The long and short flavodoxins: II. The role of the differentiating loop in apoflavodoxin stability and folding mechanism

Flavodoxins are classified in two groups according to the presence or absence of a approximately 20-residue loop of unknown function. In the accompanying paper (36), we have shown that the differentiating loop from the long-chain Anabaena PCC 7119 flavodoxin is a peripheral structural element that can be removed without preventing the proper folding of the apoprotein. Here we investigate the role played by the loop in the stability and folding mechanism of flavodoxin by comparing the equilibrium and kinetic behavior of the full-length protein with that of loop-lacking, shortened variants. We show that, when the loop is removed, the three-state equilibrium thermal unfolding of apoflavodoxin becomes two-state. Thus, the loop is responsible for the complexity shown by long-chain apoflavodoxins toward thermal denaturation. As for the folding reaction, both shortened and wild type apoflavodoxins display three-state behavior but their folding mechanisms clearly differ. Whereas the full-length protein populates an essentially off-pathway transient intermediate, the additional state observed in the folding of the shortened variant analyzed seems to be simply an alternative native conformation. This finding suggests that the long loop may also be responsible for the accumulation of the kinetic intermediate observed in the full-length protein. Most revealing, however, is that the influence of the loop on the overall conformational stability of apoflavodoxin is quite low and the natively folded shortened variant Delta(120-139) is almost as stable as the wild type protein. The fact that the loop, which is not required for a proper folding of the polypeptide, does not even play a significant role in increasing the conformational stability of the protein supports our proposal (36) that the differentiating loop of long-chain flavodoxins may be related to a recognition function, rather than serving a structural purpose.     

Allozymic variation of the endangered killifishAphanius iberus and its application to conservation

Synopsis Genetic differentiation and patterns of variability in the endangered Iberian endemic,Aphanius iberus, were analyzed by allozyme electrophoresis as a valuable database for conservation purposes. Genetic variability values expressed as heterozygosity (H = 0.015–0.097) were close to the values found in other members of Cyprinodontidae (H = 0.012–0.123). Polymorphism values (P = 0.125–0.542) were higher than reported in the literature (P = 0.036–0.150) for Cyprinodontidae. Significant correlation existed between salinity values and genetic variability expressed as heterozygosity (r = – 0.76, p < 0.01) and polymorphism (r = – 0.60, p = 0.04). Low genetic variability values (H = 0.024–0.055, P = 0.125–0.292) were exhibited by populations which inhabit salty lagoons. The highest values were found in populations occurring in marshes and irrigation channels (H = 0.051–0.097, P = 0.250–0.542). Associations among genetic, geographic and ecological parameters were tested using a Mantel test indicated that most of the genetic distances were explained by geographic distances but not by ecological factors, suggesting that isolation by distance could be the main factor explaining the differentiation between sites. According to the genetic distances obtained, two mainA. iberus groups were discernible: the Atlantic and Mediterranean. Genetic distances between both groups (DRogers = 0.179–0.261) were higher than values between recognized species of other cyprinodontids (DRogers = 0.11–0.27). On the basis of genetic distances we have dated the fragmentation of both populations to the Upper Miocene-Pliocene when most of the Mediterranean sea dried up. Subsequently, gene flow between Mediterranean and Atlantic populations was interrupted. The results of our genetic analyses suggested the existence of five operational conservation units (OCUs) forA. iberus. These units are defined as a continuous area limited by geographical boundaries, and inhabited by one or more populations sharing the same genetic pattern.     

Estradiol: A key biological substrate mediating the response to cocaine in female rats

A consistent finding in drug abuse research is that males and females show differences in their response to drugs of abuse. In women, increased plasma estradiol is associated with increased vulnerability to the psychostimulant and reinforcing effects of drugs of abuse. Our laboratory has focused on the role of estradiol in modulating the response to cocaine. We have seen that ovariectomy increases the locomotor response to a single cocaine injection, whereas estradiol exacerbates the locomotor response to repeated cocaine administration. Cocaine-induced sensitization of brain activity, as measured by fMRI, is also dependent on plasma estradiol. Moreover, we observed that although all ovariectomized rats show conditioned place preference to cocaine, it is more robust in ovariectomized rats with estradiol.Opioid receptors are enriched in brain regions associated with pleasure and reward. We find that in females, the effectiveness of kappa opioid agonists in decreasing the locomotor response to repeated cocaine varies with plasma estradiol. We also find that estradiol regulates the density of mu opioid receptors in brains areas associated with reward. These data hint that in females, estradiol modulates the behavioral effects of cocaine by regulating mu and kappa opioid signaling in mesocorticolimbic brain structures. Identifying the mechanisms that mediate differences in vulnerability to drugs of abuse may lead to effective therapeutic strategies for the treatment and prevention of addiction and relapse. We encourage health practitioners treating persons addicted to drugs to consider gender differences in response to particular pharmacotherapies, as well the sex steroid milieu of the patient.     

Safety and preliminary immunogenicity of MenC/P64k, a meningococcal serogroup C conjugate vaccine with a new recombinant carrier

This study reports the preliminary assessment of the safety and immunogenicity of the first serogroup C conjugate vaccine candidate that includes meningococcal P64k recombinant protein as the carrier (MenC/P64k). Twenty volunteers were recruited for a double-blind, randomized, controlled phase I clinical trial, receiving a single dose of MenC/P64k (study group) and a single dose of the commercial polysaccharide vaccine AC (control group). Only mild reactions were observed. No statistical differences were detected between the antipolysaccharide C IgG responses of both groups as well as between bactericidal serum titre (P > 0.05). The MenC/P64k vaccine was found to have a good safety profile, to be well tolerated and immunogenic.     

Functional interaction of common gamma-chain and growth hormone receptor signaling apparatus

We previously reported on an X-linked SCID (X-SCID) patient, who also had peripheral growth hormone (GH) hyporesponsiveness and abnormalities of the protein phosphorylation events following GH receptor (GHR) stimulation. In the present study, we examined a potential role of common cytokine receptor gamma-chain (gammac) in GHR signaling using EBV-transformed lymphocytes from healthy subjects and gammac-negative X-SCID patients. We demonstrated that the proliferative response to GH stimulation of the B cell lines of gammac-negative patients was impaired despite a comparable cellular expression of GHR molecules to controls. In patients, after GH stimulation, no phosphorylation of STAT5 was observed. In addition, the molecule localization through confocal microscopy revealed that in B cell lines of patients no nuclear translocation of STAT5b following GH stimulation occurred differently from controls. Biochemical analysis of the nuclear extracts of gammac-negative cell lines provided further evidence that the amount of STAT5b and its phosphorylated form did not increase following GH stimulation. In patients, cells reconstituted with wild-type gammac abnormal biochemical and functional events were restored resulting in nuclear translocation of STAT5. Confocal experiments revealed that GHR and gammac were colocalized on the cell membrane. Our study demonstrates the existence of a previously unappreciated relationship between GHR-signaling pathway and gammac, which is required for the activation of STAT5b in B cell lines. These data also confirm that growth failure in X-SCID is primarily related to the genetic alteration of the IL2RG gene.