This study analyzed whether therapy with CAMEL, an antimicrobial peptide (KWKLFKKIGAVLKVL), possess anticancer benefits. Although the peptide was cytotoxic for all the cell lines tested, it did not cause hemolysis, which suggests that CAMEL does not damage cell membranes. After cellular internalization, CAMEL localized to mitochondria and lowered the mitochondrial potential, resulting in the organelles' swelling, a decrease in cellular ATP level and, finally, cellular breakdown. High mobility group box 1 (HMGB1) protein, a necrotic death marker, was shown to be released from cells treated with CAMEL. Growth of B16-F10 melanoma tumors was clearly restrained after injections with CAMEL and could be kept in check throughout the period of peptide administration. However, if therapy was stopped, tumors started to grow again 3-4 days later. To reduce tumor volume and block tumor relapse, a combined therapy was required involving CAMEL and plasmid DNA carrying the interleukin-12 (IL-12) gene. The two therapeutic agents used in combination (a series of CAMEL injections first, followed by daily administration of plasmid DNA) delayed tumor growth and extended survival of treated animals in a statistically significant manner. Complete tumor regression was found in 60% of cases.Laboratory Investigation (2010) 90, 940-952;
Atrial septal defect (ASD) is an incomplete septation of atria in human heart causing circulatory problems. Its frequency is estimated at one per 10 000. Actions of numerous genes have been linked to heart development. However, no single gene defect causing ASD has yet been identified. Incomplete heart septation similar to ASD was reported in transgenic mice with both inactive alleles of gene encoding mammalian zinc metalloprotease a mammalian tolloid-like 1 (tll1). Here, we have screened 19 ASD patients and 15 healthy age-matched individuals for mutations in TLL1 gene. All 22 exons were analyzed exon by exon for heteroduplex formation. Subsequently, DNA fragments forming heteroduplexes were sequenced. In four nonrelated patients, three missense mutations in coding sequence, and one single base change in the 5 0 UTR have been detected. Two mutations (Met182Leu, and Ala238Val) were detected in ASD patients with the same clinical phenotype. As the second mutation locates immediately upstream of the catalytic zinc-binding signature, it might change the enzyme substrate specificity. The third change, Leu627Val in the CUB3 domain, has been found in an ASD patient with interatrial septum aneurysm in addition to ASD. The CUB3 domain is important for substrate-specific recognition. In the remaining 15 patients as well as in 15 reference samples numerous base substitutions, deletions, and insertions have been detected, but no mutations changing the coding sequence have been found. Lack of mutations in relation to ASD of these patients could possibly be because of genetic heterogeneity of the syndrome.
Osteogenesis imperfecta (OI) is a bone dysplasia caused by mutations in the COL1A1 and COL1A2 genes. Although the condition has been intensely studied for over 25 years and recently over 800 novel mutations have been published, the relation between the location of mutations and clinical manifestation is poorly understood. Here we report missense mutations in COL1A1 of several OI patients. Two novel mutations were found in the D1 period. One caused a substitution of glycine 200 by valine at the N-terminus of D1 in OI type I/IV, lowering collagen stability by 50% at 34 degrees C. The other one was a substitution of valine 349 by phenylalanine at the C-terminus of D1 in OI type I, lowering collagen stability at 37.5 degrees C. Two other mutations, reported before, changed amino residues in D4. One was a lethal substitution changing glycine 866 to serine in genetically identical twins with OI type II. That mutated amino acid was near the border of D3 and D4. The second mutation changed glycine 1040 to serine located at the border of D4 and D0.4, in a proband manifesting OI type III, and lowered collagen stability at 39 degrees C (2 degrees C lower than normal). Our results confirm the hypothesis on a critical role of the D1 and D4 regions in stabilization of the collagen triple-helix. The defect in D1 seemed to produce a milder clinical type of OI, whereas the defect in the C-terminal end of collagen type caused the more severe or lethal types of OI.
This study revealed inconsistencies in the fluid orders as well as mistakes in the fluid monitoring, which illustrates the difficulties of fluid therapy and reinforces the need for strong evidence-based guidelines for hypervolemic therapy in SAH.
Mutations in COL1A1 or COL1A2 genes lead to osteogenesis Imperfecta (OI) in humans. There are three possiblities to successfully treat OI including (1) gene therapy, (2) mesenchymal stem cell (MSC) therapy, or (3) a combination of both. The aim of this study was to develop a model for combined gene/cell OI therapy by targeting Col1a1 and Col1a2 genes with isogenic sequences from corresponding human genes in rat bone marrow (BM)-derived MSCs. The recombination efficacy was tested for five different rat-human-rat hybrid DNAs with rat fragments that were 1 to 4 kb long. For selection of transfected clones a neomycine resistance gene was cotransfected, and clones resistant to G418 (G418(+)) were recovered and screened for integration of specific gene loci in the rat genome. Over 90% of G418(+) clones correctly integrated the rat-human-rat hybrid DNAs, and both OI loci in the rat genome were targeted to a similar degree. Longer homologous sequences integrated into rat collagen genes approximately 10 times more efficiently. Based on our data the nonviral gene targeting technology could be potentially employed to repair collagen genes in OI patients.
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