Early kinetics of calprotectin in plasma following inguinal hernia surgery

Calprotectin is one of the most abundant proteins of neutrophil granulocytes. It is released upon neutrophil activation and is considered a sensitive and clinically useful marker for neutrophil-mediated inflammation, including bacterial infections. However, early kinetics of calprotectin activation following inflammatory activation has hitherto been unknown. The aim of the present study was to determine the early phase of the kinetics of calprotectin, in comparison with the inflammatory markers CRP, IL-6, TNF-α, and procalcitonin, in plasma following a standardized temporary mild inflammatory response, using uncomplicated inguinal hernia surgery as a model. The study cohort consisted of 17 adult patients (15 male and 2 female) undergoing elective surgery for hernia. Values of calprotectin increased significantly at 2 h following surgery, and continued to increase to reach the highest level at 24–36 h after surgery, values still not exceeding upper normal reference level. This contrasts to IL-6 and CRP, for which an elevation was found first later, 4 h and 24–36 h post-surgery, respectively, for IL-6, and CRP. No significant increase was seen for TNF-α, or procalcitonin. The data demonstrate a very rapid and significant but modest increase in calprotectin following induction of mild inflammation, supporting that calprotectin can be useful for early detection of inflammatory response.     

A cell cycle study of human mammary epithelial cells.

The growth regulation of human mammary epithelial cells (HMEC) cultured in a growth factor/hormone-enriched (e.g. EGF, insulin) medium with bovine pituitary extract as the only undefined supplement was studied. The doubling times of the cultures, in which the cells appear in colonies, was 55-72 h, and a considerable intercolonial heterogenecity in proliferative activity could be demonstrated. However, every colony, irrespective of the size of the growth fraction, comprised a sub-population of rapidly growing cells which had a mean generation time of approximately 22 h. When insulin was removed from the culture medium, HMEC proliferation was inhibited. This growth inhibition was shown to be a result of a cell cycle-specific block.     

Methylation changes in the human IGF2 p3 promoter parallel IGF2 expression in the primary tumor, established cell line, and xenograft of a human hepatoblastoma

Hepatoblastoma (HB) is a rare malignant embryonal liver tumor. Its pathogenesis has been associated with altered regulation of the IGF2 and H19 genes, and previous studies have suggested a correlation between abnormal methylation and altered expression of these genes in hepatoblastoma. Upregulation of the activity of the IGF2 promoter P3 has previously been shown to be tightly correlated with demethylation in hepatoblastoma. Here, we have used bisulfite genomic sequencing to characterize the methylation pattern of the IGF2 promoter P3 in the hepatoblastoma-derived cell line Hep T1, in the original tumor from which Hep T1 is derived, and in nude mouse xenografts of the Hep T1 cell line. The results show a clear difference in methylation pattern of the most proximal region of the IGF2 P3 promoter between the primary tumor, the cell line, and the xenografts. RNase protection and mRNA in situ hybridization revealed that variations in methylation patterns was paralleled by the levels of IGF2 P3 mRNA, which was detectable in the primary tumor and xenografts, but not in the cell line. Furthermore, it was demonstrated that H19 was reactivated and demethylated in the HepT1 cell line by 5-azaCytidine, in contrast to IGF2 P3, which was not demethylated or reactivated. We suggest that methylation of the proximal IGF2 P3 is important for its regulation.     

Characterization of a novel human putative mitochondrial transporter homologous to the yeast mitochondrial RNA splicing proteins 3 and 4

We report here a novel human gene, hMRS3/4, encoding a putative mitochondrial transporter structurally and functionally homologous to the yeast mitochondrial RNA splicing proteins 3 and 4. These proteins belong to the family of mitochondrial carrier proteins (MCF) and are likely to function as solute carriers. hMRS3/4 spans approximately 10 kb of genomic DNA on chromosome 10q24 and consists of four exons that encode a 364-aa protein with six transmembrane domains. A putative splice variant, encoding a 177-aa protein with three transmembrane domains, was also identified. hMRS3/4 has a well-conserved signature sequence of MCF and is targeted into the mitochondria. When expressed in yeast, hMRS3/4 efficiently restores the mitochondrial functions in mrs3(o)mrs4(o) knock-out mutants. Ubiquitous expression in human tissues and a well-conserved structure and function suggest an important role for hMRS3/4 in human cells.     

The C-terminus of dUTPase: observation on flexibility using NMR

The dynamics of the C-terminus of the dUTPases from Escherichia coli and equine infectious anaemia virus (EIAV) were studied by 1H-(15)N nuclear magnetic resonance spectroscopy. The two enzymes differ with regard to flexibility in the backbone of the 15 most C-terminal amino acid residues, some of which are conserved and essential for enzymic activity. In the bacterial enzyme, the residues closest to the C-terminus are highly flexible and display a correlation time in the nanosecond time range. No similar high flexibility could be detected for the C-terminal part of EIAV dUTPase, indicating a different time range of flexibility.     

Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes

We describe and validate a new membrane protein topology prediction method, TMHMM, based on a hidden Markov model. We present a detailed analysis of TMHMM's performance, and show that it correctly predicts 97-98 % of the transmembrane helices. Additionally, TMHMM can discriminate between soluble and membrane proteins with both specificity and sensitivity better than 99 %, although the accuracy drops when signal peptides are present. This high degree of accuracy allowed us to predict reliably integral membrane proteins in a large collection of genomes. Based on these predictions, we estimate that 20-30 % of all genes in most genomes encode membrane proteins, which is in agreement with previous estimates. We further discovered that proteins with N(in)-C(in) topologies are strongly preferred in all examined organisms, except Caenorhabditis elegans, where the large number of 7TM receptors increases the counts for N(out)-C(in) topologies. We discuss the possible relevance of this finding for our understanding of membrane protein assembly mechanisms. A TMHMM prediction service is available at http://www.cbs.dtu.dk/services/TMHMM/.     

Characterization of a molten globule state of bovine carbonic anhydrase III: loss of asymmetrical environment of the aromatic residues has a profound effect on both the near- and far-UV CD spectrum

Bovine muscle carbonic anhydrase (isoenzyme III; BCAIII) exhibited a three-state unfolding process at equilibrium upon denaturation in guanidine hydrochloride (GuHCl). The stable folding intermediate appeared to be of molten globule type. The stability towards GuHCl in terms of mid-point concentrations of denaturation were very similar for BCAIII and human CAII (HCAII). It was further demonstrated that the aromatic amino acid residues contributed significantly to the circular dichroism (CD) spectrum in the far-UV wavelength region during the native-->molten globule state transition. Thus, the ellipiticity change at 218 nm was shown to monitor the loss of tertiary interactions of aromatic side chains at the first unfolding transition as well as the rupture of secondary structure at the second unfolding transition. Similar aromatic contributions to the far-UV CD spectrum, but with varying magnitudes, were also noted for BCAII and HCAII, further emphasizing that interference of aromatic residues should not be neglected at wavelengths that normally are assigned to secondary structural changes.     

Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis

Here, we describe a new approach for mutational scanning of PCR products through hybridization analysis between complementary oligonucleotides. Sets of overlapping probe oligonucleotides complementary to wild-type (WT) sequence are hybridized to microbead-immobilized PCR products under solution-like conditions. Mismatch-hybridization situations between a mutant sample and probe oligonucleotides result in higher remaining concentrations in solution of involved probe oligonucleotides. Post-hybridization supernatants are subsequently analyzed for their probe oligonucleotide compositions using surface plasmon resonance-based biosensor technology. Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridization analysis between probe oligonucleotides and their corresponding sensor-chip immobilized complementary counterparts. This allows for the construction of composition diagrams revealing the existence and approximate location of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, single mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized.     

Precultivation technique for studies of microorganisms exhibiting overflow metabolism

A precultivation technique for microorganisms exhibiting overflow metabolism is presented. It is based on a low initial medium volume in the fermenter. The medium feed contains all the nutrients, but is diluted with respect to sugar, the concentration of which determines the biomass concentration at the end of the preculture. By this method, effects of varying activity of the inocula from shake flask cultures are minimised and the metabolic state, i.e. oxido-reductive, oxidative growth on sugar plus overflow metabolite or oxidative growth on sugar alone, can be controlled at the start of the main fermentation.