Differences in protein patterns of gill epithelial cells of the fish Gillichthys mirabilis after osmotic and thermal acclimation

Different protein patterns in gill epithelium of a euryhaline and eurythermal teleost fish (Gillichthys mirabilis, Family Gobiidae) in response to long-term (2 months) osmotic and thermal acclimation were found for the first time. Gill epithelial cells were isolated to remove extracellular proteins and quantify specialized cell types. Chloride cells were identified on the basis of size (>10 m) and bright appearance after [2-(p-dimethylaminostyryl)-1-methyl-pyridinium-iodine] staining. Small mitochondria-rich cells were <5 m in diameter and showed intermediate fluorescence. Abundance of chloride cells and small mitochondria-rich cells was significantly influenced by osmotic but not thermal acclimation (dilute seawater/25°C: 1.4±0.2% chloride cells, 11.9±4.6% small mitochondria-rich cells; seawater/25°C: 2.4±0.6% chloride cells, 2.2±1.3% small mitochondria-rich cells; seawater/10°C: 2.9±0.3% chloride cells, 1.2±0.7% small mitochondria-rich cells). Pavement cells, identified by low fluorescence and intermediate size (5–10 m), largely predominated under all conditions (>85% of cells). Thus, they represented the major protein source in gill epithelium. Differences in protein patterns were detectable using two-dimensional but not one-dimensional electrophoresis. Of 602 proteins identified by charge and molecular weight properties, only two were induced by high temperature (25°C) and three in response to cold acclimation (10°C). Nine proteins were induced in diluted seawater-acclimated fish, whereas no seawater-induced proteins were found. We hypothesize that proteins induced under dilute seawater conditions are important for the function of pavement cells in gills of hyper-osmoregulating G. mirabilis.Abbreviations BCA bicinchoninic acid - BSS balanced salt solution - CC chloride cells - CLB cell lysis buffer - DASPMI [2-(p-dimethylaminostryryl)-1-methylpyridinium-iodine] - DSW diluted sea water - DTT dithiothreitol - EDTA ethylene-diaminetetraacetate - FW fresh water - IEF isoelectric focusing - PC pavement cells - PDA diacrylpiperazine - pI isoelectric point(s) - PMSF phenylmethanesulphonylfluoride - SDS sodium dodecyl sulfate - SMRC small mitochondria-rich cells - SW sea water - TEMED tetramethylenediamine     

Melting profile and temperature dependent binding constant of an anticancer drug daunomycin-DNA complex

We calculate thermal fluctuational base pair opening probability and the drug binding constant of a daunomycin-bound Poly d(CGTA) · Poly d(TACG) at temperatures from room temperature to its melting temperature. For comparison we also carry out a calculation on a drug-free DNA with the same sequence. Our calculations are carried out by means of a statistical approach using microscopic structures and established force fields and with cooperative effects incorporated into the algorithm. Both hydrogen bond disruption probabilities and drug unstacking probability are determined self-consistently. These probabilities are then used to determine temperature dependent base pair opening probabilities and the drug binding constant. The calculated base pair opening probabilities and drug binding constant are found to be in fair agreement with experiments carried out at room temperature. Our calculation shows cooperative base pair disruption and drug dissociation at certain critical temperatures close to the observed melting temperatures for similar helices. We find that the temperature dependence of the drug binding constant fits well to the van't Hoff relation, in agreement with observations. Our calculation indicates the occurrence of a premelting transition in the drug-bound DNA helix. Some comments are made about this premelting transition.     

Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells

Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and Δ2-tubulin variants were detected on α-tubulin subunits; polyglutamylation was also found on β-tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and Δ2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and Δ2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of α- and β-tubulin molecules, respectively, revealed that 11 isoforms belonged to the α-subunit and 11 isoforms to the β-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several α-tubulin isoforms, antibodies against nontyrosinated and Δ2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism. Received: 2 May 1996 / Accepted: 16 September 1996     

Mitochondrial structure and dynamics as critical factors in honey bee (Apis mellifera L.) caste development

The relationship between nutrition and phenotype is an especially challenging question in cases of facultative polyphenism, like the castes of social insects. In the honey bee, Apis mellifera, unexpected modifications in conserved signaling pathways revealed the hypoxia response as a possible mechanism underlying the regulation of body size and organ growth. Hence, the current study was designed to investigate possible causes of why the three hypoxia core genes are overexpressed in worker larvae. Parting from the hypothesis that this has an endogenous cause and is not due to differences in external oxygen levels we investigated mitochondrial numbers and distribution, as well as mitochondrial oxygen consumption rates in fat body cells of queen and worker larvae during the caste fate-critical larval stages. By immunofluorescence and electron microscopy we found higher densities of mitochondria in queen larval fat body, a finding further confirmed by a citrate synthase assay quantifying mitochondrial functional units. Oxygen consumption measurements by high-resolution respirometry revealed that queen larvae have higher maximum capacities of ATP production at lower physiological demand. Finally, the expression analysis of mitogenesis-related factors showed that the honey bee TFB1 and TFB2 homologs, and a nutritional regulator, ERR, are overexpressed in queen larvae. These results are strong evidence that the differential nutrition of queen and worker larvae by nurse bees affects mitochondrial dynamics and functionality in the fat body of these larvae, hence explaining their differential hypoxia response.     

The differential effects produced by cis- and trans-DDP on DNA in calf thymus nucleosomes

Calf thymus nucleosomes containing H1 were treated with dichlorodiammineplatinum (DDP) at low binding ratios (r = 0.05–0.15). Change in the electrophoretic mobility of the extracted nucleosomal DNA was observed following treatment with cis-DDP and little change with trans-DDP. There was a decrease in the electrophoretic mobility of the nucleosomal DNA as well as obliteration of the nucleosomal repeat distance. The fluorescence intensity of terbium binding to the extracted DNA showed minimal change following drug treatment. However, the thermal melting behavior of the nucleosomal DNA was altered to a greater extent following cis-DDP treatment at 280 rather than 260 nm and a destabilization of the DNA helix was observed. These data suggest that in the whole nucleosome, cis-DDP produces greater structural effects on the packaged DNA than trans-DDP, although similar amounts of drug are bound with both isomers.     

Studies on the interaction between nanodiamond and human hemoglobin by surface tension measurement and spectroscopy methods

In this study, a novel method to probe molecular interactions and binding of human hemoglobin (Hb) with nanodiamond (ND) was introduced based on the surface tension measurement. This method complements conventional techniques, which are basically done by zeta potential and dynamic light scattering (DLS) measurements, near and far circular dichroism (CD) spectroscopy, intrinsic and extrinsic fluorescence spectroscopy. Addition of ND to Hb solution increased the surface tension value of Hb–ND complex relative to those of Hb and ND molecules. The zeta potential values reveled that Hb and ND provide identical charge distribution at pH 7.5. DLS measurements demonstrated that Hb, ND, and ND–Hb complex have hydrodynamic radiuses of 98.37 ± 4.57, 122.07 ± 7.88 nm and 62.27 ± 3.70 at pH of 7.5 respectively. Far and near UV-CD results indicated the loss of α-helix structure and conformational changes of Hb, respectively. Intrinsic fluorescence data demonstrated that the fluorescence quenching of Hb by ND was the result of the static quenching. The hydrophobic interaction plays a pivotal role in the interaction of ND with Hb. Fluorescence intensity changes over time revealed conformational change of Hb continues after the mixing of the components (Hb–ND) till 15 min, which is indicative of the denaturation of the Hb relative to the protein control. Extrinsic fluorescence data showed a considerable enhancement of the ANS fluorescence intensity of Hb–ND system relative to the Hb till 60 nM of ND, likely persuaded by greater exposure of nonpolar residues of Hb hydrophobic pocket. The remarkable decrease in T_m value of Hb in Hb–ND complex exhibits interaction of Hb with ND conducts to conformational changes of Hb. This study offers consequential discrimination into the interaction of ND with proteins, which may be of significance for further appeal of these nanoparticles in biotechnology prosecution.     

Thermal melting of chromatin from the pancreas and liver of guinea pigs treated with 1-methyl-1-nitrosourea

The carcinogen 1-methyl-1-nitrosourea (MNU) can cause pancreatic cancer in guinea pigs. We have examined the relative damage produced by MNU treatment on the chromatin from pancreas and liver of these animals. Thermal denaturation of chromatin from guinea pig pancreas and liver was studied following parenteral administration of MNU in several doses. Estimates of single strand breakage were also obtained by examination of the fluorescence of intercalated ethidium bromide. Oligomeric chromatin melted with a main Tm at 78 degrees C, with additional components at 48 degrees C, 55 degrees C and 65 degrees C. Repetitive treatment with MNU at several doses between 20 mg/kg and 70 mg/kg produced destabilization of pancreatic chromatin as shown by a shift from 78 degrees C to lower melting components. The liver by contrast was relatively unaffected. In addition, pancreatic chromatin showed an increase in alkali-induced strand unwinding with MNU treatment, probably due to an increase in single strand breaks, while there was no change in this regard in the liver. The data indicate that the pancreas is more susceptible to damage by MNU than the liver.     

Hereditary tyrosinemia type 1 metabolites impair DNA excision repair pathways

Hereditary tyrosinemia type 1 is an autosomal recessive metabolic disorder, which is caused by a defective fumarylacetoacetate hydrolase enzyme, and consequently metabolites such as succinylacetone and p-hydroxyphenylpyruvate accumulate. We used a modified comet assay to determine the effect of these metabolites on base- and nucleotide excision repair pathways. Our results indicate that the metabolites affected the repair mechanisms differently, since the metabolites had a bigger detrimental effect on BER than on NER.     

Variant rs2237892 of KCNQ1 Is Potentially Associated with Hypertension and Macrovascular Complications in Type 2 Diabetes Mellitus in A Chinese Han Population

KCNQ1 has been identified as a susceptibility gene of type 2 diabetes mellitus(T2DM) in Asian populations through genome-wide association studies. However, studies on the association between gene polymorphism of KCNQ1 and T2 DM complications remain unclear. To further analyze the association between different alleles at the single nucleotide polymorphism(SNP) rs2237892 within KCNQ1 and TD2 M and its complications, we conducted a case-control study in a Chinese Han population. The C allele of rs2237892 variant contributed to susceptibility to T2DM(odds ratio [OR], 1.45; 95% confidence interval [CI], 1.20–1.75). Genotypes CT(OR, 1.97; 95% CI,1.24–3.15) and CC(OR, 2.49; 95% CI, 1.57–3.95) were associated with an increased risk of T2 DM. Multivariate regression analysis was performed with adjustment of age, gender, and body mass index. We found that systolic blood pressure(P = 0.015), prevalence of hypertension(P = 0.037), and risk of macrovascular disease(OR, 2.10; CI, 1.00–4.45) were significantly higher in subjects with the CC genotype than in the combined population with genotype either CT or