British infection association guidelines for the diagnosis and management of enteric fever in England.

Enteric fever (EF) is an infection caused by the bacteria called Salmonella Typhi or Paratyphi. Infection is acquired through swallowing contaminated food or water. Most EF in England occurs in people returning from South Asia and other places where EF is common; catching EF in England is rare. The main symptom is fever, but stomach pain, diarrhoea, muscle aches, rash and other symptoms may occur. EF is diagnosed by culturing the bacteria from blood and/or stool in a microbiology laboratory. EF usually responds well to antibiotic treatment. Depending on how unwell the individual is, antibiotics may be administered by mouth or by injection. Over the past several years, there has been an overall increase in resistance to antibiotics used to treat enteric fever, in all endemic areas. Additionally, since 2016, there has been an ongoing outbreak of drug-resistant EF in Pakistan. This infection is called extensively drug-resistant, or XDR, EF and only responds to a limited number of antibiotics. Occasionally individuals develop complications of EF including confusion, bleeding, a hole in the gut or an infection of the bones or elsewhere. Some people may continue to carry the bacteria in their stool for a longtime following treatment for the initial illness. These people may need treatment with a longer course of antibiotics to eradicate infection. Travellers can reduce their risk of acquiring EF by following safe food and water practices and by receiving the vaccine at least a few weeks before travel. These guidelines aim to help doctors do the correct tests and treat patients for enteric fever in England but may also be useful to doctors and public health professionals in other similar countries.

The misogyny of iron deficiency.

Anaemia is common, particularly in women and the commonest underlying cause, iron deficiency, is often overlooked. Anaemia is associated with increased morbidity and mortality in patients undergoing anaesthesia; however, women are defined as being anaemic at a lower haemoglobin level than men. In this narrative review, we present the history of iron deficiency anaemia and how women's health has often been overlooked. Iron deficiency was first described as 'chlorosis' and a cause of 'hysteria' in women and initial treatment was by iron filings in cold wine. We present data of population screening demonstrating how common iron deficiency is, affecting 12-18% of apparently 'fit and healthy' women, with the most common cause being heavy menstrual bleeding; both conditions being often unrecognised. We describe a range of symptoms reported by women, that vary from fatigue to brain fog, hair loss and eating ice. We also describe experiments exploring the physical impact of iron deficiency, showing that reduced exercise performance is related to iron deficiency independent of haemoglobin concentration, as well as the impact of iron supplementation in women improving oxygen consumption and fitness. Overall, we demonstrate the need to single out women and investigate iron deficiency rather than accept the dogma of normality and differential treatment; this is to say, the need to change the current standard of care for women undergoing anaesthesia.

Role of c-Jun N-terminal kinase (JNK) activation in micturition reflexes in cyclophosphamide (CYP)-induced cystitis in female rats.

c-Jun N-terminal kinase (JNK) is member of the mitogen-activated protein kinase (MAPK) family, activated through phosphorylation following cytokine exposure and stress. In this study, phosphorylation of JNK was examined in the urinary bladder with cyclophosphamide (CYP)-induced cystitis and the effects of SP600125, a selective inhibitor of phosphorylation of JNK, on urinary bladder function were assessed using conscious, open outlet, cystometry with continuous instillation of intravesical saline. We induced bladder inflammation in adult female Wistar rats by injecting CYP intraperitoneally to produce acute (150 mg/kg; 4 h), intermediate (150 mg/kg; 48 h), and chronic (75 mg/kg; every third day for 10 days) treatments. Western blotting of urinary bladder demonstrated a significant (p ≤ 0.01) increase (i.e., phosphorylation) in JNK activation with 4- and 48-h CYP-induced cystitis. Immunohistochemistry and image analyses demonstrated a significant (p ≤ 0.01) increase in JNK activation in the urothelium with 4- and 48-h CYP-induced cystitis. Blockade of JNK phosphorylation significantly (p ≤ 0.01) increased bladder capacity and intercontraction void intervals in CYP-treated rats (4 and 48 h). Furthermore, blockade of JNK phosphorylation reduced (p ≤ 0.01) neuropeptide (substance P, calcitonin gene-related peptide) expression in the urinary bladder with CYP-induced cystitis (4 and 48 h). In contrast, blockade of JNK phosphorylation was without effect on bladder function or neuropeptide expression in urinary bladder in control (no inflammation) rats. Blockade of JNK phosphorylation may represent a novel target for improving urinary bladder function with CYP-induced cystitis.

Improving adherence to medications in pediatric liver transplant recipients.

We describe results from a clinical program, which aimed at improving adherence to medications in children who had a liver transplant. We followed the medical outcomes of 23 children and adolescents who participated in a clinical adherence-improvement protocol during the years 2001-2002. The protocol included identification of non-adherent patients by examining tacrolimus blood levels and intervention by increasing the frequency of clinic visits for non-adherent patients. In the two-yr preintervention (1999-2000), there was no improvement in any of the outcomes. After the intervention, the number of patients with high alanine aminotransferase levels (100 and above) decreased significantly, from eight before the intervention to four afterwards. Other outcomes, including the number of rejection episodes (three before, none after) and the degree of adherence to tacrolimus, also improved, but the improvement did not reach statistical significance. Although non-adherent patients were called to clinic more often under the protocol, the intervention did not lead to increased outpatient costs. This adherence--improvement intervention appears to be promising in improving outcomes in pediatric liver transplant recipients. Larger, controlled studies are needed to establish the efficacy of this or other approaches.

Human semaphorin K1 is glycosylphosphatidylinositol-linked and defines a new subfamily of viral-related semaphorins.

The semaphorin family contains a large number of secreted and transmembrane proteins, some of which are known to act as repulsive axon guidance cues during development or to be involved in immune function. We report here on the identification of semaphorin K1 (sema K1), the first semaphorin known to be associated with cell surfaces via a glycosylphosphatidylinositol linkage. Sema K1 is highly homologous to a viral semaphorin and can interact with specific immune cells, suggesting that like its viral counterpart, sema K1 could play an important role in regulating immune function. Sema K1 does not bind to neuropilin-1 or neuropilin-2, the two receptors implicated in mediating the repulsive action of several secreted semaphorins, and thus it likely acts through a novel receptor. In contrast to most previously described semaphorins, sema K1 is only weakly expressed during development but is present at high levels in postnatal and adult tissues, particularly brain and spinal cord.

Integration of foreign DNA in an intergenic region of the archaeon Methanosarcina mazei without effect on transcription of adjacent genes.

Transformation systems for methanogenic archaea are scarce, none has been reported for the genus Methanosarcina, and plasmids useful as vectors for cloning foreign DNA into methanogens that stably replicate as extrachromosomal elements are not available. We developed an integration vector for transformation of a member of the genus Methanosarcina, i.e. Methanosarcina mazei, using a segment (Int alpha; 1015 bp) which encompasses the intergenic region (431 bp) between the stress (heat-shock) genes grpE and dnaK. This segment also includes the 3' end (270 bp) of the grpE protein-coding region and the 5' end (314 bp) of the dnaK protein-coding region. Int alpha has an EcoRI site, useful for cloning, situated in the 3' direction beyond the grpE transcription termination region, and far upstream from the dnaK promoter. This location of the site, and the monocistronic mode of transcription of grpE and dnaK in M. mazei, suggested to us that a foreign insert in the site would not affect transcription of either flanking gene. A puromycin-resistance cassette (pac cassette) was inserted in the EcoRI site of Int alpha already inserted in pUC18, to obtain a vector which integrated the pac cassette in the chromosome between grpE and dnaK. The pac gene was transcribed and the transformants acquired puromycin resistance. Constitutive and heat-shock-induced transcription of grpE and dnaK in the transformants was the same as in wild-type cells. The two vectors found with transforming ability differed in the orientation of the pac cassette but both had M. mazei's DNA on each flank of the cassette, with the same orientation as that of the homologous segments in the chromosome.

Activation of the high-affinity immunoglobulin E receptor Fc epsilon RI in RBL-2H3 cells is inhibited by Syk SH2 domains.

Antigen-mediated aggregation of the high-affinity receptor for immunoglobulin E, Fc epsilon RI, results in the activation of multiple signaling pathways, leading to the release of mediators of the allergic response. One of the earliest responses to receptor stimulation is the tyrosine phosphorylation of the beta and gamma subunits of Fc epsilon RI and the association of the tyrosine kinase Syk with the phosphorylated receptor. This association is mediated by the SH2 domains of Syk and is believed to be critical for activating signaling pathways resulting in mediator release. To examine the importance of the interaction of Syk with Fc epsilon RI in signaling events following receptor activation, we introduced a protein containing the SH2 domains of Syk into streptolysin O-permeabilized RBL-2H3 cells. The Syk SH2 domains completely inhibited degranulation and leukotriene production following receptor aggregation, and they blocked the increase in protein tyrosine phosphorylation observed after receptor activation. Inhibition was specific for Fc epsilon RI-mediated signaling, since degranulation of cells activated by alternative stimuli was not blocked by the Syk SH2 domains. A protein containing a point mutation in the carboxy-terminal SH2 domain which abolishes phosphotyrosine binding was not inhibitory. In addition, inhibition of degranulation was reversed by pretreatment of the SH2 domains with a tyrosine phosphorylated peptide corresponding to the tyrosine-based activation motif found in the gamma subunit of Fc epsilon RI, the nonphosphorylated peptide had no effect. The association of Syk with the tyrosine-phosphorylated gamma subunit of the activated receptor was blocked by the Syk SH2 domains, and deregulation in cells activated by clustering of Syk directly without Fc epsilon RI aggregation was not affected by the Syk SH2 domains. These results demonstrate that the association of Syk with the activated Fc epsilon RI is critical for both early and late events following receptor activation and confirm the key role Syk plays in signaling through the high-affinity IgE receptor.

Identification of a grpE heat-shock gene homolog in the archaeon Methanosarcina mazei.

A grpE heat-shock gene was found by sequencing in the genome of the methanogenic archaeon Methanosarcina mazei S-6. It is the first example of grpE from the phylogenetic domain Archaea. Since the other seven sequenced homologs are from the domain Bacteria, it may be concluded that grpE appeared early in evolution, before the two domains separated. The archaeal grpE is located in the dnaK locus, 431 base-pairs upstream of dnaK, which is followed downstream by the dnaJ gene. The organization of these three genes is known for Bacillus subtilis, Clostridium acetobutylicum, Borrelia burgdorferi and Mycobacterium tuberculosis. The archaeal locus organization, grpE-dnaK-dnaJ, is similar to that of the former three bacteria, but different from that of M. tuberculosis. This, and sequence homologies, suggest that the M. tuberculosis GrpE belongs, together with the Streptomyces coelicolor homolog, to a subgroup of the GrpE proteins. The M. mazei grpE gene encodes a protein of 209 amino acid residues. The deduced amino acid sequence shows 28.2 to 34.6% identities, and 50.3 to 58.9 similarities (identities plus conservative substitutions) with the other six complete GrpE sequences available. These percentages fall within the range observed for the other GrpEs. Two regions in the second and fourth quarters of the GrpE molecule show higher homology, particularly in three stretches of nine, six and nine amino acid residues, respectively. The archaeal gene uses all codons but three, whereas the bacterial homologs lack higher numbers of codons. The M. mazei grpE responded to heat-shock by increasing transcription, in a manner similar to that of the nearby heat-shock gene dnaK.

Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains.

Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.

An archaeal trkA homolog near dnaK and dnaJ.

The first trkA gene homolog in the phylogenetic domain Archaea is reported. The gene is located near the dnaK-dnaJ gene cluster in the genome of Methanosarcina mazei S-6, and encodes a protein homologous to the only other TrkA known, i.e., that of the bacterium Escherichia coli, involved in K+ transport. This finding supports an essential, evolutionarily early, and conserved role for this gene in cell survival and adaptation.

Detection of Src homology 3-binding proteins, including paxillin, in normal and v-Src-transformed Balb/c 3T3 cells.

The Src homology 3 (SH3) domain, located in the amino-terminal, noncatalytic half of pp60src, is highly conserved among members of the Src family of tyrosine kinases. SH3 domains have also been identified in a variety of proteins otherwise unrelated to protein-tyrosine kinases. The presence of SH3 domains in proteins with diverse functions suggests this domain may be important for directing protein-protein interactions necessary for protein function or cellular localization. To explore possible interactions between the SH3 domain and cellular proteins, we have established conditions for the isolation of proteins that bind in solution to the Src SH3 domain. A 67-amino acid fragment of c-Src containing either the entire glutathione S-transferase-SH3 domain (GST-SH3) or the SH3 domain from the neuronal form of c-Src (GST-SH3+) was expressed as a glutathione S-transferase fusion protein. The GST fusion proteins were incubated with lysates from [35S]methionine-labeled Balb/c 3T3 cells or v-Src-transformed Balb/c 3T3 cells. We found that GST-SH3, but not wild-type GST, specifically interacted with multiple cellular proteins, whereas GST-SH3+ only weakly associated with a small subset of these proteins. The majority of the SH3-binding proteins were found in particulate and detergent-insoluble cell fractions. Anti-phosphotyrosine immunoblots of the SH3-binding proteins revealed that several of the SH3-binding proteins are phosphorylated on tyrosine in v-Src-transformed cells. In addition, a number of the SH3-binding proteins were phosphorylated on serine and/or threonine in in vitro kinase assays, suggesting that one or more of the SH3-binding proteins has kinase activity. We identified paxillin, a vinculin-binding protein, as one of the Src SH3-binding proteins. This finding strongly supports the hypothesis that SH3 domains may be involved in subcellular localization of proteins to cytoskeleton and/or cellular membranes.

Solution structure of the SH3 domain of Src and identification of its ligand-binding site.

The Src homology 3 (SH3) region is a protein domain of 55 to 75 amino acids found in many cytoplasmic proteins, including those that participate in signal transduction pathways. The solution structure of the SH3 domain of the tyrosine kinase Src was determined by multidimensional nuclear magnetic resonance methods. The molecule is composed of two short three-stranded anti-parallel beta sheets packed together at approximately right angles. Studies of the SH3 domain bound to proline-rich peptide ligands revealed a hydrophobic binding site on the surface of the protein that is lined with the side chains of conserved aromatic amino acids.

Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src.

The amino-termina, noncatalytic half of Src contains two domains, designated the Src homology 2 (SH2) and Src homology 3 (SH3) domains, that are highly conserved among members of the Src family of tyrosine kinases. The SH2 domain (which can be further divided into the B and C homology boxes) and the SH3 domain (also referred to as the A box) are also found in several proteins otherwise unrelated to protein tyrosine kinases. It is believed that these domains are important for directing specific protein-protein interactions necessary for the proper functioning of Src. To determine the importance of the SH2 and SH3 domains in regulating the functions of c-Src, we evaluated mutants of c-Src lacking the A box (residues 88 to 137), the B box (residues 148 to 187) or the C box (residues 220 to 231). Each of these deletions caused a 14- to 30-fold increase in the in vitro level of kinase activity of c-Src. Chicken embryo fibroblasts expressing the deletion mutants displayed a transformed cell morphology, formed colonies in soft agar, and contained elevated levels of cellular phosphotyrosine-containing proteins. Src substrates p36, p85, p120, p125, the GTPase-activating protein (GAP), and several GAP-associated proteins were phosphorylated on tyrosine in cells expressing the A, B, or C box deletion mutant. p110 was highly phosphorylated in cells expressing the C box mutant, was weakly phosphorylated in cells expressing the B box mutant, and was not phosphorylated in cells expressing the A box mutant. Expression of the mutant proteins caused a reorganization of the actin cytoskeleton similar to that seen in v-Src-transformed cells. In addition, deletion of the A, B, or C box did not diminish the transforming or enzymatic activity of an activated variant of c-Src, E378G. These data indicate that deletion of the A, B, or C homology box causes an activation of the catalytic and transforming potential of c-Src and that while these mutations caused subtle differences in substrate phosphorylation, the homology boxes are not required for many of the phenotypic changes associated with transformation by Src.

A dnaK homolog in the archaebacterium Methanosarcina mazei S6.

A fragment of genomic DNA cloned from the methanogenic archaebacterium, Methanosarcina mazei strain S6, was found to contain an 1857-bp open reading frame (ORF). A sequence matching the consensus ribosome-binding sequence determined for other methanogens was found upstream from the ORF. The amino acid (aa) sequence encoded by the ORF was compared with reference sequences and was found to be related to six DnaK sequences determined for five species of eubacteria (none exist for archaebacteria). The M. mazei S6 aa sequence was over 61% identical and over 77% similar (identities plus conservative substitutions) to the closest four reference sequences, which were all DnaKs. The gene described here is therefore proposed to be the first member of the dnaK family sequenced from the archaebacterial kingdom (Archaea). This finding confirms that DnaK proteins are highly conserved, occurring not only in eubacteria (Bacteria) and eukaryotes (Eucaria), but also in archaebacteria (Archaea).

Herpes simplex virus glycoprotein C is a receptor for complement component iC3b.

Herpes simplex virus type 1-infected cells bind C3b and iC3b, but not C3d, at the cell surface. Herpes simplex virus type 2 (HSV-2)-infected cells bind none of these C3 fragments. A transfection assay was used to demonstrate that binding of iC3b was to gC1. Although iC3b did not bind to HSV-2-infected cells, it did bind to mammalian cells transfected with the gC2 gene. Using linker insertion mutants, three domains on gC2 that are important for binding iC3b were mapped; these regions were similar to previously defined regions involved in binding C3b. These results suggest that some of the functions served by gC may be similar to those of CR3, the mammalian receptor for iC3b.

Identification of C3b-binding regions on herpes simplex virus type 2 glycoprotein C.

Glycoprotein C from herpes simplex viruses types 1 and 2 (gC-1 and gC-2) acts as a receptor for the C3b fragment of the third component of complement. Our goal is to identify domains on gC involved in C3b receptor activity. Here, we used in-frame linker-insertion mutagenesis of the cloned gene for gC-2 to identify regions of the protein involved in C3b binding. We constructed 41 mutants of gC-2, each having a single, double, or triple insertion of four amino acids at sites spread across the protein. A transient transfection assay was used to characterize the expressed mutant proteins. All of the proteins were expressed on the transfected cell surface, exhibited processing of N-linked oligosaccharides, and bound one or more monoclonal antibodies recognizing distinct antigenic sites on native gC-2. This suggested that each of the mutant proteins was folded into a native structure and that a loss of C3b binding by any of the mutants could be attributed to the disruption of a specific functional domain. When the panel of insertion mutants was assayed for C3b receptor activity, we identified three distinct regions that are important for C3b binding, since an insertion within those regions abolished C3b receptor activity. Region I was located between amino acids 102 and 107, region II was located between residues 222 and 279, and region III was located between residues 307 and 379. In addition, region III has some structural features similar to a conserved motif found in complement receptor 1, the human C3b receptor. Finally, blocking experiments indicated that gC-1 and gC-2 bind to similar locations on the C3b molecule.

Ultrastructural and morphometric features of poorly differentiated and undifferentiated lung tumors.

Initial results are reported from a study of lung carcinomas and carcinoid tumors performed with electron microscopy and morphometric analysis of electron micrographs. The ultrastructural findings are being used to define the range of ultrastructures of the different tumor categories diagnosed by light microscopy using the revised World Health Organization classification. Morphometric data reveal a considerable degree of overlap in cell and nuclear size among different lung tumor types.

Use of a glucocorticoid-inducible promoter for expression of herpes simplex virus type 1 glycoprotein gC1, a cytotoxic protein in mammalian cells.

Abundant expression of herpes simplex virus type 1 glycoprotein gC (gC1) in transfected mammalian cells has not previously been achieved, possibly because gC1 protein is toxic to cells. To approach this problem, the gC1 coding sequence was placed under the control of the weak but inducible glucocorticoid-responsive promoter from the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). As controls to evaluate for gC1 cytotoxicity, the MMTV LTR promoter was used to express glycoprotein gD1, and a strong, constitutive promoter from the Moloney murine sarcoma virus LTR was used to express gC1. L cells were transfected with these constructs, and a clone expressing gC1 from the inducible MMTV LTR promoter was analyzed. In the absence of glucocorticoid (dexamethasone) stimulation, only a low level of gC1 mRNA expression was detected; after overnight stimulation with dexamethasone, transcription increased approximately 200-fold. Abundant gC1 protein that was functionally active in that it bound complement component C3b, was produced. From passages 5 through 26 (70 cell population doublings), the gC1-producing clone became less responsive to overnight dexamethasone stimulation. The block to gC1 expression occurred at the level of transcription and was associated with hypermethylation of the MMTV LTR DNA. Treatment of the clone with 5-aza-2'-deoxycytidine partially reversed the block in gC1 protein production. Late-passage cells assumed a gC1-negative phenotype that appeared to offer a selective growth advantage, which suggested that gC1 was cytotoxic. Several findings support this view: (i) some cells expressing gC1 after overnight stimulation with dexamethasone assumed bizarre, syncytial shapes; (ii) continuous stimulation with dexamethasone for 5 weeks resulted in death of most cells; (iii) cells transfected with gC1 under the control of the strong Moloney murine sarcoma virus promoter assumed bizarre shapes, and stable gC1-expressing clones could not be established; and (iv) cells induced to express gD1 retained a normal appearance after overnight stimulation or 15 weeks of continuous stimulation with dexamethasone. The inducible MMTV LTR promoter is useful for expressing gC1 and may have applications for expressing other cytotoxic proteins.