Psychological stress affects the severity of radiation-induced acute skin reactions in breast cancer patients.

Psychological stress exacerbates many pathological conditions including inflammatory skin conditions. The effect of psychological stress on acute radiation-induced skin reactions has not been documented before. Here, we aimed to explore if psychological stress could aggravate skin reaction severity in breast cancer patients. We conducted a secondary analysis of patient data obtained during a randomised, controlled clinical trial for acute radiation-induced skin reaction severity in 78 breast cancer patients. Patients were assessed three times a week during treatment. Skin reaction severity was measured using the modified Radiation-Induced Skin Reaction Assessment Scale (RISRAS) and Radiation Therapy Oncology Group grades. Stress levels were determined using a 5-point LIKERT scale to rate physical well-being, managing stress levels, house, family, work and other commitments. A total of 20 patients (26%) of the 78-patient cohort were considered stressed. Skin reaction severity in stressed patients was twice that of non-stressed patients (p < 0.001) and stressed patients were five times more likely to develop moist desquamation. Our results show that psychological stress aggravates skin reaction severity during radiation therapy. This research needs to be validated in a more rigorous manner by incorporating a validated scale such as the Distress Thermometer and Impact Thermometer in future skin trials.

Glucocorticoid treatment does not alter early cardiac adaptations to growth restriction in preterm sheep fetuses.

OBJECTIVE: To study the consequences of glucocorticoid treatment in fetal growth restriction (FGR) on cardiac function. SETTING: Laboratory. SAMPLE: Sheep. METHODS: Growth restriction was induced in sheep fetuses using single umbilical artery ligation (SUAL) on days 105-110 of gestation (term 147). Control fetuses were not ligated. Betamethasone (BM) (11.4 mg intramuscularly) or saline was administered to ewes on days 5 and 6 after surgery. Ewes were anaesthetised on day 7, the fetuses were removed, and their hearts were mounted on a Langendorff apparatus. Balloon catheters were inserted into the right and left ventricles. OUTCOME MEASURES: Ventricular contractile function and infarct area following ischaemia/reperfusion. RESULTS: The SUAL resulted in FGR (body weight 77% of control). The FGR was associated with increases in basal left ventricular pressure development and rates of contraction and relaxation. Right ventricular contraction was unaffected. Following brief ischaemia/reperfusion, the infarct area in FGR hearts was increased four-fold compared with controls. Antenatal BM resulted in a proportional increase in heart size and coronary flow, especially in FGR fetuses, and left ventricular pressure and heart rate responses to ß-adrenoceptor activation were increased. CONCLUSIONS: Fetal hearts rapidly adapt to FGR to maintain substrate delivery to the brain and heart. The FGR greatly enhanced the area of ischaemia, with implications for susceptibility in postnatal life. Antenatal BM treatment does not interfere with these cardiac changes but appears to increase left ventricle ß-adrenoceptor responsiveness, which may render the offspring vulnerable to subsequent cardiac dysfunction.

Trophoblast giant-cell differentiation involves changes in cytoskeleton and cell motility.

Trophoblast giant-cell differentiation is well-characterized at the molecular level, yet very little is known about how molecular changes affect the cellular functions of trophoblast in embryo implantation. We have found, using both explanted E7.5 mouse embryo ectoplacental cone and the rat choriocarcinoma (Rcho-1) cell line, that trophoblast differentiation is distinguished by dramatic changes in cytoarchitecture and cell behavior. Undifferentiated trophoblast cells contain little organized actin and few small, peripheral focal complexes and exhibit high membrane protrusive activity, while differentiated trophoblast giant cells contain prominent stress fibers, large internal as well as peripheral focal adhesions, and become immotile. The dramatic changes in cell behavior and cytoskeletal organization of giant cells correlate with changes in the activities of the Rho family of small GTPases and a decrease in tyrosine phosphorylation of focal adhesion kinase. Together, these data provide detailed insight into the cellular properties of trophoblast giant cells and suggest that giant-cell differentiation is characterized by a transition from a motile to a specialized epithelial phenotype. Furthermore, our data support a phagocytic erosion, rather than a migratory infiltration, mechanism for trophoblast giant-cell invasion of the uterine stroma.

Exogenous amino acids regulate trophectoderm differentiation in the mouse blastocyst through an mTOR-dependent pathway.

At the late blastocyst stage, the epithelial trophectoderm cells of the mammalian embryo undergo a phenotypic change that allows them to invade into the uterine stroma and make contact with the maternal circulation. This step can be regulated in vitro by the availability of amino acids. Embryos cultured in defined medium lacking amino acids cannot form trophoblast cell outgrowths on fibronectin, an in vitro model of implantation, but remain viable for up to 3 days in culture and will form outgrowths when transferred into complete medium. The amino acid requirement is a developmentally regulated permissive event that occurs during a 4- to 8-h period at the early blastocyst stage. Amino acids affect spreading competence specifically by regulating the onset of protrusive activity and not the onset of integrin activation. Rapamycin, a specific inhibitor of the kinase mTOR/FRAP/RAFT1, blocks amino acid stimulation of embryo outgrowth, demonstrating that mTOR is required for the initiation of trophectoderm protrusive activity. Inhibition of global protein translation with cycloheximide also inhibits amino acid-dependent signals, suggesting that mTOR regulates the translation of proteins required for trophoblast differentiation. Our data suggest that mTOR activity has a developmental regulatory function in trophectoderm differentiation that may serve to coordinate embryo and uterus at the time of implantation.

Laminins promote the locomotion of skeletal myoblasts via the alpha 7 integrin receptor.

The alpha 7 beta 1 integrin is specifically expressed by skeletal and cardiac muscles, and its expression and alternative mRNA splicing at the cytoplasmic domain are developmentally regulated. We analyzed the role of alpha 7 integrin in mediating myoblast adhesion and motility on different laminin isoforms. Mouse C2C12 and MM14 myoblast cell lines were found by flow cytometry and immunoprecipitation to express high levels of the alpha 7 integrin. Overall expression of alpha 7 increased as the C2C12 myoblasts differentiated; myoblasts expressed only the alpha 7B cytoplasmic variant whereas in differentiating myotubes alpha 7A increased markedly. Function-perturbing monoclonal antibodies generated to alpha 7 integrin efficiently blocked both adhesion and migration of MM14 and C2C12 mouse myoblasts on laminin 1. Other studies with MM14 myoblasts showed that alpha 7 is also a receptor for laminin 2/4 (human placental merosins) but not for epithelial-cell-specific laminin 5. Blocking antibody to alpha 7 only partially inhibited adhesion to laminin 2/4 but almost completely blocked motility on this substrate. Finally, to assess the potential role of the alpha 7 cytoplasmic domain, CHO cells were stably transfected to expressed chimeric alpha 5 cDNA constructs containing the wild-type alpha 5 or the alpha 7A or alpha 7B cytoplasmic domain; all forms of the integrin showed identical activities for adhesion, migration, proliferation, and matrix assembly on fibronectin substrates. These results established that alpha 7 beta 1 receptor can promote myoblast adhesion and motility on a restricted number of laminin isoforms and may be important in myogenic precursor recruitment during regeneration and differentiation.

Deletion of beta 1 integrins in mice results in inner cell mass failure and peri-implantation lethality.

Integrin receptors for extracellular matrix receptors are important effectors of cell adhesion, differentiation, and migration in cultured cells and are believed to be critical effectors of these processes during development. To determine when beta 1 integrins become critical during embryonic development, we generated mutant mice with a targeted disruption of the beta 1 integrin subunit gene. Heterozygous mutant mice were normal. Homozygous loss of beta 1 integrin expression was lethal during early postimplantation development. Homozygous embryos lacking beta 1 integrins formed normal-looking blastocysts and initiated implantation at E4.5. However, the E4.5 beta 1-null embryos in situ had collapsed blastocoeles, and whereas the trophoblast penetrated the uterine epithelium, extensive invasion of the decidua was not observed. Laminin-positive endoderm cells were detected in the inner cell mass area, but endoderm morphogenesis and migration were defective. By E5.5 beta 1-null embryos had degenerated extensively. In vitro analysis showed that trophoblast function in beta 1-null peri-implantation embryos was largely normal, including expression of tissue-specific markers, and outgrowth on fibronectin- and vitronectin-coated, although not on laminin-coated substrates. In contrast, the inner cell mass region of beta 1-null blastocyst outgrowths, and inner cell masses isolated from beta 1-null blastocysts, showed highly retarded growth and defective extraembryonic endoderm morphogenesis and migration. These data suggest that beta 1 integrins are required for normal morphogenesis of the inner cell mass and are essential mediators of growth and survival of cells of the inner cell mass. Failure of continued trophoblast development in beta 1-null embryos after inner cell mass failure could be attributable to either an intrinsic requirement for beta 1 integrins for later stages of trophoblast development, or to the lack of trophic signals from the beta 1-null inner cell mass.

Mouse egg integrin alpha 6 beta 1 functions as a sperm receptor.

Binding between sperm and egg plasma membranes is an essential step in fertilization. Whereas fertilin, a mammalian sperm surface protein, is involved in this crucial interaction, sperm receptors on the egg plasma membrane have not been identified. Because fertilin contains a predicted integrin ligand domain, we investigated the expression and function of integrin subunits in unfertilized mouse eggs. Polymerase chain reactions detected mRNAs for alpha 5, alpha 6, alpha v, beta 1, beta 3, and beta 5. Immunofluorescence revealed alpha 6 beta 1 and alpha v beta 3 on the plasma membrane. GoH3, a function-blocking anti-alpha 6 monoclonal antibody, abolished sperm binding, but a nonfunction-blocking anti-alpha 6 monoclonal antibody, a function-blocking anti-alpha v beta 3 polyclonal antibody, and an RGD peptide had no effect. Somatic cells bound sperm avidly, but only if they expressed alpha 6 beta 1. A peptide analog of the fertilin integrin ligand domain inhibited sperm binding to eggs and alpha 6 beta 1+ cells and diminished GoH3 staining of eggs. Our results indicate a novel role for the integrin alpha 6 beta 1 as a cell-cell adhesion receptor that mediates sperm-egg binding.

Developmental regulation of integrin expression at the time of implantation in the mouse embryo.

The trophectoderm layer of the mouse blastocyst differentiates at the late blastocyst stage to form the invasive trophoblast that mediates implantation of the embryo into the uterine wall. The first sign that trophoblast cells have developed an invasion-specific cell behavior appears about 10-15 hours after the embryo hatches from the zona pellucida, when the quiescent, non-adherent trophectoderm cells initiate protrusive activity and become adhesive to extracellular matrix. Our previous findings that trophoblast outgrowth on extracellular-matrix-coated substrata involves the integrin family of adhesion receptors (Sutherland, A. E., Calarco, P. G. and Damsky, C. H., 1988, J. Cell Biol. 106, 1331-1348), suggested that the onset of trophoblast adhesive and migratory behavior at the time of implantation may be due to changes in expression or distribution of integrin receptors. We have thus examined the mRNA and protein expression of individual integrin subunits during pre- and periimplantation development (E0-E7.5). A basic repertoire of integrins, including receptors for fibronectin (alpha 5 beta 1), laminin (alpha 6B beta 1) and vitronectin (alpha v beta 3), was expressed continuously throughout this period, whereas the expression of five other integrin subunits was developmentally regulated. The mRNA for three of these (alpha 2, alpha 6A and alpha 7) was first detected in the late blastocyst, coincident with endoderm differentiation and development of attachment competence. The mRNA for another (alpha 1) was not detected until after trophoblast outgrowth had begun, suggesting that its expression may be induced by contact with matrix. At E7.5, three of the temporally regulated integrins (alpha 1, apha 6A, alpha 7), all of which can form receptors for laminin, were detected only in the ectoplacental cone (differentiating trophoblast), and may thus play specific roles in trophoblast adhesion and/or differentiation. Because laminin expression is upregulated in decidualized uterine stroma in response to the implanting embryo, we examined trophoblast-laminin interactions, using laminin fragments and integrin antibodies to determine which integrin receptors were involved. Trophoblast cells attached and spread on both the E8 and P1' fragments of laminin; however, the P1' binding site was cryptic in intact laminin. Interaction with P1' was RGD- and alpha v beta 3-dependent, whereas outgrowth on E8 was RGD-independent and not inhibited by antibodies to the laminin receptor alpha 6 beta 1, suggesting that alpha 7 beta 1 is the major trophoblast integrin E8 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

A comparison of methods for sample clean-up prior to quantification of metal-binding proteins.

1. The recovery of rabbit metallothionein and metallothionein-like proteins has been examined under different conditions. 2. After heat or ethanol denaturation, recovery of rabbit MT-II was quantitative. 3. Recovery of rabbit MT was not affected by the presence or absence of dithiothreitol (DTT) after heat treatment. However, recovery from ethanolic solution decreased in buffers that did not contain the antioxidant. 4. Acetone precipitation of rabbit MT resulted in lower yields which approached 90% only in the presence of DTT. 5. Recovery of metallothionein-like proteins from cytosols of the hepatopancreas of the tanner crab, Chionoecetes bairdi, were examined using pulse polarography and HPLC. Relative to heat denaturation, ethanol and acetone yielded recoveries of 66 and 28%, respectively, in the presence of DTT. However, acetone did yield a solution which contained less extraneous protein of molecular mass greater than 43 kDa than heat denaturation. 6. We concluded that heat denaturation is the preferred treatment for quantitative recovery of metal-binding proteins. Acetone precipitation is useful for the purification of MT.

Expression of syndecan, a putative low affinity fibroblast growth factor receptor, in the early mouse embryo.

Syndecan is an integral membrane proteoglycan that binds cells to several interstitial extracellular matrix components and binds to basic fibroblast-growth factor (bFGF) thus promoting bFGF association with its high-affinity receptor. We find that syndecan expression undergoes striking spatial and temporal changes during the period from the early cleavage through the late gastrula stages in the mouse embryo. Syndecan is detected initially at the 4-cell stage. Between the 4-cell and late morula stages, syndecan is present intracellularly and on the external surfaces of the blastomeres but is absent from regions of cell-cell contact. At the blastocyst stage, syndecan is first detected at cell-cell boundaries throughout the embryo and then, at the time of endoderm segregation, becomes restricted to the first site of matrix accumulation within the embryo, the interface between the primitive ectoderm and primitive endoderm. During gastrulation, syndecan is distributed uniformly on the basolateral cell surfaces of the embryonic ectoderm and definitive embryonic endoderm, but is expressed with an anteroposterior asymmetry on the surface of embryonic mesoderm cells, suggesting that it contributes to the process of mesoderm specification. In the extraembryonic region, syndecan is not detectable on most cells of the central core of the ectoplacental cone, but is strongly expressed by cells undergoing trophoblast giant cell differentiation and remains prominent on differentiated giant cells, suggesting a role in placental development. Immunoprecipitation studies indicate that the size of the syndecan core protein, although larger than that found in adult tissues (75 versus 69 x 10(3) Mr), does not change during peri-implantation development. The size distribution of the intact proteoglycan does change, however, indicating developmental alterations in its glycosaminoglycan composition. These results indicate potential roles for syndecan in epithelial organization of the embryonic ectoderm, in differential axial patterning of the embryonic mesoderm and in trophoblast giant cell function.

Inner cell allocation in the mouse morula: the role of oriented division during fourth cleavage.

Two populations of blastomeres become positionally distinct during fourth cleavage in the mouse embryo; the inner cells become enclosed within the embryo and the outer cells form the enclosing layer. The segregation of these two cell populations is important for later development, because it represents the initial step in the divergence of placental and fetal lineages. The mechanism by which the inner cells become allocated has been thought to involve the oriented division of polarized 8-cell blastomeres, but this has never been examined in the intact embryo. By using the technique of time-lapse cinemicrography, we have been able for the first time to directly examine the division planes of 8-cell blastomeres during fourth cleavage, and find that there are three, rather than two, major division plane orientations; anticlinal (perpendicular to the outer surface of the blastomere), periclinal (parallel to the outer surface of the blastomere), and oblique (at an angle between the other two). The observed frequencies of each type of division plane orientation provide evidence that the inner cells of the morula must derive from oriented division of 8-cell blastomeres, in accordance with the polarization hypothesis. Analysis of fourth cleavage division plane orientation with respect to either lineage or division order reveals that it is not associated with lineage from either the 2- or the 4-cell stage, but has a slight statistical association with fourth cleavage division order. The lack of association between division plane orientation and lineage supports the prediction that packing patterns and intercellular interactions within the 8-cell embryo during compaction play a role in determining fourth cleavage division plane orientation and thus, the positional fate of the daughter 16-cell blastomeres.

Expression and function of cell surface extracellular matrix receptors in mouse blastocyst attachment and outgrowth.

Mouse-hatched blastocysts cultured in vitro will attach and form outgrowths of trophoblast cells on appropriate substrates, providing a model for implantation. Immediately after hatching, the surfaces of blastocysts are quiescent and are not adhesive. Over the period 24-36 h post-hatching, blastocysts cultured in serum-free medium become adhesive and attach and spread on the extracellular matrix components fibronectin, laminin, and collagen type IV in a ligand specific manner. Attachment and trophoblast outgrowth on these substrates can be inhibited by addition to the culture medium of an antibody, anti-ECMr (anti-extracellular matrix receptor), that recognizes a group of 140-kD glycoproteins similar to those of the 140-kD extracellular matrix receptor complex (integrin) recognized in avian cells by CSAT and JG22 monoclonal antibodies. Addition to the culture medium of a synthetic peptide containing the Arg-Gly-Asp tripeptide cell recognition sequence of fibronectin inhibits trophoblast outgrowth on both laminin and fibronectin. However, the presence of the peptide does not affect attachment of the blastocysts to either ligand. Immunoprecipitation of 125I surface-labeled embryos using anti-ECMr reveals that antigens recognized by this antibody are exposed on the surfaces of embryos at a time when they are spreading on the substrate, but are not detectable immediately after hatching. Immunofluorescence experiments show that both the ECMr antigens and the cytoskeletal proteins vinculin and talin are enriched on the cell processes and ventral surfaces of trophectoderm cells in embryo outgrowths, in patterns similar to those seen in fibroblasts, and consistent with their role in adhesion of the trophoblast cells to the substratum.

Analysis of compaction in the preimplantation mouse embryo.

An SEM analysis of the effects of tunicamycin, cytochalasin B, and colcemid has yielded insights into the process of compaction in the early mouse embryo. All three reagents block or reverse compaction and decrease the number of microvilli (MV), although some MV polarization is permitted. In addition, tunicamycin is shown to lessen cell adhesion even in compacted embryos. Cytochalasin B causes the formation of MV clumps some of which are preferentially localized to the apex or lateral ring region. Colcemid reverses compaction and, coupled with Pronase treatment, completely blocks compaction of uncompacted 8-cell embryos. Observations also suggest that MV polarization can occur only once but compaction (the close adherance and flattening of blastomeres) can be reversed and reinduced. Evidence is consistent with a three-step compaction process involving (1) cell surface recognition and attachment of a ring of lateral microvilli to adjacent blastomeres, (2) subsequent microfilament shortening in these lateral MV, and (3) maintenance of the compacted and polarized state by microtubules.