Fatty acid composition indicates two types of metabolic syndrome independent of clinical and laboratory parameters.

Dietary composition and metabolism of fatty acids (FA) influence insulin resistance, atherogenic dyslipidemia and other components of the metabolic syndrome (MS). It is known that patients with MS exhibit a heterogeneous phenotype; however, the relationships of individual FA to MS components have not yet been consistently studied. We examined the plasma phosphatidylcholine FA composition of 166 individuals (68F/98M) with MS and of 188 (87F/101M) controls. Cluster analysis of FA divided the groups into two clusters. In cluster 1, there were 65.7 % of MS patients and 37.8 % of controls, cluster 2 contained 34.3 % of patients and 62.2 % of controls (P<0.001). Those with MS within cluster 1 (MS1) differed from individuals with MS in cluster 2 (MS2) by concentrations of glucose (P<0.05), NEFA (P<0.001), HOMA-IR (P<0.05), and levels of conjugated dienes in LDL (P<0.05). The FA composition in MS1 group differed from MS2 by higher contents of palmitoleic (+30 %), gamma-linolenic (+22 %), dihomo-gamma-linolenic (+9 %) acids and by a lower content of linoleic acid (-25 %) (all P<0.01). These FA patterns are supposed to be connected with the progression and/or impaired biochemical measures of MS (lipolysis, oxidative stress, dysglycidemia, and insulin resistance).

Crystal structure of a heat and protease-stable part of the bacteriophage T4 short tail fibre.

Adsorption of T4 bacteriophage to the Escherichia coli host cell is mediated by six long and six short tail fibres. After at least three long tail fibres have bound, short tail fibres extend and bind irreversibly to the core region of the host cell lipopolysaccharide (LPS), serving as inextensible stays during penetration of the cell envelope by the tail tube. The short tail fibres consist of a parallel, in-register, trimer of gene product 12 (gp12). The 1.9 A crystal structure of a heat and protease-stable fragment of gp12 reveals three new folds: a central right-handed triple beta-helix, a globular C-terminal domain containing a beta-sandwich and an N-terminal beta-structure reminiscent of but different from the adenovirus triple beta-spiral. The centre of the C-terminal domain shows weak homology to gp11, a trimeric protein connecting the short fibre to the base-plate, suggesting that the trimerisation motifs of gp11 and gp12 are similar. Repeating sequence motifs suggest that the N-terminal beta-structure extends further towards the N terminus and is conserved in the long tail fibre proteins gp34 and gp37.

Woodchuck hepatocytes remain permissive for hepadnavirus infection and mouse liver repopulation after cryopreservation.

Isolated hepatocytes represent a relevant model of the liver and are highly required both for research and therapeutic applications. However, sources of primary liver cells from human beings and from some animal species are limited. Therefore, cryopreservation of hepatocytes could greatly facilitate advances in various research areas. The aim of this study was to evaluate whether cryopreserved primary woodchuck hepatocytes could be used for woodchuck hepatitis B virus (WHV) infection studies, and whether they could maintain their regenerative potential in vivo after thawing. Critical steps for good quality of cryopreserved hepatocytes included the use of University of Wisconsin (UW) solution as a main component of the freezing medium, stepwise reduction of dimethylsulfoxide (DMSO) to avoid osmotic shock, and maintenance of low concentrations of DMSO in the culture medium. After cryopreservation, cell viability was still high (70% to 80%), and 50% to 60% of thawed cells attached to the plates. The appearance of covalently closed circular (ccc)DNA and of WHV-replicative forms a few days after in vitro infection demonstrated that thawed woodchuck hepatocytes were still susceptible to viral infection, thus proving maintenance of a very high hepatocyte-specific differentiation status. Furthermore, transplantation of woodchuck hepatocytes into the liver of urokinase-type plasminogen activator (uPA)/recombination activation gene-2 (RAG-2) mice, a model of liver regeneration, demonstrated that cryopreserved cells retained the ability to divide and to extensively repopulate a xenogenic liver. Notably, in vivo susceptibility to infection with WHV and proliferative capacity of frozen/thawed woodchuck hepatocytes in recipient mice were identical to those observed by transplanting fresh hepatocytes.

Structural and functional heterogeneity of naturally occurring hepatitis B virus variants.

Most organisms have developed sophisticated machineries to preserve their genomic integrity. On the contrary hepatitis B virus (HBV), like a lot of other viruses can undergo rapid and drastic sequence changes, especially if the virus has to cope with natural or therapy induced antiviral mechanisms in the host. Here, we try to summarize possible implications for the molecular pathogenesis of HBV based on the extensive research on the genetic variants of HBV.

Identification and crystallisation of a heat- and protease-stable fragment of the bacteriophage T4 short tail fibre.

Irreversible binding of T-even bacteriophages to Escherichia coli is mediated by the short tail fibres, which serve as inextensible stays during DNA injection. Short tail fibres are exceptionally stable elongated trimers of gene product 12 (gp12), a 56 kDa protein. The N-terminal region of gp12 is important for phage attachment, the central region forms a long shaft, while a C-terminal globular region is implicated in binding to the bacterial lipopolysaccharide core. When gp12 was treated with stoichiometric amounts of trypsin or chymotrypsin at 37 degrees C, an N-terminally shortened fragment of 52 kDa resulted. If the protein was incubated at 56 degrees C before trypsin treatment at 37 degrees C, we obtained a stable trimeric fragment of 3 x 33 kDa lacking residues from both the N- and C-termini. Apparently, the protein unfolds partially at 56 degrees C, thereby exposing protease-sensitive sites in the C-terminal region and extra sites in the N-terminal region. Well-diffracting crystals of this fragment could be grown. Our results indicate that gp12 carries a stable central region, consisting of the C-terminal part of the shaft and the attached N-terminal half of the globular region. Implications for structure determination of the gp12 protein and its folding are discussed.

Hepatic tissue engineering on 3-dimensional biodegradable polymers within a pulsatile flow bioreactor.

BACKGROUND: An optimal method for hepatocyte transplantation is not yet determined. With the principles of tissue engineering in vitro conditioning of hepatocytes on biodegradable polymer in a flow bioreactor before implantation forming spheroids may achieve increased cell mass and function to replace lost organ function in vivo. METHODS: Biodegradable poly-L-lactic (PLLA) polymer discs were seeded with rat hepatocytes in a concentration of 10 x 10(6) cells per ml and exposed to a medium flow of 24 ml/min for 1, 2, 4 and 6 days. The number and diameter of spheroidal aggregates was measured by phase-contrast microscopy. H&E histology was performed. Albumin production as hepatocyte specific function was determined by ELISA. RESULTS: Spheroids of viable hepatocytes of 50-200 microm in diameter were formed. Both the number and diameter of the spheroids increased during the first 2 days and then remained constant until day 6. Albumin production was maintained throughout the culture period. CONCLUSION: Short (2- 3 days) pre-transplant conditioning of hepatocytes in a flow bioreactor on biodegradable PLLA resulted in formation of spheroids with a liver-like morphology and preserved specific metabolic function. Tissue engineered hepatocyte spheroids on polymer may represent a functionally active and easy transplantable neotissue and may serve as an in vivo substitute for lost liver function.

Optimization of hepatocyte spheroid formation for hepatic tissue engineering on three-dimensional biodegradable polymer within a flow bioreactor prior to implantation.

We hypothesize that in vitro conditioning of hepatocytes within biodegradable poly-L-lactic acid (PLLA) polymer matrices prior to implantation may increase hepatocyte survival and function after transplantation. The purpose of this study was to optimize the culture conditions of hepatocytes in a pulsatile flow bioreactor. PLLA discs were seeded with rat hepatocytes in a concentration of 2.5, 5, 10, 20 and 40 x 10(6) cells/ml. Seeded discs were exposed to recirculated perpendicular flow of 0, 7, 15, 24, 32, 52 ml/min of supplemented Williams' Medium E and harvested after 6 days in flow culture. Only under flow conditions the hepatocytes formed spheroidal aggregates (SphA) of 50-260 microm in diameter with a liver-like morphology and active metabolic function. The number of SphA was examined by phase contrast microscopy and the reductive enzyme function of the hepatocytes was tested using MTT. Hematoxylin and eosin histology showed vital hepatocytes within the SphA less than 200 microm in diameter but central necrosis in the SphA exceeding this size. Immunohistochemical staining confirmed albumin production of hepatocytes within the SphA. The optimal cell seeding concentration was 10 x 10(6) cells/ml with a flow speed of 24 ml/min. SphA of hepatocytes cultured with this flow bioreactor method may prove useful as a functional unit for tissue engineering of an in vivo liver substitute.

Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.

Mice containing livers repopulated with human hepatocytes would provide excellent in vivo models for studies on human liver diseases and hepatotropic viruses, for which no permissive cell lines exist. Here, we report partial repopulation of the liver of immunodeficient urokinase-type plasminogen activator (uPA)/recombinant activation gene-2 (RAG-2) mice with normal human hepatocytes isolated from the adult liver. In the transplanted mice, the production of human albumin was demonstrated, indicating that human hepatocytes remained functional in the mouse liver for at least 2 months after transplantation. Inoculation of transplanted mice with human hepatitis B virus (HBV) led to the establishment of productive HBV infection. According to human-specific genomic DNA analysis and immunostaining of cryostat liver sections, human hepatocytes were estimated to constitute up to 15% of the uPA/RAG-2 mouse liver. This is proof that normal human hepatocytes can integrate into the mouse hepatic parenchyma, undergo multiple cell divisions, and remain permissive for a human hepatotropic virus in a xenogenic liver. This system will provide new opportunities for studies on etiology and therapy of viral and nonviral human liver diseases, as well as on hepatocyte biology and hepatocellular transplantation.

Increased hepatocyte turnover and inhibition of woodchuck hepatitis B virus replication by adefovir in vitro do not lead to reduction of the closed circular DNA.

The aim of this study was to evaluate the inhibitory effect of the nucleotide analogue adefovir on woodchuck hepatitis B virus (WHV) replication and, in particular, to determine whether the pool of covalently closed circular DNA (cccDNA) could be reduced by adefovir treatment in primary cultures of woodchuck hepatocytes isolated from a chronic carrier. Strong reduction of WHV-DNA synthesis (90%) and secretion (up to 98%) was observed with all 3 doses of adefovir used (1, 10, and 100 micromol/L), whereas in the absence of the drug, high amounts of viral particles were continuously secreted in the culture medium until the end of the study (27 days). Secretion of envelope proteins and viral RNA levels remained constant both in the adefovir-treated and -untreated cultures for the entire course of the study. Intracellular core protein levels declined by approximately 50% in all the cultures, independent of adefovir treatment. There was no indication of cccDNA loss in the adefovir-treated hepatocyte cultures even when cell turnover was induced for 14 days by the addition of epidermal growth factor (EGF) to the culture medium. Our data show that adefovir has a very strong inhibitory effect on WHV-DNA synthesis in chronically infected primary hepatocyte cultures and indicate that cccDNA is a very stable molecule that appears to be efficiently transmitted to the dividing hepatocytes.

Stability of bacteriophage T4 short tail fiber.

Adsorption of T-even bacteriophages to the E. coli host cell is mediated by long and short tail fibers. Bacteriophage T4 short tail fiber protein p12 was used to investigate the stability against thermal and chemical denaturation. Purified p12 is thermostable with a melting point of 78 degrees C. Guanidinium chloride-induced denaturation displayed strong hysteresis and an intermediate between 2 and 3 M denaturant. The transitions occur at 1.5 and 3.2 M denaturant as revealed by fluorescence spectroscopy and circular dichroism. The data suggest an equilibrium unfolding intermediate with a separate unfolding of the C-terminal knob domain and the shaft region.

Folding of coliphage T4 short tail fiber in vitro. Analysing the role of a bacteriophage-encoded chaperone.

The morphogenesis of the Escherichia coli bacteriophage T4 depends on the presence of helper proteins which are not components of the mature virion. Two bacteriophage-encoded proteins, p57 and p38, are required for the assembly of the bacteriophage T4 tail fibers. In the absence of p57, two polypeptides of the long fiber (p34 and p37) and that of the short tail fiber (p12) fail to trimerize. Instead they form water-insoluble aggregates. Co-expression of the genes 12 and 57 in vivo caused the formation of only trimeric, water-soluble p12. The function of g57 cannot be replaced by overexpression of the host proteins GroEL/ES or parvulin. The mechanism of action of this helper protein has remained unknown, mainly because it has not been possible to determine its activity in vitro. Purified p12, denatured in 7 M urea, trimerized spontaneously in a slow reaction (half-time approximately 6 h) and with low yield. Upon renaturation, p12 forms native SDS-resistant trimers as indicated by spectroscopic and hydrodynamic measurements. Addition of p57 increased the rate of folding threefold and nearly doubled the yield. These experiments demonstrate that p57 acts as a molecular chaperone during folding of T4 tail fibers.

The determinants of East-West German migration: some first results.

Determinants of the propensity to migrate are explored using data collected in a survey of East German residents following German reunification in 1991. The author notes that in the period 1989-1992, some 870,000 individuals migrated to West Germany, representing about 5% of the total East German population and 10% of the work force. He suggests that "recent developments in the literature on the option value of waiting may yield important insights into these determinants."

[Determination of drugs using gas chromatography]. / Urceni léciv plynovou chromatografií.

There are introduced values of retention time of cca 30 pure drugs used in human medicine in CSSR. The values are expressed in Kovats indices for several temperatures and types of columns. It was found, that the indices are depending on lengths of columns.