Foodborne botulism, a forgotten yet life-threatening disease: a case report.

OBJECTIVE: Botulism is a very rare disease in Switzerland, with less than one case per year, an incidence of 0.01 cases for 100,000 inhabitants. Indeed, over the past ten years, 9 cases have been reported to Public Health registry. Foodborne botulism (FB) is caused by ingestion of preformed botulinum neurotoxin. Characteristic features should be rapidly recognized, and prompt treatment should be administered to avoid further progression towards respiratory failure and death. CASE REPORT: We report the case of a patient who developed gastrointestinal symptoms just after a sandwich consumption followed by rapidly progressive cranial nerve impairment, truncal muscle weakness in a descending pattern and respiratory failure requiring mechanical ventilation. The diagnosis of foodborne botulism was delayed due to differential diagnosis considerations. Specific antitoxin therapy was administered immediately after firm clinical conviction of botulism, without waiting for serologic results that later confirmed the diagnosis. As expected, muscle weakness recovery was slow, with persistent chronic deficits nine years later. CONCLUSIONS: This case highlights differential diagnosis issues of botulism. These include acute neuromuscular disorders such as myasthenia gravis, Guillain-Barré syndrome, or tick-borne encephalitis. The importance of careful medical history and repeated clinical evaluation to avoid misdiagnosis can be lifesaving. Our case highlights the typical warning signs.

Focal adhesion kinase and associated proteins.

Focal adhesion kinase (pp125FAK), a protein tyrosine kinase, has recently been suggested to regulate aspects of signalling induced by integrins and by certain growth factor and hormone receptors. New studies indicate that the domains of pp125FAK regulate its interactions with integrin subunits, other protein tyrosine kinases, and the focal adhesion associated protein paxillin. Through these interactions, pp125FAK may regulate signalling via different extracellular ligands.

Focal adhesion kinase: an integrin-linked protein tyrosine kinase.

Recent evidence has shown that binding of integrins to components of the extracellular matrix serves to initiate cellular signals, resulting in changes in cytoskeletal architecture and activation of intracellular signalling pathways. Here we review the evidence that protein tyrosine kinases, including focal-adhesion-associated kinases, play a central role in the generation of some of these molecular signals.

Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions.

The integrin family of heterodimeric cell surface receptors play critical roles in multiple biological processes by mediating cellular adhesion to the extracellular matrix (ECM). Adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation and elevation of [Ca2+]i. The Focal Adhesion Kinase (FAK or pp125FAK), a protein tyrosine kinase that colocalizes with integrins in cellular focal adhesions, is a prime candidate for a mediator of integrin signaling events. Here we report an analysis of the domain structure of FAK in which we have identified a contiguous stretch of 159 amino acids within the COOH terminus essential for correct subcellular localization. When placed in the context of an unrelated cytosolic protein, this Focal Adhesion Targeting (FAT) sequence functions to efficiently mediate the focal adhesion localization of this fusion protein. Furthermore, this analysis suggests that pp125FAK cannot be activated oncogenically by mutation. This result could be explained if pp125FK either exhibits a narrow substrate specificity or is diametrically opposed by cellular phosphatases or other cellular processes.

Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains.

The integrins have recently been implicated in signal transduction. A likely mediator of integrin signaling is focal adhesion kinase (pp125FAK or FAK), a structurally distinct protein tyrosine kinase that becomes enzymatically activated upon engagement of integrins with their ligands. A second candidate signaling molecule is paxillin, a focal adhesion associated, cytoskeletal protein that coordinately becomes phosphorylated on tyrosine upon activation of pp125FAK. Paxillin physically complexes with two protein tyrosine kinases, pp60src and Csk (COOH-terminal src kinase), and the oncoprotein p47gag-crk, each of which could function as part of a paxillin signaling complex. Using an in vitro assay we have established that the cytoplasmic domain of the beta 1 integrin can bind to paxillin and pp125FAK from chicken embryo cell lysates. The NH2-terminal, noncatalytic domain of pp125FAK can bind directly to the cytoplasmic tail of beta 1 and recognizes integrin sequences distinct from those involved in binding to alpha-actinin. Paxillin binding is independent of pp125FAK binding despite the fact that both bind to the same region of beta 1. These results demonstrate that the cytoplasmic domain of the beta subunits of integrins contain binding sites for both signaling molecules and structural proteins suggesting that integrins can coordinate the generation of cytoplasmic signals in addition to their role in anchoring components of the cytoskeleton.

Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets.

We have investigated mechanisms involved in integrin-mediated signal transduction in platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125FAK (FAK, focal adhesion kinase). This kinase was previously shown to be localized in focal adhesions in fibroblasts, and to be phosphorylated on tyrosine in normal and Src-transformed fibroblasts. We show that thrombin and collagen activation of platelets causes an induction of tyrosine phosphorylation of pp125FAK and that pp125FAK molecules isolated from activated platelets display enhanced levels of phosphorylation in immune-complex kinase assays. pp125FAK was not phosphorylated on tyrosine after thrombin or collagen treatment of Glanzmann's thrombasthenic platelets deficient in the fibrinogen receptor GPIIb-IIIa, or of platelets pretreated with an inhibitory monoclonal antibody to GP IIb-IIIa. Fibrinogen binding to GP IIb-IIIa was not sufficient to induce pp125FAK phosphorylation because pp125FAK was not phosphorylated on tyrosine in thrombin-treated platelets that were not allowed to aggregate. These results indicate that tyrosine phosphorylation of pp125FAK is dependent on platelet aggregation mediated by fibrinogen binding to the integrin receptor GP IIb-IIIa. The induction of tyrosine phosphorylation of pp125FAK was inhibited in thrombin- and collagen-treated platelets preincubated with cytochalasin D, which prevents actin polymerization following activation. Under all of these conditions, there was a strong correlation between the induction of tyrosine phosphorylation of pp125FAK in vivo and stimulation of the phosphorylation of pp125FAK in vitro in immune-complex kinase assays. This study provides the first genetic evidence that tyrosine phosphorylation of pp125FAK is dependent on integrin-mediated events, and demonstrates that there is a strong correlation between tyrosine phosphorylation of pp125FAK in platelets, and the activation of pp125FAK-associated phosphorylating activity in vitro.

Thymoma, immunodeficiency, and herpes simplex virus infections.

Hypogammaglobulinemia develops in 3 to 6% of patients with thymoma and this association is commonly referred to as thymoma with immunodeficiency (formerly Good syndrome). Recurrent infections with encapsulated bacteria and opportunistic infections associated with disorders of both humoral and cell mediated immunity frequently occur in this rare primary, adult-onset immunodeficiency. We report a case of thymoma with immunodeficiency complicated by disseminated herpes simplex virus (HSV) infection and review five additional cases of HSV-related infections reported since 1966 in patients presenting with thymoma with immunodeficiency. Patients presented with epiglottitis, keratitis, recurrent genital herpes, ulcerative dermatitis, and acute hepatitis. Four of the six cases had a fatal outcome, two of which were directly attributable to HSV infection. Since the risk of invasive opportunistic infections is high and the presentation atypical, lymphocyte count and total serum immunoglobulin should be measured regularly in all patients presenting with thymoma with immunodeficiency.

pp125FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk.

Paxillin, a focal-adhesion-associated protein, becomes phosphorylated in response to a number of stimuli which also induce the tyrosine phosphorylation of the focal-adhesion-associated protein tyrosine kinase pp125FAK. On the basis of their colocalization and coordinate phosphorylation, paxillin is a candidate for a substrate of pp125FAK. We describe here conditions under which the phosphorylation of paxillin on tyrosine is pp125FAK dependent, supporting the hypothesis that paxillin phosphorylation is regulated by pp125FAK. pp125FAK must localize to focal adhesions and become autophosphorylated to induce paxillin phosphorylation. Phosphorylation of paxillin on tyrosine creates binding sites for the SH2 domains of Crk, Csk, and Src. We identify two sites of phosphorylation as tyrosine residues 31 and 118, each of which conforms to the Crk SH2 domain binding motif, (P)YXXP. These observations suggest that paxillin serves as an adapter protein, similar to insulin receptor substrate 1, and that pp125FAK may regulate the formation of signaling complexes by directing the phosphorylation of paxillin on tyrosine.

Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK.

Integrins play a central role in cellular adhesion and anchorage of the cytoskeleton and participate in the generation of intracellular signals, including tyrosine phosphorylation. We have recently isolated a cDNA encoding a unique, focal adhesion-associated protein tyrosine kinase (FAK) that is a component of an integrin-mediated signal transduction pathway. Here we report the isolation of cDNAs encoding the C-terminal, noncatalytic domain of the FAK kinase, termed FRNK (FAK-related nonkinase). Both the FAK- and FRNK-encoded polypeptides, pp125FAK and p41/p43FRNK, are expressed in normal chicken embryo cells. pp125FAK and p41/p43FRNK were localized to focal adhesions, suggesting that pp125FAK is directed to the focal adhesions by sequences within its C-terminal domain. We also show that the fibronectin-dependent increase in tyrosine phosphorylation of pp125FAK is accompanied by a concomitant posttranslational modification of p41FRNK.

Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK.

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.

Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.

The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.

Focal adhesion targeting: the critical determinant of FAK regulation and substrate phosphorylation.

The focal adhesion kinase (FAK) is discretely localized to focal adhesions via its C-terminal focal adhesion-targeting (FAT) sequence. FAK is regulated by integrin-dependent cell adhesion and can regulate tyrosine phosphorylation of downstream substrates, like paxillin. By the use of a mutational strategy, the regions of FAK that are required for cell adhesion-dependent regulation and for inducing tyrosine phosphorylation of paxillin were determined. The results show that the FAT sequence was the single region of FAK that was required for each function. Furthermore, the FAT sequence of FAK was replaced with a focal adhesion-targeting sequence from vinculin, and the resulting chimera exhibited cell adhesion-dependent tyrosine phosphorylation and could induce paxillin phosphorylation like wild-type FAK. These results suggest that subcellular localization is the major determinant of FAK function.

Complex formation with focal adhesion kinase: A mechanism to regulate activity and subcellular localization of Src kinases.

Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60(c-src) or p59(fyn) results in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60(c-src) or p59(fyn) to induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60(c-src) is hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60(c-src) from a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60(c-src) to focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.

Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase.

Focal adhesion kinase (pp125FAK or FAK) and paxillin colocalize with integrins in structures called focal adhesions. pp125FAK plays an important role in the transmission of integrin-induced cytoplasmic signals. Paxillin has also been implicated in cell signaling by virtue of its association with the protein tyrosine kinases pp60src and Csk (C-terminal Src kinase) as well as with the adapter/oncoprotein p47gag-crk. In this report we show that endogenous pp125FAK and paxillin form a stable complex both in vivo and in vitro and that this interaction is direct, requiring only pp125FAK and paxillin. The paxillin binding site on pp125FAK has been localized to the carboxy-terminal 148 residues of pp125FAK, but appears to be distinct from the previously identified focal adhesion-targeting sequence also present in the carboxy-terminal domain of pp125FAK. The interaction of paxillin and pp125FAK is independent of the adhesion of cells to the extracellular matrix, as the association can be detected in suspension cells as well as those attached to fibronectin.

Paxillin binding is not the sole determinant of focal adhesion localization or dominant-negative activity of focal adhesion kinase/focal adhesion kinase-related nonkinase.

The carboxy-terminal 150 residues of the focal adhesion kinase (FAK) comprise the focal adhesion-targeting sequence, which is responsible for its subcellular localization. The mechanism of focal adhesion targeting has not been fully elucidated. We describe a mutational analysis of the focal adhesion-targeting sequence of FAK to further examine the mechanism of focal adhesion targeting and explore additional functions encoded by the carboxy-terminus of FAK. The results demonstrate that paxillin binding is dispensable for focal adhesion targeting of FAK. Cell adhesion-dependent tyrosine phosphorylation strictly correlated with the ability of mutants to target to focal adhesions. Focal adhesion targeting was also a requirement for maximal FAK-dependent tyrosine phosphorylation of paxillin and FAK-related nonkinase (FRNK)-dependent inhibition of endogenous FAK function. However, there were additional requirements for these latter functions because we identified mutants that target to focal adhesions, yet are defective for the induction of paxillin phosphorylation or the dominant-negative function of FRNK. Furthermore, the paxillin-binding activity of FRNK mutants did not correlate with their ability to inhibit FAK, suggesting that FRNK has other targets in addition to paxillin.

Impact of the reduction of the recommended energy target in the ICU on protein delivery and clinical outcomes.

BACKGROUND & AIMS: Energy targets are a matter of debate for intensive care (ICU) patients. As the guidelines have evolved, energy targets have been reduced, while the protein intake objectives have increased. The impact of these changes remains largely unknown. This quality study aimed at investigating the clinical impact of these changes in patients with an ICU stay >3 days. METHODS: Observational cohort study over two 3 months periods (A, B), with distinct prevailing nutrition recommendations in patients admitted consecutively to a multidisciplinary ICU. Inclusion criterion: ICU stay >3 days. Recorded variables: severity scores, energy target and delivery, protein delivery, feeding route, length of stay (ICU, hospital) and hospital outcome. Data as mean, SD and IQR. RESULTS: The analysis included 389 patients, and 3920 observation days. Except for patient age (A versus B: 57.8 and 62.3 years; p = 0.010) and NRS (4.3 vs 3.9 respectively p = 0.002), the cohorts were similar. Compared to A, the mean prescribed energy target decreased by 125 kcal (1947 kcal/d vs. 1822 kcal*day-1 respectively), resulting in lower energy delivery (1353 kcal*day-1 vs. 1238 kcal*day-1; p < 0.0001), and reduced protein delivery (81 g*day-1 vs. 65 g*day-1: p < 0.0001). These differences were associated in survivors with prolonged mechanical ventilation (5.0 days vs. 6.7 days; p = 0.004), extended ICU stay (8.5 vs. 9.9 days; p = 0.0036), and longer hospital stay (23.4 vs. 26.4 days respectively; p = 0.028). Mortality was unchanged. CONCLUSIONS: A linear reduction in energy target recommendation without changing the feed composition led to an unplanned and significant reduction in protein delivery, which was associated with a prolonged duration of ventilation and an extended hospital stay.

[Spirituality in critical care]. / Le médecin face au besoin spirituel en médecine intensive.

Taking care of a critically-ill patient is of high complexity level, as it includes bio-pschosocial and cultural aspects. Until recently, physicians have paid little attention to spirituality, although it constitutes the essential of human being. Spiritual need may reveal to be of utmost importance and has influence on bio-psycho-social aspects for a patient experiencing severe disease with threatening outcome and near death. A physician may transfer the problem to a specialist, a chaplain, or may personally be able to assess this need. To put a bio-psycho-social-spiritual model of care into practice, health care givers including chaplains should set up a team work. Their educational programs should include spirituality care training. Swiss intensivists should acquire the competency to take care of their patients in a holistic manner.

[Merging of two intensive care units: preliminary observations]. / Fusion de deux services de médecine intensive: quelques observations préliminaires.

The merging of two intensive care units is a time of profound change, and constitutes a risk of mishaps. We report some aspects of such a project in our institution. The evaluation of various indicators reflecting the activity, patient's hospital pathways, mortality, as well as the use of specific techniques, has shown that no particular problem was observed during the first 9 months. Improvements in performance or productivity have not been demonstrated so far. The follow-up will permit to demonstrate long-term benefits. We believe that these observations may be of interest for other departmental or hospital reorganisations.

Paxillin: a focal adhesion-associated adaptor protein.

Paxillin is a focal adhesion-associated, phosphotyrosine-containing protein that may play a role in several signaling pathways. Paxillin contains a number of motifs that mediate protein-protein interactions, including LD motifs, LIM domains, an SH3 domain-binding site and SH2 domain-binding sites. These motifs serve as docking sites for cytoskeletal proteins, tyrosine kinases, serine/threonine kinases, GTPase activating proteins and other adaptor proteins that recruit additional enzymes into complex with paxillin. Thus paxillin itself serves as a docking protein to recruit signaling molecules to a specific cellular compartment, the focal adhesions, and/or to recruit specific combinations of signaling molecules into a complex to coordinate downstream signaling. The biological function of paxillin coordinated signaling is likely to regulate cell spreading and motility.

Tyrosine phosphorylation of a 120,000 dalton membrane-associated protein by the neural form of pp60c-src, pp60c-src+.

The c-src proto-oncogene encodes a 60,000 dalton tyrosine kinase, pp60c-src, which is the prototype member of the family of non-receptor tyrosine kinases. A neural-specific form of pp60c-src, pp60c-src+, is detected only in neurons of the central nervous system. pp60c-src+ contains a six amino acid insert (neural insert) in the SH3 region that is generated by alternative splicing. Previous reports indicate that the profiles of proteins phosphorylated on tyrosine in chick embryo fibroblast (CEF) cells by pp60c-src+ or pp60c-src are equivalent. In this report, the activities of pp60c-src+ and pp60c-src, as well as the activated variants, pp60(527F+) and pp60(527F), were compared in CEF cells by examining the steady-state levels of tyrosine phosphorylation of several known pp60src substrates. Most substrates examined were phosphorylated on tyrosine to equivalent levels in CEF cells expressing either the neural- or fibroblast-specific src gene products. However, the relative extent of tyrosine phosphorylation of a 120 kDa protein (p120) was increased in cells expressing the neuronal forms of either c-src or c-src527F. The increased tyrosine phosphorylation of p120 did not appear to be caused by the neural insert facilitating a specific interaction between pp60c-src+ and p120. These data indicate that preferential phosphorylation of p120 in neural cells may contribute to the specialized function of pp60c-src+ in neural cells.