Identification of a deletion in the LDL receptor gene A Finnish type of mutation

A cDNA probe for the low density lipoprotein (LDL) receptor gene was used to screen DNA samples from 52 unrelated Finnish patients with the heterozygous form of familial hypercholesterolemia (FH) and 51 healthy controls. Southern blot analysis using the restriction enzyme PvuII revealed an abnormal 11 kb (kilo base-pair) restriction fragment in 16 (31%) of the patients but none of the controls. A more detailed restriction enzyme analysis of the DNA from patients revealed a mutation which apparently is due to an 8 kb deletion extending from intron 15 to exon 18 of the LDL receptor gene. Co-segregation of FH with the mutated gene was demonstrated in three families. These data are consistent with a ‘founder gene effect’ and support the assumption that recombinant DNA methods may have great impact on the diagnostics of FH in genetically homogeneous populations.     

Long-term needle litterfall of a Scots pine Pinus sylvestris stand: relation to temperature factors

Summary Needle litterfall of a Scots pine was caught over 24 years (1962–1986) with litter-traps in a Scots pine stand in southeastern Finland. The age of the trees averaged 111 years in 1962. The stand was naturally recruited and only minor silvicultural treatments occurred during its history. Litterfall showed great year-to-year variation, the minimum being 18 g/m² (in 1968) and maximum 213 g/m² (in 1973). There was no overall trend in the amount of litterfall, and the age of the stand was thus not important in determining the needle fall. We used time domain time series analysis (ARIMA) and standard climatic data (temperature, precipitation) to investigate the relationship of litterfall to climatic factors. Mean July temperature was clearly correlated with needle litterfall. High temperature in July coincided with enhanced litterfall in the same and the next year. Litterfall enhanced litterfall in the same and the next year. Litterfall increased also after high temperatures during March–April, but only in the same year. In addition to these the litterfall had a 4-year self-dependency. This is approximately the same as the mean longevity of needles in the study area. Altogether the time series model we propose covers about 90% of the variance of the original time series.     

Exome Sequencing of Index Patients with Retinal Dystrophies as a Tool for Molecular Diagnosis

Background

Retinal dystrophies (RD) are a group of hereditary diseases that lead to debilitating visual impairment and are usually transmitted as a Mendelian trait. Pathogenic mutations can occur in any of the 100 or more disease genes identified so far, making molecular diagnosis a rather laborious process. In this work we explored the use of whole exome sequencing (WES) as a tool for identification of RD mutations, with the aim of assessing its applicability in a diagnostic context.

Methodology/Principal Findings

We ascertained 12 Spanish families with seemingly recessive RD. All of the index patients underwent mutational pre-screening by chip-based sequence hybridization and resulted to be negative for known RD mutations. With the exception of one pedigree, to simulate a standard diagnostic scenario we processed by WES only the DNA from the index patient of each family, followed by in silico data analysis. We successfully identified causative mutations in patients from 10 different families, which were later verified by Sanger sequencing and co-segregation analyses. Specifically, we detected pathogenic DNA variants (∼50% novel mutations) in the genes RP1, USH2A, CNGB3, NMNAT1, CHM, and ABCA4, responsible for retinitis pigmentosa, Usher syndrome, achromatopsia, Leber congenital amaurosis, choroideremia, or recessive Stargardt/cone-rod dystrophy cases.

Conclusions/Significance

Despite the absence of genetic information from other family members that could help excluding nonpathogenic DNA variants, we could detect causative mutations in a variety of genes known to represent a wide spectrum of clinical phenotypes in 83% of the patients analyzed. Considering the constant drop in costs for human exome sequencing and the relative simplicity of the analyses made, this technique could represent a valuable tool for molecular diagnostics or genetic research, even in cases for which no genotypes from family members are available.     

In vitro differentiation of human mesenchymal stem cells to epithelial lineage

Our study examined whether human bone marrow-derived MSCs are able to differentiate, in vitro, into functional epithelial-like cells. MSCs were isolated from the sternum of 8 patients with different hematological disorders. The surface phenotype of these cells was characterized.To induce epithelial differentiation, MSCs were cultured using Epidermal Growth Factor, Keratinocyte Growth Factor, Hepatocyte Growth Factor and Insulin-like growth Factor-II. Differentiated cells were further characterized both morphologically and functionally by their capacity to express markers with specificity for epithelial lineage. The expression of cytokeratin 19 was assessed by immunocytochemistry, and cytokeratin 18 was evaluated by quantitative RT-PCR (Taq-man). The data demonstrate that human MSCs isolated from human bone marrow can differentiate into epithelial-like cells and may thus serve as a cell source for tissue engineering and cell therapy of epithelial tissue.     

Inheritance of cholesterol metabolism of probands with high or low cholesterol absorption

Heredity of cholesterol absorption and synthesis was studied in siblings of hypercholesterolemic probands with low and high serum cholestanol to cholesterol ratio (assumed to indicate low and high absorption of cholesterol, respectively). Cholesterol synthesis was assayed with sterol balance technique and measuring serum cholesterol precursor to cholesterol ratios (synthesis markers of cholesterol), and cholesterol absorption with measuring dietary cholesterol absorption percentage and serum plant sterol and cholestanol to cholesterol ratios (absorption markers of cholesterol). In the siblings of the low absorption families, cholesterol absorption percentage and ratios of absorption markers were significantly lower, and cholesterol and bile acid synthesis, cholesterol turnover, fecal steroids and ratios of synthesis markers significantly higher than in the siblings of the high absorption families. The ratios of absorption and synthesis markers were inversely interrelated, and they were correlated with cholesterol absorption and synthesis in the siblings. In addition, low absorption was associated with high body mass index, low HDL cholesterol, and serum sex hormone binding globulin levels, suggesting that low absorption was associated with metabolic syndrome. Intrafamily correlations were significant for serum synthesis markers, cholestanol, triglycerides, and blood glucose level. In conclusion, cholesterol absorption efficiency and synthesis are partly inherited phenomena, and they can be predicted by the ratios of non-cholesterol sterols to cholesterol in serum.     

Stress protein flux during recovery from simulated ischemia: induced heat shock protein 70 confers cytoprotection by suppressing JNK activation and inhibiting apoptotic cell death

Multiple stress proteins are recruited in response to stress in living cells. There are limited reports in the literature analyzing multiple stress protein shifts and their functional consequences on stress response. Using two-dimensional electrophoresis we have analyzed shifts in stress protein profiles in response to energy deprivation as a model of ischemic injury to kidneys. A group of chaperones and stress-induced mitogen activated protein (MAP) kinases were analyzed. In addition to examining stress protein induction and phosphorylation we have also examined the mechanism of cytoprotection by heat shock protein 70 (Hsp70). Our results show that, of the different stress proteins examined, only binding protein (BiP) and Hsp70 were significantly induced upon energy deprivation. Other stress proteins, including Hsp27, calnexin, Hsp90 and ERp57 showed alterations in their phosphorylation profiles. Three different MAP kinases, namely p38, extracellular signal regulated kisase and c-jun N-terminal kinase (JNK) were activated in response to energy deprivation. While JNK activation was linked to apoptosis, activated-p38 was involved in phosphorylation of Hsp27. Study of inhibitors of Hsp70 induction or pre-induction of Hsp70 indicated that induced Hsp70 was involved in the suppression of JNK activation thereby inhibiting apoptotic cell death. Our results provide important insights into the flux in stress protein profiles in response to simulated ischemia and highlight the antiapoptotic, cytoprotective mechanism of Hsp70 action.     

Three-dimensional structure of heat shock protein 90 from <Emphasis Type="Italic">Plasmodium falciparum</Emphasis>: molecular modelling approach to rational drug design against malaria

We have recently implicated heat shock protein 90 from Plasmodium falciparum (PfHsp90) as a potential drug target against malaria. Using inhibitors specific to the nucleotide binding domain of Hsp90, we have shown potent growth inhibitory effects on development of malarial parasite in human erythrocytes. To gain better understanding of the vital role played by PfHsp90 in parasite growth,we have modeled its three dimensional structure using recently described full length structure of yeast Hsp90.S equence similarity found between PfHsp90 and yeast Hsp90 allowed us to model the core structure with high confidence. The superimposition of the predicted structure with that of the template yeast Hsp90 structure reveals an RMSD of 3.31 Angstrom. The N-terminal and middle domains showed the least RMSD (1.76 Angstrom) while the more divergent C-terminus showed a greater RMSD (2.84 Angstrom) with respect to the template. The structure shows overall conservation of domains involved in nucleotide binding, ATPase activity, co-chaperone binding as well as inter-subunit interactions. Important co-chaperones known to modulate Hsp90 function in other eukaryotes are conserved in malarial parasite as well. An acidic stretch of amino acids found in the linker region, which is uniquely extended in PfHsp90 could not be modeled in this structure suggesting a flexible conformation. Our results provide a basis to compare the overall structure and functional pathways dependent on PfHsp90 in malarial parasite. Further analysis of differences found between human and parasite Hsp90 may make it possible to design inhibitors targeted specifically against malaria.