Research Rept.
Biotechnical Fac. University of Ljubljana
Agricultural Issue. Zootechnica
74(December 1999)2
REGULATION OF UBIQUITIN-PROTEASOME-DEPENDENT PROTEIN BREAKDOWN IN
EXPERIMENTAL MODELS IN MUSCLE ATROPHY *
a), Didier
ATTAIX and Jasna M. A. STEKAR a) Present address: RCP Emona, Kavčičeva 72, SI-1000
Ljubljana, Slovenia, M.Sc.
Review paper
Language: Slovene
ABSTRACT There is more and more evidence that ATP-ubiquitin-dependent proteolytic
system has a key role in muscle protein breakdown. In different experimental
models in muscle atrophy we try to define factors involved in regulation of ATP-ubiquitin-dependent
proteolytic pathway. If we use a simplified division, we can talk about
experimental models in which we use regulation by nutrients (fasting, protein
inadequate diet), by disuse of muscle (denervation, weightlessness), by
pathological conditions (sepsis, acidosis, diabetes, burn injury, cachexia), by
hormones (insulin, IGF-1, glucocorticoids) and by cytokines (tumor necrosis
factor-a, interleukin-1, interleukin-6).
a)
and Andrej OREŠNIK a) Univ. of Ljubljana, Biotechnical Fac., Zootechnical Dept., Groblje
3, SI-1230 Domžale, Slovenia, M.Sc.
Original scientific paper
Language: Slovene
ABSTRACT Weende analysis and analysis of minerals and amino acid composition were
done on the defined cereal and maize samples harvested in Slovenia in 1997.
Analyses were done on 5 varieties of wheat: Pinka, Ana, Marija, Reska and
Žitarka; 2 varieties of winter barley Alpha in Robur and 2 varietis of spring
one: Rex and Gotic; 3 varieties of oats: Valiant, Pram and Leanda and 5 hybrids
of maize: Fanion, Dea; Helga, Alberta and Lotus. The results of chemical
analyses showed a relatively small degree of variability among wheat varieties.
The same was found for barley varieties. Valiant is the variety that has less
crude protein and ether extract and more crude fibre than the other varieties of
oats. The maize hybrid Fanion is the one that has more crude protein and ether
extract than the others. Cereals harvested in Slovenia have different average
values for individual nutritive substance as they are cited in literature. The
highert difference is found in lower crude protein content in our samples.
Mineral (P, Ca, Mg, K, Na, Zn and Mn) composition is similar among the varieties
of the cereals or maize and the average values are in the accordance with the
literature, except lower Na concentrations. Amino acid composition shows a low
degree of variability among the varieties of the same cereal or maize. The
concentration of some essential amino acids in protein is superior to the data
cited in literature.
a), Mojca KOMAN RAJŠP and Antonija HOLCMAN
a) Institut “Jožef Stefan”,
Jamova 39, SI-1000 Ljubljana, Slovenia, Ass.Prof., Ph.D.
Original scientific paper
Language: English
Dedicated to Professor Jasna
Stekar, University of Ljubljana, on the occasion of her 70th
birthday.
ABSTRACT The aim of our work was to determine fatty acid content in eggs enriched
with omega-3 polyunsaturated fatty acids from various producers on the market.
Due to simplicity, speed and reduced organic solvent usage, we used the in
situ trans-esterification ISTE method for the determination of the fatty
acid composition in eggs. The method was checked by the analysis of certified
reference material CRM 164 (Anhydrous Milk Fat). The analytical results are in
good accordance with certified values. The obtained results show that enriched
eggs have a better fatty acid composition than average table eggs, regardless
the producer. The ratio of FAs w -6 to w -3 in enriched eggs is less than 5,
being 10 in ordinary eggs. An approximately equal content of alpha linolenic
acid (18:3, n-3) was found in the enriched eggs from Jata Reja and Maia, 1.29 wt
% and 1.25 wt % respect., while it was 11.28 wt % in the Fisher-Weppler’s eggs.
The comparison of obtained results, the fatty acid content per 100 g egg
content, with literature data is quite difficult, because authors calculate the
content of FA in various ways. According to recent data from the literature
(Weihrauch
et al., 1977, Guardiola et al., 1994, Fatty acids, 1998,
Kunachowicz et al., 1998), we have chosen a conversion factor of 0.83 in
order to calculate the total fatty acids content in a 100 g egg content. The
producers should declare the fatty acid composition, i.e. the content of w -3
PUFA-s, EPA and DHA on the package so that consumers are informed about the
nutritional value of the food.
a), Berthold KOLETZKO and Thorsten U. SAUERWALD
a) Univ.
of Ljubljana, Biotechnical Fac., Zootechnical Dept., Groblje 3, SI-1230 Domžale,
Slovenia, Ph.D., M.Sc.
Review paper
Language: English
ABSTRACT Microorganisms have been receiving increased attention as sources of novel
lipids. Those that accumulate more than 20-25 % of their biomass as oil may be
termed oleaginous and their oils single cell oils (SCOs), unicellular oils or
microbial oils. For the lipid accumulation in yeasts, moulds and eukaryotic
algae, but not in bacteria, the presence of enzyme ATP-cytrate lyase is of vital
importance. This enzyme serves to produce acetyl-CoA, which is the substrate for
fatty acid biosynthesis. Nitrogen limitation is the most frequently used
condition to favour lipid accumulation. Oleaginous organisms differ from
nonoleaginous ones in being able to convert carbon from the growth medium into
the intracellular lipid, after the nitrogen has been depleted from the medium,
provided that the supply of carbon stays plentiful. Biosynthetic pathways of n-6
and n-3 polyunsaturated fatty acids from the saturated and monounsaturated
precursors with the chain elongations and desaturations are presented. The
suitability of an microalgal triglyceride-SCO highly enriched in docosahexaenoic
acid (DHASCOŇ) as a source for nutritional supplementation for
formula milk is compared to fish oil. Some safety evaluation studies of SCOs are
presented. For the safe use of SCOs in infant formulas even further safety
studies should be performed. By growing microalgal strains in a medium
containing D-[1-13C]glucose, SCOs enriched with the stable isotope
13C can be produced. Some examples of recent research and diagnostic
applications of 13C-labelled SCOs to study fatty acid metabolism are
outlined. In conclusion, SCOs in combination with stable isotopes have become
indispensable to study metabolic pathways.
Marija RAJČEVIČa),
Jože LEVSTEK, Uroš RAJČEVIČ and Tone ILC a) Poslovni sistem Mercator, d.d., Dunajska 107, SI-1113 Ljubljana,
Slovenia, Ph.D., M.Sc., senior scientist
Original scientific paper
Language: English
ABSTRACT On a farm with 230 Friesian cows, winter and summer nutrition of highly
pregnant dry cows was studied. Dietary cation-anion difference in the rations
(DCAD) was calculated to estimate its possible connection with the incidence of
milk fever and hypocalcaemia in cows. In winter season daily ration was composed
of 3 kg of hay, 10-13 kg of grass silage and 10-13 kg of maize silage, with 1 kg
of concentrate and 50 g of min.-vit. mixture. During summer season cows grazed
and were additionally fed 3 kg of hay and 8 kg of maize silage till mid June,
then until the end of season the maize silage was replaced by 10 of grass
silage. A week before expected date of calving, cows were fed an additional 2 kg
of concentrate. Throughout the dry period the cows were also fed 100 g of
min.-vit. mixture. Dietary cation-anion difference was estimated as
milliequivalent (meq) per kg of DM [(K++Na+) – (Cl-+S--)]
(Oetzel, 1993). In winter months (November-February) dietary cation-anion
difference ranged from 365 to 373 meq kg-1 DM. In studied months of
winter season 83 cows calved, 9 of which were treated for milk fever and 5 for
hypocalcaemia. In summer season (May-September) dietary cation-anion difference
ranged from 27 to 238 meq -1kg DM. In studied months of summer season
73 cows calved, 6 of which were treated for milk fever and 3 for hypocalcaemia.
a), Andrej
LAVRENČIČ and Jože STOPAR
a) Univ. of Ljubljana, Biotechnical Fac., Zootechnical Dept., Groblje
3, SI-1230 Domžale, Slovenia, Prof., Ph.D.
Original scientific paper
Language: Slovene
ABSTRACT In 1997 on 7 different plots of the same ground in four periods and in
different stages of growth in each period 10 samples of red clover (Trifolium
pratense) and Italian rygrass (Lolium multiflorum) and 8 samples of
timothy (Phleum pratense), perennial rygrass (Lollium perenne),
meadow fescue (Festuca pratensis), red fescue (Festuca rubra) and
orchardgrass (Dactylis glomerata) were collected. Differences in manganese
content have been found among different species grown and cut at the same site.
The concentration of manganese in dry matter was the highest in orchardgrass
samples (215.3 ± 54.9 mg kg-1 DM) and lower in red fescue (55.7 ± 7.8
mg kg-1 DM). Other grasses and red clover contained significantly
less manganese (33.1 mg to 42.0 mg kg-1 DM). In grasses with low
manganese contents and in red clover samples from the first cut the manganese
concentrations were significantly higher in the vegetative shoot (25.2 mg to
61.6 mg kg-1 DM) than in the reproductive shoot (16.6 mg to 47.8 mg
kg-1 DM). In the samples of orchardgrass the manganese concentration
in the first cut raised with the stage of grass maturity (from 145.5 mg to 179.5
mg kg-1 DM). The lowest average manganese concentration in dry matter
was found in all grass species and in red clover in samples from the first cut
(21.0 mg to 53.0 mg kg-1 DM) and the highest in autumn samples (42.2
mg to 68.7 mg kg-1 DM) with exception of orchardgrass with the lowest
value in the first cut (160.0 mg kg-1 DM) where the highest manganese
concentration was attained in the second cut samples (272.0 mg kg-1
DM) with lower values in summer (209.6 mg kg-1 DM) and in autumn cut
(215.4 mg kg-1
DM).
INTRODUCTION OF THE COMET
ASSAY FOR THE EVALUATION OF OXIDATIVE STRESS, ANTIOXIDANT EFFECTS AND
ENVIRONMENTAL POLLUTANTS EFFECTS ON ANIMAL CELLS *
a) a) Univ. of Ljubljana,
Biotechnical Fac., Zootechnical Dept., Groblje 3, SI-1230 Domžale, Slovenia,
Ph.D., M.Sc.
Original scientific paper
Language: English
ABSTRACT DNA damage within single mammalian cells can be one of the earliest signs of
a whole range of health problems, including disease, diet and exposure to
occupational or environmental toxins. The Comet assay is a relatively simple and
inexpensive method for measuring this damage. It works by determining the number
of breaks in the strands of DNA within the cell. Cells are embedded in agarose
gel on a microscopic slide and washed to remove the cell membranes, soluble cell
contents and histones from the nucleus. An electric field is then activated
across and the loops of damaged DNA are pulled towards the anode.
Epifluorescence microscopy of stained gels shows the image which gives the
techique its name: a clump of undamaged DNA (the head) with the loops pulled
away, forming a tail of a comet. We report herein the checking of the Comet
assay for the evaluation of oxidative stress on chicken, mouse and pig blood
cells, pig sperm cells and chicken hepatocytes. We intend to use the Comet assay
as a quantitative method in some nutritional experiments with domestic animals
including oxidative food components and antioxidants and for the evaluation of
some environmental pollutants effects on animal and human cells.
The work is a
part of CRP project entitled Feeding influences on the quality of food of
animal origin and the nutritional complementary value of the foods of plant
origin (project leader: Karel Salobir, Prof., Ph. D.)
INFLUENCE OF INCREASED
CONCENTRATION OF SinI AND SinR PROTEINS ON THE LEVEL OF THE BACIRACIN
BIOSYNTHESIS AT Bacillus licheniformis *
a)
a) Univ.
of Ljubljana, Biotechnical Fac., Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
Original scientific paper
Language: Slovene
ABSTRACT Bacteria of the genus Bacillus are Gram positive sporulating rods.
They are highly adaptable to different environmental changes concerning
substrate depletion, pH changes and strong temperature oscilations. Production
of peptide antibiotic bacitracin in
B. licheniformis starts in the late logarithmic growth phase and ends
during the stationary phase. Its biosynthesis strongly correlates with an
alkaline protease production. To define possible positively acting factors in
terms of bacitracin production in B. licheniformis we performed an
experiment expressing genes involved in the process of sporulation and
competence in B. subtilis. B. licheniformis was transformed with plasmids
pIS74 (sinIR) and pIS119 (sinR). Recombinant strains BA1 pIS74 and
BA1 pIS119 produced less bacitracin and alkaline protease, because both plasmids
in bacteria B. subtilis and B. licheniformis cause spo-
fenotype.
The paper is a
part of graduation thesis (justification 14. 05. 1999), supervisor Prof.
Franc Viktor Nekrep, Ph.D., co-advisor Aleš Gasparič, Ph.D.
THE INFLUENCE OFBacillus subtilis PROTEIN DegU ON BACITRACIN
BIOSYNTHESIS IN Bacillus licheniformis *
a)
a) Univ. of Ljubljana,
Biotechnical Fac., Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
Original scientific paper
Language: Slovene
ABSTRACT Peptide antibiotics, which are synthesised by many microorganisms, display a
great structural diversity. Among them is bacitracin that is produced by the
Bacillus licheniformis. These peptides are not coded by genes; their
biosynthesis takes place with the aid of peptide synthetases. The biosynthesis
level of bacitracin has a positive correlation with the level of alkaline
proteinase synthesis. Our aim was to raise the level of late enzymes and
consequently also the level of bacitracin biosynthesis. We transformed cells
from the B. licheniformis BA1 using the gene degU of the B.
subtilis, which we believed to be an overall positive regulator in processes
of competence and sporulation. We expected that an increased concentration of
protein DegU would raise the level of bacitracin biosynthesis. We analysed the
clones of the recombinant strains and found them to have proteolytic activity.
In two clones of the bacitracin producer B. licheniformis BA1 we
ascertained a higher level of production of the peptide antibiotic bacitracin.
The paper is a
part of graduation thesis (justification 10. 06. 1999), supervisor:
Ass.Prof. Blagajana Herzog Velikonja, Ph.D., co-advisor Aleš Gasparič,
Ph.D.
a) and
Gorazd AVGUŠTIN a) Univ. of Ljubljana, Biotechnical Fac., Zootechnical Dept., Groblje
3, SI-1230 Domžale, Slovenia
Original scientific paper
Language: English
ABSTRACT Twenty-nine strains belonging to four species of the anaerobic bacterial
genus Prevotella
were tested for deoxyribonuclease activity against the nonmethylated l phage
DNA and circular plasmid pBR322 DNA. The rate of DNA degradation when comparing
different species and even strains within the tested species showed large
variation. The strains of the species P. brevis and P.bryantii
(with exception of the strain TC1-1) all exhibit high activity and completely
degraded l DNA after 30 seconds. No DNase activity could however be detected
with 14 of the 29 tested strains. The survey of the DNase activities confirmed
the validity of the recent reclassification of rumen bacteria from the genus
Prevotella.
A simple method allowing the screening of DNase activities of anaerobic rumen
microorganisms on agar plates is described too.
DEVELOPMENT OF cPCR TECHNIQUE FOR DETECTION AND ENUMERATION OF Prevotella
bryantii
a) and Gorazd AVGUŠTIN a) Univ. of Ljubljana, Biotechnical Fac., Zootechnical Dept., Groblje 3, SI-1230 Domžale, Slovenia, M.Sc.
Original scientific paper
Language: English
ABSTRACT Competitive PCR (cPCR) system for the detection and enumeration of
Prevotella bryantii cells in rumen samples was developed. PBB14
primer, specific for P. bryantii was used as the reverse PCR primer and
EUB 338 bacterial primer as the forward primer. An internal DNA control,
containing primer specific sequences at 5’ and 3’ ends, but lacking 41 bp at the
middle of the sequence, was constructed. C-PCR products were quantified by
capillary electrophoresis and the results were calculated with regression
equation (Reilly and Attwood, 1998). By coamplification of P.bryantii B14
genomic DNA extracted from known numbers of cells and internal control, standard
curve was constructed which enables quantification of P.bryantii cells in
the range of 2.15 x 103 to 4.3 x 104 cells.
