Effect of cadmium on collagen content and solubility in rat bone

The toxic action of cadmium in the bone tissue is known, but its mechanisms are still unexplained. We examined whether Cd influences collagen content and its solubility in the femoral bone of three-week-old female rats exposed to 5 or 50 mg Cd/l in drinking water. Non-cross linked collagen was extracted with 0.5 M acetic acid, and two acid-insoluble collagen fractions were extracted with pepsin and 4.0 M guanidine hydrochloride, respectively. SDS/PAGE showed the presence of two collagen types, I and V, in all three extracted fractions. Exposure of rats to Cd for 6 months increased the amount of acid-soluble collagens type I and V and decreased the level of acid-insoluble collagens. The amount of total collagen extracted from the bones of rats exposed to 50 mg Cd/l was reduced by about 14% as compared to control and those intoxicated with 5 mg Cd/l. The solubility of type I bone collagen (determined as the percentage of acetic-soluble fraction of total collagen) was increased 2.9- and 3.0-fold in rats intoxicated with 5 and 50 mg Cd/l, respectively. Similarly, the solubility of type V collagen was increased 2.3- and 2.7-fold, respectively. Our results indicate that Cd treatment affects bone collagen by decreasing its content and increasing its solubility.     

Direct sequencing of PCR products for mutation detection in osteogenesis imperfecta

This work present a short and simple method for mutation detection in type I collagen genes, based on the direct sequencing of single-stranded DNA. The sequencing of type I collagen genes is complicated and difficult because of their large size and highly repetitive and GC-rich coding regions. Although many techniques have been developed for mutation screening in osteogenesis imperfecta (OI), they represent different degrees of sensitivity and are difficult to reproduce and too expensive for application in each laboratory. The method described here is short, easy and especially useful for sequencing of collagen genes in OI cases, in which the region with a suspected structural defect is localized by collagen analysis.     

A long-term study (1985–1995) of fish populations in the impounded Warta River,Poland

Three sites, two in the tailwater and one upstream of the backwater of a large man-made reservoir in the middle course of the 808 km long lowland Warta River (Oder catchment), were studied. The pre-impoundment study lasted one (1985), the post-impoundment one 10 yr (1986–1995). The dam contributed to the extinction of the anadromous Vimba vimba, and of the rheophilous Chondrostoma nasus, both cyprinids. A number of directly or indirectly negative changes related to the construction of the reservoir were recorded in the tailwater. These changes affected the species number, spawning groups, diversity, density, standing crop, mean body weight, ABC index, and proportional stock density – PSD. Numerous impacts related to engineering, water chemistry and climate were more negative for fish at the tailwater than at the backwater sites. The hydrology of the site upstream of the backwater continued to be dependent on natural factors. Fish populations there suffered only from accidental impacts of the construction of the dam, such as bank revetment or the clearance of trees and shrubs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytoplasmic inhibitor of eEF-2 ADP-ribosylation catalyzed by diphtheria toxin or endogenous transferase in rat liver cells

eEF-2 (100 kDa) isolated from rat liver cells undergo ADP-ribosylation in the presence of diphtheria toxin or endogenous ADP-ribosyltransferase, which was co-purified with the factor. We separated the fraction free of elongation factor and endogenous transferase, which strongly inhibited the ADP-ribosylation of eEF-2. This fraction did not affect the activity of the elongation factor. The lack of endogenous transferase activity (which is potentially lethal for the cell) in the postribosomal supernatant could be the result of its inhibition. eEF-2 (65 kDa) which is probably responsible for the process of translocation (Gajko, A. et al. (1999) Biochem. Biophys. Res. Commun. 255, 535-538) was protected from ADP-ribosylation and its irreversible inactivation in the presence of the rat liver extract fraction.     

Using anglers' records in Poland as a tool for assessing changes in fish body weight caused by fluctuations of inland water quality

Anglers' records of fish weight (ARFW) were analysed in Poland for the periods 1965–1989 (successive increases in inland water pollution), and 1990–2010 (considerable improvements in water quality). For the initial ARFW analysis, the entire study was divided into nine intervals of 5 years each; the period that followed was divided into three catchments: the Vistula, Oder and Pomeranian rivers, whereby 10 species were chosen to represent obligatory riverine and facultative species. With a few exceptions, the investigated species showed unexpected and varying reactions to the changes in water quality. The ARFW of obligatory riverine barbel Barbus barbus (L.) decreased during the first periods (1965–1989), then increased significantly from 1990 to 2010. For obligatory riverine ide Leuciscus idus (L.), tendencies were less clear but as significant. However, other obligatory riverine species have not shown a relationship between ARFW and water quality. Discrepancies were also recorded in the facultative riverine pike (Esox lucius L.) and brown trout (Salmo trutta L.). Considering the number of medals recorded, there were some interesting tendencies related to the decline or improvement in the water quality, although not in all investigated species. The species displaying a decrease in medals awarded from 1966 to 1989 and increasing after 1990, was chub Leuciscus cephalus (L.), but only until 2005. Species which showed successive increases in awards from 1989 to 2010 were: pike, perch (Perca fluviatilis L.), ide, and wels (Silurus glanis L.). However, for species in the period when the water quality actually deteriorated (1966–1990), the number of medals awarded increased: barbel, brown trout (S. trutta L.), and grayling Thymallus thymallus (L.).     

Anethole prevents hydrogen peroxide-induced apoptosis and collagen metabolism alterations in human skin fibroblasts

Thyroid hormone stimulates erythropoietic differentiation. However, severe anemia is sometimes seen in patients with hyperthyroidism, and the mechanisms have not been fully elucidated. Bone marrow is comprised about 2–8 % oxygen, and the characteristics of hematopoietic stem cells have been shown to be influenced under hypoxia. Hypoxia-inducible factor-1 is a critical mediator of cellular responses to hypoxia and an important mediator in signal transduction of thyroid hormone [triiodothyronine (T3)]. The aim of this study was to investigate the effect of T3 on erythropoiesis under hypoxia mimicking physiological conditions in the bone marrow. We maintained human erythroleukemia K562 cells under hypoxic atmosphere (2 % O₂) and examined their cellular characteristics. Compared to that under normal atmospheric conditions, cells under hypoxia showed a reduction in the proliferation rate and increase in the hemoglobin content or benzidine-positive rate, indicating promotion of erythroid differentiation. T3 had no effect on hypoxia-induced erythroid differentiation, but significantly inhibited activin A/erythroid differentiation factor-induced erythroid differentiation. Moreover, GATA2 mRNA expression was suppressed in association with erythroid differentiation, while T3 significantly diminished that suppression. These results suggest that T3 has a direct suppressive effect on erythroid differentiation under hypoxic conditions.     

Inhibitory effects of pentamidine analogues on protein biosynthesis in vitro

Pentamidine despite its rather high toxicity, is currently in clinical use. For development of new drugs of this type it is important to know the mechanism of their action. Two new amidines (I and II) and 4',6-diamidino-2-phenylindole (DAPI) were found in preliminary experiments to inhibit protein synthesis in vitro in the cell-free rat liver system. The three compounds differed in the precise mode of action. The inhibitory effect of I on the activity of the eukaryotic elongation factor eEF-2 and ribosomes seems to suggest that the binding site of eEF-2 on the ribosome was blocked by this compound. eEF-2 has been identified as the primary target of II and eEF-1 as the primary target of DAPI in the system studied.     

Studies on type I collagen in skin fibroblasts cultured from twins with lethal osteogenesis imperfecta

Studies on type I procollagen produced by skin fibroblasts cultured from twins with lethal type II of osteogenesis imperfecta (OI) showed that biosynthesis of collagen (measured by L-[5-(3)H]proline incorporation into proteins susceptible to the action of bacterial collagenase) was slightly increased as compared to the control healthy infant. SDS/PAGE showed that the fibroblasts synthesized and secreted only normal type I procollagen. Electrophoretic analysis of collagen chains and CNBr peptides showed the same pattern of electrophoretic migration as in the controls. The lack of posttranslational overmodification of the collagen molecule suggested a molecular defect near the amino terminus of the collagen helix. Digestion of OI type I collagen with trypsin at 30 degrees C for 5 min generated a shorter than normal alpha2 chain which melted at 36 degrees C. Direct sequencing of an asymmetric PCR product revealed a heterozygous single nucleotide change C-->G causing a substitution of histidine by aspartic acid in the alpha2 chain at position 92. Pericellular processing of type I procollagen by the twin's fibroblasts yielded a later appearance of the intermediate pC-alpha1(I) form as compared with control cells.     

A novel Gly to Arg substitution at position 388 of the alpha1 chain of type I collagen in lethal form of osteogenesis imperfecta

Cultured skin fibroblasts from a proband with a lethal form of osteogenesis imperfecta produce two forms of type I collagen chains, with normal and delayed electrophoretic migration; collagen of the proband's mother was normal. Peptide mapping experiments localized the structural defect in the proband to alpha1(I) CB8 peptide in which residues 123 to 402 are spaned. Direct sequencing of amplified cDNA covering this region revealed a G to A single base change in one allele of the alpha1(I) chain, that converted glycine 388 to arginine. Restriction enzyme digestion of the RT-PCR product was consistent with a heterozygous COL1A1 mutation. The novel mutation conforms to the linear gradient of clinical severity for the alpha1(I) chain and results in reduced thermal stability by 3 degrees C and intracellular retention of abnormal molecules.     

Fish assemblage changes relative to environmental factors and time in the Warta River, Poland, and its oxbow lakes

Four oxbow lakes and two neighbouring sections of their parent Warta River (Odra River system, Poland) were sampled to investigate differences in fish assemblages between habitats in 1999–2000. Additional comparisons were made with 12 other oxbow lakes in this section of the river that were sampled 30–40 years ago. Downstream of a man‐made reservoir, higher species number, diversity and evenness were recorded in oxbow lakes than in the river channel. Upstream of the reservoir, differences in these variables from both habitats were insignificant. Fluvial and stagnant water samples were clearly separated in the multivariate space of a detrended correspondence analysis (DCA). For two oxbow lakes and two neighbouring sections of the Warta River, 12 abiotic and biotic environmental variables were available, and only velocity, water temperature and conductivity were significantly correlated with canonical correspondence analysis (CCA) axes. Fish assemblages of four recently investigated oxbow lakes were clearly separated in the multivariate space of DCA from other neighbouring oxbows sampled 30–40 years ago. Species previously subdominant were becoming rare. Oxbow lakes that are continuously or at least periodically connected with the channel are indispensable for maintaining high biodiversity and a sustainable fishery in the river system.