Effect of alanyl-glutamine on leucine and protein metabolism in irradiated rats

Summary. The mechanism by which glutamine produces a favorable effect in the treatment of sepsis, injury, burns and abdominal irradiation is not completely understood. The main aim of this study was to evaluate the effect of alanyl-glutamine (AlaGln) administration on the metabolism of proteins in irradiated rats. The rats were exposed to whole-body irradiation (8 Gy) and then fed intragastrically with a mixture of glucose and amino acids either with AlaGln or without AlaGln. At 48 hours after irradiation, parameters of whole-body protein metabolism and DNA synthesis in intestinal mucosa were investigated using a primed, continuous infusion of [1-¹⁴C]leucine and [³H]thymidine. In addition, we evaluated the effect of irradiation and AlaGln on gut morphology, blood count and amino acid concentrations in blood plasma and skeletal muscle. Control rats were not irradiated but were given identical treatment. An increase in whole-body leucine oxidation, and insignificant changes in whole-body proteolysis and in protein synthesis were observed after irradiation. In irradiated rats we observed a decrease in muscle glutamine concentration, a decrease in protein synthesis in jejunum, colon and heart, and an increase in synthesis of proteins of blood plasma and spleen. Morphological examination and measurement of DNA synthesis failed to demonstrate any favorable effect of AlaGln supplementation on irradiated gut. However, administration of AlaGln resulted in a decrease in whole-body proteolysis and leucine oxidation which caused an increase in the fraction of leucine incorporated into the pool of body proteins. We conclude that the data obtained demonstrate that irradiation induces metabolic derangement associated with increased oxidation of essential branched-chain amino acids (valine, leucine and isoleucine) and that these disturbances can be ameliorated by administration of AlaGln. Received February 14, 2000 Accepted July 12, 2000     

Osteometric dimensions of metacarpal bones with respect to the professional exposure to vibration

The comparative analysis of the osteometric dimensions of metacarpal bones in three groups of males (aged from 23 to 63 years) differing with respect to the presence of the professionally connected long-term exposure to the vibration is performed. The sample encompasses forest workers employed in wood industry (exposed to heavy physical work and to the daily use of hand-held vibrating tools) from two regions of Croatia: Podravina (n = 192) and Gorski Kotar (n = 115). The control group is formed using the random sample (selected according to age criterion) of phenotypically healthy male inhabitants of rural communities of Eastern Adriatic islands and peninsula (n = 200). Additionally, the influence of the level of calcium in diet, as a regional nutritional characteristic of particular regions of Croatia, is also considered. The regression of the percent cortical area (PCA) of the second left metacarpal bone and age showed that significant decrease of PCA in older age can be observed only in males from Eastern Adriatic (the control group), while that is not so in either group of males professionally exposed to vibration. Authors conclude that the analysis of the osteometric dimensions performed on males professionally daily exposed to vibration missed to provide evidence to support the hypothesis of long-term exposure to vibration as a risk for accelerated osteoporosis of metacarpal bones.     

Peptidomic analysis of skin secretions from the bullfrog Lithobates catesbeianus (Ranidae) identifies multiple peptides with potent insulin-releasing activity

Using a combination of reversed-phase HPLC and electrospray mass spectrometry, peptidomic analysis of norepinephrine-stimulated skin secretions of the American bullfrog Lithobates catesbeianus Shaw, 1802 led to the identification and characterization of five newly described peptides (ranatuerin-1CBb, ranatuerin-2CBc, and -CBd, palustrin-2CBa, and temporin-CBf) together with seven peptides previously isolated on the basis of their antimicrobial activity (ranatuerin-1CBa, ranatuerin-2CBa, brevinin-1CBa, and -1CBb, temporin-CBa, -CBb, and -CBd). The abilities of the most abundant of the purified peptides to stimulate the release of insulin from the rat BRIN-BD11 clonal β cell line were evaluated. Ranatuerin-2CBd (GFLDIIKNLGKTFAGHMLDKIRCTIGTCPPSP) was the most potent peptide producing a significant stimulation of insulin release (119% of basal rate, P < 0.01) from BRIN-BD11 cells at a concentration of 30 nM, with a maximum response (236% of basal rate, P < 0.001) at a concentration of 3 μM. Ranatuerin-2CBd did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3 μM, indicating that the integrity of the plasma membrane had been preserved. Brevinin-1CBb (FLPFIARLAAKVFPSIICSVTKKC) produced the maximum stimulation of insulin release (285% of basal rate, P < 0.001 at 3 μM) but the peptide was cytotoxic at this concentration.     

Environmental correlates of the Late Quaternary regional extinctions of large and small Palaearctic mammals

Most studies of mammal extinctions during the Pleistocene–Holocene transition explore the relative effects of climate change vs human impacts on these extinctions, but the relative importance of the different environmental factors involved remains poorly understood. Moreover, these studies are strongly biased towards megafauna, which may have been more influenced by human hunting than species of small body size. We examined the potential environmental causes of Pleistocene–Holocene mammal extinctions by linking regional environmental characteristics with the regional extinction rates of large and small mammals in 14 Palaearctic regions. We found that regional extinction rates were larger for megafauna, but extinction patterns across regions were similar for both size groups, emphasizing the importance of environmental change as an extinction factor as opposed to hunting. Still, the bias towards megafauna extinctions was larger in southern Europe and smaller in central Eurasia. The loss of suitable habitats, low macroclimatic heterogeneity within regions and an increase in precipitation were identified as the strongest predictors of regional extinction rates. Suitable habitats for many species of the Last Glacial fauna were grassland and desert, but not tundra or forest. The low‐extinction regions identified in central Eurasia are characterized by the continuous presence of grasslands and deserts until the present. In contrast, forest expansion associated with an increase in precipitation and temperature was likely the main factor causing habitat loss in the high‐extinction regions. The shift of grassland into tundra also contributed to the loss of suitable habitats in northern Eurasia. Habitat loss was more strongly related to the extinctions of megafauna than of small mammals. Ungulate species with low tolerance to deep snow were more likely to go regionally extinct. Thus, the increase in precipitation at the Pleistocene–Holocene transition may have also directly contributed to the extinctions by creating deep snow cover which decreases forage availability in winter.     

Experimental evidence for parasite‐induced over‐winter mortality in juvenile Rhodeus amarus

In this study, the effects of the eye fluke Diplostomum pseudospathaceum (Trematoda) infection on over‐winter survival of young‐of‐the‐year (YOY) European bitterling Rhodeus amarus (Cyprinidae) were examined between September 2010 and April 2011. The fish were reared in semi‐natural conditions to ensure that results were not confounded by other parasite infections. The cumulative mortality of R. amarus from November until April was significantly higher in D. pseudospathaceum‐infected fish (57·3%) compared to controls (42·1%). Infection of the parental generation did not have any effect on the mortality of juveniles. The results indicate that D. pseudospathaceum infection increases over‐winter mortality of YOY R. amarus. The possible mechanisms causing mortality are discussed.     

Activation of protein kinase C suppresses fragmentation of pig oocytes aged in vitro

When cultured for an extended time, pig oocytes that matured in vitro to the stage of metaphase II undergo the complex process designated as ageing. Under our conditions, some pig oocytes aged 3 days remained at the stage of metaphase II (22%), but others underwent spontaneous parthenogenetic activation (45%), and still others perished through fragmentation (28%) or lysis (5%). Activation of protein kinases C (PKCs) using phorbol-12-myristate-13-acetate (PMA) protects oocytes from fragmentation. None of the oocytes were fragmented after 3 days of aging in 50 nM of PMA. A similar effect (8% of fragmented oocytes) was observed after a 3-day treatment of aging oocytes with 100 μM of 1-stearoyl-2arachidonoyl-sn-glycerol (STEAR). PMA and STEAR activate both calcium-dependent and calcium-independent PKCs. This combined effect on PKCs seems to be essential for the protection of oocytes from fragmentation. Neither the specific activator of calcium-dependent PKCs 1-oleoyl-2-acetyl-sn-glycerol (OLE) nor the specific activator of calcium-independent PKCs dipalmitoyl-l-α-phosphatidylinositol-3,4,5-triphosphate heptaammonium salt (DIPALM) suppressed the fragmentation of aging pig oocytes. Twenty-one percentage of oocytes fragmented when aged for 3 days in 10 μM OLE and 26% of aged oocytes fragmented in 100 nM of DIPALM. However, fragmentation was significantly suppressed to 7% when the oocytes were exposed to the combination of both 10 μM OLE and 100 nM DIPALM. Aging pig oocytes cultured for 1 day with PMA maintained a high capability of being parthenogenetically activated (86% of activated oocytes), using calcium ionophore with 6-dimethylaminopurine. Ageing oocytes treated with PMA also had high capability of cleavage (82%) after their artificial parthenogenetic activation. However, their ability to develop to the stage of blastocyst (12%) was suppressed when compared with oocytes activated immediately after their maturation (29%).     

In vitro reactivation of sarin-inhibited brain acetylcholinesterase from different species by various oximes

In vitro as well as in vivo evaluation of the reactivating efficacy of various oximes against nerve agent-inhibited acetylcholinesterase has been usually done with the help of animal experiments. Nevertheless, previously published data indicate that the reactivation potency of oximes may be different in human and animal species, which may hamper the extrapolation of animal data to human data. Therefore, to better evaluate the efficacy of various oximes (pralidoxime, obidoxime, HI-6, K033) to reactivate brain acetylcholinesterase inhibited by sarin by in vitro methods, human, rat and pig brain acetylcholinesterase were used to calculate kinetic parameters for the reactivation. Our results show differences among the species, depending on the type of oxime, and indicate that data from animal experiments needs to be carefully evaluated before extrapolation to humans.     

Comparative research on substance abuse and self-perception among adolescents with physical handicap

The research on substance (alcohol, tobacco and drug) abuse and on self-perception was done by comparing a test group of physically disabled adolescents and a test group of non-disabled adolescents. The respondents of the experimental group were students of the only special high school for physically handicapped persons in Croatia, Zagreb. The respondents of the control group were the students of two regular high schools in the capital of Croatia. The instrument used in this research was a self-reported, anonymous questionnaire. The respondents completed the questionnaire in the classroom. The data analysis regarding alcohol abuse indicated that physically disabled adolescents drink more often and out of quite different motives than their non-disabled peers. Regarding the prevalence, frequency, quantity and motives for smoking, no statistically significant difference has been found between the tested groups. On the contrary, significant differences between handicapped and non-disabled adolescents were evident regarding drug abuse. Only one physically disabled examinee used a drug--marijuana, only a few times a year. On the other hand, almost one quarter of the non-disabled adolescents use at least one, five at the most, type of drug sometimes or often. The results on the self-perception scale show that adolescent with physical disabilities have a much more negative attitude toward themselves than non-disabled controls. Their self-esteem and self-confidence are seriously diminished. Described findings could have a mighty impact on ways of preventing substance abuse, and on ways of increasing self-esteem among disabled and non-disabled adolescents.     

A Simple and Efficient DNA Isolation Method for Salvia officinalis

We report an efficient, simple, and cost-effective protocol for the isolation of genomic DNA from an aromatic medicinal plant, common sage (Salvia officinalis L.). Our modification of the standard CTAB protocol includes two polyphenol adsorbents (PVP 10 and activated charcoal), high NaCl concentrations (4?M) for removing polysaccharides, and repeated Sevag treatment to remove proteins and other carbohydrate contaminants. The mean DNA yield obtained with our Protocol 2 was 330.6?μg DNA g^?1 of dry leaf tissue, and the absorbance ratios 260/280 and 260/230?nm averaged 1.909 and 1.894, respectively, revealing lack of contamination. PCR amplifications of one nuclear (26S rDNA) and one chloroplast (rps16-trnK) locus indicated that our DNA isolation protocol may be used in common sage and other aromatic and medicinal plants containing essential oil for molecular biologic and biotechnological studies and for population genetics, phylogeographic, and conservation surveys in which nuclear or chloroplast genomes would be studied in large numbers of individuals.