Differential analysis of serum and urine S100 proteins in juvenile-onset systemic lupus erythematosus (jSLE).

Up to 80% of juvenile-onset systemic lupus erythematosus (jSLE) patients develop lupus nephritis (LN) that affects treatment and prognosis. Easily accessible biomarkers do not exist to reliably diagnose LN, leaving kidney biopsies as the gold-standard. Calcium-binding S100 proteins are expressed by innate immune cells and epithelia and may act as biomarkers in systemic inflammatory conditions. We quantified S100 proteins in the serum and urine of jSLE patients, matched healthy and inflammatory (IgA vasculitis) controls. Serum S100A8/A9, and serum and urine S100A12 are increased in jSLE patients when compared to controls. Furthermore, serum S100A8/A9, and serum and urine S100A12 are increased in jSLE patients with active as compared to patients with inactive/no LN. No differences in S100A4 levels were seen between groups. This study demonstrates potential promise for S100A8/A9 and S100A12 as biomarkers for jSLE and active LN. Findings require to be confirmed and tested prospectively in independent and larger multi-ethnic cohorts.

MLL-AF9 and FLT3 cooperation in acute myelogenous leukemia: development of a model for rapid therapeutic assessment.

Human leukemias harboring chromosomal translocations involving the mixed lineage leukemia (MLL, HRX, ALL-1) gene possess high-level expression, and frequent activating mutations of the receptor tyrosine kinase FLT3. We used a murine bone marrow transplant model to assess cooperation between MLL translocation and FLT3 activation. We demonstrate that MLL-AF9 expression induces acute myelogenous leukemia (AML) in approximately 70 days, whereas the combination of MLL-AF9 and FLT3-ITD does so in less than 30 days. Secondary transplantation of splenic cells from diseased mice established that leukemia stem cells are present at a very high frequency of approximately 1:100 in both diseases. Importantly, prospectively isolated granulocyte macrophage progenitors (GMPs) coinfected with MLL-AF9 and FLT3-ITD give rise to a similar AML, with shorter latency than from GMP transduced with MLL-AF9 alone. Cooperation between MLL-AF9 and FLT3-ITD was further verified by real-time assessment of leukemogenesis using noninvasive bioluminescence imaging. We used this model to demonstrate that MLL-AF9/FLT3-ITD-induced leukemias are sensitive to FLT3 inhibition in a 2-3 week in vivo assay. These data show that activated FLT3 cooperates with MLL-AF9 to accelerate onset of an AML from whole bone marrow as well as a committed hematopoietic progenitor, and provide a new genetically defined model system that should prove useful for rapid assessment of potential therapeutics in vivo.

The use of buck and ram extenders and two packaging systems to cryopreserve aoudad (Ammotragus lervia) spermatozoa.

Because the aoudad has been hunted to near extinction, cryopreservation of their semen would be useful for DNA conservation and for the possible re-establishment of captive bred animals to their former ranges. This study was conducted to investigate the effectiveness of cryopreserving aoudad spermatozoa. Semen samples from four post-pubertal animals were collected using electro-ejaculation. Microscopic analysis was performed to assess the percentages of progressively and non-progressively motile spermatozoa as well as intact acrosomes in samples prior to freezing and post-thaw. Extended samples (0.2 mL) were frozen using 2 different extenders and packaging systems and stored in LN2 Post-thaw data were arcsine-transformed and analyzed using ANOVA, 2 x 2 factorial. Samples that were processed using the ram/straw method had a significantly higher percentage (P < 0.05) of spermatozoa with intact acrosomes than did any other system. In addition, samples that were processed with the buck/pellet system had significantly greater percentages (P < 0.05) of progressive and non-progressively motile spermatozoa than the samples processed using either extender and packaged in straws. This study illustrates that some aoudad spermatozoa may be cryopreserved using the extender/processing systems developed for the domestic buck and ram.

Characterization of a newly discovered T-cell receptor beta-chain heterodimer expressed on a CD8+ bone marrow subpopulation that promotes allogeneic stem cell engraftment.

The facilitating cell is a rare CD8+ bone marrow subpopulation that can enhance allogeneic hematopoietic stem cell engraftment across complete major histocompatibility complex barriers without inducing acute graft-versus-host disease. Here we describe a CD3epsilon-associated complex on the facilitating cell surface that consists of the T-cell receptor beta-chain disulfide-linked to a previously unknown 33-kilodalton glycoprotein. Provisionally called FCp33, this glycoprotein does not represent any of the known protein chains or surrogates associated with CD3-T-cell receptor beta. Expression of this CD3-T-cell receptor beta-FCp33 complex directly correlates with the facilitating cell's functional ability to enhance allogeneic stem cell engraftment in vivo.

Location cue validity affects inhibition of return of visual processing.

Inhibition-of-return is the process by which visual search for an object positioned among others is biased toward novel rather than previously inspected items. It is thought to occur automatically and to increase search efficiency. We examined this phenomenon by studying the facilitative and inhibitory effects of location cueing on target-detection response times in a search task. The results indicated that facilitation was a reflexive consequence of cueing whereas inhibition appeared to depend on cue informativeness. More specifically, the inhibition-of-return effect occurred only when the cue provided no information about the impending target's location. We suggest that the results are consistent with the notion of two levels of visual processing. The first involves rapid and reflexive operations that underlie the facilitative effects of location cueing on target detection. The second involves a rapid but goal-driven inhibition procedure that the perceiver can invoke if doing so will enhance visual search performance.

Further studies of elevated cerebrospinal fluid neuronal cell adhesion molecule in schizophrenia.

BACKGROUND: The purposes of the present study were to attempt to replicate a previous finding of increased cerebrospinal fluid (CSF) neuronal cell adhesion molecule (N-CAM) in schizophrenia, and to assess whether the increases could be related to medication, clinical state effects, or brain structural measures. METHODS: CSF N-CAM was measured by the Western blot technique in 45 DSM-III-R diagnosed male schizophrenic patients both on and off haloperidol treatment and in 20 healthy male control subjects. RESULTS: CSF N-CAM was significantly increased in schizophrenic patients, with no overlap in the ranges, when compared to controls. There were no significant effects of medication or exacerbation on CSF N-CAM. No associations with measures of brain structure were found. CONCLUSIONS: Because N-CAM levels were not shown to be different on and off treatment or in exacerbated versus nonexacerbated patients, the higher levels seen in schizophrenic patients may be inherent to the disorder and possibly related to neurodevelopment.

A truncated SV40 large T antigen lacking the p53 binding domain overcomes p53-induced growth arrest and immortalizes primary mesencephalic cells.

As an alternative to primary fetal tissue, immortalized central nervous system (CNS)-derived cell lines are useful for in vitro CNS model systems and for gene manipulation with potential clinical use in neural transplantation. However, obtaining immortalized cells with a desired phenotype is unpredictable, because the molecular mechanisms of growth and differentiation of CNS cells are poorly understood. The SV40 large T antigen is commonly used to immortalize mammalian cells, but it interferes with multiple cell-cycle components, including p53, p300, and retinoblastoma protein, and usually produces cells with undifferentiated phenotypes. In order to increase the phenotypic repertoire of immortalized CNS cells and to address the molecular mechanisms underlying immortalization and differentiation, we constructed an expression vector containing a truncated SV40 large T gene that encodes only the amino-terminal 155 amino acids (T155), which lacks the p53-binding domain. Constructs were first transfected into a p53-temperature-sensitive cell line, T64-7B. Colonies expressing T155 proliferated at the growth-restrictive temperature. T155 was then transfected into primary cultures from embryonic day-14 rat mesencephalon. Two clonal cell lines were derived, AF-5 and AC-10, which co-expressed T155 and mature neuronal and astrocytic markers. Thus, the amino-terminal portion of SV40 large T is sufficient to: (1) overcome p53-mediated growth arrest despite the absence of a p53-binding region, and (2) immortalize primary CNS cells expressing mature markers while actively dividing. T155 and T155-transfectants may be useful for further studies of cell-cycle mechanisms and phenotyic expression in CNS cells or for further gene manipulation to produce cells with specific properties.

Inhibition-of-return at multiple locations in visual space.

Inhibition-of-return is thought to be a visual search phenomenon characterized by delayed responses to targets presented at recently cued or recently fixated locations. We studied this inhibition effect following the simultaneous presentation of multiple location cues. The results indicated that response inhibition can be associated with as many as four locations at the same time. This suggests that a purely oculomotor account of inhibition-of-return is oversimplified. In short, although oculomotor processes appear to play a role in inhibition-of-return they may not tell the whole story about how it occurs because we can only program and execute eye movements to one location at a time.

Inattention and the perception of visual feature conjunctions.

Visual processing of objects in the absence of focused attention appears to be limited. We varied the degree of attention, or visual processing, that observers paid to objects using an instruction set manipulation. In 2 experiments, subjects performed tasks that required superficial or detailed visual analysis of the objects involved. In subsequent recognition tests, information about conjunctions of shape and internal color/texture pattern was limited when only superficial visual analysis was required to encode the object. This implies that the degree of visual processing, during object encoding affects the likelihood that feature conjunctions are incorporated into the visual representation of these objects.

Neutral location cues and cost/benefit analysis of visual attention shifts.

The effects of location cuing on target responses can be examined by comparing informative and neutral cuing conditions. In particular, the magnitudes of costs of invalid location cuing and of benefits of valid location cuing can be determined by comparing invalid and valid cue responses to location-nonspecific neutral cue responses. Cost/benefit analysis is based on the assumption that neutral baseline measures reflect a general warning effect about the impending target's onset but no other specific target information. The experiments we report were carried out to determine the appropriateness of two baseline measures for cost/benefit analyses of direct (nonsymbolic) location cuing effects. We found that a multiple-cue baseline attenuated the benefits of valid cuing, and that a background-flash baseline arbitrarily attenuated costs or benefits depending on flash intensity. It is proposed that a background flash is the more suitable neutral cue because it is target-location-nonspecific, but that its intensity should be adjusted to elicit a target-onset warning signal of the same magnitude as the location cues with which it will be compared.

Blood supply of the ovine adrenal gland and its relevance in adrenal autotransplantation.

The arterial supply and venous drainage of 62 left and 5 right ovine adrenal glands is described, and the contribution of individual arteries to successful adrenal gland autotransplantation was evaluated. Arterial flow was measured by direct collection from the draining adrenal vein. Assessment of function of the transplanted adrenal gland was made from survival of the sheep and by the cortisol response to infusion of ACTH and the aldosterone secretory response to infusion of angiotensin II or potassium. For the left adrenal, the principal arterial supply was from the renal artery in 21 (34%), a lumbar artery in 32 (52%), and the anterior mesenteric artery in 3. The total blood flow was 5.0 +/- SEM 0.4 mL/min, the flow from the renal branch 2.3 +/- 0.3 mL/min, and the principal lumbar branch 2.6 +/- 0.3 mL/min. Venous drainage from the left adrenal was via a major adrenal vein to the left renal vein, but additional tributaries to the renal vein were present in 26%. The arterial supply to the adrenal is regional and omission of a branch at transplantation could result in infarction of portion of the gland. By defining arterial supply and measuring blood flow, selection of the appropriate artery or multiple arteries can achieve an adrenal gland autotransplant survival of 90%.

Polarity matching in the Ternus configuration.

Two experiments were conducted to examine the effect of changes in the sign of element contrast on perceptions of the Ternus apparent motion display. In the first experiment, the contrast polarity of all three elements in the display were alternated from the first frame of view to the second. At short durations, this increased perceptions (relative to a control condition) of simultaneity in the display, decreased perceptions of element motion, and did not significantly affect perceptions of group motion. At long durations, this manipulation did not affect performance. In a second experiment, patterns of element polarity were manipulated to favour perceptions of either element motion or of group motion relative to a control condition in which all elements had identical contrast polarity. At a long duration, this manipulation affected perceptions of the configuration; this manipulation did not affect the appearance of the display at a short duration. Together, these results are inconsistent with the predictions of Grossberg and Rudd's [Psychological Review, 99, 78-121 (1992)] motion oriented contrast filter. However, they are consistent with a model of motion correspondence processing that includes a polarity matching constraint.

Shifts of visual attention to multiple simultaneous location cues.

Single and simultaneous multiple location cues were presented a brief period of time before the onset of targets that observers were required to identify. Single and multiple cueing effects were similar, even when targets were presented at locations "in between" pairs of cues. This implies that pairs of direct stimulus cues are not processed by an attentional focus of variable spatial extent that encompasses both cued locations. If this were the case, then targets presented in between cued locations would fall within such an attentional focus and identification response times should have been reduced. The increase in these response times that actually occurred when a target was presented at uncued locations in between a pair of cued locations suggests that pairs of cued locations are encoded independently by an operation called spatial indexing. A multiple indexing proposal and its role in mediating visual attention shifts initiated by direct stimulus cues is described.

Simultaneity in the Ternus configuration: psychophysical data and a computer model.

The Ternus configuration is an apparent motion display which is typically described as being bistable; subjects usually describe seeing either element motion or group motion, depending upon temporal properties of the display. The results of an experiment are reported in which subjects are also permitted to report seeing four stationary display elements (simultaneity). It was found that simultaneity was produced when both frame durations and interstimulus intervals (ISIs) were brief. A weaker than expected effect of stimulus onset asynchrony (SOA) was revealed because this third type of judgment was obtained. Furthermore, statistical analyses indicated that SOA was not by itself the best predictor of judgment type. The interaction between duration and ISI was also an important predictor. This suggested that a complete account of the Ternus configuration requires two mechanisms: a visible persistence mechanism, governed by an SOA law, and a motion correspondence mechanism, governed by an ISI law. These two mechanisms were added to Dawson's (1991) [Psychological Review, 98, 569-603] autoassociative network for motion correspondence processing. The resulting model could generate each of the three interpretations of the Ternus configuration at appropriate combinations of frame duration and ISI.

Inattention and the perception of visual features.

Subjects selectively attended to one of two interleaved, novel figures while ignoring the other figure. In subsequent tests administered to determine the extent to which the ignored figure was perceived, recognition of shape and the location of contour gaps was at the chance level. Moreover, recognition of the presence of contour gaps was significantly below the chance level. These results indicate that preattentive visual processing of unattended objects is too crude to encode global shape and local features such as contour gaps. It is suggested that preattentive processing creates visual representations of unattended objects that contain very limited information about features.

Effect of depth information on a bistable spiral-motion aftereffect.

Two types of spiral-motion aftereffects were elicited by a single pattern: subjects reported seeing the pattern expand in the two-dimensional viewing plane or bulge toward them in three-dimensional space. Under binocular-viewing conditions reports of two-dimensional translations predominated. But when depth information was restricted under monocular-viewing conditions, reports of three-dimensional translations were more frequent. It appears that the bistability of these aftereffects can be influenced by the degree of depth information available about a stimulus pattern.

Evidence against the involvement of vasoactive intestinal peptide in ovine parotid secretion and blood flow.

1. The proposition that stimulation of the secretomotor nerve to the ovine parotid gland might involve co-release of vasoactive intestinal peptide (VIP) was tested by studying responses to infusion of VIP directly into the gland's arterial blood supply and by assay of VIP in parotid venous blood. 2. In unstimulated glands, an arterial blood concentration of 1.5 - 2.5 X 10(-9) mol/L VIP did not evoke fluid secretion but it increased K+ and phosphate secretion and glandular blood flow. The same blood concentration of VIP potentiated the stimulation of salivary flow rate caused by intraarterial infusion of bethanechol but nerve stimulation was not potentiated. VIP increased glandular blood flow in both conditions of stimulation. 3. Atropine blocked neurally stimulated salivary secretion but an increase in glandular blood flow was still detectable. There was therefore no evidence for a non-cholinergic neural mechanism for salivary secretion. 4. Furthermore, VIP concentrations in glandular venous blood were not increased by nerve stimulation. 5. The results indicate that exogenous VIP can affect the flow and composition of ovine parotid secretion but was not involved in the response to secretomotor nerve stimulation.

Radioimmunoassay of carbonic anhydrase VI in saliva and sheep tissues.

A specific and sensitive radioimmunoassay has been developed for the measurement of the secreted carbonic anhydrase isoenzyme (CA VI) in sheep saliva and tissues. The assay can detect as little as 75 pg of CA VI, and the antibody used does not cross-react with CA II or CA III. The intra-assay variation, measured using a saliva sample, was 3.0%, whereas the inter-assay variation was 10.5%. The concentration of CA VI in parotid saliva from normal, resting sheep was 5.6 +/- 3.0 micrograms.ml-1 (n = 42) or 79.4 +/- 35.7 micrograms.mg of total protein-1. With feeding, the CA VI concentrations increased an average of 6-fold. The secretion rate of CA VI from the vascularly isolated neurotomized parotid gland of the anaesthetized sheep was 0.62 +/- 0.40 micrograms.min-1, compared with a rate of 11.7 +/- 7.8 micrograms.min-1 from the parotid gland of normal conscious sheep. Stimulation of the parotid-gland preparation by the muscarinic agent bethanechol increased the secretion rate to 438 +/- 172 microgram.min-1 (n = 8), and electrical stimulation of the secretomotor Moussu nerve increased CA VI secretion rate to 634 +/- 330 micrograms.min-1 (n = 4). Submandibular saliva from anaesthetized sheep contained 6.9 +/- 2.1 micrograms of CA VI.ml-1 (n = 3). The only tissues found to contain measurable amounts of CA VI were the parotid (6.4 micrograms.mg of protein-1) and submandibular (1.8 micrograms.mg of protein-1) salivary glands. The sublingual salivary gland, kidney, lung, adrenal, brain, skeletal muscle, liver, heart, pancreas, small intestine and cerebrospinal fluid did not have a measurable CA VI content.

Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry.

Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CA VI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension [Fernley, R. T., Wright, R. D., & Coghlan, J. P. (1988b) Biochemistry 27, 2815]. Overall the human CA VI protein has a sequence identity of 35% with human CA II, while residues involved in the active site of the enzymes have been conserved. The human sheep secreted carbonic anhydrases have a sequence identity of 72%. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

The effects of K+ channel blockers on ovine parotid secretion depend on the mode of stimulation.

The vascularly isolated parotid glands of sheep were submaximally stimulated for 4 min by nerve stimulation, or by infusion of acetylcholine (ACh) or bethanechol directly into the artery supplying the gland. The three modes of stimulation caused almost equal increases in the rate of salivary flow, initial losses of K+ and phosphate from the gland and total glandular deficits of K+ and phosphate. Concurrent arterial infusion of the K+ channel blocker tetraethylammonium (TEA), at 1.5-3.3 mM in blood, almost abolished these responses to bethanechol but did not alter the responses to nerve stimulation or ACh. The responses to bethanechol were restored by increasing the concentration fourfold. Concurrent arterial infusion of 4-aminopyridine (4-AP), at 0.1-2.3 mM in blood, partially inhibited the increase in salivary flow due to bethanechol but not the response to nerve stimulation or ACh. The specificity and competitive nature of the action of TEA and 4-AP on responses to direct muscarinic stimulation by bethanechol are consistent with blockade of K+ channels in secretory end-piece cells. The failure of TEA and 4-AP to inhibit responses to nerve stimulation and ACh may be due to the blocking agents potentiating the release of neurotransmitter ACh. It is also possible that nerve stimulation and ACh may cause the release of a co-transmitter which bethanechol does not.