Motor asymmetry and estimation of body-scaled aperture width in Parkinson's disease.

The present study examined how asymmetrical motor symptomatology helps predict the pattern of perceptual judgements of body-scaled aperture width in lateralised Parkinson's disease (PD). Eleven patients with PD predominantly affecting the left side of their body (LPD), 16 patients with PD predominantly affecting their right side (RPD), and 16 healthy controls made forced-choice judgements about whether or not they would fit without turning their shoulders through a life-sized schematic doorway shown on a large screen. Whereas control and LPD groups made accurate estimations of body-scaled aperture width, RPD patients significantly underestimated aperture width relative to their body, perceiving doorways on average that were 12% narrower than their bodies as wide enough to allow them to pass through without rotation. Across all patients, estimates of body-scaled aperture width correlated with ratio of right-to-left symptom severity. These perceptual errors may indicate a mismatch between the neural representation of external space and that of body size in PD.

Hemispace differences in the visual perception of size in left hemiParkinson's disease.

The visual perception of size in different regions of external space was studied in Parkinson's disease (PD). A group of patients with worse left-sided symptoms (LPD) was compared with a group with worse right-sided symptoms (RPD) and with a group of age-matched controls on judgements of the relative height or width of two rectangles presented in different regions of external space. The relevant dimension of one rectangle (the 'standard') was held constant, while that of the other (the 'variable') was varied in a method of constant stimuli. The point of subjective equality (PSE) of rectangle width or height was obtained by probit analysis as the mean of the resulting psychometric function. When the standard was in left space, the PSE of the LPD group occurred when the variable was smaller, and when the standard was in right space, when the variable was larger. Similarly, when the standard rectangle was presented in upper space, and the variable in lower space, the PSE occurred when the variable was smaller, an effect which was similar in both left and right spaces. In all these experiments, the PSEs for both the controls and the RPD group did not differ significantly, and were close to a physical match, and the slopes of the psychometric functions were steeper in the controls than the patients, though not significantly so. The data suggest that objects appear smaller in the left and upper visual spaces in LPD, probably because of right hemisphere impairment.

Dopamine and the representation of the upper visual field: evidence from vertical bisection errors in unilateral Parkinson's disease.

It has been suggested that dopamine is an important neurotransmitter in the brain mechanisms which represent the upper visual field. This idea was tested with a vertical line bisection task in unilateral Parkinson's disease. Stimuli of a range of lengths were presented on a large screen in three positions (left, centre and right) and at two viewing distances (0.6 and 1.5m). The patients, who were compared with a group of normal age-matched controls, comprised 16 sufferers from predominantly unilateral disease, 8 with more severe left-sided symptoms (LPD) and 8 with more severe right-sided symptoms (RPD). The LPD group consistently set the bisecting cursor below the midpoint of the stimulus lines, and their bisection error became larger as the length of the line increased. In contrast, the controls set the cursor above the midpoint of the line, an error which also increased with line length. The settings of the RPD group were similar to those of the controls. The results suggest altitudinal neglect in left unilateral PD, and support the hypothesis of dopaminergic involvement in the coding of upper visual space, with the proviso that the perceptual component of this involves the right hemisphere in humans.

Evidence from a line bisection task for visuospatial neglect in left hemiparkinson's disease.

The perception of extrapersonal space in Parkinson's disease was examined with two line bisection tasks. One was a conventional pencil and paper test, the line bisection section of the Behavioural Inattention Test. In the other, the stimuli were displayed on a large (2x2.4 m) screen and varied in length (48-480 mm) and also in location on the screen (left, centre and right). They were presented at two viewing distances (0.6 and 1.5 m). Subjects remotely adjusted the position of a cursor until it appeared to bisect the stimulus line, using two push-buttons, one in each hand. The PD participants (n=18) had a marked asymmetry of motor symptoms. They were divided into two groups, those with predominantly left-sided motor symptoms (LPD, n=9), and those with predominantly right-sided motor symptoms (RPD, n=9). The control group (n=9) were all right-handed. No significant differences between the groups were found on the BIT bisection task. In contrast, when the stimuli were presented on the screen, LPD subjects showed a significant rightward bias in their settings of the cursor, particularly for lines on the left and centre of the screen, which was greater, the longer the stimulus line. The RPD group bisected lines slightly to the left, in common with the control group (pseudo-neglect). In a second experiment, Parkinsonian subjects repeated this task, but with the buttons reversed between the hands, so that the cursor was moved to the left by the right hand, and vice versa, but the pattern of results was the same as in the first experiment. The data suggest a small but reliable neglect in left hemiparkinson's disease, which is contralateral to the non-dominant (and probably worse affected) hemisphere. The dissociation between the response and the bisection error suggests a visuospatial impairment in LPD.

Disruption of estimation of body-scaled aperture width in Hemiparkinson's disease.

A group of patients with left-sided symptoms of Parkinson's disease (LPD) was compared with a group of patients with right-sided symptoms (RPD) and with a group of healthy age-matched controls on body-scaled judgements of aperture width. Participants judged whether or not they would fit through a life-sized schematic doorway shown on a large screen. A staircase technique was used to find the door width for which 50% of the judgements were positive. The ratio between this measure and the width of the participant's body at the shoulders (the aperture to shoulder - A/S ratio) was calculated. The A/S ratio was approximately 1.5 in the LPD group, approximately 0.9 in the RPD group, and approximately 1.1 in the control group, suggesting that the visual representation of the doorway (or that of its relationship to perceived body-size) is compressed in LPD (and perhaps expanded in RPD). The A/S ratios were invariant with viewing distance (0.6 or 1.5 m), the contrast polarity of the display (white on dark, or vice versa) and the doorway surround (blank, or vertical or horizontal stripes). The findings are discussed with reference to the neural representation of external space and of the body, and to the motor problems of Parkinson's disease.

Cytotoxic T lymphocyte-assisted suicide. Caspase 3 activation is primarily the result of the direct action of granzyme B.

Cytototoxic T lymphocyte-induced apoptosis can occur either through the directed exocytosis of granzyme B and perforin or via ligation of Fas. Both pathways involve the activation of a family of cysteine proteinases, the caspases, that cleave substrates at aspartic acid and are themselves activated by cleavage at internal aspartate residues. Fas recruits caspase 8, which initiates the death program through the subsequent activation of caspase 3. Granzyme B can process both caspase 8 and 3 in vitro, suggesting that both Fas and granzyme B access the apoptotic program in the same way. Here we demonstrate that although the two mechanisms are similar, the events that lead to activation of caspase 3 can be distinguished in vivo on the basis of their sensitivities to both pharmacological and virus-encoded caspase inhibitors. In cytotoxic T lymphocytes-mediated death the initial cleavage event on caspase 3 is insensitive to benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (zVAD-fmk) inhibition in both mouse and human systems. During Fas-mediated death, however, activation of caspase 3 is completely inhibited to zVAD-fmk. In addition, the viral serpin SPI-2, a homologue of cytokine response modifier A (crmA), is an effective inhibitor of the Fas but not the granzyme pathway. Our results demonstrate that whereas Fas-mediated activation of caspase 3 requires an upstream caspase activity that is zVAD-fmk-sensitive, the initial cleavage of caspase 3 during granule-mediated cell death is insensitive to zVAD-fmk, suggesting that caspase 3 is cleaved directly by granzyme B in vivo.

Interaction between a Ca2+-binding protein calreticulin and perforin, a component of the cytotoxic T-cell granules.

Calreticulin is a component of cytotoxic T-lymphocyte and NK lymphocyte granules. We report here that granule-associated calreticulin terminates with the KDEL endoplasmic reticulum retrieval amino acid sequence and somehow escapes the KDEL retrieval system. In perforin knock-out mice calreticulin is still targeted into the granules. Thus, calreticulin will traffic without perforin to cytotoxic granules. In the granules, calreticulin and perforin are associated as documented by (i) copurification of calreticulin with perforin but not with granzymes and (ii) immunoprecipitation of a calreticulin-perforin complex using specific antibodies. By using calreticulin affinity chromatography and protein ligand blotting we show that perforin binds to calreticulin in the absence of Ca2+ and the two proteins dissociate upon exposure to 0.1 mM or higher Ca2+ concentration. Perforin interacts strongly with the P-domain of calreticulin (the domain which has high Ca2+-binding affinity and chaperone function) as revealed by direct protein-protein interaction, ligand blotting, and the yeast two-hybrid techniques. Our results suggest that calreticulin may act as Ca2+-regulated chaperone for perforin. This action will serve to protect the CTL during biogenesis of granules and may also serve to regulate perforin lytic action after release.

A physical interaction between the cell death protein Fas and the tyrosine kinase p59fynT.

The Fas antigen (Apo1/CD95) is a transmembrane protein belonging to the nerve growth factor receptor family. It is expressed on a variety of cells, including activated T lymphocytes. Ligation of Fas with its natural ligand or with anti-Fas antibodies often results in the apoptotic death of the cell, making Fas an important mediator of down-regulating immune responses. The signal transduction pathways utilized by Fas are currently unknown, although tyrosine kinase activity has recently been strongly implicated. Here, we report that the tyrosine kinase p59fyn physically associates with Fas in Fas-sensitive cells. In addition, we show that activated T lymphocytes from fyn knockout mice exhibit elevated lifespans and reduced apoptosis in vitro compared to their normal counterparts. Furthermore, activated T lymphocytes from the fyn-deficient mice are less sensitive to killing by both anti-Fas antibody and Fas-ligand cytotoxic T cells. These results suggest that p59fyn plays an important role in Fas signal transduction.

Proteinases are involved in both DNA fragmentation and membrane damage during CTL-mediated target cell killing.

A number of inhibitors with different specificities were used to probe the involvement of proteinases in the mechanism of cytotoxic T lymphocyte (CTL)-mediated lysis. N-Acetyl-L-tyrosine ethyl ester (ATEE) and N-benzoyl-L-arginine ethyl ester (BAEE) are reversible substrate inhibitors of proteinases with trypsin-like and chymotrypsin-like specificities, respectively. BAEE did not prevent either the chromium release or DNA fragmentation induced in mouse tumor target cells by a mixed lymphocyte population. In contrast, ATEE inhibited both processes. The irreversible proteinase inhibitor 3,4-dichloroisocoumarin (DCI) also blocked both chromium release and DNA fragmentation, but at significantly lower concentrations than ATEE. More importantly, chromium release was more susceptible to inhibition by DCI than DNA fragmentation. Addition of a combination of the endonuclease inhibitor aurintricarboxylic acid plus DCI resulted in virtually complete inhibition of both DNA fragmentation and chromium release when the drugs were added at the beginning of the incubation period. In contrast addition of DCI 15 or 30 min following initiation of the lytic cycle abolished the affect of DCI on fragmentation, but not lysis. A model which suggests a dual role for the proteinases in CTL-mediated target cell death is presented. First, proteinases are involved in the initiation of DNA fragmentation. Second, they have an ongoing function in membrane damage.

Mechanisms of lysis by cytotoxic T cells.

When a cytotoxic lymphocyte binds to an antigenic cell, there is a reorientation of the Golgi apparatus and a directed exocytosis of cytoplasmic granules toward the offending cell. In the model originally proposed by Henkart, these granules were hypothesized to contain lytic molecules that contribute to the demise of the target cell. Initially, perforin/cytolysin was believed to be the major player in the mechanism of lysis. Recent work, however, using a variety of biochemical, molecular biological, and genetic approaches, has provided convincing evidence that other granule-associated molecules contribute to the lytic pathway. Of particular note are the granzymes, whose proteolytic activity is intimately associated with the induction of DNA fragmentation and apoptosis. In this review we summarize these recent experiments and present an updated view of the granule-mediated killing mechanism. For some years the universality of the granule-killing mechanism has been challenged. Experiments reported in the last few years on the characteristics of target cell death, detailed studies with cytolytic T cell lines, and, ultimately, with lymphocytes from mice that have been genetically altered using homologous recombination, have proven the existence of a second pathway. We discuss whether the existence of this alternate, Fas antigen/Fas ligand, mechanism really deals a knock-out blow to the granule exocytosis followers. Most likely it represents an important immunoregulatory system rather than a defense against pathogenesis. The granule-mediated and Fas-dependent mechanisms of cytolysis represent, at first sight, completely different pathways. However, it is becoming increasingly apparent that once the kiss of death has been received, through either granules or Fas, the events within the target cell are remarkably similar. Considerable attention is now being focussed on a family of interleukin-1 beta-converting enzymes (ICEs) that become activated within the target cell after attack by a cytotoxic lymphocyte. These enzymes are particularly interesting as they have now been shown to be directly involved in developmentally controlled, programmed cell death.

Characterization of a granule-independent lytic mechanism used by CTL hybridomas.

The mechanism(s) by which CTL induce target cell lysis have not been clearly elucidated. Perforin and the cytotoxic cell proteinases (granzymes) contained within the granules of CTL and NK, have been implicated, but abundant evidence for the existence of alternate lytic pathways has accumulated. In this report we characterize the mechanism of killing used by two cytolytic hybridomas (PMM-1 and MD90) that express neither perforin nor the granzymes. These characteristics are compared with results obtained by using a representative Ag-dependent, granule-containing T cell clone in cytolysis assays. The major differences were that the granule-negative hybridomas could lyse a variety of target cells in the presence of cyclosporin and the absence of calcium. All the effectors could kill in the presence of protein synthesis inhibitors (cycloheximide and emetine) and induced DNA fragmentation in the target cells. The cytolytic hybridomas had to be stimulated to be cytolytic and this activation required the presence of calcium, was dependent on protein synthesis, and inhibited by the addition of cyclosporin. Although TNF was shown not be involved, the sensitivity of the target cells to lysis by the granule-negative killers correlated with the level of expression of Fas Ag. With the use of L1210 and an L1210 cell line transfected with Fas cDNA we demonstrated that these MD90 and PMM-1 kill the latter much more effectively and that this increase was effectively inhibited with anti-Fas Ab. Furthermore the lack of sensitivity to cyclosporin, cycloheximide, emetine, and EGTA was confirmed with these targets. We conclude that these two cytolytic hybridomas use the Fas lytic pathway to induce lysis in target cells.

Calreticulin: from Ca2+ binding to control of gene expression.

Calreticulin is a highly conserved Ca(2+)-binding/storage protein of the endoplasmic reticulum (ER). Recently, it has been shown to play a role in the control of gene expression by interacting with the DNA-binding domain of various steroid receptors. How does this ER protein gain access to the nuclear steroid receptors? We propose that calreticulin undergoes unique intracellular trafficking that allows it to colocalize with and bind to steroid receptors.

Modulation of gene expression by calreticulin binding to the glucocorticoid receptor.

Calreticulin is a multifunctional protein that acts as a major Ca(2+)-binding (storage) protein in the lumen of the endoplasmic reticulum. It is also found in the nucleus, suggesting that it may have a role in transcription regulation. Calreticulin has been reported to bind to the synthetic peptide KLGFFKR, which is almost identical to an amino-acid sequence in the DNA-binding domain of the superfamily of nuclear receptors. Could calreticulin interact with the DNA-binding domain of these receptors and affect their function? Here we report that the amino terminus of calreticulin interacts with the DNA-binding domain of the glucocorticoid receptor and prevents the receptor from binding to its specific glucocorticoid response element. Overexpression of calreticulin in mouse L fibroblasts inhibits glucocorticoid-response-mediated transcriptional activation of a glucocorticoid-sensitive reporter gene and of the endogenous, glucocorticoid-sensitive gene encoding cytochrome P450. Together these results indicate that calreticulin may be important in gene transcription, regulating the glucocorticoid receptor and perhaps other members of the super-family of nuclear receptors.

Epidemiology of Blastocystis hominis infection in Papua New Guinea: age-prevalence and associations with other parasites.

A community-based study of Blastocystis and other intestinal parasites in the Asaro Valley, Papua New Guinea showed an extraordinary high prevalence and variety of protozoan infections. Apart from infants, nearly everybody had at least one infection, and the mean number of infections per person was around 2.7. The graph of age-specific prevalence for Blastocystis is similar in shape to those for Entamoeba coli and Endolimax nana, indicating probable similarity in transmission patterns and host response. There was no evidence for pathogenicity of Blastocystis at the community level. Three methods are compared for the measurement of association between infections. Two show strong associations, but these are considered to be the result of parallel age-prevalence curves and environmental factors at the village level. When age- and village-matched pairs were considered, only a weak positive association with E. nana was detectable.

Effects of hypothyroidism and high-fat feeding on mRNA concentrations for the low-density-lipoprotein receptor and on acyl-CoA:cholesterol acyltransferase activities in rat liver.

1. Induction of hypothyroidism in rats by feeding propylthiouracil (PTU) significantly increased serum cholesterol concentrations, and the effect was more pronounced for cholesterol in low-density lipoproteins (LDL) rather than high-density lipoproteins (HDL). The concentrations of serum triacylglycerol were decreased in hypothyroidism. These effects on serum lipids were also seen when the normal rats were pair-fed with the PTU-treated group. 2. Feeding a diet rich in saturated fat and cholesterol further increased cholesterol concentrations in LDL and also elevated that in very-low-density lipoprotein (VLDL) of hypothyroid rats. In euthyroid rats such a diet resulted in a relatively small increase in VLDL cholesterol, whereas LDL cholesterol was decreased. 3. Steady-state concentrations of mRNA for the hepatic LDL receptor were significantly decreased in the livers of hypothyroid rats, but were not significantly changed by high-fat feeding in euthyroid or hypothyroid rats. 4. The expression of the LDL receptor in hepatocytes cultured from hypothyroid rats was decreased relative to the euthyroid controls. 5. Whereas the esterification of cholesterol with oleate in hepatocytes cultured from hypothyroid rats was decreased, the activity of acyl-CoA:cholesterol acyltransferase (ACAT) in the livers of these animals was not changed. 6. High-fat feeding increased the hepatic ACAT activity in normal and hypothyroid rats. 7. Incubation of rat hepatocytes with 10 nM-tri-iodothyronine for 4 h increased the relative concentration of the mRNA for the LDL receptor by 25%. 8. It is therefore concluded that thyroid hormones stimulate the synthesis and expression of the hepatic LDL receptor. Elevated cholesterol concentrations in LDL in hypothyroidism probably result from a primary defect in the expression of the hepatic receptor, rather than indirectly via changes in ACAT activity.

Cloning of a gene that encodes a new member of the human cytotoxic cell protease family.

A family of unusual serine proteases that are believed to be involved in the effector mechanism of cell-mediated cytotoxicity have previously been described in the mouse. However, in the human only one gene encoding a member has been isolated. By use of a mixture of murine cDNAs as probes, a second human gene has now been isolated. The primary structures of the gene and the predicted protein are very similar to those of the mouse. In addition, in keeping with the postulated involvement in cytolysis, transcripts were detected only in cytotoxic cells. The organization of the coding and noncoding regions of the gene, the clustering of family members, and the chromosomal location, close to the alpha chain of the T cell antigen receptor, are all conserved between human and mouse.

Studies of the mechanism of natural killer (NK) degranulation and cytotoxicity.

Exocytosis of cytotoxic granule contents towards bound target cells is thought to be of central importance in natural killer (NK) cell-mediated killing. Although cellular cytotoxicity involving degranulation is thought to be calcium-dependent, the biochemical mechanisms that mediate this granule mobilization are unknown. Inositol-1,4,5-tris-phosphate (IP3), which acts to elevate internal calcium levels, and 1,2-diacylglycerol (DAG), which activates protein kinase C, are potent second messengers that have been shown to synergistically mediate secretion in other cell types. Production of these products of inositol phospholipid metabolism has previously been demonstrated in a rat NK cell line RNK upon exposure to susceptible tumor targets. We therefore investigated the role of IP3 and DAG in NK-mediated cytotoxicity, specifically at the level of degranulation. Pretreatment of RNK cells with neomycin, a drug that interferes with the hydrolysis of inositol phospholipids and thus inhibits the formation of second messengers, inhibited RNK cytotoxicity against a susceptible tumor target and also inhibited RNK production of DAG in response to a similar target. Natural killing exhibited by normal rat nylon wool-nonadherent splenocytes was also inhibited by neomycin. Phorbol-12-myristate, 13-acetate (PMA), a phorbol ester that acts like DAG to activate protein kinase C, markedly enhanced lysis of a susceptible target cell by RNK. We evaluated whether modulation of lysis by these drugs was associated with effects on RNK degranulation by assaying the release of a granule-specific serine esterase (BLTE) in response to PMA and the calcium ionophore A23187. These agents synergized to promote the release of BLTE, and the extent of release was dependent on the concentrations of both agents. D2O and cytochalasin B, which enhance secretion in other cells, both enhanced BLTE release from RNK cells, indicating that we were detecting BLTE released via granule secretion and not due to nonspecific causes such as cell lysis. Our findings lead us to propose that NK cells form IP3 and DAG in response to susceptible target cells and that a major function of these second messengers is to mediate the exocytosis of cytotoxic granules towards the bound target cells.

Production of inositol-phospholipid-derived second messengers in a rat natural killer cell line exposed to susceptible tumor targets.

It is currently believed that natural killer (NK) cells kill bound target cells by exocytosis of cytotoxic granules via a calcium-dependent process. After confirming that NK-mediated killing was indeed dependent upon extracellular calcium, we investigated the production of inositol-phospholipid-derived second messengers in a rat NK cell line, RNK, upon exposure to susceptible target cells. These messengers, inositol trisphosphate (IP3) and diacylglycerol (DAG), are associated with calcium-dependent secretory processes in a number of cell types. When RNK cells were exposed to susceptible YAC-1 tumor targets significant amounts of both IP3 and DAG were produced. The levels of the membrane phospholipid parent molecules of these second messengers declined in similarly stimulated RNK cells over a comparable time period. Using three different target cell lines, it was found that the levels of DAG that RNK produced in response to the different targets followed the same rank order as their susceptibility to RNK-mediated lysis. These data suggest that IP3 and DAG are produced in NK cells in response to tumor target cells, and these second messengers may have a functional role in NK-mediated killing.