Solid-state cultivation of Grifola frondosa (Dicks: Fr) S.F. Gray biomass and immunostimulatory effects of fungal intra- and extracellular beta-polysaccharides

Grifola frondosa strain GF3, was cultivated on solid-state substrate consisting of milled whole corn plant (Zea mays) and olive press cake supplemented with mineral additives and olive oil. Maintenance of the moisture content in the solid substrate is of crucial importance. Moistures higher than 70% promote growth of G. frondosa mycelium and polysaccharide production. Four fractions of pure extracellular beta-D-glucans with total mass 127.2mg and four fractions of intracellular polysaccharides with total mass 47.2mg were isolated. Polysaccharides were further separated by ion-exchange, gel and affinity chromatography. Isolated polysaccharide fractions from fungal mycelium proved to induce moderate amounts of TNF-alpha in PBMC cells in vitro. The extent of TNF-alpha induction was up to 322pgmL(-1) at a polysaccharide concentration of 200microgmL(-1) for the intracellular fraction. The TNF-alpha inducing activity is comparable to romurtide, which has been used as a supporting therapy in cancer patients treated with radiotherapy and/or chemotherapy.     

Genotype frequencies of UDP-glucuronosyltransferase 1A1 promoter gene polymorphism in the population of healthy Croatian pre-scholars

Increased serum bilirubin levels in patients with Gilbert syndrome (GS) are caused by reduction of hepatic activity of bilirubin glucuronosyltranferase to about 30% of normal. UGT1A1 genetic polymorphism with absent or very low bilirubin UDP-glucuronosyltransferase (B-UGT) activity is associated with Gilbert's syndrome (GS) and other hyperbilirubinemias. The genetic basis of GS is the insertion of two additional TA nucleotides (resulting in seven repeats of TA) in the TATAA box, present in proximal promoter of UGT1A1 gene. This study included 323 Croatian pre-scholars, including 164 boys and 159 girls. Statistical analysis showed significant difference for total bilirubin concentration between different genotypes (p < 0.001). Also, statistically significant difference for total bilirubin concentration was emphasized between genotypes 6/6 and 7/7 (p < 0.001) as well as 6/7 and 7/7 (p < 0.001). Higher total plasma bilirubin concentrations are significantly correlated with 7/7 genotype which is present in 9.8% of population studied.     

Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa (Medicago sativa L.)

Peroxidase (POD) and superoxide dismutase (SOD) enzyme activities were analyzed in non-regenerative transformed embryogenic lines of alfalfa (Medicago sativa L.) carrying wound-inducible oryzacystatin I (OC-I), wound-inducible oryzacystatin I antisense (OC-Ias), or hygromycin phosphotransferase (hpt) genes. All of the transformed lines analyzed had elevated levels of all POD isoforms. Three POD isoforms with pI values of approximately 4.5, 4.8, and 8.4, and one additional pair of isoforms with a pI value of approximately 8.8 were separated from tissue extracts of all transgenic lines. Isoelectrofocusing patterns revealed the induction of one isoform of SOD with a pI of about 5.6 in all transgenic lines compared with non-transformed embryogenic tissue. These results indicate that the process of transformation may disrupt redox homeostasis in alflalfa tissues.     

Importance of NKT cells in resistance to herpes simplex virus, fate of virus-infected neurons, and level of latency in mice

Herpes simplex virus type 1 (HSV-1) produces acute mucocutaneous infections, spread to sensory ganglia, and establishment of latency. In addition, neurovirulent strains have potential to invade the central nervous system (CNS), with potentially a lethal outcome. Early activation of defenses at all stages is essential to limit virus load and reduce the risk of neuronal damage, extensive zosteriform skin lesions, and catastrophic spread to the CNS. NKT cells respond rapidly, and we have shown previously that CD1d-deficient mice are compromised in controlling a neuroinvasive isolate of HSV-1. We now compare infection in Jα18 GKO and CD1d GKO mice, allowing direct assessment of the importance of invariant Vα14⁺ NKT cells and deduction of the role of the CD1d-restricted NKT cells with diverse T-cell receptors. The results indicate that both subsets of NKT cells contribute to virus control both in the afferent phase of infection and in determining the mortality, neuroinvasion, loss of sensory neurons, size of zosteriform, lesions and levels of latency. In particular, both are crucial determinants of clinical outcome, providing protection equivalent to a 1-log dose of virus. These NKT cells can be expected to provide protection at doses of virus that might be encountered naturally.     

Production of phenolic compounds,antioxidant and antimicrobial activities in hairy root and shoot cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L.

Three hairy root clones of Hypericum perforatum (HR 2, HR 15 and HR 27) transformed with Agrobacterium rhizogenes A4M70GUS and their corresponding regenerated shoot culture clones (HRRS) were compared for differences in growth, production of phenolic compounds, antioxidant and antimicrobial activities. Transgenic clones were selected on the basis of morphological evaluation, genetic and molecular analyses. The clone HR 2 had the highest biomass accumulation, while HR 27 showed the highest shoot regeneration potential. The total phenolics and flavan-3-ols were enhanced in all tested transgenic cultures, while total flavonoids and hypericins were augmented in HRRS clones compared to non-transformed shoots. The HRRS clones produced substantial amounts of chlorogenic acid and 3-p-coumaroylquinic acid. Regarding the flavonoids, they produced significant contents of luteolin hexoside (HRRS 2), quercitrin and quercetin (HRRS 15) and isoquercetin (HRRS 27), while HR 2 and 15 accumulated 4-O-methylkaempferol-O-hexoside and quercetin 6-C-glucoside, respectively. The HR 15 was promising for the production of catechin and procyanidin derivatives and together with its HRRS clone exhibited a high potential for hyperforin and adhyperforin production. All identified naphtodianthrones were confirmed in HRRS 2 and 15 clones. Among xanthones, mangiferin was found as the major compound in HRRS, while trihydroxy-1-metoxy-C-prenyl xanthone was dominant in HR clones. Antimicrobial activity of transgenic cultures revealed that HRRS 15 strongly inhibited the growth of Bacillus cereus, Micrococcus flavus, Pseudomonas aeruginosa and Escherichia coli. Altogether, H. perforatum HR and HRRS cultures could be proposed as promising experimental systems for enhanced production of phenolic compounds with antioxidant and antibacterial properties.     

Cytogenetic stability of <Emphasis Type="Italic">Centaurea ragusina</Emphasis> long-term culture

Centaurea ragusina L. (Asteraceae) is an endemic Croatian plant species, which developed xeromorphic characteristics as a consequence of its natural environment – vertical limestone cliffs above the Adriatic sea. Cytogenetic status of C. ragusina long-term culture (94th subculture) and C. ragusina seedlings was analysed and compared after 4 weeks of growth. Cytogenetic stability was investigated in root meristem cells of C. ragusina cultured plants originated from Pen đa (cliffs near Dubrovnik) and seedlings originated from three different localities in south Adriatic (Penđa, Pasjača – cliffs near Dubrovnik and island of Komiža) using mitotic index and mitotic and chromosomal abnormalities as parameters. Mitotic indices of cultured plants and ‘Penđa’ seedlings were similar and showed significant increase compared to mitotic indices of ‘Komiža’ and ‘Pasjača’ seedlings. Although the highest number of mitotic abnormalities was recorded in root meristem cells of cultured plants, it was only a bit higher than in root tips of ‘Pasjača’ and ‘Penđa’ seedlings, while that of ‘Komiža’ was two times lower compared to cultured plants. Pattern of analysed mitotic abnormalities was very similar in root tips of cultured plants and ‘Pasjača’ and ‘Penđa’ seedlings, with exception of ‘Komiža’ seedlings. Presented results suggest that long-term cultivation of C. ragusina has almost no effect on culture ageing considering similar distribution of scored mitotic abnormalities as in ‘Penđa’ and ‘Pasjača’ seedlings.     

Gemcitabine in the first and second-line chemotherapy of advanced non-small cell lung cancer

Aim of this study was to estimate efficacy of gemcitabine in first and the second-line chemotherapy for patients with advanced non-small cell lung cancer (stage III and IV). In first-line chemotherapy, 120 patients were treated with different chemotherapy regimens. Fifty-nine patients were treated with gemcitabine / cisplatin (PG), 41 with cisplatin / etoposide (PE) and 20 with mitomycin / ifosfamide / cisplatin (MIC). Forty patients, unsuccessfully treated with PE and MIC in first-line therapy were treated with PG (24 pts) and with best supportive care (BSC) (16 pts). In first-line therapy PG was superior to PE and MIC protocol (mean survival (MS) 10 vs. 7 vs. 8.5 months). Response rate (RR) for PG in first-line therapy was 46% and 21% in second-line. We showed also significantly better survival in patients treated with PG in second-line chemotherapy comparing to best supportive care (MS 9 vs. 5.5 months). Toxic side effects for combination PG was acceptable. This study confirmed that PG combination is safe and effective as first and second-line chemotherapy for patients with advanced non-small cell lung cancer.     

Inbreeding and the genetic complexity of human hypertension

Considerable uncertainty exists regarding the genetic architecture underlying common late-onset human diseases. In particular, the contribution of deleterious recessive alleles has been predicted to be greater for late-onset than for early-onset traits. We have investigated the contribution of recessive alleles to human hypertension by examining the effects of inbreeding on blood pressure (BP) as a quantitative trait in 2760 adult individuals from 25 villages within Croatian island isolates. We found a strong linear relationship between the inbreeding coefficient (F) and both systolic and diastolic BP, indicating that recessive or partially recessive quantitative trait locus (QTL) alleles account for 10-15% of the total variation in BP in this population. An increase in F of 0.01 corresponded to an increase of approximately 3 mm Hg in systolic and 2 mm Hg in diastolic BP. Regression of F on BP indicated that at least several hundred (300-600) recessive QTL contribute to BP variability. A model of the distribution of locus effects suggests that the 8-16 QTL of largest effect together account for a maximum of 25% of the dominance variation, while the remaining 75% of the variation is mediated by QTL of very small effect, unlikely to be detectable using current technologies and sample sizes. We infer that recent inbreeding accounts for 36% of all hypertension in this population. The global impact of inbreeding on hypertension may be substantial since, although inbreeding is declining in Western societies, an estimated 1 billion people globally show rates of consanguineous marriages >20%.     

Entrapment of peptidoglycans and adamantyltripeptides into liposomes: an HPLC assay for determination of encapsulation efficiency

The encapsulation of different immunomodulating peptides, the peptidoglycan monomer, its semisynthetic derivatives (Adamant-1-yl)-acetyl-peptidoglycan monomer and Boc-Tyr-peptidoglycan monomer, respectively, and of two diastereoisomers of adamantyltripeptides into the large negatively charged multilamellar liposomes was investigated. The reproducible quantitative method using HPLC was established for the determination of the entrapped compounds. It was shown that the tested compounds could be efficiently incorporated into liposomes using either the film or modified film method. The results confirmed that the peptidoglycans with lipophilic substituents and particularly the adamantyltripeptides were incorporated into liposomes with higher efficiency than the peptidoglycan monomer using either of the described methods. Liposome preparations were stable at 4 degrees C up to seven days as shown by minimal leaking of the entrapped material.