Biomedical subjects
Publications and source records attributed to A Meyer.
Four years after unexplained left eye visual loss and 15 days after surgical procedure on the other eye, a 93-year-old female developed a pigmented left conjunctival lesion. Ultrasound examination demonstrated this lesion to be in fact an extraocular extension of uveal melanoma. The patient was treated by external radiotherapy after expanded enucleation.
A phylogeny of the principal lineages of cichlid fishes and two other fish families of the suborder Labroidei was based on phylogenetic information from DNA sequences of the flanking region of a (CA)n microsatellite locus. Microsatellite (CA)n containing clones from a genomic library of an African cichlid fish from Lake Tanganyika, Tropheus moorii, were sequenced and primers for the polymerase chain reaction designed. All primers amplified the homologous microsatellite loci in many more than the source species and one microsatellite flanking locus (TmoM27) was particularly conserved and amplified in several lineages of perciform fishes that diverged more than 80-100 million years ago. Despite the extensive level of evolutionary conservation of this microsatellite flanking region (MFR), this nuclear region contained reliable phylogenetic information in the form of both point and length mutations. A phylogeny of cichlids based on this MFR agrees with other phylogenetic hypotheses based on morphological, mitochondrial, and anonymous nuclear DNA. Madagascan and Indian cichlids are found to be paraphyletic and the most basal group in the family Cichlidae. African and Neotropical cichlids are both monophyletic and sistergroups. Within African lineages, the East African cichlids are most likely to be monophyletic and the West African cichlids are probably paraphyletic and basal to all African species. The focal microsatellite locus contained much variation in (CA)n repeats in African cichlids and in surfperches (up to 64 repeats), but was short (with only 2-4 repeats) and almost invariant in Neotropical cichlids. The design of phylogenetically highly versatile MFR-primers will be of use not only for phylogeny reconstruction among families of perciform fishes, but also for population-level work in the thousands of species belonging to this highly species-rich suborder of fishes.
The study of development has relied primarily on the isolation of mutations in genes with specific functions in development and on the comparison of their expression patterns in normal and mutant phenotypes. Comparative evolutionary analyses can complement these approaches. Phylogenetic analyses of Sonic hedgehog (Shh) and Hoxd-10 genes from 18 cyprinid fish species closely related to the zebrafish provide novel insights into the functional constraints acting on Shh. Our results confirm and extend those gained from expression and crystalline structure analyses of this gene. Unexpectedly, exon 1 of Shh is found to be almost invariant even in third codon positions among these morphologically divergent species suggesting that this exon encodes for a functionally important domain of the hedgehog protein. This is surprising because the main functional domain of Shh had been thought to be that encoded by exon 2. Comparisons of Shh and Hoxd-10 gene sequences and of resulting gene trees document higher evolutionary constraints on the former than on the latter. This might be indicative of more general evolutionary patterns in networks of developmental regulatory genes interacting in a hierarchical fashion. The presence of four members of the hedgehog gene family in cyprinid fishes was documented and their homologies to known hedgehog genes in other vertebrates were established.
Prohormone convertases (PCs) belong to the mammalian family of subtilisin/kexin-like enzymes which have been implicated in the posttranslational processing of precursor proteins. Several PCs are produced in the central and peripheral nervous system, and only a few specific precursor-substrates have been identified in vivo. In the nervous system, PCs may be involved in intracellular processing of precursors for neuropeptides, hormones and neurotrophic factors, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). To study the interrelationships between the convertases furin, PC1 and PC2, and the neurotrophins NGF, BDNF and NT-3, we compared their mRNA distribution in different tissues. We also examined their expression in the hippocampus of mice undergoing kainic acid-induced seizures. In this experiment, in situ hybridization (ISH) demonstrated that the levels of mRNA for furin, PC1 and BDNF increased maximally at 3 h after kainic acid administration, followed by a decline to normal levels by 96 h. NGF showed small changes, while NT-3 was downregulated with minimal expression levels between 3 to 12 h. Double ISH with radioactively-labeled riboprobes and digoxigenin-labeled riboprobes demonstrated colocalization of furin with NGF and BDNF in the mouse submaxillary gland, and of furin and PC1 with BDNF in the trigeminal ganglion. Based on colocalization studies and evidence of coordinate expression with NGF and BDNF, we suggest the involvement of furin in processing of proNGF, and of both furin and PC1 in processing of proBDNF.
The East African Lakes Tanganyika, Malawi and Victoria each harbour hundreds of endemic invertebrate and vertebrate species. Inferences about the ecological and evolutionary processes responsible for the origin of these species flocks will only be possible when they are made within historical and comparative frameworks. Specifically, the relative importance of intrinsic characteristics and extrinsic factors may offer information about the processes that drive diversification and speciation in these species. We investigated the sequence variation of a segment of the mitochondrial DNA control region of 32 populations representing all four nominal species in the three genera of eretmodine cichlids from Lake Tanganyika. Based on a phylogenetic analysis of these data we attempted to evaluate the importance of major lake level fluctuations on patterns of intralacustrine speciation. The geography of genetic variation reveals a high degree of within-lake endemism among genetically well-separated lineages distributed along the inferred shore lines of three historically intermittent lake basins. Seismic data indicate that extreme lowering of water levels in the Pleistocene caused the single Lake Tanganyika basin to split into three isolated ones. The strong phylogeographic structure of the Eretmodini, and the observation that some closely related populations occur on opposite shores of the lake, agree with this geological scenario. The three-clade-three-basin phylogeographic pattern was repeated twice within this tribe of cichlids. The phylogeographic pattern of eretmodine cichlids suggests that major fluctuations in the level of the lake have been important in shaping their adaptive radiation and speciation. The mitochondrially defined clades are in conflict with the current taxonomy of the group and suggest that there has been convergent evolution in trophic morphology, particularly in the shapes of oral teeth, taxonomically the most diagnostic characters of the three genera.
We have cloned a novel CC chemokine receptor cDNA from mouse thymus. The deduced amino acid sequence shows 74% identity to the human monocyte chemotactic protein (MCP)-1 receptor (CC CKR-2b) and 54% to a recently cloned murine macrophage inflammatory protein (MIP)-1alpha receptor (Gao, J. L., and Murphy, P. M.(1995) J. Biol. Chem. 270, 17494-17501). Northern blot analysis of mouse tissues showed that the mRNA was also expressed in heart, spleen and liver, and to a lesser extent in lung and brain. The rank order of CC chemokine competition for 125I-labeled human RANTES (regulated on activation, normal T-cell expressed and secreted) binding to human embryonic kidney (HEK) 293 cells stably transfected with the receptor cDNA was murine MIP-1alpha >> human MIP-1beta > human RANTES > murine RANTES > murine MIP-1beta > human MCP-2 > murine MCP-1 (JE) > human MIP-1alpha > human MCP-3 > human MCP-1. Of the chemokines tested, only murine MIP-1alpha, human and murine MIP-1beta and RANTES, human MCP-2, and JE were able to induce mobilization of intracellular Ca2+ from fura-2-loaded HEK 293 cells expressing the receptor. These results suggest that this receptor functions as a high affinity murine MIP-1alpha receptor; however, it is likely to be an important target for the biological activities of several CC chemokines in mouse.
The origin of land vertebrates was one of the major transitions in the history of vertebrates. Yet, despite many studies that are based on either morphology or molecules, the phylogenetic relationships among tetrapods and the other two living groups of lobe-finned fishes, the coelacanth and the lungfishes, are still unresolved and debated. Knowledge of the relationships among these lineages, which originated back in the Devonian, has profound implications for the reconstruction of the evolutionary scenario of the conquest of land. We collected the largest molecular data set on this issue so far, about 3,500 base pairs from seven species of the large 28S nuclear ribosomal gene. All phylogenetic analyses (maximum parsimony, neighbor-joining, and maximum likelihood) point toward the hypothesis that lungfishes and coelacanths form a monophyletic group and are equally closely related to land vertebrates. This evolutionary hypothesis complicates the identification of morphological or physiological preadaptations that might have permitted the common ancestor of tetrapods to colonize land. This is because the reconstruction of its ancestral conditions would be hindered by the difficulty to separate uniquely derived characters from shared derived characters in the coelacanth/lungfish and tetrapod lineages. This molecular phylogeny aids in the reconstruction of morphological evolutionary steps by providing a framework; however, only paleontological evidence can determine the sequence of morphological acquisitions that allowed lobe-finned fishes to colonize land.
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DNA gyrase is the target of the coumarin group of antibacterial agents. The drugs are known to inhibit the ATPase activity of gyrase and bind to the 24-kDa N-terminal subdomain of gyrase B protein. Supercoiling assays with intact DNA gyrase and ATPase assays with a 43-kDa N-terminal fragment of the B protein suggest that the drugs bind tightly, with Kd values <10(-7) M. In addition, the ATPase data suggest that 1 coumermycin molecule interacts with 2 molecules of the 43-kDa protein while the other coumarins form a 1:1 complex. This result is confirmed by cross-linking experiments. Rapid gel-filtration experiments show that the binding of ADPNP(5'-adneylyl beta,gamm-imidodiphosphate) and coumarins to the 43-kDa protein is mutally exclusive, consistent with a competitive mode of action for the drugs. Rapid gel-filtration binding experiments using both the 24-and 43-kDa proteins also show that the drugs bind with association rate constants of >10(5) M-1.s-1, and dissociation rate constants of approximately 3x10(-3)s-1 and approximately 4x10(-3)s-1 for the 43-and 24-kDa proteins, respectively. Titration calorimetry shows that the Kd values for coumarins binding to both proteins are approximately 10-8M and that binding is enthalpy driven.
In work performed by a number of laboratories, it has become quite clear that the oral administration of autoantigens exerts a profoundly suppressive effect on the development and long-term clinical course of autoimmune disease. Specific peptide sequences derived from the autoantigens are similarly suppressive. An interesting sidelight to emerge from specificity studies is that oral administration of a self-protein or peptide sequence (i.e., rat MBP peptide administered to a rat) is markedly less tolerogenic than oral administration of a non-self or even closely related sequence (guinea pig MBP peptide administered to a rat). The dose of oral antigen is now known to play a critical role in determination of the mechanism of oral tolerance, with low doses of antigen causing active suppression with concomitant release of TGFbeta1. Studies outlined here suggest that oral administration of higher antigen doses (e.g., 20 mg MBP to rats or mice) results in deletion of specific antigen-reactive T lymphocytes. This conclusion stems from the fact that injections of IL-2 could not reverse high-dose tolerance while reversing low-dose oral tolerance. Moreover, feeding MBP to MBP-TCR transgenic mice caused trafficking of transgenic cells to the intestine followed by a profound depletion of transgene-positive cells and reduction in proliferative function in all peripheral lymphoid organs. Oral tolerance has proven to be of therapeutic benefit in other animal models of autoimmune disease as well, including uveitis, collagen-induced arthritis, adjuvant arthritis, thyroiditis, myasthenia gravis, and diabetes. Initial human trials in multiple sclerosis, rheumatoid arthritis, and uveitis show promising results.
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Multiple sclerosis (MS) is a chronic demyelinating disease of the human central nervous system (CNS) which can be characterized clinically by a remitting-relapsing or a chronic progressive course. There is a striking similarity between the clinical and histopathological features of MS and the experimentally induced disease, experimental autoimmune encephalomyelitis (EAE). Induced by the injection of myelin basic protein (MBP) and adjuvants, EAE is characterized by clinical neurologic signs of paralysis and histopathologic changes consisting of perivascular mononuclear infiltration and demyelination. We have reported that the oral administration of MBP exerts a profoundly suppressive effect on EAE induced in the Lewis rat. This MBP-induced oral tolerance is characterized by an inhibition of EAE clinical neurologic signs, reduced CNS histopathologic changes, a profound decrease in the T-lymphocyte proliferative response specific for the fed antigen, and a decrease in serum antibody specific for MBP. In a chronic relapsing model of EAE in the B10.PL mouse, we have shown that the oral administration of MBP either prior to MBP challenge or on the first day of clinical signs results in a decreased number and severity of EAE relapses. The oral tolerance approach has also proven effective in the suppression of other organ-specific autoimmune diseases including collagen-induced arthritis, adjuvant arthritis, uveoretinitis, experimental myasthenia gravis, diabetes, and thyroiditis as well as graft rejection. Two primary mechanisms have been proposed to explain oral tolerance in EAE-active suppression following feeding of lower doses of antigen and clonal anergy or deletion following administration of higher doses. In vivo approaches in rats and transgenic mice have been used to further explore the mechanisms underlying oral tolerance. Administration of recombinant interleukin (IL)-2 was shown to reverse the tolerance induced by feeding low doses of MBP, but not the tolerance induced by feeding high doses of MBP, indicating that deletion had occurred in the high-dose group. Moreover, the oral administration of MBP to MBP-specific T-cell receptor (TCR) transgenic mice resulted in a profound decrease of the transgenic T cells in the blood, lymph node cells (LNC), mesenteric LNC, and spleen compartments. The proliferative response to MBP was also profoundly reduced in these organs, indicating that the cells had been deleted from these sites. The results achieved in animal models have led to clinical trials of oral tolerization in three human autoimmune diseases--MS, uveoretinitis, and rheumatoid arthritis--with promising results.
The subfamily of fishes Trematominae is endemic to the subzero waters of antarctica and is part of the longer notothenioid radiation. Partial mitochondrial sequences from the 12S and 16S ribosomal RNA (rRNA) genes and a phylogeny for 10 trematomid species are presented. As has been previously suggested, two taxa, Trematomus scotti and T. newnesi, do not appear to be part of the main trematomid radiation. The genus Pagothenia is nested within the genus Trematomus and has evolved a unique cyropelagic existence, an association with pack ice. Using a mitochondrial rRNA molecular clock rate of 0.14% transversion changes per million years, the average age of the trematomids is estimated at 3.4 million years (MY). If the age of the trematomids is approximately 3.4 MY, this group could have speciated during the period of deglaciation in Antarctica 2.5-4.8 million years ago. This era was marked by significant changes on the Antarctic shores, such as the opening of fjords, which might have provided a stimulus for specification.
Germ cell tumours in children are more often extragonadal than in adults and the most frequent type is the yolk sac tumour. Limited cytogenetic data exist on extragonadal yolk sac tumours in children. We applied in situ hybridization (ISH) to interphase cell nuclei of four paediatric extragonadal pure yolk sac tumours and one yolk sac tumour component of a mixed germ cell tumour using paraffin-embedded tissue sections. The panel of chromosome-specific DNA probes was selected on the basis of their relevance in adult germ cell tumours and consisted of five DNA probes specific for the (peri)centromeric regions of chromosomes 1, 8, 12, and/or 17, X and/or one DNA probe specific for the subtelomeric region of chromosome 1 (p36.3). Only one tumour failed to show numerical and structural chromosome aberrations with the DNA probes used. The other four had an increased incidence of numerical chromosome aberrations with an over-representation of at least one chromosome. The DNA indices determined in the paraffin-embedded tumour material correlated well with the in situ hybridization findings. In only a few cases were chromosomes over-represented, when compared with the corresponding DNA indices. Recently, we have shown that the short arm of chromosome 1 is a non-random site of deletion in paediatric gonadal pure yolk sac tumours. The occurrence of similar deletions in one extragonadal pure yolk sac tumour and in one yolk sac tumour component, in conjunction with two further ISH reports, suggests that the loss of gene(s) in this region is an important event in the pathogenesis of paediatric malignant germ cell tumours of nearly all sites.
The patterns and rates of nucleotide substitution in mitochondrial ribosomal RNA genes are described and applied in a phylogenetic analysis of fishes of the subfamily Serrasalminae (Teleostei, Characiformes, Characidae). Fragments of 345 bp of the 12S and 535 bp of the 16S genes were sequenced for 37 taxa representing all but three genera in the subfamily. Secondary-structure models based on comparative sequence analysis were derived to characterize the pattern of change among paired and unpaired nucleotides, forming stem and loop regions, respectively. Base compositional biases were in the direction of A-rich loops and G-rich stems. Ninety-five percent of substitutions in stem regions were compensatory mutations, suggesting that selection for maintenance of base pairing is strong and that independence among characters cannot be assumed in phylogenetic analyses of stem characters. The relative rate of nucleotide substitution was similar in both fragments sequenced but higher in loop than in stem regions. In both genes, C-T transitions were the most common type of change, and overall transitions outnumbered transversions by a factor of two in 16S and four in 12S. Phylogenetic analysis of the mitochondrial DNA sequences suggests that a clade formed by the genera Piaractus, Colossoma, and Mylossoma is the sister group to all other serrasalmins and that the genera Myleus, Serrasalmus, and Pristobrycon are paraphyletic. A previous hypothesis concerning relationships for the serrasalmins, based on morphological evidence, is not supported by the molecular data. However, phylogenetic analysis of host-specific helminth parasites and cytogenetic data support the phylogeny of the Serrasalminae obtained in this study and provide evidence for coevolution between helminth parasites and their fish hosts.
A characteristic feature of the major histocompatibility complex (Mhc) polymorphism in mammals is the existence of allelic lineages shared by related species. This trans-species polymorphism has thus far been documented only in primates, rodents, and artiodactyls. In this communication we provide evidence that it also exists in cyprinid (bony) fishes at the class II A and B loci coding for the alpha and beta polypeptide chains of the class II alpha:beta heterodimers. The study has focused on three species of the family Cyprinidae, subfamily Rasborinae: the zebrafish (Danio rerio), the giant danio (D. malabaricus), and the pearl danio (D. albolineatus). The polymerase chain reaction was used to amplify and then sequence intron 1 and exon 2 of the class II B loci and exon 2 of the class II A loci in these species. Phylogenetic analysis of the sequences revealed the existence of allelic lineages whose divergence predates the divergence of the three species at both the A and B loci. The lineages at the B locus in particular are separated by large genetic distances. The polymorphism is concentrated in the peptide-binding region sites and is apparently maintained by balancing selection. Sharing of this unique Mhc feature by both bony fishes and mammals suggests that the main function of the Mhc (presentation of peptides to T lymphocytes) has not changed during the last 400 million years of its evolution.