8 hours following in vivo exposure but occurred to a much lesser extent and at later times following in vitro exposure. FAA was inactive against LLTC cells growing in vivo in diffusion chambers, suggesting that a host cellular component was necessary for activity. FAA was found to induce hemorrhagic necrosis in the advanced LL tumors, as well as in a number of human tumor xenografts growing in athymic mice. The human cell lines from which the xenografts were derived, as well as the LL tumor lines and P388 leukemia lines, were inhibited by FAA in vitro. However, the ranking of FAA activity in vivo did not parallel that observed in vitro. Together, these observations strongly suggest that FAA has an indirect mode of antitumor action.

The demonstration of B-cell, T-cell and myeloid antigens in paraffin sections.

The monoclonal antibodies MB1 and MT1, which detect B cells and T cells respectively, have been applied to human lymphoid tissues. The distribution of staining within paraffin sections was compared with that observed using frozen sections and was found to be identical. The antibodies were then applied to paraffin sections of 19 B-cell lymphomas and 10 T-cell lesions in which full immunophenotyping had been performed. The B lymphomas all consisted of a large majority of MB1 positive cells with a variable infiltrate of small MT1 positive lymphocytes. The T cell lesions consisted of MT1 positive cells with few MB1 positive cells except in residual B cell areas of lymph nodes. In paraffin sections from cases of Hodgkin's disease anti-Leu M1 identified Reed-Sternberg cells and their variants and MB1 and MT1 showed a similar distribution of B cells and T cells to that demonstrated in previous studies using frozen sections. The results show that MB1 and MT1 are useful markers for B and T cells in routinely fixed paraffin embedded tissue. In conjunction with anti-Leu M1 they provide a valuable panel of antisera for the examination of lymph nodes and other biopsies when frozen tissue is not available.

A longitudinal serological survey of parvovirus infection in dogs.

Serum samples collected from dogs routinely presented at a clinic between June 1974 and October 1980 were tested for the presence of haemagglutination inhibition (HI) titres to canine parvovirus. The first positive titre (>1:320) was demonstrated in serum collected in October 1979. The first confirmed clinical case of canine parvovirus enteritis was diagnosed by the authors in July 1979. In addition, between 1st December 1980 and 1st March 1981, serum samples were collected from 106 healthy dogs which were presented for canine parvovirus vaccination for the first time. Twenty-four dogs (approx. 23%) showed HI titres >1:320 indicating probable previous canine parvovirus infection. Therefore approx. 80% of dogs in the clinic area were at risk at that time and vaccination should have protected them from infection.