Following a study of oxidative tryptophan metabolism to kynurenines, we have now analysed the blood of patients with either Huntington's disease or traumatic brain injury for levels of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and melatonin. There were no differences in the baseline levels of these compounds between patients and healthy controls. Tryptophan depletion did not reduce 5-HT levels in either the controls or in the patients with Huntington's disease, but it increased 5-HT levels in patients with brain injury and lowered 5-HIAA in the control and Huntington's disease groups. An oral tryptophan load did not modify 5-HT levels in the patients but increased 5-HT in control subjects. The tryptophan load restored 5-HIAA to baseline levels in controls and patients with brain injury, but not in those with Huntington's disease, in whom 5-HIAA remained significantly depressed. Melatonin levels increased on tryptophan loading in all subjects, with levels in patients with brain injury increasing significantly more than in controls. Baseline levels of neopterin and lipid peroxidation products were higher in patients than in controls. It is concluded that both groups of patients exhibit abnormalities in tryptophan metabolism, which may be related to increased inflammatory status and oxidative stress. Interactions between the kynurenine, 5-HT and melatonin pathways should be considered when interpreting changes of tryptophan metabolism.

The kynurenine pathway generates the excitotoxic N-methyl-(D)-aspartate receptor agonist, quinolinic acid and the glutamate antagonist, kynurenic acid, as well as free-radical generators. We investigated the status of the pathway following severe brain injury sustained at least 1 year previously in 15 patients compared with controls. At baseline, patients with brain injury showed increased levels of neopterin, erythrocyte sedimentation rate, C-reactive protein and peroxidation products in the blood compared with controls, indicating persistent inflammation and oxidative stress. At baseline and following tryptophan depletion, more tryptophan was converted to kynurenine in patients than controls, but less kynurenine was converted into the neuroprotectant, kynurenic acid. This suggests that neuroprotection by kynurenic acid may be inadequate in brain-damaged patients even many years after injury. On tryptophan loading, patients metabolized more kynurenine into kynurenic acid than controls, a process which may be neuroprotective. In addition, lower levels of 3-hydroxykynurenine and 3-hydroxyanthranilic acid in patients after tryptophan loading should be protective since these compounds generate free radicals. The results suggest that for brain-damaged patients, increased activation of the kynurenine pathway, oxidative stress and raised levels of inflammation continue many years after the original insult, possibly contributing to the continuing cerebral dysfunction in these patients.

Abnormalities in the kynurenine pathway may play a role in Huntington's disease (HD). In this study, tryptophan depletion and loading were used to investigate changes in blood kynurenine pathway metabolites, as well as markers of inflammation and oxidative stress in HD patients and healthy controls. Results showed that the kynurenine : tryptophan ratio was greater in HD than controls in the baseline state and after tryptophan depletion, indicating increased indoleamine dioxygenase activity in HD. Evidence for persistent inflammation in HD was provided by elevated baseline levels of C-reactive protein, neopterin and lipid peroxidation products compared with controls. The kynurenate : kynurenine ratio suggested lower kynurenine aminotransferase activity in patients and the higher levels of kynurenine in patients at baseline, after depletion and loading, do not result in any differences in kynurenic acid levels, providing no supportive evidence for a compensatory neuroprotective role for kynurenic acid. Quinolinic acid showed wide variations in blood levels. The lipid peroxidation data indicate a high level of oxidative stress in HD patients many years after disease onset. Levels of the free radical generators 3-hydroxykynurenine and 3-hydroxyanthranilic acid were decreased in HD patients, and hence did not appear to contribute to the oxidative stress. It is concluded that patients with HD exhibit abnormal handling of tryptophan metabolism and increased oxidative stress, and that these factors could contribute to ongoing brain dysfunction.

In previous studies tryptophan loads have been administered to human subjects in order to raise central levels of 5-hydroxytryptamine (5HT) and assess the effects of 5HT on behaviour and mood. However, tryptophan is metabolised primarily along the oxidative kynurenine pathway. In this study a 6 g oral tryptophan load was administered to 15 healthy volunteers and the levels of kynurenines and lipid peroxidation products (indicative of oxidative stress) were measured. The results demonstrate that tryptophan loading produces a highly significant increase in lipid peroxidation products in parallel with increased kynurenines. The oxidative stress may result from the generation of quinolinic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid, all of which are known to have the ability to generate free radicals. The results may have implications for the use of tryptophan loading in psychiatric practice, and for the chronic use of diets high in tryptophan.

The extracellular signal-regulated kinase (ERK) signaling pathway is strongly activated in response to TCR stimulation in normal T cells. However, the extent to which activation of the ERK pathway is necessary for TCR-stimulated cytokine production is not clear. We have addressed this question by use of two separate methods to interfere with TCR activation of the ERK cascade. The first approach utilized transient expression of a catalytically inactive form of mitogen-activated/ERK 1 (CI-MEK1), while the second involved using the MEK1- and MEK2-specific inhibitor PD98059 to block ERK activation by the TCR. In order to assess the requirement for ERK activation in T cell cytokine production, we have measured the effect of ERK inhibition upon the production of six cytokines, IL-3, IL-4, IL-5, IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and IFN-gamma, by newly activated normal mouse T cells in response to TCR stimulation. The results of experiments using both methods to block ERK activation have revealed a requirement for intact ERK signaling for the full elicitation of TCR-stimulated cytokine production. Dose-response analyses using the MEK inhibitor PD98059 showed that the TCR-stimulated production of all cytokines measured was affected by this treatment. However, the production of IL-3 and IL-4 was only partially dependent upon ERK activation, whereas IL-5, IL-10, IFN-gamma and GM-CSF production was severely affected by diminished ERK activation. We conclude that the ERK pathway is differentially involved in the activation of different cytokine genes in normal T cells.

We have characterised the promoters of the human and murine VRF (vascular endothelial growth factor (VEGF) related factor) gene. A series of deletions were made of a 553-bp region 5' of the VRF initiation codon and were used in a luciferase reporter gene assay to determine the minimal promoter of the VRF gene. The region between base pairs -443 and -195 was sufficient to mediate transcription in lymphocytes and the region between -550 and -443 enhanced this promoter activity. Primer extension studies identified two regions of transcription initiation, both of which are preceded by Sp1, AP-2 and Egr-1 transcription factor binding sites. The VRF promoter is similar to VEGF in that it is associated with a CpG island, contains sites for Sp1 and AP-2, and lacks a TATA box. However, it has marked differences in that the promoter contains Egr-1 sites and lacks both hypoxia-inducible factor-1 and AP-1 sites. These data may indicate that expression of these two growth factors is regulated by different physiological stimuli.

We previously showed that T cells from the mediastinal lymph nodes (MLN) and lung parenchyma of influenza virus-infected mice were functionally remarkably different. Here we demonstrate that the differences in cytokine production are due to differences in the frequencies of T cells within the activated pool able to produce cytokines after TCR stimulation. FACS analysis of T cells from MLN and lung tissue demonstrated that T cells expressing any of the activation markers tested (LFA-1, CD25, CD44, CD45RB, CD49d, CD62L) always expressed high levels of CD44 and LFA-1. These double-high T cells produced >99% of all anti-CD3 mAb-induced IL-4 and IFN-gamma. Separation of T cells employing mAb against the other activation markers in combination with anti-CD44 mAb did not enable further fractionation into cytokine producers and nonproducers. Despite their similar phenotype, purified double-high lung parenchyma T cells produced markedly higher levels of IL-2, IL-4, and IFN-gamma, and contained a higher frequency of cytokine producers than their MLN counterparts. Activation of the extracellular signal-regulated kinase (ERK)-2 in response to TCR cross-linking was detected in double-high T cells from lung tissue but not MLN. The requirement for ERK signaling for maximal IFN-gamma synthesis could nevertheless be demonstrated in both populations by blockade with the inhibitor PD98509. Collectively, the data suggest that inductive and effector sites differ in the frequency of activated T cells able to induce ERK-2-regulated cytokine production after TCR ligation.

The yeast genome sequencing project predicts an open reading frame (YKL073) that would encode a novel member of the Hsp70 family of molecular chaperones. We report that this 881 codon reading frame represents a functional gene expressing a 113-119 kDa glycoprotein localized within the lumen of the endoplasmic reticulum (ER). We therefore propose to designate this gene LHS1 (Lumenal Hsp Seventy). Our studies indicate that LHS1 is regulated by the unfolded protein response pathway, as evidenced by its transcriptional induction in cells treated with tunicamycin, and in various mutants defective in precursor processing (sec11-7, sec53-6 and sec59-1). LHS1 is not essential for viability, but an Lhs1 null mutant strain exhibits a coordinated induction of genes regulated by the unfolded protein response indicating a role for Lhs1p in protein folding in the ER. Furthermore, the null mutation is synthetically lethal in combination with (delta)ire1, thus activation of the unfolded protein response pathway is essential for cells to tolerate loss of Lhs1p. Synthetically lethality is also seen with mutations in KAR2, strongly suggesting that Kar2p and Lhs1p have overlapping functions. The Lhs1 null mutant exhibits a severe constitutive defect in the translocation of several secretory preproteins. We therefore propose that Lhs1p is a molecular chaperone of the ER lumen involved in both polypeptide translocation and subsequent protein folding.

Stimulation of T cells via the T cell receptor (TCR) activates a number of signaling pathways that are potentially involved in the elicitation of physiological responses, such as the production of cytokines. The extracellular signal‐regulated kinases (ERK) are a group of molecules activated in response to TCR ligation, whose role in T cell cytokine production is controversial. In this study, we have asked whether ERK activation is coupled to the production of a number of T cell‐derived cytokines, and whether particular cytokines are differentially affected by ERK activation. To address these questions, we have utilized a constitutively active version of the immediate upstream activator of both ERK1 and ERK2, mitogen‐activated/extracellular signal‐regulated kinase 1 (MEK1), to activate ERK signaling selectively in the absence of other TCR‐activated signaling pathways. The effect of constitutive MEK/ERK activation on T cell cytokine production was measured by transiently co‐transfecting newly activated mouse T cells with DNA encoding constitutively active MEK1 (CA‐MEK1) and the human interleukin‐2 (IL‐2) receptor α chain (hCD25), purifying hCD25+ transfectants by flow‐cytometric cell sorting, and measuring the production of IL‐3, IL‐4, interferon (IFN)‐γ and granulocyte/macrophage‐colony‐stimulating factor (GM‐CSF) either in the presence or absence of ionomycin stimulation. Newly activated T cells were used in these experiments as they more closely resemble T cells activated in vivo than do transformed T cells or long‐term established T cell clones. CA‐MEK1 expression led to constitutive ERK activation, which acted synergystically with ionomycin treatment to stimulate cytokine production. Furthermore, these experiments revealed a hierarchy of cytokine responsiveness to MEK/ERK activation, such that the production of IL‐3 was most affected, followed by GM‐CSF, IFN‐γ, and IL‐4.

T cell antigen receptor (TCR) ligation results in the tyrosine phosphorylation of numerous intracellular protein substrates, and the identification of these substrates has been a major undertaking by several groups. We have used pervanadate treatment to artificially increase cellular phosphotyrosine levels and immobilized anti-phosphotyrosine monoclonal antibodies to partially purify tyrosine phosphorylated proteins in quantities suitable for amino acid sequencing. This strategy was used to identify three phosphotyrosine containing proteins, with relative molecular masses of 105, 81, and 76 kD by amino acid sequencing. Here we report the identification of pp105 as alpha-actinin-1, pp81 as the murine equivalent of the HS1 gene product, and pp76 as Hsc70. This is the first report that alpha-actinin-1 and Hsc70 are targets of activated tyrosine kinases. Furthermore, we show that Hsc70 is tyrosine phosphorylated in response to TCR ligation, which constitutes the first evidence that Hsc70 might be subject to regulation by tyrosine kinase signaling pathways.

Anchoring of proteins to membranes by glycosylphosphatidylinositols (GPIs) is ubiquitous among all eukaryotes and heavily used by parasitic protozoa. GPI is synthesized and transferred en bloc to form GPI-anchored proteins. The key enzyme in this process is a putative GPI:protein transamidase that would cleave a peptide bond near the COOH terminus of the protein and attach the GPI by an amide linkage. We have identified a gene, GAA1, encoding an essential ER protein required for GPI anchoring. gaal mutant cells synthesize the complete GPI anchor precursor at nonpermissive temperatures, but do not attach it to proteins. Overexpression of GAA1 improves the ability of cells to attach anchors to a GPI-anchored protein with a mutant anchor attachment site. Therefore, Gaa1p is required for a terminal step of GPI anchor attachment and could be part of the putative GPI:protein transamidase.

Engagement of the T cell antigen receptor (TCR) leads to activation of multiple tyrosine kinases and rapid tyrosine phosphorylation of intracellular protein substrates. A number of these substrates have been identified and they include TCR subunits, phospholipase C-gamma1, p95vav, and ezrin. In a recent study we have demonstrated that VCP (valosin-containing protein) becomes tyrosine phosphorylated upon TCR cross-linking. Analysis of the predicted amino acid sequence of this protein indicates that it is a member of a family of oligomeric proteins containing duplicated domains with predicted ATPase activity. In the current study we determine the site of tyrosine phosphorylation in VCP, demonstrate that murine VCP indeed is an oligomeric ATPase, and show that the tyrosine phosphorylation of the protein has no effect on VCP ATPase activity. Recent evidence suggests that VCP associates with clathrin. A possible role of tyrosine phosphorylation in regulating this protein-protein interaction is discussed.

Data from two case control studies in Oxfordshire were combined and analysed. The combined study covered 1940 subjects, 723 cases, and 1217 controls, between the ages of 50 and 79 with a response rate of 97% for cases and 94% for controls. Diabetes was shown to be a powerful and highly significant risk factor for cataract with a relative risk of 5.04. More than 11% of cataracts in Oxfordshire are attributable to diabetes. The relative risk did not increase significantly with age within the range 50 to 79 years but was higher in females than in males. For females with diabetes the relative risk was 7.85 with 95% confidence interval from 4.30 to 14.3 compared with 3.42 with confidence interval from 2.05 to 5.71 for males with diabetes. Diabetes remained a powerful risk factor when other identified risk factors had been controlled for. No known mechanism for the development of diabetic complications provides an explanation for the excess risk in females. Combination of the two studies led to better estimates of the relative risk of glaucoma as a risk factor for cataract (3.96 with 95% confidence interval from 2.35 to 6.68). The relative risk appeared to be greater in women than in men but this difference was not statistically significant. There was no significant change in risk with age. Glaucoma is a powerful and independent risk factor for cataract in both sexes and may be responsible for 5% of all cataracts in our area.

It is now well accepted that T cell antigen receptor (TCR) engagement induces a cascade of protein phosphorylation with activation of both tyrosine and serine/threonine kinases. The most proximal events in this cascade appear to be the tyrosine phosphorylation of multiple intracellular proteins. This paper summarizes new data from our laboratory and places it in the context of observations made over the past several years. The initial problems that we and others have focused on are the identification and characterization of the kinases, phosphatases, and substrates that make up the elements of this pathway. With this information in hand, one can begin to understand the mechanism of signal transduction at a molecular level.