Mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin

The mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) to bovine serum albumin was studied by relaxation methods as well as the binding isotherm using gel chromatography. A single relaxation was observed over a wide range of HABA concentration except at the extremes of high concentration where another slow process was observed. The concentration dependence of the reciprocal relaxation time of the fast process decreased monotonically with increase in concentration of HABA at constant polymer concentration. The data were analyzed on the basis of Brown's domain structure model and were found to be consistent with a sequential binding mechanism. The azohydrazon tautomerism of HABA was identified with the intramolecular step of the complex. The activation parameters of the step, determined from the temperature dependence of the relaxation time of the fast process, showed that this step is rate limited by an enthalpy barrier in both forward and backward directions. Comparison of the activation parameters with those of other serum albumin-ligand systems suggests that there is an enthalpy-entropy compensation in the activation process of the intramolecular step with the compensation temperature at about 270 K; the enthalpy-entropy compensation is thought to be related to the hydrophobic nature of the ligand.     

Collagen receptors mediate early events in the attachment of epithelial (MDCK) cells

Summary Madin-Darby canine kidney (MDCK) cells kept in suspension culture for 12–15 hr displayed high-affinity binding sites for¹²⁵I-lathyritic (soluble) collagen (120,000/cell,K _D =30nm) and preferred collagens types I and IV over laminin or fibronectin as substrates during the first hour of attachment. On the other hand, after 4 hr, attachment to all four substrates was equally efficient. Upon challenge with a collagen substrate, the high-affinity sites were rapidly recruited on it (T1/2=6 min). Their occupancy by soluble collagen triggered the exocytosis of a second large population of low-affinity collagen binding sites that included laminin and seems to be involved in a second cell-attachment mechanism. These results are compatible with a twostep model of MDCK cell attachment to the substrate: first, via high-affinity collagen binding sites, and second, via laminin of cellular origin.     

Thermal inhibition of repair of methylmethane sulfonate-damaged DNA in chick embryo fibroblasts

Chick embryo fibroblasts were treated with the monofunctional alkylating agent methylmethane sulfonate at various concentrations for 1 h at 42°C, rinsed and then incubated post-treatment at various temperatures at which the kinetics of alkali-labile bond disappearance was followed. Growth experiments showed that these cells grew similarly at temperatures of either 37°C or 42°C. Repair as assessed by removal of alkali-labile bond was also similar for postincubation in the temperature range 37–42°C for damage due to methylmethane sulfonate treatment at concentrations less than 1.5 mM. When the postincubation temperature was raised higher than 42.5–43°C, this type of repair was stopped. The normal internal body temperature of adult chickens is about 41.6°C. Hence the present finding indicates that chick cells are much more severely restricted in DNA repair at temperatures above normal than are mammalian cells, which can function in this respect for several deg. C above 37°C.     

Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

Plant intraspecific functional trait variation is related to within‐habitat heterogeneity and genetic diversity in Trifolium montanum L.

Intraspecific trait variation (ITV), based on available genetic diversity, is one of the major means plant populations can respond to environmental variability. The study of functional trait variation and diversity has become popular in ecological research, for example, as a proxy for plant performance influencing fitness. Up to now, it is unclear which aspects of intraspecific functional trait variation (iFD_CV) can be attributed to the environment or genetics under natural conditions. Here, we examined 260 individuals from 13 locations of the rare (semi‐)dry calcareous grassland species Trifolium montanum L. in terms of iFD_CV, within‐habitat heterogeneity, and genetic diversity. The iFD_CV was assessed by measuring functional traits (releasing height, biomass, leaf area, specific leaf area, leaf dry matter content, F_v/F_m, performance index, stomatal pore surface, and stomatal pore area index). Abiotic within‐habitat heterogeneity was derived from altitude, slope exposure, slope, leaf area index, soil depth, and further soil factors. Based on microsatellites, we calculated expected heterozygosity (H_e) because it best‐explained, among other indices, iFD_CV. We performed multiple linear regression models quantifying relationships among iFD_CV, abiotic within‐habitat heterogeneity and genetic diversity, and also between separate functional traits and abiotic within‐habitat heterogeneity or genetic diversity. We found that abiotic within‐habitat heterogeneity influenced iFD_CV twice as strong compared to genetic diversity. Both aspects together explained 77% of variation in iFD_CV ( = .77, F_{2, 10} = 21.66, p < .001). The majority of functional traits (releasing height, biomass, specific leaf area, leaf dry matter content, F_v/F_m, and performance index) were related to abiotic habitat conditions indicating responses to environmental heterogeneity. In contrast, only morphology‐related functional traits (releasing height, biomass, and leaf area) were related to genetics. Our results suggest that both within‐habitat heterogeneity and genetic diversity affect iFD_CV and are thus crucial to consider when aiming to understand or predict changes of plant species performance under changing environmental conditions.     

MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25 years, and lack of auto-antibodies. It accounts for 2–5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil.     

Free Radicals and Carcinogenesis

The role of free radicals and active states of oxygen in human cancer is as yet unresolved. Various lines of evidence provide strong but inferential evidence that free radical reactions can be of crucial importance in certain carcinogenic mechanisms. A central point in considering free radical reactions in carcinogenesis is that human cancer is really a group of highly diverse diseases for which the initial causation and the progression to clinical disease occur through a wide variety of mechanisms. Furthermore, for many human cancers it appears that there are alternate pathways capable of tumor initiation and tumor progression. While for certain of these pathways free radical reactions appear necessary, it is unlikely that there are human cancers for which free radicals, or any other mechanism, are sufficient for the entire processbeginning with the genetic alteration leading to a somatic mutation and eventually resulting in clinically overt disease. It is crucial that we view free radical reactions as aong a panoply of mechanisms leading to human cancer, and consider research about the role of free radicals in cancer as opportunities to prevent the initiation or progression of human cancer.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

A DNA Damage Checkpoint Pathway Coordinates the Division of Dikaryotic Cells in the Ink Cap Mushroom Coprinopsis cinerea

The fungal fruiting body or mushroom is a multicellular structure essential for sexual reproduction. It is composed of dikaryotic cells that contain one haploid nucleus from each mating partner sharing the same cytoplasm without undergoing nuclear fusion. In the mushroom, the pileus bears the hymenium, a layer of cells that includes the specialized basidia in which nuclear fusion, meiosis, and sporulation occur. Coprinopsis cinerea is a well-known model fungus used to study developmental processes associated with the formation of the fruiting body. Here we describe that knocking down the expression of Atr1 and Chk1, two kinases shown to be involved in the response to DNA damage in a number of eukaryotic organisms, dramatically impairs the ability to develop fruiting bodies in C. cinerea, as well as other developmental decisions such as sclerotia formation. These developmental defects correlated with the impairment in silenced strains to sustain an appropriated dikaryotic cell cycle. Dikaryotic cells in which chk1 or atr1 genes were silenced displayed a higher level of asynchronous mitosis and as a consequence aberrant cells carrying an unbalanced dose of nuclei. Since fruiting body initiation is dependent on the balanced mating-type regulator doses present in the dikaryon, we believe that the observed developmental defects were a consequence of the impaired cell cycle in the dikaryon. Our results suggest a connection between the DNA damage response cascade, cell cycle regulation, and developmental processes in this fungus.