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Biomedical subjects

M Brand

Publications and source records attributed to M Brand.

At least 73 records · Page 4Linked to original sources
PubMed1996

Zebrafish pigmentation mutations and the processes of neural crest development.

Neural crest development involves cell-fate specification, proliferation, patterned cell migration, survival and differentiation. Zebrafish neural crest derivatives include three distinct chromatophores, which are well-suited to genetic analysis of their development. As part of a large-scale mutagenesis screen for embryonic/early larval mutations, we have isolated 285 mutations affecting all aspects of zebrafish larval pigmentation. By complementation analysis, we define 94 genes. We show here that comparison of their phenotypes permits classification of these mutations according to the types of defects they cause, and these suggest which process of neural crest development is probably affected. Mutations in eight genes affect the number of chromatophores: these include strong candidates for genes necessary for the processes of pigment cell specification and proliferation. Mutations in five genes remove part of the wild-type pigment pattern, and suggest a role in larval pigment pattern formation. Mutations in five genes show ectopic chromatophores in distinct sites, and may have implications for chromatophore patterning and proliferation. 76 genes affect pigment or morphology of one or more chromatophore types: these mutations include strong candidates for genes important in various aspects of chromatophore differentiation and survival. In combination with the embryological advantages of zebrafish, these mutations should permit cellular and molecular dissection of many aspects of neural crest development.

Adaptation, Physiological↗
PubMed1996

Mutations affecting xanthophore pigmentation in the zebrafish, Danio rerio.

In a large-scale screen for mutants with defects in embryonic development we identified 17 genes (65 mutants) specifically required for the development of xanthophores. We provide evidence that these genes are required for three different aspects of xanthophore development. (1) Pigment cell formation and migration (pfeffer and salz); (2) pigment synthesis (edison, yobo, yocca and brie) and (3) pigment translocation (esrom, tilsit and tofu). The number of xanthophore cells that appear in the body is reduced in embryos with mutations in the two genes, salz and pfeffer. In heterozygous and homozygous salz and pfeffer adults, the melanophore stripes are interrupted, indicating that xanthophore cells have an important function in adult melanophore pattern formation. Most other genes affect only larval pigmentation. In embryos mutant for edison, yobo, yocca and brie, differences in pteridine synthesis can be observed under UV light and by thin-layer chromatography. Homozygous mutant females of yobo show a recessive maternal effect. Embryonic development is slowed down and embryos display head and tail truncations. Xanthophores in larvae mutant in the three genes esrom, tilsit and tofu appear less spread out. In addition, these mutants display a defect in retinotectal axon pathfinding. These mutations may affect xanthophore pigment distribution within the cells or xanthophore cell shape. Mutations in seven genes affecting xanthophore pigmentation remain unclassified.

Animals↗
PubMed1996

Genes controlling and mediating locomotion behavior of the zebrafish embryo and larva.

Zebrafish embryos and larvae have stage-specific patterns of motility or locomotion. Two embryonic structures accomplish this behavior: the central nervous system (CNS) and skeletal muscles. To identify genes that are functionally involved in mediating and controlling different patterns of embryonic and larval motility, we included a simple touch response test in our zebrafish large-scale genetic screen. In total we identified 166 mutants with specific defects in embryonic motility. These mutants fall into 14 phenotypically distinct groups comprising at least 48 genes. Here we describe the various phenotypic groups including mutants with no or reduced motility, mechanosensory defective mutants, 'spastic' mutants, circling mutants and motor circuit defective mutants. In 63 mutants, defining 18 genes, striation of somitic muscles is reduced. Phenotypic analysis provides evidence that these 18 genes have distinct and consecutive functions during somitic muscle development. The genes sloth (slo) and frozen (fro) already act during myoblast differentiation, while 13 genes appear to function later, in the formation of myofibers and the organization of sarcomeres. Mutations in four other genes result in muscle-specific degeneration. 103 mutations, defining at least 30 genes, cause no obvious defects in muscle formation and may instead affect neuronal development. Analysis of the behavioral defects suggests that these genes participate in the diverse locomotion patterns observed, such as touch response, rhythmic tail movements, equilibrium control, or that they simply confer general motility to the animal. In some of these mutants specific defects in the developing nervous system are detected. Mutations in two genes, nevermind (nev) and macho (mao), affect axonal projection in the optic tectum, whereas axon formation and elongation of motorneurons are disrupted by mutations in the diwanka (diw) and the unplugged (unp) genes.

Animals↗
PubMed1996

Zebrafish mutations affecting retinotectal axon pathfinding.

We have isolated mutants in the zebrafish Danio rerio that have defects in axonal connectivity between the retina and tectum. 5-day-old fish larvae were screened by labeling retinal ganglion cells with DiI and DiO and observing their axonal projections to and on the tectum. 82 mutations, representing 13 complementation groups and 6 single allele loci, were found that have defects in retinal ganglion cell axon pathfinding to the tectum. These pathfinding genes fall into five classes, based on the location of pathfinding errors between eye and tectum. In Class I mutant larvae (belladonna, detour, you-too, iguana, umleitung, blowout) axons grow directly to the ipsilateral tectal lobe after leaving the eye. Class II mutant larvae (chameleon, bashful) have ipsilaterally projecting axons and, in addition, pathfinding mistakes are seen within the eye. In Class III mutant larvae (esrom, tilsit, tofu) fewer axons than normal cross the midline, but some axons do reach the contralateral tectal lobe. Class IV mutant larvae (boxer, dackel, pinscher) have defects in axon sorting after the midline and retinal axons occasionally make further pathfinding errors upon reaching the contralateral tectal lobe. Finally, Class V mutant larvae (bashful, grumpy, sleepy, cyclops, astray) have anterior-posterior axon trajectory defects at or after the midline. The analysis of these mutants supports several conclusions about the mechanisms of retinal axon pathfinding from eye to tectum. A series of sequential cues seems to guide retinal axons to the contralateral tectal lobe. Pre-existing axon tracts seem not to be necessary to guide axons across the midline. The midline itself seems to play a central role in guiding retinal axons. Axons in nearby regions of the brain seem to use different cues to cross the ventral midline. Mutant effects are not all-or-none, as misrouted axons may reach their target, and if they do, they project normally on the tectum. The retinotectal pathfinding mutants reveal important choice points encountered by neuronal growth cones as they navigate between eye and tectum.

Animals↗
PubMed1996

The zebrafish epiboly mutants.

Epiboly, the enveloping of the yolk cell by the blastoderm, is the first zebrafish morphogenetic movement. We isolated four mutations that affect epiboly: half baked, avalanche, lawine and weg. Homozygous mutant embryos arrest the vegetal progress of the deep cells of the blastoderm; only the yolk syncytial layer of the yolk cell and the enveloping layer of the blastoderm reach the vegetal pole of the embryo. The mutations half baked, avalanche and lawine produce a novel dominant effect, termed a zygotic-maternal dominant effect: heterozygous embryos produced from heterozygous females slow down epiboly and accumulate detached cells over the neural tube; a small fraction of these mutant individuals are viable. Heterozygous embryos produced from heterozygous males crossed to homozygous wild-type females complete epiboly normally and are completely viable. Additionally, embryos heterozygous for half baked have an enlarged hatching gland, a partial dominant phenotype. The phenotypes of these mutants demonstrate that, for the spreading of cells during epiboly, the movement of the deep cells of the blastoderm require the function of genes that are not necessary for the movement of the enveloping layer or the yolk cell. Furthermore, the dominant zygotic-maternal effect phenotypes illustrate the maternal and zygotic interplay of genes that orchestrate the early cell movements of the zebrafish.

Animals↗
PubMed1996

The zebrafish early arrest mutants.

This report describes mutants of the zebrafish having phenotypes causing a general arrest in early morphogenesis. These mutants identify a group of loci making up about 20% of the loci identified by mutants with visible morphological phenotypes within the first day of development. There are 12 Class I mutants, which fall into 5 complementation groups and have cells that lyse before morphological defects are observed. Mutants at three loci, speed bump, ogre and zombie, display abnormal nuclei. The 8 Class II mutants, which fall into 6 complementation groups, arrest development before cell lysis is observed. These mutants seemingly stop development in the late segmentation stages, and maintain a body shape similar to a 20 hour embryo. Mutations in speed bump, ogre, zombie, specter, poltergeist and troll were tested for cell lethality by transplanting mutant cells into wild-type hosts. With poltergeist, transplanted mutant cells all survive. The remainder of the mutants tested were autonomously but conditionally lethal: mutant cells, most of which lyse, sometimes survive to become notochord, muscles, or, in rare cases, large neurons, all cell types which become postmitotic in the gastrula. Some of the genes of the early arrest group may be necessary for progression though the cell cycle; if so, the survival of early differentiating cells may be based on having their terminal mitosis before the zygotic requirement for these genes.

Animals↗
PubMed1996

Genes establishing dorsoventral pattern formation in the zebrafish embryo: the ventral specifying genes.

We identified 6 genes that are essential for specifying ventral regions of the early zebrafish embryo. Mutations in these genes cause an expansion of structures normally derived from dorsal-lateral regions of the blastula at the expense of ventrally derived structures. A series of phenotypes of varied strengths is observed with different alleles of these mutants. The weakest phenotype is a reduction in the ventral tail fin, observed as a dominant phenotype of swirl, piggytail, and somitabun and a recessive phenotype of mini fin, lost-a-fin and some piggytail alleles. With increasing phenotypic strength, the blood and pronephric anlagen are also reduced or absent, while the paraxial mesoderm and anterior neuroectoderm is progressively expanded. In the strong phenotypes, displayed hy homozygous embryos of snailhouse, swirl and somitabun, the somites circle around the embryo and the midbrain region is expanded laterally. Several mutations in this group of genes are semidominant as well as recessive indicating a strong dosage sensitivity of the processes involved. Mutations in the piggytail gene display an unusual dominance that depends on both a maternal and zygotic heterozygous genotype, while somitabun is a fully penetrant dominant maternal-effect mutation. The similar and overlapping phenotypes of mutants of the 6 genes identified suggest that they function in a common pathway, which begins in oogenesis, but also depends on factors provided after the onset of zygotic transcription, presumably during blastula stages. This pathway provides ventral positional information, counteracting the dorsalizing instructions of the organizer, which is localized in the dorsal shield.

Animals↗
PubMed1996

dino and mercedes, two genes regulating dorsal development in the zebrafish embryo.

We describe two genes, dino and mercedes, which are required for the organization of the zebrafish body plan. In dino mutant embryos, the tail is enlarged at the expense of the head and the anterior region of the trunk. The altered expression patterns of various marker genes reveal that, with the exception of the dorsal most marginal zone, all regions of the early dino mutant embryo acquire more ventral fates. These alterations are already apparent before the onset of gastrulation. mercedes mutant embryos show a similar but weaker phenotype, suggesting a role in the same patterning processes. The phenotypes suggests that dino and mercedes are required for the establishment of dorsal fates in both the marginal and the animal zone of the early gastrula embryo. Their function in the patterning of the ventrolateral mesoderm and the induction of the neuroectoderm is similar to the function of the Spemann organizer in the amphibian embryo.

Animals↗
PubMed1996

Proinflammatory cytokines interleukin 1 beta and tumor necrosis factor alpha inhibit growth hormone stimulation of insulin-like growth factor I synthesis and growth hormone receptor mRNA levels in cultured rat liver cells.

Low levels of insulin-like growth factor I (IGF-I) in critical illness are observed despite increased or normal levels of growth hormone (GH). The mechanisms for this apparent GH resistance have not been elucidated. As many of the acute inflammatory responses in critical illness are mediated by the proinflammatory cytokines interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha), the present studies evaluated IL-1 beta and TNF-alpha effects on steady-state and GH-stimulated IGF-I synthesis and GH receptor mRNA levels. In rat hepatocytes in primary culture, IGF-I released into culture medium was determined by radioimmunoassay, and quantitative competitive polymerase chain reaction was used to measure IGF-I mRNA and GH receptor mRNA concentrations. Growth hormone increased GH receptor mRNA, IGF-I mRNA and IGF-I protein secreted into the culture medium. In cells not stimulated with GH, modest inhibitory effects of IL-1 beta on GH receptor mRNA, IGF-I mRNA and IGF-I protein levels were seen. However, the stimulatory effects of GH were inhibited in a dose-dependent manner both by IL-1 beta and TNF-alpha, and at higher cytokine concentrations no stimulatory effects of GH were observed. Both IL-1 beta and TNF-alpha in submaximal dose had additive inhibitory effects on IGF-I protein concentrations but these effects did not result in irreversible damage to cells, as indicated by restoration of IGF-I and GH receptor mRNA levels to normal after withdrawal of cytokines. In conclusion, we demonstrated that in rat hepatocytes in primary culture IL-1 beta and TNF-alpha inhibited GH-stimulated IGF-I synthesis. Diminished GH receptor mRNA concentrations in response to IL-1 beta and TNF-alpha indicate that low IGF-I levels during severe illness, despite high circulating GH levels, may at least partially be a consequence of suppression of hepatic GH receptor synthesis by IL-1 beta and TNF-alpha.

Animals↗
PubMed1995

Use of laparoscopic techniques in oncologic right colectomy in a canine model.

BACKGROUND: The purpose of this study was to evaluate the feasibility of laparoscopic oncologic righ colectomy (RC) with intraperitoneal ileocolic anastomosis (ICA) in a canine model. METHODS: In 21 dogs a laparoscopic RC with transection of the main right colic artery and removal of adjacent lymph nodes (LN) was carried out using an Nd:YAG contact laser and endoscopic stapler. Two weeks after surgery, all animals were killed. The number of remaining right colon mesenteric LN, length of remaining right colic artery, bursting pressure (BP) of ICA, and postoperative morbidity were evaluated. RESULTS: No major intraoperative complications were recorded. One dog died of pneumonia and heartworms. There were no postoperative septic or anastomotic complications. All dogs passed feces within the first 24 h postoperatively. Median operative time was 135 min (range 105-180 min). Length of remaining right colic artery after oncologic resection was 4.5 mm (range 3-7 mm), the number of remaining LN was 0, and the median anastomotic BP was 232 mm Hg (range 132-312 mm Hg). CONCLUSION: Oncologic resection of the right colon with high vascular ligation, wide mesenteric clearance of LN, and intraperitoneal anastomosis is feasible and safe in a canine model.

Anastomosis, Surgical↗
PubMed1994

Monopolar electrosurgery and Nd:YAG Contact Laser in laparoscopic intestinal surgery.

In a prospective randomized study using a canine model, we compared the use of monopolar electrosurgery (EC) (n = 23) and the Nd:YAG Contact Laser (CL) (n = 21) on intra- and postoperative morbidity in laparoscopic large-bowel resection. In EC, cutting was performed with scissors and coagulation was performed with electrosurgery. In CL, cutting and coagulation were carried out with the Nd:YAG Contact Laser. Laparoscopic oncologic right colectomy with intraperitoneal ileocolic stapled anastomosis was performed in all dogs. Intraoperative smoke development, difficulty of dissection, hemostasis, and postoperative adhesions were judged using a five-point score. Two weeks after surgery, all dogs were sacrificed and zoopsy was carried out. Three dogs died postoperatively from pneumonia and one from an anastomotic leak. There were no other postoperative complications. Operative time was 135 min (range 105-180) in the CL group and 145 min (range 60-210) in the EC group. Intraoperative smoke development, difficulty of dissection, and postoperative amount of adhesions were not different between groups (P > 0.05). Hemostasis in the CL group (median score of 1, range 1-2) was significantly better (P = 0.01) than in the EC group (median score of 2, range 1-5). Scissors and electrosurgery as well as Nd:YAG Contact Laser can be used successfully in intestinal laparoscopic surgery. Although the use of the Contact Laser did not cause less postoperative morbidity than the conventional method, there was significantly better hemostasis using the Nd: YAG Contact Laser.

Animals↗
PubMed1994

Laparoscopic oncologic total abdominal colectomy with intraperitoneal stapled anastomosis in a canine model.

The purpose of this study was to evaluate the feasibility of performing an oncologic type of colectomy using laparoscopic techniques with intraperitoneal stapled ileorectal anastomosis in a canine model. In 21 dogs, laparoscopic oncologic colectomy was carried out using dissection with a contact Nd:YAG laser. The right, middle colic, and inferior mesenteric arteries were transected close to their origins, and all adjacent mesenteric lymph nodes were removed. Ileorectal anastomosis was performed using a circular end-to-end stapler especially designed for laparoscopic surgery. Two weeks after surgery, all animals were killed. The number of remaining mesenteric lymph nodes, bursting pressure of ileorectal anastomosis, and postoperative morbidity were evaluated. No major intraoperative complications were recorded. One dog died postoperatively from an anastomotic leak. All dogs passed feces in the first 24 postoperative hours. Median operative time was 145 min (90-240 min). The number of remaining colonic mesenteric lymph nodes was 0, and median anastomotic bursting pressure was 325 mm Hg (183-415 mm Hg). The median distance from ileocolic anastomosis to anal verge was 8.5 cm (6.0-11.0 cm). Laparoscopic oncologic colectomy with wide mesenteric clearance of lymph nodes and intraperitoneal stapled ileorectal anastomosis is feasible and safe in a canine model.

Anastomosis, Surgical↗
PubMed1994

daughterless is essential for neuronal precursor differentiation but not for initiation of neuronal precursor formation in Drosophila embryo.

The first steps of neuronal precursor formation require several genes that encode transcription regulators with the helix-loop-helix (HLH) motif, including the proneural genes of the achaete-scute complex AS-C (achaete (ac), scute (sc) and lethal of scute (l'sc)) and daughterless (da). The da protein dimerizes with AS-C products in vitro to form DNA-binding proteins. Previous studies have shown that the AS-C genes are expressed initially in discrete clusters of ectodermal cells (the proneural clusters) and then more strongly in the neuronal precursors that arise from these clusters and delaminate from the epidermal layer. In this paper, we studied the distribution of da protein with an antibody raised against Da. We found that Da is ubiquitously but non-uniformly distributed. Within the ectodermal layer, its level is neither elevated (as in the case of AS-C genes) nor reduced (as in the case of emc product) in the proneural cluster. It is, however, at higher levels in many neuronal precursors. We further studied the requirement of da in neuronal precursor development by using a variety of markers for neuronal precursors. Our results reveal the existence of at least two stages in neuronal precursor formation. da is not required for the initial appearance of nascent neuronal precursors but is required for these cells to express multiple neuronal precursor genes and to produce the normal number of neurons.

Animals↗
PubMed1993

The lytic transition of Epstein-Barr virus is imitated by recombinant B-cells.

Lytic transition of Epstein-Barr virus (EBV) is initiated by distinct immediate early regulators of the viral cycle, in synchronization to temporary, permissive conditions during host cell differentiation. We developed eukaryotic vectors suitable to imitate the processes involved in lytic transition in cell culture systems. Two stable B cell lines were established: R59Z activator cells were used to induce lytic EBV expression in a constitutive manner by the production of the BZLF 1 trans-activator (Zta). R7-57 reporter cells, on the other hand, signaled induced activity of the lytic origin of EBV replication (ori Lyt). Different modes, like chemical induction, lytic superinfection with EBV and single gene trans-activation converted the recombinant ori Lyt element in R7-57 reporter cells. BZLF 1, transiently expressed in R7-57 reporter cells, was the only EBV trans-activator found, sufficient in inducing the viral lytic cycle. Basing on these experiments, trans-cellular activation of EBV was tested by cocultivation of BZLF 1-expressing R59Z activator cells with the R7-57 reporter line. No lytic effect on the reporter cells could be measured, neither by cocultivation of activator cells nor by coincubation of BZLF 1-containing cell lysates. Latency breaking activity, however, was transferred from activator to reporter cells when active, exogenous virus was added. The cell system described in these experiments provides a tool for the detection of EBV reactivation and demonstrates the potential of the lytic regulatory gene BZLF 1.

Animals↗
PubMed1993

Principal neutralizing domain of HIV-1 is highly immunogenic when expressed on the surface of hepatitis B core particles.

The development of subunit vaccines against HIV requires the identification of immunologically relevant antigens and a suitable method of antigen delivery. Ideally, defined epitopes with neutralizing activity should be included in a vaccine preparation. The carrier for such peptide sequences should enhance the immunogenicity of the selected epitopes. In this study hepatitis B virus core antigen (HBcAg) was used as a carrier moiety for the principal neutralizing domain (PND, V3-loop) of HIV-1. A 25 amino acid V3-loop sequence was fused to HBcAg at various positions by genetic engineering. The resulting hybrid HBcAg/HIV polypeptides were analysed for particle formation and immunogenicity. Fusion of the PND to an internal position replacing an immunodominant antibody-binding region of HBcAg or to a C-terminally truncated HBcAg resulted in the formation of hybrid particles with biochemical and biophysical properties similar to those of wild-type HBcAg particles. Both types of hybrids are recognized by monoclonal and polyclonal antisera raised against PND peptides of various HIV-1 isolates. Hybrid particles with a C-terminal fusion but not an internal fusion are also recognized by a polyvalent anti-HBcAg serum. In both cases the V3 domain is surface accessible. Immunization of mice with hybrid particles induces an enhanced antibody response against the V3 sequence. The internal fusion is more immunogenic than the C-terminal fusion.

AIDS Vaccines↗
PubMed1993

asense is a Drosophila neural precursor gene and is capable of initiating sense organ formation.

Neural precursor cells in Drosophila arise from the ectoderm in the embryo and from imaginal disc epithelia in the larva. In both cases, this process requires daughterless and the proneural genes achaete, scute and lethal-of-scute of the achaete-scute complex. These genes encode basic helix-loop-helix proteins, which are nuclear transcription factors, as does the asense gene of the achaete-scute complex. Our studies suggest that asense is a neural precursor gene, rather than a proneural gene. Unlike the proneural achaete-scute gene products, the asense RNA and protein are found in the neural precursor during its formation, but not in the proneural cluster of cells that gives rise to the neural precursor cell. Also, asense expression persists longer during neural precursor development than the proneural gene products; it is still expressed after the first division of the neural precursor. Moreover, asense is likely to be downstream of the proneural genes, because (1) asense expression is affected in proneural and neurogenic mutant backgrounds, (2) ectopic expression of asense protein with an intact DNA-binding domain bypasses the requirement for achaete and scute in the formation of imaginal sense organs. We further note that asense ectopic expression is capable of initiating the sense organ fate in cells that do not normally require the action of asense. Our studies therefore serve as a cautionary note for the inference of normal gene function based on the gain-of-function phenotype after ectopic expression.

Amino Acid Sequence↗
PubMed1993

The regulation and function of the helix-loop-helix gene, asense, in Drosophila neural precursors.

asense is a member of the achaete-scute complex (AS-C) of helix-loop-helix genes involved in Drosophila neurogenesis. Unlike the other AS-C members, which are expressed in subsets of the ectodermal areas (proneural clusters) that give rise to neural precursors, asense is one of a number of genes that are specifically expressed in the neural precursors themselves (neural precursor genes). We have identified a mutant asense phenotype that may reflect this later expression pattern. As a step in understanding the determination of neural precursors from the proneural clusters, we have investigated the potential role of the AS-C products as direct transcriptional activators of neural precursor genes by analysing the regulation of asense. Using genomic rescues and asense-lacZ fusion genes, the neural precursor regulatory element has been identified. We show that this element contains binding sites for AS-C/daughterless heterodimers. Delection of these sites reduces the expression from the fusion gene, but significant expression is still achieved, pointing to the existence of other regulators of asense in addition to the AS-C. asense differs from the other AS-C members in its expression pattern, regulation, mutant phenotype and some DNA-binding properities.

Animals↗