Human Lipoproteins at Model Cell Membranes: Effect of Lipoprotein Class on Lipid Exchange.

High and low density lipoproteins (HDL and LDL) are thought to play vital roles in the onset and development of atherosclerosis; the biggest killer in the western world. Key issues of initial lipoprotein (LP) interactions at cellular membranes need to be addressed including LP deposition and lipid exchange. Here we present a protocol for monitoring the in situ kinetics of lipoprotein deposition and lipid exchange/removal at model cellular membranes using the non-invasive, surface sensitive methods of neutron reflection and quartz crystal microbalance with dissipation. For neutron reflection, lipid exchange and lipid removal can be distinguished thanks to the combined use of hydrogenated and tail-deuterated lipids. Both HDL and LDL remove lipids from the bilayer and deposit hydrogenated material into the lipid bilayer, however, the extent of removal and exchange depends on LP type. These results support the notion of HDL acting as the 'good' cholesterol, removing lipid material from lipid-loaded cells, whereas LDL acts as the 'bad' cholesterol, depositing lipid material into the vascular wall.

Genetic and morphological variability of the European mudminnow Umbra krameri (Teleostei, Umbridae) in Serbia and in Bosnia and Herzegovina, a basis for future conservation activities.

As a basis for future conservation activities, the genetic and external body morphology variability of the European mudminnow Umbra krameri, a highly endangered fish species in Serbia and in Bosnia and Herzegovina, was determined for existing populations with the use of molecular markers (mitochondrial and microsatellite DNA) and geometric morphometric methods. Mitochondrial DNA cytochrome b gene analysis revealed two previously undescribed haplotypes: Da1 (the Lugomir population from the Danube River basin) and Sa1 (the Bakreni Batar and the Gromizelj populations from the Sava River system), with a corresponding genetic distance of 0·7%. Paired values of FST and DAS distances for microsatellite marker data show that the difference between the Danube and the Sava populations is seven to nine times higher than the difference between the populations within the Sava River system. Geometric morphometric analyses also support a clear separation of the Lugomir population from the Bakreni Batar and the Gromizelj populations. The analysis of the body shape variation, however, indicates a significant difference between the two genetically indistinguishable Sava populations. The observed genetic and phenetic relationships of the analysed mudminnow populations most probably represent a consequence of historical, geographical and ecological factors. These results will offer guidelines for future protection, conservation and sustainable management of this species in the region.

Kidney length in healthy members of Balkan endemic nephropathy families.

BACKGROUND: Kidney size may differ between healthy members of Balkan endemic nephropathy (BEN) and non-BEN families. The present study was designed to elucidate this, in comparison with values for BEN patients. METHODS: A total of 71 BEN patients (34 males, 64.4 ± 12.0 years), 74 healthy BEN family members (39 males, 49.1 ± 12.2 years), and 59 non-BEN family members (19 males, 49.2 ± 12.3 years) were involved. We measured the longest craniocaudal length and minimal parenchymal thickness on each kidney of all examined subjects using ultrasound. RESULTS: No significant difference was found between the kidney length of healthy subjects from BEN (11.0 ± 0.8 cm) and non-BEN families (10.9 ± 0.8 cm), but kidneys were significantly longer than in BEN patients (9.9 ± 1.3 cm). Minimal parenchymal thickness was similar in all three groups. When subjects from each group were divided according to estimated glomerular filtration rate (eGFR), kidney length of the healthy groups was significantly longer than in BEN patients both in stage 1 (p =0.039) and stage 2 (p =0.044) of chronic kidney disease. The parental history of BEN was not associated with kidney dimensions, eGFR, or urinary excretion of albumin and alpha1-microglobulin. CONCLUSION: Kidneys of BEN patients were significantly shorter than in healthy members of both BEN and non-BEN families, but no difference was found in kidney length and parenchymal thickness between healthy members of BEN and non-BEN families. No significant association was found between parental history of BEN and kidney size and function either in BEN patients or in healthy members from BEN families. Hippokratia 2015; 19 (4): 304-308.

First mtDNA sequencing of Volga and Ob basin taimen Hucho taimen: European populations stem from a late Pleistocene expansion of H. taimen out of western Siberia and are not intermediate to Hucho hucho.

New concatenated mtDNA sequences (three genes; n = 22) of Siberian taimen Hucho taimen primarily from west Siberian and European regions of the species' range were added to 12 previously published sequences to provide a phylogeographic overview of the species. European samples show only very minor divergence from west Siberian populations, supporting a late Pleistocene expansion from Siberia into the Urals, with no particular relation to the Danube River basin huchen Hucho hucho as once hypothesized. The disjunct distribution of the genus is most likely based on an early Pleistocene vicariant event.

New distribution data and population structure of the European mudminnow Umbra krameri in Serbia and Bosnia and Herzegovina.

Seventy-six individuals of the European mudminnow Umbra krameri from two recent populations from Serbia (Bakreni Batar and Lugomir) and one from Bosnia and Herzegovina (Gromizelj) were analysed for habitat preferences and population structure. The population from Lugomir is a newly recorded population in Serbia. Besides this new record, it is noteworthy that all three studied locations are outside the currently known species distribution range limits.

Evidence for two transferrin loci in the Salmo trutta genome.

To determine the organization of transferrin (TF) locus in the Salmo trutta genome, partial DNA and cDNA sequencing, fluorescent in situ hybridization (FISH) and Salmo salar BAC analysis were performed. TF expression levels and copy number prediction were assessed using real-time PCR. In addition to two previously reported DNA TF variant sequences of S. trutta and Salmo marmoratus (TF1), two novel variant sequences (TF2) were revealed in both species. Variant-specific sequence tags, characterizing two variants for each TF type (TF1 and TF2), were identified in genomic clones from each of the F1 hybrids between S. trutta and S. marmoratus. These clearly documented double heterozygote status at the TF loci. The real-time PCR data showed that each of the two TF types (TF1 and TF2) existed in one copy only and that the transcription of TF2 was considerably lower compared with TF1. Using FISH, hybridization signals were observed on two medium-sized acrocentric chromosomes of S. trutta karyotype. A TF type-specific PCR followed by a restriction analysis revealed the presence of two TF loci in the majority of analysed BAC clones. It was concluded that the TF gene is duplicated in the genome of S. trutta, and that the two TF loci are located adjacent to one another on the same chromosome. The differing transcription levels of TF1 and TF2 appear to depend on the corresponding promoter activity, which at least for TF2 seems to vary between different Salmo congeners.

Expression of the proliferating cell nuclear antigen and protein products of tumour suppressor genes in the human foetal testis.

Tumour suppressor genes retinoblastoma (Rb1) and adenomatous polyposis coli (Apc) as well as the proliferating cell nuclear antigen (PCNA) are involved in embryonic development. The purpose of the present study was to investigate the expression of Rb1 protein, APC protein and PCNA during development of the human foetal testis. Qualitative analysis of their expression at the single-cell level was performed using immunohistochemistry on archive samples of the foetal testis (18-37 gestation week). Stereological parameters (volume density, absolute volume, numerical density, absolute number) were calculated for quantification of the overall expression of those proteins that were expressed frequently enough for such an analysis. PCNA was frequently expressed in nuclei of immature Sertoli cells and prospermatogonia and less frequently in surrounding peritubular (myoid) and interstitial cells. The pRb1 protein was present in nuclei of prospermatogonia and Sertoli cells but was absent from the interstitial tissue. APC protein was expressed in the cytoplasm of a very small number of prospermatogonia and interstitial (Leydig) cells. The overall expression of PCNA in all stages of development was higher than pRb1 expression.

Differentiation of rat neural tissue in a serum-free embryo culture model followed by in vivo transplantation.

AIM: To analyze neural tissue differentiation in a unique, chemically-defined in vitro culture model of gastrulating rat embryo proper by use of transmission electron microscopy (TEM), proliferating cell nuclear antigen (PCNA) expression, and in vivo transplantation after a 2-week culture in serum-free or serum-supplemented media. Influence of protein-free medium unfavorable for differentiation of neural tissue in vitro was compared with favorable serum-free media enriched with transferrin or albumin. Differentiation of mesodermal derivatives in transplants was also investigated. METHODS: We cultivated 9.5-day-old Fischer rat embryos on the gas-liquid interface in the protein-free Eagle's Minimum Essential Medium (MEM), in MEM with either iron-saturated holotransferrin (50 microg/mL) or iron-free apotransferrin (50 microg/mL), and in medium saturated with either bovine serum albumin (BSA) (4 mg/mL or 400 microg/mL) or rat serum (50%). After the two-week culture period, light microscopy, TEM, and immunohistochemical method for detection of PCNA were done. Some explants were transplanted under the kidney capsule of adult male rats to be cultured in vivo for additional two weeks. Chi-square test or Fisher exact test were used to compare the proportion of tissues developed. RESULTS: Proportion of differentiated neural tissue was similar in explants cultivated in apotransferrin- and holotransferrin-supplemented media (13/33 and 9/20, respectively), but higher than in explants cultivated in protein-free medium (1/13). Neurons and glia cells produced a neuropil structure. Myelinization occurred only in serum-supplemented medium. PCNA expression was detected in a small number of neural tissue cells, even in serum-free cultivated embryos. Differentiation of brain-like tissue, cerebrospinal, and vegetative ganglionic cells occurred in all groups of transplants. However, in the transplants derived from protein-free medium, the proportion of neural tissue, cartilage, bone, skeletal and smooth muscle was significantly lower than in transferrin-supplemented media (p<0.01). Albumin seemed to promote differentiation of all tissues except vegetative ganglionic cells. CONCLUSION: Nerve tissue differentiated to a rather high degree in a two-week in vitro postimplantation embryo culture. Transferrin or albumin, as the only proteins used for serum-free precultivation, significantly improved subsequent differentiation of nerve tissue and mesodermal derivatives in transplants in vivo.

Microcirculation is similar in ischemic and venous ulcers.

Microcirculation of 15 ischemic and 15 venous ulcers, their scars, and intact surrounding skin were examined in order to demonstrate their similarities in the development and healing process. Subpapillary and nutritive perfusion of four areas were investigated by a laser Doppler perfusion imager (arbitrary units) and capillary microscopy (capillaries/mm2): one ulcer area without granulation tissue (no wound healing) and one with granulation tissue (ulcer healing); one skin area adjacent to the ulcer (1-8 mm) (scar developed from ulcer areas) and one distant (12-25 mm; intact skin). Areas without granulation tissue in ischemic and venous ulcers were similar, demonstrating a lack of capillaries (0.13 +/- 0.52; 0.93 +/- 2.09) and low laser Doppler flux (0.81 +/- 0.69; 1.47 +/- 1.17; P > 0.05 for each). In granulation tissue of both ulcers there was a tendency to a higher capillary density (0.67 +/- 1.40; 5.60 +/- 2.32; P < 0.0001 for venous ulcers) and a higher laser Doppler flux (1.15 +/- 0.67; 4.04 +/- 1.62; P < 0.0001 for venous ulcers) than in areas without granulation tissue. In scars of ischemic and venous ulcers capillary density (8.18 +/- 8.84; 13.60 +/- 5.45) and laser Doppler flux (1.72 +/- 1.00; 1.94 +/- 1.45) were similar (P > 0.05). In skin distant from ischemic ulcers very high capillary density (24.63 +/- 1.89) was associated with low laser Doppler flux (0.99 +/- 0.59); distant from venous ulcer capillary density was moderate (10.47 +/- 3.42) while laser Doppler flux was high (3.77 +/- 1.62; P < 0.0001 between both groups). The development and healing process of ischemic and venous ulcers is similar. Nutritive and subpapillary perfusion are involved in ulcer healing. In intact skin surrounding ischemic and venous ulcers, microcirculation is different due to the underlying pathophysiology.

Model of interstitial pressure as a result of cyclical changes in the capillary wall fluid transport.

Reported interstitial pressures range from -8 to +6 mm Hg in different tissues and from <-20 mm Hg in burned tissue or more than +30 mm Hg in tumors. We have tried to link interstitial pressure to the here proposed cyclical changes in the fluid transport across the capillary wall. In the presented model interstitial pressure is considered as an average of pressures in numerous pericapillary spaces. A single pericapillary pressure is a dynamic difference between the net outward (hydraulic pressure+interstitial colloid osmotic pressure) and inward (plasma colloid oncotic pressure) forces. Hence, dominating net outward forces would result in a positive pericapillary interstitial pressure, while stronger inward forces would produce negative pressures in the pericapillary space. All interruptions of blood flow leave some blood in capillaries with a normal oncotic pressure and no hydrostatic pressure that might act as a strong absorber of interstitial fluid until the blood flow is reestablished. Model assumptions for the systemic circulation capillaries include (a) precapillary sphincters can almost entirely stop the capillary flow, (b) only a minority of sphincters are normally open in the tissue, and (c) hydrostatic pressures in unperfused capillaries are similar to the pressures at their venous ends. The key proposal is that capillaries with closed precapillary sphincters along their entire length have low hydrostatic pressure of 10 to 15 mm Hg. This pressure cannot force filtration, so these capillaries reabsorb interstitial fluid from the pericapillary space along their entire length. In the open capillaries, hydrostatic pressure filtrates fluid to the pericapillary space along most of their length. Fluid enters, moves some 20 or 30 micrometers away and back to be reabsorbed at the same point. Closed periods are periods of intense fluid reabsorption, while the short open periods refill the space with fresh fluid. It can be calculated that subcutaneous tissue interstitial pressure values might develop if the closed periods are 1.14 to 2.66 times longer than the open periods. Positive interstitial pressures observed in some organs might develop if open periods are longer than the closed periods. High interstitial colloid pressure in lungs makes both perfused and unperfused capillaries absorptive, resulting in more negative values of lung interstitial pressure. The same model is used to explain interstitial pressure values in tumors, burned tissue and intestinal villi.

Chemically defined protein-free in vitro culture of mammalian embryo does not restrict its developmental potential for differentiation of skin appendages.

In a unique serum- and protein-free chemically defined in vitro culture model of postimplantation mammalian development the epidermis differentiates regularly, although the differentiation of other tissues is impaired due to the lack of the serum. The present study in that model was done to estimate more carefully the degree of epidermal differentiation in defined media supplemented with some growth- or differentiation-stimulating substances. The main objective was to discover by grafting in vivo to the richer environment whether simple protein-free culture conditions restrict an inherent embryonic potential for differentiation of skin appendages. Embryonic parts of E9.5 gastrulating Fischer rat embryos were cultivated for 2 weeks in the protein-free Eagle's minimum essential medium supplemented with holotransferrin, apotransferrin, insulin and/or Na(2)SeO(3) and in controls cultivated in protein-free medium or in serum-supplemented medium. In all experiments there was a high incidence of differentiation of the epidermis. A high level of epidermal differentiation was confirmed for the first time at the ultrastructural level. A well-differentiated cornified layer and cells connected with desmosomes containing keratohyaline masses and cytokeratin filaments were found. A strong immunohistochemical signal for the proliferating cell nuclear antigen was always detected in the basal layer of the epidermis showing that those cells were still able to proliferate. Finally, embryos precultivated for 1 or 2 weeks in the protein-free medium and media supplemented with apotransferrin or serum were grafted under the kidney capsule for an additional 2 weeks. It was discovered that even after spending 2 weeks in the simple protein-free medium in vitro, embryos retained their developmental potential for differentiation of skin appendages (hair and sebaceous glands).

A monoclonal antibody to the DEC-205 endocytosis receptor on human dendritic cells.

DEC-205 is a multilectin receptor for adsorptive endocytosis, expressed in mouse dendritic cells (DC) and some epithelia. DEC-205 is homologous to the macrophage mannose receptor (MMR). A cDNA for murine DEC-205 was used to identify 3 overlapping human DEC-205 clones from a lymphocyte library. The human homologue is a transmembrane protein of 1722 aminoacids with 10 externally disposed C-type lectin domains having 77% identity to the mouse counterpart. The NH(2) terminal cysteine-rich and fibronectin type II domains were expressed and used to immunize mice. A hybridoma, MG38, which specifically recognized the immunogen was obtained from a DEC-205 knockout mouse. The antibody precipitated a 205 kD protein from metabolically labeled, monocyte-derived DCs. MG38 labeled mature monocyte-derived DCs but showed weak or no labeling of other peripheral blood mononuclear cells. In tissue sections, MG38 identified DEC-205 on thymic cortical epithelium and DCs in the thymic medulla and tonsillar T cell areas. In contrast, an anti-MMR antibody stained DEC-205 negative, macrophages in the thymus cortex, the trabeculae of the thymus and tonsil, as well as efferent lymphatics in the tonsil. Therefore, the MG38 anti-DEC-205 antibody is useful for identifying DCs and reveals clear differences in sites where MMR and DEC-205 are expressed in lymphoid tissues.

The cysteine-rich domain of the macrophage mannose receptor is a multispecific lectin that recognizes chondroitin sulfates A and B and sulfated oligosaccharides of blood group Lewis(a) and Lewis(x) types in addition to the sulfated N-glycans of lutropin.

The mannose receptor (MR) is an endocytic protein on macrophages and dendritic cells, as well as on hepatic endothelial, kidney mesangial, tracheal smooth muscle, and retinal pigment epithelial cells. The extracellular portion contains two types of carbohydrate-recognition domain (CRD): eight membrane-proximal C-type CRDs and a membrane-distal cysteine-rich domain (Cys-MR). The former bind mannose-, N-acetylglucosamine-, and fucose-terminating oligosaccharides, and may be important in innate immunity towards microbial pathogens, and in antigen trapping for processing and presentation in adaptive immunity. Cys-MR binds to the sulfated carbohydrate chains of pituitary hormones and may have a role in hormonal clearance. A second feature of Cys-MR is binding to macrophages in marginal zones of the spleen, and to B cell areas in germinal centers which may help direct MR-bearing cells toward germinal centers during the immune response. Here we describe two novel classes of carbohydrate ligand for Cys-MR: chondroitin-4 sulfate chains of the type found on proteoglycans produced by cells of the immune system, and sulfated blood group chains. We further demonstrate that Cys-MR interacts with cells in the spleen via the binding site for sulfated carbohydrates. Our data suggest that the three classes of sulfated carbohydrate ligands may variously regulate the trafficking and function of MR-bearing cells.

Introducing ropivacaine into a department's epidural analgesic practice. Improving acute pain service practice.

The results of introducing a new licensed local anaesthetic drug, ropivacaine, into routine practice were evaluated by measuring the efficacy and adverse effects of patient controlled epidural analgesia (PCEA), using ropivacaine 2 mg/ml (R), or the mixtures in current use: fentanyl 5 (micrograms/ml with bupivacaine 1 mg/ml (BF5) and fentanyl 10 (micrograms/ml) with bupivacaine 1 mg/ml (BF10). All patients were nursed on general wards after surgery. For two months, 102 consecutive patients were studied. Pain scores at rest were significantly better in the fentanyl and bupivacaine groups, (mean rank R: 35.5, BF5: 22.7, BF10: 26.9, P < 0.05). There was a significant correlation between patient controlled boluses and pain at rest and (p < 0.001), and pain on moving (p < 0.001). Nausea and vomiting was worse in the BF10 (p < 0.05). Older patients demanded less analgesia (p < 0.001). Postoperatively BF5 provided better pain relief with trends demonstrating fewer side-effects and complications than BF10 or R. We now use fentanyl 5 (micrograms/ml and bupivacaine 1 mg/ml as our standard epidural infusion mixture.

Changes in the placenta and in the rat embryo caused by the demethylating agent 5-azacytidine.

DNA methylation is an important mechanism for regulation of gene expression during vertebrate development. 5-azacytidine is used as an experimental tool for demethylation. In this work, a single dose of 5-azacytidine (5 mg/kg body weight) was administered to rats at different stages of development. After 5-azacytidine administration on the first or third day of pregnancy, no changes were detected. After administration on the fourth day of pregnancy or later, a reduction in growth was observed. After treatment on day five and on any other day till day eleven of pregnancy, no living fetuses were found. Of those treated on day twelve, 24% of fetuses survived, but forelimb and hindlimb malformations were present. Administered on day thirteen, 5-azacytidine did not interfere with survival, but malformations were still present. From day fourteen on, 5-azacytidine caused no gross external malformations. Placentas were also influenced by 5-azacytidine. They were significantly smaller and histological evaluation showed the labyrinthine part to be severely reduced. In contrast, trophoblast giant cells were more abundant than in controls.

Anthropogenetic variability of populations of the north-eastern part of the Slavonia and Baranya Region--Croatia.

In this work we studied the anthropogenetic structure of eight populations in the north-eastern part of the Slavonia-Baranya region. The results are based on the analysis of ABO and Rh systems of erythrocytic antigens i.e. analysis of A1, A2, B and O genes, and in Rh systems D and d genes. The analysis was carried out on 7,860 grown-ups chosen by a random sample method among blood donors in Clinical Hospital in Osijek. The gene frequencies, "genetic distances", coefficients of kinship and heterozygosity have been calculated for examined populations. The results show that the present anthropologenetic population structure reflects the common origin of the examined populations, but also that the numerous changes throughout their ethno-history, caused first of all by population migrations, which is one of the basic mechanisms that have shaped the population structure of the north-eastern part of Slavonia-Baranya region.

The human S-adenosylmethionine decarboxylase gene: nucleotide sequence of a pseudogene and chromosomal localization of the active gene (AMD1) and the pseudogene (AMD2).

S-adenosylmethionine decarboxylase (AdoMet-DC) is a key enzyme in polyamine biosynthesis. The human genome contains at least two loci for the AdoMetDC gene (AMD), one of which (AMD1) has previously been mapped to chromosome 6 and the other (AMD2) to the X chromosome. The locus on chromosome 6 is the transcriptionally active gene. We now report characterization of the AMD2 locus (GenBank Accession No. U02035) on the X chromosome, which contains sequences that cross-hybridize with human AdoMetDC cDNA. This DNA lacks all of the introns present in AMD1 and has numerous mutations in the protein-coding region. Its overall nucleotide sequence identity with AdoMetDC cDNA is about 90%. AMD2 is therefore a processed pseudogene, which, because of multiple mutations, cannot be translated to an active AdoMetDC enzyme, even if it were transcribed. Chromosomal loci for human AMD sequences were determined by in situ hybridization to metaphase chromosomes, with genomic DNAs from the active gene and the pseudogene loci as probes. AMD1 was localized to chromosome region 6q21-->q22 and AMD2 to band Xq28.

Structure and organization of the human S-adenosylmethionine decarboxylase gene.

Genomic clones for the S-adenosylmethionine (AdoMet) decarboxylase gene were isolated from a human chromosome 6 DNA library. In addition, polymerase chain reaction and specific primers were used to amplify fragments from chromosomal DNA covering exonic regions not found in the screening of DNA libraries with AdoMet decarboxylase cDNA. The gene encompasses at least 22 kilobases of chromosome 6 DNA and comprises nine exons and eight introns, in contrast to the corresponding rat gene that has only eight exons (Pulkka, A., Ihalainen, R., Aatsinki, J., and Pajunen, A. (1991) FEBS Lett. 291, 289-295). Exon-intron junctions in the human and rat AdoMet decarboxylase genes were in identical positions except that exons 6 and 7 of the human gene formed a single exon in the rat gene. Alu-like sequences are present in four introns and the 5'-flanking region of the human gene. The promoter region contains a TATA box adjacent to the cap site; in addition, DNA elements for binding of transcription factors AP-1, AP-2, CREB, SP-1, and multiple steroid receptors are present between position -3,158 and the transcription start site. Two AdoMet decarboxylase promoter-reporter gene constructs with about 170 and 1,500 nucleotides of the 5'-flanking DNA were used in transient expression studies. AdoMet decarboxylase promoter was capable of driving reporter gene expression, but it was less active than the murine ornithine decarboxylase promoter. There are at least three potential polyadenylation signals at the 3'-end of the gene, and utilization of the first two results in the formation of the 2.0- and 3.6-kilobase AdoMet decarboxylase mRNA species present in human tissues and cell lines. AdoMet decarboxylase gene-related sequences were also present in a human X chromosome-specific DNA library. Partial nucleotide sequencing of this DNA revealed a lack of introns present in the gene located on chromosome 6, suggesting that the locus on the X chromosome contains a processed AdoMet decarboxylase pseudogene.