Convergent structure and function of mycelial galleries in two unrelated Neotropical plant-ants.

The construction process and use of galleries by Azteca brevis (Myrmicinae: Dolichoderinae) inhabiting Tetrathylacium macrophyllum (Salicaceae) were compared with Allomerus decemarticulatus (Myrmicinae: Solenopsidini) galleries on Hirtella physophora (Chrysobalanaceae). Though the two ant species are phylogenetically distant, the gallery structure seems to be surprisingly similar and structurally convergent: both are pierced with numerous holes and both ant species use Chaetothyrialean fungi to strengthen the gallery walls. Al. decemarticulatus is known to use the galleries for prey capture and whether this is also the case for Az. brevis was tested in field experiments. We placed Atta workers as potential prey/threat on the galleries and recorded the behaviour of both ant species. We found considerable behavioural differences between them: Al. decemarticulatus was quicker and more efficient at capture than was Az. brevis. While most Atta workers were captured after the first 5 min by Al. decemarticulatus, significantly fewer were captured by Az. brevis even after 20 min. Moreover, the captured Atta were sometimes simply discarded and not taken to the nest by Az. brevis. As a consequence, the major function of the galleries built by Az. brevis may, therefore, be defense against intruders in contrast to Al. decemarticulatus which uses them mainly for prey capture. This may be due to a higher need for protein in Al. decemarticulatus compared to coccid-raising Az. brevis.

[Punctate maculae on the back of the hands of a 44-year-old woman]. / Punktförmige Makulae an den Handrücken einer 44-jährigen Frau.

A 44-year-old woman developed punctate erythematous maculae on the backs of her hands, arms and shoulders following a pregnancy. Laboratory evaluation was unremarkable. Our differential diagnosis includes idiopathic teleangiectases, teleangiectasia eruptiva perstans, angioma serpiginosum and angiokeratoma corporis diffusum Fabry. Microscopic examination showed increased numbers of the small vessels of the upper vascular plexus with dilated capillaries. This coupled with the clinical findings led us to the diagnosis of angioma serpiginosum with symmetrical distribution involving the shoulder girdle, upper aspects of the arms, and the backs of the hands. We treated with a pulsed dye laser and noted some regression after two sessions.

Induction of mitotic crossing over in Saccharomyces cerevisiae by breakdown products of dimethylnitrosamine, diethylnitrosamine, 1-naphthylamine and 2-naphthylamine formed by an in vitro hydroxylation system.

Dimethylnitrosamine and diethylnitrosamine, two potent carcinogens, are nonmutagenic when tested directly in microorganisms. Likewise 1-naphthylamine and 2-naphthylamine are also nonmutagenic but the N-hydroxy derivatives are mutagenic in microorganisms. Apparently these compounds require metabolism to breakdown products which are then the proximately active agents, and microorganisms lack the enzymes necessary to effect this conversion. These compounds are mutagenic in Saccharomyces after conversion to breakdown products in an in vitro hydroxylation medium. The induction of mitotic crossing over in Saccharomyces cerevisiae by breakdown products of dimethylnitrosamine, diethylnitrosamine, 1-naphthylamine and 2-naphthylamine formed in the Udenfriend hydroxylation medium is reported in this communication. Mitotic crossing over was detected as red sectored colonies resulting from induced homozygosity of the ade2 marker. Dimethylamine and diethylamine, which lack the nitroso group of the nitrosamines, did not induce mitotic crossing over under any of the test conditions. To further confirm that the induced sectored colonies were the result of mitotic crossing over they were tested for the presence of reciprocal products. The expected reciprocal products were found in over 67% of the isolates tested. The significance and practicality of using mitotic recombination as an indicator of genetic damage potential of chemicals is discussed.

Semidominance of rad18-2 for several phenotypic characters in Saccharomyces cerevisiae.

Inbred diploid yeast strains heterozygous or homozygous for the rad18-2 allele and carrying markers for detection of mitotic recombination were constructed. The homozygous rad18-2/rad 18-2 strain was highly sensitive to killing by UV light, showed greatly elevated frequencies of spontaneous and induced mitotic recombination and was more sensitive to trimethoprim than the wild-type diploid. The heterozygous strain RAD18/rad 18-2 was intermediate in its response for these same phenotypic characters. These findings are discussed in the light of other studies in which incomplete dominance of genes involved in some aspect of DNA repair has been reported.

Finite-element-modeling of egg white as a substitute for tissue coagulation during bipolar radiofrequency-induced thermofusion.

Radiofrequency-induced thermofusion is a frequently used electrosurgical procedure for the sealing of blood vessels. A disadvantage of vessel sealing instruments is that the generated thermal energy spreads to the surrounding tissue and may irreversibly damage it. This is particularly problematic when operating close to sensitive structures such as nerves. Given their advantages, there is nonetheless a lot of interest in using bipolar vessel sealing for surgical procedures. To select instruments that may be safely used in such cases, it is important to reliably quantify the thermal spread to the surrounding tissue. Mathematical models can help to evaluate the transient behavior, that is the evolution of the thermal spread over time, more precisely. A finite element model allows for a detailed analysis of inhomogeneities in the spatial temperature distribution. As a first step towards a finite model of the bipolar vessel sealing process, a model of the coagulation of chicken egg white is presented here. Egg white has thermal and electrical properties that are very similar to tissue, making it suitable as a substitute for the analysis of the coagulation process. It has the additional advantage, that the spatial and temporal evolution of the thermal spread can be visually gauged. The presented model describes the experimentally observed spatial temperature distribution, the shape of the coagulated egg white, and the formation of hotspots. Furthermore, it is shown that the model can correctly predict the shape of the coagulated egg white in further experiments.

Clinical and immunological changes in AIDS patients following adoptive therapy with activated autologous CD8 T cells and interleukin-2 infusion.

OBJECTIVES: (1) To determine the safety and feasibility of repetitive reinfusions of activated autologous CD8 cells followed by low-dose continuous interleukin (IL)-2 infusion in patients with AIDS. (2) To study the relationships between clinical responses, surface marker phenotypic distributions and cytokine expression patterns of both cultured CD8 cells and lymphocytes in the peripheral blood compartment. DESIGN: Six adult patients with Centers for Disease Control and Prevention group IV HIV-1 disease ranging from mild to severe, were studied. All patients were receiving zidovudine prior to and during the study period, and had initial CD4 and CD8 cell counts > 50 and 200 x 10(6)/l, respectively. METHODS: Autologous CD8 T cells (10(8)-10(10)) were reinfused five times after ex vivo culture and stimulation with phytohemagglutinin and recombinant (r) IL-2. The fifth such infusion was followed by 5 days of rIL-2 infusion. Phenotypes and cytokine expression patterns of the expanded cells were determined as well as serum levels of immune mediators throughout the study. RESULTS: Patients showed stable CD4 and CD8 cell counts, p24 antigenemia, and minimal toxicity over the 24-week protocol study. Clinical improvement was observed in lymphadenopathy (six out of six), oral hairy leukoplakia (three out of four), and Kaposi's sarcoma (KS; two out of two) in the patients studied. In vivo induction of detectable levels of bioactive acid-stable interferon (IFN)-alpha, but not of other cytokines studied, upon activated CD8 cell reinfusion was associated consistently with improvement of oral hairy leukoplakia. However, partial regression of KS was observed after the CD8 cell infusion cycles and without IFN-alpha induction. In one of the two patients studied, KS regression was associated with decreased IL-1 alpha serum levels. In the other patient, who had failed previous IFN-alpha therapy, KS regression was observed after a decline in reinfused CD8 cell-associated gene expression of tumor necrosis factor (TNF)-beta. Both IL-1 alpha and TNF-beta are growth factors for KS cells. CONCLUSIONS: These observations demonstrate the feasibility and safety of ex vivo CD8 cell activation, expansion, and reinfusion, and rIL-2 infusion in AIDS patients. The findings in this Phase I trial suggest potential clinical efficacy and encourage Phase II trials. The correlations obtained between clinical and immunological states could contribute to an understanding of the relationship between CD8 T-cell function and HIV-1-associated disease progression.

Different types of false positive anti-HIV reactions in patients on haemodialysis.

Serum samples of 589 haemodialysis patients were screened for HIV antibody by ELISA methods. Of these, 36 samples were found to be repeatedly reactive. None of the 36, however, could be confirmed by competitive enzyme immunoassays and Western blot; therefore, they were considered to be false positive. The sera could be divided in two groups. The sera of Group 1 were designated as the usual type of false positivity, caused most probably by anti-lymphocyte antibodies. In 19 sera, however, a special type of false positivity was found. These sera reacted strongly with the plates coated with the supernatants of HIV-infected cells but not with those of uninfected H9 cells. Three and two sera showed, respectively, positive immunofluorescence reaction with the HIV-infected, but not with the uninfected, H9 and CEM cells. Reactivity to HIV-infected H9 cells could be adsorbed from a part of these samples with lesser amounts of HIV-infected than uninfected H9 cells. This special type of false positivity was observed frequently (7/65) in patients who rejected a kidney graft. These findings suggest that this type of anti-HIV false positivity is due to antibodies reacting with cellular antigens present in HIV-infected but not in uninfected lymphocytes. Their appearance seems to be associated with the immunological activation occurring at graft rejection.

Immunological alterations in anti-HTLV-III negative haemophiliacs and homosexual men in Hungary.

Hungary can be considered as a low risk area for AIDS since no patient with full-blown AIDS or AIDS-related complex has been found in the country. A complex clinical and immunological (T cell subsets, DNCB sensitization test, circulating immune complexes, acid-labile alpha interferon) investigation was performed between November, 1983 and June, 1984 in order to study whether alterations found in symptom-free homosexuals and haemophiliacs in the risk areas for AIDS can be observed in Hungary as well. 38 patients with mild haemophilia, 35 patients with severe haemophilia and 40 homosexual men were investigated in parallel to 37 heterosexual blood donors as controls. Anti-HTLV-III antibodies were measured later in the stored serum aliquots from the same subjects by the indirect membrane immunofluorescence assay. Although specific anti-HTLV-III antibodies were not detected in the haemophiliacs or homosexuals, immunological alterations characteristic for the members of AIDS risk groups in the high risk areas (decrease in the percentage of OKT4 cells and/or decrease of the OKT4/8 ratio) were found in one-third of the homosexuals and haemophiliacs tested. In addition, a significant part of these subjects did not develop delayed type hypersensitivity skin reaction on DNCB rechallenge. These findings indicate that an immunodeficiency independent of HTLV-III infection can be present in two major AIDS risk groups, in homosexual men and haemophiliacs.

Enhanced chemiluminescence-based hybridization analysis for PCR-mediated HIV-1 DNA detection offers an alternative to 32P-labelled probes.

The efficiency of enhanced chemiluminescence based on a novel generation substrate for alkaline phosphatase, adamantyl-1,2-dioxetane phosphate, was compared with that of 32P-labelled probe for visualization of human immunodeficiency virus type 1 (HTV-1)-specific DNA-DNA hybrids. The probe used for nonisotopic detection was digoxigenin labelled and targeted by anti-digoxigenin antibody Fab-fragments conjugated to alkaline phosphatase. The dot-blot hybridization analysis performed on a dilution series of HIV-1 proviral DNA demonstrated a lower sensitivity limit of 0.5 pg with the nonisotopic method. However, one order of magnitude less DNA could still be detected by a random-primed 32P-labelled probe. The ability of nonradioactive and radioactive probes to detect 590-bp gag gene-specific target sequences generated by the polymerase chain reaction (PCR)-mediated amplification of HIV-1 DNA was also compared. Analysis of 20 samples from individuals at increased risk for HIV infection by using the two assayed systems produced virtually equivalent signal images on corresponding specimens. Furthermore, complete concordance in the performance was found when HIV-1 proviral DNA was investigated by PCR in additional 50 samples of human blood mononuclear cells.

Effect of treatment medium on induction of aneuploidy by nocodazole in Saccharomyces cerevisiae.

While studying ways to improve responsiveness of Saccharomyces cerevisiae strain D61.M to agents that induce aneuploidy, we noted that nocodazole, which strongly induces aneuploidy when yeast cells are treated in yeast extract-peptone-dextrose (YEPD) medium, had no effect when a synthetic complete (SC) medium was used. Further study revealed that the presence of peptone was necessary for induction. Other aneuploidy-inducing agents, including ethyl acetate, acetone, and methyl benzimidazole-2-yl-carbamate (MBC), were equally active in either medium. Benomyl, which degrades to MBC, was less active in SC than in YEPD medium.

Aneuploidy induction in Saccharomyces cerevisiae by two solvent compounds, 1-methyl-2-pyrrolidinone and 2-pyrrolidinone.

A number of solvent compounds that were tested in Saccharomyces cerevisiae were potent inducers of aneuploidy, although they did not induce any other genetic effects. As an extention of these earlier findings, 1-methyl-2-pyrrolidinone was tested and was found to induce aneuploidy. Several structurally related compounds were also tested; 2-pyrrolidinone induced aneuploidy, but succinimide, pyrrolidine, 1-methylpyrrolidine, 1-methyl-3-pyrrolidinol, and 2-pyrrolidineethanol did not. Maleimide and its N-hydroxy, N-methyl, and N-ethyl derivatives were also negative for aneuploidy induction.

Persistent viral infections in human carcinogenesis.

Several taxonomically distinct human pathogenic viruses capable of upholding persistent infections have been recognized as important carcinogens. Jointly they are characterized by going into decade-long interactions with host cells and/or tissues. Tumours arise after a long latent period in a few infected individuals. The cellular changes necessary for malignancy are only in part directly or indirectly caused by virus-cell interactions. Cofactors are assumed to be involved. The different routes to malignancy reflect the distinct strategies of each virus in its interaction with the host, which for the upkeep of chronic infections requires a tight control of both virus and cell multiplication and the extent to which an immune response is provoked. The size of the virus-cancer problem and the possibility of prevention makes virology one of the most promising areas of cancer prevention on a global scale. A much wider use of the vaccination against hepatitis B, especially in children, is warranted in developed countries.

Programmed cell death: will it become a factor in cancer prevention?

Among the factors triggering programmed cell death (PCD) are a number of known carcinogens, and several consequences of DNA abnormalities characteristic of cancer have been shown capable of eliciting the PCD response. So although elimination of a potentially malignant cell is likely to be a rare consequence of PCD it could turn out to be important for cancer development. A brief survey is given of the most well-known triggering factors, the molecular mechanisms of the pathways involved and the emerging experimental and clinical data relating capacity of PCD to cancer initiation and progression. It is suggested that future cancer prevention will have to consider also those factors which may abrogate normal PCD.

A biometrical view on normal values of CD4 and CD8 lymphocyte counts in peripheral blood.

Statistical methods have been used to determine an optimal approach to the definition of the reference range for CD4 ("helper") and CD8 ("suppressor/cytotoxic") T cell numbers and the CD4/CD8 ratio in the peripheral blood. A graphical presentation of the absolute values for CD4 and CD8 for 85 healthy blood donors showed that a reference ellipse defined by fitting a gaussian distribution to logarithmically transformed data for absolute counts of CD4 and CD8 cells was superior to the fitting of an ellipse to untransformed data. Further analysis for another 147 subjects showed that the 95% tolerance prediction for the CD4/CD8 ratio in health could be stated with 95% confidence as 0.6 to 5.0. This approach allows clear definition of reference ranges for T cell tests in health and would also be applicable to results for patients with a disease such as HIV 1 infection in which a reference range for "well" patients exists and a change in the T cell ratio is of prognostic significance.

Legislative and technical aspects of mutagenicity testing.

A brief account is given of the history of the legislative acts that give responsibility to the U.S. Food and Drug Administration (FDA) for ensuring the safety of foods, drugs, and cosmetics. Within the present legislative framework the FDA has the authority to impose regulations which are designed to ensure the safety of all foods, drugs, and cosmetics. The existing legislative authority is adequate for this purpose; however, the difficulty lies instead with technology and the inadequacy of scientific perspective in the emerging area of mutagenicity testing. Earlier efforts in development of mutagenicity screening systems culminated only a few years ago in the proposal to use the host-mediated assay, somatic cell cytogenetics, and dominant lethal tests collectively. Subsequent research efforts indicated that there were serious practical and scientific deficiencies in using this approach. More recently a new proposal, the tier system, has been suggested as an alternative measure. The proposed tier system at FDA consists of three testing levels of increasing complexity. The first tier is an initial screening effort using techniques having maximum sensitivity that are also useful for large-scale, rapid testing. The second tier is designed to identify and confirm that the presumptive mutagens detected in the first tier are truly mutagenic for higher organisms, most especially, for mammals. The third tier would be devoted to explicit genetic tests in mammals designed to ascertain the imposed risk to man by the introduction of a mutagen in our environment. The FDA is currently involved in a number of research activities in the area of mutagenicity safety screening which will explore the adequacies and possible deficiencies of the tier system approach. These efforts are described for our in-house activities, our contract activities, and our cooperative and collaborative activities with other government agencies and institutions.

Induction of chromosome loss in Saccharomyces cerevisiae strain D61.M by selected benzimidazole compounds.

Twenty-two benzimidazole compounds were tested for induction of chromosome loss (CHRL) in the diploid yeast Saccharomyces cerevisiae strain D61.M. Six compounds tested positive for CHRL induction: mebendazole, albendazole, RS-9237-000, fenbendazole, 2-benzimidazolylacetonitrile, and thiabendazole. Mebendazole, albendazole, RS-9237-000, and fenbendazole were strongly positive only after modified testing media were used to enhance solubility. The compounds that tested negative for CHRL were 2-phenylbenzimidazole, 2-(2-pyridyl)benzimidazole, benzimidazole, 2-aminobenzimidazole, 2-amino-5,6-dimethylbenzimidazole, 2-(aminomethyl)benzimidazole dihydrochloride hydrate, 5,6-dimethylbenzimidazole, 2-guanidinobenzimidazole, 2-methylbenzimidazole, 2-(methylmercapto) benzimidazole, 1-methyl-2-phenylbenzimidazole, 2-benzimidazolylurea, RS-65255-000, oxibendazole, and RS-95005-000. One chemical, cambendazole, tested negative or only marginally positive. Modified testing medium was also used to enhance the solubility of 2-phenylbenzimidazole, oxibendazole, and RS-95005-000. Because no toxicity was observed with oxibendazole or RS-95005-000, the negative results obtained with these two compounds could not be considered definitive.

Induction of chromosome loss in yeast by combined treatment with neurotoxic hexacarbons and monoketones.

The neurotoxic hexacarbon compounds n-hexane, 2-hexanone and 2,5-hexanedione were tested in combination with acetone and methyl ethyl ketone for the potential to induce chromosome loss in strain D61.M of Saccharomyces cerevisiae. n-Hexane and 2-hexanone, alone or in combination, induced only marginally positive chromosome loss, whereas the metabolite and presumed proximal genetically active agent 2,5-hexanedione was strongly positive when tested alone and in combination. These observations are discussed in relation to the reported potentiation of the neurotoxic effects of these hexacarbons when exposure results from combinations with other solvents, e.g., acetone and methyl ethyl ketone. Treatments that result in neurotoxicity in experimental animals and humans and those that result in chromosome loss in a yeast genetic test system may be correlated by their activity on a common intracellular target.

Observations on chromosome loss detection by multiple recessive marker expression in strain D61.M of Saccharomyces cerevisiae.

Since chromosomes of fungi are difficult to observe directly, strains have been developed in which chromosome loss can be detected by the use of genetic markers. In the diploid D61.M strain of Saccharomyces cerevisiae, the loss of a copy of chromosome VII that carries 3 dominant wild-type alleles is measured by expression of 3 recessive mutant alleles carried on the other remaining copy of chromosome VII. We have tested the hypothesis that expression of the 3 recessive alleles might be due to 3 simultaneous independent genetic events other than chromosome loss, such as mutation or recombination. We have measured, when possible, the frequencies of expression for each of these recessive alleles, independently and in combination one with another, under both selective and non-selective conditions. Our results show that simultaneous expression of these 3 recessive alleles is attributable to chromosome loss (greater than 98%). Similarly, at least 99% of the nocodazole-induced events are attributable to chromosome loss. In contrast, most if not all of the apparent chromosome loss induced by ethyl methanesulfonate is due to multiple events of mutation or recombination.

High levels of chromosome instability in polyploids of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was used to study the genetic consequences of polyploidy in a unicellular organism. Isogenic diploid (2N), triploid (3N) and tetraploid (4N) strains with a genetically marked chromosome VII (cyh2-leu1-CEN7-ade6) were constructed and were used to follow the loss of one, two or three chromosome VII's during mitosis. We found that as ploidy increased, the frequency of loss of a single chromosome VII increased: Loss of one copy of chromosome VII occurred at a rate nearly 30-fold higher in triploids and approximately 1000-fold higher in tetraploids than in the diploid. Loss of two or three copies occurred at an even greater frequency. These findings suggest either that aneuploidy (3N-1, 3N-2, 4N-1, 4N-2, 4N-3) increases genome instability or that multiple chromosome loss events occur at high frequency. Polyploidy appears to dramatically increase chromosome loss, presumably due to the inability of the cell to undergo proper chromosome segregation. The biological significance and possible causes for the instability of polyploidy in unicellular organisms such as yeast are discussed.

Investigations of aneuploidy-inducing chemical combinations in Saccharomyces cerevisiae.

For several years we have been investigating combinations of chemicals for their ability to induce aneuploidy. Earlier published results indicated that combinations of certain chemicals showed a potentiation effect while other combinations did not. We have continued to explore this phenomenon and report additional findings in this communication. Combinations of ethyl acetate and methyl ethyl ketone showed a potentiation effect as did 1-methyl-2-pyrrolidinone-nocodazole combinations. Combinations that did not show a potentiation effect were 2-pyrrolidinone-nocodazole and 1-methyl-2-pyrrolidinone-ethyl acetate. We also found that nocodazole, which is a potent inducer of aneuploidy in yeast extract-peptone-dextrose (YEPD) medium but not in synthetic complete (SC) medium, showed a potentiation effect with ethyl acetate in SC medium. This effect in SC medium is similar to that previously reported for nocodazole with ethyl acetate in YEPD medium. When nocodazole was dissolved in 1-methyl-2-pyrrolidinone as a concentrated stock solution, a potentiation effect occurred even at low concentrations of the solvent.