Photochemical reaction of 9-cis-retro-γ-rhodopsin at low temperatures

9-cis-Retro-γ;rhodopsin (λ_max = 420 nm) was prepared from 9-cis-retro-γ-retinal and cattle opsin. After cooling to liquid nitrogen temperature (77 K), the pigment was irradiated with light at 380 nm. The spectrum shifted to the longer wavelengths, owing to formation of a batho product. This fact indicates that the conjugated double bond system from C-5 to C-8 of the chromophoric retinal in rhodopsin was not necessary for formation of bathorhodopsin. Reirradiation of the batho product with light at wavelengths longer than 520 nm yielded a mixture composed of presumably 9- or 11-cis forms of retro-γ-rhodopsin. These three isomers are interconvertible by light at liquid nitrogen temperature. Thus the retro-γ-rhodopsin system is similar in photochemical reaction at 77 K to cattle rhodopsin system. Each system has its own batho product. Based on these results, it was infered that the formation of bathorhodopsin is due to photoisomerization of the chromophoric retinal of rhodopsin and is not due to translocation of a proton on the ring or on the side chain from C-6 to C-8 of the chromophoric retinal to the Schiff-base nitrogen.     

Fluorescence recovery kinetic analysis of gamma-tubulin binding to the mitotic spindle

Fluorescence recovery after photobleaching has been widely used to study dynamic processes in the cell, but less frequently to analyze binding interactions and extract binding constants. Here we use it to analyze γ-tubulin binding to the mitotic spindle and centrosomes to determine the role of γ-tubulin in microtubule nucleation in the spindle. We find rapid γ-tubulin turnover in mitotic spindles of Drosophila early embryos, characterized by diffusional interactions and weak binding, differing from centrosomes with tight binding interactions. The diffusion coefficient of γ-tubulin is consistent with a major species existing in the cytoplasm as the less efficiently nucleating γ-tubulin small complex (γTuSC) or γ-tubulin, rather than γ-tubulin ring complex (γTuRC). The fluorescence recovery kinetics we observe implies that γ-tubulin functions by binding weakly to spindle microtubules. γ-Tubulin may interact transiently with the spindle, nucleating microtubules very rapidly, differing from centrosomes, where γ-tubulin binds tightly to nucleate microtubules.     

Intrinsic bending and structural rearrangement of tubulin dimer: molecular dynamics simulations and coarse-grained analysis

Microtubules are long polymers of αβ-tubulin heterodimers. They undergo a process known as dynamic instability, in which the ends of a microtubule switch stochastically between phases of slow growth and rapid shrinkage. The molecular mechanisms inducing the depolymerization of microtubules were attributed to the hydrolysis of the guanosine triphosphate (GTP) nucleotide bound to the β-tubulin. The hydrolysis of GTP is thought to cause microtubule instability by promoting outward curving of the protofilaments constituting the microtubule lattice. The bending of protofilaments is associated with the structural transformation of a tubulin dimer from straight to curved conformations. However, the nature of intrinsic bending of the dimer remains elusive. This study uses molecular dynamics (MD) simulations and coarse-grained analysis to reveal the intrinsic bending, as well as the local structural rearrangements, of the unassembled tubulin dimer as the dimer relaxes from its lattice-constrained, straight conformation of a zinc-induced tubulin sheet. The effect of the nucleotide state on dimer-bending is investigated by the introduction of γ-phosphate into the β-tubulin to form GTP-bound tubulin. In agreement with recent experimental studies that proposed nucleotide-independent curved conformations, both guanosine diphosphate (GDP)-bound and GTP-bound tubulin dimers were found to have curved conformations, but with a tendency toward smaller bending in the GTP-tubulin than in the GDP-tubulin. The perturbation induced through the introduction of γ-phosphate is posited to play a role in straightening the intradimer bending. The local structural rearrangements of GDP-tubulin because of the bending mode of motion of the dimer reveal that one of the three functional domains, the intermediate domain, exhibits significantly lower bending deformation compared with the others, signifying a dynamic connection to the functionally defined domains.     

Expression patterns of peroxisome proliferator-activated receptor gamma 1 versus gamma 2, and their association with intramuscular fat in goat tissues

Intramuscular fat (IMF) shortage causes the lack of juiciness and tenderness of goat meat, while peroxisome proliferator-activated receptor gamma 1 (PPARγ1) and gamma 2 (PPARγ2) play key roles in lipid metabolism. Nevertheless, their expression patterns and the relationship with IMF have been poorly exposed. Using quantitative polymerase chain reaction (qPCR), classical Soxhlet extraction, and in situ hybridization, we demonstrated that among 13 goat tissues, expression of PPARγ1 was dramatically higher than that of PPARγ2 except for lung. We further demonstrated the expression patterns of PPARγ1 and PPARγ2 and their negative association with intramuscular fat content in three goat muscles with kids growing. Meanwhile, PPARγ expression was located in the connective tissues. These results suggest that PPARγ1 is rather active for most tissues of goat, and closely related with the muscular fat metabolism during early postnatal life, but a more direct proof remains to be provided.     

Characterization of γ-crystallin from the eye lens of bullfrog: Complexity of γ-crystallin multigene family as revealed by sequence comparison among different amphibian species

γ-Crystallin is the major and most abundant lens protein present in the eye lens of lower vertebrates such as amphibian and piscine species. To facilitate structural characterization ofγ-crystallins isolated from the lens of the bullfrog (Rana catesbeiana), a cDNA mixture was synthesized from the poly(A)⁺mRNA isolated from fresh eye lenses. cDNA encodingγ-crystallin was then amplified using polymerase chain reaction (PCR) based on two primers designed according to the relatively conserved N- and C-terminal sequences of knownγ-crystallins from teleostean fishes. PCR-amplified product corresponding toγ-crystallin isoforms was obtained, which was then subcloned in pUC18 vector and transformed intoEscherichia coli strain JM109. Plasmids containing amplifiedγ-crystallin cDNAs were purified and prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing several clones containing DNA inserts of about 0.54 kb revealed the presence of two isoforms with an open reading frame of 534 base pairs, covering twoγ-crystallins each with a deduced protein sequence of 177 amino acids including the translation-initiating methionine. Theseγ-crystallins of pI 6.364 and 6.366 contain a low-methionine content of 2.81%, in contrast to 11–16% obtained for thoseγ-crystallins with high-methionine content from most teleostean lenses. Pairwise sequence comparison of bullfrogγ-crystallins with those published sequences ofγ-crystallins from carp, shark,Xenopus and anotherRana frog, bovine, and human lenses indicates that there is only 46–63% sequence similarity among these species, revealing that amphibians possess a very complex and heterogeneous group ofγ-crystallins even from closely related species ofRana frogs. The sequence analysis and comparison of various isoforms of the frogγ-crystallin family provide a firm basis for identifying these lens proteins as members of a multigene family more complex than that reported for mammalianγ-crystallins.     

Correlation of Peroxisome Proliferator-Activated Receptor (PPAR-γ) mRNA Expression with Pro12Ala Polymorphism in Obesity

Our study aimed to analyze whether the expression of PPARγ mRNA in subcutaneous adipocyte tissue correlates with Pro12Ala PPARγ2 polymorphism in the obesity context. We found that mRNA expression of PPARγ in subcutaneous adipose tissue was greater in obese subjects (P < 0.05) than in the nonobese control group. Concurrently, genotyping of the Pro12Ala polymorphism showed that obese subjects possess a significantly higher frequency of the Pro/Pro genotype than nonobese controls (90.5 vs 79.5%; P = 0.03), suggesting that this genotype is involved in an increased risk of obesity in the Tunisian population. Taken together, our results demonstrate that the Pro12 allele is accompanied by an overexpression of PPARγ mRNA in subcutaneous adipocyte tissue, suggesting that the PPARγ Pro12Ala variant may contribute to the observed variability in PPARγ mRNA expression and consequently in body mass index and insulin sensitivity in the general population.     

A mathematical model for the territorial competition of the subterranean termites Coptotermes formosanus and Reticulitermes flavipes (Isoptera: Rhinotermitidae)

The foraging territories of two subterranean termites, Coptotermes formosanus Shiraki and Reticulitermes flavipes (Kollar), were simulated using a two-dimensional model to explore how territorial competition changes according to two variables characterizing territory formation: the total number of territories, and the blocking probability. Meanwhile, the blocking probability quantitatively describes the likelihood that a tunnel will be terminated when another tunnel is encountered. In our previous study, we introduced an interference coefficient γ to characterize territorial competition, and obtained γ as a function of the total number of territories and the blocking probability for a single termite species by model simulation. In the field, the territorial competition of more than two termite species is frequently observed. Here, we extended the γ function to be able to explain the competition between the two species by applying statistical regression to the simulation data. Further, we statistically checked the extended γ function by comparing the γ function for a single species. We also discuss another approach to mathematically derive the extended γ function, which can be easily generalized for use in cases of territorial competition involving more than two termite species.     

Development of water stress in kale leaves of different insertion levels

The mutual relationship between the water potential (γ _w ), its components, namely the osmotic potential (γ _s ) and the pressure potential (γ _p ), and the water saturation deficit (ΔW _sat ) were determined in the leaves of different insertion levels. During the water stress development in kale plants induced by decreasing soil moisture theγ _w decreased, parallely in all the leaves but the same decrease ofγ _q was accompanied by the highest decrease of theγ _p , probably due to the accumulation of osmotically active solutes, and the lowest decrease ofγ _p in the upper leaves and with the lowest decrease ofγ _s and the highest decrease ofγ _p in the lower leaves. Also the corresponding values of the ΔW _sat were always lower in the upper than in the middle and lower leaves. Thus the upper leaves wilted at more negative values ofγ _w than the other leaves. On the contrary, during the wilting of the cut off leaves the relationship betweenγ _w and ΔW _sat in the upper, middle and lower leaves was practically the same. The very slightly higher decrease ofγ _s in the upper leaves in comparison with the other leaves was compensated by a lower deerease of theirγ _p . These changes in the ratios ofγ _w ,γ _s ,γ _p and ΔW _sat with the leaf insertion levels enabled the preference of the upper leaves in retaining the necessary water supply during the wilting of plantsin situ.     

Human αB-crystallin discriminates between aggregation-prone and function-preserving variants of a client protein

BackgroundThe eye lens crystallins are highly soluble proteins that are required to last the lifespan of an organism due to low protein turnover in the lens. Crystallin aggregation leads to formation of light-scattering aggregates known as cataract. The G18V mutation of human γS-crystallin (γS-G18V), which is associated with childhood-onset cataract, causes structural changes throughout the N-terminal domain and increases aggregation propensity. The holdase chaperone protein αB-crystallin does not interact with wild-type γS-crystallin, but does bind its G18V variant. The specific molecular determinants of αB-crystallin binding to client proteins is incompletely charcterized. Here, a new variant of γS, γS-G18A, was created to test the limits of αB-crystallin selectivity.MethodsMolecular dynamics simulations were used to investigate the structure and dynamics of γS-G18A. The overall fold of γS-G18A was assessed by circular dichroism (CD) spectroscopy and intrinsic tryptophan fluorescence. Its thermal unfolding temperature and aggregation propensity were characterized by CD and DLS, respectively. Solution-state NMR was used to characterize interactions between αB-crystallin and γS-G18A.ResultsγS-G18A exhibits minimal structural changes, but has compromised thermal stability relative to γS-WT. The placement of alanine, rather than valine, at this highly conserved glycine position produces minor changes in hydrophobic surface exposure. However, human αB-crystallin does not bind the G18A variant, in contrast to previous observations for γS-G18V, which aggregates at physiological temperature.ConclusionsαB-crystallin is capable of distinguishing between aggregation-prone and function-preserving variants, and recognizing the transient unfolding or minor conformers that lead to aggregation in the disease-related variant.General significanceHuman αB-crystallin distinguishes between highly similar variants of a structural crystallin, binding the cataract-related γS-G18V variant, but not the function-preserving γS-G18A variant, which is monomeric at physiological temperature.     

Inhibition of human folylpolyglutamate synthetase by diastereomeric phosphinic acid mimics of the tetrahedral intermediate

Phosphorus-containing pseudopeptides, racemic at the C-terminal α-carbon, are potent mechanism-based inhibitors of folylpolyglutamate synthetase (FPGS). They are mimics of the tetrahedral intermediate postulated to form during FPGS-catalyzed biosynthesis of poly(γ-l-glutamates). In the present paper, the FPGS inhibitory activity of each diastereomer coupled to three heterocycles is reported. The high R_f pseudopeptide containing the 5,10-dideazatetrahydropteroyl (DDAH₄Pte) heterocycle is most potent (K_is = 1.7 nM). While the heterocyclic portion affects absolute FPGS inhibitory potency, the high R_f species is more potent in each pair containing the same heterocycle. This species presumably has the same stereochemistry as the natural folate polyglutamate, i.e., (l-Glu-γ-l-Glu). Unexpectedly, the low R_f (presumed l-Glu-γ-d-Glu) species are only slightly less potent (<30-fold) than their diastereomers. Further study of this phenomenon comparing l-Glu-γ-l-Glu and l-Glu-γ-d-Glu dipeptide-containing FPGS substrates shows that <1% contamination of commercial d-Glu precursors by l-Glu may give misleading information if l-Glu-γ-l-Glu substrates have low K_m values.     

Identification and characterization of TCRγ and TCRδ chains in channel catfish,Ictalurus punctatus

Channel catfish, Ictalurus punctatus, T cell receptors (TCR) γ and δ were identified by mining of expressed sequence tag databases, and full-length sequences were obtained by 5′-RACE and RT-PCR protocols. cDNAs for each of these TCR chains encode typical variable (V), diversity (D), joining (J), and constant (C) regions. Three TCRγ V families, seven TCRγ J sequences, and three TCRγ C sequences were identified from sequencing of cDNA. Primer walking on bacterial artificial chromosomes (BACs) confirmed that the TRG locus contained seven TRGJ segments and indicated that the locus consists of (Vγ3-Jγ6-Cγ2)–(Vγ1_n-Jγ7-Cγ3)–(Vγ2-Jγ5-Jγ4-Jγ3-Jγ2-Jγ1-Cγ1). In comparison for TCRδ, two V families, four TCRδ D sequences, one TCRδ J sequence, and one TCRδ C sequence were identified by cDNA sequencing. Importantly, the finding that some catfish TCRδ cDNAs contain TCR Vα-D-Jδ rearrangements and some TCRα cDNAs contain Vδ-Jα rearrangements strongly implies that the catfish TRA and TRD loci are linked. Finally, primer walking on BACs and Southern blotting suggest that catfish have four TRDD gene segments and a single TRDJ and TRDC gene. As in most vertebrates, all three reading frames of each of the catfish TRDD segments can be used in functional rearrangements, and more than one TRDD segment can be used in a single rearrangement. As expected, catfish TCRδ CDR3 regions are longer and more diverse than TCRγ CDR3 regions, and as a group they utilize more nucleotide additions and contain more nucleotide deletions than catfish TCRγ rearrangements.     

Spontaneous chromosomal instability in peripheral blood lymphocytes from two molecularly confirmed Italian patients with Hereditary Fibrosis Poikiloderma: insights into cancer predisposition

Two Italian patients with the initial clinical diagnosis of Rothmund-Thomson syndrome were negative for RECQL4 mutations but showed in peripheral blood cells a spontaneous chromosomal instability significantly higher than controls. Revisiting after time their clinical phenotype, the suggestive matching with the autosomal dominant syndrome Poikiloderma, Hereditary Fibrosing with Tendon Contracture, Myopathy and Pulmonary fibrosis (POIKTMP) was confirmed by identification of the c.1879A>G (p.Arg627Gly) alteration in FAM111B. We compare the overall clinical signs of our patients with those of reported carriers of the same mutation and present the up-to-date mutational repertoire of FAM111B and the related phenotypic spectrum. Our snapshot highlights the age-dependent clinical expressivity of POIKTMP and the need to follow-up patients to monitor the multi-tissue impairment caused by FAM111B alterations. We link our chromosomal instability data to the role of FAM111B in cancer predisposition, pointed out by its implication in DNA-repair pathways and the outcome of pancreatic cancer in 2 out of 17 adult POIKTMP patients. The chromosomal instability herein highlighted well connects POIKTMP to cancer-predisposing syndromes, such as Rothmund-Thomson which represents the first hereditary poikiloderma entering in differential diagnosis with POIKTMP.     

Effect of an external RF field on the beam-plasma discharge in a magnetic field

The effect of an RF field on a steady-state beam-plasma discharge with a plane electrode placed parallel to a sheetlike electron beam is studied experimentally. The plasma parameters were measured by a single probe, and the electron distribution function was determined with the use of an electrostatic analyzer. The energy and current of the electron beam were E _B=2.5 keV and J _B=0.05–1.5 A, respectively. The working pressure was p=2×10^?5–10^?3 torr. The frequency of the external RF field was 13.56 MHz. Both the steady-state regimes in which the RF field had no effect on the plasma parameters and regimes with a pronounced effect of the RF field were observed. The experiments show that the regime of the discharge depends strongly on the plasma density and the magnetic field. The parametric instability is studied theoretically in the weak-turbulence approximation. It is shown that, due to the decay nature of the spectrum of plasma oscillations, the onset of instability is accompanied by the transfer of the energy of fluctuations over the spectrum, from the pump frequency toward its harmonics.     

Phenazine ethosulfate as a preferred electron acceptor to phenazine methosulfate in dye-linked enzyme assays.

Rapid nonenzymatic reduction of 2,6-dichlorophenolindophenol by N-methyl phenazonium methosulfate has been observed in aqueous solution and has been found to increase with increasing pH and ionic strength. The instability of N-methyl phenazonium methosulfate in aqueous solution has been explored in terms of change of absorption spectrum and formation of free radicals as evidenced by EPR spectroscopy. N-Ethyl phenazonium ethosulfate has been found to be much more stable than the methyl analog and did not reduce dichlorophenolindophenol nonenzymatically. The implications of these findings with respect to use of these dyes as artificial electron acceptors are discussed and the recommendation made that, wherever possible, use of N-methyl phenazonium methosulfate be discontinued in favor of use of the N-ethyl analog.     

Replication-mediated instability of the GAA triplet repeat mutation in Friedreich ataxia

Friedreich ataxia is caused by the expansion of a polymorphic and unstable GAA triplet repeat in the FRDA gene, but the mechanisms for its instability are poorly understood. Replication of (GAA•TTC)_n sequences (9–105 triplets) in plasmids propagated in Escherichia coli displayed length- and orientation-dependent instability. There were small length variations upon replication in both orientations, but large contractions were frequently observed when GAA was the lagging strand template. DNA replication was also significantly slower in this orientation. To evaluate the physiological relevance of our findings, we analyzed peripheral leukocytes from human subjects carrying repeats of similar length (8–107 triplets). Analysis of 9400 somatic FRDA molecules using small-pool PCR revealed a similar mutational spectrum, including large contractions. The threshold length for the initiation of somatic instability in vivo was between 40 and 44 triplets, corresponding to the length of a eukaryotic Okazaki fragment. Consistent with the stabilization of premutation alleles during germline transmission, we also found that instability of somatic cells in vivo and repeats propagated in E.coli were abrogated by (GAGGAA)_n hexanucleotide interruptions. Our data demonstrate that the GAA triplet repeat mutation in Friedreich ataxia is destabilized, frequently undergoing large contractions, during DNA replication.     

Role of the DELSEED Loop in Torque Transmission of F1-ATPase

F₁-ATPase is an ATP-driven rotary motor that generates torque at the interface between the catalytic β-subunits and the rotor γ-subunit. The β-subunit inwardly rotates the C-terminal domain upon nucleotide binding/dissociation; hence, the region of the C-terminal domain that is in direct contact with γ—termed the DELSEED loop—is thought to play a critical role in torque transmission. We substituted all the DELSEED loop residues with alanine to diminish specific DELSEED loop-γ interactions and with glycine to disrupt the loop structure. All the mutants rotated unidirectionally with kinetic parameters comparable to those of the wild-type F₁, suggesting that the specific interactions between DELSEED loop and γ is not involved in cooperative interplays between the catalytic β-subunits. Glycine substitution mutants generated half the torque of the wild-type F₁, whereas the alanine mutant generated comparable torque. Fluctuation analyses of the glycine/alanine mutants revealed that the γ-subunit was less tightly held in the α₃β₃-stator ring of the glycine mutant than in the wild-type F₁ and the alanine mutant. Molecular dynamics simulation showed that the DELSEED loop was disordered by the glycine substitution, whereas it formed an α-helix in the alanine mutant. Our results emphasize the importance of loop rigidity for efficient torque transmissions.     

Intracellular neutralization of SV40 tumor antigens following microinjection of specific antibody

We present the nucleotide sequences of the ^Gγ- and ^Aγ-globin genes from one chromosome (A) and of most of the ^Aγ gene from the other chromosome (B) of the same individual. All three genes have a small, highly conserved intervening sequence (IVS1) of 122 bp located between codons 30 and 31 and a large intervening sequence (IVS2) of variable length (866–904 bp) between codons 104 and 105. A stretch of simple sequence DNA occurs in IVS2 which appears to be a hot spot for recombination. On the 5′ side of this simple sequence, the allelic ^Aγ genes differ considerably in IVS2 whereas the nonallelic ^Gγ- and ^Aγ genes from chromosome A differ only slightly. Yet on the 3′ side of the simple sequence, the allelic genes differ only slightly whereas the nonallelic genes differ considerably. We hypothesize that the 5′ two thirds of the ^Aγ gene on chromosome A has been “converted” by an intergenic exchange to become more like the ^Gγ gene on its own chromosome A than it is like the allelic ^Aγ gene on the other chromosome B. Our sequence data suggest that intergenic conversions occur in the germ line. The DNA sequence differences between two chromosomes from a single individual strongly suggest that DNA sequence polymorphisms for localized deletions, additions and base substitutions are very common in human populations.