Ectopic expression of hoxb2 after retinoic acid treatment or mRNA injection: disruption of hindbrain and craniofacial morphogenesis in zebrafish embryos

To investigate pattern formation in the vertebrate hindbrain, we isolated a full length hoxb2 cDNA clone from zebrafish. In a gene phylogeny, zebrafish hoxb2 clusters with human HOXB2, and it maps on linkage group 3 along with several other loci whose orthologues are syntenic with human HOXB2. In the hindbrain, hoxb2 is expressed at high levels in rhombomere 3 (r3), lower levels in r4, still lower in r5, and at undetectable levels in r6. In r7, r8, and the rostral spinal cord, hoxb2 is expressed at a lower level than in r5. Lateral cells appearing to emanate from r4 express both hoxb2 and dlx2, suggesting that they are neural crest. Overexpression of hoxb2 by mRNA injections into early cleavage stage embryos resulted in abnormal morphogenesis of the midbrain and rostral hindbrain, abnormal patterning in r4, fusion of cartilage elements arising from pharyngeal arches 1 and 2, and ectopic expression of krx20 and valentino (but not pax2, rtk1, or hoxb1) in the rostral hindbrain, midbrain, and, surprisingly, the eye. Treatments with retinoic acid produced a phenotype similar to that of ectopic hoxb2 expression, including ectopic krx20 (but not valentino) expression in the eye, and fusion of cartilages from pharyngeal arches 1 and 2. The results suggest that hoxb2 plays an important role in the patterning of hindbrain and pharyngeal arches in the zebrafish.     

Early expression of a novel nucleotide receptor in the neural plate of Xenopus embryos

Extracellular ATP functions as a neurotransmitter and neuromodulator in the adult nervous system, and a signaling molecule in non-neural tissue, acting either via ligand-gated ion channels (P2X) or G-protein-coupled receptors (P2Y). ATP can cause an increase in intracellular Ca2+ (Ca2+i) in embryonic cells and so regulate cell proliferation, migration, and differentiation. We have isolated a Xenopus cDNA encoding a novel P2Y receptor, XlP2Y, which is expressed abundantly in developing embryos. Recombinant XlP2Y responds equally to all five naturally occurring nucleoside triphosphates (ATP, UTP, CTP, GTP, and ITP), which elicit a biphasic Ca2+-dependent Cl- current (ICl,Ca) where the second phase persists for up to 60 min. XlP2Y also causes a continuous release of Ca2+i and a low level persistent activation of ICl,Ca in Xenopus oocytes through the spontaneous efflux of ATP. mRNAs for XlP2Y are expressed transiently in the neural plate and tailbud during Xenopus development, coincident with neurogenesis. This restricted pattern of expression and novel pharmacological features confer unique properties to XlP2Y, which may play a key role in the early development of neural tissue.     

pXen, a utility vector for the expression of GST-fusion proteins in Xenopus laevis oocytes and embryos

We describe a plasmid, pXen, designed for the optimized expression of proteins fused to glutathione-S-transferase (GST) in Xenopus laevis oocytes and embryos. The Xenopus model system permits the biochemical analysis of signaling pathways and analysis of embryo phenotype in response to manipulation of proto-oncogene expression. pXen is a modified pSP64T vector which contains an SP6 RNA polymerase promoter followed by the translational initiation sequence of Xenopus beta-globin and the glutathione binding domain of GST. The Xenopus 3' beta-globin untranslated region and polyadenylation site immediately follow the multiple cloning site to permit the efficient translation of in vitro transcribed RNA in oocytes and embryos. The utility of pXen is demonstrated by cloning the catalytic domain of the serine/threonine kinase proto-oncogene Raf-1 into this vector and injecting the corresponding in vitro transcribed RNA into oocytes. Catalytically active GST-vRaf fusion protein was expressed in the injected oocytes and induced oocyte maturation. Moreover, the GST-vRaf fusion protein could be readily purified from Xenopus extracts using glutathione Sepharose. We demonstrate that the Raf-1 catalytic domain retains activity when fused with the N-terminal GST moiety and is subject to negative regulation by the cyclic AMP-dependent protein kinase (PKA). The pXen vector will be useful for an in vivo analysis of the physiological role and regulation of a wide variety of signaling molecules when expressed in Xenopus oocytes and embryos.     

Collagen mRNA expression during tissue development: the temporospacial order coordinates bone morphogenesis with collagen fiber formation

Urinary incontinence is a prevalent and distressing condition that affects > 30% of elderly individuals. A wide variety of treatment modalities is available, and can be effective in reducing or eliminating the symptoms and adverse consequences of urinary incontinence. Pharmacological therapy is an important component of the successful management of this condition, but the agents currently used do not act selectively on the lower urinary tract. Adverse effects of drug treatment are common, and are especially problematic in the elderly. A careful assessment of the type of urinary incontinence and the institution of a rational management programme are the keys to improvement or even cure in patients with this condition.     

Ectopic expression of Fgf-4 in chimeric mouse embryos induces the expression of early markers of limb development in the lateral ridge

To determine if estrogen would protect treated rats from deficits in performance on a working memory task across time, 18 female 6-month-old Sprague-Dawley rats were trained to a criterion on a water-escape spatial delayed matching-to-sample problem. Following training, rats were ovariectomized, and nine were maintained on estrogen (polyestradiol-phosphate, 0.5 mg every 3 weeks) and nine on its vehicle for 200 days. After recovery from surgery, the rats were tested for performance every 6 weeks under three conditions: 5 min retention interval (RI); 30 min RI; and 30 min RI with an emotional experience during the RI. Analysis of correct choices revealed that estrogen-treated rats made more correct choices (p < .05) than controls on the 5 min undisturbed interval; estrogen tended to impair performance on the emotionally distracting interval. Estrogen apparently protected working memory on the undisturbed trials and might be pertinent to the maintenance of memory in female mammals.     

Sizzled: a secreted Xwnt8 antagonist expressed in the ventral marginal zone of Xenopus embryos

An expression cloning screen was used to isolate a novel gene homologous to the extracellular cysteine-rich domain of frizzled receptors. The gene (which we called sizzled for secreted frizzled) was shown to encode a soluble secreted protein, containing a functional signal sequence but no transmembrane domains. Sizzled (szl) is capable of inhibiting Xwnt8 as assayed by (1) dose-dependent inhibition of siamois induction by Xwnt8 in animal caps, (2) rescue of embryos ventralized by Xwnt8 DNA and (3) inhibition of XmyoD expression in the marginal zone. Szl can dorsalize Xenopus embryos if expressed after the midblastula transition, strengthening the idea that zygotic expression of wnts and in particular of Xwnt8 plays a role in antagonizing dorsal signals. It also suggests that inhibiting ventralizing wnts parallels the opposition of BMPs by noggin and chordin. szl expression is restricted to a narrow domain in the ventral marginal zone of gastrulating embryos. szl thus encodes a secreted antagonist of wnt signaling likely involved in inhibiting Xwnt8 and XmyoD ventrally and whose restricted expression represents a new element in the molecular pattern of the ventral marginal zone.     

Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta

X-Delta-1, a Xenopus homologue of the Drosophila Delta gene, is expressed in the early embryonic nervous system in scattered cells that appear to be the prospective primary neurons. Ectopic X-Delta-1 activity inhibits production of primary neurons and interference with endogenous X-Delta-1 activity results in overproduction of primary neurons. These results indicate that the X-Delta-1 protein mediates lateral inhibition delivered by prospective neurons to adjacent cells, and that commitment to a neural fate in vertebrates is regulated by Delta-Notch signalling as in Drosophila.     

The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco

The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed.     

Developmental expression of thrombin in zebrafish embryos: a novel model to study hemostasis

Oxidative stress causes modification of cellular macromolecules and leads to cell damage. The objective of this study was to identify protein modifications that relate to thiol groups in human red blood cells under oxidative stress. With t-butyl hydroperoxide (t-BH) treatment, results of isoelectric focusing (IEF) analysis showed that two dithiothreitol-reversible modifications are observed, one toward the cathode and the other to the anode. Protein change toward the cathode was demonstrated to be hemoglobin oxidation, which gains a net positive charge, based on the same focus on IEF gels as hemoglobin and methemoglobin and molecular weight analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Otherwise, the change toward the anode was the result of mixed disulfide formation between GSH and protein thiols. Based on the results of molecular weight analysis and its reversion from methemoglobin, protein formed mixed disulfides with GSH were also regarded as hemoglobin. As red blood samples were treated with diamide or GSSG, in addition to the mixed disulfides observed in t-BH-treated cells, additional hemoglobin-GSH mixed disulfide appeared. But the disappearance of this diamide-induced additional mixed disulfide by treating cells with t-BH after diamide treatment suggests that the increase of negative charges from GSH are offset by ferrohemoglobin oxidation to ferrihemoglobin. Additionally, other dithiothreitol-reversible modifications of one cell membrane protein, spectrin, were also observed from the formation of high molecular weight molecules as detected by SDS-PAGE. Results indicate that protein thiols in human red blood cells are susceptible to modification under oxidative stress. IEF analysis provides a useful tool to measure methemoglobin and hemoglobin GSH mixed disulfide formation.     

Developmental expression in the rat cerebellum of SC1, a putative brain extracellular matrix glycoprotein related to SPARC

In the nervous system, extracellular matrix (ECM) molecules have been shown to have effects on cell migration, process outgrowth and the survival of neurons. Recently we have described the molecular cloning of SC1, a putative brain extracellular matrix glycoprotein, showing partial similarity to the ECM glycoprotein SPARC/osteonectin. We have now examined the expression of SC1 during the development of the rat cerebellum at both the protein and mRNA levels. Our results indicate that SC1 is both temporally and spatially regulated during this process. Bergmann glial cells express SC1 mRNA and the resultant protein is deposited along the length of their radial fibres during the process of granule cell migration in the developing cerebellum. SC1 mRNA and protein is also found in the adult cerebellum, concentrated in the Bergmann glial cells and their radial processes, indicating that this putative ECM molecule continues to play roles in the central nervous system after migration and proliferative events have ceased.     

Development of the cardiac conduction tissue in human embryos using HNK-1 antigen expression: possible relevance for understanding of abnormal atrial automaticity

BACKGROUND: Abnormal atrial automaticity in young patients with structurally normal hearts is often located around the pulmonary veins and in sinus venosus-related parts of the right atrium. We hypothesize that these ectopic pacemaker sites correspond to areas of embryonic myocardium with an early phenotypic differentiation, as indicated by differences in antigen expression during normal cardiac development. METHODS AND RESULTS: In human embryos ranging in age from 42 to 54 days of gestation, the development of the cardiac conduction system was studied with the use of HNK-1 immunohistochemistry. HNK-1 stains the developing atrioventricular conduction system, ie, the bundle branches, His bundle, right atrioventricular ring, and retroaortic ring. In addition, the myocardium around the common pulmonary vein showed transient HNK-1 antigen expression. In the right atrium, 3 HNK-1-positive connections were demonstrated between the sinoatrial node and the right atrioventricular ring. An anterior tract through the septum spurium connects the sinoatrial node with the anterior right atrioventricular ring, and 2 posterior tracts connect the sinoatrial node with the posterior right atrioventricular ring through the right venous valve (future crista terminalis) and sinus septum, encircling the coronary sinus. The medioposterior part of the right atrioventricular ring connected to the His bundle and the medioanterior part form 2 node-like structures. CONCLUSIONS: In patients with abnormal atrial automaticity, the distribution of left and right atrial pacemaker foci correspond to areas of the embryonic myocardium that temporarily express the HNK-1 antigen.     

Ectopic pregnancy associated with the intrauterine device: a study of seventy cases

Seventy cases of ectopic pregnancy associated with an IUD comprised 10% of all ectopics in a 9 year period. This increased to 15% in the last 19 months as more IUD's were in use. In two thirds of the ectopics the IUD had been in situ more than 1 year. Unusual bleeding and cramping attributed to the IUD obscured the diagnosis and resulted in removal or replacement of the IUD in over one half the cases 1 to 8 weeks before surgery. The episodic nature of the abdominal hemorrhage in two thirds of all ectopics resulted in surgery on day 44 average gestational age. The IUD is probably not causal in ectopic pregnancy but does not protect the predisposed patient from ectopic pregnancy which should be suspected in any patient with an IUD who has irregular bleeding and abdominal pain.     

Cloning and nucleotide sequence of a complementary DNA encoding Xenopus laevis metallothionein: mRNA accumulation in response to heavy metals

This investigation studied the effect of 50 Hz electric and magnetic fields on the human heart. The electrocardiograms of 27 transmission-line workers and 26 male volunteers were recorded with a Holter recorder both in and outside the fields. The measurements took from half an hour to a few hours. The electric field strength varied from 0.14 to 10.21 kV/m and the magnetic flux density from 1.02 to 15.43 microT. Analysis of the ECG recordings showed that extrasystoles or arrhythmias were as frequent outside the field as in the field. In some cases a small decrease in heart rate was observed after field exposure.     

Our experience with the use of the dorsal ulnar artery flap in hand and wrist tissue cover

A field study of 1146 drivers and passengers of vehicles equipped with motorized passive belts was conducted in shopping malls and other locations in the states of Arizona and Indiana. The Indiana data was collected the summer of 1994 and the Arizona data the summer of 1995. Shoulder belt use by drivers and passengers was 93.4% in Indiana and 87.8% in Arizona. Lap belt use was 65% in Indiana and 69.9% in Arizona. Over 99% of drivers in both states knew that a manually fastened lap belt was provided along with the motorized shoulder belt. Most drivers agreed that they are supposed to wear the lap belt (96.3% in Indiana and 94.3% in Arizona) and said that the vehicle they were driving provided a warning signal when the lap belt was not fastened (75.7% in Indiana and 79.4% in Arizona). Most drivers were also aware of warning labels telling them to use their lap belt (63.9% in Indiana and 68.2% in Arizona).     

Monkey leptin receptor mRNA: sequence, tissue distribution, and mRNA expression in the adipose tissue of normal, hyperinsulinemic, and type 2 diabetic rhesus monkeys

We investigated the discharge morphology and propagation patterns of electroencephalographic seizures of temporal lobe onset in 21 children and young adults who underwent invasive long-term EEG monitoring (LTM). Of those, 15 subsequently underwent anterior temporal lobectomy. The onset was focal in 63%. The most frequent discharge morphology was low amplitude beta (30%) or rhythmic/semirhythmic theta discharge (30%). Thirteen patients displayed several sequences of propagation with different spreading stages along a fixed path. Initial spreading to the ipsilateral frontal lobe was associated with a higher frequency of secondary generalization than initial spreading to the contralateral temporal lobe (P = 0.18). A comparison of 13 patients older than 18 years of age with 8 patients younger than 14 years showed a trend towards a lower rate of propagating from the temporal lobe (P = 0.13) in the younger age group. Discharge morphology was not correlated with age, focality, or outcome of surgery.     

Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms

Traditional and molecular typing schemes for the characterization of pathogenic microorganisms are poorly portable because they index variation that is difficult to compare among laboratories. To overcome these problems, we propose multilocus sequence typing (MLST), which exploits the unambiguous nature and electronic portability of nucleotide sequence data for the characterization of microorganisms. To evaluate MLST, we determined the sequences of approximately 470-bp fragments from 11 housekeeping genes in a reference set of 107 isolates of Neisseria meningitidis from invasive disease and healthy carriers. For each locus, alleles were assigned arbitrary numbers and dendrograms were constructed from the pairwise differences in multilocus allelic profiles by cluster analysis. The strain associations obtained were consistent with clonal groupings previously determined by multilocus enzyme electrophoresis. A subset of six gene fragments was chosen that retained the resolution and congruence achieved by using all 11 loci. Most isolates from hyper-virulent lineages of serogroups A, B, and C meningococci were identical for all loci or differed from the majority type at only a single locus. MLST using six loci therefore reliably identified the major meningococcal lineages associated with invasive disease. MLST can be applied to almost all bacterial species and other haploid organisms, including those that are difficult to cultivate. The overwhelming advantage of MLST over other molecular typing methods is that sequence data are truly portable between laboratories, permitting one expanding global database per species to be placed on a World-Wide Web site, thus enabling exchange of molecular typing data for global epidemiology via the Internet.