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L E Kelly

Publications and source records attributed to L E Kelly.

At least 19 recordsLinked to original sources
PubMed2001

Drosophila stoned proteins regulate the rate and fidelity of synaptic vesicle internalization.

At an initial step during synaptic vesicle recycling, dynamin and adaptor proteins mediate the endocytosis of synaptic vesicle components from the plasma membrane. StonedA and stonedB, novel synaptic proteins encoded by a single Drosophila gene, have predicted structural similarities to adaptors and other proteins implicated in endocytosis. Here, we test possible roles of the stoned proteins in synaptic vesicle internalization via analyses of third instar larval neuromuscular synapses in two Drosophila stoned (stn) mutants, stn(ts) and stn(8P1). Both mutations reduce presynaptic levels of stonedA and stonedB, although stn(ts) has relatively weak effects. The mutations cause retention of synaptic vesicle proteins on the presynaptic plasma membrane but do not alter the levels or distribution of endocytosis proteins, dynamin, alpha-adaptin, and clathrin. In addition, stn(8P1) mutants exhibit depletion and enlargement of synaptic vesicles. To determine whether these defects arise from altered synaptic vesicle endocytosis or from defects in synaptic vesicle biogenesis, we implemented new methods to assess directly the efficiency of synaptic vesicle recycling and membrane internalization at Drosophila nerve terminals. Behavioral and electrophysiological analyses indicate that stn(ts), an allele with normal evoked release and synaptic vesicle number, enhances defects in synaptic vesicle recycling shown by Drosophila shi(ts) mutants. A dye uptake assay demonstrates that slow synaptic vesicle recycling in stn(ts) is accompanied by a reduced rate of synaptic vesicle internalization after exocytosis. These observations are consistent with a model in which stonedA and stonedB act to facilitate the internalization of synaptic vesicle components from the plasma membrane.

Animals↗
PubMed2000

The products of the Drosophila stoned locus interact with synaptic vesicles via synaptotagmin.

The stoned locus of Drosophila melanogaster encodes two novel proteins, stonedA (STNA) and stonedB (STNB), both of which are expressed in the nervous system. Flies with defects at the stoned locus have abnormal behavior and altered synaptic transmission. Genetic interactions, in particular with the shibire (dynamin) mutation, indicated a presynaptic function for stoned and suggested an involvement in vesicle cycling. Immunological studies revealed colocalization of the stoned proteins at the neuromuscular junction with the integral synaptic vesicle protein synaptotagmin (SYT). We show here that stoned interacts genetically with synaptotagmin to produce a lethal phenotype. The STNB protein is found by co-immunoprecipitation to be associated with synaptic vesicles, and glutathione S-transferase pull-downs demonstrate an in vitro interaction between the micro2-homology domain of STNB and the C2B domain of the SYTI isoform. The STNA protein is also found in association with vesicles, and it too exhibits an in vitro association with SYTI. However, we find that the bulk of STNA is in a nonmembranous fraction. By using the shibire mutant to block endocytosis, STNB is shown to be present on some synaptic vesicles before exocytosis. However, STNB is not associated with all synaptic vesicles. We hypothesize that STNB specifies a subset of synaptic vesicles with a role in the synaptic vesicle cycle that is yet to be determined.

Animals↗
PubMed1998

A product of the Drosophila stoned locus regulates neurotransmitter release.

The Drosophila stoned locus encodes two novel gene products termed stonedA and stonedB, which possess sequence motifs shared by proteins involved in intracellular vesicle traffic. A specific requirement for stoned in the synaptic vesicle cycle has been suggested by synthetic genetic interactions between stoned and shibire, a gene essential for synaptic vesicle recycling (Petrovich et al., 1993). A synaptic role of stoned gene products also is suggested by altered synaptic transients in electroretinograms recorded from stoned mutant eyes (Petrovich et al., 1993). We show here that the stonedA protein is highly enriched at Drosophila nerve terminals. Mutant alleles that affect stonedA disrupt the normal regulation of synaptic vesicle exocytosis at neuromuscular synapses of Drosophila. Spontaneous neurotransmitter release is enhanced dramatically, and evoked release is reduced substantially in such stoned mutants. Ultrastructural studies reveal no evidence of major disorganization at stoned mutant nerve terminals. Thus, our data indicate a direct role for stonedA in regulating synaptic vesicle exocytosis. However, genetic and morphological observations suggest additional, subtle effects of stoned mutations on synaptic vesicle recycling. Remarkably, almost all phenotypes of stoned mutants are similar to those previously described for mutants of synaptotagmin, a protein postulated to regulate both exocytosis and the recycling of synaptic vesicles. We propose a model in which stonedA functions together with synaptotagmin to regulate synaptic vesicle cycling.

Amino Acid Substitution↗
PubMed1998

Embryonic expression and activity of doughnut, a second RYK homolog in Drosophila.

In the Drosophila embryo, a subset of muscles require expression and function of the RYK subfamily RTK gene derailed (drl) for correct attachment. We have isolated a second RYK homolog, doughnut (dnt), from Drosophila. The DNT protein exhibits 60% amino acid identity to DRL, and is structurally as similar to the mammalian RYK proteins as is DRL, indicating an ancient duplication event. dnt is expressed in dynamic patterns in the embryonic epidermis, being found at high level in epithelia adjacent to cells that are invaginating into the interior of the embryo, including ventral furrow, cephalic furrow, fore- and hindgut, optic lobe and tracheal pits. dnt is capable of a partial rescue of the muscle attachment defect of drl-/- embryos, indicating that it encodes a receptor with a related and significantly overlapping biochemical function.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of two distinct calmodulin-binding sites in the Trpl ion-channel protein of Drosophila melanogaster.

Two putative light-sensitive ion channels have been isolated from Drosophila, encoded by the transient-receptor-potential (trp) and transient-receptor-potential-like (trpl) genes. The cDNA encoding the Trpl protein was initially isolated on the basis that the expressed protein binds calmodulin. Using both fusion proteins and a synthetic peptide, we now show that two calmodulin-binding sites are present in the C-terminal domain of the Trpl protein, CBS-1 and CBS-2. CBS-1 binds calmodulin in a Ca2+-dependent fashion, requiring Ca2+ concentrations above 0.3-0.5 microM for calmodulin binding. In contrast, CBS-2 binds the Ca2+-free form of calmodulin, with dissociation occurring at Ca2+ concentrations between 5 and 25 microM. Phosphorylation of a serine residue within a peptide encompassing CBS-1 by cyclic AMP-dependent protein kinase (PKA) abolishes calmodulin binding, and phosphorylation of the adjacent serine by protein kinase C appears to modulate this phosphorylation by PKA. Interpretation of these findings provides a novel model for ion-channel gating and modulation in response to changing levels of intracellular Ca2+.

Amino Acid Sequence↗
PubMed1996

The stoned locus of Drosophila melanogaster produces a dicistronic transcript and encodes two distinct polypeptides.

The stoned gene of Drosophila melanogaster is required for normal neuronal function in both adult and larva. We have identified DNA sequences that lie within a genetic region that is known to include the stoned gene and that also reveal restriction site variations in two stoned lethal mutants. This genomic region contains a single transcription unit coding for an approximately 8.4-kb transcript. The transcript is preferentially expressed in the head of adult flies. The isolation and sequencing of cDNA and genomic clones reveals that stoned appears to encode a dicistronic mRNA, although the possible existence of other forms of mRNA cannot be excluded. Antibody cross-reactivity shows that two proteins are translated from the stoned locus in vivo. Both open reading frames (ORFs) encode novel proteins. The protein encoded by the first ORF contains four tandemly repeated motifs, and one domain of the protein encoded by the second ORF shows similarity to a family of proteins (AP50s) associated with clathrin assembly protein complexes.

Amino Acid Sequence↗
PubMed1996

A study of role ambiguity and role strain among school nurses.

Two hundred fifty-one school nurses practicing in pennsylvania were surveyed regarding perceptions of their professional role. Findings demonstrated that role ambiguity and role strain occur when school nurses attempt to practice in a setting where two disciplines, education and nursing, may have different expectations regarding role performance.

Adult↗
PubMed1995

Adolescent mothers: what factors relate to level of preventive health care sought for their infants?

This exploratory study investigated the level of preventive health care services that adolescent mothers (ages 14 to 17) sought for their infants during the first 2 years of the infants' lives. Findings showed that mothers who maintained a relationship with their child's father or exhibited a high Powerful Others Locus of Control were more likely to practice better preventive health care. The role that social support and locus of control play in mediating parental stress is discussed. Implications for nurses working with adolescent mothers are presented.

Adolescent↗
PubMed1995

Effects of quadriceps femoris muscle strengthening on crouch gait in children with spastic diplegia.

BACKGROUND AND PURPOSE: Despite evidence of weakness in children with cerebral palsy, the use of strength training in this population remains controversial. SUBJECTS: Fourteen children with spastic diplegia, ranging in age from 6 to 14 years (X = 9.1, SD = 2.5), participated in a bilateral quadriceps femoris muscle strengthening program. METHODS: The children exercised three times per week for 6 weeks using ankle weights at loads of approximately 65% of each child's maximum isotonic force production. The maximal voluntary contraction of the quadriceps femoris muscles was measured before, at the midpoint of, and immediately following the exercise program at 30, 60, and 90 degrees of knee flexion. Gait analyses were performed before and after the strengthening program to determine whether quadriceps femoris muscle strengthening influenced gait. RESULTS: Children with spastic diplegia can increase quadriceps femoris muscle strength through heavy resistance exercise. Repeated-measures analysis of variance and multivariate analysis of variance procedures were used to assess changes in force and in gait variables. Improvement in the degree of crouch at initial floor contact at the freely selected speed and an increase in stride length at free and fast speeds were found. CONCLUSION AND DISCUSSION: These findings suggest that resistance exercise is an effective treatment strategy and as such should be considered as one component in the habilitation of children with cerebral palsy.

Adolescent↗
PubMed1995

The Age Group Evaluation and Description (AGED) Inventory: a new instrument for assessing stereotypes of and attitudes toward age groups.

The AGED Inventory was designed to overcome shortcomings identified in the Aging Semantic Differential, a frequently used measure of how age groups are perceived. The new instrument was developed to allow assessment both of age stereotypes and of attitudes toward age-specified targets. The Inventory was developed with data from 300 male and 300 female respondents. Two seven-item evaluative factors (the Goodness and Positiveness dimensions) resulted from a series of factor analyses used to reduce an initial set of thirty-five evaluative adjective pairs. Two seven-item descriptive factors (the Vitality and Maturity dimensions) resulted from a separate series of factor analyses used to reduce an initial set of fifty adjective pairs judged to differentiate age groups. Using confirmatory factor analyses and coefficients of congruence on data from an additional 800 respondents, the factor structures of the two evaluative factors and of the two descriptive factors were found to be replicable for young, middle-aged, and old targets assessed in either between or within subject designs. Its ease of administration, multidimensionality, flexibility of targets specification, and capability for assessing attitude and/or stereotype in a manner congruent with current conceptualizations of these constructs make the AGED Inventory useful in a variety of contexts.

Adolescent↗
PubMed1993

A genetic analysis of the stoned locus and its interaction with dunce, shibire and Suppressor of stoned variants of Drosophila melanogaster.

The genetic complementation patterns of both behavioral and lethal alleles at the stoned locus have been characterized. Mosaic analysis of a stoned lethal allele suggests that stoned functions either in the nervous system or in both the nervous system and musculature, but is not required for gross neural development. The behavioral alleles stnts and stnC, appear to be defective in a diametrically opposite sense, show interallelic complementation, and indicate distinct roles for the stoned gene product in the visual system and in motor coordination. A number of other neurological mutations have been investigated for their possible interaction with the viable stoned alleles. Mutations at two loci, dunce and shibire, act synergistically with the stnts mutations to cause lethality, but fail to interact with stnC. A third variant (Suppressor of stoned) has been identified which can suppress the debilitation associated with the stnts mutations. These data, together with a previously identified interaction between the stnts and tan mutants, indicate a central role for the stoned gene product in neuronal function, and suggests that the stoned gene product interacts, either directly or indirectly, with the neural cAMP second messenger system, with the synaptic membrane recycling pathway via dynamin, and with biogenic amine metabolism.

Alleles↗
PubMed1993

Cyromazine resistance in Drosophila melanogaster (Diptera: Drosophilidae) generated by ethyl methanesulfonate mutagenesis.

Flies resistant to cyromazine (CGA-72662) were selected in susceptible laboratory populations of Drosophila melanogaster (Meigen) treated with ethyl methane-sulfonate after growth on cyromazine concentrations > LC99. Two resistant lines were obtained. In each case, resistance was a result of a mutation in a single, but different, gene. The resistance genes, designated Rst(2)Cyr and Rst(3)Cyr, were localized to map positions 64 on chromosome II and 47 on chromosome III, respectively. Concentration-mortality analysis of each mutant revealed that both genes conferred a low level (< 5 times) of resistance to cyromazine. Rst(2)Cyr produced LC99s of 1.3 x 10(-4)% (wt/vol) for heterozygotes and 2.7 x 10(-4)% for homozygotes; Rst(3)Cyr values were 1.6 x 10(-4) and 1.8 x 10(-4)%, respectively. These values compare with an LC99 of 5 x 10(-5)% for wild-type. The role of D. melanogaster as a model for insecticide resistance studies is discussed, especially the comparison of laboratory-generated cyromazine resistance in D. melanogaster with field resistance in Musca domestica L.

Animals↗
PubMed1993

A neural gene from Drosophila melanogaster with homology to vertebrate and invertebrate glutamate decarboxylases.

Cross-species hybridization has been used to isolate a second Drosophila gene, with homology to a feline glutamate decarboxylase (Gad) cDNA. The gene differs in sequence, chromosomal location, and spatial expression from the previously reported Drosophila Gad gene, but both encode proteins of 58 kDa. The derived amino acid sequence reveals a typical pyridoxal phosphate binding site and sequence homology consistent with a glutamate decarboxylase function. The protein includes an amino-terminal polyasparagine sequence, and a beta-pleated sheet region, with regularly spaced glutamine and arginine residues, not found in other decarboxylases. Expression in the adult is limited to the neuropil of the first optic ganglion and to regions of the thoracic musculature that may correspond to the location of motor neuron axons. This is consistent with a glial localization for the transcript. There is no overlap with the reported expression of Drosophila Gad. Although the molecular evidence suggests that this gene encodes a pyridoxal phosphate-dependent decarboxylase, glutamate decarboxylase activity associated with this gene could not be demonstrated, and the in vivo substrate is unknown. It is possible that the protein encoded by this gene is novel, not only in sequence and spatial expression, but also in substrate specificity.

Amino Acid Sequence↗
PubMed1992

Identification of a Drosophila gene encoding a calmodulin-binding protein with homology to the trp phototransduction gene.

We have isolated a number of Drosophila cDNAs on the basis of their encoding calmodulin-binding proteins. A full-length cDNA clone corresponding to one of these genes has been cloned and sequenced. Conservation of amino acid sequence and tissue-specific expression are observed between this gene and the transient receptor potential (trp) gene. We propose the name transient receptor potential-like (trpl) to describe this newly isolated gene. The trpl protein contains two possible calmodulin-binding sites, six transmembrane regions, and a sequence homologous to an ankyrin-like repeat. Structurally, the trpl and trp proteins resemble cation channel proteins, particularly the brain isoform of the voltage-sensitive Ca2+ channel. The identification of a protein similar to the trp gene product, yet also able to bind Ca2+/calmodulin, allows for a reinterpretation of the phenotype of the trp mutations and suggests that both genes may encode light-sensitive ion channels.

Amino Acid Sequence↗
PubMed1991

Physical activity and attitudes in lean and obese children and their mothers.

Forty pairs of biologically related mothers and children were recruited to determine (1) if differences existed in physical activity levels of obese and nonobese children and (2) if their activity or attitude toward it was influenced by maternal activity and attitudes. Ultimately, the goal was to identify whether certain maternal factors (her adiposity, activity level, and/or attitude toward activity) related to the child's adipose level. Children were classified as obese or nonobese by skinfold thickness. Mothers and children completed either the ATPA or CATPA, attitude measurement instruments. Each wore a Caltrac activity monitor for two consecutive days. No significant difference was found between obese and nonobese boys' and girls' physical activity level nor their total attitude toward physical activity. However, significant gender differences were found for the vertigo and aesthetic dimensions. When children's adiposity and gender were considered as interacting factors, differences were also found for the attitude dimensions of health and fitness, and catharsis. There were no significant relationships between child and maternal activity level, attitude toward physical activity, and adipose level. Obese and nonobese children had similar levels of physical activity and attitudes toward activity, unrelated to the maternal factors measured. Although attitudes were positive, physical activity levels for both groups were rather low -- and not likely to help in weight control efforts.

Child↗
PubMed1990

Sequence analysis, and chromosomal localization of a gene encoding a cystatin-like protein from Drosophila melanogaster.

Using polyclonal antibodies raised against a Drosophila Ca2(+)-binding protein (DCABP-23), clones were isolated from a Drosophila head cDNA library constructed in the expression vector lambda gt11. Two non-homologous clones have been isolated and are being subjected to sequence analysis. One of these clones, though not encoding DCABP-23, does encode a Drosophila cystatin-like protein. This presumed Drosophila cystatin shows homology to mammalian cystatins, chicken egg white cystatin and the rice oryzacystatin. The Drosophila cystatin has been mapped, by in situ hybridization, to region 88C on the right arm of the third chromosome.

Amino Acid Sequence↗
PubMed1990

Purification and properties of a 23 kDa Ca2(+)-binding protein from Drosophila melanogaster.

Recent reports have shown that there exists in mammalian brain a number of heat-stable Ca2(+)-binding proteins that are distinct from calmodulin [McDonald & Walsh (1985) Biochem. J. 232, 559-567]. We have attempted to characterize equivalent Ca2(+)-binding proteins from Drosophila. Affigel-phenothiazine chromatography, which can be used to purify calmodulin and other Ca2(+)-binding proteins, allowed the identification of a possible heat-stable 23 kDa Ca2(+)-binding protein. A purification procedure for this protein has been devised. Purified 23 kDa protein shows characteristics typical of a Ca2(+)-binding protein; there is a mobility shift on SDS/polyacrylamide gels in the presence of EGTA, and Western blotting, followed by the use of the 45Ca2+ overlay technique, confirms that the 23 kDa protein does bind Ca2+. 45Ca2+ binding studies indicate that this protein binds 1 mol of Ca2+/mol of protein, with Kd 1.9 microM. A single band with pI 5.2 is obtained on isoelectric focusing. Analysis of Western blots of Drosophila tissues probed with antibodies to the Ca2(+)-binding protein indicates that it has a widespread distribution, but is absent from muscle tissue. The antibodies also cross-react with a protein of identical molecular mass in extracts of sheep brain. The possible similarity between this Drosophila Ca2(+)-binding protein and mammalian proteins is discussed, and comparison is made between this Drosophila protein and other Ca2(+)-binding proteins purified from vertebrates.

Amino Acids↗