Age at diagnosis as an indicator of eligibility for BRCA1 DNA testing in familial breast cancer

We searched for criteria that could indicate breast cancer families with a high prior probability of being caused by the breast/ovarian cancer susceptibility locus BRCA1 on chromosome 17. To this end, we performed a linkage study with 59 consecutively collected Dutch breast cancer families, including 16 with at least one case of ovarian cancer. We used an intake cut-off of at least three first-degree relatives with breast and/or ovarian cancer at any age. Significant evidence for linkage was found only among the 13 breast cancer families with a mean age at diagnosis of less than 45 years. An unexpectedly low proportion of the breast-ovarian cancer families were estimated to be linked to BRCA1, which could be due to a founder effect in the Dutch population. Given the expected logistical problems in clinical management now that BRCA1 has been identified, we propose an interim period in which only families with a strong positive family history for early onset breast and/or ovarian cancer will be offered BRCA1 mutation testing. More recent work has indicated that RUL09 is probably due to BRCA2 (multipoint lod score of 1.17), while in families RUL47 and RUL49 a frameshift mutation in BRCA1 has been evidenced. Each of these two latter families contain an early-onset sporadic breast cancer patient, explaining their negative lod scores with 17q-markers.     

alpha6 Integrin is regulated with lens cell differentiation by linkage to the cytoskeleton and isoform switching.

The developing chicken embryo lens provides a unique model for examining the relationship between alpha6 integrin expression and cell differentiation, since multiple stages of differentiation are expressed concurrently at one stage of development. We demonstrate that alpha6 integrin is likely to mediate the inductive effects of laminin on lens differentiation as well as to function in a matrix-independent manner along the cell-cell interfaces of the differentiating cortical lens fiber cells. Both alpha6 isoform expression and its linkage to the cytoskeleton were regulated in a differentiation-specific manner. The association of alpha6 integrin with the Triton-insoluble cytoskeleton increased as the lens cells differentiated, reaching its highest levels in the cortical fiber region where the lens fiber cells are formed. In this region of the lens alpha6 integrin was uniquely localized along the cell-cell borders of the differentiating fiber cells, similar to beta1. alpha6beta4, the primary transmembrane protein of hemidesmosomes, is also expressed in the lens, but in the absence of hemidesmosomes. Differential expression of alpha6A and alpha6B isoforms with lens cell differentiation was seen at both the mRNA and the protein levels. RT-PCR studies demonstrated that alpha6B was the predominant isoform expressed both early in development, embryonic day 4, and in the epithelial regions of the day 10 embryonic lens. Isoform switching, with alpha6A now the predominant isoform, occurred in the fiber cell zones. Immunoprecipitation studies showed that alpha6B, which is characteristic of undifferentiated cells, was expressed by the lens epithelial cells but was dramatically reduced in the lens fiber zones. Expression of alpha6B began to drop as the cells initiated their differentiation and then dropped precipitously in the cortical fiber zone. In contrast, expression of the alpha6A isoform remained high until the cells became terminally differentiated. alpha6A was the predominant isoform expressed in the cortical fiber region. The down-regulation of alpha6B relative to alpha6A provides a developmental switch in the process of lens fiber cell differentiation.     

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells’ native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens¹. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.     

Modulation of N-cadherin junctions and their role as epicenters of differentiation-specific actin regulation in the developing lens

Extensive elongation of lens fiber cells is a central feature of lens morphogenesis. Our study investigates the role of N-cadherin junctions in this process in vivo. We investigate both the molecular players involved in N-cadherin junctional maturation and the subsequent function of these junctions as epicenters for the assembly of an actin cytoskeleton that drives morphogenesis. We present the first evidence of nascent cadherin junctions in vivo, and show that they are a prominent feature along lateral interfaces of undifferentiated lens epithelial cells. Maturation of these N-cadherin junctions, required for lens cell differentiation, preceded organization of a cortical actin cytoskeleton along the cells' lateral borders, but was linked to recruitment of α-catenin and dephosphorylation of N-cadherin-linked β-catenin. Biochemical analysis revealed differentiation-specific recruitment of actin regulators cortactin and Arp3 to maturing N-cadherin junctions of differentiating cells, linking N-cadherin junctional maturation with actin cytoskeletal assembly during fiber cell elongation. Blocking formation of mature N-cadherin junctions led to reduced association of α-catenin with N-cadherin, prevented organization of actin along lateral borders of differentiating lens fiber cells and blocked their elongation. These studies provide a molecular link between N-cadherin junctions and the organization of an actin cytoskeleton that governs lens fiber cell morphogenesis in vivo.     

β1 Integrins Mediate Chondrocyte Interaction with Type I Collagen, Type II Collagen, and Fibronectin

Chondrocytes isolated from the cephalic region of sterna from 14-day-old chick embryos used β1 integrins and required either Mg²⁺ or Mn²⁺ for attachment to plates coated with type I collagen, type II collagen, and fibronectin. β1 integrin was concentrated in adhesion plaques of the chondrocytes plated on type I collagen, type II collagen, and fibronectin substrates. Chondrocytes expressed at least 3 α-subunits, including α3, α5, and putative α2. α5, but not α3, had a higher molecular weight in chondrocytes than in fibroblasts. Levels of α3 and α5 were about 25-30% of that in fibroblasts. When the chondrocytes were cultured in the presence of ascorbate in suspension, the cells aggregated into clusters. This aggregation was dependent on β1 integrin and type II collagen.     

Intrachromosomal insertions: a case report and a review

Summary We describe the phenotype of a child having a recombinant chromosome 3 with a duplication 3q13.2 q25 derived from a paternal inv ins(3)(p25.3q25q13.2). A review of 27 reported cases of intrachromosomal insertions has revealed that for a carrier of intrachromosomal insertion the risk of a child with an unbalanced karyotype is 15%. This risk may be higher for particular insertions. The recombinant chromosome can have a duplication or a deletion of different segments depending on whether the insertion is direct or inverted, paracentric or pericentric, and whether there is meiotic crossing over in the inserted or the interstitial non-inserted segment. Several of the insertions have been difficult to interpret and some of them have been mistaken for paracentric inversions. Caution is therefore indicated in interpreting parental karyotypes of a child with a deletion or a duplication, particularly if it is interstitial. This is because, whereas a risk of recurrence of a child with an unbalanced karyotype is low in de novo cases and for carriers of paracentric inversions, it is high for carriers of insertions.     

Neuroadaptive responses in brainstem noradrenergic nucleic following chronic morphine exposure

Opiate dependence and withdrawal involve neuroadaptive responses in the central nervous system. A host of studies have previously implicated the A6 noradrenergic neurons of the pontine nucleus locus coeruleus (LC) as an important mediator of somatic signs observed upon withdrawal from opiates. Recent studies, however, are showing that noradrenergic neurons of the LC may not be solely involved in mediating somatic signs of withdrawal. The A2 noradrenergic neurons of the nucleus of the solitary tract (nucleus tractus solitarius [NTS]) in the caudal brainstem may be another possible site. Neurons in the nucleus paragigantocellularis lateralis (PGi), located in the rostral ventral medulla, which are known to send collateral projections to both the LC and the NTS, may co-modulate both noradrenergic nuclei in a parallel fashion, which may represent an anatomical substrate underlying the behavioral expression of opiate withdrawal. The PGi provides glutamatergic and opioid innervation to LC neurons. Hyperactivity of LC during opiate withdrawal arises, in part, from increased glutamate transmission in this pathway. The authors have recently shown that the excitatory transmitter, glutamate, co-exists with the endogenous opioid peptide, enkephalin, in a subset of axon terminals in the LC. Decreases in endogenous opioids in afferents to LC and NTS, following chronic opiate administration, may be equally important in modulating noradrenergic neurons following chronic opiate exposure, by removing a neurochemical system that would inhibit noradrenergic neurons. A persistent decrease in opioid peptide release from afferents during withdrawal would result in glutamate acting on postsynaptic targets, in an unopposed fashion. A parallel effect in opioid projections from PGi to the NTS would potentially support similar actions in this noradrenergic nucleus. The authors' recent data show that opioid-containing neurons in the PGi project to the NTS, and that enkephalin levels are decreased in opioid afferents to the NTS. This review summarizes data that the authors have collected regarding opioid expression changes in brainstem circuits (PGi-LC and PGi-NTS), following chronic morphine treatment, which may represent a model for understanding of adaptations in endogenous opioid circuits during drug dependence and withdrawal.     

Suppression of MAPK/JNK-MTORC1 signaling leads to premature loss of organelles and nuclei by autophagy during terminal differentiation of lens fiber cells

Although autophagic pathways are essential to developmental processes, many questions still remain regarding the initiation signals that regulate autophagy in the context of differentiation. To address these questions we studied the ocular lens, as the programmed elimination of nuclei and organelles occurs in a precisely regulated spatiotemporal manner to form the organelle-free zone (OFZ), a characteristic essential for vision acuity. Here, we report our discovery that inactivation of MAPK/JNK induces autophagy for formation of the OFZ through its regulation of MTORC1, where MAPK/JNK signaling is required for both MTOR activation and RPTOR/RAPTOR phosphorylation. Autophagy pathway proteins including ULK1, BECN1/Beclin 1, and MAP1LC3B2/LC3B-II were upregulated in the presence of inhibitors to either MAPK/JNK or MTOR, inducing autophagic loss of organelles to form the OFZ. These results reveal that MAPK/JNK is a positive regulator of MTORC1 signaling and its developmentally regulated inactivation provides an inducing signal for the coordinated autophagic removal of nuclei and organelles required for lens function.     

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