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A research study was undertaken to examine the working practices of four bridewells in Hampshire in relation to mentally disordered offenders (MDOs) and diversion services. A consecutive sample of individuals detained in cells and not identified by the police as having a mental disorder were screened for the presence of mental disorder and their suitability for diversion. Custody and detention staff were observed and interviewed to elicit their views and working practices in relation to MDOs. The findings revealed that in bridewells with diversion schemes an average of about 7% of detained individuals had mental disorders who were suitable for diversion but were not detected by the police. In the bridewell without a diversion scheme the figure was 14%. Conversely, many individuals without a formal mental disorder were inappropriately referred to diversion schemes. The effectiveness of screening processes by custody staff was variable. Facilities in the bridewells were not suitable for containing disturbed individuals. Delays in obtaining mental health assessments caused considerable concern for police officers and prolonged the discomfort of vulnerable individuals. Further preparation and training of custody staff is needed to improve screening procedures. Reception and detention facilities for mentally disordered individuals should be reviewed and response times for approved social workers (ASWs) and psychiatrists would benefit from improvement.

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beta-Amyloid protein has been implicated as a potential causative agent in the neuropathology associated with Alzheimer's disease. This possibility is supported by observations that beta-amyloid induces neuronal degeneration and astrocyte reactivity in vitro by as yet undefined mechanism(s). In this report, we present data demonstrating that the pathological effects of beta-amyloid on cultured cells are modulated by activation of the thrombin receptor. At concentrations between 50 and 500 nM, thrombin pretreatment significantly attenuates neurotoxicity mediated by fibrillar aggregates of beta 1-42 and beta 25-35 peptides. In cultured astrocytes, the stellate morphology induced by beta 1-42 and beta 25-35 aggregates can be prevented and reversed by thrombin exposures between 10 pM and 1 microM. In contrast, thrombin potentiates rather than attenuates the beta-amyloid-induced increased expression of basic fibroblast growth factor, suggesting that thrombin differentially modulates the effects of beta-amyloid on astrocytes. Thrombin's effects on both neurons and astrocytes are mimicked by thrombin receptor-activating peptide and inhibited by two potent thrombin inhibitors, hirudin and protease nexin-1. These data provide both new insight into the signaling pathways underlying the cellular effects of beta-amyloid and additional support for the role of thrombin as an important mediator of neuropathological events.

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Thrombin is a multifunctional serine protease that is rapidly produced from prothrombin at sites of tissue injury and catalyzes the final steps in blood coagulation. Thrombin also regulates gene expression and process outgrowth in neurons and astrocytes and stimulates proliferation of astrocytes. Since thrombin is produced immediately upon breakdown of the blood-brain barrier we examined its effects on astrocytes and neurons cultured under conditions which resemble those found in vivo following cerebrovascular injury. These studies showed that thrombin markedly protected rat primary astrocytes from cell death induced by hypoglycemia or oxidative stress. Thrombin also protected rat primary hippocampal neurons from cell death produced by hypoglycemia or growth supplement deprivation. Synthetic peptides which directly activate the thrombin receptor also protected astrocytes and neurons from these environmental insults, demonstrating that the thrombin effects were mediated through the thrombin receptor. In contrast to these results with stressed cells, high concentrations of thrombin killed both astrocytes and neurons cultured under normal conditions. All of the effects of thrombin on astrocytes and neurons were blocked by the brain thrombin inhibitor, protease nexin-1 (PN-1). This shows that the effects required the proteolytic activity of thrombin and is consistent with the known proteolytic mechanism by which thrombin activates its receptor. These results indicate that thrombin and PN-1 may regulate the viability of both astrocytes and neurons in early moments following trauma to the CNS or other conditions that alter the blood-brain barrier.

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A marked and significant reduction of protease nexin-1 (PN-1) and PN-2/amyloid beta protein precursor (A beta PP) was observed in selected regions of Alzheimer's disease (AD) brains as compared to those of aged-matched controls. Correlative analysis indicated a relationship between PN-1 reduction and the severity of pathologic alterations. A statistically significant inverse correlation was noted between the level of PN-1 activity and the density of tau-positive dystrophic neurites in the hippocampus. In view of the ability of thrombin and PN-1 activity to regulate neurite outgrowth, it is possible that abnormal thrombin and PN-1 interactions may play a role in dystrophic neurite formation. The presence of clusters of dystrophic neurites around the capillaries suggests that blood-brain barrier (BBB) dysfunction may enhance such abnormal interactions. The decrease in PN-2/A beta PP levels in AD brains could possibly contribute to neuronal degeneration in AD in view of the ability of PN-2/A beta PP to protect neurons against the toxic effects of the A beta.

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The clotting protease thrombin might contribute to the pathophysiology of central nervous system (CNS) injury and certain diseases by its ability to retract processes on neurons and astrocytes and to stimulate astrocyte proliferation. Protease nexin-1 (PN-1) is a 43 kDa thrombin inhibitor found predominantly in the brain where much of it resides around capillaries and large blood vessels. This location of PN-1 prompted the hypothesis that it may play a protective role against extravasated thrombin released following cerebrovascular injury or under certain pathological conditions. Recent studies indicated that the levels of PN-1 are markedly reduced in the postmortem brains of patients with Alzheimer's disease (AD). It was suggested that this reduction in PN-1 levels was due to the sequestration of PN-1 by extravasated thrombin. In the present study we examined the specific nature of this reduction by immunohistochemical staining of sections from control and AD brains using PN-1 specific antibodies. We show that the levels of PN-1 immunoreactivity around blood vessels and the number of blood vessels exhibiting PN-1 immunoreactivity were markedly reduced in the brains of patients with AD compared to age-matched controls; this reduction was reflected by a decrease in the levels of PN-1 activity and PN-1 protein. Thus an imbalance between PN-1 and thrombin may be a contributing factor in the pathology of AD.

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Recent studies have shown that serine protease inhibitors can be regulated in their activity, specificity, and location by glycoprotein or extracellular matrix (ECM) co-factors. Protease nexin-1 (PN-1) is a member of the serpin superfamily of serine protease inhibitors which can rapidly inhibit thrombin, urokinase, and plasmin. PN-1 binds tightly to and is regulated by the ECM. This interaction accelerates the inhibition of thrombin by PN-1 and blocks urokinase and plasmin inhibition by PN-1. Previous work showed that heparan sulfate proteoglycan is largely responsible for the acceleration of thrombin inhibition by PN-1. Our current studies were directed at identifying ECM component(s) that decreased the ability of PN-1 to inhibit urokinase and plasmin. These studies showed that collagen type IV decreased the formation of SDS-stable complexes between urokinase or plasmin and PN-1 without affecting formation of complexes between thrombin and PN-1. The second order rate constant for inhibition of urokinase by PN-1 was markedly decreased with increasing collagen type IV, whereas the second order rate constant for inhibition of thrombin by PN-1 was unaffected by addition of collagen type IV. Other ECM components (collagen type I, vitronectin, fibronectin, and heat-denatured collagen type IV) did not affect complex formation or the rate of inhibition of proteases by PN-1, indicating that these effects were specific to collagen type IV. Binding of PN-1 to immobilized collagen type IV was demonstrated using an enzyme-linked immunosorbent assay; the concentration of PN-1 necessary to obtain 50% saturation of the immobilized collagen type IV binding sites was approximately 15 nM. Collagen type IV was also copurified with PN-1 from fibroblast-conditioned medium. These results demonstrate a novel regulation of serpin specificity in which an ECM co-factor decreased the inhibition of certain proteases by the serpin without affecting the inhibition of its target protease.

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The clotting protease thrombin might contribute to cell damage following brain injury by its ability to retract processes on neurons and astrocytes. Protease nexin-1 (PN-1), a potent inhibitor of thrombin, is localized around cerebral blood vessels where it may protect these cells from extravasated thrombin during injury or alteration of the blood-brain barrier. Here we examined the effects of several injury-related factors on the regulation of PN-1 in cultured brain cells. Interleukin-1, tumor necrosis factor-alpha, and transforming growth factor-beta stimulated the secretion of PN-1 by the neuroblastoma cell line SK-N-SH. This cell line comprises both neuronal and glial cells. Analyses using cloned derivatives of these two cell types showed that PN-1 was secreted by the glial cells; PN-1 secretion was stimulated 90-fold by interleukin-1, 15-fold by tumor necrosis factor-alpha, 10-fold by tumor growth factor-beta, and 4-fold by platelet-derived growth factor. Measurements of newly synthesised PN-1 demonstrated that these factors produced an equivalent stimulation of PN-1 synthesis. The neuronal cells secreted two thrombin-binding proteins distinct from PN-1. Interactions between these two cell types regulated the secretion of PN-1 and the two thrombin-binding proteins.

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The herpes simplex virus type 1 (HSV-1) immediate-early regulatory proteins ICP27 and ICP0 each encode putative zinc-finger metal-binding domains. We utilized the technique of metal chelate affinity chromatography to demonstrate that ICP27 and ICP0 were able to bind to zinc in vitro. This property was further exploited to purify ICP27 from extracts of HSV-1-infected cells. The purification procedure also revealed that ICP27 possessed single-stranded DNA-binding activity. Analysis of ICP27 truncated peptides produced by in vitro translation verified that the zinc-binding region of ICP27 resides in the carboxy terminal 105 amino acids spanning the putative metal binding motif. However, a specific configuration of cysteine and histidine residues in this region was not required for binding to occur as demonstrated by the ability of a frame-shift mutation to bind with an efficiency similar to wild type. The mutated peptide retained four histidine and cysteine residues but in a configuration different from the consensus proposed for zinc-finger motifs. Therefore, while the region spanning the metal binding domain of ICP27 is essential for both the activator and repressor functions, and ICP27 binds zinc in vitro, it is not clear whether zinc binding in vivo is necessary for function.

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The herpes simplex virus type 1 (HSV-1) alpha or immediate-early proteins ICP4 (IE175), ICP0 (IE110), and ICP27 (IE63) are trans-acting proteins which affect HSV-1 gene expression. We previously showed that ICP27 in combination with ICP4 and ICP0 could act as a repressor or an activator in transfection assays, depending on the target gene (R. E. Sekulovich, K. Leary, and R. M. Sandri-Goldin, J. Virol. 62:4510-4522, 1988). To investigate the regions of the ICP27 protein which specify these functions, we constructed a series of in-frame insertion and deletion mutants in the ICP27 gene. These mutants were analyzed in transient expression assays for the ability to repress or to activate two different target genes. The target plasmids used consisted of the promoter regions from the HSV-1 beta or early gene which encodes thymidine kinase and from the beta-gamma or leaky late gene. VP5, which encodes the major capsid protein, each fused to the chloramphenicol acetyltransferase gene. Our previous studies showed that induction of pTK-CAT expression by ICP4 and ICP0 was repressed by ICP27, whereas the stimulation of pVP5-CAT expression seen with ICP4 and ICP0 was significantly increased when ICP27 was also added. In this study, a series of transfection assays was performed with each of the ICP27 mutant plasmids in combination with plasmids containing the ICP4 and ICP0 genes with each target. The results of these experiments showed that mutants containing insertions or deletions in the region from amino acids 262 to 406 in the carboxy-terminal half of the protein were unable to stimulate expression of pVP5-CAT but were able to repress induction of pTK-CAT activity by ICP4 and ICP0. Mutants in the carboxy-terminal 78 amino acids lost both activities; that is, these mutants did not show repression of pTK-CAT activity or stimulation of pVP5-CAT activity, whereas mutants in the hydrophilic amino-terminal half of ICP27 were able to perform both functions. These results show that the carboxy-terminal half of ICP27 is important for the activation and repression functions. Furthermore, the carboxy-terminal 62 amino acids are required for the repressor activity, because mutants with this region intact were able to repress. Analysis of the DNA sequence showed that there are a number of cysteine and histidine residues encoded by this region which have some similarity to zinc finger metal-binding regions found in other eucaryotic regulatory proteins. These results suggest that the structural integrity of this region is important for the function of ICP27.

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Purified preparations of herpes simplex virus type 2 DNA polymerase made by many different laboratories always contain at least two polypeptides. The major one, of about 150,000 molecular weight, has been associated with the polymerase activity. The second protein, of about 54,000 molecular weight, which we previously designated ICSP 34, 35, has now been purified. The purified protein has been used to prepare antisera (both polyclonal rabbit serum and monoclonal antibodies). These reagents have been used to characterize the protein, to demonstrate its quite distinct map location from that of the DNA polymerase on the herpes simplex virus genome, and to demonstrate the close association between the two polypeptides.

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Monoclonal antibodies directed against several herpes simplex virus (HSV)-induced DNA-binding proteins were used to investigate protein interactions in HSV-infected cells. Q1 monoclonal antibody, which is specific for the HSV-induced alkaline nuclease, when used in an immunoadsorbant column resulted in the purification of the alkaline nuclease, to which large quantities of the major DNA-binding protein were bound. Conversely, when a monoclonal antibody to the major DNA-binding protein was used in affinity chromatography other polypeptides (including the DNA polymerase and alkaline nuclease) were eluted in addition to the major DNA-binding protein. Similar results were obtained when the experiment was performed using a monoclonal antibody to another HSV-2 DNA-binding protein. These results suggest the possibility that these polypeptides interact as part of the HSV DNA replication complex, and this hypothesis is discussed.

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Purified herpes simplex virus thymidine kinase has been used to immunize mice for the production of monoclonal antibodies to the enzyme. Monoclonal antibodies were successfully produced against both herpes simplex virus type 1 and type 2 enzymes. These antibodies should prove useful for detecting the enzyme under a variety of experimental conditions. We also demonstrate that the antibodies can provide an alternative method for obtaining large amounts of purified thymidine kinase.

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