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G M Kelly

Publications and source records attributed to G M Kelly.

33 records · Page 2Linked to original sources
PubMed1995

Zebrafish wnt8 and wnt8b share a common activity but are involved in distinct developmental pathways.

The specification of the vertebrate body plan is dependent on numerous signaling molecules, including members of the Wnt family. We have identified two zebrafish wnt8 paralogs related to Xwnt-8B and Xwnt-8, respectively. A RT-PCR assay demonstrated that wnt8 is expressed maternally, with transcripts detected throughout embryogenesis, whereas wnt8b transcripts were first detected during late gastrulation. The wnt8 transcripts at 50% epiboly are spatially restricted to those cells at the blastoderm margin, overlying gsc-expressing cells in the axial hypoblast. During late gastrulation, wnt8 was no longer detected in the marginal cells at the dorsal midline and by mid-segmentation, transcripts were found in the presumptive tail bud. In contrast, wnt8b expression is spatially restricted to prospective neuroepithelium, and later to neural-specific structures. Overexpression of both wnts results in two major phenotypes: radialized embryos and embryos with anterior defects. These phenotypes were preceded by significant changes in the spatial expression patterns of gsc and ntl transcripts, reminiscent of activities of Xwnt-8 in Xenopus, and consistent with a role for wnt8 in the specification or patterning of mesoderm.

Amino Acid Sequence↗
PubMed1995

The CNS-specific hyaluronan-binding protein BEHAB is expressed in ventricular zones coincident with gliogenesis.

Hyaluronan (HA) is a ubiquitous component of extracellular matrices, and in several systems it plays a central role in regulating cellular proliferation and differentiation. Cell, or tissue,-specific functions of HA are likely to be mediated by cell, or tissue,-specific HA-binding proteins. We previously hyaluronan-binding protein from rat and cat (Jaworski et al., 1994). In view of the potential role of HA in neural differentiation, we examined the expression of BEHAB during late embryonic and early postnatal development of the rat. BEHAB is expressed at very high levels in ventricular zones throughout the neuraxis. Expression is first detected at embryonic day 15 (E15) in the spinal cord, and is detected at progressively more rostral levels at later ages. BEHAB expression, like other features of neural development, follows both caudal-to-rostral and ventral-to-dorsal gradients. The timing of BEHAB expression parallels the timing of the generation of glial cells. In all areas of the CNS examined, BEHAB expression begins after the peak of neurogenesis and coincident with gliogenesis. The regulation of proliferation and differentiation by HA in other tissues, together with the expression of BEHAB in zones of mitotic activity coincident with the generation of glia, suggests that the extracellular matrix protein encoded by BEHAB could play a role in the generation or differentiation of CNS glia.

Aging↗
PubMed1994

BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan-binding proteins that is restricted to the brain.

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix of all tissues. In the mammalian central nervous system (CNS) HA is present throughout development and into adulthood. While the functions of HA are likely to be mediated by HA-binding proteins, no cell or tissue specific HA-binding proteins have been reported. In an effort to characterize the composition of the extracellular matrix of the CNS, we sought to identify neural HA-binding proteins. We report here the isolation and characterization of a cDNA with a high degree of sequence homology to members of the proteoglycan tandem repeat (PTR) family of HA-binding proteins. Unlike other HA-binding proteins, the expression of this cDNA is restricted to the CNS. We propose the name BEHAB, Brain Enriched HyAluronan Binding protein, for this gene. The expression of BEHAB mRNA is developmentally regulated; expression is first detected in the late embryonic period and peaks during the first two postnatal weeks. In the embryo, BEHAB is expressed at highest levels in mitotically active cells. The sequence of BEHAB has long stretches of identity between rat and cat, suggesting that the encoded protein is functionally important. The size and sequence of BEHAB are consistent with the possibility that it could serve a function like link protein, stabilizing interactions between HA and brain proteoglycans. These observations suggest that existence of other tissue specific HA-binding proteins.

Amino Acid Sequence↗
PubMed1993

Expression of wnt10a in the central nervous system of developing zebrafish.

A polymerase chain reaction-derived clone with sequence homology to partial-length shark and salamander Wnt-10a was used to screen a zebrafish cDNA library, yielding a cDNA encoding a full-length Wnt polypeptide. Transcripts encoding zebrafish wnt10a are first detected during the segmentation period of embryogenesis, at a time when there is extensive development and differentiation of the central nervous system. wnt10a transcripts are confined to differentiating brain regions, with expression first appearing diffusely in the floor of the hindbrain ventricle and proceeding anteriorly to the mid- and forebrain. Comparatively, zebrafish wnt1 transcripts are expressed in a stripe at the future midbrain-hindbrain boundary and at the future forebrain-midbrain border. With the differentiation of the neural tube into distinct regions, wnt1 signals are also detected along the dorsal midline of the midbrain, the mid- and posterior hindbrain, and the spinal cord. These patterns suggest roles for wnt10a in the regionalization of the brain.

Amino Acid Sequence↗
PubMed1993

Dissecting Wnt signalling pathways and Wnt-sensitive developmental processes through transient misexpression analyses in embryos of Xenopus laevis.

We review evidence that Xenopus Wnts (Xwnts) have activities consistent with their hypothesized roles as secreted signalling factors involved in multiple developmental processes. Transient misexpression of different Xwnts has distinct effects upon early development, and upon the formation of tissues in UV-irradiated embryos. Misexpression of Xwnts also has distinct effects on the in vitro differentiation of blastula cap explants. Cellular responses to Xwnt signals include changes in gap junctional permeability, altered responsiveness to growth factors, and possibly changes in cell adhesion. Current data suggest that a maternal Xwnt- or noggin-like activity is involved in the Nieuwkoop center activity during mesoderm induction, that Xwnt-8 participates in a pathway of differentiation as ventral mesoderm, and that Xwnt-5A is a potential modulator of morphogenetic movements.

Animals↗
PubMed1992

The high molecular weight Cat-301 chondroitin sulfate proteoglycan from brain is related to the large aggregating proteoglycan from cartilage, aggrecan.

Monoclonal antibodies Cat-301 and Cat-304 recognize a neuronal cell surface-associated chondroitin sulfate proteoglycan (CSPG), which is expressed during critical periods of postnatal development in the mammalian central nervous system (CNS). In the present study we show that the CNS CSPG identified by Cat-301/304 is similar to aggrecan, the high molecular weight CSPG from cartilage. By Western blot analysis, cartilaginous tissues, which are rich sources of aggrecan, have a high concentration of a high molecular weight CSPG which is immunoreactive with Cat-301 and 304. The Cat-301 and 304 epitopes, however, are partially masked by chondroitin sulfate glycosamino-glycan and are unmasked by digestion of the antigen with chondroitinase ABC. Although the antigen from both cartilage and CNS can be purified by CsCl buoyant density gradient centrifugation, a standard technique for purifying aggrecan, most of the antigen from the CNS has a lower buoyant density than that of cartilage. This may be due, in part, to the paucity of keratan sulfate substitution on the CNS antigen compared with that of the cartilage antigen. Both the CNS and cartilage antigens bind to hyaluronic acid, a feature characteristic of aggrecan. The physiochemical, biochemical, and functional properties of the Cat-301/304 antigen from cartilage are identical to aggrecan. The CNS antigen is similar, but not identical, to the cartilage antigen, and may thus represent another member of the family of high molecular weight CSPGs which bind to and aggregate with hyaluronic acid.

Aggrecans↗
PubMed1991

Identification of a calcium-dependent calmodulin-binding domain in Xenopus membrane skeleton protein 4.1.

Xenopus membrane skeleton protein 4.1 is expressed constitutively during embryonic development and accumulates to high levels within the retina during normal morphogenesis. There exists a high degree of amino acid identity between Xenopus protein 4.1 and human protein 4.1, suggesting that the mechanisms known to modulate the function(s) of human protein 4.1 may also serve to regulate Xenopus protein 4.1. Calmodulin (CaM) is one regulatory protein known to affect membrane-cytoskeletal interactions. An in vitro binding assay was used to test the ability of Xenopus protein 4.1 to bind CaM. Two independent approaches, involving protein 4.1 synthesized in vitro from synthetic RNA or a partial length protein 4.1 fusion protein expressed in Escherichia coli, demonstrate calcium-dependent, CaM binding. Both approaches demonstrate that the CaM-binding site is within the amino-terminal region of Xenopus protein 4.1. Results of this calmodulin binding activity suggest a possible regulatory mechanism by which calcium and calmodulin may affect the function of protein 4.1 during development.

Amino Acid Sequence↗
PubMed1990

Membrane skeleton protein 4.1 in developing Xenopus: expression in postmitotic cells of the retina.

Membrane skeleton protein 4.1 plays a key role in modulating the interactions of spectrin, actin, and integral membrane proteins in erythroid and nonerythroid cells. We have investigated its structure and expression during embryonic development of Xenopus laevis. An analysis of the complete 2758-nucleotide sequence and predicted translation of 801 amino acids (85.5 kDa) of X. laevis oocyte protein 4.1 reveals that, within overlapping regions, oocyte protein 4.1 is 74% identical to a composite amino acid sequence of human erythroid and lymphoid protein 4.1 and has an identity similar to that of amino acid motifs variably expressed in either human erythroid or lymphoid protein 4.1 S1 nuclease protection analysis demonstrates the presence of a single species of protein 4.1 transcript in embryos. Antibodies produced against X. laevis protein 4.1 fusion protein recognize two bands of 180 and 115 kDa on Western blots of X. laevis embryos and retina and, using immunocytochemical techniques, label the developing retina most intensely. In vitro transcription of a cDNA construct fully encoding X. laevis protein 4.1 yields a synthetic mRNA which, when translated in vitro, produces a polypeptide that comigrates on SDS-polyacrylamide gels with the 115-kDa form of embryos and retina. Protein 4.1 is found exclusively in photoreceptors following the terminal mitosis of retinal neurons. When retinal synaptogenesis is complete, protein 4.1 is also expressed in the inner retina. In adult amphibian retinas, protein 4.1 is detected in photoreceptors, bipolar cells, and ganglion cell axons. As these cell types have previously been shown to express spectrin, actin, and ankyrin, it is likely that the membrane skeleton of erythrocytes and retinal cells share functional similarities.

Amino Acid Sequence↗
PubMed1988

Reduction studies on bacterial recombinant somatomedin C/insulin-like growth factor-1.

N-Met-Somatomedin C/insulin-like growth factor-1 (rSmC) had been produced in recombinant Escherichia coli in monomeric form. The intact rSmC peptide is initially synthesized in E. coli cells in denatured form as inclusion bodies. The rSmC peptide in these inclusion bodies was found in reduced form. Isolation of this rSmC peptide was accomplished by separation and dissolution of the inclusion bodies, with dissociation of non-covalently aggregated species. The reduced rSmC was converted to a metastable state, termed un-refolded rSmC. Further processing of this rSmC generated two other isomers, termed refolded rSmC. The transitions of the peptide among these different states, reduced rSmC, un-refolded rSmC, and refolded rSmC can be readily monitored by reversed-phase high-performance liquid chromatography. By reduction and re-oxidation of the purified individual isomers we found that they are likely to be related to each other as conformation isomers which appear to be stabilized by disulfide bonds.

Animals↗
PubMed1986

Maternal ethanol consumption: binding of L-glutamate to synaptic membranes from whole brain, cortices, and cerebella of offspring.

We examined the influence of chronic maternal ethanol consumption on the Na+- and Ca2+-independent binding of L-glutamate to synaptic plasma membranes from whole brain as well as from cortices and cerebella of developing offspring. The maximum specific binding (Bmax) of L-glutamate to the Na+- and Ca2+-independent binding sites in synaptic plasma membranes of brain peaked at 17 days of age in the offspring of both control and ethanol-fed rats, although at that age there were significantly fewer binding sites in the brains of the offspring of ethanol-fed rats. The regional localization of this deficit is not now known. However, it appears that one major glutamatergic region (the cortex) does not reflect the transient deficiency of L-glutamate sites in brain. In fact, the concentration of L-glutamate binding sites in cortical synaptic plasma membranes was significantly increased in the 20-day-old offspring of ethanol-fed rats. In contrast to the cortex, binding to cerebellar synaptic plasma membranes was comparable in 20-day-old offspring of control and ethanol-fed rats. Despite transient alterations in the concentrations of L-glutamate binding sites in brain and synaptic plasma membranes, the affinity of the sites for L-glutamate (Kd) was consistently normal in the 14- to 26-day-old offspring of ethanol-fed rats.

Alcohol Drinking↗
PubMed1985

Gangliosides in axolemmal and synaptic membrane fractions from developing rats: effects of maternal ethanol consumption on offspring.

Previous work from this laboratory has shown that in utero exposure to ethanol significantly alters the synthesis of glycoproteins in synaptic, axolemmal, and myelin membranes from developing rats. In an attempt to determine whether in utero exposure to ethanol similarly alters the synthesis of other glycoconjugates involved with cell-cell interactions, the present study examined the influence of chronic maternal ethanol consumption prior to parturition on the content and synthesis of gangliosides in axolemmal and synaptic plasma membranes from developing rats. The results demonstrate that, in contrast to central nervous system glycoproteins, synaptic and axolemmal glycolipids are minimally affected by in utero exposure to ethanol. At all ages examined (17 to 34 days of age), the offspring of control and ethanol-treated rats had a comparable distribution of radiolabel among synaptic and axolemmal gangliosides, a normal concentration of ganglioside sialic acid in synaptic plasma membranes, and a near-normal distribution of sialic acid among synaptic gangliosides. The present study provides evidence which indicates that the radiolabeling patterns of axolemmal and synaptic membrane gangliosides are similar. Specifically, the most heavily labeled synaptic and axolemmal gangliosides were GT1b (20-37% of the total radioactivity) and GD1a (20-32%). A smaller proportion of radioactivity was associated with GD1b (approximately 11-16%), GM1 (5-10%), and GQ1b (4-11%), as well as with GD3 and the other monosialogangliosides (less than 5%). During the age period examined the proportion of radioactive GT1b increased in both membrane fractions.

Animals↗
PubMed1981

Effects of [Na+], [Cl-], carbonic anhydrase, and intracellular pH on corneal endothelial bicarbonate transport.

Sodium removal from the solution bathing the isolated rabbit corneal endothelium caused a reduction in both unidirectional and net flux of bicarbonate, whereas chloride withdrawal from the solution had no effect on the net bicarbonate flux but increased the unidirectional fluxes. These data correlate with previously published data on the effects of similar solution manipulations on both fluid movement and potential difference across the endothelium and strongly implicate bicarbonate as the primary ion involved in the maintenance of corneal hydration. Carbonic anhydrase (1 mg/ml) added to the solution bathing both sides of the endothelium markedly increased unidirectional and net bicarbonate fluxes, possibly by maintaining a high bicarbonate/CO2 concentration close to the membrane and thereby eliminating chemical gradients in the unstirred layer adjacent to the membrane. Determinations of intracellular pH with the 5,5-dimethyl-2,4-oxazolidine-dione method indicate that at more acid ambient conditions there is a lesser gradient between cell and bathing medium for H+; similar ambient conditions in other experiments resulted in larger unidirectional bicarbonate fluxes than at neutral pH. The data are suggestive of a nonvectorial H+-HCO3- exchange occurring across the endothelial cellular membranes.

Animals↗
PubMedDate not supplied

Maternal ethanol consumption: effect on (Na+-K+)-ATPase in rat offspring.

The activity of Mg2+-activated, ouabain-sensitive adenosine triphosphatase, (Na+-K+)-ATPase, was determined in homogenates of hypothalamus, cortex, cerebellum, and brain stem from the 19-day-old offspring of rats that were pair-fed control or (6.6%, v/v) ethanol liquid diets on a chronic basis prior to parturition. In the offspring of both control and ethanol-fed rats the specific activity of (Na+-K+)-ATPase was significantly (p less than 0.01) greater in the cortex than it was in the hypothalamus, brain stem or cerebellum (hypothalamus approximately brain stem approximately cerebellum). When the offspring of ethanol-fed and control rats were compared we observed no significant (p greater than 0.05) differences in the activity of (Na+-K+)-ATPase in any of the four brain regions examined. In addition, the results of kinetic analyses of cortical (Na+-K+)-ATPase were similar in the 19-day-old offspring of ethanol-fed rats and those whose mothers consumed either the control liquid diet or standard laboratory chow. The results of these studies suggest that the activity of the plasma membrane enzyme, (Na+-K+)-ATPase, was not affected in the 19-day-old offspring of ethanol-fed rats.

Adenosine Triphosphate↗