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Biomedical subjects

S A Lloyd

Publications and source records attributed to S A Lloyd.

At least 19 recordsLinked to original sources
PubMed2005

Adult and in utero exposure to cocaine alters sensitivity to the Parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Cocaine abuse is a significant problem in the United States, including its use by approximately 1% of pregnant women. Cocaine acts as an indirect agonist of dopamine at the dopamine transporter, resulting in the presence of excess dopamine in the synapse. Since synaptic dopamine can rapidly oxidize to form free radicals, it was hypothesized that exposure to this drug might produce damage in dopaminergic systems such as the substantia nigra pars compacta, damage to which is a hallmark of Parkinson's disease. To test this hypothesis we exposed mice both in utero and as adults to cocaine and examined its effects on the nigrostriatal system. We found that exposure to cocaine both in utero or as adults did not affect substantia nigra cell number, but did make these neurons more susceptible to the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. We also found long-lasting changes in D2 receptor mRNA levels as well as changes in the monoamine transport system and several growth factors. This work suggests that use of cocaine might be a predisposing factor for development of Parkinson's disease in both adults exposed chronically as well as in individuals exposed prenatally.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2001

Targeting exported substrates to the Yersinia TTSS: different functions for different signals?

Many Gram-negative pathogens utilize a type III secretion system (TTSS) to inject toxins into the cytosol of eukaryotic cells. Previous studies have indicated that exported substrates are targeted to the Yersinia TTSS via the coding regions of their 5' mRNA sequences, as well as by their cognate chaperones. However, recent results from our laboratory have challenged the role of mRNA targeting signals, as we have shown that the amino termini of exported substrates are crucial for type III secretion. Here, we discuss the nature of these amino-terminal secretion signals and propose a model for the secretion of exported substrates by amino-terminal and chaperone-mediated signals. In addition, we discuss the roles of chaperones as regulators of virulence gene expression and present models suggesting that such regulation can occur independently of the delivery of their substrates to the secretion apparatus.

Bacterial Outer Membrane Proteins↗
PubMed2001

Yersinia YopE is targeted for type III secretion by N-terminal, not mRNA, signals.

Pathogenic Yersinia species inject virulence proteins, known as Yops, into the cytosol of eukaryotic cells. The injection of Yops is mediated via a type III secretion system. Previous studies have suggested that YopE is targeted for secretion by two signals. One is mediated by its cognate chaperone YerA, whereas the other consists of either the 5' end of yopE mRNA or the N-terminus of YopE. In order to characterize the YopE N-terminal/5' mRNA secretion signal, the first 11 codons of yopE were systematically mutagenized. Frameshift mutations, which completely alter the amino acid sequence of residues 2-11 but leave the mRNA sequence essentially intact, drastically reduce the secretion of YopE in a yerA mutant. In contrast, a mutation that alters the yopE mRNA sequence, while leaving the amino acid sequence of YopE unchanged, does not impair the secretion of YopE. Therefore, the N-terminus of YopE, and not the 5' end of yopE mRNA, serves as a targeting signal for type III secretion. In addition, the chaperone YerA can target YopE for type III secretion in the absence of a functional N-terminal signal. Mutational analysis of the YopE N-terminus revealed that a synthetic amphipathic sequence of eight residues is sufficient to serve as a targeting signal. YopE is also secreted rapidly upon a shift to secretion-permissive conditions. This 'rapid secretion' of YopE does not require de novo protein synthesis and is dependent upon YerA. Furthermore, this burst of YopE secretion can induce a cytotoxic response in infected HeLa cells.

5' Untranslated Regions↗
PubMed2001

The type III secretion chaperone LcrH co-operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis.

The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens secrete and subsequently translocate antihost effector proteins into target eukaryotic cells by a common type III secretion system (TTSS). In this process, YopD (Yersinia outer protein D) is essential to establish regulatory control of Yop synthesis and the ensuing translocation process. YopD function depends upon the non-secreted TTSS chaperone LcrH (low-calcium response H), which is required for presecretory stabilization of YopD. However, as a new role for TTSS chaperones in virulence gene regulation has been proposed recently, we undertook a detailed analysis of LcrH. A lcrH null mutant constitutively produced Yops, even when this strain was engineered to produce wild-type levels of YopD. Furthermore, the YopD-LcrH interaction was necessary to regain the negative regulation of virulence associated genes yops). This finding was used to investigate the biological significance of several LcrH mutants with varied YopD binding potential. Mutated LcrH alleles were introduced in trans into a lcrH null mutant to assess their impact on yop regulation and the subsequent translocation of YopE, a Rho-GTPase activating protein, across the plasma membrane of eukaryotic cells. Two mutants, LcrHK20E, E30G, I31V, M99V, D136G and LcrHE30G lost all regulatory control, even though YopD binding and secretion and the subsequent translocation of YopE was indistinguishable from wild type. Moreover, these regulatory deficient mutants showed a reduced ability to bind YscY in the two-hybrid assay. Collectively, these findings confirm that LcrH plays an active role in yop regulation that might be mediated via an interaction with the Ysc secretion apparatus. This chaperone-substrate interaction presents an innovative means to establish a regulatory hierarchy in Yersinia infections. It also raises the question as to whether or not LcrH is a true chaperone involved in stabilization and secretion of YopD or a regulatory protein responsible for co-ordinating synthesis of Yersinia virulence determinants. We suggest that LcrH can exhibit both of these activities.

Animals↗
PubMed2001

Development of memory for pattern and path: further evidence for the fractionation of visuo-spatial memory.

Evidence from a number of sources now suggests that the visuo-spatial sketchpad (VSSP) of working memory may be composed of two subsystems: one for maintaining visual information and the other for spatial information. In this paper we present three experiments that examine this fractionation using a developmental approach. In Experiment 1, 5-, 8-, and 10-year old children were presented with a visuo-spatial working memory task (the matrices task) with two presentation formats (static and dynamic). A developmental dissociation in performance was found for the static and dynamic conditions of both tasks, suggesting that the activation of separable subsystems of the VSSP is dependent upon a static/dynamic distinction in information content rather than a visual/spatial one. A highly similar pattern of performance was found for a mazes task with static and dynamic formats. However, one strategic activity, the use of simple verbal recoding, may also have been responsible for the observed pattern of performance in the matrices task. In Experiments 2 and 3 this was investigated using concurrent articulatory suppression. No evidence to support this notion was found, and it is therefore proposed that static and dynamic visuo-spatial information is maintained in working memory by separable subcomponents of the VSSP.

Child↗
PubMed1999

Structure of the C-terminal domain of FliG, a component of the rotor in the bacterial flagellar motor.

Many motile species of bacteria are propelled by flagella, which are rigid helical filaments turned by rotary motors in the cell membrane. The motors are powered by the transmembrane gradient of protons or sodium ions. Although bacterial flagella contain many proteins, only three-MotA, MotB and FliG-participate closely in torque generation. MotA and MotB are ion-conducting membrane proteins that form the stator of the motor. FliG is a component of the rotor, present in about 25 copies per flagellum. It is composed of an amino-terminal domain that functions in flagellar assembly and a carboxy-terminal domain (FliG-C) that functions specifically in motor rotation. Here we report the crystal structure of FliG-C from the hyperthermophilic eubacterium Thermotoga maritima. Charged residues that are important for function, and which interact with the stator protein MotA, cluster along a prominent ridge on FliG-C. On the basis of the disposition of these residues, we present a hypothesis for the orientation of FliG-C domains in the flagellar motor, and propose a structural model for the part of the rotor that interacts with the stator.

Amino Acid Sequence↗
PubMed1998

Electrostatic interactions between rotor and stator in the bacterial flagellar motor.

Bacterial flagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the flagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor-stator interface.

Bacterial Physiological Phenomena↗
PubMed1998

Function of protonatable residues in the flagellar motor of Escherichia coli: a critical role for Asp 32 of MotB.

Rotation of the bacterial flagellar motor is powered by a transmembrane gradient of protons or, in some species, sodium ions. The molecular mechanism of coupling between ion flow and motor rotation is not understood. The proteins most closely involved in motor rotation are MotA, MotB, and FliG. MotA and MotB are transmembrane proteins that function in transmembrane proton conduction and that are believed to form the stator. FliG is a soluble protein located on the cytoplasmic face of the rotor. Two other proteins, FliM and FliN, are known to bind to FliG and have also been suggested to be involved to some extent in torque generation. Proton (or sodium)-binding sites in the motor are likely to be important to its function and might be formed from the side chains of acidic residues. To investigate the role of acidic residues in the function of the flagellar motor, we mutated each of the conserved acidic residues in the five proteins that have been suggested to be involved in torque generation and measured the effects on motility. None of the conserved acidic residues of MotA, FliG, FliM, or FliN proved essential for torque generation. An acidic residue at position 32 of MotB did prove essential. Of 15 different substitutions studied at this position, only the conservative-replacement D32E mutant retained any function. Previous studies, together with additional data presented here, indicate that the proteins involved in motor rotation do not contain any conserved basic residues that are critical for motor rotation per se. We propose that Asp 32 of MotB functions as a proton-binding site in the bacterial flagellar motor and that no other conserved, protonatable residues function in this capacity.

Aspartic Acid↗
PubMed1997

Charged residues of the rotor protein FliG essential for torque generation in the flagellar motor of Escherichia coli.

The FliG protein of Escherichia coli is essential for assembly and function of the flagellar motor. Certain mutations in FliG give a non-motile, or Mot-, phenotype, in which flagella are assembled but do not rotate. Mutations with this property are clustered in a C-terminal segment of FliG that is stable when expressed alone, and thus probably constitutes an independently folded domain. Previously, we suggested that this domain forms the rotor portion of the active site for torque generation in the motor. In this work, we have used a mutational approach to identify the amino acid residues in the C-terminal domain of FliG that are most important for motor function. Site-directed mutagenesis was used to replace each of the conserved residues in this domain with alanine, and the effects on motor function were measured. Because charged residues have often been suggested to have important roles in torque generation, conserved charged residues were changed individually and in all pairwise combinations. The results show that three charged residues of FliG, Arg279, Asp286 and Asp287, are directly involved in torque generation. Mutations in these residues cause motility defects that suggest that they function jointly, in an active site whose most important property is a specific arrangement of charges. Two other charged residues, Lys262 and Arg295, may also be involved in torque generation, but are less critical than Arg279, Asp286 or Asp287. Unchanged residues of the FliG motility domain do not appear to have direct roles in torque generation, although some are needed for the stability of the protein or for normal clockwise/ counter-clockwise switching. The Mot- mutations of fliG isolated previously by random mutagenesis do not alter the putative active-site residues, but render the proteins abnormally susceptible to proteolysis, suggesting significantly altered conformations or reduced stabilities.

Bacterial Proteins↗
PubMed1996

Torque generation in the flagellar motor of Escherichia coli: evidence of a direct role for FliG but not for FliM or FliN.

Among the many proteins needed for assembly and function of bacterial flagella, FliG, FliM, and FliN have attracted special attention because mutant phenotypes suggest that they are needed not only for flagellar assembly but also for torque generation and for controlling the direction of motor rotation. A role for these proteins in torque generation is suggested by the existence of mutations in each of them that produce the Mot- (or paralyzed) phenotype, in which flagella are assembled and appear normal but do not rotate. The presumption is that Mot- defects cause paralysis by specifically disrupting functions essential for torque generation, while preserving the features of a protein needed for flagellar assembly. Here, we present evidence that the reported mot mutations in fliM and fliN do not disrupt torque-generating functions specifically but, instead, affect the incorporation of proteins into the flagellum. The fliM and fliN mutants are immotile at normal expression levels but become motile when the mutant proteins and/or other, evidently interacting flagellar proteins are overexpressed. In contrast, many of the reported fliG mot mutations abolish motility at all expression levels, while permitting flagellar assembly, and thus appear to disrupt torque generation specifically. These mutations are clustered in a segment of about 100 residues at the carboxyl terminus of FliG. A slightly larger carboxyl-terminal segment of 126 residues accumulates in the cells when expressed alone and thus probably constitutes a stable, independently folded domain. We suggest that the carboxyl-terminal domain of FliG functions specifically in torque generation, forming the rotor portion of the site of energy transduction in the flagellar motor.

Bacterial Proteins↗
PubMed1994

Glucocorticoid receptor and interleukin-1 receptor messenger RNA expression in corneal cells.

Interleukin-1 receptor and glucocorticoid receptor messenger ribonucleic acid (RNA) sequences coding for the corresponding proteins were detected in corneal epithelium, stromal fibroblast, and endothelial cells using the polymerase chain reaction and hot blotting. Identification of interleukin-1 receptor mRNA in each of the three major cell types of the cornea suggests that interleukin-1 alpha has autocrine and/or paracrine roles in the cornea, since previous studies have found that interleukin-1 alpha mRNA is produced in corneal epithelial, stromal fibroblast, and endothelial cells. Further investigation is needed to determine the functions regulated by the interleukin-1 receptor and glucocorticoid receptor in the cornea and the role of each in corneal wound healing.

Adolescent↗
PubMed1993

Fibroblast growth factor-1 receptor messenger RNA expression in corneal cells.

The polymerase chain reaction (PCR) and hot-blotting methods were used to identify fibroblast growth factor (FGF) receptor-1-specific messenger ribonucleic acid (mRNA) sequences in cDNA samples prepared from human corneal endothelial cell cultures with proliferative and senescent morphology, an ex vivo corneal epithelium sample, two primary corneal epithelial cell cultures, two third-passage corneal epithelial cell cultures, and two stromal fibroblast cultures. The PCR primers used in this study distinguished mRNAs coding for three aminoterminal motifs (alpha, beta, and gamma) of the FGF receptor-1 that are derived by alternative splicing from a single genomic sequence. Messenger RNA molecules coding for FGF receptor-1 amino-terminal motif were detected in corneal endothelial and epithelial cells. The alpha and beta amino-terminal motif, but not the gamma amino-terminal motif, mRNAs were detected in stromal fibroblasts. The gamma motif lacks a known signal sequence for membrane translocation and is thought to represent an intracellular form of the FGF receptor-1. Identification of mRNA coding for FGF receptor-1 along with the previous identification of basic FGF mRNA and protein in corneal endothelial, epithelial, and stromal fibroblast cells suggests an autocrine and/or paracrine role for basic FGF in the physiology of the cornea.

Base Sequence↗
PubMed1993

Fibroblast growth factor receptor-1, interleukin-1 receptor, and glucocorticoid receptor messenger RNA production in the human lacrimal gland.

PURPOSE: To determine whether messenger RNA coding for fibroblast growth factor receptor-1, interleukin-1 receptor, and glucocorticoid receptor messenger RNA are produced in human lacrimal tissue. METHODS: Total cellular RNA was isolated from three specimens of normal human lacrimal tissue and complementary DNA was synthesized. The polymerase chain reaction and sequence-specific primers were used to amplify the sequences of interest from the complementary DNA. Hot blotting and sequence-specific probes were used to demonstrate that the expected amplification products were specific. RESULTS: Data demonstrated that fibroblast growth factor receptor-1, interleukin-1 receptor, and glucocorticoid receptor messenger RNA are produced in human lacrimal tissue. CONCLUSIONS: These results and the previous identification of basic fibroblast growth factor in the lacrimal gland suggest that basic fibroblast growth factor has autocrine or paracrine functions in lacrimal tissue. More study is needed to determine whether the corresponding proteins are produced and, if so, what functions are regulated by fibroblast growth factor receptor-1, interleukin-1 receptor, and glucocorticoid receptor in the lacrimal gland.

Base Sequence↗
PubMed1993

Extended life of human corneal endothelial cells transfected with the SV40 large T antigen.

PURPOSE: To transfect human corneal endothelial cells with a plasmid vector coding for the SV40 large T antigen to extend the life of the cells in culture. METHODS: Human corneal endothelial cells were transfected with the SV40 large T antigen-coding plasmid pSV3neo using the electroporation method. Transfected and control cells were propagated in culture until senescence. Polymerase chain reaction and immunofluorescence were used to demonstrate messenger RNA and protein, respectively, for the Simian virus 40 large T antigen in the transfected cells. Polymerase chain reaction and hot blotting were used to demonstrate messenger RNA coding for several growth factors and receptors in transfected and control cells. RESULTS: The transfected cells continued to proliferate to 38 passages (more than 120 population doublings) in culture (control cells, 8 population doublings). Transfected cells, but not control cells, expressed messenger RNA coding for the Simian virus 40 large T antigen. Similarly, immunofluorescent staining with monoclonal antibodies demonstrated that the Simian virus 40 large T antigen protein was present in the nucleus of the transfected cells. Transfected cells were shown to produce messenger RNA coding for epidermal growth factor, epidermal growth factor receptor, basic fibroblast growth factor, fibroblast growth factor receptor-1, interleukin-1 alpha, the interleukin-1 receptor, transforming growth factor beta-1, and the glucocorticoid receptor. Qualitative expression of the messenger RNA coding for each of these modulators was similar in proliferating primary corneal endothelial cells and proliferating or confluent transfected corneal endothelial cells. CONCLUSIONS: In culture, the life of human corneal endothelial cells transfected with a plasmid vector coding for the Simian virus 40 large T antigen is extended. This study suggests that human corneal endothelial cells have the capacity for extensive proliferation, but the proliferation of untransfected cells is regulated through mechanisms that have not yet been characterized.

Adult↗
PubMed1992

Two-dimensional gel electrophoretic comparison of endothelial cell-Descemet's membrane proteins in Fuchs' dystrophy and normal corneas.

Two-dimensional polyacrylamide gel electrophoresis was used to compare the proteins isolated from the combined corneal endothelial cell-Descemet's membrane complex of three pairs of corneas with Fuchs' dystrophy with three pairs of normal corneas. Normal or Fuchs' dystrophy endothelium and Descemet's membrane was documented by pathologic analysis of each cornea. Proteins were separated by isoelectric point in the first dimension and molecular weight in the second dimension. Over 300 proteins were resolved from each sample, and similar patterns were noted in both groups. No consistent differences were detected between the corneas with Fuchs' dystrophy and the normal corneas. Allelic variations of some proteins were detected in both groups.

Cornea↗
PubMed1992

EGF, EGF receptor, basic FGF, TGF beta-1, and IL-1 alpha mRNA in human corneal epithelial cells and stromal fibroblasts.

We sought to determine whether human corneal epithelial cells and stromal fibroblasts synthesize messenger RNAs (mRNA) coding for epidermal growth factor (EGF), EGF receptor, basic fibroblast growth factor (basic FGF), transforming growth factor-beta 1 (TGF-beta 1), and interleukin-1 alpha (IL-1 alpha). Total cellular RNA was extracted from cultured stromal fibroblasts and ex vivo and cultured corneal epithelial cells. Oligo dt-primed complementary DNA (cDNA) was synthesized from each RNA sample. The polymerase chain reaction (PCR) was used to amplify sequences for EGF, EGF receptor, basic FGF, TGF-beta 1, IL-1 alpha, and beta actin from cDNA samples from each cell type. Southern blots of the PCR products were probed with oligonucleotides complementary to internal sequences within each of the amplified products. The amplification products were shown to be specific. For each modulator, the amplification product of the expected size was identified with at least one specific, alternative amplification product. The alternative splicing products suggest that there may be alternative mRNA splicing for each of the modulators studied. Differences were noted in the IL-1 alpha specific amplification products in stromal fibroblasts compared to corneal epithelial cells. EGF and EGF receptor mRNA production in human corneal epithelial cells and stromal fibroblasts suggest an autocrine role for EGF in the physiology of each of these cell types.

Adolescent↗
PubMed1992

EGF, basic FGF, and TGF beta-1 messenger RNA production in rabbit corneal epithelial cells.

The polymerase chain reaction (PCR) was used to demonstrate that rabbit corneal epithelial cells produce messenger RNAs coding for epidermal growth factor (EGF), basic fibroblast growth factor (FGFb), and transforming growth factor beta-1 (TGF beta 1) ex vivo and in primary culture. EGF, FGFb, and TGF beta 1 mRNAs were detected in central and peripheral ex vivo epithelial tissue in wounded and unwounded rabbit corneas. Southern blots of the PCR products were probed with oligonucleotides to demonstrate that the appropriately sized amplification products were specific. These results suggest that corneal epithelial cells produce growth factors that may have autocrine or paracrine effects on epithelial cells, and possibly other cells of the cornea. The functions, if any, performed by these growth factors in corneal epithelial wound healing are yet to be elucidated.

Animals↗