Biomedical subjects
Publications and source records attributed to C Brennan.
Mutant analysis in the zebrafish is revealing the genes that are expressed in the early neuroepithelium and that regulate factors responsible for the guidance of commissural axons. We review work on the developing zebrafish brain illustrating the way in which territories of regulatory gene expression influence the formation and positioning of axon pathways.
No-isthmus (Noi) is a member of the zebrafish Pax family of transcriptional regulators that is expressed in restricted domains of the developing CNS. In the developing eye and optic nerve, the Noi+ cells are primitive glial cells that line the choroid fissure and optic stalk/nerve to its junction with the optic tract. This pattern of Noi expression is retained in the adult, defining the optic nerve astroglia, which wrap the left and right nerves separately at the midline, thus forming the bodily crossed optic chiasm found in fish. In embryos carrying mutations in the noi gene, the choroid fissure fails to close, glial cells of the optic nerve fail to differentiate and optic axons exhibit abnormal trajectories exiting the eye and at the midline of the diencephalon. Optic axons select inappropriate pathways into the contralateral optic nerve, rostrally towards the anterior commissure and along the ipsilateral optic tract. Noi+ cells also border the pathway of axons in the postoptic commissure, which is located adjacent to the optic chiasm. These postoptic commissural axons are defasciculated and also exhibit pathfinding defects in noi- embryos. These results indicate that Noi is required in cells that line the pathways taken by optic and non-optic commissural axons for guidance across the midline of the diencephalon. We find that expression of two members of the Netrin family of axon guidance molecules and the signalling protein Sonic hedgehog is disturbed in noi- embryos, whereas several members of the Eph family of receptors and ligands show no obvious alterations in expression at the diencephalic midline.
The isolation and characterisation of two zebrafish Eph receptor ligand cDNAs which we have called zfEphL3 and zfEphL4 is described. These genes are expressed in the presumptive midbrain of developing embryos from 6 somites. By 24 hours L3 is expressed throughout the midbrain including the region of the presumptive tectum whereas L4 is strongly expressed in the midbrain caudal to the presumptive tectum. At later stages of development L3 is expressed in a graded fashion throughout the tectum and L4 is maintained at its posterior margin. Growth cone collapse and pathway selection assays demonstrate that both these proteins have a collapse activity for retinal ganglion cells. When faced with a choice of substrate on which to grow, temporal axons from chick retinal ganglion cells selectively avoided membranes from Cos cells transfected with L3, whereas nasal axons did not. Both temporal and nasal axons avoided membranes from Cos cells transfected with L4. The expression patterns together with the functional data suggest that although both ligands may be able to guide retinal ganglion cells axons in vitro, they have different roles in the guidance of retinotectal projections in vivo. The expression of L3 is consistent with a role in the guidance of retinal ganglion cells to their targets on the tectum whereas that of L4 suggests a role in delineating the posterior boundary of the optic tectum.
This study examines the activity of two key enzymes of reverse cholesterol transport, cholesterol ester transfer protein (CETP) and lecithin:cholesterol acyl transferase (LCAT) in 21 patients with non-insulin dependent diabetes mellitus (NIDDM) and 21 control subjects. Serum CETP was assessed by measuring plasma-mediated cholesteryl ester transfer between pooled exogenous lipoprotein with endogenous LCAT inhibited--an estimate of CETP mass. CETP activity was determined as cholesteryl ester transfer in the presence of the patients' lipoproteins and LCAT (endogenous assay). LCAT activity was determined in the same assay. There was no significant difference in CETP mass between the diabetic and non-diabetic subjects and there was no correlation between CETP mass and LCAT activity. Using the endogenous lipoprotein assay, CETP was elevated in serum from diabetic patients compared to control subjects (10.05 +/- 1.89 vs. 5.50 +/- 0.53 nmol/ml/h P < 0.05). LCAT was also increased in the diabetic patients (53.63 +/- 4.70 vs. 41.22 +/- 3.40 nmol/ml/h P < 0.05). Serum free cholesterol from diabetic and control subjects correlated with CETP activity measured using endogenous lipoprotein assay (r = 0.77, P < 0.001 and r = 0.82, P < 0.001), and also with LCAT activity (r = 0.76, P < 0.01 and r = 0.79, P < 0.01). There was a negative correlation between CETP activity with the endogenous lipoprotein assay and serum high density lipoprotein (HDL) cholesterol in the diabetic patients (r = -0.38, P < 0.01), but not in control subjects. In a subgroup of 10 control subjects, there was a positive correlation between LCAT activity and apolipoprotein (apo) A-I (r = 0.49, P < 0.05) and apo A-II (r = 0.51, P < 0.05) and also between CETP activity (endogenous assay) and apo A-I (r = 0.87, P = 0.001) and apo A-II (r = 0.63, P < 0.05). No relationship was observed between CETP activity and apo A-I or apo A-II in the diabetic subjects. Thus, serum CETP mass was normal in Type 2 diabetes but CETP activity (endogenous assay) was increased and was related to free cholesterol levels and LCAT activity in both diabetic and non-diabetic subjects.
Perinatal exposure of the developing brain to gonadal steroids during a limited critical period induces permanent, organizational differences in neural structures between male and female animals. These differences are believed to underlie the manifestation of sexually dimorphic behaviors. Gamma-aminobutyric acid type A (GABAA) receptors expressed in the ventromedial nucleus (VMN) of the hypothalamus appear to be a key component underlying the expression of sexually dimorphic, and GABAA-mediated transmission within the VMN is critical for the expression of sexual behavior in female, but not male, rats. Here we report that analysis of VMN neurons from neonatal rats revealed significant sex-specific differences in GABAA receptor channel properties. Specifically, GABAA-mediated currents elicited by direct agonist application decayed more rapidly in VMN neurons from females than from males. Kinetic differences became more pronounced during the first 2 weeks of postnatal development. Analysis of small, spontaneous inhibitory postsynaptic currents recorded in intact slices indicated a trend towards slower responses in neurons from males than females, but the differences in decay kinetics were not significant. Sex-specific differences in GABAA receptor kinetics may arise from activation of receptors not receiving synaptic contacts in the slice preparation or may become apparent at intact synapses under conditions of increased activity and evoked release.
Here we present the refined crystal structures of three different conformational states of the Asp153-->Gly mutant (D153G) of alkaline phosphatase (AP), a metalloenzyme from Escherichia coli. The apo state is induced in the crystal over a 3 month period by metal depletion of the holoenzyme crystals. Subsequently, the metals are reintroduced in the crystalline state in a time-dependent reversible manner without physically damaging the crystals. Two structural intermediates of the holo form based on data from a 2 week (intermediate I) and a 2 month soak (intermediate II) of the apo crystals with Mg2+ and Zn2+ have been identified. The three-dimensional crystal structures of the apo (R = 18.1%), intermediate I (R = 19.5%), and intermediate II (R = 19.9%) of the D153G enzyme have been refined and the corresponding structures analyzed and compared. Large conformational changes that extend from the mutant active site to surface loops, located 20 A away, are observed in the apo structure with respect to the holo structure. The structure of intermediate I shows the recovery of the entire enzyme to an almost native-like conformation, with the exception of residues Asp 51 and Asp 369 in the active site and the surface loop (406-410) which remains partially disordered. In the three-dimensional structure of intermediate II, both Asp 51 and Asp 369 are essentially in a native-like conformation, but the main chain of residues 406-408 within the loop is still not fully ordered. The D153G mutant protein exhibits weak, reversible, time dependent metal binding in solution and in the crystalline state.(ABSTRACT TRUNCATED AT 250 WORDS)
Specific forelimb muscles in anurans are sexually dimorphic and underlie the androgen-dependent clasping response of males during amplexus. Previous studies have reported that androgen treatment slows the contractile properties of these sexually dimorphic forelimb muscles. In amphibians, the expression of functionally distinct acetylcholine (ACh) receptors, the levels of acetylcholinesterase (AChE), the extent of multiple innervation, and the structure of individual end plates vary with the contractile properties of the muscle fibers. In higher vertebrates, androgens have been reported to alter the expression of ACh receptors, AChE, and the neuromodulator, calcitonin gene-related peptide (CGRP). To determine whether the known androgen-dependent changes in contraction of androgen-sensitive forelimb muscles are accompanied by concomitant changes in synaptic structure or function, we have compared functional neuromuscular transmission, the pattern of innervation, and CGRP immunoreactivity in nerve or muscle preparations from castrated (C) and castrated and testosterone-treated (CT) adult male Xenopus laevis. CGRP expression in androgen receptor (AR)-immunopositive neurons was increased in CT animals. However, no significant differences were found in ACh-mediated single channel or macroscopic currents, the extent of multiple end plates, or end plate morphology for forelimb fibers isolated from C and CT Xenopus. In contrast, analysis of forelimb fibers from gonadally intact adult females and juvenile animals of both sexes revealed that macroscopic synaptic currents were significantly shorter in these animals than in either C or CT adult males. Our data suggest that forelimb fibers in sexually dimorphic muscles of Xenopus do show significant differences in synaptic transmission; however, neither end-plate organization nor functional neuromuscular transmission are subject to activational effects of androgens in adult male frogs.
An important component of neuronal differentiation is the tightly controlled expression of a spectrum of ion channel proteins. Ion channels play a critical role in the generation and propagation of action potentials as well as in the cellular response to neurotransmitters, and thus are central in the transfer and integration of information in the nervous system. A model system amenable to analysis of ion channel expression and neuronal differentiation is the rat pheochromocytoma (PC12) cell line. Here, we have used electrophysiological and molecular biological approaches to analyze the expression of voltage-dependent sodium (Na) channels and nicotinic acetylcholine receptors (nAChR) in mutagenized variants (nnr cells) of the PC12 cell line. Our data reveal striking differences in the expression of these channels when compared to wild-type PC12 cells. Even in the absence of nerve growth factor (NGF), nnr cells express functional Na channels and Na channel mRNA at levels exceeding those in wild-type PC12 cells differentiated with NGF. In contrast, acetylcholine-induced currents were evident in only a small proportion of cells, presumably due to the altered pattern of expression of mRNAs encoding individual nAChR subunits. The altered ion channel expression in these variants provides an avenue for analyzing Na channel and nAChR channel function, as well as for identifying mechanisms governing their expression.
Explore the source record for details and available documents.
The substitution of aspartate at position 153 in Escherichia coli alkaline phosphatase by glycine results in a mutant enzyme with 5-fold higher catalytic activity (kcat) but no change in Km at pH 8.0 in 50 mM Tris-HCl. The increased kcat is achieved by a faster release of the phosphate product as a result of the lower phosphate affinity. The mutation also affects Mg2+ binding, resulting in an enzyme with lower metal affinity. The 3-D X-ray structure of the D153G mutant has been refined at 2.5 A to a crystallographic R-factor of 16.2%. An analysis of this structure has revealed that the decreased phosphate affinity is caused by an apparent increase in flexibility of the guanidinium side chain of Arg166 involved in phosphate binding. The mutation of Asp153 to Gly also affects the position of the water ligands of Mg2+, and the loop Gln152-Thr155 is shifted by 0.3 A away from the active site. The weaker Mg2+ binding of the mutant compared with the wild type is caused by an altered coordination sphere in the proximity of the Mg2+ ion, and also by the loss of an electrostatic interaction (Mg2+.COO-Asp153) in the mutant. Its ligands W454 and W455 and hydroxyl of Thr155, involved in the octahedral coordination of the Mg2+ ion, are further apart in the mutant compared with the wild type.
A 32-kDa protein (HLC-32) was purified from sea urchin embryos and characterized. In indirect immunofluorescence analysis this species was found to be distributed throughout the cytoplasm of the unfertilized egg but within 1 hr following fertilization was localized to the hyaline layer with some residual immunofluorescence remaining within the cytoplasm of the single cell embryo. By the early blastula stage the antigen was also detected in the basal lamina, indicating a bidirectional transport mode for this protein. A cDNA clone was isolated from a lambda Zap expression library. Sequence analysis revealed a protein rich in acidic (12.4 mole%) and basic (14.3 mole%) residues. Comparative sequence analysis revealed 41 and 47% amino acid sequence homology with two butanol-extractable proteins previously isolated from blastula stage embryos of the sea urchin Paracentrotus lividus. DNA gel blot analysis indicated that the gene encoding HLC-32 belonged to a small multigenic family, while in RNA gel blot analyses a 1.4-kB transcript was detected in ovaries but not gut, testes, mature eggs, or various stage embryos. At the early blastula stage of development this protein underwent a post-translational modification reaction which resulted in the loss of a 4-amino-acid peptide fragment from the amino-terminal end of this species.
An epitope from the HIV-1 gp120 protein V3 loop has been inserted onto the surface of bacterial alkaline phosphatase at different positions in the vicinity of the enzyme active site, creating hybrid proteins that can bind to an anti-gp120 monoclonal antibody. One of the hybrid proteins, API1, has a 13 amino acid V3 loop sequence inserted between residues 407 and 408 of alkaline phosphatase. The enzymatic activity of this protein is modulated upon antibody binding. API1 maintains the full activity of the wild type alkaline phosphatase but in the presence of the anti-gp120 antibody, the enzyme activity is inhibited by 40-50%. Thus, the hybrid enzyme can be used to detect the presence of antibody in solution. The concept of signalling proteins may have a wide application. Two models for the mechanism of modulation, steric hindrance and allosteric regulation, are discussed.
Neuromuscular transmission in dystrophic mice has been extensively studied through analysis of nerve-evoked endplate potentials. In the present study, single channel ACh-induced activity recorded from endplates of fast twitch muscle fibers from mdx and dy dystrophic mice, was compared with activity recorded from wild-type fiber endplates to ascertain whether expression of these phenotypes leads to changes in ACh receptor properties and synaptic transmission. An 89 pS class of ACh-induced events predominated in recordings from wild-type and both strains of dystrophic mice. The mean open times for this class of events was well described by two exponential components, one with a voltage-independent time constant of approximately 0.3 ms and the other with a time constant of 2-5 ms which increased e-fold with approximately 120 mV of hyperpolarization. The expression of the mdx or the dy phenotype was not associated with significant differences in the conductance, distribution of open durations, or the voltage-dependence of the mean open time for this class of ACh-induced events.
The current study was undertaken to determine prospectively the risk of cerebral thromboembolism and the prognostic significance of left ventricular thrombus in ambulatory patients with chronic congestive heart failure. A total of 264 ambulatory patients (mean age 62 years, mean left ventricular ejection fraction 27%) were followed prospectively for 24 +/- 9 months to determine the incidence of nonhemorrhagic stroke, transient ischemic attack, and mortality. Two-dimensional echocardiographic studies, performed for clinical indications other than previous systemic thromboembolism in 109 patients, were analyzed to relate the presence of left ventricular thrombus to subsequent outcome. Nine cerebral thromboembolic events occurred in 264 patients during the two-year mean follow-up period, yielding a rate of 1.7 thromboembolic events per 100 patient-years of follow-up. Known risk factors for stroke (hypertension, diabetes mellitus, and/or atrial fibrillation) were present in all nine patients with cerebral thromboembolic events. The 109 patients with echocardiographic studies had more severe heart failure than patients without echocardiographic studies (functional class 2.6 vs 2.1, p < 0.01), greater risk of a thromboembolic event (2.4 vs 1.4 events/100 patient-years of follow-up, p < 0.01), and higher mortality (21.3 vs 5.5 deaths/100 patient-years, p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenal-derived cells in culture slowly upregulate numbers of putative calcium channel proteins in response to inhibitory drugs including ethanol, benzodiazepines and morphine. This appears to be an adaptive response to cell inhibition involving second messengers and protein phosphorylation and culminating in alterations in expression of the calcium channel gene (Fig. 1). These increases in calcium channels lead to rebound increases in cell excitability when the depressant drugs are withdrawn (a characteristic feature of chemical dependence on depressant drugs) so that this change in numbers of calcium channels may provide a model for one mechanism of chemical dependence. Similar findings have been reported in the brains of animals made dependent on depressant drugs, and genetic and pharmacological manipulations of dependence and withdrawal further support a role for calcium channels in these phenomena.
The protein hyalin, a major component of the sea urchin extraembryonic hyaline layer, was previously shown to undergo a Ca(2+)-induced self-association into large aggregates (gelation). This reaction represented a major step in assembly of the layer. In the experiments reported here, digestion with trypsin resulted in a rapid dissociation of hyalin into a mixture of peptides which retained the capacity to bind Ca2+. However, unlike intact hyalin, none of these peptides associated into large aggregates (gelation) in the presence of Ca2+, Mg2+, and NaCl. Loss of the ability to undergo gelation was not accompanied by any significant change in the content of acidic plus amide amino acid residues. Decreasing the pH to 5.6 resulted in a loss of 25% of hyalin's Ca(2+)-binding capacity but had no effect on the ability of the protein to undergo gelation. Peptide fragments were only partially effective at inhibiting hyalin gelation. Clearly, not all the Ca(2+)-binding sites were required for hyalin gelation and Ca2+ binding alone was insufficient to drive this reaction. In addition, hyalin appeared to possess two classes of protein-protein interaction domains, one of which was essential for gelation.
The nicotinic acetylcholine (ACh) receptor is an integral membrane protein which mediates synaptic transmission at the skeletal neuromuscular junction. A key event in the development of the neuromuscular junction is the formation of high density aggregates of ACh receptors in the postsynaptic membrane. Receptor clustering has been attributed, in part, to their association with a peripheral membrane protein of Mr 43,000 (43K protein). We have addressed whether the association of the 43K protein can alter the single channel properties of the ACh receptor, and thus influence neuromuscular transmission at developing synapses, by expressing ACh receptors with and without the 43K protein in heterologous expression systems. We found that coexpression of the 43K protein with the receptor did not significantly alter either its single channel conductance or its mean channel open time. This was true in oocytes and also in COS cells where it was possible to localize 43K-induced clusters by fluorescence microscopy and to record from those clustered receptors. These data are in agreement with previous single channel studies which have shown that the properties of diffusely distributed and clustered receptors in native muscle cells from both mice and Xenopus do not differ.
Four hundred and sixty-five college women were evaluated to determine if specific variables of social and sexual behavior correlated with the presence of human papillomavirus (HPV) DNA in the genital tract, and if these associations differed between women who were HPV DNA positive, HPV DNA positive/clinically (cytologically) negative, or who reported previous HPV-related disease by a history of an abnormal Papanicolaou (Pap) smear or genital warts. HPV positive women had more sexual partners in the recent past, more sexual episodes per month, and used spermacides less commonly than controls. Similarly, self-reporters were more likely to have more lifetime sexual partners and earlier age of onset for sexual activity. Cytologically negative HPV positive women were distinguished only by more sexual episodes per month and sexual partners in the past year (borderline significance). Alcohol use was significantly more frequent in all groups, underscoring this variable as a risk factor for both HPV DNA positivity and related disease in young women. Potential explanations for differences between women with clinically and non-clinically related HPV positivity are discussed, with emphasis on the need for followup studies to determine if an epidemiologically distinct subset of HPV DNA positive but clinically negative women are at risk for subsequent cervical disease.