Maturation-promoting factor governs mitogen-activated protein kinase activation and interphase suppression during meiosis of rat oocytes

Meiosis is a particular example of a cell cycle, characterized by two successive divisions without an intervening interphase. Resumption of meiosis in oocytes is associated with activation of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). The activity of MPF declines during the transition between the two meiotic divisions, whereas the activity of MAPK is sustained. Attempts to disclose the interplay between these key regulators of meiosis in both amphibian and mammalian oocytes generated contradictory results. Furthermore, the enzyme that governs the suppression of interphase in mammals is still unidentified. To our knowledge, we provide herein the first demonstration in a mammalian system that inhibition of MPF at reinitiation of meiosis abrogated Mos expression and MAPK activation. We also show that oocytes, in which reactivation of MPF at completion of the first telophase was prevented, exhibited an interphase nucleus with decondensed chromosomes. Inhibition of MAPK did not interfere with the progression to the second meiotic metaphase but, rather, resulted in parthenogenic activation. We conclude that in rat oocytes, MPF regulates MAPK activation and its timely reactivation prevents the oocytes from entering interphase.     

No evidence for consistent virus-specific immunity in simian immunodeficiency virus-exposed, uninfected rhesus monkeys

Defining the immune correlates of the protection against human immunodeficiency virus type 1 (HIV-1) acquisition in individuals who are exposed to HIV-1 but do not become infected may provide important direction for the creation of an HIV-1 vaccine. We have employed the simian immunodeficiency virus (SIV)/rhesus monkey model to determine whether monkeys can be repeatedly exposed to a primate lentivirus by a mucosal route and escape infection and whether virus-specific immune correlates of protection from infection can be identified in uninfected monkeys. Five of 18 rhesus monkeys exposed 18 times by intrarectal inoculation to SIVmac251 or SIVsmE660 were resistant to infection, indicating that the exposed/uninfected phenotype can be reproduced in a nonhuman primate AIDS model. However, routine peripheral blood lymphocyte gamma interferon enzyme-linked immunospot (ELISPOT), tetramer, and intracellular cytokine staining assays, as well as cytokine-augmented ELISPOT and peptide-stimulated tetramer assays, failed to define a systemic antigen-specific cellular immune correlate to this protection. Further, local cell-mediated immunity could not be demonstrated by tetramer assays of these protected monkeys, and local humoral immunity was not associated with protection against acquisition of virus in another cohort of mucosally exposed monkeys. Therefore, resistance to mucosal infection in these monkeys may not be mediated by adaptive virus-specific immune mechanisms. Rather, innate immune mechanisms or an intact epithelial barrier may be responsible for protection against mucosal infection in this population of monkeys.     

Membrane targeting of ribosomes and their release require distinct and separable functions of FtsY

The mechanism underlying the interaction of the Escherichia coli signal recognition particle (SRP) receptor FtsY with the cytoplasmic membrane is not fully understood. We investigated this issue by utilizing active (NG+1) and inactive (NG) mutants of FtsY. In solution, the mutants comparably bind and hydrolyze nucleotides and associate with SRP. In contrast, a major difference was observed in the cellular distribution of NG and NG+1. Unlike NG+1, which distributes almost as the wild-type receptor, the inactive NG mutant accumulates on the membrane, together with ribosomes and SRP. The results suggest that NG function is compromised only at a later stage of the targeting pathway and that despite their identical behavior in solution, the membrane-bound NG-SRP complex is less active than NG+1-SRP. This notion is strongly supported by the observation that lipids stimulate the GTPase activity of NG+1-SRP, whereas no stimulation is observed with NG-SRP. In conclusion, we propose that the SRP receptor has two distinct and separable roles in (i) mediating membrane targeting and docking of ribosomes and (ii) promoting their productive release from the docking site.     

Can the Red-Green Duochrome Test Be Used Prior to Correcting the Refractive Cylinder Component?

Purpose

A primary task of the eye care professional is determining the refraction, or optical correction, of a patient. The duochrome red-green test is a standard tool for verification of the final refraction. Traditionally, it is recommended for use both prior to and subsequent to determining the cylindrical or astigmatic component of the refraction. In order for it to be effective when used before correcting the cylinder it is necessary that the COLC (Circle of Least Confusion) be on the retina. This study examined whether it is necessarily true that the duochrome response in uncorrected astigmatism will be as trust-worthy as it is with corrected cylinders.

Methods

The red-green examination was performed monocularly under the following three conditions: a. fully corrected refraction for the subgroup of eyes that had spherical refractions and for the subgroup of eyes with sphero-cylindrical refractions. b. best sphere-only correction without cylinder correction in sphero-cylindrical eyes c. an induced cylinder error in spherical eyes. The interval between the last “red” response and the first “green” response for the right eyes as a group and separately for the physiological cylinder and induced cylinder correction sub-groups was calculated and compared using a paired, two-tailed t-test.

Results

The intervals between “red” and “green” responses were not significantly different in the population as a whole and in the uncorrected physiological cylinder and induced cylinder subgroups examined.

Conclusion

Based on the finding that the interval of red-green equality with fully corrected cylinder and without the cylindrical correction are not significantly different, the red-green duochrome test can indeed be used both before and after cylindrical correction.     

Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 3. Short- and long-time dynamics of the excited-state proton transfer

Steady-state emission, femtosecond pump-probe spectroscopy, and time-correlated single-photon counting (TCSPC) measurements were used to study the photophysics and the excited-state proton transfer (ESPT) reactions in the green fluorescent protein (GFP) variant S65T/H148D at three pH values: 6.0, 7.9, and 9.5. Selective mutation of GFP caused a dramatic change in the steady-state and excited-state behavior as compared to the wild-type GFP (wt-GFP) studied earlier. An excitation wavelength dependence of the quantum yield of the strong emission band at 510 nm (I* band) indicates the competition between adiabatic and non-adiabatic excited-state proton-transfer reactions. Pump-probe measurements show that the signal buildup probed at 510 nm is biphasic, where 0.8 of the signal amplitude is ultrashort, <150 fs, and 0.2 of the signal decreases with a approximately 10 ps time constant. This effect is a summary result of adiabatic ESPT to the carboxylate group of Asp148 and nonradiative processes. When compared with the luminescence of wt-GFP, the steady-state intensity at 450 nm is lower by a factor of about 10. This very weak emission at 450 nm has a nonexponential decay. It is relatively pH insensitive and, at about 25 ps, is almost twice as long as in wt-GFP. The former exhibits a surprisingly small kinetic deuterium isotope effect (KDIE), compared with the KDIE of about 5 for wt-GFP. Such weak proton dependence may indicate that this emission comes from the species not directly involved in the ESPT. In contrast, pH and H/D isotope dependence of the intense nonexponential luminescence decay of the S65T/H148D deprotonated form measured at 510 nm may result from an isomerization-induced deactivation that is accompanied by the proton recombination quenching. The data are complementary to the femtosecond time-resolved emission data obtained by ultrafast fluorescence up-conversion spectroscopy, found in the preceding paper (Shi et al.). The spectroscopic results are discussed on the basis of the detailed X-ray structure of the mutant published in the preceding paper (Shu et al.).     

Cloning and characterization of the tomato karyopherin alpha1 gene promoter

The karyopherin alpha1 (LeKAPalpha 1) gene of tomato (Lycopersicon esculentum) encodes a receptor involved in nuclear import. To analyze the expression pattern of this gene, a genomic clone containing its upstream region was isolated and sequenced. To study the promoter functionality, a 2170 bp fragment (LM1), was fused to glucuronidase (GUS) and introduced into petunia cells by particle bombardment. For further characterization of the promoter, one inverse and three deletion constructs were studied in cell suspension. To follow its expression in tobacco leaves, transgenic plants expressing GUS under the control of the LM1 promoter were made. Expression of LM1-GUS was largely restricted to actively growing leaf regions, suggesting possible involvement of active cell division and plant growth regulators in LeKAPalpha 1 expression.     

Sequential ATP-induced allosteric transitions of the cytoplasmic chaperonin containing TCP-1 revealed by EM analysis

The eukaryotic cytoplasmic chaperonin containing TCP-1 (CCT) is a hetero-oligomeric complex that assists the folding of actins, tubulins and other proteins in an ATP-dependent manner. To understand the allosteric transitions that occur during the functional cycle of CCT, we imaged the chaperonin complex in the presence of different ATP concentrations. Labeling by monoclonal antibodies that bind specifically to the CCTalpha and CCTdelta subunits enabled alignment of all the CCT subunits of a given type in different particles. The analysis shows that the apo state of CCT has considerable apparent conformational heterogeneity that decreases with increasing ATP concentration. In contrast with the concerted allosteric switch of GroEL, ATP-induced conformational changes in CCT are found to spread around the ring in a sequential fashion that may facilitate domain-by-domain substrate folding. The approach described here can be used to unravel the allosteric mechanisms of other ring-shaped molecular machines.