Direct observation of iron-induced conformational changes of mitochondrial DNA by high-resolution field-emission in-lens scanning electron microscopy.

When respiring rat liver mitochondria are incubated in the presence of Fe(III) gluconate, their DNA (mtDNA) relaxes from the supercoiled to the open circular form dependent on the iron dose. Anaerobiosis or antioxidants fail to completely inhibit the unwinding. High-resolution field-emission in-lens scanning electron microscopy imaging, in concert with backscattered electron detection, pinpoints nanometer-range iron colloids bound to mtDNA isolated from iron-exposed mitochondria. High-resolution field-emission in-lens scanning electron microscopy with backscattered electron detection imaging permits simultaneous detailed visual analysis of DNA topology, iron dose-dependent mtDNA unwinding, and assessment of iron colloid formation on mtDNA strands.

Heteromeric connexons in lens gap junction channels.

Gap junction channels are formed by paired oligomeric membrane hemichannels called connexons, which are composed of proteins of the connexin family. Experiments with transfected cell lines and paired Xenopus oocytes have demonstrated that heterotypic intercellular channels which are formed by two connexons, each composed of a different connexin, can selectively occur. Studies by Stauffer [Stauffer, K. A. (1995) J. Biol. Chem. 270, 6768-6772] have shown that recombinant Cx26 and Cx32 coinfected into insect cells may form heteromeric connexons. By solubilizing and subfractionating individual connexons from ovine lenses, we show by immunoprecipitation that connexons can contain two different connexins forming heteromeric assemblies in vivo.

Lens complementation system for the genetic analysis of growth, differentiation, and apoptosis in vivo.

A genetic approach has been established that combines the advantages of blastocyst complementation with the experimental attributes of the developing lens for the functional analysis of genes governing cellular proliferation, terminal differentiation, and apoptosis. This lens complementation system (LCS) makes use of a mutant mouse strain, aphakia (ak), homozygotes of which fail to develop an ocular lens. We demonstrate that microinjection of wild-type embryonic stem (ES) cells into ak/ak blastocysts produces chimeras with normal ES-cell-derived lenses and that microinjection of Rb-/- ES cells generates an aberrant lens phenotype identical to that obtained through conventional gene targeting methodology. Our determination that a cell autonomous defect underlies the aphakia condition assures that lenses generated through LCS are necessarily ES-cell-derived. LCS provides for the rapid phenotypic analysis of loss-of-function mutations, circumvents the need for germ-line transmission of null alleles, and, most significantly, facilitates the study of essential genes whose inactivation is associated with early lethal phenotypes.

Phase separation in aqueous solutions of lens gamma-crystallins: special role of gamma s.

We have studied liquid-liquid phase separation in aqueous ternary solutions of calf lens gamma-crystallin proteins. Specifically, we have examined two ternary systems containing gamma s--namely, gamma IVa with gamma s in water and gamma II with gamma s in water. For each system, the phase-separation temperatures (Tph (phi)) alpha as a function of the overall protein volume fraction phi at various fixed compositions alpha (the "cloud-point curves") were measured. For the gamma IVa, gamma s, and water ternary solution, a binodal curve composed of pairs of coexisting points, (phi I, alpha 1) and (phi II, alpha II), at a fixed temperature (20 degrees C) was also determined. We observe that on the cloud-point curve the critical point is at a higher volume fraction than the maximum phase-separation temperature point. We also find that typically the difference in composition between the coexisting phases is at least as significant as the difference in volume fraction. We show that the asymmetric shape of the cloud-point curve is a consequence of this significant composition difference. Our observation that the phase-separation temperature of the mixtures in the high volume fraction region is strongly suppressed suggests that gamma s-crystallin may play an important role in maintaining the transparency of the lens.

Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated.

Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.

An apoptotic defect in lens differentiation caused by human p53 is rescued by a mutant allele.

If deprived of wild-type p53 function, the body loses a guardian that protects against cancer. Restoration of p53 function has, therefore, been proposed as a means of counteracting oncogenesis. This concept of therapy requires prior knowledge with regard to proper balance of p53 function in a given target tissue. We have addressed this problem by targeting expression of the wild-type human p53 gene to the lens, a tissue entirely composed of epithelial cells that differentiate into elongated fiber cells. Transgenic mice expressing wild-type human p53 develop microphthalmia as a result of a defect in fiber formation that sets in shortly after birth. We see apoptotic cells that fail to undergo proper differentiation. In an effort to directly link the observed lens phenotype to the activity of the wild-type human p53 transgene, we also generated mice expressing a mutant human p53 allele that lacks wild-type function. A normal lens phenotype is restored in double transgenic animals that carry both wild-type and mutant human p53 alleles. Our study highlights the difficulties that can arise if p53 levels are improperly balanced in a differentiating tissue.

Expression of pax-6 during urodele eye development and lens regeneration.

Regeneration of eye tissues, such as lens, seen in some urodeles involves dedifferentiation of the dorsal pigmented epithelium and subsequent differentiation to lens cells. Such spatial regulation implies possible action of genes known to be specific for particular cell lineages and/or axis. Hox genes have been the best examples of genes for such actions. We have, therefore, investigated the possibility that such genes are expressed during lens regeneration in the newt. The pax-6 gene (a gene that contains a homeobox and a paired box) has been implicated in the development of the eye and lens determination in various species ranging from Drosophila to human and, because of these properties, could be instrumental in the regeneration of the urodele eye tissues as well. We present data showing that pax-6 transcripts are present in the developing and the regenerating eye tissues. Furthermore, expression in eye tissues, such as in retina, declines when a urodele not capable of lens regeneration (axolotl) surpasses the embryonic stages. Such a decline is not seen in adult newts capable of lens regeneration. This might indicate a vital role of pax-6 in newt lens regeneration.

Pax-6 is essential for lens-specific expression of zeta-crystallin.

Pax-6 is essential for normal eye development and has been implicated as a "master gene" for lens formation in embryogenesis. Guinea pig zeta-crystallin, a taxon-specific enzyme crystallin, achieves high expression specifically in lens through use of an alternative promoter. Here we show that Pax-6 binds a site in this promoter, which is essential for lens-specific expression. Lens and lens-derived cells exhibit a tissue-specific pattern of alternative splicing of Pax-6 transcripts and Pax-6 is expressed in adult lenses and cells that support zeta-crystallin expression. These results suggest that zeta-crystallin is a natural target gene for Pax-6 and that this Pax family member has a direct role in the continuing expression of tissue-specific genes.

Pax-6 and lens-specific transcription of the chicken delta 1-crystallin gene.

The abundance of delta-crystallin in the chicken eye lens provides an advantageous marker for tissue-specific gene expression during cellular differentiation. The lens-specific expression of the delta 1-crystallin gene is governed by an enhancer in the third intron, which binds a positive (delta EF2) and negative (delta EF1) factor in its core region. Here we show by DNase I footprinting, electrophoretic mobility-shift assays, and cotransfection experiments with the delta 1-promoter/enhancer fused to the chloramphenicol acetyltransferase reporter gene that the delta 1-crystallin enhancer has two adjacent functional Pax-6 binding sites. We also demonstrate by DNase I footprinting that the delta EF1 site can bind the transcription factor USF, raising the possibility that USF may cooperate with Pax-6 in activation of the chicken delta 1- and alpha A-crystallin genes. These data, coupled with our recent demonstration that Pax-6 activates the alpha A-crystallin gene, suggest that Pax-6 may have been used extensively throughout evolution to recruit and express crystallin genes in the lens.

Comparison between clinical results of two diffractive multifocal lenses with the same platform but different additions

PURPOSE: To evaluate visual results with two multifocal diffractive lenses designed with the same platform but with different additions. SETTING: Grupo Innova Ocular clinics. METHODS: A total of 50 eyes from 50 patients were included. Group 1 (n = 25) was implanted with the TECNIS® 1 ZLB +3.25 and group 2 (n = 25) with the TECNIS® 1 ZKB +2.75. Patients were assessed at 24 hours, 1 week and 1 month postoperatively. At surgical discharge, corrected (CDVA) and uncorrected distance visual acuity (UCDVA), near visual acuity (VA) at 25, 40 and 80 cm, visual quality and the defocus curve were measured. RESULTS: Changes in sphere and spherical equivalent were statistically significant (p<0.01) in both groups at 1 week and 1 month compared to preoperative values. In group 1, UCDVA logMAR at 1 month was 0.06 ± 0.02. In group 2, UCDVA at 1 month was 0.03 ± 0.03. In near vision, the TECNIS® 1 ZLB group obtained a VA logMAR of 0.35 ± 0.02 at 25 cm, 0.13 ± 0.02 at 40 cm and 0.27 ± 0.02 at 80 cm, while in the TECNIS® 1 ZKB group, the values were 0.38  ± 0.03, 0.14 ± 0.03 and 0.23 ± 0.06, respectively. No statistically significant differences were found either when results for visual quality were compared. CONCLUSION: Both the TECNIS® 1 ZLB and TECNIS® 1 ZKB are excellent options for obtaining good distance and near vision, in addition to providing good intermediate vision, especially at distances such as those required for working with computers.

Method for the characterization of Fresnel lens flux transfer performance

Fresnel lenses and other faceted or micro-optic devices are increasingly used in multiple applications like solar light concentrators and illumination devices, just to name some representative. However, it seems to be a certain lack of adequate techniques for the assessment of the performance of final fabricated devices. As applications are more exigent this characterization is a must. We provide a technique to characterize the performance of Fresnel lenses, as light collection devices. The basis for the method is a configuration where a camera images the Fresnel lens aperture. The entrance pupil of the camera is situated at the focal spot or the conjugate of a simulated solar source. In this manner, detailed maps of the performance of different Fresnel lenses are obtained for different acceptance angles.

The Importance of the First Psychotherapy Case in the Development of the Therapist's Professional Self as Viewed Through the Lens of Self Psychology

This paper explores the gap in the literature between what is herein referred to as the "first psychotherapy case" and its impact on the development of the trainee psychotherapist's professional self. The self psychology concepts of identity development, selfobject needs and fulfillment, narcissism, shame, countertransference, and structuralization are incorporated into the theoretical framework from which this developmental milestone is viewed. The theory's emphasis on early experiences and the development of self highlight the distinctiveness of the first case for the therapist. The beginning psychotherapy case poses a unique context for selfobject experiences and the developing self, involving both the therapist's presumably mature needs (assuming an existing cohesive nuclear self) and more infantile needs as the professional, peripheral self develops. As a result, the potential and important implications for the psychotherapist, the patient, training implications for the supervisor, and the ensuing treatment through termination are identified. The intent is to shed light on an area that is understudied thus far, and to begin a conversation as to why and how the impact of the first case on the psychotherapist should be examined. Implications, limitations, and ideas for future exploratory and qualitative research are also discussed.

Examining the Impact of Child Psychologists' Stigmatization of Parents on Treatment Outcomes Through a Developmental Lens

The purpose of this paper is to examine how child psychologists' specialized training inhuman development may make them more prone to stigmatize the parents of their young clients. The stigmatization of parents may lead to fewer parents seeking treatment for their children and to poorer treatment outcomes for those who work with a child psychologist. The process of stigmatization is summarized to provide context for the method through which parents receive stigma. A commonly used theory of child development, Erik Erikson's stages of ego development, is outlined to provide background on how child psychologists may interpret and evaluate a child'sdevelopment. Child psychologists' may identify parenting practices that seem to hinder or stunt children's emotional development, which would make the psychologist more aptto stigmatize and isolate parents from the treatment process. To demonstrate the unique ways in which a child psychologist may stigmatize parents of children at different developmental stages two case studies are included. Finally, a theoretical model of treatment is described that may be more inclusive, and less stigmatizing of parents. This model outlines how the parents' concerns about and observations of their children should be validated and reflected in the treatment process. This treatment modality would allow for child psychologists to more actively involve parents in treatment and provide more education and support around their children's unique emotional development needs. Through this treatment model and child psychologists' awareness of and attempts to reduce the stigmatization of parents, treatment outcomes for young clients may improve.

Conceptualizing The Miscarriage Experience Through The Lens Of Ghosts In The Nursery

The purpose of this doctoral paper was to use the “Ghosts in the Nursery” theory (Fraiberg, Adelson, & Shapiro, 1975) as a framework for understanding clinicians’ perceptions of women’s experience of miscarriage. Specific attention was paid to the experience of becoming pregnant with a subsequent child. Professionals who work in the field of infant mental health were asked to explore the theory’s utility in conceptualizing the experience of becoming pregnant after a miscarriage. Results indicated that the perceptions of women’s experiences of miscarriage and subsequent pregnancy are congruent with previous research findings. Further elaboration and information are provided to illustrate the experience of having a child and being a parent after experiencing a loss, and to explore the idea of understanding miscarriage as a “ghost”. This study applies a new perspective to the theory of “Ghosts in the Nursery” (Fraiberg et al., 1975) to children born after a loss.