bluuebunny/crossref_metadata_2025 huggingface dataset:
Variability of Vascular Endothelial Growth Factor in Normal Human Breast Tissue in Vivo during the Menstrual Cycle
Authors: Dabrosin Charlotta
Published: June 2003

Exposure to sex steroids increases the risk of breast cancer, but the mechanisms are poorly understood. Angiogenesis is crucial in tumor development and progression. Very little is known about the regulation of angiogenesis in the normal breast. Vascular endothelial growth factor (VEGF) has a key stimulatory role in angiogenesis. Interferon-inducible protein 10 (IP-10) is a potent inhibitor of angiogenesis in vivo. These factors function in autocrine/paracrine pathways; therefore, direct measurements in the target tissue are needed. I measured VEGF and IP-10 in normal human breast tissue in situ in healthy women, using microdialysis, in the follicular and luteal phase of the menstrual cycle. In breast tissue, VEGF levels increased in the luteal phase, compared with the follicular phase (17.8 ± 4 pg/ml to 34 ± 9 pg/ml, P < 0.05). Plasma VEGF did not show a cyclic variation (10.6 ± 2.8 pg/ml vs. 14.6 ± 3.5 pg/liter, P = 0.3). IP-10 levels did not vary during the menstrual cycle either in breast tissue (65 ± 17 pg/ml vs. 75 ± 21 pg/ml, P = 0.6) or in plasma (64 ± 7 pg/ml vs. 81 ± 10 pg/ml, P = 0.06). The data suggests that, in the luteal phase, VEGF and IP-10, in the normal human breast, exhibit a proangiogenic profile. This may be one mechanism by which sex steroids contribute to breast cancer development.
Endocrine and Metabolic Responses to Long-Term Monotherapy with the Antiepileptic Drug Valproate in the Normally Cycling Rhesus Monkey
Authors: Ferin Michel , Morrell Martha , Xiao Ennian , Kochan Lisa , Qian Fang , Wright Thomas , Sauer Mark
Published: June 2003

An association between epilepsy and reproductive disturbances with an apparent increase in a polycystic ovarian syndrome (PCOS) has been reported. Whether this association can be attributed to epilepsy itself or is related to antiepileptic drug therapy, in particular valproate (VPA), remains controversial. We studied effects of a long-term VPA treatment on cycling monkeys, postulating that, if VPA monotherapy were to promote abnormal endocrine and metabolic parameters that are characteristic of PCOS, changes in cyclicity would be readily demonstrated.

After a 2-month control, a 12- to 15-month VPA monotherapy was initiated in 7 regularly cycling rhesus monkeys. Overall mean levels of VPA were 88.7 ± 4.0 (se) μg/ml. Mean body weight increased progressively during VPA treatment from 8.5 ± 0.5 kg before treatment to 9.6 ± 0.7 kg in the last week of treatment (P < 0.05). Monkeys continued to have regular ovulatory menstrual cycles throughout VPA monotherapy. Length of the cycles was 28 ± 0.58 d in control and 28.4 ± 1.18 d in the last 3 months of VPA treatment. Follicular and luteal lengths and peak preovulatory estradiol and integrated luteal progesterone levels did not differ between control and treatment. Ovaries from VPA-treated monkeys showed histological evidence of ovulation, and none had characteristic features of PCOS. Endocrine PCOS markers, such as increased early follicular LH/FSH ratio and androgen levels were not different in control and VPA treatment cycles. LH and 17-hydroxyprogesterone responses to GnRH agonist challenges and the insulin response to glucose tolerance tests were similar in control and VPA groups. Lipid profiles were not affected by VPA treatment. The data indicate that a 12- to 15-month therapeutic exposure to VPA does not induce cyclic hormonal or morphological ovarian abnormalities or characteristics of the PCOS when administered to nonepileptic normally cycling nonhuman primates.
Serum Androgen Bioactivity in Cryptorchid and Noncryptorchid Boys during the Postnatal Reproductive Hormone Surge
Authors: Raivio Taneli , Toppari Jorma , Kaleva Marko , Virtanen Helena , Haavisto Anne-Maarit , Dunkel Leo , Jänne Olli A.
Published: June 2003

The first postnatal months of life in boys are characterized by activation of the hypothalamic-pituitary-testicular axis that results in the well depicted surge of reproductive hormones. Serum testosterone levels at that time are high, but infants do not display signs of virilization, and subsequently there is only indirect evidence that circulating androgens during the surge are biologically active. We used a recombinant cell bioassay to determine serum androgen bioactivity in 80 3-month-old boys born after full-term pregnancies (37–42 wk) in whom localization of the testes was determined by palpation after birth and at a mean age of 3 months. At that age, serum androgen bioactivity ranged from less than 0.8 to 1.9 nm testosterone equivalents and correlated with serum testosterone concentration (r = 0.71; P < 0.0001; n = 34), free androgen index (r = 0.80; P < 0.0001; n = 34), age (r = −0.29; P < 0.01; n = 80), and localization of the testes (r = −0.24; P < 0.05; n = 80). Moreover, all boys in this study with detectable androgen bioactivity (n = 26) had testes located in scrotal or high scrotal position (n = 64), whereas all boys (n = 16) with at least 1 suprascrotal, inguinal, or nonpalpable testis had nonmeasurable androgen bioactivity in serum (P < 0.01). We conclude that 3-month-old boys are exposed to biological effects of androgens during the postnatal activation of the hypothalamic-pituitary-testicular axis, and that this exposure may be reduced in boys with at least 1 testis located superior to the scrotum.
Two Homozygous Mutations in the 11β-Hydroxysteroid Dehydrogenase Type 2 Gene in a Case of Apparent Mineralocorticoid Excess
Authors: Carvajal Cristian A. , Gonzalez Alexis A. , Romero Damian G. , González Angel , Mosso Lorena M. , Lagos Elizabeth T. , Hevia Maria del P. , Rosati Maria P. , Perez-Acle Tomás O. , Gomez-Sanchez Celso E. , Montero Joaquín A. , Fardella Carlos E.
Published: June 2003

The human microsomal 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) metabolizes active cortisol into cortisone and protects the mineralocorticoid receptor from glucocorticoid occupancy. In a congenital deficiency of 11β-HSD2, the protective mechanism fails and cortisol gains inappropriate access to mineralocorticoid receptor, resulting in low-renin hypertension and hypokalemia. In the present study, we describe the clinical and molecular genetic characterization of a patient with a new mutation in the HSD11B2 gene. This is a 4-yr-old male with arterial hypertension. The plasma renin activity and serum aldosterone were undetectable in the presence of a high cortisol to cortisone ratio. PCR amplification and sequence analysis of HSD11B2 gene showed the homozygous mutation in exon 4 Asp223Asn (GAC→AAC) and a single nucleotide substitution C→T in intron 3. Using site-directed mutagenesis, we generated a mutant 11βHSD2 cDNA containing the Asp223Asn mutation. Wild-type and mutant cDNA was transfected into Chinese hamster ovary cells and enzymatic activities were measured using radiolabeled cortisol and thin-layer chromatography. The mRNA and 11βHSD2 protein were detected by RT-PCR and Western blot, respectively. Wild-type and mutant 11βHSD2 protein was expressed in Chinese hamster ovary cells, but the mutant enzyme had only 6% of wild-type activity. In silico 3D modeling showed that Asp223Asn changed the enzyme’s surface electrostatic potential affecting the cofactor and substrate enzyme-binding capacity. The single substitution C→T in intron 3 (IVS3 + 14 C→T) have been previously reported that alters the normal splicing of pre-mRNA, given a nonfunctional protein. These findings may determine the full inactivation of this enzyme, explaining the biochemical profile and the early onset of hypertension seen in this patient.
Low-Dose Flutamide-Metformin Therapy Reverses Insulin Resistance and Reduces Fat Mass in Nonobese Adolescents with Ovarian Hyperandrogenism
Authors: Ibáñez Lourdes , Ong Ken , Ferrer Angela , Amin Rakesh , Dunger David , de Zegher Francis
Published: June 2003

Ovarian hyperandrogenism is a common disorder often presenting post menarche with anovulatory oligomenorrhea and signs of androgen excess. Associated hyperinsulinemic insulin resistance, dyslipidemia, and central fat excess herald long-term disease risk. Combined antiandrogen (flutamide 250 mg/d) and insulin-sensitizing (metformin) therapy has beneficial effects, in particular on dyslipidemia and androgen excess in young women. We studied the effects of low-dose flutamide-metformin combination on metabolic variables and body composition in adolescent girls with ovarian hyperandrogenism. Thirty teenage girls (age range, 13.6–18.6 yr) with hyperinsulinemic hyperandrogenism participated in a 12-month pilot study with a 3-month off-treatment phase and a 9-month treatment phase (randomized sequence) on combined flutamide (125 mg/d) and metformin (1275 mg/d). Body composition was assessed by dual-energy x-ray absorptiometry; endocrine-metabolic state and ovulation rate were screened every 3 months. Insulin sensitivity was assessed by homeostasis model assessment (HOMA). Overnight GH and LH profiles were obtained pretreatment and after 6 months on treatment (n = 8). Over the 3-month pretreatment control phase (n = 14) all study indices were unchanged. Flutamide-metformin treatment (n = 30) was followed within 3 months by marked decreases in hirsutism score and serum androgens, by a more than 50% increase in insulin sensitivity and by a less atherogenic lipid profile (all P < 0.0001). After 9 months on flutamide-metformin, body fat decreased by 10%, with a preferential 20% loss of abdominal fat; conversely lean body mass increased, and total body weight remained unchanged; ovulation rate increased from 7–87% after 9 months. Baseline GH hypersecretion and elevated serum IGF-1 normalized after 6 months on flutamide-metformin. Within 3 months post treatment (n = 16), a rebound was observed for all assessed indices. In conclusion, in teenage girls with ovarian hyperandrogenism, low-dose combined flutamide-metformin therapy attenuated a spectrum of abnormalities, including insulin resistance and hyperlipidemia. Improved insulin sensitivity and reduced androgen activity led to a marked redistribution of body fat and lean mass, resulting in a more feminine body shape.
Mutation of IVS2 –12A/C>G in Combination with 707–714delGAGACTAC in the CYP21 Gene Is Caused by Deletion of the C4-CYP21 Repeat Module with Steroid 21-Hydroxylase Deficiency
Authors: Lee Hsien-Hsiung , Chang Shwu-Fen , Tsai Fuu-Jen , Tsai Li-Ping , Lin Ching-Yu
Published: June 2003

More than 90% of the cases of congenital adrenal hyperplasia are caused by mutations of the CYP21 gene. Approximately 75% of the defective CYP21 genes are generated through intergenic recombination, termed apparent gene conversion, from the neighboring CYP21P pseudogene. Among them, mutation of the aberrant splicing donor site of IVS2 –12A/C>G at nucleotide (nt) 655 is believed to be a result derived from this mechanism and is the most prevalent case among all ethnic groups. However, mutation of 707–714delGAGACTAC rarely exists alone, although this locus is a distance of 53 nt away from IVS2 –12A/C>G. From the molecular characterization of the mutation of IVS2 –12A/C>G combined with 707–714delGAGACTAC in patients with congenital adrenal hyperplasia, we found that it appeared to be in a 3.2-rather than a 3.7-kb fragment generated by Taq I digestion in a PCR product of the CYP21 gene. Interestingly, the 5′ end region of such a CYP21 haplotype had CYP21P-specific sequences. Our results indicate that the coexistence of these two mutations is caused by deletion of the CYP21P, XA, RP2, and C4B genes and intergenic recombination in the C4-CYP21 repeat module. Surprisingly, this kind of the haplotype of the mutated CYP21 gene has not been reported as a gene deletion.
The Rationale and Management of Hyperglycemia for In-Patients with Cardiovascular Disease: Time for Change
Authors: Trence Dace L. , Kelly Janet L. , Hirsch Irl B.
Published: June 2003

There is increasing evidence that aggressive glycemic control for patients admitted into the hospital improves clinical outcomes, especially for patients with cardiovascular disease. There appear to be a variety of mechanisms for this. Although hyperglycemia has been shown to result in poor wound healing and more infectious complications, especially after cardiac surgical procedures, what has become clear is that the treatment of hyperglycemia with iv glucose, insulin, and potassium (GIK) results in better clinical outcomes even in patients without diabetes. The mechanisms for this are not year clear, but could be related to the insulin administration, perhaps due to suppression of various cytokines or free fatty acids. The practical use of insulin in these patients requires basic understanding of the use of both iv and sc insulin. Although there are several appropriate options for both of these routes of administration, it is critical that all caregivers involved in this population’s care are knowledgeable about insulin strategies.
A Compound Heterozygote Case of Type II Aldosterone Synthase Deficiency
Authors: Dunlop Felicity M. , Crock Patricia A. , Montalto Joseph , Funder John W. , Curnow Kathleen M.
Published: June 2003

An infant with failure to thrive, persistent hyponatremia and episodic vomiting and diarrhea was admitted to hospital at 9 months of age, and the diagnosis of type II aldosterone synthase deficiency was confirmed by plasma and urinary steroid determinations. The entire coding sequence of the aldosterone synthase gene (CYP11B2) was determined (both strands) in the affected infant, an unaffected sibling, and both parents. An exon 3 mutation (C554T, leading to amino acid T185I) was found in the father and both siblings, and an exon 9 mutation (A1492G, leading to T498A) was found in the affected infant and the mother. Expression of the mutant sequences in COS cells showed steroidogenic patterns typical of aldosterone synthase type II deficiency, including very low levels of aldosterone synthesis (≤0.5% of wild-type enzyme) consistent with the low aldosterone levels in the patient’s plasma. Both mutations in this compound heterozygote localize to the β3-sheet in the cytochrome P450 enzyme structure, as does the previously characterized R181W mutation. This region of the enzyme is not part of the putative structural core, but mutations to this region suggest that it is important for conferring the unique ability of aldosterone synthase to catalyze efficient oxygenation of the C18 carbon of steroid substrates.
Microarray Analysis of Uterine Gene Expression in Mouse and Human Pregnancy
Authors: Bethin Kathleen E. , Nagai Yoshihiko , Sladek Robert , Asada Minoru , Sadovsky Yoel , Hudson Thomas J. , Muglia Louis J.
Published: June 2003

Improved care of infants born prematurely has increased their survival. However, the incidence of preterm labor has not changed. To understand the processes involved in preterm labor, we used oligonucleotide microarrays to study gene expression in murine and human uterus during pregnancy. The induction of enzymes for prostaglandin synthesis was used as a marker for important changes during pregnancy because prostaglandins strongly contribute to both human and murine labor. We identified 504 genes that changed at least 2-fold between d 13.5 and 19.0 in the gravid mouse uterus. In the pregnant human myometrium, we found 478 genes that changed at least 2-fold in either term or preterm labor compared with preterm nonlabor specimens and 77 genes that significantly varied in both preterm and term labor. Patterns of gene regulation within functional groups comparing human preterm and term labor were similar, although the magnitude of change often varied. Surprisingly, few genes that changed significantly throughout pregnancy were the same in the mouse and human. These data suggest that functional progesterone withdrawal in human myometrium may not be the primary mechanism for labor induction, may implicate similar mechanisms for idiopathic preterm and term labor in humans, and may identify novel targets for further study.
Laminin α subunits and their role inC. elegansdevelopment
Authors: Huang Cheng-chen , Hall David H. , Hedgecock Edward M. , Kao Gautam , Karantza Vassiliki , Vogel Bruce E. , Hutter Harald , Chisholm Andrew D. , Yurchenco Peter D. , Wadsworth William G.
Published: June 2003

Laminins are heterotrimeric (α/β/γ) glycoproteins that form a major polymer within basement membranes. Different α, β andγ subunits can assemble into various laminin isoforms that have different, but often overlapping, distributions and functions. In this study,we examine the contributions of the laminin α subunits to the development of C. elegans. There are two α, one β and oneγ laminin subunit, suggesting two laminin isoforms that differ by theirα subunit assemble in C. elegans. We find that near the end of gastrulation and before other basement membrane components are detected, theα subunits are secreted between primary tissue layers and become distributed in different patterns to the surfaces of cells. Mutations in either α subunit gene cause missing or disrupted extracellular matrix where the protein normally localizes. Cell-cell adhesions are abnormal: in some cases essential cell-cell adhesions are lacking, while in other cases,cells inappropriately adhere to and invade neighboring tissues. Using electron microscopy, we observe adhesion complexes at improper cell surfaces and disoriented cytoskeletal filaments. Cells throughout the animal show defective differentiation, proliferation or migration, suggesting a general disruption of cell-cell signaling. The results suggest a receptor-mediated process localizes each secreted laminin to exposed cell surfaces and that laminin is crucial for organizing extracellular matrix, receptor and intracellular proteins at those surfaces. We propose this supramolecular architecture regulates adhesions and signaling between adjacent tissues.
Semaphorin3a1 regulates angioblast migration and vascular development in zebrafish embryos
Authors: Shoji Wataru , Isogai Sumio , Sato-Maeda Mika , Obinata Masuo , Kuwada John Y.
Published: June 2003

Semaphorins are a large family of secreted and cell surface molecules that guide neural growth cones to their targets during development. Some semaphorins are expressed in cells and tissues beyond the nervous system suggesting the possibility that they function in the development of non-neural tissues as well. In the trunk of zebrafish embryos endothelial precursors(angioblasts) are located ventral and lateral to the somites. The angioblasts migrate medially and dorsally along the medial surface of the somites to form the dorsal aorta just ventral to the notochord. Here we show that in zebrafish Sema3a1 is involved in angioblast migration in vivo. Expression of sema3a1 in somites and neuropilin 1, which encodes for a component of the Sema3a receptor, in angioblasts suggested that Sema3a1 regulates the pathway of the dorsally migrating angioblasts. Antisense knockdown of Sema3a1 inhibited the formation of the dorsal aorta. Induced ubiquitous expression of sema3a1 in hsp70:gfpsema3a1myc transgenic embryos inhibited migration of angioblasts ventral and lateral to the somites and retarded development of the dorsal aorta, resulting in severely reduced blood circulation. Furthermore, analysis of cells that express angioblast markers following induced expression of sema3a1 or in a mutant that changes the expression of sema3a1 in the somites confirmed these results. These data implicate Sema3a1, a guidance factor for neural growth cones, in the development of the vascular system.
Suppression of macho-1-directed muscle fate by FGF and BMP is required for formation of posterior endoderm in ascidian embryos
Authors: Kondoh Keisuke , Kobayashi Kenji , Nishida Hiroki
Published: June 2003

Specification of germ layers is a crucial event in early embryogenesis. In embryos of the ascidian, Halocynthia roretzi, endoderm cells originate from two distinct lineages in the vegetal hemisphere. Cell dissociation experiments suggest that cell interactions are required for posterior endoderm formation, which has hitherto been thought to be solely regulated by localized egg cytoplasmic factors. Without cell interaction,every descendant of posterior-vegetal blastomeres, including endoderm precursors, assumed muscle fate. Cell interactions are required for suppression of muscle fate and thereby promote endoderm differentiation in the posterior endoderm precursors. The cell interactions take place at the 16- to 32-cell stage. Inhibition of cell signaling by FGF receptor and MEK inhibitor also supported the requirement of cell interactions. Consistently, FGF was a potent signaling molecule, whose signaling is transduced by MEK-MAPK. By contrast, such cell interactions are not required for formation of the anterior endoderm. Our results suggest that another redundant signaling molecule is also involved in the posterior endoderm formation, which is likely to be mediated by BMP.

Suppression of the function of macho-1, a muscle determinant in ascidian eggs, by antisense oligonucleotide was enough to allow autonomous endoderm specification. Therefore, the cell interactions induce endoderm formation by suppressing the function of macho-1, which is to promote muscle fate. These findings suggest the presence of novel mechanisms that suppress functions of inappropriately distributed maternal determinants via cell interactions after embryogenesis starts. Such cell interactions would restrict the regions where maternal determinants work, and play a key role in marking precise boundaries between precursor cells of different tissue types.
Neural crest induction by paraxial mesoderm inXenopusembryos requires FGF signals
Authors: Monsoro-Burq Anne-Hélène , Fletcher Russell B. , Harland Richard M.
Published: June 2003

At the border of the neural plate, the induction of the neural crest can be achieved by interactions with the epidermis, or with the underlying mesoderm. Wnt signals are required for the inducing activity of the epidermis in chick and amphibian embryos. Here, we analyze the molecular mechanisms of neural crest induction by the mesoderm in Xenopus embryos. Using a recombination assay, we show that prospective paraxial mesoderm induces a panel of neural crest markers (Slug, FoxD3, Zic5 and Sox9),whereas the future axial mesoderm only induces a subset of these genes. This induction is blocked by a dominant negative (dn) form of FGFR1. However,neither dnFGFR4a nor inhibition of Wnt signaling prevents neural crest induction in this system. Among the FGFs, FGF8 is strongly expressed by the paraxial mesoderm. FGF8 is sufficient to induce the neural crest markers FoxD3, Sox9 and Zic5 transiently in the animal cap assay. In vivo, FGF8 injections also expand the Slug expression domain. This suggests that FGF8 can initiate neural crest formation and cooperates with other DLMZ-derived factors to maintain and complete neural crest induction. In contrast to Wnts, eFGF or bFGF, FGF8 elicits neural crest induction in the absence of mesoderm induction and without a requirement for BMP antagonists. In vivo, it is difficult to dissociate the roles of FGF and WNT factors in mesoderm induction and neural patterning. We show that, in most cases, effects on neural crest formation were parallel to altered mesoderm or neural development. However, neural and neural crest patterning can be dissociated experimentally using different dominant-negative manipulations:while Nfz8 blocks both posterior neural plate formation and neural crest formation, dnFGFR4a blocks neural patterning without blocking neural crest formation. These results suggest that different signal transduction mechanisms may be used in neural crest induction, and anteroposterior neural patterning.
Six1is required for the early organogenesis of mammalian kidney
Authors: Xu Pin-Xian , Zheng Weiming , Huang Li , Maire Pascal , Laclef Christine , Silvius Derek
Published: June 2003

The murine Six gene family, homologous to Drosophila sine oculis(so) which encodes a homeodomain transcription factor, is composed of six members (Six1-6). Among the six members, only the Six2gene has been previously shown to be expressed early in kidney development,but its function is unknown. We have recently found that the Six1gene is also expressed in the kidney. In the developing kidney, Six1is expressed in the uninduced metanephric mesenchyme at E10.5 and in the induced mesenchyme around the ureteric bud at E11.5. At E17.5 to P0, Six1 expression became restricted to a subpopulation of collecting tubule epithelial cells. To study its in vivo function, we have recently generated Six1 mutant mice. Loss of Six1 leads to a failure of ureteric bud invasion into the mesenchyme and subsequent apoptosis of the mesenchyme. These results indicate that Six1 plays an essential role in early kidney development. In Six1-/- kidney development, we have found that Pax2, Six2 and Sall1expression was markedly reduced in the metanephric mesenchyme at E10.5,indicating that Six1 is required for the expression of these genes in the metanephric mesenchyme. In contrast, Eya1 expression was unaffected in Six1-/- metanephric mesenchyme at E10.5,indicating that Eya1 may function upstream of Six1. Moreover, our results show that both Eya1 and Six1expression in the metanephric mesenchyme is preserved in Pax2-/- embryos at E10.5, further indicating that Pax2 functions downstream of Eya1 and Six1 in the metanephric mesenchyme. Thus, the epistatic relationship between Pax, Eya and Six genes in the metanephric mesenchyme during early kidney development is distinct from a genetic pathway elucidated in the Drosophila eye imaginal disc. Finally, our results show that Eya1 and Six1genetically interact during mammalian kidney development, because most compound heterozygous embryos show hypoplastic kidneys. These analyses establish a role for Six1 in the initial inductive step for metanephric development.
Direct and concentration-dependent regulation of the proneural geneNeurogenin2by Pax6
Authors: Scardigli Raffaella , Bäumer Nicole , Gruss Peter , Guillemot François , Le Roux Isabelle
Published: June 2003

Expression of the proneural gene Neurogenin2 is controlled by several enhancer elements, with the E1 element active in restricted progenitor domains in the embryonic spinal cord and telencephalon that express the homeodomain protein Pax6. We show that Pax6 function is both required and sufficient to activate this enhancer, and we identify one evolutionary conserved sequence in the E1 element with high similarity to a consensus Pax6 binding site. This conserved sequence binds Pax6 protein with low affinity both in vitro and in vivo, and its disruption results in a severe decrease in E1 activity in the spinal cord and in its abolition in the cerebral cortex. The regulation of Neurogenin2 by Pax6 is thus direct.

Pax6 is expressed in concentration gradients in both spinal cord and telencephalon. We demonstrate that the E1 element is only activated by high concentrations of Pax6 protein, and that this requirement explains the restriction of E1 enhancer activity to domains of high Pax6 expression levels in the medioventral spinal cord and lateral cortex. By modifying the E1 enhancer sequence, we also show that the spatial pattern of enhancer activity is determined by the affinity of its binding site for Pax6. Together, these data demonstrate that direct transcriptional regulation accounts for the coordination between mechanisms of patterning and neurogenesis. They also provide evidence that Pax6 expression gradients are involved in establishing borders of gene expression domains in different regions of the nervous system.
SDF-9, a protein tyrosine phosphatase-like molecule, regulates the L3/dauer developmental decision through hormonal signaling inC. elegans
Authors: Ohkura Kiyotaka , Suzuki Norio , Ishihara Takeshi , Katsura Isao
Published: June 2003

The dauer larva of the nematode Caenorhabditis elegans is a good model system for investigating the regulation of developmental fates by environmental cues. Here we show that SDF-9, a protein tyrosine phosphatase-like molecule, is involved in the regulation of dauer larva formation. The dauer larva of sdf-9 mutants is different from a normal dauer larva but resembles the dauer-like larva of daf-9 and daf-12 dauer-constitutive mutants. Like these mutants, the dauer-constitutive phenotypes of sdf-9 mutants were greatly enhanced by cholesterol deprivation. Epistasis analyses, together with the relationship between sdf-9 mutations and daf-9 expression, suggested that SDF-9 increases the activity of DAF-9 or helps the execution of the DAF-9 function. SDF-9 was expressed in two head cells in which DAF-9 is expressed. By their position and by genetic mosaic experiments, we identified these cells as XXXL/R cells, which are known as embryonic hypodermal cells and whose function at later stages is unknown. Killing of the sdf-9-expressing cells in the wild-type first-stage larva induced formation of the dauer-like larva. Since this study on SDF-9 and former studies on DAF-9 showed that the functions of these proteins are related to those of steroids, XXXL/R cells seem to play a key role in the metabolism or function of a steroid hormone(s)that acts in dauer regulation.
The T-box-encoding Dorsocross genes function in amnioserosa development and the patterning of the dorsolateral germ band downstream of Dpp
Authors: Reim Ingolf , Lee Hsiu-Hsiang , Frasch Manfred
Published: June 2003

Dpp signals are responsible for establishing a variety of cell identities in dorsal and lateral areas of the early Drosophila embryo, including the extra-embryonic amnioserosa as well as different ectodermal and mesodermal cell types. Although we have a reasonably clear picture of how Dpp signaling activity is modulated spatially and temporally during these processes, a better understanding of how these signals are executed requires the identification and characterization of a collection of downstream genes that uniquely respond to these signals. In the present study, we describe three novel genes, Dorsocross1, Dorsocross2 and Dorsocross3, which are expressed downstream of Dpp in the presumptive and definitive amnioserosa,dorsal ectoderm and dorsal mesoderm. We show that these genes are good candidates for being direct targets of the Dpp signaling cascade. Dorsocross expression in the dorsal ectoderm and mesoderm is metameric and requires a combination of Dpp and Wingless signals. In addition, a transverse stripe of expression in dorsoanterior areas of early embryos is independent of Dpp. The Dorsocross genes encode closely related proteins of the T-box domain family of transcription factors. All three genes are arranged in a gene cluster, are expressed in identical patterns in embryos, and appear to be genetically redundant. By generating mutants with a loss of all three Dorsocross genes, we demonstrate that Dorsocross gene activity is crucial for the completion of differentiation, cell proliferation arrest, and survival of amnioserosa cells. In addition, we show that the Dorsocross genes are required for normal patterning of the dorsolateral ectoderm and, in particular, the repression of wingless and the ladybird homeobox genes within this area of the germ band. These findings extend our knowledge of the regulatory pathways during amnioserosa development and the patterning of the dorsolateral embryonic germ band in response to Dpp signals.
Arabidopsis CROOKEDencodes for the smallest subunit of the ARP2/3 complex and controls cell shape by region specific fine F-actin formation
Authors: Mathur Jaideep , Mathur Neeta , Kirik Victor , Kernebeck Birgit , Srinivas Bhylahalli Purushottam , Hülskamp Martin
Published: June 2003

The generation of a specific cell shape requires differential growth,whereby specific regions of the cell expand more relative to others. The Arabidopsis crooked mutant exhibits aberrant cell shapes that develop because of mis-directed expansion, especially during a rapid growth phase. GFP-aided visualization of the F-actin cytoskeleton and the behavior of subcellular organelles in different cell-types in crooked and wild-type Arabidopsis revealed that localized expansion is promoted in cellular regions with fine F-actin arrays but is restricted in areas that maintain dense F-actin. This suggested that a spatiotemporal distinction between fine versus dense F-actin in a growing cell could determine the final shape of the cell. CROOKED was molecularly identified as the plant homolog of ARPC5, the smallest sub-unit of the ARP2/3 complex that in other organisms is renowned for its role in creating dendritic arrays of fine F-actin. Rescue of crooked phenotype by the human ortholog provides the first molecular evidence for the presence and functional conservation of the complex in higher plants. Our cell-biological and molecular characterization of CROOKED suggests a general actin-based mechanism for regulating differential growth and generating cell shape diversity.
The meiotic protein SWI1 is required for axial element formation and recombination initiation inArabidopsis
Authors: Mercier Raphael , Armstrong Susan. J. , Horlow Christine , Jackson Neil P. , Makaroff Christopher A. , Vezon Daniel , Pelletier Georges , Jones Gareth H. , Franklin F. Christopher H.
Published: June 2003

We report the detailed characterization of SWITCH1 (SWI1) an Arabidopsis thaliana protein that has been linked with the establishment of sister chromatid cohesion during meiosis. Using a combination of cytological methods including immunolocalization of meiotic chromosome-associated proteins we show that SWI1 is required for formation of axial elements. Our studies reveal that the swi1-2 mutation prevents the formation of RAD51 foci during meiotic prophase and suppresses the chromosome fragmentation phenotype of the recombination-defective dif1-1 mutant. Together, these data suggest that SWI1 may be required for meiotic recombination initiation. Finally we raised an antibody against SWI1 and showed, by immunolocalization coupled with bromodeoxyuridine incorporation experiments, that SWI1 is expressed exclusively in meiotic G1 and S phase. Thus, SWI1 appears to be required for early meiotic events that are at the crossroad of sister chromatid cohesion, recombination and axial element formation. The possible inter-relationship between these processes and the function of SWI1 are discussed.
Myf5 expression in somites and limb buds of mouse embryos is controlled by two distinct distal enhancer activities
Authors: Buchberger Astrid , Nomokonova Natalia , Arnold Hans-Henning
Published: June 2003

The initiation of skeletal muscle development in the mouse embryo is strictly associated with the expression of the muscle-specific transcription factor Myf5, the first of four myogenic regulatory factors (MRFs) to be expressed in muscle progenitors, and ablation of the Myf5 gene prevents myogenesis. The complex spatiotemporal expression pattern of Myf5 depends on many discrete regulatory elements that are dispersed over long distances throughout the gene locus. These multiple control modules act differently in the various muscle precursor populations, presumably in response to diverse signals that control myogenesis. A potent enhancer region regulating Myf5 expression in limb muscles and somites has been identified previously at –58/–48 kb upstream of the transcriptional start site (Hadchouel et al., 2000). Here, we focus on the physical and functional dissection of this control region. We demonstrate that a conserved sequence of 270 bp located around –57 kb is required and sufficient to drive Myf5 expression in limbs and to maintain it in somites. A second enhancer nearby is responsible for Myf5 transcription in occipital/cranial somites. This enhancer activity also directs expression accurately to the myotome, preventing ectopic expression in the dermomyotome during the second phase of Myf5 gene activation in somites.

Our data suggest that the enhancer identified here collaborates with other somitic enhancers to ensure correct myotomal Myf5 expression. Moreover, it constitutes an important element that mediates somitic expression after the initial and transient Myf5 activation through a previously described sonic hedgehog-dependent early epaxial enhancer.
lin-35/Rb andubc-18, an E2 ubiquitin-conjugating enzyme, function redundantly to control pharyngeal morphogenesis inC. elegans
Authors: Fay David S. , Large Edward , Han Min , Darland Monica
Published: June 2003

The retinoblastoma gene product has been implicated in the regulation of multiple cellular and developmental processes, including a well-defined role in the control of cell cycle progression. The Caenorhabditis elegansretinoblastoma protein homolog, LIN-35, is also a key regulator of cell cycle entry and, as shown by studies of synthetic multivulval genes, plays an important role in the determination of vulval cell fates. We demonstrate an additional and unexpected function for lin-35 in organ morphogenesis. Using a genetic approach to isolate lin-35 synthetic-lethal mutations, we have identified redundant roles for lin-35 and ubc-18, a gene that encodes an E2 ubiquitin-conjugating enzyme closely related to human UBCH7. lin-35 and ubc-18 cooperate to control one or more steps during pharyngeal morphogenesis. Based on genetic and phenotypic analyses, this role for lin-35 in pharyngeal morphogenesis appears to be distinct from its cell cycle-related functions. lin-35 and ubc-18 may act in concert to regulate the levels of one or more critical targets during C. elegans development.
Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-α
Authors: Maas-Szabowski Nicole , Stärker Anja , Fusenig Norbert E.
Published: June 2003

The human keratinocyte cell line HaCaT expresses essentially all epidermal differentiation markers but exhibits deficiencies in tissue organization as surface transplants in nude mice and even more so in organotypic co-cultures with fibroblasts. Whereas tissue differentiation by normal keratinocytes(NEKs) is regulated by stromal interactions, this mechanism is impaired in HaCaT cells. This regulatory process is initiated by interleukin-1 (IL-1)release in keratinocytes, which induces expression of keratinocyte growth factor (KGF/FGF-7) and granulocyte macrophage-colony stimulating factor(GM-CSF) in fibroblasts. Production and release of IL-1 is very low and,consequently, expression of the fibroblast-derived growth factors KGF/FGF-7 and GM-CSF is absent in HaCaT-fibroblast co-cultures. However, addition of KGF and GMCSF, respectively, is inefficient to improve stratification and differentiation by HaCaT cells due to the low expression of their cognate receptors. More importantly, expression and release of the autocrine keratinocyte growth factor TGF-α is dramatically decreased in HaCaT cells. Addition of TGF- α or EGF stimulated HaCaT cell proliferation but, even more effectively, suppressed apoptosis, thus facilitating the formation of a regularly stratified epithelium. Furthermore, TGF-αenhanced the expression of the receptors for KGF and GM-CSF so that addition of these growth factors, or of their inducer IL-1, further improved epidermal tissue differentiation leading to in vitro skin equivalents comparable with cultures of NEKs. Thus, supplementing TGF-α normalized epidermal tissue regeneration by immortal HaCaT keratinocytes and their interaction with stromal cells so that regular skin equivalents are produced as standardized in vitro models.
BTG2 antiproliferative protein interacts with the human CCR4 complex existing in vivo in three cell-cycle-regulated forms
Authors: Morel Anne-Pierre , Sentis Stéphanie , Bianchin Claire , Le Romancer Muriel , Jonard Laurence , Rostan Marie-Claude , Rimokh Ruth , Corbo Laura
Published: June 2003

The yeast CCR4-NOT complex exists in two forms (1.0 and 1.9 MDa) that share several common subunits, including yCCR4, yCAF1 and five NOT proteins(NOT1-5). Here, we report that different complexes containing mammalian homologs of CCR4-NOT subunits exist in mammalian cells, with estimated sizes of ∼1.9 MDa, ∼1 MDa and ∼650 kDa, and that BTG2, a member of a protein family with antiproliferative functions, can associate with these complexes. Immunoprecipitation and gel filtration experiments established that BTG2 interacts in vivo with hCCR4 protein via hCAF1 and hPOP2. Moreover, we show that hCCR4, as well as hCAF1 and BTG2, modulate the transcription regulation mediated by ERα. Finally, we demonstrate that the cellular localization of hCAF1 and the cell content in hCAF1-containing complexes change as cells progress from quiescence to S phase. These findings suggest that the different regulatory pathways in which hCAF1 is involved, notably transcription regulation and mRNA turnover, may occur through distinct CCR4 complexes in the course of cell-cycle progression.
Phagosomal oxidative activity during β2 integrin (CR3)-mediated phagocytosis by neutrophils is triggered by a non-restricted Ca2+signal: Ca2+ controls time not space
Authors: Dewitt Sharon , Laffafian Iraj , Hallett Maurice B.
Published: June 2003

The temporal and spatial relationship between particle binding to the neutrophil by β2 integrin (CR3), the Ca2+ elevation and subsequent oxidase activation has been unclear. This is because of the difficulty in studying the time course of individual phagocytic events in individual neutrophils. Here, we have used a micromanipulation technique to present C3bi-opsonised zymosan particles to the neutrophil under observation. In this way, the moment of particle contact, pseudopod formation and internalisation has been established and cytosolic free Ca2+ and oxidation of dichlorodihydrofluorescein (DCDHF)-labelled particles determined simultaneously. Using this approach, we have found that the Ca2+signal, which is triggered by CR3-mediated phagocytosis, can be resolved into two temporally separated components. The first Ca2+ signal occurs during β2 integrin engagement as the phagocytic cup forms but does not trigger oxidation of the particle. The second global Ca2+ signal,which is triggered about the time of phagosomal closure, causes an abrupt activation of the oxidase. This second Ca2+ signal was not restricted to the region of the phagosome yet only triggered the oxidase activation locally in the phagosome, with no evidence of activation at other sites in the neutrophil. This points to a dual control of oxidase activation,with Ca2+ controlling the timing of oxidase activation but slower and more localised molecular events, perhaps involving oxidase assembly and phosphatidylinositol 3-phosphate generation, determining the site of oxidase activation.
Plastid ultrastructure defines the protein import pathway in dinoflagellates
Authors: Nassoury Nasha , Cappadocia Mario , Morse David
Published: June 2003

Eukaryotic cells contain a variety of different compartments that are distinguished by their own particular function and characteristic set of proteins. Protein targeting mechanisms to organelles have an additional layer of complexity in algae, where plastids may be surrounded by three or four membranes instead of two as in higher plants. The mechanism of protein import into dinoflagellates plastids, however, has not been previously described despite the importance of plastid targeting in a group of algae responsible for roughly half the ocean's net primary production. Here, we show how nuclear-encoded proteins enter the triple membrane-bound plastids of the dinoflagellate Gonyaulax. These proteins all contain an N-terminal leader sequence with two distinct hydrophobic regions flanking a region rich in hydroxylated amino acids (S/T). We demonstrate that plastid proteins transit through the Golgi in vivo, that the first hydrophobic region in the leader acts as a typical signal peptide in vitro, and that the S/T-rich region acts as a typical plastid transit sequence in transgenic plants. We also show that the second hydrophobic region acts as a stop transfer sequence so that plastid proteins in Golgi-derived vesicles are integral membrane proteins with a predominant cytoplasmic component. The dinoflagellate mechanism is thus different from that used by the phylogenetically related apicomplexans, and instead, is similar to that of the phylogenetically distant Euglena,whose plastids are also bound by three membranes. We conclude that the protein import mechanism is dictated by plastid ultrastructure rather than by the evolutionary history of the cell.
SETA/CIN85/Ruk and its binding partner AIP1 associate with diverse cytoskeletal elements, including FAKs, and modulate cell adhesion
Authors: Schmidt Mirko H. H. , Chen Baihua , Randazzo Lisa M. , Bögler Oliver
Published: June 2003

The adaptor protein SETA/CIN85/Ruk is involved in regulating diverse signal transduction pathways, including the internalization of tyrosine kinase receptors via the Cbl ubiquitin ligases, and attenuating PI3K activity by interaction with its regulatory subunit. Here we present evidence for a new aspect of SETA function, based on the initial observation that it co-localizes with actin in microfilaments and at focal adhesions, and with microtubules. Although there was no evidence for direct molecular interactions between SETA and cytoskeletal proteins, the SETA-interacting protein AIP1, which is a rat ortholog of the Xenopus src substrate Xp95, strongly interacted with structural proteins of the cytoskeleton, including actin and tubulins. Both SETA and AIP1 interacted with focal adhesion kinase (FAK) and proline rich tyrosine kinase 2 (PYK-2), and c-Cbl interacted with PYK-2. AIP1, which interacted more strongly than either SETA or c-Cbl, required an intact consensus tyrosine kinase phosphorylation sequence at Y319 to bind to focal adhesion kinases, which suggests that phosphorylation is an important mediator of this complex. SETA, which interacted as a dimer with focal adhesion kinases, promoted the interaction between PYK-2 and AIP1. Direct analysis of the impact of these proteins on cell adhesion, by use of an electrical cell-substrate impedance sensor (ECIS), showed that SETA promoted cell adhesion while AIP1 and c-Cbl reduced it. Furthermore, the ability of AIP1 and AIP1 mutants to decrease cell adhesion in ECIS analysis correlated with their presence in PYK-2 complexes, providing a direct link between AIP1-mediated molecular interactions and cellular behavior. Transfection of AIP1 also reduced the level of phosphorylation of endogenous PYK-2 and FAK, suggesting that this protein may directly regulate focal adhesion kinases, and thereby cell adhesion. These data are the first to implicate the adaptor protein SETA and its binding partner AIP1 as being involved with the cytoskeleton and in the regulation of cell adhesion, and suggest that they may be part of the focal adhesion kinase regulatory complex.
Inhibition of p38 MAPK signaling promotes late stages of myogenesis
Authors: Weston Andrea D. , Sampaio Arthur V. , Ridgeway Alan G. , Underhill T. Michael
Published: June 2003

Signaling through the p38 mitogen-activated protein kinases (MAPKs) is essential for cartilage formation in primary cultures of limb mesenchyme. Here we show that, concurrent with a decrease in chondrogenesis, inhibition of p38 in limb bud cultures dramatically promotes muscle development. Specifically,treatment of primary limb bud cultures with p38 inhibitors increases the expression of myogenic markers and causes a striking increase in formation of myotubes, which were detected using antibodies specific for myosin heavy chain. These results are surprising in that they contrast with several previous reports describing a requirement for p38 during myogenesis. Nonetheless, the enhanced myogenesis leads to the formation of an extensive network of contractile myofibers, and this enhanced myogenesis can be conferred upon myogenic cells from clonal populations, such as G8 or C2C12 cells, if they are co-cultured with the limb mesenchymal cells. We provide evidence for the maintenance and rapid organization of existing,somitic-derived limb myoblasts in response to p38 inhibitors. These findings imply a novel and unexpected role for p38 MAPK inhibition in myogenesis and highlight the importance of the limb bud microenvironment in promoting the progression of limb myoblasts.