Ischemic preconditioning protects against paraplegia after transient aortic occlusion in the rat.

BACKGROUND: Paraplegia can result from operations requiring transient occlusion of the thoracic aorta. A rat model of paraplegia with the characteristics of delayed paraplegia and transient ischemic dysfunction was developed to determine whether ischemic preconditioning (IPC) improved neurologic outcome. METHODS: Rats underwent balloon occlusion of the upper descending thoracic aorta. One group (2 minute IPC, n = 19) underwent 2 minutes of IPC and a second group (5 minute IPC, n = 19) had 5 minutes of IPC 48 hours before 10 minutes of occlusion. The control group (n = 31) had no IPC prior to 10 minutes of occlusion. RESULTS: Paraplegia occurred in 68% of the control animals (21 of 31 paraplegic: 6 delayed and 15 immediate paraplegia). Both the 2-minute IPC and 5-minute IPC groups had a decreased incidence of paraplegia when compared to controls (32%, p = 0.011 and 26%, p = 0.009, respectively). CONCLUSIONS: A rat model of spinal cord ischemia demonstrating both delayed paraplegia and transient ischemic dysfunction was characterized. Both 2-minute and 5-minute periods of IPC were found to protect against paraplegia.

Phosphorylation of Tyr319 in ZAP-70 is required for T-cell antigen receptor-dependent phospholipase C-gamma1 and Ras activation.

Accumulating evidence indicates that the interdomain B regions of ZAP-70 and Syk play pivotal roles in the coupling of T-cell antigen receptor (TCR) stimulation to the activation of downstream signaling pathways. The interdomain B region of ZAP-70 contains at least three candidate sites of tyrosine phosphorylation. In this report, we identify Tyr319 as a functionally important phosphorylation site in the ZAP-70 interdomain B region. TCR crosslinkage triggered a rapid increase in the phosphorylation of Tyr319 in Jurkat T cells. Although mutation of Tyr319 to Phe had no effect on the tyrosine kinase activity of ZAP-70, the resulting ZAP(Y319-->F) mutant failed to reconstitute TCR-dependent Ca2+ mobilization, Ras activation, CD69 expression and NFAT-dependent transcription in ZAP-70-deficient Jurkat cells. These defects were correlated with reduced tyrosine phosphorylation of phospholipase C (PLC)-gamma1 and the LAT adapter protein in the ZAP(Y319-->F)-expressing cells. On the other hand, ZAP(Y319-->F)-expressing cells displayed normal increases in SLP-76 phosphorylation and ERK activation during TCR stimulation. Phosphorylation of Tyr319 promoted the association of ZAP-70 with the SH2 domains of two key signaling molecules, Lck and PLC-gamma1. These studies suggest that Tyr319 phosphorylation is required for the assembly of a ZAP-70-containing signaling complex that leads to the activation of the PLC-gamma1- and Ras-dependent signaling cascades in antigen-stimulated T cells.

Interdomain B in ZAP-70 regulates but is not required for ZAP-70 signaling function in lymphocytes.

The protein tyrosine kinase ZAP-70 plays an important role in T-cell activation and development. After T-cell receptor stimulation, ZAP-70 associates with the receptor and is phosphorylated on many tyrosines, including Y292, Y315, and Y319 within interdomain B. Previously, we demonstrated that Y292 negatively regulates ZAP-70 function and that Y315 positively regulates ZAP-70 function by interacting with Vav. Recent studies have suggested that Y319 also positively regulate ZAP-70 function. Paradoxically, removal of interdomain B (to create the construct designated Delta), containing the Y292, Y315, and Y319 sites, did not eliminate the ability of ZAP-70 to induce multiple gene reporters in Syk-deficient DT-40 B cells and ZAP-70/Syk-deficient Jurkat cells. Here we show that Delta still utilizes the same pathways as wild-type ZAP-70 to mediate NF-AT induction. This is manifested by the ability of Delta to restore induction of calcium fluxes and mitogen-activated protein kinase activation and by the ability of dominant negative Ras and FK506 to block the induction of NF-AT activity mediated by Delta. Biochemically we show that the stimulated tyrosine phosphorylation of Vav, Shc, and ZAP-70 itself is diminished, whereas that of Slp-76 is increased in cells reconstituted with Delta. Deletion of interdomain B did not affect the ability of ZAP-70 to bind to the receptor. The in vitro kinase activity of ZAP-70 lacking interdomain B was markedly reduced, but the kinase activity was still required for the protein's in vivo activity. Based on these data, we concluded that interdomain B regulates but is not required for ZAP-70 signaling function leading to cellular responses.

SLP-76 is a direct substrate of SHP-1 recruited to killer cell inhibitory receptors.

Activation of immune system cells via antigen-, Fc-, or natural killer cell-triggering-receptor stimulation is aborted by co-engagement of inhibitory receptors. Negative signaling by killer cell inhibitory receptors and related receptors depends on the Src homology 2 (SH2)-containing protein tyrosine phosphatase SHP-1. Using a combination of direct binding and functional assays, we demonstrated that the SH2 domain-containing leukocyte protein 76 (SLP-76) is a specific target for dephosphorylation by SHP-1 in T cells and natural killer cells. Furthermore, we showed that tyrosine-phosphorylated SLP-76 is required for optimal activation of cytotoxic lymphocytes, suggesting that the targeted dephosphorylation of SLP-76 by SHP-1 is an important mechanism for the negative regulation of immune cell activation by inhibitory receptors.

Antiproliferative action of interferon-alpha requires components of T-cell-receptor signalling.

Signal transduction through both cytokine and lymphocyte antigen receptors shares some common pathways by which they initiate cellular responses, such as activation of mitogen-activated protein kinase(s). However, other signalling components appear to be uniquely coupled to each receptor. For example, the interferon receptors transduce regulatory signals through the JAK/STAT pathway, resulting in an inhibition of growth and of antiviral effects, whereas this pathway apparently plays no role in T-cell-receptor (TCR)-dependent gene expression. Conversely, signal transduction through the TCR requires the tyrosine kinases Lck and ZAP-70 and the tyrosine phosphatase CD45. Here we show that, unexpectedly, transmission of growth-inhibitory signals by interferon-alpha (IFN-alpha) in T cells requires the expression and association of CD45, Lck and ZAP-70 with the IFN-alpha-receptor signalling complex.

Regulation of ZAP-70 intracellular localization: visualization with the green fluorescent protein.

To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

ZAP-70 tyrosine kinase is required for the up-regulation of Fas ligand in activation-induced T cell apoptosis.

Activation-induced cell death (AICD) is initiated by the TCR-dependent up-regulation of Fas ligand (FasL) mRNA. The subsequently generated soluble or cell-associated FasL gene products bind Fas, leading to apoptosis of the T cells. Although TCR stimulation is essential to initiate AICD, little is known about which TCR-initiated second messengers are required for FasL expression. We provide evidence in this work that T cells lacking the tyrosine kinase ZAP-70 are unable to up-regulate FasL and undergo AICD. Transfection of wild-type ZAP-70 into the ZAP-70-deficient T cells restores their sensitivity to TCR-induced apoptosis, whereas transfection of catalytically inactive ZAP-70 does not. These results provide clear evidence that ZAP-70 tyrosine kinase is essential in up-regulating FasL for TCR-induced apoptosis.

Sequential involvement of Lck and SHP-1 with MHC-recognizing receptors on NK cells inhibits FcR-initiated tyrosine kinase activation.

Recognition of major histocompatibility (MHC) class I complexes on target cells by killer cell inhibitory receptors (KIR) blocks natural killer (NK) and T cell cytotoxic function. The inhibitory effect of KIR ligation requires the phosphotyrosine-dependent association of KIR with the cytoplasmic SH2-containing protein tyrosine phosphatase SHP-1. Using a somatic genetic model, we first define a requirement for the Src family protein tyrosine kinase (PTK) Lck in mediating KIR tyrosine phosphorylation. We then investigate how KIR ligation interrupts PTK-dependent NK cell activation signals. Specifically, we show that KIR ligation inhibits the Fc receptor (FcR)-induced tyrosine phosphorylation of the FcR-associated zeta signaling chain, the PTK ZAP-70, and phospholipase C gamma. Overexpression of catalytically inactive SHP-1 (acting as a dominant negative) restores the tyrosine phosphorylation of these signaling events and reverses KIR-mediated inhibition of NK cell cytotoxic function. These results suggest sequential roles for Lck and SHP-1 in the inhibition of PTK following MHC recognition by NK cells.

Characterization of the human XPA promoter.

We cloned the human xeroderma pigmentosum group A gene (XPA) and characterized the XPA promoter (pXPA) by transient cat expression. The pXPA is extraordinarily weak in human fibroblasts (1% of RSV-LTR) and appears to function without any of the usual promoter elements. Regions containing positive and negative control elements were localized.

Global intellectual deficits in cystinosis.

Fourteen families of children with infantile nephropathic cystinosis were evaluated using the Stanford-Binet Intelligence Scale, Fourth Edition [Thorndike et al., 1986: Stanford-Binet Intelligence Scale, Fourth Ed.]. The IQs of 15 children with cystinosis, their 23 sibs and 24 parents were compared in order to evaluate a potential effect of cystinosis on intelligence. Children with cystinosis had a significantly lower mean IQ than their sibs and their parents (P = .001). Thus, even though the mean IQ of the children with cystinosis (94.4 +/- 10) was within the average range, there is evidence that these children have a mild global intellectual deficit relative to their expected IQ based upon the IQs of other relatives. In addition, to a subset of the subjects we administered a measure of scholastic ability, the Wide Range Achievement Test-Revised [Jastak and Wilkinson, 1984: The Wide Range Achievement Test-Revised], which consists of spelling, reading, and arithmetic subtests. The 11 cystinosis subjects scored significantly lower (P = .01) than their 16 sibs and their 14 parents in the area of spelling, whereas they did not significantly differ in their performance in the areas of reading and arithmetic.

The effect of day of the estrous cycle, location of ovulatory structure, and progesterone on in vitro bovine endometrial secretions.

Endometrial tissue was collected by biopsy from mature Holstein cows either on d 0 (estrus) or on d 9, 14, or 18 of the estrous cycle to determine the effects of day of the cycle, uterine horn, and in vitro progesterone on endometrial protein secretion. Tissue was incubated for 24 h in supplemented media containing 14C amino acids and either 0, 4.7, or 47 ng of progesterone/ml. Media were analyzed for total protein, radiolabeled protein, and profile of protein released during incubation. Endometrial tissue at d 0 released more protein than did tissue collected on all other days. Radiolabeled proteins were greater on d 0 and 18 than on d 9 and 14. Endometrium from the uterine horn contralateral to the corpus luteum released more radiolabeled protein than endometrium from the uterine horn ipsilateral to the corpus luteum. Seventeen protein bands were identified by electrophoresis. Proximity of the uterine horn to the site of ovulation affected the distribution of specific bands 21,400, 55,000, 74,600, and 88,100 molecular weight. The proportion of released proteins represented by proteins of molecular weights 12,700, 19,100, 21,400, 32,000, and 66,500 was affected either by day of the estrous cycle, proximity of uterine horn to the site of ovulation, or progesterone concentration. The results show that day of the estrous cycle and uterine horn not only alter overall endometrial protein secretion and synthetic activity but also have specific effects on distribution of individual proteins.

Influence of cornual insemination on endometrial damage and microbial flora in the bovine uterus.

The objectives were to determine effects of cornual insemination on endometrial characteristics and uterine bacterial flora. Forty cows were assigned randomly (10/group) to each of the following treatments: 1) control--no uterine manipulation; 2) uterine body insemination--deposition of the inseminate at the internal cervical os; 3) cornual insemination--one-half of inseminate deposited into each uterine horn anterior to the internal bifurcation and 4) cornual insemination plus endometrial biopsy--insemination into each uterine horn and trauma induced by endometrial biopsy. Animals were slaughtered and reproductive tracts were removed 5 d after treatment. Bacterial populations of the uterine lumen were evaluated by both flushings and swabbings of each horn. Treatment did not influence the presence of luminal bacteria as determined by flushing or swab samples. Gross endometrial characteristics were quantitated in all tracts. Characteristics considered were total endometrial surface area, discolored regions that appeared to be distinctly darker than the remaining portion of the endometrium and obvious surface damage. Discolored regions were evaluated as both total areas and percentage of endometrium. Treatment did not influence total or percent discolored areas. Microscopic evaluation of these tissues revealed increased edema when compared with nondiscolored regions but no disruption of the luminal epithelium was found. Surface damage was found only in the biopsied treatment group. Observation of the endometrium revealed that extensive regeneration occurred by 5 d after treatment. The data suggest that cornual insemination does not result in uterine trauma or bacterial presence 5 d after breeding, and the endometrium is capable of regeneration after trauma during estrus.

Use of an immunoperoxidase method for identification of Bacteroides fragilis.

An indirect immunoperoxidase (IP) slide test was evaluated for the laboratory identification of Bacteroides fragilis. Antigen-antibody complexes were detected with goat anti-rabbit immunoglobulin G-peroxidase conjugate with 3-amino-9-ethyl-carbazole as the peroxidase substrate. Ninety-one percent of 44 B. fragilis strains tested were IP positive (3+ to 4+ reactions) with greater than or equal to 1:160 dilutions of rabbit antiserum produced against whole cells of B. fragilis ATCC 23745. The antiserum was species specific. No cross-reactions were observed with 35 Bacteroides strains of other species or with a variety of facultative or aerobic gram-negative bacilli. Four B. fragilis strains were IP negative. One of these (VPI 2393) was the deoxyribonucleic acid (DNA) homology group II reference strain. The other three were clinical isolates. IP-negative and representative IP-positive strains were tested for DNA homology with the type strains for DNA homology groups I and II (VPI 2553 and VPI 2393). Two of the three clinical isolates were classified as DNA homology group II, and the remaining strain was classified as a group I. Capsular material known to be unique to B. fragilis was common to both DNA homology groups as indicated by reactions with purified anticapsular antiserum. The IP technique provides a suitable alternative to fluorescent microscopy for the rapid immunological identification of B. fragilis.