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Biomedical subjects

C L Harris

Publications and source records attributed to C L Harris.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1998

A randomized controlled trial evaluating nutrition counseling with or without oral supplementation in malnourished HIV-infected patients.

OBJECTIVE: To evaluate the effects of nutrition counseling with or without oral supplementation in malnourished patients infected with the human immunodeficiency virus (HIV). DESIGN: Randomized controlled trial. SUBJECTS: HIV-infected men (n=118) who were less than 90% of usual weight for height or who had lost more than 10% of body weight. INTERVENTION: Nutrition counseling alone (control group) vs nutrition counseling plus enteral supplementation (supplement group) for 6 weeks. All patients were instructed to consume a diet that exceeded estimated total energy expenditure by 960 kcal/day. MAIN OUTCOME MEASURES: Weight, skinfold thickness, fat-free mass, grip strength, quality of life, and cognitive function (Buschke test). STATISTICAL ANALYSES: Differences in baseline variables and outcomes were evaluated using analysis of variance or the Wilcoxon rank sum test. RESULTS: Ninety-nine men completed at least 4 weeks of treatment, 49 in the supplement group and 50 in the control group. Half the patients in each treatment group achieved at least 80% of their energy target. No differences in weight, skinfold thickness measurements, or quality of life were observed. Compared with the control group, the supplement group had larger increases in fat-free mass and grip strength, although the differences did not reach statistical significance. APPLICATIONS: In the short term, nutrition counseling with or without oral supplementation can achieve a substantial increase in energy intake in about 50% of malnourished HIV-infected patients. Although further study is needed to evaluate long-term effects, these findings suggest that nutrition counseling has an important role in the management of malnourished HIV-infected patients.

Adult↗
PubMed1997

Evidence that levels of presenilins (PS1 and PS2) are coordinately regulated by competition for limiting cellular factors.

Mutations in two related genes, PS1 and PS2, account for the majority of early onset cases of familial Alzheimer's disease. PS1 and PS2 are homologous polytopic membrane proteins that are processed endoproteolytically into two fragments in vivo. In the present report we examine the fate of endogenous PS1 and PS2 after overexpression of human PS1 or PS2 in mouse N2a neuroblastoma cell lines and human PS1 in transgenic mice. Remarkably, in N2a cell lines and in brains of transgenic mice expressing human PS1, accumulation of human PS1 derivatives is accompanied by a compensatory, and highly selective, decrease in the steady-state levels of murine PS1 and PS2 derivatives. Similarly, the levels of murine PS1 derivatives are diminished in cultured cells overexpressing human PS2. To define the minimal sequence requirements for "replacement" we expressed familial Alzheimer's disease-linked and experimental deletion variants of PS1. These studies revealed that compromised accumulation of murine PS1 and PS2 derivatives resulting from overexpression of human PS1 occurs in a manner independent of endoproteolytic cleavage. Our results are consistent with a model in which the abundance of PS1 and PS2 fragments is regulated coordinately by competition for limiting cellular factor(s).

Animals↗
PubMed1997

Processing of presenilin 1 in brains of patients with Alzheimer's disease and controls.

Mutations in the presenilin genes are involved in the aetiology of the majority of familial early-onset Alzheimer disease (AD) cases. Presenilin 1 (PS1) is proteolytically processed in vivo, resulting in the accumulation of N-terminal approximately 27-28 kDa and C-terminal approximately 18 kDa derivatives. To examine the metabolism of PS1 in brains of patients with AD harbouring PS1 mutations I143T and G384A, we performed immunoblot analyses of brain homogenates using well characterized antibodies. We document that approximately 27-28 kDa N-terminal and approximately 18 kDa C-terminal PS1 proteolytic fragments accumulate in brain of these individuals, and that in large part the accumulation pattern is indistinguishable from that observed in brains from individuals with sporadic AD or controls. Notably, little, if any, full-length PS1 was detected in brain tissue of patients carrying PS1 mutations or in those with sporadic AD, indicating that failed proteolysis of PS1 is not a central feature of pathogenesis in these patients.

Adult↗
PubMed1997

Tumour cell killing using chemically engineered antibody constructs specific for tumour cells and the complement inhibitor CD59.

Immunotherapy using MoAbs is inefficient due to limited activation of human effectors by mouse antibodies and multiple protective mechanisms available to host cells against autologous complement. We have used chemically engineered antibody constructs and human complement in vitro to specifically target and kill neoplastic B lymphoid cells (Raji). Fab'gamma Fc gamma2 chimaeric antibody (specific for human CD37) was used to activate the classical pathway of human complement on Raji cells, whilst CD59 was neutralized using one of two different bispecific F(ab'gamma)2 antibody constructs which contained both cell-targeting (anti-CD19 or anti-CD38) and CD59-neutralizing moieties. When either bispecific construct was used to neutralize CD59, 15-25% of cells were lysed. If CD55 was also neutralized using specific antibody, Raji cells were efficiently killed (70% lysis). When added to a mixture of target (Raji) and bystander (K562) cells, one bispecific antibody (anti-CD38 x anti-CD59) could be specifically delivered to Raji, avoiding significant uptake on CD59-expressing bystander cells (K562). The second bispecific antibody (anti-CD19 x anti-CD59) bound equally well to either cell type. Cell-specific targeting was dependent upon combination of a low-affinity anti-CD59 Fab'gamma with a high-affinity anti-tumour cell Fab'gamma. When Raji and K562 cells were mixed and incubated with a combination of the engineered constructs and anti-CD55 antibodies, Raji cell lysis (30-40%) was observed in the absence of K562 killing. We propose that combinations of these constructs may be of use for treatments such as ex vivo purging of autologous bone marrow or in vivo targeting of tumour cells.

Animals↗
PubMed1995

Lead inhibits Ca(2+)-stimulated nitric oxide synthase activity from rat cerebellum.

Pb2+ is reported to cause cognitive dysfunctions in children and to inhibit long-term potentiation (LTP), a model form of synaptic plasticity that involves nitric oxide (NO). Since Pb2+ interacts with Ca(2+)-calmodulin, and brain nitric oxide synthase (NOS) is Ca(2+)-calmodulin regulated, we examined the effects of Pb2+ on NOS activity prepared from rat cerebellum. NOS required NADPH and was inhibited by monomethylarginine. Full NOS activity required 0.6 microM free Ca2+ and was inhibited 50% by 17 nM and 100% by 80 nM free Pb2+. NOS inhibition by Pb2+ was reversible by increasing free Ca2+ concentrations. Evaluation of other divalent cations resulted in the following ranked order of potencies: Cu2+ > Pb2+ >> Zn2+; Fe2+, Ba2+, Mg2+, Mn2+, and Sr2+ were ineffective. These results suggest that Pb2+ inhibition of brain NOS activity may account for some of the effects of Pb2+ on the CNS.

Animals↗
PubMed1995

Aminoacyl-tRNA synthetase complex in Saccharomyces cerevisiae.

The size distribution of aminoacyl-tRNA synthetase activity was investigated in cell extracts prepared from Saccharomyces cerevisiae. Bio-Gel A-5M chromatography of 105,000 g supernatants separated isoleucyl-tRNA synthetase activity into three peaks, with apparent molecular masses (Da) of about 100,000, 350,000 and 10(6) or greater. Similar results were obtained with synthetases specific for glutamic acid, serine and tyrosine. Sucrose-density-gradient centrifugation of yeast supernatants also provided evidence for the existence of synthetase complexes. These data provide the first evidence for the existence of a high-molecular-mass aminoacyl-tRNA synthetase complex in yeast, perhaps similar to those reported in higher eukaryotes.

Amino Acyl-tRNA Synthetases↗