Thr373, Asp375, and Lys260 are in the coenzyme site of porcine NADP-dependent isocitrate dehydrogenase

Thr(373), Lys(374), Asp(375), and Lys(260) were chosen as site-directed mutagenesis targets within porcine NADP-dependent isocitrate dehydrogenase based on structurally corrected sequence alignment among prokaryotic and eukaryotic NADP-isocitrate dehydrogenases. Wild-type and all mutant enzymes were expressed in Escherichia coli and purified to homogeneity. These mutations do not alter the secondary structure or dimerization state of the mutants. The D375N and K260Q mutants exhibit, respectively, a 15- and 28-fold increase in K(m) for NADP, along with marked decreases in V(max) as compared to wild-type enzyme. In contrast, replacing Lys(374), which was previously proposed to contribute to apparent coenzyme affinity, does not change the enzyme's kinetic parameters. T373S exhibits similar kinetic parameters to those of wild-type while T373A and T373V mutations reduce the V(max) values of the resulting enzymes to 1 and 20%, respectively of that of wild-type. We conclude that a hydroxyl group at position 373 is required for effective enzyme function and that Asp(375) and Lys(260) are critical amino acids contributing to coenzyme affinity as well as catalysis by porcine NADP-isocitrate dehydrogenase.     

Communication between the two active sites of glutathione S-transferase A1-1, probed using wild-type-mutant heterodimers

To study the communication between the two active sites of dimeric glutathione S-transferase A1-1, we used heterodimers containing one wild-type (WT) active site and one active site with a single mutation at either Tyr9, Arg15, or Arg131. Tyr9 and Arg15 are part of the active site of the same subunit, while Arg131 contributes to the active site of the opposite subunit. The V(max) values of Tyr9 and Arg15 mutant enzymes were less than 2% that of WT, indicating their importance in catalysis. In contrast, V(max) values of Arg131 mutant enzymes were about 50-90% of that of WT enzyme while K(m)(GSH) values were approximately 3-8 times that of WT, suggesting that Arg131 plays a role in glutathione binding. The mutant enzyme (with a His(6) tag) and the WT enzyme (without a His(6) tag) were used to construct heterodimers (WT-Y9F, WT-Y9T, WT-R15Q, WT-R131M, WT-R131Q, and WT-R131E) by incubation of a mixture of wild-type and mutant enzyme at pH 7.5 in buffer containing 1,6-hexanediol, followed by dialysis against buffer lacking the organic solvent. The resultant heterodimers were separated from the wild-type and mutant homodimers using chromatography on nickel-nitrilotriacetic acid agarose. The V(max) values of all heterodimers were lower than expected for independent active sites. Our experiments demonstrate that mutation of an amino acid residue in one active site affects the activity in the other active site. Modeling studies show that key amino acid residues and water molecules connect the two active sites. This connectivity is responsible for the cross-talk between the active sites.     

Functional plasticity of dendritic cell subsets as mediated by CD40 versus B7 activation

Murine dendritic cells (DCs) can present Ag in an immunogenic or tolerogenic fashion, the distinction depending on either the occurrence of specialized DC subsets or the maturation or activation state of the DC. Although DC subsets may be programmed to direct either tolerance or immunity, it is not known whether appropriate environmental stimulation can result in complete flexibility of a basic program. Using splenic CD8(-) and CD8(+) DCs that mediate the respective immunogenic and tolerogenic presentation of self peptides, we show that both the in vivo and in vitro activities of either subset can be altered by ligation of specific surface receptors. Otherwise immunogenic CD8(-) DCs become tolerogenic upon B7 ligation by soluble CTLA-4, a maneuver that initiates immunosuppressive tryptophan catabolism. In contrast, CD40 ligation on tolerogenic CD8(+) DCs makes these cells capable of immunogenic presentation. Thus, environmental conditioning by T cell ligands may alter the default function of DC subsets to meet the needs of flexibility and redundancy.     

CXCL12 chemokine and CXCR4 receptor: association with susceptibility and prognostic markers in triple negative breast cancer

CXCL12/CXCR4 signaling has been implicated in breast carcinogenesis, and genetic polymorphisms in these molecules have been associated with different types of cancer. The present study analyzed genetic polymorphisms in CXCL12 (rs1801157, G?>?A) and CXCR4 (rs2228014, C?>?T) and CXCR4 immunostaining in tumor tissues from patients with triple negative breast cancer (TNBC) aiming to evaluate their possible role in its’ susceptibility and prognosis. Genetic polymorphisms were analyzed in 59 TNBC patients and 150 control women; age-adjusted logistic regression showed no association when variants were considered in isolation; however, a statistically significant interaction was noted for heterozygosis for both allelic variants increasing the odds for TNBC (CXCL12-GA by CXCR4-CT: OR 7.23; 95% CI 1.15–45.41; p?=?0.035). CXCL12 polymorphism was correlated negatively with proliferation index (Ki67) (Tau-b?=???0.406; p?=?0.006). CXCR4 immunostaining was evaluated in 37 TNBC patients (22 with paired tumor-normal adjacent tissue). CXCR4 was detected more intensely in cell cytoplasm than in membrane, and was more expressed in tumor than in normal adjacent tissues, although not statistically significant. CXCR4 expression on the membrane of tumor cells was correlated positively with histopathological grade (Tau-b?=?0.271; p?=?0.036) and negatively with lymph node metastasis (Tau-b?=???0.478; p?=?0.036). The present study indicates that CXCL12 and CXCR4 polymorphisms and CXCR4 immunostaining might have susceptibility and prognostic roles in TNBC pathogenesis.     

Multiple homoplasious insertions and deletions of a Triticeae (Poaceae) DNA transposon: a phylogenetic perspective

Background  

Stowaway elements are short, non-autonomous DNA transposons categorized as miniature inverted-repeat transposable elements (MITEs). The high MITE copy number in grass genomes suggests an active history of amplification and insertion, but ongoing MITE activity has only rarely been seen, and ongoing Stowaway activity has never been observed. Thus, a phylogenetic perspective on presence vs. absence of elements in an aligned data set can provide valuable historical insights into the dynamics of MITE acquisition and loss.     

Spatial segregation among age classes in cave salamanders: habitat selection or social interactions?

Within species, individuals with different sexes, morphs and age classes often show spatial segregation. Both habitat selection and social processes have been proposed to explain intraspecific spatial segregation, but their relative importance is difficult to assess. We investigated spatial segregation between age classes in the cave salamander Hydromantes (Speleomantes) strinatii, and used a hypothetico-deductive approach to evaluate whether social or ecological processes explain segregation pattern. We recorded the location and age class of salamanders along multiple caves; we measured multiple microhabitat features of different sectors of caves that may determine salamander distribution. We assessed age-class segregation, and used generalized mixed models and an information-theoretic framework, to test if segregation is explained by social processes or by differences in habitat selection. We found significant age-class segregation, juveniles living in more external cave sectors than adults. Multiple environmental features varied along caves. Juveniles and adults showed contrasting habitat selection patterns: juveniles were associated with sectors having high invertebrate abundance, while adults were associated with scarce invertebrates and low temperature. When the effect of environmental features was taken into account, the relationship between juveniles and adults was non negative. This suggests that different habitat preferences, related to distinct risk-taking strategies of age classes, can explain the spatial segregation. Juveniles require more food and select more external sectors, even if they may be risky. Conversely, adults may trade off food availability in favour of safe areas with stable micro-climate.     

Apoptosis and atrophy in rat slow skeletal muscles in chronic heart failure

Congestive heart failure is characterized by a skeletal musclemyopathy with muscle bulk loss. The mechanisms responsible for thesechanges are not clear at present. We have investigated the role ofapoptosis in the rat "slow" soleus muscle during the developmentof heart failure, which was induced by injection of monocrotaline (30 mg/kg). We looked at the time course of apoptosis by studying sixanimals at each of the following time points: 0, 17, 24, and 30 days.We found a decreased expression of the antiapoptotic protein Bcl-2,which was accompanied by a rise of proapoptotic caspase-3. Ubiquitinlevels did not change. DNA nick-end labeling showed an increased numberof apoptotic nuclei both in myofibers and interstitial cells when heartfailure occurred. At variance with previous observations in thefast-twitch tibialis anterior muscle in the same animals, in whichtumor necrosis factor- (TNF-) increased at the time thatapoptosis occurred, the magnitude of apoptosis is lower in soleusmuscle and there is no appearance of muscle atrophy. In soleus muscle,apoptosis is accompanied by activation of the caspase-3 system. Thereis no activation of the TNF-- and ubiquitin-dependent protein waste.In conclusion, slow muscles are less prone to develop apoptosis thanfast muscles. Muscle atrophy appears earlier in these latter ones.

     

Adaptive Modifications in the Calpain/Calpastatin System in Brain Cells after Persistent Alteration in Ca2+ Homeostasis

Persistent dysregulation in Ca²⁺ homeostasis is a pervasive pathogenic mechanism in most neurodegenerative diseases, and accordingly, calpain activation has been implicated in neuronal cells dysfunction and death. In this study we examined the intracellular functional state of the calpain-calpastatin system in −G93A(+) SOD1 transgenic mice to establish if and how uncontrolled activation of calpain can be prevented in vivo during the course of prolonged [Ca²⁺]_i elevation. The presented data indicate that 1) calpain activation is more extensive in motor cortex, in lumbar, and sacral spinal cord segments compared with the lower or almost undetectable activation of the protease in other brain areas, 2) direct measurements of the variations of Ca²⁺ levels established that the degree of the protease activation is correlated to the extent of elevation of [Ca²⁺]_i, 3) intracellular activation of calpain is always associated with diffusion of calpastatin from perinuclear aggregated forms into the cytosol and the formation of a calpain-calpastatin complex, and 4) a conservative fragmentation of calpastatin is accompanied by its increased expression and inhibitory capacity in conditions of prolonged increase in [Ca²⁺]_i. Thus, calpastatin diffusion and formation of the calpain-calpastatin complex together with an increased synthesis of the inhibitor protein represent a cellular defense response to conditions of prolonged dysregulation in intracellular Ca²⁺ homeostasis. Altogether these findings provide a new understanding of the in vivo molecular mechanisms governing calpain activation that can be extended to many neurodegenerative diseases, potentially useful for the development of new therapeutic approaches.