Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development

Incomplete urethral tubularization (hypospadias) and anorectal abnormalities are two common and poorly understood birth defects that affect the extreme caudal midline of the human embryo. We now show that cell surface molecules essential for proper axon pathfinding in the developing nervous system, namely ephrin-B2 and the ephrin receptors EphB2 and EphB3, also play major roles in cell adhesion events that tubularize the urethra and partition the urinary and alimentary tracts. Mice carrying mutations which disrupt the bidirectional signals that these molecules transduce develop with variably penetrant severe hypospadias and incomplete midline fusion of the primitive cloaca. We further show that animals completely lacking ephrin-B2 reverse signaling present a fully penetrant failure in cloacal septation. This results in severe anorectal malformations characterized by an absence of the terminal-most hindgut (rectum) and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder. Consistent with an apparent requisite for both forward and reverse signaling in these caudal remodeling events, EphB2 and ephrin-B2 are coexpressed at the midline in the fusing urethral/cloacal endoderm and underlying lateral mesoderm of the urorectal septum that migrates toward the caudal midline as the cloaca septates. Our data thus indicate that B-subclass Eph and ephrin molecules play an important role in these clinically significant midline cell-cell adhesion and fusion events.     

The normal development of the anorectum in the pig

The development of the anorectum was studied in forty-four embryos and foetuses of pig varying in length from 9 mm total length to 210 mm crownrump length and in three newborn pigs. The presence of some features during critical stages in the development of the cloaca in the pig such as an epithelial mass protruding into the dorsal cloaca near its intestinal orifice and distinct differences in the type of epithelium in different regions of the cloacal system greatly facilitated the study of the developmental process. Thus it could be established that a change in position of the dorsal cloaca and adjacent structures such as the distal part of the gut and the urorectal septum via the dorsal part of the cloacal plate towards the tailgroove is of major importance for the partition of the cloaca into a separate intestinal and urogenital division. A subsequent disintegration of the dorsal part of the cloacal plate results in two separate openings for both the systems at the same time. Disintegration of the ventral part of the cloacal plate leads only to a further widening of the external opening of the urogenital system. In the cloacal system three distinct zones were discerned, a dorsal and ventral cloacal and a cloacal plate region. From the dorsal cloacal epithelium the whole anorectal segment between the intestinal mucosa and the anal epidermis develops. The epithelium of ventral cloacal origin seems to disappear completely . Cloacal plate epithelium forms the epithelial lining of distal parts of the urogenital system. The penile urethra in the male is formed by a ventralward movement of the urogenital opening by the growing perineum and not by fusion of genital folds.     

Interaction of Hsp90AA1 with phospholipids stabilizes membranes under stress conditions

During heat shock conditions, structural changes in cellular membranes may lead to cell death. Hsp90AA1 and other heat shock proteins involved in membranes are responsible for protecting membrane stabilization. However, the membrane binding mechanism of Hsp90AA1 remains largely uncharacterized. In this study, we showed Hsp90AA1 interacts with phospholipid membrane with high affinity. Using the depth-dependent fluorescence-quenching with brominated lipids, we found Hsp90AA1 penetrated 10.7?Å into the hydrocarbon core of the lipid bilayer. Circular dichroism spectra studies showed Hsp90AA1 lost part of its α-helical structures upon interaction with phospholipid membrane. By assessing binding properties of the three Hsp90AA1 domains, we found Hsp90AA1 interacted into the lipid bilayer mainly toward its C-terminus domain (CTD). Using scanning electron microscopy, we examined the protection on host cell membrane by overexpressing Hsp90AA1. The results indicated Hsp90AA1 or Hsp90AA1-CTD expressing E. coli cells exhibited better membrane integrity compared to the control after thermal treatment. The following liposome leakage assay suggested the protection of Hsp90AA1 might due to its stabilization of the membrane lipid. Collectively, the present study demonstrates Hsp90AA1 embeds into the lipid bilayer through its C-terminal domain and the Hsp90AA1-lipid association potentially has a significant function in keeping membranes stabilization during stress conditions.     

Normal development of the male anterior urethra

A histological study was performed on serially sectioned human and mouse embryos to study the influences of programmed cell death (PCD) during morphogenesis for clarifying the existing controversies on the morphology and basic processes involved in the embryonic development of the male anterior urethra. The following new insights into the development of the anterior urethra could be established. The formation of the urethra starts with the early adhesion of the arms of the genital tubercle. In this way an epithelial plate is formed, located in the ventral midline, that is in continuity with the cloacal membrane. Male sex differentiation takes place following rupture of this cloacal membrane through programmed cell death. Fusion of the urogenital swellings with primary luminization gives rise to the penile urethra, whereas the glandular part of the urethra is formed through secondary luminization of the epithelial cord that is formed during fusion of the arms of the genital tubercle, i.e., the glans. In both fusion processes, apoptosis plays a key role. The consequence of fusion of the urogenital swellings is that their mesodermal cores unite on the ventral aspect of the penile urethra, where they differentiate into the integumental structures. The prepuce starts to develop as a fold of ectoderm with a mesodermal core after complete fusion of the entire urethra. Finally, the scrotum was found to develop through merging of the labioscrotal swellings and not by fusion.     

Bmp7 functions via a polarity mechanism to promote cloacal septation

Background

During normal development in human and other placental mammals, the embryonic cloacal cavity separates along the axial longitudinal plane to give rise to the urethral system, ventrally, and the rectum, dorsally. Defects in cloacal development are very common and present clinically as a rectourethral fistula in about 1 in 5,000 live human births. Yet, the cellular mechanisms of cloacal septation remain poorly understood.

Methodology/Principal Findings

We previously detected Bone morphogenetic protein 7 (Bmp7) expression in the urorectal mesenchyme (URM), and have shown that loss of Bmp7 function results in the arrest of cloacal septation. Here, we present evidence that cloacal partitioning is driven by Bmp7 signaling in the cloacal endoderm. We performed TUNEL and immunofluorescent analysis on cloacal sections from Bmp7 null and control littermate embryos. We found that loss of Bmp7 results in a dramatic decrease in the endoderm survival and a delay in differentiation. We used immunological methods to show that Bmp7 functions by activating the c-Jun N-terminal kinase (JNK) pathway. We carried out confocal and 3D imaging analysis of mitotic chromosome bundles to show that during normal septation cells in the cloacal endoderm divide predominantly in the apical-basal direction. Loss of Bmp7/JNK signaling results in randomization of mitotic angles in the cloacal endoderm. We also conducted immunohistochemical analysis of human fetal sections to show that BMP/phospho-SMAD and JNK pathways function in the human cloacal region similar as in the mouse.

Conclusion/Significance

Our results strongly indicate that Bmp7/JNK signaling regulates remodeling of the cloacal endoderm resulting in a topological separation of the urinary and digestive systems. Our study points to the importance of Bmp and JNK signaling in cloacal development and rectourethral malformations.     

Genome-wide Association Study and Meta-Analysis Identify ISL1 as Genome-wide Significant Susceptibility Gene for Bladder Exstrophy

The bladder exstrophy-epispadias complex (BEEC) represents the severe end of the uro-rectal malformation spectrum, and is thought to result from aberrant embryonic morphogenesis of the cloacal membrane and the urorectal septum. The most common form of BEEC is isolated classic bladder exstrophy (CBE). To identify susceptibility loci for CBE, we performed a genome-wide association study (GWAS) of 110 CBE patients and 1,177 controls of European origin. Here, an association was found with a region of approximately 220kb on chromosome 5q11.1. This region harbors the ISL1 (ISL LIM homeobox 1) gene. Multiple markers in this region showed evidence for association with CBE, including 84 markers with genome-wide significance. We then performed a meta-analysis using data from a previous GWAS by our group of 98 CBE patients and 526 controls of European origin. This meta-analysis also implicated the 5q11.1 locus in CBE risk. A total of 138 markers at this locus reached genome-wide significance in the meta-analysis, and the most significant marker (rs9291768) achieved a P value of 2.13 × 10⁻¹². No other locus in the meta-analysis achieved genome-wide significance. We then performed murine expression analyses to follow up this finding. Here, Isl1 expression was detected in the genital region within the critical time frame for human CBE development. Genital regions with Isl1 expression included the peri-cloacal mesenchyme and the urorectal septum. The present study identified the first genome-wide significant locus for CBE at chromosomal region 5q11.1, and provides strong evidence for the hypothesis that ISL1 is the responsible candidate gene in this region.     

Transmission of circulating cell-free AA amyloid oligomers in exosomes vectors via a prion-like mechanism

Recent studies clearly demonstrated that several types of pathogenic amyloid proteins acted as agents that could transmit amyloidosis by means of a prion-like mechanism. Systemic AA amyloidosis is one of the most severe complications of chronic inflammatory disorders, particularly rheumatoid arthritis. It is well known that, similar to an infectious prion protein, amyloid-enhancing factor (AEF) acts as a transmissible agent in AA amyloidosis. However, how AEF transmits AA amyloidosis in vivo remained to be fully elucidated. In the present study, we focused on finding cell-free forms of AEF and its carriers in circulation by using the murine transfer model of AA amyloidosis. We first determined that circulating cell-free AEF existed in blood and plasma in mice with systemic AA amyloidosis. Second, we established that plasma exosomes containing AA amyloid oligomers derived from serum amyloid A had AEF activity and could transmit systemic AA amyloidosis via a prion-like mechanism. These novel findings should provide insights into the transmission mechanism of systemic amyloidoses.     

The Cu,Zn superoxide dismutase isoenzymes of Xenopus laevis: purification, identification of a heterodimer and differential heat sensitivity

The three Cu,Zn superoxide dismutase electromorphs of the amphibian Xenopus laevis were purified by an original procedure. N-terminal sequence analysis demonstrated that they are two different homodimers (AA and BB) and a hybrid heterodimer (AB), arising from the co-expression of duplicated genes. The three forms have the same pI, same enzyme activity and EPR spectra, but different heat-sensitivity, form BB being more resistant than form AA, with form AB showing intermediate sensitivity. Thermostability of BB and the control bovine enzyme was enhanced by a tenfold increase in protein concentration. It is suggested that the higher heat sensitivity of the AA isoenzyme is related to the presence of an extra Cys residue and to an easier dissociation of the protein dimer into monomers.     

Congenitally caused fused labia in the common marmoset (Callithrix jacchus)

In this paper, the occurrence of an external genital abnormality in female marmoset monkeys (fused labia) is discussed. This malformation was detected, for the first time, in a group of animals at the German Primate Center (GPC), Goettingen. The malformed vulva was completely sealed except for an opening of 1.5-2.5 mm around the urethra sufficient for urination. Because of this defect the animals were not able to copulate. As a consequence, the affected females were functionally infertile although they had a normal genital tract and a regular cycle. This vulvar abnormality was found in 12 females, offspring of 10 pairs in which either one or both came to the German Primate Center from two genetically related colonies in Munich, Germany, and one colony in Basel, Switzerland. The abnormality appeared to be recessive and inheritable from either parent. In pairs in which both animals were from one of the mentioned colonies, 45% of the female offspring were affected. In pairs where only one partner came from these colonies, 26% of female offspring had the malformation. These results indicate that avoidance of inbreeding, which is frequently performed in primate colonies, may reduce, but not eliminate the expression of abnormalities of genetic origin. Therefore selective breeding is required, and, in colonies where these recessive mutations are widespread, the development of genetic screening tests would be advantageous.     

Putative gamma-subunit of the IL-2 receptor is detected in low, intermediate, and high affinity IL-2 receptor-bearing cells

Three forms of the IL-2R have been reported: low, intermediate, and high affinity. Each form correlates with the expression of two different chains: p55 (alpha-subunit) and p75 (beta-subunit). We report here a putative new subunit of the IL-2R, termed p95-110 or gamma-subunit. This new subunit has a molecular mass of 95 to 110 kDa and is expressed in low, intermediate, and high affinity IL-2R-bearing cells. We propose that p95-110 is the partner of p75 in the formation of intermediate affinity IL-2R, inasmuch as neither p75 nor p95-110 alone can bind IL-2.     

Investigations into the membrane activity of arenicin antimicrobial peptide AA139

Arenicin-3 is an amphipathic β-hairpin antimicrobial peptide that is produced by the lugworm Arenicola marina. In this study, we have investigated the mechanism of action of arenicin-3 and an optimized synthetic analogue, AA139, by studying their effects on lipid bilayer model membranes and Escherichia coli bacterial cells. The results show that simple amino acid changes can lead to subtle variations in their interaction with membranes and therefore alter their pre-clinical potency, selectivity and toxicity. While the mechanism of action of arenicin-3 is primarily dependent on universal membrane permeabilization, our data suggest that the analogue AA139 relies on more specific binding and insertion properties to elicit its improved antibacterial activity and lower toxicity, as exemplified by greater selectivity between lipid composition when inserting into model membranes i.e. the N-terminus of AA139 seems to insert deeper into lipid bilayers than arenicin-3 does, with a clear distinction between zwitterionic and negatively charged lipid bilayer vesicles, and AA139 demonstrates a cytoplasmic permeabilization dose response profile that is consistent with its greater antibacterial potency against E. coli cells compared to arenicin-3.     

Effect of 12 hr temporal relation of serotonin and dopamine precursor drugs (5-HTP and L-DOPA) on photosexual responses of immature Japanese quail

To study the interaction of photoperiod and circadian neurotransmitter activity, L-DOPA L-dihydroxyphenylalanine, dopamine precursor) was administered daily, 12 hr after 5-HTP (5-hydroxy tryptophan, serotonin precursor) in sexually immature Japanese quail, raised under short photoperiod (LD8:16) since hatching. The 12 hr treatment of 5-HTP and L-DOPA was given under continuous condition of light for 11 days. After treatment the quail were transferred to intermediate day length (LD 13.5:10.5). The cloacal gland size of drug treated group increased significantly in comparison to control. The quail were then transferred to short photoperiod (LD 8:16). The cloacal gland size of both the groups started decreasing gradually but the rate was significantly low in drug treated quail in comparison to control. The results indicate that the endogenous mechanism controlling seasonality may be reset by drugs that influence serotonergic and dopaminergic activity. The 12 hr relation between the two drugs is stimulatory for gonadal growth under intermediate day length and retards the rate of regression when transferred to short days.     

Nutrient levels in malformed and healthy tissues of mango (Mangifera indica L.)

Samples of malformed and healthy panicles of mango (Mangifera indica L.) as well as leaves and shoots bearing them were collected at different stages of development (fully swollen buds, bud inception, fully grown panicles prior to full bloom and at full bloom) over two consecutive years and were analysed for their macro- and micronutrient status. In addition, malformed and healthy seedlings were collected and analysed. Malformed panicles were found to be significantly higher in N at all the developmental stages except at bud inception. Phosphorus and K also tended to accumulate in malformed panicles at later stages of their development. In general, malformed panicles exhibited lower levels of P, K and Ca than healthy panicles. The differences in levels of Mg and S in malformed and healthy panicles were not significant. All micronutrients were in much lower concentrations in malformed panicles except for Mn which appears to accumulate in malformed panicles particularly at the early stages of development. The leaves on the shoots bearing malformed panicles also showed a tendency to accumulate N, while P, Mg and S were always higher in leaves on shoots bearing healthy panicles. The leaves on shoots bearing healthy panicles had lower levels of Fe, Cu and Mn, whereas levels of Zn and B tended to be higher in leaves on shoots bearing malformed panicles. The nutrient concentration differences between the two kinds of shoots were generally nonsignificant (P=0.05), except for K and S which were significantly lower in shoots bearing malformed panicles. The shoots bearing malformed panicles showed significantly (P=0.05) higher levels of almost all nutrients compared with shoots bearing healthy panicles. Vegetative malformation was found to be associated significantly (p=0.05) with higher amounts of all nutrients except Ca which was significantly higher in healthy seedlings. The present study, therefore, seems to point to lower Ca as one of the pre-disposing factors causing malformation in mango.A part of Ph.D. thesis of the senior author.A part of Ph.D. thesis of the senior author.     

Amino acid sequence variations in protein AA of cats with high and low incidences of AA amyloidosis

1. Amyloid isolated from the liver of a domestic short-haired (DSH) cat was dissolved and purified by gel filtration for amino acid sequence analysis. 2. Sequences of two major peptides corresponding to positions 18-23 and 25-75 of human amyloid protein AA were obtained when cyanogen bromide-cleaved protein was applied to an amino acid sequenator. 3. Comparison of these regions of amyloid protein from the Abyssinian cat (high incidence of AA amyloidosis) and DSH cat (low incidence of AA amyloidosis) revealed three amino acid differences, two of which occurred within regions that are completely conserved in the Abyssinian cat and all other species. 4. Secondary prediction plots showed less potential for amyloidogenicity (i.e., less beta-sheet conformation) in protein AA of the DSH cat as compared to the Abyssinian cat and other animal species. 5. These differences in protein AA of the DSH cat may, therefore, be linked to the comparatively uncommon occurrence of AA amyloidosis in the DSH cat as compared to the Abyssinian cat and other animals species.     

RAS2 protein of Saccharomyces cerevisiae undergoes removal of methionine at N terminus and removal of three amino acids at C terminus

RAS2 protein of Saccharomyces cerevisiae undergoes post-translational modifications involving methyl esterification and palmitic acid addition, resulting in their association with the plasma membrane. In this paper, we provide evidence that two kinds of proteolytic events accompany the biosynthesis. This is shown by separating and characterizing three intracellular forms of RAS2 protein: precursor, intermediate, and mature (fatty acid-acylated) forms. N-Terminal sequencing has revealed that all three forms start with proline, which is the second amino acid expected from the RAS2 gene sequence. Thus, the first methionine is removed very early during the biosynthesis. Isolation and sequencing of C-terminal peptides indicate that three C-terminal amino acids present in the precursor form are removed in the intermediate and in the fatty acid acylated forms. C-Terminal proteolysis appears to accompany methyl esterification, since the methylation occurs with the intermediate and the fatty acid-acylated forms, but not with the precursor. Palmitic acid is identified as the major fatty acid attached to the fatty acid-acylated form.     

Birth rate and mortality rate of infants with congenital malformations of the limbs in the Awajishima free-ranging group of Japanese monkeys (Macaca fuscata)

The birth rate and mortality rate of infants with congenital malformations of the limbs were examined in the Awajishima free-ranging group of Japanese macaques (Macaca fuscata). Of the 606 infants born between 1978 and 1995, 86 (14.2%) were malformed. The male-female ratio did not differ between malformed and normal infants. Most kin-groups included females who gave birth to malformed infants at least once. The mortality rate within the first year after birth for malformed infants (28.2%) was significantly higher than that for normal infants (10.0%). However, this indicates that more than 70% of malformed infants were able to survive for the first year of life, even though they were unable to cling to their mothers ventrum due to their limb deformities. This finding indicates that maternal care-taking is sufficient to enable malformed infants to survive during the early stages of development and that clinging by the infant is not necessary for the display of maternal care. Am. J. Primatol. 42:225–234, 1997. © 1997 Wiley-Liss, Inc.     

The origin of the ectodermal ring in staged human embryos of the first 5 weeks

Seven embryos of stages 10-16 (3-5 weeks) were studied for their external form by means of graphic reconstructions. This is the first systematic report on the general surface anatomy of the early human embryo. The brain has been described and illustrated in a previous publication, and the present article is concerned particularly with an important although neglected feature: the ectodermal ring, described by Schmitt [Morph. Arb., Jena 8: 236-303, 1898] and named by Blechschmidt [Funktionsentwicklung. I. Mechanische Gegenwirkungen. Musterschmidt, G?ttingen 1948]. Late in stage 10, three longitudinal bands can be seen on the surface: somitic, intermediate, and lateral plate. The intermediate band, which partly overlies nephrogenic tissue, is the site of the future ectodermal ring, which begins as an inverted U at stage 11 and is completed caudally in stage 12. The ectodermal ring, which may well be an important example of epithelial-mesenchymal interaction, comprises six parts: (a) rostral part, containing the situs neuroporicus, and olfactory and lens discs; (b) pharyngeal part, the covering of the pharyngeal arches; (c) occipital and cervicothoracic parts, related at first to the four occipital somites and later to the cervicothoracic junction; (d) membral part, represented by a preliminary ectodermal thickening, followed within 2 days by the apical ectodermal ridge; (e) intermembral part, related at first to the underlying coelom, and mesonephric duct and ridge; (f) caudal part, containing the cloacal membrane and a temporary 'caudal ectodermal ridge'. It is stressed that the incorrectly named 'Milchstreifen' is merely the intermembral part, in which the mammary crest ('Milchlinie' or 'Milchleiste') appears 1 week later.     

Enhanced Anticancer Effect of Adding Magnesium to Vitamin C Therapy: Inhibition of Hormetic Response by SVCT-2 Activation

l-Ascorbic acid (vitamin C, AA) is known as an antioxidant, but at high concentrations, AA can kill cancer cells through a prooxidant property. Sodium-dependent vitamin C transporter family-2 (SVCT-2) determines the cellular uptake of AA, and the activity of SVCT-2 is directly related to the anticancer activity of AA. Cancer cells that showed high SVCT-2 expression levels were more sensitive to AA treatment than cancer cells with low SVCT-2 expression levels. Cells with low SVCT-2 expression showed a hormetic response to a low dose of AA. Magnesium ions, which are known to activate SVCT-2, could increase the V_max value of SVCT-2, so we investigated whether providing magnesium supplements to cancer cells with low SVCT-2 expression that had shown a hormetic response to AA would elevate the V_max value of SVCT-2, allowing more AA to accumulate. To evaluate the effects of magnesium on cancer cells, MgSO₄ and MgCl₂ were screened as magnesium supplements; both forms showed synergistic anticancer effects with AA. Taken together, the results of this study suggest that magnesium supplementation enhanced the anticancer effect of AA by inhibiting the hormetic response at a low dose. This study has also demonstrated that AA treatment with magnesium supplementation provided more effective anticancer therapy than AA treatment alone.     

Immunological relationships among subunits of glutathione S-transferases A, AA, B and ligandin and hybrid formation between AA and ligandin by guanidine hydrochloride

Immunological properties of ligandin(Lig) and glutathione S-transferase(GST)-A, -AA and -B were investigated for elucidating their subunit relationships. By using either anti-Lig or -AA antibody, GST-B made a clear common precipitin line with Lig or AA in double immunodiffusion and the activity was inhibited intermediately between Lig and AA, whereas Lig and AA reacted very weakly with antibodies to each other. A hybrid between Lig and AA formed by guanidine hydrochloride treatment was identified immunochemically to be GST-B. GST-A had no immunological relationship with any of other three forms.     

Vegetation composition of a maritime salt marsh in Qatar in relation to edaphic features

Abstract. Coverpercentage values for plant species were estimated using the line-intercept method along two belt transects starting at the lower mangrove frontier and ending at the upper height of the salt marsh. Twelve perennial species representing five growth forms were recorded with a total cover ranging between 10 and 50 %, depending on the type of substrate. Principal components analysis of the soil data indicated an array of stands, on the basis of which four vegetation zones couldbe distinguished. The procumbent growth form predominating on the salt marsh is related to high salinity levels. The procumbent vegetation shows a zonation related to the degree of waterlogging. The rhizomatous growth form is found on sites with a low bicarbonate content and saturation percentage levels, while the undershrub- and stoloniferous growth forms occupy sites of low salinity level, linked with intermediate values of the other edaphic factors.