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Biomedical subjects

C L Harris

Publications and source records attributed to C L Harris.

At least 19 recordsLinked to original sources
PubMed2001

PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER.

Glycosylphosphatidylinositol (GPI) acts as a membrane anchor of many cell surface proteins. Its structure and biosynthetic pathway are generally conserved among eukaryotic organisms, with a number of differences. In particular, mammalian and protozoan mannosyltransferases needed for addition of the first mannose (GPI-MT-I) have different substrate specificities and are targets of species- specific inhibitors of GPI biosynthesis. GPI-MT-I, however, has not been molecularly characterized. Characterization of GPI-MT-I would also help to clarify the topology of GPI biosynthesis. Here, we report a human cell line defective in GPI-MT-I and the gene responsible, PIG-M. PIG-M encodes a new type of mannosyltransferase of 423 amino acids, bearing multiple transmembrane domains. PIG-M has a functionally important DXD motif, a characteristic of many glycosyltransferases, within a domain facing the lumen of the endoplasmic reticulum (ER), indicating that transfer of the first mannose to GPI occurs on the lumenal side of the ER membrane.

Amino Acid Sequence↗
PubMed2001

Enhancement of complement-mediated lysis by a peptide derived from SCR 13 of complement factor H.

Complement factor H (fH) is an important regulator of complement activation. It contributes to protection of cells against homologous complement attack. In this study we tested the effect of fH-depletion of normal human serum (NHS) on lysis of antibody-coated sheep and human erythrocytes (EshA and EhuA). In the absence of fH, lysis of sensitised Esh and Ehu was clearly increased. Addition of fH to fH-depleted serum re-established protection of cells against complement similar to that seen with NHS. A fH-derived peptide (pepAred), covering the N-terminal half of SCR 13 in fH, was able to enhance complement-mediated lysis of EhuA significantly. However, the oxidised form of this peptide (pepAox) had no effect. Biotinylated pepAred, but not pepAox, was able to directly bind to cells. Additionally, pepAred competed with direct fH-cell interaction which was observable only after treatment of purified fH with mercaptoethanol. Only pepAred increased the amount of C3 fragments and reduced levels of fH detectable on cells as shown by FACS analysis and radio-immuno assay. Furthermore, fH and factor I (fI)-mediated cleavage of agarose bound C3b into iC3b was decreased in the presence of pepAred. These data indicate that a fH-derived peptide can enhance complement-mediated lysis. We will continue to investigate whether the use of a fH peptide can be exploited for therapeutical purposes.

Amino Acid Sequence↗
PubMed2001

Illusory words created by repetition blindness: a technique for probing sublexical representations.

When two orthographically similar words are displayed using rapid serial visual presentation (RSVP), the repeated letters in the second critical word (W2) are not detected, leading to a deficit in reporting this word, a phenomenon known as repetition blindness (RB). The unrepeated letters in W2 do appear to be detected and available to feed activation to words compatible with them (Morris & Harris, 1999). When a fragment was strategically placed in the RSVP stream, as in GROW throw ank, observers reported seeing thank more often than in the control condition BEAT throw ank. Illusory words were facilitated by repetition blindness only when the recombining letters maintained their position in the words. Illusory word report was insensitive to the phonological similarity of the recombining letters; equal quantities of illusory words were created by sequences like china CHEAT THR (-->threat) and swung SWEAT THR (-->threat). In addition to being an interesting phenomenon in its own right, the illusory words paradigm may have considerable use as a tool for probing the perceptual units underlying visual word recognition.

Adult↗
PubMed2001

Are individual or consecutive letters the unit affected by repetition blindness?

When 2 similar words (e.g., react reach) are briefly sequentially displayed, the 2nd word may be omitted from the report, a phenomenon known as repetition blindness (RB). Previous researchers have suggested that consecutive letters are the unit affected by RB. Six experiments provided new data on orthographic RB. Two letters at the beginning or end of words resulted in RB, as did alternating interior letters (tactile earthly) and 3 letters with different relative positions (arid bird). However, no RB was found with a single final letter (show view). Observed RB may reflect pattern completion because RB for pairs like throat theory was reduced when the nonrepeated letters (eory) were consistent with only a single word. The experiments point to a model of orthographic RB in which both individual letters and letter sequences of length 2 or more play a role.

Adult↗
PubMed2000

Neurocognitive function in antisocial personality disorder.

Recent neuroimaging studies and neuropsychological test findings support the contention that prefrontal dysfunction is associated with psychopathic personality traits and antisocial behavior. However, conflicting results have arisen regarding performance on measures of frontal executive function. We administered a neuropsychological test battery consisting of measures sensitive to frontal lobe dysfunction and a battery of personality questionnaires and clinical scales sensitive to antisocial personality disorder (APD) subjects presenting with prominent psychopathic personality features and matched control subjects. We also monitored the subjects' electrodermal activity during the presentation of emotionally charged stimuli. APD subjects showed greater neuropsychological deficits on measures sensitive to orbitofrontal dysfunction in comparison to control participants. Moreover, APD subjects were electrodermally hyporesponsive to aversive stimuli relative to control group members. APD subjects did not demonstrate performance deficits on classical tests of frontal executive function. Participants also underwent clinical assessment. As expected, APD subjects were less conscientious, self-reproaching, guilt-prone, and socially anxious than matched control subjects. Moreover, the scores indicated that APD subjects were more venturesome and uninhibited relative to control subjects. Contrary to expectations, APD subjects and community control subjects did not differ on a self-report measure of sensitivity to specific phobic situations.

Antisocial Personality Disorder↗
PubMed2000

Pigs express multiple forms of decay-accelerating factor (CD55), all of which contain only three short consensus repeats.

We report the cloning of cDNAs encoding multiple isoforms of the pig analogue of human decay-accelerating factor (DAF; CD55). Screening of a pig muscle cDNA library using a human DAF probe identified a single clone that encoded a DAF-like molecule comprising three short consensus repeats (SCR) homologous with the amino-terminal three SCR in human DAF, a serine/threonine-rich (ST) region, and sequence compatible with a transmembrane domain and cytoplasmic tail. Northern blot and RT-PCR analysis showed that pig DAF was expressed in a wide range of tissues. Additional isoforms of DAF were sought using RT-PCR and 3'-rapid amplification of cDNA ends followed by sequencing. Isoforms containing a GPI anchor and with differing lengths of ST region were identified; no isoform containing a fourth SCR was found. Cloning of the GPI-anchored isoform from granulocytes confirmed that it was identical with the original transmembrane isoform through the three SCR and first portion of ST and was derived from a frame shift caused by splicing out 176 bp of sequence. A panel of mAbs was generated and used to analyze the distribution and anchoring of pig DAF in circulating cells. Pig DAF was expressed on all circulating cells and was transmembrane anchored on erythrocytes, but completely or partially GPI anchored on granulocytes and mononuclear cells. The transmembrane isoform of pig DAF was expressed on Chinese hamster ovary cells and was shown to affect regulatory activity for the classical pathway of human complement, but was only marginally active against pig serum.

Amino Acid Sequence↗
PubMed2000

Human and rodent decay-accelerating factors (CD55) are not species restricted in their complement-inhibiting activities.

Homologous complement activation is restricted on cells by the complement regulators, decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. These proteins act in concert with other membrane structures to protect cells from homologous complement attack. In contrast, cells are usually sensitive to heterologous complement attack. It has been suggested that species-specific restriction of complement activation can be attributed to the inability of regulators to inhibit across species. We have investigated the capacities of human, rat and mouse analogues of DAF to regulate homologous and heterologous complement. Cells transfected with cDNA encoding these analogues were protected from heterologous complement attack. C3b-deposition experiments indicated that whilst cells were best protected by DAF from the same species, all three analogues inhibited human, rat and mouse complement. Comparable results were obtained in haemolysis assays using soluble, recombinant forms of the proteins. Inhibition of the classical pathway (CP) was best achieved with homologous DAF, although human DAF also inhibited rat complement, rat DAF also inhibited human complement and mouse DAF inhibited complement from all species. Human DAF was the best inhibitor of alternative pathway (AP)-mediated attack, inhibiting complement from all species. Mouse DAF inhibited mouse and rat AP, whilst rat DAF inhibited only rat AP. These data indicate that human and rodent analogues of DAF are not species restricted and highlights interesting differences in the capacity to regulate AP and CP. This has implications in broader fields of research, such as xenotransplantation, where cross-species regulation of complement is of paramount importance.

Animals↗
PubMed2000

Effect of subarachnoid morphine administration on extubation time after coronary artery bypass graft surgery.

OBJECTIVE: To determine the effects of 2 low doses of intrathecal morphine on extubation time and on postoperative analgesic requirements after coronary artery bypass graft (CABG) surgery. DESIGN: A prospective, randomized, double-blind, placebo-controlled study. SETTING: Tertiary-care university hospital. PARTICIPANTS: Fifty adult patients scheduled for elective primary CABG surgery. INTERVENTIONS: Patients were randomized to receive placebo, 250 microg, or 500 microg intrathecal morphine, preoperatively. Intraoperative fentanyl and midazolam were limited to 15 microg/kg and 20 microg/kg intravenously. Patients were extubated in the intensive care unit by a blinded observer using predefined extubation criteria. MEASUREMENTS AND MAIN RESULTS: Time to extubation and postoperative requirements for morphine, midazolam, nitroglycerin, and sodium nitroprusside were recorded by a blinded observer. Extubation times were 441 +/- 207 minutes versus 325 +/- 188 minutes versus 409 +/- 245 minutes for the placebo, 250-microg, and 500-microg groups (p = 0.27). Postoperative morphine requirements in the 250-microg and 500-microg groups were 13.6 +/- 7.8 mg and 11.7 +/- 7.4 mg, compared with 21.3 +/- 6.2 mg in the placebo group (p = 0.001). There were no differences among the study groups with regard to postoperative midazolam, nitroglycerin, and sodium nitroprusside requirements. CONCLUSIONS: Despite decreased postoperative morphine requirements, intrathecal morphine administration did not have a clinically relevant effect on extubation time after CABG surgery. This study suggests that 250 microg is the optimal dose of intrathecal morphine to provide significant postoperative analgesia without delaying tracheal extubation.

Adolescent↗
PubMed2000

Orthographic repetition blindness.

Repetition blindness (RB) is the failure to report the second occurrence of a repeated word, when words are sequentially and briefly displayed (Kanwisher, 1987). RB is also observed for non-identical words, such as home, dome. Explanations for non-identity RB assume that similarity at the level of the whole word causes the second word to be suppressed ("similarity inhibition"). Three experiments demonstrate that RB is robust for diverse types of orthographic relatedness, including critical words that share only their first initial letter, their last two letters, first three letters, middle three letters, beginning and final letters, three alternating letters, and three non-aligned letters (as in chance hand). The theoretical construct of similarity inhibition may be able to account for these data, although one mechanism previously proposed in the literature, neighbourhood inhibition, is probably not a useful way to explain the data on RB for words sharing only one or two letters. We introduce an alternative explanation for orthographic RB: Only the repeated letters are suppressed, and amount of RB depends on how easily the perceiver can reconstruct the target word from the non-suppressed letters.

Adult↗
PubMed1999

Molecular and functional analysis of mouse decay accelerating factor (CD55).

Molecular cloning of mouse decay accelerating factor (DAF; CD55) predicted two forms of the molecule, one transmembrane (TM) and the other glycosylphosphatidylinositol (GPI)-anchored; these are encoded by separate genes termed Daf-GPI and Daf-TM. In the present study several additional isoforms of mouse DAF, generated by alternative splicing from these genes, are described. Northern-blot analysis of RNA and reverse transcriptase-PCR from various tissues indicated that spleen and testis expressed high levels of DAF, which comprised several species. These species were cloned and sequence analysis revealed various novel forms in addition to those previously reported. Two novel forms were derived from the Daf-TM gene but the transmembrane sequence defined previously was replaced by a unique GPI-anchor addition sequence; one clone also had part of the serine/threonine/proline (STP) region deleted. A third clone, encoding a transmembrane protein, was also derived from this gene but the entire STP region was deleted. A fourth clone, derived from the Daf-GPI gene, contained a novel C-terminal sequence, suggestive of a secreted form of the protein. Two DAF cDNAs (TM and GPI-anchored) were stably expressed in Chinese hamster ovary cells. When these cells were attacked with mouse or rat complement and analysed for C3b deposition, DAF-transfected cells had greatly reduced C3b deposition compared with controls. Transfection with DAF also conferred protection from complement in a cell-lysis assay, and a soluble, recombinant form of mouse DAF inhibited complement in a haemolytic assay.

Alternative Splicing↗
PubMed1999

Evidence that intramolecular associations between presenilin domains are obligatory for endoproteolytic processing.

Mutations in genes encoding presenilins (PS1 and PS2) cosegregate with the majority of early onset cases of familial Alzheimer's disease. PS1 and PS2 are polytopic membrane proteins that undergo endoproteolytic cleavage to generate stable NH2- and COOH-terminal derivatives (NTF and CTF, respectively). Several lines of evidence suggest that the endoproteolytic derivatives are likely the functional units of PS in vivo. In the present report, we examine the disposition of PS NTF and CTF assemblies in stable mouse N2a neuroblastoma cell lines expressing human PS polypeptides. We show that exogenous expression of PS1 NTFs neither assemble with endogenous CTF nor exhibit dominant negative inhibitory effects on the endogenous PS1 cleavage and the accumulation of derivatives. In cells co-expressing PS1 and PS2, PS1- and PS2-derived fragments do not form mixed assemblies. In contrast, cells expressing a chimeric PS1/PS2 polypeptide form stable PS1 NTF-PS2 CTF assemblies. Moreover, expression of chimeric PS1/PS2 polypeptides harboring a familial early onset AD-linked mutation (M146L) elevates the production of Abeta42 peptides. Our results provide evidence that assembly of structural domains contained within NH2- and COOH-terminal regions of PS occur prior to endoproteolytic cleavage.

Alzheimer Disease↗
PubMed1999

Efficient generation of monoclonal antibodies against surface-expressed proteins by hyperexpression in rodent cells.

The generation of antibodies is one of the first requirements in the characterisation of a newly cloned protein. However, this requires expression and purification of the protein in sufficient yield and purity for immunisation of animals and screening of fusion wells. Even with the development of highly efficient protocols based upon incorporation of specific peptide tags, this can be a tedious and time-consuming process. In an effort to improve the speed and efficiency of obtaining antibodies reactive with newly cloned proteins we have developed an approach based upon the expression of the protein at high level in cell lines originating from the species to be used for immunisation. To illustrate this approach we describe the generation of antibodies against two recently cloned proteins that are normally expressed at the membrane, the rat and mouse analogues of human decay accelerating factor (DAF; CD55). However, the strategy is applicable not only to membrane proteins but also to other proteins which can be expressed on the cell membrane by incorporating at the carboxy terminus the signal sequence for glycosyl phosphatidylinositol (GPI) anchor addition derived from DAF or another GPI-anchored protein. The strategy also permits rapid and efficient screening using flow cytometry on expressing cells.

3T3 Cells↗
PubMed1999

A sublexical locus for repetition blindness: evidence from illusory words.

Six experiments used an illusory words paradigm to demonstrate that repetition blindness (RB) in orthographically similar words affects only the words' shared letters. Rapid serial visual presentation streams of words and word fragments allowed the unique letters of the 2nd critical word to combine with a subsequent fragment to create a word, as in rock shock ell. The illusory word shell was reported 2-3 times as frequently in RB conditions as in control conditions. Further experiments ruled out letter migration, contour summation, and differences in processing load as explanations for the results. These findings are inconsistent with current proposals that orthographic RB represents similarity inhibition or lexical competition or that it reflects problems with word-level token individuation.

Humans↗
PubMed1999

Posttraumatic obsessive-compulsive disorder: a three-factor model.

This paper presents a three-factor causal model of obsessive-compulsive disorder (OCD), which posits that exposure to long-term traumatic stress generates an inordinate degree of anxiety during the psychological development of the premorbid OCD child. In response to these conditions the child evolves a distinct cognitive style characterized by exaggerated threat appraisal and magical beliefs, and experiences alterations in brain metabolism. An entire functional brain system (a basal ganglia-orbitofrontal circuit) enters into a state of enhanced responsiveness following exposure to protracted threat. Over time the threshold for stimulation is dramatically lowered, resulting in a hypersensitivity to cues that signify potential harm. Individuals adapt to this hypersensitivity through a variety of strategies, which constitute OCD.

Adaptation, Psychological↗
PubMed1998

A bispecific monoclonal antibody directed against both the membrane-bound complement regulator CD55 and the renal tumor-associated antigen G250 enhances C3 deposition and tumor cell lysis by complement.

Tumor cells may inhibit the induction of a complement-mediated inflammatory response through overexpression of membrane-bound regulators of complement activation. Therefore, it is of interest to determine the most efficient approach to block these membrane-bound complement regulators on tumor cells. In the present study, we first generated a bispecific mAb directed against both CD55, using the functional blocking mAb MBC1, and the highly expressed HLA class I molecule as a model for a tumor-associated Ag, using the mAb W6/32. Tumor cells opsonized with bispecific mAb W6/32*MBC1, then exposed to complement and subsequently stained for C3 deposition, were assessed by flow cytometric analysis. We found that opsonization with W6/32*MBC1 resulted in a 92% enhancement of C3 deposition on renal tumor cells as compared with opsonization with W6/32 alone and a 17% enhancement of the C3 deposition as compared with incubation with a mixture of both parental mAb. Based on these results, we developed a bispecific mAb recognizing both CD55 and the relatively low expressed renal tumor-associated Ag G250. Increasing concentrations of the bispecific mAb G250*MBC1 resulted in a 25 to 400% increase in C3 deposition on renal tumor cells as compared with C3 deposition in the presence of the parental mAb G250 alone. G250*MBC1 enhanced C3 deposition by 21% in comparison with a mixture of both parentals. Furthermore, opsonization of tumor cells with G250*MBC1 rendered these cells more sensitive to complement-mediated lysis. In conclusion, the bispecific mAb G250*MBC1 induces deposition of C3 and tumor cell lysis more efficiently than G250 alone.

Antibodies, Bispecific↗
PubMed1998

Relative resistance of striatal neurons containing calbindin or parvalbumin to quinolinic acid-mediated excitotoxicity compared to other striatal neuron types.

To evaluate the relative ability of those striatal neuron types containing calbindin or parvalbumin to withstand a Ca(2+)-mediated excitotoxic insult, we injected the NMDA receptor-specific excitotoxin quinolinic acid (QA) into the striatum in mature adult rats and 2 months later examined the relative survival of striatal interneurons rich in parvalbumin and striatal projection neurons rich in calbindin. To provide standardization to the survival of striatal neuron types thought to be poor in Ca2+ buffering proteins, the survival was compared to that of somatostatin-neuropeptide Y (SS/NPY)-containing interneurons and enkephalinergic projection neurons, which are devoid of or relatively poorer in such proteins. The various neuron types were identified by immunohistochemical labeling for these type-specific markers and their relative survival was compared at each of a series of increasing distances from the injection center. In brief, we found that parvalbuminergic, calbindinergic, and enkephalinergic neurons all showed a generally comparable gradient of neuronal loss, except just outside the lesion center, where calbindin-rich neurons showed significantly enhanced survival. In contrast, striatal SS/NPY interneurons were more vulnerable to QA than any of these three other types. These observed patterns of survival following intrastriatal QA injection suggest that calbindin and parvalbumin content does not by itself determine the vulnerability of striatal neurons to QA-mediated excitotoxicity in mature adult rats. For example, parvalbuminergic striatal interneurons were not impervious to QA, while cholinergic striatal interneurons are highly resistant and SS/NPY+ striatal interneurons are highly vulnerable. Both cholinergic and SS/NPY+ interneurons are devoid of any known calcium buffering protein. Similarly, calbindin does not prevent striatal projection neuron vulnerability to QA excitotoxicity. Nonetheless, our data do suggest that calbindin may offer striatal neurons some protection against moderate excitotoxic insults, and this may explain the reportedly slightly greater vulnerability of striatal neurons that are poor in calbindin to ischemia and Huntington's disease.

Animals↗
PubMed1998

Stable association of presenilin derivatives and absence of presenilin interactions with APP.

Mutations in two related genes, presenilin 1 and 2 presenilin 2 (PS1 and PS2), cosegregate with Alzheimer's disease. PS1 and PS2 are highly homologous polytopic membrane proteins that are subject to endoproteolytic cleavage in vivo. The resulting N- and C-terminal derivatives are the preponderant PS-related species that accumulate in cultured cells and tissue. In earlier studies, we demonstrated that PS1 N- and C-terminal derivatives accumulate to 1:1 stoichiometry and that the absolute levels of fragments are established by a tightly regulated and saturable mechanism. These findings led to the suggestion that the levels of PS1 derivatives might be determined by their association with limiting cellular components. In this study, we use in situ chemical cross-linking and coimmunoprecipitation analyses to document that the N- and C-terminal derivatives of either PS1 or PS2 can be coisolated. Moreover, and in contrast to published reports which documented that PS1 and PS2 form stable heteromeric assemblies with the beta-amyloid precursor protein (APP), we have failed to provide evidence for physiological complexes between PS1 and PS2 holoproteins or their derivatives with APP.

Alzheimer Disease↗