ULTRASTRUCTURE OF THE SECONDARY PHLOEM OF TILIA AMERICANA

Summer and winter (July and January) samples of secondary phloem of Tilia americana were studied with the electron microscope. Parenchyma cells contain: nuclei, endoplasmic reticulum, ribosomes, plastids, mitochondria and occasional dictyosomes. Well-defined tonoplasts separate vacuoles from cytoplasmic ground substance. Vacuoles often contain tannins. Lipid droplets are common in cytoplasm. Endoplasmic reticulum–connected plasmodesmata are aggregated in primary pit fields. Companion cells differ from parenchyma cells in having numerous sieve-element connections, possibly slime, and in lacking plastids. Mature, enucleate sieve elements possess 1–4 extruded nucleoli. Numerous vesicles occupy a mostly parietal position in association with plasmalemma. The mature sieve element lacks endoplasmic reticulum, organelles (except for few mitochondria) and tonoplast. In OsO_4– and glutaraldehyde-fixed elements, slime has a fine, fibrillar appearance. Normally, these fine fibrils are organized into coarser ones which form strands that traverse the cell and the plasmalemma-lined pores of sieve plates and lateral sieve areas.     

Hepatoprotective effects of melatonin against pronecrotic cellular events in streptozotocin-induced diabetic rats

Oxidative stress-mediated damage to liver tissue underlies the pathological alterations in liver morphology and function that are observed in diabetes. We examined the effects of the antioxidant action of melatonin against necrosis-inducing DNA damage in hepatocytes of streptozotocin (STZ)-induced diabetic rats. Daily administration of melatonin (0.2 mg/kg) was initiated 3 days before diabetes induction and maintained for 4 weeks. Melatonin-treated diabetic rats exhibited improved markers of liver injury (P?<?0.05), alkaline phosphatase, and alanine and aspartate aminotransferases. Melatonin prevented the diabetes-related morphological deterioration of hepatocytes, DNA damage (P?<?0.05), and hepatocellular necrosis. The improvement was due to containment of the pronecrotic oxygen radical load, observed as inhibition (P?<?0.05) of the diabetes-induced rise in lipid peroxidation and hydrogen peroxide increase in the liver. This was accompanied by improved necrotic markers of cellular damage: a significant reduction in cleavage of the DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP-1) into necrotic 55- and 62-kDa fragments, and inhibition of nucleus-to-cytoplasm translocation and accumulation in the serum of the high-mobility group box 1 (HMGB1) protein. We conclude that melatonin is hepatoprotective in diabetes. It reduces extensive DNA damage and resulting necrotic processes. Melatonin application could thus present a viable therapeutic option in the management of diabetes-induced liver injury.     

Microsatellite inferred genetic diversity and structure of Western Balkan grapevines (Vitis vinifera L.)

A collection of 196 grapevine samples from five countries of the Western Balkan region, representing local and traditional cultivars, was genotyped with 22 SSR markers. Identity analysis revealed 125 unique genotypes, which were further used for diversity assessment. The average number of alleles per locus detected was 11?±?3.53, ranging from 6 to 21. The low cumulative probability of identical genotypes (2.96?×?10^?20) shown in this study implies an even distribution of alleles within the analyzed set of grapevines and a sufficient number of loci. On the basis of the discriminatory power of each SSR, a set of five markers (VVMD5, VVMD7, VVMD28, VChr3a, and VChr8b) was determined as sufficient for high-throughput discrimination of the target cultivars. The maximum discriminating power was evidenced for loci VVMD28 and Vchr8b (0.96, 0.94, respectively). A core collection covering the entire genetic diversity resulted in a set of 60 genotypes representing approximately 50 % of the samples from each country. Structure clustering of Balkan and West European cultivars resulted in four well distinct groups identified according to the classification of Negrul (1946). The lowest level of admixed genotypes was assigned for grapevines from Bosnia and Herzegovina (61 %) and the highest for Serbian (87 %) grapevines. In terms of grape use, the wine cultivars were divided into three groups and the fourth group was intermixed, with half wine and half table grapes. The highest Nei’s genetic distance (0.22) was discovered between Slovenian and Macedonian cultivars, while the lowest (0.09) was between Slovenian and Serbian cultivars. Macedonian cultivars were genetically most distant from the others (0.17). A similar pattern of differentiation among populations is seen with distance-based clustering. Analysis of molecular variance revealed only 1 % of genetic variation among groups of different origin, while the variation among individuals within geographical groups and within individuals explained 13 and 86 % of the total variation, respectively.     

Biologically active constituents of a polyphenol extract from Geranium sanguineum L. with anti-influenza activity

From the aerial roots of the medicinal plant Geranium sanguineum L. a polyphenol-rich extract with strong anti-influenza activity has been isolated. To investigate its active fractions, the extract was partitioned by solvents with increasing polarity. The n-BuOH fraction contained the majority of the in vitro antiviral activity; the EtOAc fraction was the most effective one in vivo. A bioassay-directed fractionation of the n-BuOH and EtOAc fractions was performed to obtain information about the nature of the chemical components of the plant extract, responsible for the antiviral effect. The individual constituents were identified by spectroscopic methods and comparison with authentic samples and by HPLC. The cell-toxic and virus-inhibitory effects of the fractions and some individual polyphenol compounds, found in Geranium sanguineum L., were studied using the replication of representative influenza viruses in cell cultures. This study showed that the presence of a variety of biologically active compounds as well as the possible synergistic interactions between them seem to be decisive for the overall antiviral effect.     

Klebsiella Phage vB_KleM-RaK2 — A Giant Singleton Virus of the Family Myoviridae

At 346 kbp in size, the genome of a jumbo bacteriophage vB_KleM-RaK2 (RaK2) is the largest Klebsiella infecting myovirus genome sequenced to date. In total, 272 out of 534 RaK2 ORFs lack detectable database homologues. Based on the similarity to biologically defined proteins and/or MS/MS analysis, 117 of RaK2 ORFs were given a functional annotation, including 28 RaK2 ORFs coding for structural proteins that have no reliable homologues to annotated structural proteins in other organisms. The electron micrographs revealed elaborate spike-like structures on the tail fibers of Rak2, suggesting that this phage is an atypical myovirus. While head and tail proteins of RaK2 are mostly myoviridae-related, the bioinformatics analysis indicate that tail fibers/spikes of this phage are formed from podovirus-like peptides predominantly. Overall, these results provide evidence that bacteriophage RaK2 differs profoundly from previously studied viruses of the Myoviridae family.     

Airborne pollen of Betula,Corylus and Alnus in Zagreb,Croatia. A three‐year record

The aim of this study was to construct a picture of the influence of meteorological conditions on the start and duration of the airborne Betulaceae pollen season and the pollen concentrations in the atmosphere of Zagreb, Croatia. The study during three seasons (2002–2004) used a 7‐day Hirst‐type volumetric pollen and spore trap. Total annual airborne pollen of Alnus, Corylus and Betula greatly varied from year to year. The differences in the dates of onset of airborne pollen presence of Alnus, Corylus and Betula noted in Zagreb in 2002–2004 were controlled by weather conditions, particularly temperature and precipitation. In all years studied, airborne pollen peaks were recorded on days with temperature above 0°C and without or minimal precipitation. The mean number of days with airborne pollen concentrations exceeding levels which provoke symptoms of an allergic reaction was 15, 16 and 29 days for alder, hazel and birch, respectively. The results of the present study may provide useful data for allergologists to reach accurate diagnoses, and timely information on concentrations of airborne pollen types and concentrations for individuals with pollen hypersensitivity.     

Effect of Liposomal Formulations and Immunostimulating Peptidoglycan Monomer (PGM) on the Immune Reaction to Ovalbumin in Mice

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-l-Ala-d-isoGln-mesoDpm(εNH₂)-d-Ala-d-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator. The aim of this study was to investigate the influence of different liposomal formulations of OVA, with or without PGM, on the production of total IgG, as well as of IgG1 and IgG2a subclasses of OVA-specific antibodies (as indicators of Th2 and Th1 type of immune response, respectively). CBA mice were immunized s.c. with OVA mixed with liposomes, OVA with PGM mixed with liposomes, OVA encapsulated into liposomes and OVA with PGM encapsulated into liposomes. Control groups were OVA in saline, OVA with PGM in saline, and OVA in CFA/IFA adjuvant formulation. The entrapment efficacy of OVA was monitored by HPLC method. The adjuvant activity of the mixture of OVA and empty liposomes, the mixture of OVA, PGM, and liposomes and PGM encapsulated with OVA into liposomes on production of total anti-OVA IgG was demonstrated. The mixture of PGM and liposomes exhibited additive immunostimulating effect on the production of antigen-specific IgGs. The analysis of IgG subclasses revealed that encapsulation of OVA into liposomes favors the stimulation of IgG2a antibodies, indicating the switch toward the Th1 type of immune response. When encapsulated into liposomes or mixed with liposomes, PGM induced a switch from Th1 to Th2 type of immune response. It could be concluded that appropriate formulations of antigen, PGM, and liposomes differently affect the humoral immune response and direct the switch in the type of immune response (Th1/Th2).     

Age-Dependent Effects of ELF-MF on Oxidative Stress in the Brain of Mongolian Gerbils

The aim of study was to investigate the effects of extremely low frequency magnetic field (ELF-MF; 50 Hz; 0.1, 0.25 and 0.5 mT) on oxidative stress in the brain of 3- (adult) and 10-month-old (middle-aged) gerbils. Nitric oxide (NO) level, superoxide (O₂ ^?) production, superoxide dismutase (SOD) activity, and index of lipid peroxidation (ILP) were measured in the forebrain cortex, striatum, hippocampus, and cerebellum immediately and 3 days after cessation of 7-day exposure. In all gerbils, ELF-MF significantly increased oxidative stress in all tested brain regions. This effect was correlated with the value of magnetic induction and was higher in middle-aged gerbils. Three days after cessation of exposure, the values of examined parameters were closer to control levels. In adult gerbils, the effect of ELF-MF of 0.1 mT on NO level, O₂ ^? production and SOD activity was almost fully disappeared, and ILP was at the control level regardless of the value of magnetic induction. In middle-aged gerbils, the effect of ELF-MF was still present but to a lesser degree than those observed immediately after cessation of exposure. These findings pointed out the ability of ELF-MF to induce age- and magnetic induction-dependent modification of oxidative stress in the brain.