Genotoxicity of tauromustine, a new water soluble taurine-based nitrosourea. I. Mutagenic and clastogenic activity of tauromustine in vitro.

Tauromustine (TCNU) a new taurine-based nitrosourea in phase III clinical trials against colon cancer, has been tested and compared with 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (CCNU) for mutagenic potential in Salmonella typhimurium TA98 and TA100 and, for its ability to cause chromosome aberrations and sister chromatid exchanges (SCE) in human lymphocytes. A dose dependent increase in the number of mutations in S. typhimurium TA100 but not in TA98 was found from 1.6 to 1000 micrograms/plate for both TCNU and CCNU. In addition, in the presence of Aroclor activated liver microsomes, CCNU caused a further increase in the mutation frequency which was not found for TCNU. A dose dependent increase in the chromosome aberration rate in human lymphocytes was induced by both TCNU and CCNU at concentrations ranging from 3.75 to 30 micrograms/ml after 24 h treatment during the last cell cycle. When lymphocytes were treated in G0 both TCNU and CCNU induced a higher frequency of aberrations at 15 and 30 micrograms/ml than during the last cell cycle and, unexpectedly, chromosome-type, i.e. dicentrics appeared. The difference in the frequency of aberrations in these two phases may be related to the different levels of O6-methyl guanine transferase present in resting contra cycling lymphocytes. TCNU at 0.8-8 micrograms/ml also induced a dose dependent increase in the number of SCE in human lymphocytes. Neither the number of chromosomal aberrations nor the number of SCE were affected by the addition of 1000 micrograms/ml taurine to the culture medium.


Cytogenetic effects of permethrin in cultured human lymphocytes.

The pyrethroid insecticide permethrin was tested for its ability to induce sister chromatid exchanges (SCE), micronuclei (MN) and structural chromosome aberrations (CA) in cultured human peripheral blood lymphocytes. Permethrin was tested in the range of 5-500 micrograms/ml in the absence and in the presence of a rat liver activation system (S9 mix). Small elevations in the SCE frequencies were found and even though statistically significant may have no biological meaning, the more so since there was no dose-effect relationship. Permethrin induced both MN and CA when it was evaluated in the absence of a metabolic activation system. Nevertheless, it cannot be said that S9 mix suppressed the activity in itself. The effect of permethrin seemed to be time of exposure dependent. Permethrin could be characterized as a S-phase independent agent with greater potential for inducing chromosomal damage than sister chromatid exchanges.


Analysis of eight known or suspected aneugens by the in vitro human lymphocyte micronucleus test.

In the light of the CEC aneuploidy programme eight known or suspected aneugens were evaluated with the human lymphocyte 'in vitro' micronucleus test using the cytochalasin-B technique. Only colchicine, chloral hydrate and hydroquinone induced dose-dependent increases in micronucleus frequencies. The other five chemicals (cadmium chloride, econazole, pyrimethamine, thiabendazole and thimerosal) were all negative, both with and without S9, and treated in G1 or G2 phase. These data are in good agreement with results from different 'in vivo' studies. However, discrepancies were found between the results from this study of human lymphocytes and those of other cultured cell types where econazole, cadmium chloride, pyrimethamine and thiabendazole induce a significant increase of micronuclei.


Clastogenic and aneuploidogenic effects of cigarette smoke condensate, mitomycin C and vincristine sulfate.

Immunofluorescent staining of kinetochores in micronuclei (MN) of cytochalasin-B-blocked cells was used to distinguish between clastogenic and aneuploidogenic effects of cigarette smoke condensate (CSC), mitomycin C (MMC) and vincristine sulfate (VS) in Chinese hamster lung V79 cells by using an anti-kinetochore antibody. Within the base-line micronucleated cells (2%), 50 to 58% contained kinetochore-positive (KC+) MN. MMC induced a significantly higher number of MN compared to the controls (P < 0.01), and the majority of micronucleated cells (77% at 0.08 microgram/ml) contained kinetochore-negative (KC-)MN. In contrast, VS induced a large number of MN (P < 0.01) but most of the micronucleated cells contained KC+ MN (93% at 0.08 microgram/ml). Cigarette smoke condensate (CSC) induced both KC+ (P < 0.01) and KC-(P < 0.05) MN suggesting the presence of both clastogenic and aneuploidogenic agents. However, the higher frequency of KC+ than KC- micronucleated cells suggests a stronger aneuploidogenic effect of CSC.


DNA repair modifies the site and strand specificity of ethyl methanesulfonate mutagenesis in yeast.

The influence of DNA repair on the specificity of ethyl methanesulfonate (EMS) mutagenesis in a plasmid-borne copy of the Saccharomyces cerevisiae SUP4-o gene was investigated. Isogenic yeast strains that are repair-proficient (RAD) or defective for nucleotide excision (rad1), postreplication (rad18) or recombinational repair (rad52) were treated with EMS. Compared to the RAD wild-type, the maximum SUP4-o mutation frequency was 2-fold greater in the rad1 background whereas it was approximately 50% less in the rad18 and rad52 strains. The majority (779/788) of SUP4-o mutations characterized by DNA sequencing were single base pair changes, primarily (> 91%) G.C-->A.T transitions in the RAD, rad1 and rad18 strains. In the rad52 background, only 57% of the substitutions were G.C-->A.T transitions with transversions at G.C pairs accounting for almost all of the remaining changes. Comparisons of the distributions of single base pair substitutions in SUP4-o revealed that there was no excision repair-dependent bias for G.C-->A.T events to occur at sites flanked by a 5' or 3' A.T pair as observed previously for EMS mutagenesis of the lacIgene in Escherichia coli (Burns et al., 1986). These transitions also did not occur more often at sites where the guanine was flanked by a 5' purine than by a 5' pyrimidine. However, they exhibited a small preference for sites having the guanine on the transcribed strand in the RAD and rad52, but not rad1 or rad18, strains.(ABSTRACT TRUNCATED AT 250 WORDS)


Clastogenicity to the mouse bone marrow of the mouse germ cell genotoxin streptozotocin.

The methylating agent streptozotocin is active in the mouse bone marrow micronucleus assay following a single intraperitoneal injection of 150-180 mg/kg. This correlates with its previously reported toxicity to mouse germ cells when administered by the same route of exposure. The potent mutagenicity of streptozotocin to strain G46 of Salmonella typhimurium is compared with its much weaker activity in strain TA1535. The genotoxicity of streptozotocin in vivo is reviewed.


Effects of hydrodynamics and leukocyte-endothelium specificity on leukocyte-endothelium interactions.

In vivo microscopy was used to assess the relative contribution of hydrodynamic forces (network topography and shear rate) and the specificity for leukocytes to interact with venular endothelium as determinants of leukocyte-endothelium interactions. To ascertain this, microvascular networks in the rat and rabbit mesentery were examined under normograde and mechanically induced retrograde flows to determine the effect of reversed flow on leukocyte-endothelium interactions in arterioles and venules. The data indicate that retrograde perfusion under hemodynamic (red blood cell velocity and shear rate) states equivalent to normograde flow significantly increased leukocyte marginating flux in arterioles (from 0 to 0.5 cells/5 sec) and decreased flux significantly in venules (from 1.0 to 0.2 cells/5 sec). The increased flux in arterioles under retrograde conditions, however, was significantly lower than the flux in venules under normograde conditions and the decreased flux in venules during retrograde flow was significantly greater than the flux in arterioles during normograde flow. This apparent discrepancy appears to be the result of a heterogeneous distribution of adhesive receptors on vascular endothelium. Furthermore, marginating leukocytes in arterioles made only brief contact with the endothelium before being swept away while marginating leukocytes in venules during normal and retrograde perfusion rolled along the vascular wall, with similar velocities in both directions. In conclusion, although hydrodynamic forces are important in facilitating leukocyte margination through mechanisms of radial migration, it is leukocyte-endothelium specificity in venules that ultimately determines leukocyte-endothelium interactions.


Interstitial pressure, volume, and flow during infusion into brain tissue.

A model of infusion-induced swelling in the brain is presented, in which gray and white matter are treated as poroelastic media. The distributions of interstitial pressure, flow, and volume are derived for steady-state and transient infusion protocols. A significant percentage increase in interstitial volume is predicted near the injection site, despite only a modest increase in tissue-averaged fluid content there. The model also can be used to estimate mechanical parameters of brain tissue, such as its hydraulic permeability, shear modulus, and Lamé constant. A solute transport equation that incorporates tissue swelling is also presented. This work suggests that knowing the distribution of swelling induced by infusion is a prerequisite to describing interstitial transport of solutes.


Intravital videomicroscopy of the chorioallantoic microcirculation: a model system for studying metastasis.

The chick embryo is a useful model for studying hematogenous metastasis. Cancer cells injected into veins of the chorioallantoic membrane (CAM) circulate briefly through all tissues but form metastases predominantly in the CAM. This respiratory organ is particularly suitable for intravital microscope because of its accessibility without the need for surgery and the density and planar configuration of its vessels (which we confirmed by microcorrosion casting). Using an inverted microscope with oblique transillumination for high-resolution images and epifluorescence to identify labeled B16F1 melanoma cells, we studied successive stages of metastasis formation in the CAM in vivo. By 2 min postinjection (pi) all cancer cells had become arrested within the microvasculature. This initial arrest appeared to be due to size restriction, based on measurements of cell and vessel diameters. At 15-60 min pi, trapped cells were seen in tapering arterioles (27%), orifices from arterioles to the capillary plexus (61%), or in the plexus itself (12%). Some cells had extravasated into the underlying mesenchyme by 3 hr (pi), and at 24 hr all cancer cells had completed this process. The mean rate of migration out of capillary lumens was approximately 1 micron/hr. Micrometastases grew in a planar configuration just beneath the capillary plexus, with a cell doubling time of approximately 24 hr. Our technique is also applicable to other tumor types and host animals and provides a powerful tool to complement studies on the molecular basis of metastasis.


Local tissue oxygenation by statistically distributed sources.

The effect of red blood cell separation on tissue oxygenation was analyzed using a previously developed model which accounts for the particulate nature of blood at the capillary level. Results show that an empirically based RBC pattern yields the same levels of oxygenation at 20% hematocrit as an even spacing pattern. Introduction of an empirically based RBC spacing pattern during hemodilution, where capillary hematocrit is reduced by half with concomitant doubling of velocity, results in a reduction of average tissue pO2 by 23% and volume of oxygenated tissue by 28%, whereas axial delivery of oxygen along the length of the capillary was unchanged. Tissue oxygenation levels are optimized with even RBC spacing pattern but not with an empirical pattern because the tissue does not effectively use the oxygen delivered by unevenly spaced RBCs despite an equivalent time-averaged flux. Model predictions for tissue oxygenation for statistically distributed spacing under normal and hemodilution conditions show trends consistent with previously obtained results using even spacing, namely, increased axial distance and volume oxygenated, with decreased average tissue pO2. During hemodilution, the volume of tissue oxygenated above 2 mm Hg decreased to 84%, while the volume above 1 mm Hg increased to 131% relative to control. This finding suggests a redistribution of oxygen within tissue during hemodilution, causing a greater amount of tissue to be exposed, but at a lower pO2.


Application of image analysis for evaluation of red blood cell dynamics in capillaries.

We have devised a method to display and directly evaluate red blood cell (rbc) dynamics in capillaries using the same dual camera intravital video microscopy system employed to determine rbc oxygen saturation (Ellis et al., 1990). Capillary images are recorded on videotape and an interactive graphics system is used for analysis. Data are sampled once a frame for 60 sec using a window (one pixel wide (0.93 micron) and 100 pixels high) positioned along the axis of a capillary. The resulting data are displayed as sequential space-time images 100 pixels high by 300 pixels wide (10 sec). The space-time images thus created represent the dynamics of the rbc's in a single comprehensive static image in which the rbc's appear as dark, diagonal bands separated by light bands representing plasma gaps. From these images one can obtain information on velocity of individual rbc's (micron/sec), lineal density of rbc's (rbc/mm), and rbc supply rate (rbc/sec). This information can be used to delineate the temporal and spatial heterogeneity of hemodynamics in capillary networks. These data can then be combined with coincident data on red blood cell oxygenation to provide a complete picture of oxygen transport in capillaries or it can be used alone as a tool for the evaluation of basic in vivo and in vitro rheological questions.


Optically accessible microchannels formed in a single-crystal silicon substrate for studies of blood rheology.

An attempt to use microgrooves as blood flow channels in single-crystal silicon is described. Grooves were formed in the (100) crystalline surface by means of photolithography and orientation-dependent etching. This substrate surface was tightly covered with an optically flat glass plate to prevent leakage. This structure was used to microscopically observe flow behavior of blood cells. As a first design of such a cell-flow apparatus, many parallel same-size channels of relatively short length (equivalent diameter 6 microns, length 14.4 microns, number 2600) have been produced to simultaneously measure the total volume flow rate of blood under constant suction. Despite differences in cross-sectional shape and length, the ratio of blood flow velocity to pressure gradient in the channels was comparable with estimates for capillary vessels in vivo. Activated white blood cells blocked the channels, while aggregations of red blood cells showed unexpectedly small resistance to channel transit.


Production of a monoclonal antibody that recognizes the lipopolysaccharide of a Campylobacter-like organism.

A monoclonal antibody was produced to a Campylobacter-like organism (RMIT 32A) which was isolated from the terminal ileum of a pig with proliferative enteritis. Isotyping of the antibody revealed that it was an IgG2a with kappa light chains. Immunoblots using the antibody against proteinase-K-treated whole cell lysates of RMIT 32A, a selection of Campylobacter species and other enteric bacteria showed that the antibody was specific for RMIT 32A and was directed against the lipopolysaccharide. This antibody can be used for the specific detection of RMIT 32A.


Antibody response to oral streptococci in Behçet's disease.

The serum antibody titers against oral streptococci were studied by enzyme-linked immunosorbent assay (ELISA) both in patients with Behçet's disease (BD) and control groups. The patients with BD showed significantly higher antibody titers to S. sanguis strains 113-20, 114-23, and 118-1 which were isolated from patients with BD, in comparison with control groups. Also, the reactions of high-titered sera to the crude cell wall and soluble (or membrane) fractions of the 113-20 strain were observed by western blot test. The sera of the patients with BD demonstrated strong bands of approximately 36 kDa, 82 kDa, and 87 kDa in the crude cell wall fractions, and many bands of 80 kDa to 150 kDa in the membrane fractions, indicating that these proteins are the ones leading the high antibody titers to this bacterium in the sera of patients with BD.


A simple method for overproduction and purification of p24 Gag protein of human immunodeficiency virus type 1.

A simple method for the overproduction in Escherichia coli and purification of major core protein p24 of human immunodeficiency virus type 1 (HIV-1) was described. The gag-pol region encoding p24, p15, and protease was fused to 3' end of lacZ gene on plasmid. A LacZ-Gag fusion protein, the major primary product, is designed to be cleaved by the HIV-1 protease coexpressed through frameshifting. In fact, p24 and its immediate precursor, p25, were produced in the cells grown at 25C, but not at 37C. When the gag and pol frames were fused in-frame to express the protease without frameshifting, the main product, a LacZ-Gag-Pol fusion protein, was efficiently processed to give p24 exclusively both at 37C and 25C, suggesting more efficient expression of the protease. Recombinant p24 was purified to near homogeneity by a simple three-step procedure. The amino-terminal sequence of the recombinant p24 was the same as that of p24 deduced from nucleotide sequence, indicating that correct processing occurred in E. coli by the coexpressed protease. The method described here provides a means to obtain a large amount of highly pure p24, which is useful for crystallographic and functional studies, preparation of specific antibody, and diagnostic and prognostic uses.


A prospective study on correlation between the decrease in anti-P17 antibody level and progression to AIDS in asymptomatic carriers of HIV.

As the majority of human immunodeficiency virus (HIV) carriers are in asymptomatic stage for a long period of time, it is important to investigate the factors or surrogate markers for conversion from asymptomatic to symptomatic stage. Our study is designed to evaluate the relationship among virus isolation rate, anti-p17 antibody status and progression to AIDS. We studied anti-p17 antibody status along with virus isolation in 56 asymptomatic carriers and 46 AIDS cases. Progression to AIDS was markedly associated with high rate of virus isolation and loss of anti-p17 antibody. In order to know the meaning of loss of anti-p17 antibody during the clinical course, 15 anti-p17 antibody positive and 16 anti-p17 antibody negative cases were followed up prospectively for the development of AIDS. None of the anti-p17 antibody positive cases developed AIDS while 6 out of 16 anti-p17 negative cases developed AIDS during observation period (P < 0.05). Progression to AIDS was associated with loss of anti-p17 antibody. Identification of cases losing anti-p17 antibody in peripheral blood during asymptomatic period may help high-risk group who are in need of chemoprophylaxis. Moreover, study of anti-p17 antibody may be helpful in designing vaccine in future if it works as a neutralizing antibody to HIV in vivo.


Molecular analysis of immunoglobulin heavy chain genes coding for idiotypic and anti-idiotypic antibodies involved in B-B cellular interaction.

We recently reported that a unique B cell clone (B19-1d), specific for a cross-reactive idiotype (CRI) on MOPC104E myeloma protein (M104E), enhances Igh-restricted CRI+ antibody production. In this paper, we report the nucleotide sequences of immunoglobulin heavy chain variable regions (VH) of both M104E and B19-1d-derived hybridoma (HB19) antibodies. The sequence data revealed that both belong to the J558 germ line VH gene subfamily. Strikingly, not only the VH region, but also the leader sequences of M104E and HB19 are very similar to each other at 88% (VH) and 91% (leader) homology, but they use different D and J segments. The VH region sequence similarity is highest among the germ line VH gene sequences of the BALB/c J558 subfamily so far screened. Southern hybridization data, using 5'-noncoding regions of either M104E or HB19 genomic VH gene clones as probes, revealed that both VH genes are conserved in the M104E CRI producer strains of mice. Moreover, these probes show the restriction length polymorphism pattern of mouse VH genes in various strains. That the HB19 VH gene locates to the 5' upper arm of the M104E VH gene on the chromosome was suggested by Southern blot hybridization. Immunoglobulin VH gene restriction of idiotypic and antiidiotypic B-B cellular interaction is discussed from a molecular point of view.


Spontaneous proliferation of HTLV-II-infected peripheral blood lymphocytes: HLA-DR-driven, IL-2-dependent response.

Peripheral blood lymphocytes obtained from HTLV-II-infected persons (n = 13) and cultured in the absence of exogenous stimulator demonstrated augmented spontaneous proliferation (17,672 +/- 5,498 cpm) when compared with cells from healthy donors (1,921 +/- 1,306 cpm). Removal of non-T population did not abrogate the proliferative response of patients' PBMC, suggesting that the proliferation is not related to the autologous mixed lymphocyte reaction. Addition of recombinant interleukin-2 (rIL-2; 0.1 U/ml) to spontaneously proliferating cultures from HTLV-II-infected persons resulted in a 3- to 4-fold increase in proliferation (61,985 +/- 16,003); in contrast, PBMC from controls demonstrated 38- to 42-fold increase in their proliferative capacity in response to rIL-2 (77,256 +/- 13,044). Antibodies to both IL-2 receptor and HLA-DR were able to inhibit the spontaneous proliferation of PBMC from HTLV-II-infected persons in a dose-dependent manner. Furthermore, addition of cyclosporin A, which preferentially blocks accumulation of IL-2 mRNA, also inhibited spontaneous proliferation in a dose-dependent manner. These observations suggest that the spontaneous proliferation of HTLV-II-infected PBMC is at least in part an HLA-DR-driven, IL-2-dependent event, which is not analogous to the AMLR.


Influence of inoculation route on virulence of Rhodococcus equi in mice.

Virulence of R. equi ATCC 33701 was compared by the intraperitoneal (i.p.) and intravenous (i.v.) routes in mice. Strain ATCC 33701 was more virulent by the i.v. than the i.p. route. The LD50 of strain ATCC 33701 by either route correlated with the initial number of bacteria in the liver and spleen at day 0.


Prevalence of antibodies to Pseudomonas pseudomallei exotoxin and whole cell antigens in military personnel in Sabah and Sarawak, Malaysia.

Sera from 420 military personnel serving in Sabah and Sarawk, Malaysia, were tested for antibodies to Pseudomonas pseudomallei exotoxin and whole cell antigens by enzyme-linked immunosorbent assay procedure (ELISA). Data showed that 54.4% of serum samples were positive for antibodies to P. pseudomallei exotoxin and 65.7% were positive for antibodies to the whole cell antigens. Samples gave much lower titers for anti-exotoxin antibodies compared to titers against crude whole cell antigens. The incidence of antibody to exotoxin was highest in the age groups ranging from 26 to 32 years, where the positive rates were higher than 40% and 30% for military personnel serving in Sarawak and Sabah, respectively.


[Inhibition of Candida valida growth by copper ions].

The high toxicity of copper ions for Candida valida growth was established at pH-auxostat regime. The value of mu max decreased even at the residual Cu2+ concentration 1.0 mg/l. The inhibition constant (Ki) that characterized a copper ion concentration at which yeast specific growth rate was halved was equal to 7.7 mg/l. A linear dependence of 1/mu max on a residual concentration of copper ions indicates that yeast growth inhibition is due to inhibition of one enzymic reaction which is the most sensitive to copper. Yeast growth inhibition by copper was accompanied by accumulation of Cu2+ ions in biomass, a decrease in nucleic acid and true protein contents, and changes in amino acid composition of protein. The amounts of cystine and cysteine in protein increased and tryptophane content decreased with inhibition of yeast growth. Yeast growth inhibition by copper did not affect the lipid content but significantly reduced the degree of unsaturation due to a decrease in the amounts of polyunsaturated linoleic and alpha-linolenic acids.


[Influence of carbon sources on the biological activity and morphology of Bacillus thuringiensis parasporal crystals].

The influence of the carbon source on biological activity and morphological properties of the parasporal crystals of Bacillus thuringiensis subsp. kurstaki cultivated on three different media has been studied. Biological activity of delta-endotoxins contained in crystals was tested according to their impact on insects and microorganisms. Variations in carbon sources composition resulted in changes of biological activity and morphology of crystals, caused by alterations in synthesis rate of various delta-endotoxins contained in the crystals. Variations in the content of a certain carbon source (e.g. starch) in the medium produced no effect on the crystals properties, and the content variations of the delta-endotoxins content was proportional to the producer biomass. Control of the antibacterial effect of delta-endotoxins allows a valid evaluation of their biological activity under various growth conditions as well as comparison of the biological potential of entomopathogenic chemicals for plant protection containing delta-endotoxins with different specificity of insecticidal activity.


[Dynamics of the population structure of the Escherichia coli recombinant strain during continuous culture].

The populational structure of the Escherichia coli strain Z905 containing the recombinant plasmid with the phenotype AprLux+ was studied in chemostat. It was shown that the stability of the ratio of plasmid containing cells and cells without plasmids depends in the first place on the presence of the selective factor (ampicillin) in the medium and on the sources of carbon and energy limiting growth.


Total eye transplantation for the blind: a challenge for the future.

Blindness is a human and social problem of incalculable weight. In the future, artificial 'bionic' prostheses and retinal grafts could achieve a long-sought cure. Several lines of evidence led to the speculation that a total eye transplantation for the cure of retinal blindness may become feasible in the near future. It is proposed that a brain dead patient's eye, whose retinal viability has been demonstrated with an electroretinogram recording, be transplanted into the blind's voided orbital socket, through a frontoorbitotemporal craniotomy and orbitozygomatic osteotomy. Regenerating optic nerve axons are channeled in a specially constructed guide to the homolateral corpus genicolatum laterale, while the retinal ganglion cells are adequately protected during the regrowth period. Aspects of this paradigm are reviewed and discussed.


[Comparative electron microscopic study of Bacillus thuringiensis cells grown in a chemostat].

It has been shown that the culture of Bacillus thuringiensis subsp. gall. 69-6 dissociates into R- and S-variants in chemostat. Although under some conditions the sporogenesis and the synthesis of S-variant toxin began two hours earlier than these processes in R-variant cells which were observed, respectively, after 10 or 12 hours from the beginning of the experiment, the intensities of sporogenesis and toxin production as well as the exit of spores and toxin excretion from cells were similar after 24 hours. The resistance to the bacteriophage present in chemostat was the advantage of S-variant cells. The data obtained by electron microscopy indicate that the phagoresistance is caused by the structural organization of the S-variant cell wall. Its peptidoglycan component is thin and is distinguished by crumb structure. By means of negative contrast microscopy it was found that the surface T-layer of R-variant cell wall was characterized by the tetragonal packaging of protein subunits indicating the regular orientation of phagoreceptors in it. The redistribution of protein subunits in the T-layer of S-variant cell wall prevented from the adsorbance of bacteriophages on the cell surface. The adsorbance of phages on the surface of R-variant cells was observed rather often. It led to the degradation of peptidoglycan, the formation of protoplasts and lysis.


Pre-existing chronic infraepithelial Trichomonas invasion with consecutive immunodepression enables progression of human immunodeficiency virus: a new concept of acquired immunodeficiency syndrome pathogenesis.

All three existing species of human trichomonads cause similar immune and histopathological host responses. The elicited crossimmunity causes interspecific competition of trichomonal infections in their typical localizations, which results in very rare simultaneous colonization of the same host by different species. This phenomenon points to the existence of a unique immune disorder or subclinical sensitization, regardless of which trichomonas species is in question. The total percentage of infestations in all three species points to the most widespread parasitosis, i.e. the immunodepression of human beings. Trichomonaemia has a very strong immunodepressive action. All the other agents, including viruses act only as opportunists.


[Directed autoselection of bacteria during turbidostat cultivation].

A method has been proposed for directed bacteria autoselection, based on their sensitivity to growth inhibitors during continuous unlimited cultivation. A smooth increase of a growth inhibitor concentration in the medium has been used in response to the appearance and autoselection of more resistant strains. The aim can be reached by continuous measurements of the specific microbial growth rate during the process and its keeping at the required level by the inhibitor additions. The experimental data are given showing application of the method for Escherichia coli and Pseudomonas sp. turbidostat cultivation with valine, low pH and formaldehyde as inhibitors.


[Conjugative transfer of plasmid DNA between bacteria in soil].

Conjugative transfer of plasmid RP4 between populations of azospirilla and between Escherichia coli and Azospirillum brasilense in nonsterile soil has been investigated. The process of genetic exchange was realized at the early stages of interpopulational interactions, further on the process intensity was obviously rather low. Population dynamics of azospirilla transconjugates in soil depends on the presence or the absence of additional food substrate.


[Pathways of degradation of organic components of waste water of (meth)acrylate-producing factories to methane by communities of microorganisms of adapted and unadapted sludge].

Pathways of the degradation of the main compounds of (meth)acrylate-producing factories wastewater (methyl methacrylate, methyl and butyl acrylate, acrylate and methacrylate, acetone, isopropanol, butanol and methanol) by the anaerobic microbial consortium of mesophilic unadapted granulated sludge from the "UASB" reactor and of adapted activated sludge from the contact reactor were comparatively studied. It was shown that the degradation of fatty acids and alcohols took place in both types of sludge. Methacrylate, acrylate and acetone degradation occurred only in adapted sludge. Both types of sludge were characterized by the reversible conversion of acetone and isopropanol and by the presence of the isomeric transition of butyrate and isobutyrate too. The present results allow to suggest that the adaptation of activated sludge to substrate includes the accumulation of biomass of microorganisms capable of hydrolyze specific substrates into such general intermediates as low-molecular-weight fatty acid and alcohols further metabolized to methane and carbon dioxide.


Monoclonal antibody therapy in the treatment of Reye's syndrome.

A role for lipopolysaccharides (endotoxins, LPS) in 7 the pathogenesis of Reye's syndrome (RS) has previously been suggested. Impairment of hepatic LPS clearance can lead to systemic endotoxemia as previous studies by this and other laboratories have suggested for several hepatic disorders including RS. Systemic LPS may mediate many of the clinical findings associated with RS by eliciting monokines such as tumor necrosis factor-alpha, interleukin-1, interleukin-6, and interleukin-8. Monoclonal antibody therapy directed at LPS, and monokines may represent a novel approach to the treatment of RS.


Altered postural reflexes in Parkinson's disease: a reverse hypothesis.

In subjects standing on a movable platform, sudden dorsiflexion of the ankle joint elicits a set of reflexes in leg muscles. These responses include a short latency (SL) and medium latency (ML) stretch reflex in the gastrocnemius muscle and a distal to proximal innervation sequence of long latency (LL) reflexes in the shortened tibialis anterior and vastus lateralis muscles. Because of their role in maintaining upright stance these responses have been termed postural reflexes. In patients with Parkinson's disease (PD), the following abnormalities have been described: 1) enhanced ML-amplitudes; 2) a reversed LL innervation sequence; and 3) delayed onset latencies. These abnormalities are thought to be due to defective motor programming and disturbed control of spinal and supraspinal reflex centers by basal ganglia circuits. The altered reflexes have been held responsible for some of the clinical features of PD, including balance impairment and rigidity. In this paper, we argue the reverse hypothesis that postural reflexes are essentially normal in PD, and that the observed alterations are at least in part consequence rather than cause of balance impairment, the stooped parkinsonian posture and rigidity of PD patients.


[Features of the initial stage of surface colonization by Eshcherichia coli cells as a function of their mobility and chemotaxis].

Dependence of adhesion and colonization of hydrophilic and hydrophobic surfaces by Escherichia coli strains with different mobility and chemotaxis was studied using E. coli mot+che+, E. coli mot+che-, E. coli mot-che+. Primary adhesion was shown to correlate with mobility of cells and hydrophilic/hydrophobic character of their surface. Secondary adhesion correlated in addition with chemotactic characteristics of bacteria. E. coli populations were shown to vary in electrophoretic mobility and cells capability for adhesion and chemotaxis.


A proposed mechanism of anticancer activity of indomethacin.

The exact mechanism of anticancer activity of indomethacin still remains unclear. In view of the gathered observations and ideas, I hypothesize that the effect of indomethacin may be related--at least in certain cases--to its antagonism to gastrin/cholecystokinin family of peptides and/or to its agonistic activity to thromboxane A2 and melatonin. Future experiments in this direction are required.


Activation of oncogenes and/or inactivation of anti-oncogenes by reactive oxygen species.

Abundant evidence indicates that reactive oxygen species (ROS) are involved in mutagenesis and carcinogenesis. These chemical-generated or phagocyte-released ROS are known to cause a variety of genetic alterations which lie at the heart of the carcinogenic process. ROS have also been shown to cause malignant transformation of normal cells, and to increase expression of certain proto-oncogenes such as c-fos and c-jun. It is known that certain proto-oncogenes and anti-oncogenes may serve as the targets of carcinogens of various sorts. I hypothesize that ROS-mediated DNA damage may cause mutations and/or deletions in certain specific coding regions of tumor-related genes, and could be responsible for subsequent activation of oncogenes and/or inactivation of anti-oncogenes.


Calcium intake and the protein composition of mouse brain: relevance to multiple sclerosis.

Epidemiological data has suggested the involvement of calcium metabolism in the incidence of multiple sclerosis through a link with high childhood milk intake followed by a large or sudden reduction during the adolescent growth spurt. In this study polyacrylamide gel electrophoresis of SDS-solubilized proteins in the aqueous insoluble pellet fractions of laboratory mouse brain tissue at 6 months of age has shown that a dietary reduction from high calcium intake to 0.005% CaCO3 or less in early life can cause a change in the electrophoretic pattern of low molecular weight proteins. This change is similar in appearance and molecular weight to an abnormality seen in the electrophoretic pattern of SDS-solubilized proteins in the aqueous insoluble pellet fractions of multiple sclerosis plaque. It is proposed that this finding could indicate a worthwhile area for future research and that the hypothesis of a reduction from high to very low calcium intake during adolescence being involved in the pathogenesis of multiple sclerosis should not be discounted.


On the protective mechanism of the sickle cell trait (HbAS).

The sickle cell trait has been reported to offer some protection against anaemia in pregnancy. Some attribute this to the break up of the cells which release iron for the synthesis of haemoglobin. The hypothesis presented here is that the 'partial' protective mechanism of the sickle cell trait is due to increased erythropoietic activity.


Sudden infant death syndrome: the vitamin E/selenium iron hypothesis (dietary anti/pro-oxidant imbalance).

Cot death or Sudden Infant Death Syndrome (SIDS) remains a largely unexplained and unpreventable factor in infant mortality. The existence of a dietary link between SIDS and vitamin E/selenium deficiency, documented through veterinary evidence, is posited as an etiological factor. Further substantiation of this hypothesis is derived from infant nutrition practices and cases of SIDS in New Zealand.


Ageing, information and the magical number seven.

Concepts from information theory are applied to human ageing. An equation is derived, based on the general properties of information systems, which closely predicts the mortality rate in old age. The same equation predicts that infection will be increasingly severe when first exposure to micro-organisms is delayed leading to diseases which peak in early and middle age. A parameter of the equation is the level of redundancy in human information processing. Observed mortality rates show that this is sevenfold and it is suggested that seven is a fundamental dimensionless biological constant.


An inside-out theory of attention.

The brain has two attentional systems; one attentional system is devoted to intrapersonal space and the second attentional system to extrapersonal space. The development of these two attentional systems with inward or outward focus arose from the hemispheral neural systems designed for attending to extrapersonal contralateral space. As a necessary consequence of the brain changes to develop language, one of the hemispheral attentional systems was altered for intrapersonal attention while the other hemispheral attentional system became obligated to attend to all of extrapersonal space. This conceptualization of attention provides a coherent explanation for the confusing hemispheral dominance proposed for attention on the basis of clinical hemineglect, i.e. that the left hemisphere attends to right extrapersonal space and that the right hemisphere attends to both left and right extrapersonal space. This conceptualization also provides a meaningful account of denial of limbs (anosognosia) and imagery.


The threshold effect: consequence of change in the frequency of food intake in the presence of a functional threshold.

Studies on diseases thought to be diet-induced have so far assumed that the effect of a food depends solely on the amount ingested. When the metabolism of a food derivative is dependent on a threshold-dependent mechanism this single variable assumption is invalid, as the net effect of the derivative will also depend on intake frequency. In this paper the consequences of varying intake frequency in the presence of a notional threshold are analysed mathematically. It is shown that at the low frequency end of the scale relevant to human diet, if a metabolic threshold exists then the net effect of a food will be critically dependent on intake frequency, and that the effect of changing frequency may exceed that produced by even substantial changes in quantum. It is concluded that epidemiological studies which have not included data on frequency of intake involve a potential error and could be misleading.


Purification and characterization of cell wall proteins of Listeria monocytogenes.

Two proteins were purified from Listeria monocytogenes cell wall using detergent extraction, Superose 6 gel chromatography. Mono Q cation-exchange chromatography and Superose 12 gel chromatography. Proteins were shown to form complexes of ca. 300 kDa and pI 4.7. These complexes could not be dissociated in 6 M urea, however, during SDS-PAGE 79 and 39 kDa monomers were formed. Immunoblot analysis showed that the proteins under investigation were common to all listerial strains tested, but were absent in strains of other bacteria. We propose that the proteins investigated here could serve as immunoserological markers for Listeria.


[Total cholesterol, LDL-cholesterol and apoprotein B in umbilical cord blood: cross-sectional study].

The aim of the present study was to evaluate, in a large series of term newborns, the prevalence of hypercholesterolemia in cord blood, using as parameters, levels of plasma cholesterol, LDL-cholesterol and apo B higher than mean + 2 standard deviations, alone or in association. Data reported here represent the results of a transversal study. 1275 term newborns were studied. In everybody, in cord blood samples, at birth, cholesterol, triglycerides, HDL-cholesterol, apoprotein B and LDL-cholesterol have been assayed. In 3.13% of them (40 subjects), cholesterol levels > mean + 2 standard deviations were present. Moreover, only 6 subjects (0.47%) showed all three parameter levels higher than mean + 2 standard deviations, and 4 subjects (0.31%), in spite of normal cholesterol levels, presented high LDL-cholesterol and apo B levels. Integrated utilization of these parameters in the neonatal screening of familial hypercholesterolemia will have to be confirmed by follow-up studies.


[Plasma L-carnitine levels in children with celiac disease].

Carnitine is a very important co-factor for the metabolism of fatty acids, because it is the necessary carrier for the passage of acyl groups inside the mitochondria, where beta-oxidation takes place. In the human body the total pool of carnitine is made by two fractions, one being endogenous and the second exogenous. The absorption of exogenous carnitine takes place mainly at duodenal-jejunal level by an active transport mechanism based on amino acid carrier. Plasma L-carnitine concentrations have been measured in 66 coeliac patients (mean age: 7 years and 4 months). Thirty three of them were on a free diet and the other 33 were on a gluten-free diet for at last six month. In 10 patients we studied plasma L-carnitine levels both on a free diet and on a gluten-free diet. As controls we examined 33 healthy children comparable for age. Plasma L-carnitine concentrations have been measured by a spectrophotometrical method according to Marquis and Fritz's technique and subsequently modified by Pearson and Seccombe. In the 66 coeliac patients the mean values of serum L-carnitine were significantly lower than those in the controls (p < 0.001). The levels were significantly lower in patients a free diet with respect to those on gluten-free diet (p < 0.01). The 10 subjects who were examined both on free diet and on gluten-free diet showed an increase of plasma concentrations in the latter condition.(ABSTRACT TRUNCATED AT 250 WORDS)


[The pediatric emergency department: assessment of clinical activity and interaction with local health authorities].

A perspective study was carried out at the Emergency Department of the Gaslini Institute on a sample of the children admitted from May to September 1990. The study aimed to describe types and modalities of admission. "G. Gaslini" Institute is a multidisciplinary children's hospital: 45% of patients admitted in the hospital come from the southern regions of Italy, while the admission at the Emergency Department were local in 82%. Among all children considered, 2080 (71.9% were admitted on request of their parents, who did not ask previously any physician; of those cases, 356 (17%) were hospitalized, 474 (25%) were either examined by specialist or treated and then discharged. In the remaining cases, no intervention was necessary. As a consequence, 58% of children were admitted without any clinically plausible reason. Parents' anxiety clearly played an important role in most cases. 840 children were addressed to the Emergency Department by a physician, 652 (22.3%) by the doctor in charge, 213 (32.7%) of which were sent back home by the doctor on duty, who did not think it necessary any treatment. Of the 120 (4.1%) children sent by different Emergency Departments, 95 (79.1%) were hospitalized. The reason why physicians addressed quite a large number of children to the Emergency Department, when they didn't need any intervention remain nucleo. Their diagnosis, in fact, agreed with those of the doctor on duty in 83% of cases. In conclusion, it is difficult to imagine a diagnostic or therapeutic problem which needs specific structures. Cooperative programs with physicians working in our region will be carried out in order to control, where possible, admission to Emergency Departments.


[Chronic inflammatory bowel diseases in childhood. Case contribution and critical considerations].

The term "inflammatory bowel disease" describes 2 major categories of chronic disease: Crohn's disease and ulcerative colitis. Non specific chronic colitis and allergic colitis in childhood can usually be distinguished by clinical and histopathologic criteria. This study regards a retrospective analysis of 23 patients suffering from chronic inflammatory bowel disease. Clinical manifestations began between 4 and 18 years of age. The Authors report remarkable frequency in pediatric age and common diagnostic delay, especially in Crohn's disease. The possibility of extraintestinal symptoms (erythema nodosum, arthritis, iridocyclitis) is also showed. Moreover they analyse present diagnostic proceedings: in childhood the employ of 99 mTc-HmPAO-labelled granulocytes is a new and non invasive test useful in the follow-up of the patients. In the treatment corticosteroids and sulfasalazine are used in various combinations associated with general supportive measures. Finally the Authors confirm that chronic inflammatory bowel disease may cause, especially in childhood, remarkable involvement on the growth and several psychological disorders.


[Evaluation of the fluidifying effect on nasal mucus of physiologic solution combined with increasing concentrations of polysorbates].

Nasal decongestants are widely used in the treatment of rhinitis. Decongestant drugs are one of the main components, but numerous reports gave been made concerning side, local and general effects. The problem is particularly important in the treatment of infantile rhinitis for which the use of saline solution alone is often preferred. The paper evaluates the fluidifying action of a saline solution and polysorbate 20, a tensioactive molecule, on nasal mucous. Changes in mucous viscosity were assessed using a "filancemeter" and thromboelastograph. At the same time a clinical study on the efficacy of treatment and the possible presence of side effects was carried out in a population of rhinitic subjects. The study highlighted the fluidifying effect of the preparation on nasal mucous and the complete absence of side effects.


[Herpetic encephalitis with biphasic course. A case report and review of the literature].

Encephalitis represents an important event in pediatric practice. Between the different causes Herpes Simplex Virus (HSV) seems to be the most significant etiological agent, not only for its frequency but chiefly for conditioning prognosis. The Authors report a new case of HSV encephalitis with biphasic course illustrating the most recent pathologic, diagnostic and therapeutic features in the attempt to individualize those factors, between clinical data, laboratory findings and imaging that allow early diagnosis and consequently precocious specific antiviral therapy.


[Spinal lipomas in childhood].

Spinal lipomas account for 5% of the tumors of the spinal cord, frequently present already at birth. Most commonly they are associated with forms of dysraphism, but lipomas without bony involvement are considered dysembriogenetic lesions too. Children with lipoma frequently have intact neurological functions, but may become symptomatic later on. Diagnosis is possible also in neurologically intact patients because of skin lesions or subcutaneous masses. Many surgeons suggest early surgery to prevent injury to neural structures from traction due to cord tethering; others prefer to wait for the rise of any symptom before considering surgery. However, neurological recovery after surgery is rarely observed, and, when present, is always partial; the primary goal of surgery is to stop the clinical progression through the detethering of the cord.


[Non-tumoral neonatal hydrocephalus. Results of surgical treatment in the first month of life].

Two hundred eight patients with non-tumoral congenital hydrocephalus underwent CSF shunting below the age of one month. Ultrasonography was the most frequently utilized tool; hydrocephalus was recognized during pregnancy in 52% of the cases. Hydrocephalus was associated with myelomeningocele in 97 infants; in 38 subjects ventricular dilation was secondary to aqueductal stenosis. Post-hemorrhagic and post-infective hydrocephalus accounted for only 20 and 13 cases respectively. At the follow-up observation, a normal psychomotor development was recorded in 67.3% of the cases. Seventeen patients died. CSF infection was the most common cause of death (41%); in 6 patients the exitus was determined by the natural evolution of congenital associated malformations. Shunt revision did not influence the morbidity and the mortality significantly. On the other hand, CSF infections appeared to influence the prognosis negatively. In our experience, the prognosis of hydrocephalus operated on in the first month of life does not differ from that of hydrocephalus operated on late in life.


[A case of CHARGE with fibrocystic disease of the pancreas].

The Authors describe a case of CHARGE association (CA) with fibrocystic disease of the pancreas (FCP). The CA is characterized by seven mayor defects (colobomatous malformation, heart defect, atresia choanae, growth and mental deficiency, genital hypoplasia, ear anomalies and/or deafness). Four of this seven defects are necessary for the diagnosis. Other diseases and anomalies associated with CA are examined. The peculiarity of contemporaneous presence of FCP and the seriousness of the prognosis is pointed out.


[Vascular lesions in Candida albicans sepsis].

In two premature newborns affected by candida sepsis we observed at ultrasonography alterations of the heart and of the anterior cerebral artery. These alterations suggest a cardiovascular involvement that is rarely reported in the literature as a complication of systemic candidiasis.


An alternative to DNA extraction for the diagnosis of Pneumocystis carinii pneumonia by polymerase chain reaction using a new oligonucleotide probe.

We have used new specific primers and probe in a polymerase chain reaction (PCR) followed by Southern blot assays to detect Pneumocystis carinii in human bronchoalveolar lavage samples obtained from HIV-infected patients with pulmonary symptoms. To facilitate the procedure we developed a filtration technique without DNA extraction yielding a high sensitivity (18/18 positive results). The high specificity of the technique was shown by testing immunosuppressed patients without P. carinii pneumonia.


Chlamydia trachomatis confirmatory testing of PCR-positive genitourinary specimens using a second set of plasmid primers.

A plasmid-based PCR for the detection of Chlamydia trachomatis was evaluated with a confirmatory PCR employing a second set of plasmid primers. A total of 258 genitourinary specimens including 134 female endocervical and urethral specimens and 124 male urethral specimens were tested by culture, blocked EIA and PCR. Fifty-four specimens were positive by culture, 50 were positive by EIA and 71 were positive by PCR. Fourteen specimens that were PCR-positive but culture- and EIA-negative were confirmed positive by the confirmatory PCR. Two of the 187 specimens which were negative by culture and EIA were positive by PCR but failed to confirm with the second set of primers. Using an expanded gold standard of culture, blocked EIA and confirmed PCR, the overall sensitivities for culture, blocked EIA and confirmed PCR were 76.0% (54/71), 70.4% (50/71) and 100% (71/71) and the specificities were 100% (187/187), 100% (187/187), respectively. These results demonstrated that a confirmatory PCR was useful for sorting out discordant specimens and establishing the true specificity of PCR. Furthermore, these results demonstrate that PCR is more sensitive than culture and EIA and suggest that a confirmed PCR test should be included in the gold standard for the evaluation of new tests for diagnosing Chlamydia trachomatis infections.


Detection of Mycobacterium leprae and the potential for monitoring antileprosy drug therapy directly from skin biopsies by PCR.

An improved protocol for PCR analysis of Mycobacterium leprae-infected tissues, based on enzymatic lysis, has been developed and used to demonstrate the feasibility of using PCR for detecting M. leprae in routine skin biopsies taken from leprosy patients throughout the clinical spectrum. Of 92 multibacillary patients tested, 99% were PCR-positive using gel electrophoresis or DNA hybridization to detect the amplified product. Similar analysis of paucibacillary patients, in which only one of 27 biopsies had demonstrable AFB microscopically, gave a positivity rate of 74%. No PCR signals were demonstrated from skin biopsies from seven patients with non-leprosy dermatoses and one AIDS patient with a disseminated atypical mycobacteriosis. Evaluation of leprosy patients with antileprosy drug therapy prior to biopsy demonstrated that PCR signals were either greatly diminished or absent after 2 months of continuous antibiotic therapy. PCR was also able to detect the presence of M. leprae in tissues of patients receiving antibacterial therapy when patients were suspected of harbouring drug-resistant M. leprae.


Development of a DNA probe for Ureaplasma urealyticum.

Ureaplasma urealyticum has been associated with a variety of disease conditions in humans. However, its exact etiologic role has not been well established because of the difficulties encountered in cultural diagnosis and the time needed for positive identifications. A DNA probe which is specific for a target DNA sequence unique to this suspected pathogen offers a rapid, sensitive and specific means of diagnosis. This study details the development of a polymerase chain reaction system for U. urealyticum. Using conventional hybridization techniques, a cloned genomic fragment was found to be specific for this organism. Sequencing of part of this probe DNA permitted the assignment of oligonucleotide primers which amplified a 186 bp target segment. This PCR system is specific for U. urealyticum but not for other closely related species of mycoplasma. This highly sensitive diagnostic technique will aid in determining the etiologic role, tissue tropism and dynamics of pathogenesis of this organism, and thereby result in better patient care.


A species-specific DNA probe for Providencia stuartii identification.

A DNA probe is described that can be used for identification of Providencia stuartii by means of filter hybridization assays. The probe, which is a fragment of the P. stuartii phoN gene coding for an acid phosphatase, appeared to be able to recognize only P. stuartii strains in slot-blot hybridization experiments performed with total DNA extracted from 545 strains of 64 different Gram-negative bacterial species, including all the major representatives of the family Enterobacteriaceae. Owing to the problems that may be often encountered for correct identification of P. stuartii at the species level when using commercial identification systems, this probe may result useful for fast and reliable identification of P. stuartii strains for taxonomical, epidemiological and diagnostic studies.


A specific DNA probe for detecting Mycoplasma hyopneumoniae in experimentally infected piglets.

Mycoplasma hyopneumoniae is the primary agent of swine enzootic pneumonia. Because of fastidious growth requirements and its serological cross-reactions with other porcine mycoplasmas, we developed a specific DNA probe for its detection. A partial genomic library of M. hyopneumoniae was constructed in plasmid pBR 322 using Hind III chromosomal fragments. The recombinant plasmids were screened by differential hybridization with M. flocculare and M. hyorhinis genomic DNA probes. One non-hybridizing recombinant plasmid was selected and its 1.65 kbp insert (designated I141) tested for specificity against genomic DNA from numerous mycoplasmas, other bacteria species and DNA from lung tissue of specific pathogen free (SPF) piglets. The 32P labelled I141 could detect specifically down to 400 pg of M. hyopneumoniae genomic DNA. To test the suitability of the I141 probe for the laboratory diagnosis of M. hyopneumoniae infections, we used clinical tracheobronchial specimens from piglets which were experimentally infected with M. hyopneumoniae. The results with hybridization on each specimen were compared to findings with an immunofluorescence test. Of the clinical specimen tested, there was agreement in the two tests of 63%.


Candida albicans antifungal-resistant strains: studies on adherence and other pathogenicity related characteristics.

In search of adhesion-variant strains of Candida albicans the adherence of a number of polyenes and/or azole-resistant strains of this yeast was studied (C. albicans 6406, 6406/8 and 799-XL, -XS, -YS, -R and YL). For comparison C. albicans KCCC 14172, known for its high adhesion and proteinase production, was also used. All isolates showed significantly lower adhesion (P < 0.001) compared with KCCC 14172. The exception was 6406/8 which showed superior adherability to all strains tested (2.5-4.8 times more adherent). This superiority prompted us to study the possible variation between this strain and the others in parameters that contribute to pathogenicity. Strain 6406/8 had the smallest average cell size (0.5-0.75 the size of cells from other strains). Variation in proteinase production and germ-tube formation existed among strains, with strain 6406/8 producing the lowest levels of inducible proteinase (2-4-fold less than the others), as well as being the least germ-tube former (10 times less than other strains). Ultrastructural comparisons between strain 6406/8 and its parent showed that the mutant strain had a thinner cell wall with a dense floccular layer throughout the cell wall compared to the parent strain. The cytoplasmic membrane of the mutant was more conspicuous than that of the parent strain. Comparison of the pathogenicity of strain 6406/8 and its parent (6406) revealed that although the mutant strain initially showed higher colonization than the parent strain, it was cleared much faster.(ABSTRACT TRUNCATED AT 250 WORDS)


Glucose transport in Crithidia luciliae.

The glucose analogue, 2-deoxy-D-glucose, was used to characterise the glucose transport system in Crithidia luciliae choanomastigotes. Uptake was temperature dependent with a Q10 of 2, and saturable with a Km of 0.22 mM and Vmax of 5.5 nmol min-1 (mg protein)-1 at 23 degrees C. Preloaded cells showed rapid exchange of intracellular 2-deoxy-D-glucose when incubated with extracellular D-glucose or 2-deoxy-D-glucose but little exchange with L-glucose. The substrate specificity of the uptake was studied using a number of D-glucose analogues. 6-Deoxy-D-glucose, 3-fluoro-3-deoxy-D-glucose and 4-fluoro-4-deoxy-D-glucose all competed for the transporter and had significant inhibitory effects on 2-deoxy-D-glucose transport. In contrast, 1-thio-beta-D-glucose, trehalose, 3-O-methyl-D-glucose, arginine, thymidine, L-sorbose and L-glucose were not inhibitory. The results imply the existence of a glucose transporter. The transport was blocked by a number of inhibitors and ionophores, including fluoride, azide, cyanide, dinitrophenol, valinomycin and nigericin. Overall, the uptake, exchange and efflux of 2-deoxy-D-glucose is consistent with transport via facilitated diffusion.


Chemiluminescence of polymorphonuclear granulocytes in the presence of selected Candida species.

We monitored the respiratory burst of polymorphonuclear granulocytes (PMN) during infection in vitro with 25 strains of six Candida species with different extracellular proteolytic activity by chemiluminescence (CL). The CL-response of PMNs to viable Candida blastoconidia was compared to the response to ethanol-fixed cells of the same strain. Ethanol-fixed blastoconidia of all tested fungal strains uniformly increased the CL-response on contact. Viable fungal blastoconidia induced different CL-responses: (1) virulent C. albicans and C. tropicalis induced a low CL-response, (2) less virulent C. glabrata, C. parapsilosis and C. krusei induced CL-responses which reflected strain-specific differences, and (3) viable blastoconidia of attenuated C. guilliermondii uniformly caused a high PMN CL-response. After the CL-measurement, the killing of Candida by PMNs was assayed. High CL-response could be correlated to an augmented killing of the yeasts. These results were discussed with respect to a factor of fungal virulence, the secretion of acid proteinase.


Identification of a 33-kilodalton immunodominant antigen of Trypanosoma congolense as a cysteine protease.

A 33-kDa protein of Trypanosoma congolense is a major antigen in infected cattle and the production of antibody to this antigen appeared to correlate with enhanced resistance to trypanosomiasis [4]. Immunoelectron microscopy using a monoclonal antibody (mAb 4C5) raised against the 33-kDa antigen showed a lysosomal localisation, similar to that of a previously described 32-kDa cysteine protease of T. congolense. Both mAb 4C5 and anti-33 kDa antibody from infected cattle bound on Western blots to the cysteine protease that had been purified by affinity chromatography on cystatin-Sepharose. Sepharose-coupled mAb 4C5 was used to affinity purify the antigen from bloodstream forms of T. congolense. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the affinity-purified antigen had a molecular mass of 33 kDa under non-reducing conditions, and 40 kDa under reducing conditions. Anti-33-kDa antibody from infected cattle bound to both non-reduced and reduced affinity-purified antigen on Western blots. Serum from a rabbit immunised with the biochemically purified enzyme also bound the affinity-purified antigen. The affinity-purified antigen displayed proteolytic activity in fibrinogen-containing SDS-PAGE and against Azocoll. It hydrolysed benzyloxycarbonyl-Phe-Arg-7-amino-methyl coumarin (Z-Phe-Arg-NHMec) with a Km similar to that of the biochemically purified enzyme. Proteolytic and peptidolytic activities of the antigen were inhibited by the inhibitors of cysteine proteases, cystatin and trans-epoxysuccinyl-L-leucyl-amido (4-guanidino)butane (E-64). On two-dimensional gel electrophoresis, the antigen displayed similar characteristics to those of the biochemically purified enzyme. We conclude that the 33-kDa antigen of T. congolense and the cysteine protease are the same molecule.


Fusarium infections. Criteria for the identification of the responsible species.

The incidence of human infections by Fusarium is increasing in contemporary medicine. Since the recognition of the species involved in such processes is generally difficult for non-specialists, we include here the main criteria that should be used in the identification of the responsible species. The most frequent species are F. verticillioides, F. oxysporum and F. solani, which are illustrated. The differences between Fusarium and Acremonium and Cylindrocarpon, two close genera, are discussed. The latter two are also illustrated. The main morphological characteristics of Fusarium such as sporodochium, conidiophore, conidiogenous cell, phialide, locus, macroconidia, mesoconidia, microconidia and chlamydospores are defined and illustrated. A key to the identification of Fusarium species isolated from human lesions is also included.


Sequence homology and absence of mRNA defines a possible pseudogene member of the Trypanosoma cruzi gp85/sialidase multigene family.

A genomic clone, pTt21, containing DNA apparently transcribed specifically in Trypanosoma cruzi trypomastigotes, was obtained by differentially screening a genomic library with trypomastigote and epimastigote cDNA. This 3444-bp clone contained open reading frames at each end, separated by a 1.8-kb non-coding region. The translated polypeptide from the 3' open reading frame (ORF2) of 1037 bp had 25-30% identity with 5 recently published T. cruzi gp85/sialidase sequences, and 20-25% identity with bacterial sialidases. Rabbit antiserum raised against an Escherichia coli fusion protein derived from the 5' open reading frame (ORF1) identified a surface antigen of 160 kDa, specifically expressed in trypomastigotes. A probe containing the first 211 bp from ORF1 was used to obtain a complete copy (c1821) of a gene that was closely related to ORF1, and encoded another member of the gp85/sialidase family. c1821 encodes a protein of 897 amino acids, but assignment of the N-terminus of the polypeptide was not possible. The 5'-most start codon is an unfavourable context to act as a translation initiator, it does not align with the initiator methionines of other gp85/sialidase sequences, nor is it followed by a signal peptide sequence characteristically found in other gp85/sialidase sequences. Although homology with the 5' ends of other gp85/sialidase sequences decays towards the 5' end of c1821, alignment of c1821 with 4 other gp85/sialidases indicated that the coding sequence should extend upstream at least 160 amino acids. In this region of c1821 there are multiple stop codons in each frame. The presence of the stop codons, the alignment data and our inability to amplify reverse transcribed mRNA using four internal primers, suggest that c1821 may not be present as a mature mRNA and is a pseudogene. Comparison of the apparently non-repetitive 3' coding domain of c1821 with the corresponding repetitive domains of two other members of the gp85/sialidase family revealed a high degree of similarity in nucleotide but not in amino acid sequence, and c1821 may thus represent an evolutionary intermediate between sub-families of the gp85/sialidase superfamily.


Biotypes of the dermatophyte Microsporum distortum.

During a study of the epidemiology of Microsporum canis and closely related species, sub-specific variation was demonstrated within thirteen isolates of Microsporum distortum based on the pattern of carbohydrate utilization. Strains from three continents could be differentiated by their utilization of trehalose and erythritol. Isolates from New Zealand yielded the same patterns as M. canis (Arthroderma otae (-)); examination of 120 isolates indicated that M. canis could not be biotyped by carbohydrate utilization, although limited variation had been noted. Examination of total native protein patterns by gel electrophoresis showed that variations existed within the M. distortum group; the much larger number of M. canis (A. otae (-)) strains had appeared to be homogenous.


Transient inhibition of protein synthesis accompanies differentiation of Trypanosoma brucei from bloodstream to procyclic forms.

It has been widely believed that bloodstream forms of Trypanosoma brucei must be first transformed into intermediary and/or short-stumpy forms in the bloodstream of the mammalian host before differentiation to the procyclic culture form can occur. In our recent studies, the pleomorphic T. brucei strain TREU667 was found to differentiate directly from the long-slender bloodstream form to the procyclic form in Cunningham's medium at 26 degrees C [7]. In the present investigation, the same was found true for another pleomorphic strain of T. brucei, STIB366D. Four independent monomorphic strains of T. brucei were tested; two, #427 and EATRO164, were found capable of differentiating in vitro directly into procyclic forms, whereas the other two, TREU667/RP-56 and EATRO110, could not. There is thus no correlation between the capability of differentiating in vitro and the ability of being converted from long-slender to intermediary and short-stumpy bloodstream forms. Two additional markers for following differentiation, other than observing morphological changes, were tested. Assays for the emerging phosphoenolpyruvate carboxykinase (PEPCK) by immunoblottings worked well, with results agreeing closely with the morphological change. But immunoblottings of glycosomal phosphoglycerate kinase (gPGK) failed to demonstrate a significant decrease in the protein level upon completion of differentiation. Apparently, gPGK has a rather long half-life and is unsuitable as a marker of differentiation. When temperature was dropped from 37 degrees C to 26 degrees C at the starting point of in vitro differentiation, protein synthetic activity in the pleomorphic T. brucei TREU667 bloodstream form was decreased by 4-fold. When the activity was gradually brought back to and beyond the original level after a day's incubation, the profile of newly synthesized proteins was that of the procyclic form. A monomorphic variant of TREU667, RP-56, which is incapable of differentiating in vitro, has a much higher protein synthetic activity than its pleomorphic parent in the bloodstream form. This high activity and the bloodstream profile of proteins thus synthesized were unaffected by the decreased temperature in Cunningham's medium until cell death. We thus conclude that a general inhibition of protein synthesis in bloodstream forms caused by temperature drop may be among the early events triggering differentiation into the procyclic form.


A fourth family of the Plasmodium falciparum S-antigen.

The S-antigen of Plasmodium falciparum is a highly diverse heat stable protein that is located in the parasitophorous vacuole of the mature asexual intraerythrocytic parasite. The gene for S-antigen exists within the parasite population as multiple alleles at a single locus. Its sequence contains a large central block of tandemly arranged peptides that are identical or very similar in one allele but differ widely in sequence, repeat length and number among different alleles, and consequently antigenic specificity. Thus, antibodies directed against the repeat region can be used to define the serotype of an S-antigen. Flanking this repeat block are 2 short regions of non-repetitive sequence which have been described as occurring in three different forms, each of which is used to define a single S-antigen family. We present the S-antigen sequence for the isolate 3D7 which defines not only a novel serotype but also a novel S-antigen family. The central repeat block is composed of 57 copies of an 8-residue peptide with consensus sequence ED(E/K)VSNG(R/G). Comparison of the four S-antigen families reveals that they differ considerably from each other with variation being most pronounced in the carboxy terminal-flanking region. This pattern of sequence variation differs considerably from that found for MSA-1 and MSA-2, the only other diverse proteins of P. falciparum for which sequence information is available.


The effect of sulconazole on the antigenic composition of two dermatophyte species.

Trichophyton rubrum and Trichophyton interdigitale have been grown in liquid culture in the presence of sulconazole. The antigenic activity of detergent extracts of intact organisms was analysed following SDS-PAGE and the probing of Western blots with homologous antisera raised in rabbits and with sera from patients with dermatophyte infections. Differences in protein-band patterns were noted; some bands present in control samples were absent in azole-treated samples and vice versa. These differences were reflected in antigenic band patterns, especially among components of approximate molecular weight of 30-40, 50-60 and 92-100 kDa.


Membrane-associated proteases process Plasmodium falciparum merozoite surface antigen-1 (MSA1) to fragment gp41.

The Plasmodium falciparum merozoite surface antigen-1 (MSA1) undergoes stage-specific processing; this processing appears isolate-specific during cleavage to fragment gp41. Recombinant substrates were prepared from the two allelic forms of MSA1; the MAD20 substrate was cleaved at four sites in the molecule whilst the K1 form was cleaved once. However both parasite isolates, although expressing different allelic forms of MSA1, possess the same repertoire of MSA1-specific proteases. The cleavage site in native gp41 is conserved between P. falciparum isolates. The specificity of substrate cleavage was determined by N-terminal sequencing of cleaved substrate fragments; two cleavage sites, identical to native MAD20 processed fragments, were not conserved between alleles. An additional non-conserved site was cleaved by an erythrocyte protease. The MSA1-specific proteases were membrane-associated but soluble forms were purified by anion-exchange chromatography. The gp41-specific protease activity was inhibited by serine, thiol and metalloprotease inhibitors whilst the two other MSA1-specific proteases were serine proteases (as was the erythrocyte protease).


Recurrent tinea pedis: a double blind study on the prophylactic use of fenticonazole powder.

Tinea pedis is a common and frequently recurring dermatophytic infection, which is extremely difficult to eradicate. The often inevitable persistence of predisposing conditions, especially maceration, suggests that application of powders containing antifungal medication to the affected area could be effective in preventive therapy against recurrence. For this study we used one of the most recent azole antifungal agents, fenticonazole. Thirty patients affected with tinea pedis were cured with topical antifungal treatment; both the diagnosis and the cure were confirmed by microscopic and cultural mycological analyses. The results of subsequent double blind antifungal versus placebo treatment (controlled with clinical and mycological tests over a period of 4 months and with a final statistic evaluation) confirmed the effectiveness of such therapy in reducing the frequency of tiresome relapses in such patients.


Identification and partial characterization of a lipophosphoglycan from a pathogenic strain of Entamoeba histolytica.

An acidic glycoconjugate could be extracted from a delipidated residue fraction of [3H]galactose, [3H]mannose or [32P]orthophosphate metabolically labeled Entamoeba histolytica with water/ethanol/diethylether/pyridine/NH4OH (15:15:5:1:0.017). The radioactively labeled glycoconjugate comprised 50-55% of the total [3H]galactose label incorporated into macromolecules. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the radiolabeled glycoconjugate showed two diffuse smears centering around 110 kDa and 45 kDa. Similar profiles were observed for both [3H]galactose- and [32P]orthophosphate-labeled glycoconjugate. No such bands were visible in [35S]methionine-labeled material. The hydrophobic nature of this glycoconjugate was inferred from its chromatographic behavior on phenyl-Sepharose. The molecule was rendered hydrophilic after digestion with phosphatidylinositol-specific phospholipase C. It was also sensitive to deamination by nitrous acid. Mild acid hydrolysis led to its fragmentation into smaller molecules as revealed by Sepharose 4B chromatography. Paper chromatographic analysis of the depolymerized [3H]galactose- and [3H]mannose-labeled fragments revealed that each was sensitive to alkaline phosphatase. The major dephosphorylated fragment migrated as an apparent galactose and mannose containing disaccharide which migrated identically to the Gal beta 1-4Man disaccharide derived from the lipophosphoglycan of Leishmania donovani. The above data support the existence of a major acidic glycoconjugate in E. histolytica bearing striking structural similarities to the lipophosphoglycan of Leishmania.


Analysis of the genes encoding immunodominant piroplasm surface proteins of Theileria sergenti and Theileria buffeli by nucleotide sequencing and polymerase chain reaction.

The nucleotide sequences of the cDNAs encoding a 33-kDa piroplasm protein of Theileria sergenti (p33) and a similar protein of Theileria buffeli (p34) were determined. Both of the genes contained an open reading frame of 849 base pairs. The predicted amino acid sequence of p33 and p34, consisting of 283 residues, showed 82% similarity. A transmembrane hydrophobic domain and signal peptides were predicted. The polymerase chain reaction was used to amplify p33/34 genes from the piroplasm DNA of T. sergenti, T. buffeli and Theileria orientalis. Following amplification, p33 and p34 genes were clearly differentiated using the restriction enzymes sites that were not shared between them. These results indicated that p33 and p34 were conserved molecules among these Theileria species, and the genes that encode p33/34 proteins were suitable for discrimination of T. sergenti from T. buffeli/T. orientalis.


Identification and characterization of gamma-giardin and the gamma-giardin gene from Giardia lamblia.

The giardins are abundant cytoskeletal proteins that range in size from 29-38 kDa and are specific to the ventral disk of the intestinal protozoan parasite Giardia lamblia. The 29-kDa (beta and beta-1; refs. 8-10) and the 33-kDa (alpha-1 and alpha-2; refs. 3 and 7) giardins have been characterized previously. In this paper we extend the analysis of the giardins to include the 38-kDa giardin, which we have named gamma-giardin. After purifying gamma-giardin by two-dimensional electrophoresis, we raised polyclonal antibodies to the protein and used them to demonstrate that gamma-giardin shares at least one epitope with 9 other giardin polypeptides and that it localizes to the ventral disk of the parasite. We also determined an internal peptide sequence of 12 amino acid residues and used this information to construct oligonucleotide probes for the gamma-giardin gene. After cloning the gene, we determined the nucleotide sequence of its 933-bp open reading frame and 866 bp of 5' and 3' flanking sequence. We found the downstream AGTPuAAPy motif typical of all G. lamblia genes sequenced to date, and determined that the single copy of the gamma-giardin gene localizes to the same chromosome or chromosomal cluster as the alpha-giardins. Finally, we demonstrated by primer extension analysis that gamma-giardin transcripts contain a short untranslated leader characteristic of G. lamblia messenger RNAs.


Isolation of putative cysteine protease genes of Ostertagia ostertagi.

Recombinant phage containing putative Ostertagia ostertagi cysteine protease genes have been isolated from a lambda EMBL-3:genomic DNA library using a Haemonchus contortus cathepsin B-like cysteine protease cDNA as hybridization probe. Restriction enzyme maps of the phages suggest that they identify at least 3 genes, 2 of which appear to be linked in tandem. The complete nucleotide sequence of one gene, CP-1, was determined. The CP-1 gene appears to be organized into 12 exons than span 4.5 kb of DNA. The number and sizes of the exons are essentially identical to those in the H. contortus AC-2 cysteine protease gene. Partial nucleotide sequences obtained for a second O. ostertagi gene, CP-3, revealed a similar organization for exons 8-12 in this gene. Like other cathepsin B-like cysteine proteases, CP-1 appears to be synthesized initially as a preproprotein that is proteolytically processed to its mature form. The amino acid identity between the presumptive CP-1 and CP-3 proteins is 66%, which is similar to the level of homology between the presumed mature protein regions of CP-1 and AC-2. Amino acid identity between CP-1 and AC-2 is greatest in the mature protein region and lowest in the signal sequence and propeptide regions. The CP-3 protein appears to be most closely related to the H. contortus AC-5 protein. CP-1 and CP-3 display significantly greater homology to H. contortus cysteine proteases than they do to human cathepsin B or the Sm31 cysteine protease of Schistosoma mansoni (about 40% identity with each).


The facultative anaerobic energy metabolism of Schistosoma mansoni sporocysts.

Schistosoma mansoni miracidia in water are known to possess an aerobic energy metabolism, the Krebs cycle being the main terminal of the breakdown of endogenous glycogen reserves. The present study demonstrated that after in vitro transformation of miracidia into sporocysts, the organisms degraded glucose to lactate and carbon dioxide in a more anaerobic ratio than do miracidia. The occurrence of a large Pasteur effect demonstrated, however, that oxidative phosphorylation was still the major process used for energy generation. After 24 h in vitro cultivation the sporocysts had consumed more external glucose and their metabolism had shifted towards lactate production. Sporocysts could cope with inhibited respiration: they had a large anaerobic capacity and survived perfectly in the presence of cyanide, producing a large amount of succinate in addition to lactate. It was demonstrated that this succinate was largely produced via phosphoenolpyruvate carboxykinase (PEPCK). This pathway, which is known to occur in most parasitic helminths, has never been demonstrated in schistosomes, not even in the miracidial stage immediately preceding the sporocysts. It was also shown that in sporocysts part of the lactate was not formed directly by glycolysis, but via a detour including fumarate and the action of PEPCK. The results demonstrated that S. mansoni sporocysts are facultative anaerobes, fully equipped to adjust their energy metabolism to the variable conditions inside their intermediate host, the snail. In the presence of oxygen, they derive most of their energy from the aerobic degradation of glucose to carbon dioxide, but under anaerobic conditions they switch towards lactate and succinate production.


Structure of a Plasmodium chabaudi acidic phosphoprotein that is associated with the host erythrocyte membrane.

We have characterized by molecular cloning and sequencing a Plasmodium chabaudi antigen that is associated with the membrane of the infected erythrocyte throughout the entire intraerythrocytic cycle. The protein (PcEMA1) has a predicted size of 50 kDa and contains a major tandem repeat array of 16 octapeptides that constitutes almost 30% of the protein. At its amino-terminus, PcEMA1 has a string of hydrophobic residues characteristic of a secreted protein, but does not contain a hydrophobic membrane-spanning segment. The antigen appears to reside on the cytoplasmic face of the erythrocytic membrane. PcEMA1 has a predicted pI of 4.4 and is a potential phosphoprotein.


A 2359-base pair DNA fragment from Cryptosporidium parvum encoding a repetitive oocyst protein.

A Cryptosporidium parvum lambda gt11 expression library was constructed using EcoRI-digested genomic DNA extracted from in vitro-excysted oocysts. Screening of this library with rat anti-Cryptosporidium antiserum led to the isolation of a clone containing a 2359-bp EcoRI fragment. When this fragment was ligated into the EcoRI site of plasmid vector pMS1S, the resulting clone expressed a 200-kDa beta-galactosidase fusion protein. Western blot analysis using serum raised against this fusion protein indicated that the EcoRI fragment represented part of a gene encoding a 190-kDa oocyst wall protein of C. parvum. Sequencing of the fragment revealed a continuous open reading frame encoding 786 amino acids. The DNA sequence is relatively low in G+C (39.1%), and the third codon position contains only 17.9% G+C. The deduced peptide sequence has unusually high proportions of cysteine, proline, glutamine and histidine. Another striking feature of the amino acid sequence is the presence of distinctly repetitive regions based on conserved cysteine residues.


Influence of oxygen on the fermentative metabolism of metronidazole-sensitive and resistant strains of Trichomonas vaginalis.

The microaerophilic protozoon Trichomonas vaginals responds to extracellular changes in oxygen concentration: acetate, lactate, ethanol, H2 and CO2 formation, as well as glucose-depletion rates, are affected. All these variables except ethanol production rates, also differed between clinically metronidazole-sensitive (1910) and resistant (IR78 and CDC85) strains. Most interesting were the greatly increased glucose-scavenging rates of resistant isolates and their low specific activities of hydrogenase and H2 formation rates by comparison with the metronidazole-sensitive strain. Results suggest that all three strains of this parasite are well adapted to the O2 levels prevailing in situ (13-56 microM). Thus, vaginal oxygen tensions have more pronounced effects on the balances of fermentation products in the resistant strains, and results indicate that these strains may then use hydrogenosomal pathways to their advantage.


Molecular analysis of Plasmodium falciparum hexokinase.

Hexokinase, a key glycolytic enzyme, is involved in the initial phosphorylation reaction of imported glucose and specific blocking of this activity may therefore arrest the development of malaria parasites. We describe here the cloning of a single copy hexokinase gene of Plasmodium falciparum (PfHK) from cDNA or genomic DNA libraries. The deduced amino acid sequence of PfHK has 26% identity with human hexokinase I and its predicted molecular mass assigns it as an invertebrate type isoenzyme of hexokinase. A single 1.5-kb exon is translated from a 3-kb mRNA in asexual stages of the parasite. In contrast to aldolase and GPI, the gene for this glycolytic enzyme is located on chromosome 8. Poly- and monoclonal antibodies against recombinant PfHK support our cloning results at the protein level as they detect a protein of the predicted size and isoelectric point by Western blotting in parasite protein samples. Moreover, polyclonal rabbit IgG against recombinant PfHK partially inhibits the hexokinase activity of a P. falciparum lysate which provides direct proof that the gene cloned encodes hexokinase of the parasite.


Diphenylhydantoin stimulates the intrapituitary conversion of thyroxine to triiodothyronine in the rat.

Treatment of normal rats with diphenylhydantoin (DPH) decreases serum thyroxine (T4) and triiodothyronine (T3) levels without the anticipated rise in serum thyrotropin (TSH). The present work has studied the intrapituitary conversion of T4 to T3 in male Wistar rats, 200-250 g body weight (BW), treated with DPH 5 mg/100 g BW/day for 8 days. A tracer dose of 3',5'-[125I]T4 (150 microCi) was injected intravenously, and 2 h later hypophyses were removed and homogenized individually at 4 degrees C in ice-cold PBS buffer (pH 7.4). T4 and T3 were extracted in 400 microliters n-butanol:2 N HCl (9:1) and chromatographed in tertiary amyl alcohol:hexane: 1 N ammonia (5:1:6). In 11 untreated control rats, [125I]T3 generated from [125I]T4 deiodination was 35 +/- 6% and intact [125I]T4 was 49 +/- 9% of total chromatographic radioactivity. In 11 DPH-treated rats [125I]T3 increased (p < 0.001) and [125I]T4 decreased (p < 0.02). The DPH effect was blocked in rats treated for 2 days with iopanoic acid 10 mg/100 g BW, though blocking was not seen in rats treated with half the dose of iopanoic acid. In normal rats receiving supplemental doses of T4 (2 micrograms/100 g BW/day for 8 days), DPH similarly increased pituitary 5'-deiodination. Administration of propylthiouracil (PTU) to T4-supplemented rats had no effect on pituitary 5'-deiodination of T4, whereas the addition of DPH to PTU treatment increased [125I]T3 production (p < 0.01). Serum T4 (p < 0.001) and T3 (p < 0.01) were decreased after DPH therapy, while serum and pituitary TSH were not altered.(ABSTRACT TRUNCATED AT 250 WORDS)


Suppression of luteinizing hormone secretion during food restriction in male rhesus monkeys (Macaca mulatta): failure of naloxone to restore normal pulsatility.

Short periods of fasting have been shown to cause a significant slowing of pulsatile LH secretion in men and male rhesus monkeys, which appears to result from a slowing of GnRH drive to the reproductive axis. To determine whether an increased activity of endogenous opioid peptides causes this slowing of pulsatile LH secretion, the ability of naloxone administration to reverse the fasting-induced suppression of LH secretion was tested. For this study, 6 adult male rhesus monkeys, with indwelling femoral and jugular venous catheters, were maintained on tether/swivel systems. Naloxone was administered to monkeys as a continuous infusion (0.25 mg/kg/h, with an initial loading dose of approximately 1.0 mg) for 8 h (16.00 to 24.00 h) on a day of normal feeding and again on a day of fasting. The LH response to naloxone was determined by collecting blood samples every 15 min from 12.00 to 24.00 h. LH pulse frequency on a day of normal feeding was 4.0 +/- 0.52 pulses/8 h, and naloxone administration on a day of feeding increased LH pulse frequency to 6.8 +/- 0.86 pulses/8 h. On a day of fasting, LH pulse frequency was 1.67 +/- 0.67 pulses/8 h, and naloxone administration on a day of fasting slightly, but not significantly, increased LH pulse frequency to 2.5 +/- 0.51 pulses/8 h. Similar studies were performed with a higher dose of naloxone (0.625 mg/kg/h, with an initial loading dose of approximately 2.0 mg) and again naloxone administration did not reverse the effects of fasting on pulsatile LH secretion. These results suggest that the slowing of pulsatile LH release that occurs with short periods of food restriction does not result from increased secretion of endogenous opioid peptides.


Interactions of estradiol with gonadotropin-releasing hormone and thyrotropin-releasing hormone in the control of growth hormone secretion in the goldfish.

The effects of testosterone (T) and estradiol (E2) on serum growth hormone (GH) concentrations were investigated throughout the seasonal reproductive cycle of the female goldfish. Gonad-intact female goldfish were implanted intraperitoneally for 5 days with silastic pellets containing no steroid (blank), T(100 micrograms/g) or E2 (25-100 micrograms/g). In blank-implanted females, seasonal variations in serum GH were evident; maximal serum GH levels were found in spring while minimal GH levels were found in summer and early autumn. Implantation of E2-containing silastic capsules stimulated increases (2-4 times control) in serum GH levels throughout the reproductive cycle. Implantation of T did not affect serum GH at any time of the year. One possible mechanism by which E2 could exert its effects may be through alteration of pituitary sensitivity to GH-releasing factors. The decapeptide salmon gonadotropin-releasing hormone (sGnRH) is found in the brain and pituitary of goldfish and stimulates gonadotropin (GTH) and GH secretion. In contrast, thyrotropin-releasing hormone (TRH) stimulates GH, but not GTH, release from pars distalis fragments obtained from sexually regressed (ED50 = 5.7 +/- 3.8 nM; August) or sexually mature (ED50 = 0.53 +/- 0.28 nM; March) fish; in vivo E2 treatment resulted in a 3-fold increase in the in vitro GH response to TRH. Furthermore, E2 treatment increased sGnRH-stimulated GH release by approximately 4-fold. These results demonstrate that E2 but not T stimulates GH secretion throughout the reproductive cycle of female goldfish. Furthermore, sGnRH and TRH stimulate GH release in a teleost, and these stimulatory responses are enhanced by physiological levels of E2.


Estrogen receptor in rat brain: presence in the hippocampal formation.

A series of studies was done in order to fully characterize the estrogen receptor (ER) expressed in the hippocampus of adult female rat. The structural identity among the ER mRNAs expressed in the hippocampus, hypothalamus and uterus was established by polymerase chain reaction amplification of the ER cDNA. Subsequently, the ER of the hippocampus was proved to bind DNA and beta-estradiol with the same affinity as the hypothalamic receptor. Finally, it was demonstrated that systemic administration of beta-estradiol determines the nuclear increase of ER levels with a time course which appears to be almost superimposable in the hippocampus and hypothalamus. On the basis of the above-mentioned evidence, it is concluded that the ER expressed in the hippocampus is structurally and functionally indistinguishable from the receptor expressed in the other hormone target tissues.


Long-term failure of compensatory growth in rats following acute neonatal passive immunization against growth hormone-releasing hormone.

Interruption of hypothalamic growth hormone-releasing hormone (GHRH) secretion by administration of antiserum against GHRH (GHRH-ab) decreases growth hormone (GH) secretion and inhibits growth in rats. The present study was undertaken to investigate whether there is a period of accelerated or catch-up growth following a period of growth arrest induced by GHRH-ab treatment. Neonatal male and female rats were injected daily on days 1-14 of age. Animals received normal rabbit serum (NRS) or GHRH-ab subcutaneously at a dose of 5 microliters/10 g body weight. Body weight, serving as an index of somatic growth, was monitored over the next 3 months. The increase in absolute body weight and growth velocity of GHRH-ab-treated rats, regardless of gender, was lower than the increase of NRS-treated animals. Significant decreases were observed by day 13 of age in the female rats and day 17 in the male rats. The percent differences and absolute difference in weight between the two treatment groups clearly demonstrated that the GHRH-ab-treated rats did not demonstrate any period of catch-up growth. A second group of animals was treated in a similar fashion to evaluate serum GH concentrations at three months of age. Pulsatile GH secretion, as assessed by peak frequency and amplitude, was normal in all of the rats, suggesting that the failure of catch-up growth in the GHRH-ab-treated animals was not due to decreased GH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)


Phorbol esters stimulate in vitro GnRH release from the hypothalamus of the estrogen-primed, ovariectomized rat: inhibitory effects of cocaine.

The purpose of this study was (1) to characterize more fully the effects of phorbol esters to stimulate GnRH release in vitro and (2) to determine whether cocaine (which disrupts estrous cyclicity in rats) affected phorbol ester stimulation of GnRH release in vitro. Hypothalami were collected from ovariectomized rats injected subcutaneously with 50 micrograms/kg of 17 beta-estradiol benzoate the two previous mornings. Sagittal sections of this block of CNS tissue comprising the preoptic area/anterior hypothalamus and mediobasal hypothalamus/median eminence were perfused at a rate of 6.2 ml/h with a modified Krebs-Ringer buffer (pH 7.4) using a programmable perfusion system. Perfusion results showed that 10-min pulses of phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate (PDBu) increased GnRH release in dose-dependent fashion (10(-10) to 10(-6) M) but had no effect on aminergic transmitter release. The biologically inactive alpha-phorbol was without effect on GnRH release. PDBu-stimulated GnRH release was blocked by both tetrodotoxin and cocaine, known inhibitors of Na+ influx. These results suggest a role for protein kinase C in regulating the release of GnRH. Our results that cocaine and tetrodotoxin attenuated phorbol ester stimulation of GnRH, presumably through inhibition of Na+ influx, suggest a direct biochemical mechanism for cocaine disruption of hypothalamic GnRH secretion and, consequently, cyclic reproductive function.


Isolation and characterization of hypothalamic growth-hormone releasing factor from common carp, Cyprinus carpio.

A growth hormone-releasing factor (GRF)-like peptide was isolated from the hypothalamus of common carp, Cyprinus carpio, by acid extraction, gel filtration chromatography, immunoaffinity chromatography using antiserum directed against rat GRF, and multiple steps of HPLC using octadecyl columns. Based on Edman degradation and peptide mapping, this teleost GRF was established to be a 45-residue peptide with the following primary structure: His-Ala-Asp-Gly-Met-Phe-Asn-Lys-Ala-Tyr-Arg-Lys-Ala-Leu-Gly-Gln-Leu-Ser- Ala-Arg - Lys-Tyr-Leu-His-Thr-Leu-Met-Ala-Lys-Arg-Val-Gly-Gly-Gly-Ser-Met-Ile-Glu- Asp-Asp-Asn-Glu-Pro-Leu-Ser. Carp GRF is closely related structurally to peptides of the glucagon-secretin superfamily, and more particularly to mammalian vasoactive intestinal peptide (VIP) precursors and the N-terminal portion of mammalian GRFs. A synthetic replicate of this peptide is highly potent [50% effective dose (ED50) approximately 0.08 nM] in stimulating GH release from cultured goldfish pituitary glands and in elevating serum GH levels 30 min after injection (0.1 micrograms/g) in goldfish.