Liposome dehydration on nitrocellulose and its application in a biotin immunoassay.

The feasibility of utilizing dehydrated liposomes in the development of a simple immunoassay device for point-of-care diagnostics or field assays was demonstrated. The recovery of liposomes after a cycle of dehydration and rehydration was studied using biotin-tagged, dye-loaded liposomes with antibiotin antibodies immobilized in a defined zone on nitrocellulose strips. Liposomes were vacuum-dehydrated on the strip at a location below the antibiotin zone. The strip was placed in a tube containing a carrier solution and capillary action brought the solution to the dehydrated liposomes, rehydrated them, and caused them to migrate to the antibody zone where intact liposomes were captured and measured optically. High concentrations of either trehalose or sucrose external to the liposomes and both polyvinylpyrrolidone and gelatin in the membrane blocking reagent were essential for preservation of the dehydrated/rehydrated liposomes on nitrocellulose. Between 70 and 80% of the liposomes were recovered on the nitrocellulose strips after a cycle of dehydration and rehydration. The dehydrated liposomes on the strips were stable for at least 1 year when stored in vacuum-sealed plastic bags at 4 degrees C. The technique was successfully applied to the development of a rapid one-step strip immunoassay for biotin.     

A rice tubulin tyrosine ligase‐like 12 protein affects the dynamic and orientation of microtubules

The detyrosination/retyrosination cycle is the most common post‐translational modification of α‐tubulin. Removal of the conserved C‐terminal tyrosine of α‐tubulin by a still elusive tubulin tyrosine carboxypeptidase, and religation of this tyrosine by a tubulin tyrosine ligase (TTL), are probably common to all eukaryotes. Interestingly, for plants, the only candidates qualifying as potential TTL homologs are the tubulin tyrosine ligase‐like 12 proteins. To get insight into the biological functions of these potential TTL homologs, we cloned the rice TTL‐like 12 protein (OsTTLL12) and generated overexpression OsTTLL12‐RFP lines in both rice and tobacco BY‐2 cells. We found, unexpectedly, that overexpression of this OsTTLL12‐RFP increased the relative abundance of detyrosinated α‐tubulin in both coleoptile and seminal root, correlated with more stable microtubules. This was independent of the respective orientation of cortical microtubule, and followed by correspondingly changing growth of coleoptiles and seminal roots. A perturbed organization of phragmoplast microtubules and disoriented cell walls were further characteristics of this phenotype. Thus, the elevated tubulin detyrosination in consequence of OsTTLL12 overexpression affects structural and dynamic features of microtubules, followed by changes in the axiality of cell plate deposition and, consequently, plant growth.     

In vivo cervical cancer growth inhibition by genetically engineered cytotoxic T cells

Purpose: The CD44 v7/8 splice variant that is frequently expressed in cervical carcinoma and rarely expressed in normal tissues displays promising properties as a target antigen for cancer immune therapy. In this study, cytotoxic T lymphocytes (CTLs) were genetically engineered to gain CD44v7/8 target specificity. Methods: Clone 96 (CI96), an established murine cytotoxic T-cell line, and naïve murine T cells were retrovirally transduced with a fusion gene construct encoding for the single chain fragment scFv of the monoclonal antibody VFF17 and for the chain of the T-cell receptor (TCR). The therapeutic potential of genetically engineered T cells was tested in vitro and in vivo. Results: Surface expression of the chimeric TCR on infected Cl96 and naïve T cells was shown by FACS analysis. CD44v7/8-positive target cells were efficiently lysed by transduced Cl96 and naïve T cells, demonstrating the functionality and specificity of the chimeric TCR. In a xenograft BALB/c mouse model, efficient growth retardation of CD44v7/8-positive tumours was mediated by genetically engineered Cl96(VFF17)cyYZ cells. Conclusions: We were able to reprogramme the target specificity of recombinant Cl96 and naïve CTLs resulting in efficient cytolysis of CD44v7/8-positive cervical cancer cells. High transduction rates and the specific cytolysis of CD44v7/8-redirected CTLs are promising tools for an immune gene therapy approach for advanced cervical cancer.Abbreviations Ab Antibody - CTL Cytolytic T lymphocyte - mAb Monoclonal antibody - TCR T-cell receptor     

Aryl hydroxylation of the herbicide diclofop by a wheat cytochrome p-450 monooxygenase : substrate specificity and physiological activity

Wheat (Triticum aestivum L. cv Etoile de Choisy) microsomes catalyzed the cytochrome P-450-dependent oxidation of the herbicide diclofop to three hydroxy-diclofop isomers. Hydroxylation was predominant at carbon 4, with migration of chlorine to carbon 5 (67%) and carbon 3 (25%). The 2,4-dichloro-5-hydroxy isomer was identified as a minor reaction product (8%). Substrate-specificity studies showed that the activity was not inhibited or was weakly inhibited by a range of xenobiotic or physiological cytochrome P-450 substrates, with the exception of lauric acid. Wheat microsomes also catalyze the metabolism of the herbicides chlorsulfuron, chlortoluron, and 2,4-dichlorophenoxyacetic acid and of the model substrate ethoxycoumarin, as well as the hydroxylation of the endogenous substrates cinnamic and lauric acids. Treatments of wheat seedlings with phenobarbital or the safener naphthalic acid anhydride enhanced the cytochrome P-450 content of the microsomes and all related activities except that of cinnamic acid 4-hydroxylase, which was reduced. The stimulation patterns of diclofop aryl hydroxylase and lauric acid hydroxylase were similar, in contrast with the other activities tested. Lauric acid inhibited competitively (K_i = 9 μm) the oxidation of diclofop and reciprocally. The similarity of diclofop aryl hydroxylase and lauric acid hydroxylase was further investigated by alternative substrate kinetics, autocatalytic inactivation, and computer-aided molecular modelisation studies, and the results suggest that both reactions are catalyzed by the same cytochrome P-450 isozyme.     

Superproduction of heavy minicircular mitochondrial DNA in aging wheat coleoptiles.

On incubation of 7-day-old wheat seedlings in the presence of [3H]thymidine, the radioactivity incorporated into coleoptile DNA is found to be localized mainly (greater than 95%) in the fraction of heavy mitochondrial DNA (H-mt DNA; rho = 1.716 gm/cm3). Upon long (48-72 h) incubation of cut-off seedlings in water, the amount of this DNA shows a dramatic increase and corresponds to about 10% of the total coleoptile DNA. H-mtDNA is represented by open circular molecules with a contour length varying from 0.12 to 0.6 microns. The functional role of this DNA is still unknown.     

Phytochrome-mediated regulation of a monooxygenase hydroxylating cinnamic acid in etiolated pea seedlings

Cinnamic acid is hydroxylated by the mixed-function oxidase trans-cinnamic acid 4-hydroxylase (CA4H). The hydroxylation reaction involves the transfer of electrons from reduced pyridine nucleotides via the enzyme NADPH cytochrome P-450 reductase to the terminal oxidase cytochrome P-450. This multi-enzyme complex is localized in the microsomal fraction. Isopycnic and velocity gradient centrifugation suggest that in the apical bud of etiolated pea seedlings this complex is restricted to the endoplasmic reticulum membranes. CA4H activity which develops in dark germinating pea seedlings was found to be stimulated by light, an effect mediated by phytochrome. CA4H and NADPH cytochrome c reductase activities, cytochromes P-450 and b 5 contents were measured in seedlings submitted to either short pulses of red and far-red light, or to continuous far-red or blue irradiation. The results are discussed in terms of a specific effect of phytochrome on the different parts of the multi-enzyme complex.     

The dialectics of socialization: A tentative formulation

Conclusion A cognitive anthropological approach helps us to understand that children's conceptions are antithetical to adult conceptions. Furthermore, socialization is a dialectical process involving an interplay of these antithetical child conceptions and adult conceptions. As indicated in the field observations, change in the child is internal but the transition from one stage of growth to another occurs only through interaction with the environment. One sees elements of adult conceptions created in the child but they are and remain child conceptions until such time as the Leap has occurred. Babs' teacher, for example, misunderstood this antithetical quality in Babs and assumed her mature behavior indicated she was really mature, really an adult. Qualitatively and quantitatively Babs was still a child and had to initiate action on her own part to prove that. She exhibited behavior that was misconstrued as adult-like, but because she was still a child, her conceptions of herself, her environment and correlatively, of her behavior remained child-like.Carol was obviously quite resourceful and adept at moving in and out of various behavioral patterns appropriate to social situations in which she found herself. This was necessary for survival, particularly when she was forced into situations where prejudicial people controlled her life and her future. The antithetical qualities of her conceptual framework and that of her teacher are overshadowed by the real historical circumstances of race relations in this society. But those antithetical conceptual qualities are still clearly evident. Adult conceptions from the outside intrude on Carol's life and her thinking. And, elements of adult conceptions are being created within her. Yet these autonomous creations will remain child conceptions as long as she remains a child. She responds to her environment, but she responds as a child, subjectified/objectified. She is still qualitatively and quantitatively a childClearly much of what children learn from the environment — from school — is harmful to their autonomy. Through current socialization processes, the dialectics of learning remain incomplete. The interaction whereby children's categories antithetical to adult categories are transformed into a new synthesis of adult categories is missing. Instead, the adult conceptions created in and by the children merely replicate the adult categories of their socializers. Therefore, the current socialization process is a reactionary one. Children are increasingly objectified as their adult categories are less and less new categories, but merely replications of already formalized adult categories. Because the dialectical process of socialization is left incomplete, incomplete adults reach physical maturity. Such people are incapable of governing their own lives, of living out the complexities and inconsistencies of adulthood.Reformists are correct in calling for change in educational methods but not with the methods they suggest. Children should be educated for democracy, but the methods utilized should not result in chaos and disorder because they lack discipline.In fact, although ideologically at odds with each other, methodologically conventional and emancipated teachers differ only in locus of control. Conventional teachers see socialization as a unidirectional process in which they provide the stimulus and the students provide the response. Emancipated teachers see socialization as a dialectical process and deploy that process to educate children for greater autonomy, social and self-understanding. But, in rejecting conventional modes of thought, unconventional teachers do not necessarily reject sensible pedagogical methods. The processes and methods of education are more complex than a simple liberal (soft)/conservative (strict) theoretical dichotomy of education would suggest. Children can still be respected even as their teachers realize their conceptual categories are incomplete and inferior. Relativists who insist, for example, that all speech patterns, because they exhibit internal consistency and structural regularity are inherently equal, fail to recognize or to uncover the true source of social inequality. Relativistic revelations only mystify that inequality and contribute nothing to social liberation.Education for liberation is a slow, arduous task and will not occur by using short-cuts, or pop-psych approaches. It will come from strict adherence to exacting and demanding methods. It will be facilitated through a recognition of the dialectical nature of the socialization process and a cognitive anthropological investigation into the nature of conceptual categories of children. And it will have to be part of a general transformation of this society, while, at the same time, remaining an index of the succes of any such transformation. This means, among other things, that children must be taught to govern their own lives and to govern others. It does not mean, of course, that every person would govern; only that every person should understand the mechanics of governing — that every person, as Gramsci held, could govern.Maribeth Durst is Assistant Professor of Sociology, at St. John's University, Staten Island, New York.

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Interactions between muscle fibers and segment boundaries in zebrafish

The most obvious segmental structures in the vertebrate embryo are somites: transient structures that give rise to vertebrae and much of the musculature. In zebrafish, most somitic cells give rise to long muscle fibers that are anchored to intersegmental boundaries. Therefore, this boundary is analogous to the mammalian tendon in that it transduces muscle-generated force to the skeletal system. We have investigated interactions between somite boundaries and muscle fibers. We define three stages of segment boundary formation. The first stage is the formation of the initial epithelial somite boundary. The second "transition" stage involves both the elongation of initially round muscle precursor cells and somite boundary maturation. The third stage is myotome boundary formation, where the boundary becomes rich in extracellular matrix and all muscle precursor cells have elongated to form long muscle fibers. It is known that formation of the initial epithelial somite boundary requires Notch signaling; vertebrate Notch pathway mutants show severe defects in somitogenesis. However, many zebrafish Notch pathway mutants are homozygous viable suggesting that segmentation of their larval and adult body plans at least partially recovers. We show that epithelial somite boundary formation and slow-twitch muscle morphogenesis are initially disrupted in after eight (aei) mutant embryos (which lack function of the Notch ligand, DeltaD); however, myotome boundaries form later ("recover") in a Hedgehog-dependent fashion. Inhibition of Hedgehog-induced slow muscle induction in aei/deltaD and deadly seven (des)/notch1a mutant embryos suggests that slow muscle is necessary for myotome boundary recovery in the absence of initial epithelial somite boundary formation. Because we have previously demonstrated that slow muscle migration triggers fast muscle cell elongation in zebrafish, we hypothesize that migrating slow muscle facilitates myotome boundary formation in aei/deltaD mutant embryos by patterning coordinated fast muscle cell elongation. In addition, we utilized genetic mosaic analysis to show that somite boundaries also function to limit the extent to which fast muscle cells can elongate. Combined, our results indicate that multiple interactions between somite boundaries and muscle fibers mediate zebrafish segmentation.     

Arabidopsis cyp51 mutant shows postembryonic seedling lethality associated with lack of membrane integrity

CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14alpha-demethylase involved in the postsqualene sterol biosynthetic pathway. According to the current gene annotation, the Arabidopsis (Arabidopsis thaliana) genome contains two putative CYP51 genes, CYP51A1 and CYP51A2. Our studies revealed that CYP51A1 should be considered an expressed pseudogene. To study the functional importance of the CYP51A2 gene in plant growth and development, we isolated T-DNA knockout alleles for CYP51A2. Loss-of-function mutants for CYP51A2 showed multiple defects, such as stunted hypocotyls, short roots, reduced cell elongation, and seedling lethality. In contrast to other sterol mutants, such as fk/hydra2 and hydra1, the cyp51A2 mutant has only minor defects in early embryogenesis. Measurements of endogenous sterol levels in the cyp51A2 mutant revealed that it accumulates obtusifoliol, the substrate of CYP51, and a high proportion of 14alpha-methyl-delta8-sterols, at the expense of campesterol and sitosterol. The cyp51A2 mutants have defects in membrane integrity and hypocotyl elongation. The defect in hypocotyl elongation was not rescued by the exogenous application of brassinolide, although the brassinosteroid-signaling cascade is apparently not affected in the mutants. Developmental defects in the cyp51A2 mutant were completely rescued by the ectopic expression of CYP51A2. Taken together, our results demonstrate that the Arabidopsis CYP51A2 gene encodes a functional obtusifoliol 14alpha-demethylase enzyme and plays an essential role in controlling plant growth and development by a sterol-specific pathway.