Biotherapeutic effects of Bifidobacterium spp. on orogastric and systemic candidiasis in immunodeficient mice

Two commercially available Bifidobacterium spp. (Bifidobacterium infantis and Bifidobacterium lactis) were compared for their capacities to protect immunodeficient bg/bg-nu/nuand bg/bg-nu/+mice from orogastric and lethal candidiasis. Both Bifidobacterium spp. prolonged the survival of Candida albicans-colonized adult and neonatal bg/bg-nu/numice. The bifidobacteria affected the production of antibodies to C. albicans, inhibited disseminated candidiasis, suppressed weight loss associated with C. albicans infection, inhibited the growth of C. albicans in the alimentary tract, inhibited systemic candidiasis of endogenous origin, and decreased the severity of gastric candidiasis in both mouse strains. B. infantis inhibited systemic candidiasis of endogenous origin better than B. lactis; however, B. lactis was significantly more effective at inhibiting C. albicans colonization of the alimentary tract, suppressing gastric candidiasis, and protecting bg/bg-nu/numice from lethal candidiasis than B. infantis. These results show that Bifidobacterium spp. can protect immunodeficient mice from candidiasis but different species manifest quantitative and qualitative differences in their probiotic and biotherapeutic effects.     

Role of an inhibitory pyrimidine element and polypyrimidine tract binding protein in repression of a regulated alpha-tropomyosin exon.

Splicing of exons 2 and 3 of a-tropomyosin (TM) involves mutually exclusive selection of either exon 3, which occurs in most cells, or of exon 2 in smooth muscle (SM) cells. The SM-specific selection of exon 2 results from the inhibition of exon 3. At least two essential cis-acting elements are required for exon 3 inhibition, the upstream and downstream regulatory elements (URE and DRE). These elements are essential for repression of TM exon 3 in SM cells, and also mediate a low level of repression of exon 3 in an in vitro 5' splice site competition assay in HeLa extracts. Here, we show that the DRE consists of at least two discrete components, a short region containing a number of UGC motifs, and an essential pyrimidine-rich tract (DY). We show that the specific sequence of the DY element is important and that DY is able to bind to factors in HeLa nuclear extracts that mediate a low background level of exon 3 skipping. Deletion of a sequence within DY identified as an optimal binding site for PTB impairs (1) regulation of splicing in vivo, (2) skipping of exon 3 in an in vitro 5' splice site competition, (3) the ability of DY competitors to affect the 5' splice site competition in vitro, and (4) binding of PTB to DY. Addition of recombinant PTB to in vitro splicing reactions is able to partially reverse the effects of the DY competitor RNA. The data are consistent with a model for regulation of TM splicing that involves the participation of both tissue-specific and general inhibitory factors and in which PTB plays a role in repressing both splice sites of exon 3.     

Isolation of isoproturon-degrading bacteria from treated soil via three different routes

Three different isolation routes (flask enrichment/flask degradation assay, flask enrichment/microplate degradation assay, MPN assay/microplate degradation assay) were used to obtain pure cultures of bacteria which degraded isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea) as sole carbon and nitrogen source in a mineral salts medium from a field soil treated with isoproturon in the laboratory. All three isolation routes were successful, but the microplate assay of degradation was more successful than the flask assay. Characterization of 36 isolates indicated that they formed 16 distinct phenotypes (10 Gram-positive phenotypes, six Gram-negative phenotypes) which are likely to represent distinct species. Low concentrations of the degradation product 3-(4-isopropylphenyl)-1-methylurea (IPPMU) were occasionally found in the culture solutions. When provided as the sole source of carbon and nitrogen, the monomethyl degradation product was itself rapidly degraded by several of the isolates. Some isolates were also able to use the demethylated degradation product 3-(4-isopropylphenyl)-urea (IPPU) as sole source of carbon and nitrogen, although there was occasionally an extended lag-phase before rapid degradation commenced. One isolate was particularly active and degraded isoproturon, the monomethyl and demethylated degradation products of isoproturon, and demethylated the related phenylureas diuron and linuron.     

Recognition of picornavirus internal ribosome entry sites within cells; influence of cellular and viral proteins.

The ability of different picornavirus internal ribosome entry site (IRES) elements to direct initiation of protein synthesis has been assayed in different cell lines in the presence and absence of viral proteases that inhibit cap-dependent protein synthesis. Reporter plasmids that express dicistronic mRNAs, containing different IRES elements, with the general structure CAT/IRES/LUC, have been assayed. In each plasmid, the CAT sequence encodes chloramphenicol acetyl transferase and the LUC sequence encodes luciferase. The poliovirus (PV) 2A protease and the foot-and-mouth disease virus (FMDV) Lb protease induce the cleavage of the translation initiation factor elF4G and hence inhibit the activity of the cap-binding complex, elF4F. In human osteosarcoma (HTK-143) cells, each of the various IRES elements functioned efficiently. In these cells, the co-expression of the viral proteases severely inhibited the expression of CAT, but the proteases had little effect on the activities of the various IRES elements. In contrast, in baby hamster kidney (BHK) cells, the efficiencies of the different IRES elements varied significantly, whereas, in normal rat kidney (NRK) cells, each of the IRES elements was relatively inefficient. In both BHK and NRK cells, the activities of those IRES elements that functioned inefficiently were strongly stimulated by the co-expression of the PV 2A or FMDV Lb proteases. This stimulation was independent of the loss of cap-dependent protein synthesis and was not achieved by the co-expression of the C-terminal fragment of elF4G. The results suggest that the PV 2A and FMDV Lb proteases induce the cleavage of another cellular protein, in addition to elF4G, which influences IRES function.     

Simultaneous utilization of glucose and D-xylose by Candida shehatae in a chemostat

The kinetics of biomass formation, D-xylose utilization, and mixed substrate utilization were determined in a chemostat using the yeast Candida shehatae. The maximum growth rate of C. shehatae grown aerobically on D-xylose was 0.42 h⁻¹ and the Monod constant, K _s, was 0.06 g L⁻¹. The biomass yield, Y _{X/S}, ranged from 0.40 to 0.50 g g⁻¹ over a dilution rate range of 0.2–0.3 h⁻¹, when C. shehatae was grown on pure D-xylose. Mixtures of D-xylose and glucose (∼1 : 1) were simultaneously utilized over a dilution rate from 0.15 to 0.35 h⁻¹ at pH 3.5 and 4.5, but pH 3.5 reduced μ_max and reduced the dilution rate range over which D-xylose was utilized in the presence of glucose. At pH 4.5, μ_max was not reduced with the mixed sugar feed and the overall or lumped K _s value was not significantly increased (0.058 g L⁻¹ vs 0.06 g L⁻¹), when compared to a pure D-xylose feed. Kinetic data indicate that C. shehatae is an excellent candidate for chemostat production of value added products from renewable carbon sources, since simultaneous mixed substrate utilization was observed over a wide range of growth rates on a 1 : 1 mixture of glucose and D-xylose. Received 21 August 1997/ Accepted in revised form 28 May 1998     

High-performance liquid chromatographic determination of GS4071, a potent inhibitor of influenza neuraminidase, in plasma by precolumn fluorescence derivatization with naphthalenedialdehyde

GS4071 is a potent inhibitor of influenza neuraminidase. A precolumn fluorescence derivatization HPLC method is described for the analysis of GS4071 in rat plasma. Plasma samples were subjected to solid-phase extraction on C₁₈ extraction columns. After extraction, GS4071 was derivatized with naphthalenedialdehyde in the presence of potassium cyanide to produce highly fluorescent cyano[f]benzoisoindole derivatives. Derivatized samples were stable for >24 h at 4°C. The samples were analyzed by an isocratic HPLC method using fluorescence detection at 420 nm excitation and 470 nm emission wavelength. The method was validated and applied to the analysis of plasma samples from pre-clinical pharmacokinetic studies in rats. The limit of detection for GS4071 was 20 ng/ml. For five replicate samples at 50, 400, and 1000 ng/ml, the within-day precision values were 16.9, 9.4 and 4.5%, respectively, and the between-day precision values were 16.9, 7.9, and 2.1%, respectively. The method was linear from 25 to 1600 ng/ml and the total recovery was >68% over this concentration range.     

Brain stem lesion size determined by DEAD red or conjugation of neurotoxin to fluorescent beads

Neurotoxinmicroinjected into the retrotrapezoid nucleus of anesthetized ratsdecreases phrenic activity and eliminates the response toCO₂. In unanesthetized rats, suchtreatment has no effect on awake, resting breathing and decreasesCO₂ sensitivity by 40% (M. Akilesh, M. Kamper, A. Li, and E. E. Nattie. J. Appl. Physiol. 82: 469-479, 1997). One important factorin explaining these disparate results is the actual size of theanatomic lesion. In the present study, we injected ibotenic acid intothe retrotrapezoid nucleus of anesthetized rats and evaluated lesionsize by using two new approaches: 1)DEAD red, a fluorescent probe that enters impaired cells through leakymembranes and binds to nucleic acids, and2) conjugation of toxin tofluorescent beads. With the use of DEAD red, the region containinglabeled dying cells was 313 ± 104 nl(n = 4), six times larger than theinitial injected volume, and the physiological effects on phrenicamplitude, the CO₂ response, andblood pressure began within minutes and were substantial. Withconjugated toxin, in theory, neuronal damage would be limited to theregion of detectable fluorescence (49 ± 10 nl;n = 4). Effects on phrenicamplitude, CO₂ sensitivity, andblood pressure were absent until ~2 h postinjection. Controlexperiments, with 2 h of in vitro incubation of theneurotoxin-microbead conjugate and injection of the supernatant aftercentrifugation, showed similar results that suggest release ofconjugated neurotoxin. We conclude that DEAD red provides a usefulmeans to monitor neuronal impairment in acute studies in vivo.Conjugation of neurotoxin to microbeads may be less reliable in this regard.

     

Seasonal changes in glomerular filtration rate in Antechinus stuartii (Marsupialia: Dasyuridae)

The small marsupial Antechinus stuartii experiences a synchronised life cycle that culminates in complete male mortality (within 3 weeks) following the 1 week mating period in mid-August (late winter). There are pronounced physiological changes in male A. stuartii over the life cycle and renal function was assessed for correlation with these changes. Glomerular filtration rate and urine and plasma electrolytes were determined in male and female A. stuartii in February, May, July and August. Females showed little change in glomerular filtration rate, except for pre-mating values in August which decreased. In contrast, glomerular filtration rate of males decreased significantly in July and August. Plasma sodium and chloride levels were higher in males than females and were higher in animals in July and August than in February and May. Plasma potassium levels dropped in both males and females in July and August. Plasma osmolality was higher in animals in February compared to animals from May and August. However, there were no significant sex or seasonal differences in urine electrolytes, although urea concentration was higher in females than males. Urine osmolality was higher in both sexes in July and August. There were no significant differences in total excretory rates of sodium, potassium or chloride between sexes or between seasons. Many of the alterations in renal function are correlated with known physiological and hormonal profiles in A. stuartii. This is the first observation of seasonal changes in glomerular filtration rate that are unrelated to dietary and water stresses. Accepted: 8 September 1997     

Behavioural Responses to Scent Marks of Increasing Age in Klipspringer Oreotragus oreotragus

Klipspringers Oreotragus oreotragus are small antelopes which use scent marks to demarcate and defend their territory. The strength of behavioural response to the introduction of unfamiliar scent was measured in order to estimate the effective lifespan of scent secretion. The total number of scent marks deposited during the experiment was higher than control levels when fresh scent was introduced, but not when older scent was introduced. However, the proportion of marks deposited on introduced twigs remained higher than control levels until scent was aged at least 7d. The probability of licking twigs immediately before scent-marking was much higher than control levels when scent aged up to 3d was introduced, but not when scent was aged 7d or more. These results indicate that preorbital gland scent retains at least some degree of efficacy for up to 7d. The decreasing level of response to scent aged 0-7d is most likely to be due to decay of the scent signal. However, in contrast to the total number of marks deposited and the probability of licking before scent-marking, which both consistently decreased in response to older scent, the proportion of marks deposited on scent aged 3d was higher than over fresh scent. It is suggested that, in view of the role that scent-marking plays in territory defence, this difference in the timing of peak response between the three variables may represent an adaptive response to the perceived threat of encounter with an adversary, which is mediated by the age of the scent marks.     

Testing extraction and storage parameters for a fecal hormone method

Four experiments were conducted to test different aspects of a “field‐friendly” fecal hormone extraction method that utilizes methanol extraction in the field followed by storage on C18 solid‐phase extraction cartridges. Fecal samples were collected from geladas (Theropithecus gelada) housed at the Bronx Zoo, and the experiments were conducted in a laboratory setting to ensure maximum control. The experiments were designed to either simulate the conditions to which fecal samples are subjected during fieldwork or improve on an existing protocol. The experiments tested the relationship between fecal hormone metabolite preservation/recovery and: (1) the amount of time a sample is stored at ambient temperature; (2) the number of freeze/thaw cycles a sample undergoes; (3) the effectiveness of different extraction solutions; and (4) the effectiveness of different cartridge washes. For each experiment, samples were assayed by radioimmunoassay for fecal glucocorticoid (GC) and testosterone (T) metabolites. Results for each of the experiments were as follows. First, storage at ambient temperature did not affect hormone levels until 4 weeks of storage, with significant increases for both GC and T metabolites at 4 weeks. Second, hormone levels significantly decreased in samples after two freeze/thaw cycles for GCs and six freeze/thaws cycles for T. Third, for both GCs and T, hormone extraction using various methanol solutions was significantly higher than using 100% ethanol. Finally, using a 20% methanol solution to wash cartridges significantly increased GC levels but had no effect on T levels. These results suggest that, when utilizing C18 cartridges for fecal steroid storage, researchers should consider several methodological options to optimize hormone preservation and recovery from fecal samples. Am. J. Primatol. 72:934–941, 2010. © 2010 Wiley‐Liss, Inc.