Influence of probiotic and yeast culture supplementation on selected biochemical and immunological parameters of growing lambs.

This study was carried out to evaluate the potential effects of 90 days-long dietary supple- mentation of probiotic and yeast culture on immunity condition of lambs. Fifteen Rahmani growing male lambs (about 5 months old and 23.21±2.75 kg body weight) were randomly allo- cated to three equal groups consisting of 5 animals each. The animals in the first group, served as a control (group C), were fed a basal diet without any supplementation. The lambs in the second and third group were fed the basal diet supplemented with probiotic (group Y) or yeast culture (group YC), respectively. The probiotic consisted of live yeast (Saccharomyces cerevisae) alone, while the yeast culture was composed of Saccharomyces cerevisiae and the media on which it was grown. In group Y and YC, each lamb was supplemented daily with 0.5 g and 7.0 g of live yeast and yeast culture, respectively. Blood samples were collected before feeding the supplements and then every 15 days until the day 90th. Total and differential leucocytic counts, total protein, albumin, IgA, IgG and IgM levels were measured in blood. There were insignificant (p>0.05) variations in the levels of total and differential leucocytic counts and total protein among the groups throughout the experiment. However, significant differences (p⟨0.05) were found in globulin, IgA, IgG and IgM in both (Y) and (YC) groups, but the effect of yeast culture seems to be better than that of the probiotic. In conclusions, the obtained results indicate that the tested probiotic and yeast culture improve the immunological status of lambs.

Efficacy of ß-hydroxy-ß-methylbutyric acid (HMB) for growing rate and its influence for health indicators in blood test of young early-weaning goats.

The study was conducted on 26 male, 30 days-old goats, separated from their mothers, divided into two equal groups: I - control and II - experimental, consisting of 13 animals each. All animals were fed with milk replacer, experimental group received additionally 50 g/kg body weight, additive of HMB, for 60 days. The following features were analyzed: body weight, daily increases of body weight, as well as hematological and biochemical blood features. Differences in body weight were found, between experimental and control group, after 60 days of experiment 0.57 kg (p≤0.01). The daily weight gain of experimental animals was higher in comparison with control group. Significant differences were also noted in results of hematological and biochemical blood parameters. Experimental animals showed a higher level of red blood cells as well as number of lymphocytes in comparison with the control group, (p≤0.01).Significant changes were also observed in the level of triglycerides, inorganic phosphorus and protein between both groups. The acid-base balance parameters and ionogram, showed a higher pH level (p≤0.05) HCO - act., HCO - std., BE, ctCO , O sat, K+, Cl- (p≤0.01), while the anion gap (AG) and Na+ were significantly lower in control group (p≤0.01).

A novel in vitro assay for assessing efficacy and toxicity of antifungals using human leukaemic cells infected with Candida albicans.

AIMS: This study describes a novel in vitro assay that simultaneously determines antifungal efficiency and host cell toxicity using suspensions of human leukaemic cells (HL-60) infected with Candida albicans. METHODS AND RESULTS: The effect of Candida infection on host cell viability was evaluated by the microscopy of trypan blue-stained cells and lactate dehydrogenase (LDH) activity. The in vitro 'drug potency assay' utilized the Cell Counting Kit-8 and measured post-antifungal treatment viability of Candida-infected HL-60 cells and the ability of the antifungal treatment to prevent infection. LDH activity showed that 42% ± 4·0 and 85·3% ± 7·40 of HL-60 cells were killed following Candida infection at the multiplicity of infection (MOI) of 1 : 1 and 1 : 5, respectively. The antifungal nystatin (0·78-25 µmol l(-1) ) was found to inhibit C. albicans infection as seen by the significantly increased viability of HL-60 cells. Cytotoxicity of nystatin towards infected HL-60 cells was evident at higher concentrations and this was also confirmed by propidium iodide staining. CONCLUSIONS: An assay using undisturbed cell suspension conditions was successfully developed for assessing the selectivity of the antifungal therapy in the host-Candida environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay employing Candida infection of host cell suspensions represents a promising method for testing interactions of antifungal compounds with both fungal and host cells.

Chitosan-protein scaffolds loaded with lysostaphin as potential antistaphylococcal wound dressing materials.

AIMS: The development of technology for preparing chitosan-protein scaffolds loaded with lysostaphin, which potentially could be used as dressing for wound treatment and soft tissue infections caused by Staphylococcus aureus. METHODS AND RESULTS: The unique technology of chitosan solubilization using gaseous CO(2) instead of organic or inorganic acids was used for the incorporation of lysostaphin, the enzyme that exhibits bactericidal activity against staphylococci, within the structure of chitosan-protein sponges. The developed chitosan-protein scaffolds loaded with lysostaphin revealed high antistaphylococcal activity, which has been confirmed with a large (n = 143) collection of clinical (skin and wound infections) and animal (bovine mastitis) isolates of these bacteria, including MRSA. No change of bactericidal activity of the lyophilized materials has been observed during half-year storage at 4°C. CONCLUSIONS: The developed materials are potential candidates for preparing biologically active, antistaphylococcal wound dressing materials. SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococci belong to the most popular and most burdensome aetiological factors of wound and soft tissues infections. The developed chitosan-protein scaffolds loaded with lysostaphin could be a possible solution to problems associated with treatment of these infections.

Immunomodulating effect of Inter Yeast S on the non-specific and specific cellular and humoral immunity in lambs.

The objective of this study was to determine the stimulating effect of the Inter Yeast S dietary supplement on selected parameters of specific and non-specific humoral and cellular immunity in lambs. The study involved 32 lambs aged 30 +/- 3 days, divided into two equal groups: II--control, and II--experimental. Experimental group animals were fed a C-J concentrate mixed with a prebiotic, the Inter Yeast S, commercially available, containing dried brewer's yeast Saccharomyces cerevisiae in the amount of 3 g/kg of the concentrate. At the beginning of the experiment (day 0) and on the 15th, 30th and 60th day of the study, blood was sampled from the jugular vein to determine selected parameters of biochemical, specific and non-specific humoral and cellular immunity in lambs (total protein levels, gamma globulin levels, lysozyme activity, ceruloplasmin activity, proliferative response of blood lymphocytes (MTT) after stimulation with LPS or ConA, the metabolic activity (RBA) and potential killing activity (PKA) of phagocytes). As regards humoral immunity parameters, significantly higher gamma globulin levels and higher lysozyme and ceruloplasmin activity were found in blood serum of experimental lambs administered the Inter Yeast S, compared with those determined in control lambs not fed the supplement. No statistically significant differences in serum total protein were found between the control and experimental groups. An analysis of cellular immunity indicators revealed significantly higher levels of RBA and PKA, and higher proliferative response of blood lymphocytes (MTT) after stimulation with LPS and ConA in the experimental group, compared with those observed in the control group.

ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators.

We have used fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-tagged phospholipid analogues, NBD-PE (phosphatidylethanolamine), NBD-PC (phosphatidylcholine) and NBD-PS (phosphatidylserine), to demonstrate that Cdr1p and its other homologues, Cdr2p and Cdr3p, belonging to the ATP-binding cassette (ABC) superfamily behave as general phospholipid translocators. Interestingly, CDR1 and CDR2, whose overexpression leads to azole resistance in C. albicans, elicit in-to-out transbilayer phospholipid movement, while CDR3, which is not involved in drug resistance, carries out-to-in translocation of phospholipids between the two monolayers of plasma membrane. Cdr1p, Cdr2p and Cdr3p could be further distinguished on the basis of their sensitivities to different inhibitors. For example, the in-to-out activity associated with Cdr1p and Cdr2p is energy-dependent and sensitive to sulphydryl blocking agents such as N-ethylmaleimide (NEM) and cytoskeleton disrupting agent cytochalasin E, while Cdr3p-associated out-to-in activity is energy-dependent but insensitive to NEM and cytochalasin E. We found that certain drugs, such as fluconazole, cycloheximide and miconazole, to which Cdr1p confers resistance could also affect in-to-out transbilayer movement of NBD-PE, while the same drugs had no effect on Cdr3p-mediated out-to-in translocation of NBD-PE. The ineffectiveness of these drugs to affect Cdr3p mediated out-to-in phospholipid translocation further confirms the inherent difference in the directionality of phospholipid translocation between these pumps. Notwithstanding the role of some of the Cdrps in drug resistance, this study clearly demonstrates that these ABC transporters of C. albicans are phospholipid translocators and this function could represent one of the physiological functions of such large family of proteins.

Amide and ester derivatives of N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid: the selective inhibitor of glucosamine-6-phosphate synthase.

Several amide and ester derivatives of a glutamine analogue, N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid (FMDP) (1-8), were synthesized and evaluated for the inhibitory activity in regard to glucosamine-6-phosphate synthase from Candida albicans. The syntheses were accomplished by the reaction of N2-tert-butoxycarbonyl-N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid (BocFMDP) with the corresponding amines to give the FMDP amides (1-4) or with alkyl halides to give corresponding esters of FMDP (5-8). Among the synthesized compounds, the acetoxymethyl ester of FMDP was the most active inhibitor of the enzyme. Its IC50 value compared to that of FMDP (4 microM) was equal to 11.5 microM. The methyl and allyl esters and the N-hexyl-N-methyl-amide of FMDP exhibited a moderate enzyme inhibitory activity.

Unusual susceptibility of a multidrug-resistant yeast strain to peptidic antifungals.

The susceptibility of Saccharomyces cerevisiae JG436 multidrug transporter deletion mutant, Deltapdr5, to several antifungal agents was compared to that of JG436-derived JGCDR1 and JGCaMDR1 transformants, harboring the CDR1 and CaMDR1 genes, encoding the main drug-extruding membrane proteins of Candida albicans. The JGCDR1 and JGCaMDR1 yeasts demonstrated markedly diminished susceptibility to the azole antifungals, terbinafine and cycloheximide, while that to amphotericin B was unchanged. Surprisingly, JGCDR1 but not JGCaMDR1 cells showed enhanced susceptibility to peptidic antifungals, rationally designed compounds containing inhibitors of glucosamine-6-phosphate synthase. It was found that these antifungal oligopeptides, as well as model oligopeptides built of proteinogenic amino acids, were not effluxed from JGCDR1 cells. Moreover, they were taken up by these cells at rates two to three times higher than by JG436. The tested oligopeptides were rapidly cleaved to constitutive amino acids by cytoplasmic peptidases. Studies on the mechanism of the observed phenomenon suggested that an additive proton motive force generated by Cdr1p stimulated uptake of oligopeptides into JGCDR1 cells, thus giving rise to the higher antifungal activity of FMDP [N(3)-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid]-peptides.

Mechanism of antifungal action of kanosamine.

The antibiotic kanosamine inhibited growth of Saccharomyces cerevisiae and a range of human pathogenic fungi, including Candida albicans. Kanosamine was transported into C. albicans cells by the glucose transport system and subsequently phosphorylated. The product of its intracellular metabolism, kanosamine-6-phosphate, was an inhibitor of the enzyme glucosamine-6-phosphate synthase. Inhibition was competitive in respect to one of the substrates, D-fructose-6-phosphate, with Ki = 5.9 mM, and was non-competitive in respect to the second substrate, L-glutamine. On the other hand, kanosamine-6-phosphate had no effect on the enzyme catalysing the next metabolic step, namely glucosamine-6-phosphate N-acetylase. The action of kanosamine on C. albicans cells resulted in profound morphological changes, inhibition of septum formation and cell agglutination. Experiments with S. cerevisiae mutants showed that the presence of the Cdr1p drug efflux pump did not affect the antifungal activity of kanosamine.

Purification to homogeneity of Candida albicans glucosamine-6-phosphate synthase overexpressed in Escherichia coli.

The Candida albicans GFA1 gene encoding glucosamine-6-phosphate synthase, an enzyme of cell wall biosynthesis pathway in fungi and bacteria, recently an object of interest as a target for the chemotherapy of systemic mycoses, was PCR amplified and cloned to an Escherichia coli expression vector pET23b. The activity of the enzyme in the lysates from the overproducing E. coli strain was approximately 50-100 times higher than in the lysates from the control E. coli strain. This abundant overproduction allows to purify milligram amounts of the enzyme to homogeneity.

Oligomeric structure and regulation of Candida albicans glucosamine-6-phosphate synthase.

Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase was purified to apparent homogeneity with 52% yield from recombinant yeast YRSC-65 cells efficiently overexpressing the GFA1 gene. The pure enzyme exhibited Km(Gln) = 1.56 mM and Km(Fru-6-P) = 1.41 mM and catalyzed GlcN-6-P formation with kcat = 1150 min-1. The isoelectric point of 4.6 +/- 0.05 was estimated from isoelectric chromatofocusing. Gel filtration, native polyacrylamide gel electrophoresis, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the native enzyme was a homotetramer of 79.5-kDa subunits, with an apparent molecular mass of 330-340 kDa. Results of chemical modification of the enzyme by group-specific reagents established an essential role of a cysteinyl residue at the glutamine-binding site and histidyl, lysyl, arginyl, and tyrosyl moieties at the Fru-6-P-binding site. GlcN-6-P synthase in crude extract was effectively inhibited by UDP-GlcNAc (IC50 = 0.67 mM). Purification of the enzyme markedly decreased the sensitivity to the inhibitor, but this could be restored by addition of another effector, glucose 6-phosphate. Binding of UDP-GlcNAc to the pure enzyme in the presence of Glc-6-P showed strong negative cooperativity, with nH = 0.54, whereas in the absence of this sugar phosphate no cooperative effect was observed. Pure enzyme was a substrate for cAMP-dependent protein kinase, the action of which led to the substantial increase of GlcN-6-P synthase activity, correlated with an extent of protein phosphorylation. The maximal level of activity was observed for the enzyme molecules containing 1. 21 +/- 0.08 mol of phosphate/mol of GlcN-6-P synthase. Monitoring of GlcN-6-P synthase activity and its sensitivity to UDP-GlcNAc during yeast --> mycelia transformation of C. albicans cells, under in situ conditions, revealed a marked increase of the former and a substantial fall of the latter.

Antihistoplasmal in vitro and in vivo effect of Lys-Nva-FMDP.

The new synthetic antifungal agent, L-Lysyl-L-Norvalyl-FMDP, inhibits growth of the yeast form of Histoplasma capsulatum. The compound is transported into the fungal cells by peptide permeases, cleaved intracellularly to constitutive amino acids, and the released C-terminal amino acid inhibits glucosamine-6-phosphate synthase. Promising antihistoplasmal in vivo activity of the FMDP-peptide was observed in an organ load test in mice.

Cloning and sequence analysis of Histoplasma capsulatum glucosamine-6-phosphate synthase gene fragment.

The 3' part of the glucosamine-6-phosphate synthase gene from Histoplasma capsulatum was PCR amplified using degenerate primers designed from the known glucosamine-6-phosphate synthase gene sequences, cloned and sequenced. The computer analysis of the 676 bp sequence revealed the presence of two introns. The identities of the deduced amino acid sequence to the corresponding Saccharomyces cerevisiae and Candida albicans fragment are 65 and 63.8%, respectively.

The expanding clinical spectrum of unilateral acute idiopathic maculopathy.

OBJECTIVE: To report on new features of unilateral acute idiopathic maculopathy (UAIM). PATIENTS: We have evaluated an additional 17 patients with UAIM since 1991. This is a report of new features of the maculopathy noted in seven patients from this new series. RESULTS: New clinical findings in UAIM included eccentric macular lesions, subretinal exudation, papillitis, and bilaterality. The occurrence of UAIM in association with pregnancy and human immunodeficiency virus was also observed. CONCLUSIONS: The description of these newly reported features broadens our understanding of the nature of UAIM. With recognition of the expanded clinical spectrum of this disorder, a more confident approach to diagnosis and management may be achieved.

Isolation and characterization of the GFA1 gene encoding the glutamine:fructose-6-phosphate amidotransferase of Candida albicans.

Glutamine:fructose-6-phosphate amidotransferase (glucosamine-6-phosphate synthase) catalyzes the first step of the hexosamine pathway required for the biosynthesis of cell wall precursors. The Candida albicans GFA1 gene was cloned by complementing a gfa1 mutation of Saccharomyces cerevisiae (previously known as gcn1-1; W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975). GFA1 encodes a predicted protein of 713 amino acids and is homologous to the corresponding gene from S. cerevisiae (72% identity at the nucleotide sequence level) as well as to the genes encoding glucosamine-6-phosphate synthases in bacteria and vertebrates. In cell extracts, the C. albicans enzyme was 4-fold more sensitive than the S. cerevisiae enzyme to UDP-N-acetylglucosamine (an inhibitor of the mammalian enzyme) and 2.5-fold more sensitive to N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (a glutamine analog and specific inhibitor of glucosamine-6-phosphate synthase). Cell extracts from the S. cerevisiae gfa1 strain transformed with the C. albicans GFA1 gene exhibited sensitivities to glucosamine-6-phosphate synthase inhibitors that were similar to those shown by the C. albicans enzyme. Southern hybridization indicated that a single GFA1 locus exists in the C. albicans genome. Quantitative Northern (RNA) analysis showed that the expression of GFA1 in C. albicans is regulated during growth: maximum mRNA levels were detected during early log phase. GFA1 mRNA levels increased following induction of the yeast-to-hyphal-form transition, but this was a response to fresh medium rather than to the morphological change.

[Dislocation of lens particles during cataract surgery]. / Przemieszczenie fragmentów soczewki podczas zabiegu usuwania zacmy.

BACKGROUND: The charts of 46 patients with dislocated lens particles during cataract surgery were reviewed. Forty patients had trans-pars plana vitrectomy; 5 were treated medically and one had retinal detachment repair only. MATERIAL AND METHODS: The following complications were associated with dislocated lens particles: uveitis 87%, glaucoma 66%, corneal edema 71%, retinal detachment 5%. RESULTS: The vision on the initial visit was 20/200 or worse in 85%. After vitrectomy the final vision was 20/40 or better in 58%. Visual acuity of 20/40 or better was obtained in 72% of patients who had surgery the same day; in 55% who had surgery later and in 64% with initial IOL placement. Insertion of an intraocular lens at the time of cataract surgery is not contraindicated. CONCLUSION: The best visual acuity occurred in patients who had vitrectomy the same day as cataract surgery. Small lens particles may be left in the eye. Trans-pars plana vitrectomy is a successful method of treatment of dislocated lens particles during cataract surgery.

The influence of the reproductive cycle on levels of some metabolism indices in ewes.

Investigations were performed in 14 ewes of the Polish Merino race, undergoing the second reproductive cycle. Fundamental indices of the protein and energy metabolism, levels of Ca, P, Mg, Cu, Fe and beta-carotene as well as vitamins A, E and C in the blood plasma and serum were determined. The analyses were carried out during the following periods of the reproductive cycle: 3 weeks before mating, the early and late pregnancy, the culmination and close of lactation. The levels of metabolism indices, especially those of the protein and energy metabolism, were found to vary during the studied periods of the reproductive cycle. In all the periods, low levels (during lactation a deficiency) of Ca and Mg and a distinct deficiency of vitamin C were detected. It seems therefore, necessary to work out the reference values for particular periods of the reproductive cycle.

Amide and ester derivatives of N3-trans-epoxysuccinoyl-L-2,3-diaminopropanoic acid: inhibitors of glucosamine-6-phosphate synthase.

Several analogs 5, 6, 7, 8, 10 and 11 of the C-terminal fragment of a peptide antibiotic Sch 37137 were designed and tested as inhibitors of glucosamine-6-phosphate synthase from Saccharomyces cerevisiae. From IC50 values and kinetic parameters of inhibition of glucosamine-6-phosphate synthase by compounds 5-11 it has been found that the inhibitory potency of these compounds follows the order: 6 > 5 > 8 > 9 > 7, 10, 11. This suggests that an inhibitor with a primary amido group binds better to the active site of the enzyme than other inhibitors. The order of reactivity of compounds 5-11 may be attributed to a steric inability of the inhibitor to fit into the active site of the enzyme and also indicates the importance of the chirality of trans-epoxysuccinic acid on the inhibitory properties of the synthesized compounds.

[About fluorescein angiography one more time]. / O technice angiografii fluoresceinowej raz jeszcze.

The authors remind commonly known values of fluorescein angiography, techniques of its performance and the scheme of preparation of the photographic material for adequate evaluation, description and preservation.