Follow-up after emergency laparotomy suggests high one- and five-year mortality with risk stratified by ASA.

INTRODUCTION: We investigated all-cause mortality following emergency laparotomy at 1 and 5 years. We aimed to establish a basis from which to advise patients and relatives on long-term mortality. METHODS: Local data from a historical audit of emergency laparotomies from 2010 to 2012 were combined with National Emergency Laparotomy Audit (NELA) data from 2017 to 2020. Covariates collected included deprivation status, preoperative blood work, baseline renal function, age, American Society of Anesthesiologists (ASA) grade, operative time, anaesthetic time and gender. Associations between covariates and survival were determined using multivariate logistic regression and Kaplan-Meier analysis. We used patients undergoing laparoscopic cholecystectomy between 2015 and 2020 as controls. RESULTS: ASA grade was the best discriminator of long-term outcome following laparotomy (n=894) but was not a predictor of survival following cholecystectomy (n=1,834), with mortality being significantly greater in the laparotomy group. Following cholecystectomy, 95% confidence intervals for survival at 5 years were 98-99%. Following laparotomy these intervals were: ASA grade 1, 79-96%; ASA grade 2, 69-82%; ASA grade 3, 44-58%; ASA grade 4, 33-48%; and ASA grade 5, 4-51%. The majority of deaths occurred after 30 days. CONCLUSIONS: Emergency laparotomy is associated with a significantly increased risk of death in the following 5 years. The risk is strongly correlated to ASA grade. Thirty-day mortality estimation is not a good basis on which to advise patients and carers on long-term outcomes. ASA grade can be used to predict long-term outcomes and to guide patient counsel.

Detection and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa.

Enzymes of the pathway for de novo biosynthesis of pyrimidine nucleotides have been reported in spermatozoa from fruitfly and mammals. The aim of the present study was to test the hypothesis that the enzymes for biosynthesis of uridine monophosphate (UMP) are concentrated near the mitochondria, which are segregated in the mid-piece of spermatozoa. Baby hamster kidney fibroblasts were compared with spermatozoa from rams, boars, bulls and men. Antibodies raised against synthetic peptides from sequences of the multienzyme polypeptides containing glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (CAD) and UMP synthase, which catalyse reactions 1-3 and 5-6, respectively, were used, together with an affinity-purified antibody raised against dihydroorotate dehydrogenase (DHODH), the mitochondrial enzyme for step 4. Western blot analysis, immunofluorescent microscopy and immunoelectron microscopy confirmed that CAD and UMP synthase are found in the cytoplasm around and outside the mitochondria; DHODH is found exclusively inside the mitochondria. CAD was also located in the nucleus, where it has been reported in the nuclear matrix, and in the cytoplasm, apparently associated with the cytoskeleton. It is possible that CAD in the cytoplasm has a role unconnected with pyrimidine biosynthesis.

Relative labelling index: a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections.

Simple and efficient protocols for quantifying immunogold labelling of antigens localized in different cellular compartments (organelles or membranes) and statistically evaluating resulting labelling distributions are presented. Two key questions are addressed: (a) is compartmental labelling within an experimental group (e.g. control or treated) consistent with a random distribution? and (b) do labelling patterns vary between groups (e.g. control vs. treated)? Protocols rely on random sampling of cells and compartments. Numbers of gold particles lying on specified organelle compartments provide an observed frequency distribution. By superimposing test-point lattices on cell profiles, design-based stereology is used to determine numbers of points lying on those same compartments. Random points hit compartments with probabilities determined by their relative sizes and so provide a convenient internal standard, namely, the expected distribution if labelling is purely random. By applying test-line lattices, and counting sites at which these intersect membrane traces, analogous procedures provide observed and expected labelling distributions for different classes of membranes. Dividing observed golds by expected golds provides a relative labelling index (RLI) for each compartment and, for random labelling, the predicted RLI = 1. In contrast to labelling densities of organelles (golds microm(-2) or membranes (golds microm(-1)), RLI values are estimated without needing to know lattice constants (area per point or length per intersection) or specimen magnification. Gold distributions within a group are compared by chi-squared analysis to test if the observed distribution differs significantly from random and, if it is non-random, to identify compartments which are preferentially labelled (RLI > 1). Contingency table analysis allows labelling distributions in different groups of cells to be compared. Protocols are described and illustrated using worked specimen examples and real data.

Electron microscopy applications for quantitative cellular microbiology.

Recently, there has been a surge of interest in analysing intracellular trafficking of pathogenic microorganisms. This field relies heavily on a wide range of approaches developed by cell biologists. However, one well-established approach that seems to be underused in cellular microbiology is electron microscopy (EM). In this article, we emphasize the power of state-of-the-art EM approaches, especially when used in conjunction with the quantitative approaches provided by stereology. Together, this combination of techniques can be exploited to pursue kinetic analyses of the intracellular pathways followed by microorganisms. In addition, it will provide a more detailed description of the antigenic composition of relevant structures than is possible by light microscopy (LM). Here, we will explain how EM, applied in an integrated approach with LM, has distinct advantages and can provide unique insights into the cellular fate of microorganisms.

Immunolocalisation of phospholipase D1 on tubular vesicular membranes of endocytic and secretory origin.

We have examined the localisation of overexpressed phospholipase D1 (PLD1) using antibodies against its amino- and carboxyl-terminal domains. PLD1 overexpressed in COS-7 cells showed variable distribution by immunofluorescence but was mainly in punctate structures in the perinuclear region and at the plasma membrane. Downregulation by an anti-sense plasmid resulted in almost exclusively perinuclear distribution in punctate structures that contained immunoreactivity for the endogenous KDEL receptor and the early endosomal antigen EEA1 protein. Influenza haemagglutinin (HA) and HA-derived mutants designed to locate primarily to secretory or endocytic membranes were present in PLD1-positive membranes. Immunofluorescence analysis in permanent CHO cell lines that express PLD1 inducibly confirmed the presence of PLD1 on both endocytic and secretory membranes. Analysis of PLD1 distribution by immunocytochemistry and electron microscopy of intact CHO cells and of isolated membranes revealed that PLD1 was present in tubulovesicular elements and multivesicular bodies. Some of these were close to the Golgi region whereas others stained positive for endocytic cargo proteins. Morphometric analysis assigned the majority of PLD1 immunoreactivity on endosomal membranes and a smaller amount on membranes of secretory origin. PLD1, via signals that are currently not understood, is capable of localising in tubulovesicular membranes of both endocytic and secretory origin.

Characterization and regulation of constitutive transport intermediates involved in trafficking from the trans-Golgi network.

Transport vesicles or containers (TCs) mediate constitutive protein transport between the trans-Golgi network (TGN) and the plasma membrane. A key question is the nature and regulation of these transport containers or intermediates. We have used a trans-Golgi network resident, TGN38, to investigate TC formation. TGN38 is a recycling membrane glycoprotein that moves to the cell surface via constitutive membrane traffic and returns via the endosomal pathway. An in vitro assay to measure TC formation was devised using rat liver Golgi membranes, cytosolic factors and ATP. Transport intermediates containing TGN38 were produced and found to be smooth vesicles and tubules of up to 200 nm in length. These membrane-enclosed structures contain different constitutively secreted membrane glycoproteins, including molecules involved in immune functions such as MHC Class I and the polymeric Ig receptor, showing that these intermediates correspond to TCs that have been previously identified in vivo. Importantly, TC formation can be stimulated or inhibited by protein kinase and phosphatase inhibitors, showing regulation by intracellular signalling pathways.

Evidence for prebudding arrest of ER export in animal cell mitosis and its role in generating Golgi partitioning intermediates.

During mitosis the interconnected Golgi complex of animal cells breaks down to produce both finely dispersed elements and discrete vesiculotubular structures. The endoplasmic reticulum (ER) plays a controversial role in generating these partitioning intermediates and here we highlight the importance of mitotic ER export arrest in this process. We show that experimental inhibition of ER export (by microinjecting dominant negative Sar1 mutant proteins) is sufficient to induce and maintain transformation of Golgi cisternae to vesiculotubular remnants during interphase and telophase, respectively. We also show that buds on the ER, ER exit sites and COPII vesicles are markedly depleted in mitotic cells and COPII components Sec23p, Sec24p, Sec13p and Sec31p redistribute into the cytosol, indicating ER export is inhibited at an early stage. Finally, we find a markedly uneven distribution of Golgi residents over residual exit sites of metaphase cells, consistent with tubulovesicular Golgi remnants arising by fragmentation rather than redistribution via the ER. Together, these results suggest selective recycling of Golgi residents, combined with prebudding cessation of ER export, induces transformation of Golgi cisternae to vesiculotubular remnants in mitotic cells. The vesiculotubular Golgi remnants, containing populations of slow or nonrecycling Golgi components, arise by fragmentation of a depleted Golgi ribbon independently from the ER.

Characterization of a novel phosphatidylinositol 3-phosphate-binding protein containing two FYVE fingers in tandem that is targeted to the Golgi.

We have identified a novel protein of predicted molecular mass 40 kDa that contains two FYVE domains in tandem and has therefore been named TAFF1 (TAndem FYVE Fingers-1). The protein is expressed predominantly in heart and binds to PtdIns3P specifically, even though the FYVE domains in TAFF1 lacks the first Arg of the consensus sequence R(K/R)HHCR, critical for the PtdIns3P binding of other FYVE domains identified so far. The first Arg is replaced by a Thr and Ser in the N-terminal and C-terminal FYVE domains of TAFF1 respectively. Mutational analysis indicates that both FYVE domains are required for high affinity binding to PtdIns3P. Cell localization studies using a green fluorescent protein fusion show that TAFF1 is localized to the Golgi, and that the Golgi targeting sequence is located within the N-terminal 187 residues and not in either FYVE domain.

The manganese cation disrupts membrane dynamics along the secretory pathway.

The endoplasmic reticulum and Golgi apparatus play key roles in regulating the folding, assembly, and transport of newly synthesized proteins along the secretory pathway. We find that the divalent cation manganese disrupts the Golgi apparatus and endoplasmic reticulum (ER). The Golgi apparatus is fragmented into smaller dispersed structures upon manganese treatment. Golgi residents, such as TGN46, beta1,4-galactosyltransferase, giantin, and GM130, are still segregated and partitioned correctly into smaller stacked fragments in manganese-treated cells. The mesh-like ER network is substantially affected and peripheral ER elements are collapsed. These effects are consistent with manganese-mediated inhibition of motor proteins that link membrane organelles along the secretory pathway to the cytoskeleton. This divalent cation thus represents a new tool for studying protein secretion and membrane dynamics along the secretory pathway.

Merkel cells in the human oesophagus.

Merkel cells (MCs) are well recognized in the basal layers of the skin and oral mucosa, but this paper describes for the first time the presence of MCs in the human oesophagus. These cells are not identified in neonatal oesophagus, but are seen singly and in clusters in adult specimens. Application of stereological techniques shows that MCs are more numerous in the mid-oesophageal region. Cells expressing established markers of MCs have also been demonstrated in two out of six primary small cell carcinomas of the oesophagus. Further investigation of the role of MCs in oesophageal innervation and epithelial biology will be of interest.

Forward and retrograde trafficking in mitotic animal cells. ER-Golgi transport arrest restricts protein export from the ER into COPII-coated structures.

Protein transport arrest occurs between the ER and Golgi stack of mitotic animal cells, but the location of this block is unknown. In this report we use the recycling intermediate compartment protein ERGIC 53/p58 and the plasma membrane protein CD8 to establish the site of transport arrest. Recycled ERGIC 53/p58 and newly synthesised CD8 accumulate in ER cisternae but not in COPII-coated export structures or more distal sites. During mitosis the tubulovesicular ER-related export sites were depleted of the COPII component Sec13p, as shown by immunoelectron microscopy, indicating that COPII budding structures are the target for mitotic inhibition. The extent of recycling of Golgi stack residents was also investigated. In this study we used oligosaccharide modifications on CD8 trapped in the ER of mitotic cells as a sensitive assay for recycling of Golgi stack enzymes. We find that modifications conferred by the Golgi stack-resident GalNac transferase do occur on newly synthesised CD8, but these modifications are entirely due to newly synthesised transferase rather than to enzyme recycled from the Golgi stack. Taken together our findings establish for the first time that the site of ER-Golgi transport arrest of mitotic cells is COPII budding structures, and they clearly speak against a role for recycling in partitioning of Golgi stack proteins via translocation to the ER.

Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) interacts stereoselectively with the d-enantiomer of PtdIns(3,4,5)P3 (KD 1.6 nM) and PtdIns(3,4)P2 (KD 5.2 nM), but binds with lower affinity to PtdIns3P or PtdIns(4,5)P2. The binding of PtdIns(3,4,5)P3 to PDK1 was greatly decreased by making specific mutations in the pleckstrin homology (PH) domain of PDK1 or by deleting it. The same mutations also greatly decreased the rate at which PDK1 activated protein kinase Balpha (PKBalpha) in vitro in the presence of lipid vesicles containing PtdIns(3,4,5)P3, but did not affect the rate at which PDK1 activated a PKBalpha mutant lacking the PH domain in the absence of PtdIns(3,4,5)P3. When overexpressed in 293 or PAE cells, PDK1 was located at the plasma membrane and in the cytosol, but was excluded from the nucleus. Mutations that disrupted the interaction of PtdIns(3,4,5)P3 or PtdIns(4,5)P2 with PDK1 abolished the association of PDK1 with the plasma membrane. Growth-factor stimulation promoted the translocation of transfected PKBalpha to the plasma membrane, but had no effect on the subcellular distribution of PDK1 as judged by immunoelectron microscopy of fixed cells. This conclusion was also supported by confocal microscopy of green fluorescent protein-PDK1 in live cells. These results, together with previous observations, indicate that PtdIns(3,4,5)P3 plays several roles in the PDK1-induced activation of PKBalpha. First, it binds to the PH domain of PKB, altering its conformation so that it can be activated by PDK1. Secondly, interaction with PtdIns(3,4,5)P3 recruits PKB to the plasma membrane with which PDK1 is localized constitutively by virtue of its much stronger interaction with PtdIns(3,4,5)P3 or PtdIns(4,5)P2. Thirdly, the interaction of PDK1 with PtdIns(3,4,5)P3 facilitates the rate at which it can activate PKB.

ERK2 signalling from internalised epidermal growth factor receptor in broken A431 cells.

Activation of extracellular signal-regulated kinase 2 (ERK2) signalling from epidermal growth factor receptors (EGFRs) is widely assumed to originate in the plasma membrane. Using an in vitro assay, we investigated whether EGF/EGFR complexes internalised by endocytosis in A431 cells can initiate signalling in the ERK2 pathway. At 0 degrees C, binding of EGF induced tyrosine phosphorlyation of EGFR and, when the cells were subsequently broken by scraping and warmed in the presence of exogenous cytosol, activation of ERK2 occurred. At 0 degrees C, washes with pH 4.5 media reversed EGF binding, tyrosine phosphorylation and ERK-2 activation in exogenous cytosol, providing conditions in which signalling from the cell surface and internalised EGFRs could be distinguished. When cells containing internalised EGF/EGFR complexes were first washed in low pH media at 0 degrees C and then broken and incubated in exogenous cytosol, substantial activation of ERK2 occurred. This activation reached a maximum after a 5-min internalisation and was almost completely prevented by incubation in 0.45 M sucrose, a known inhibitor of receptor-mediated endocytosis. These data are consistent with activation of the ERK2 signalling pathway by internalised EGRFs situated in endosomal compartments. Our observation that EGFR tyrosine dephosphorylation is incomplete above pH 5.5 suggests that signalling is initiated in early endosomes.

Quantitative immunoelectron microscopy reveals alpha2,6 sialyltransferase is concentrated in the central cisternae of rat hepatocyte Golgi apparatus.

The Golgi apparatus is a membrane bound organelle involved in synthesis of N-linked oligosaccharides which are trimmed and then lengthened by a series of sugar transferases adding N-acetylglucosamine, galactose and sialic acid in sequence. We previously published qualitative work which localized Galbeta1,4GlcNAc alpha2,6 sialyltransferase of rat hepatocytes to the trans cisternae and the trans Golgi network. We now report the use of combined stereological and immunoelectron microscopical techniques for mapping the Golgi stack composition and distribution of sialyltransferase protein in rat hepatocytes. The Golgi stack showed substantial variation in composition consisting of 1, 2, 3, 4, or 5 cisternae with an average of 2.5 cisternae. Sialyltransferase labeling was mainly located in the central cisternae of the Golgi stacks irrespective of whether the stacks were oriented in a cis/trans direction using morphological criteria. Only 20% of the total sialyltransferase labeling was present in the transmost cisterna and 2% in the trans Golgi Network. The low labeling in the transmost cisterna was essentially due to the presence of a sialyltransferase negative cisterna. These data emphasize the importance of quantitation in obtaining a representative picture of Golgi enzyme distribution in three dimensions. They indicate that central cisternae, rather than the transmost cisterna and TGN, function in sialylation along the secretory pathway of rat hepatocytes.

Okadaic acid induces selective arrest of protein transport in the rough endoplasmic reticulum and prevents export into COPII-coated structures.

Quantitative immunoelectron microscopy and subcellular fractionation established the site of endoplasmic reticulum (ER)-Golgi transport arrest induced by the phosphatase inhibitor okadaic acid (OA). OA induced the disappearance of transitional element tubules and accumulation of the anterograde-transported Chandipura (CHP) virus G protein only in the rough ER (RER) and not at more distal sites. The block was specific to the early part of the anterograde pathway, because CHP virus G protein that accumulated in the intermediate compartment (IC) at 15 degrees C could gain access to Golgi stack enzymes. OA also induced RER accumulation of the IC protein p53/p58 via an IC-RER recycling pathway which was resistant to OA and inhibited by the G protein activator aluminium fluoride. The role of COPII coats in OA transport block was investigated by using immunofluorescence and cell fractionation. In untreated cells the COPII coat protein sec 13p colocalized with p53/p58 in Golgi-IC structures of the juxtanuclear region and peripheral cytoplasm. During OA treatment, p53/p58 accumulated in the RER but was excluded from sec 13p-containing membrane structures. Taken together our data indicate that OA induces an early defect in RER export which acts to prevent entry into COPII-coated structures of the IC region.

Antigen endocytosis and presentation mediated by human membrane IgG1 in the absence of the Ig(alpha)/Ig(beta) dimer.

Membrane immunoglobulin (mIg) M and D heavy chains possess minimal (KVK) cytoplasmic tails and associate with the Ig alpha/Ig beta (CD79) dimer to achieve surface expression and antigen presentation function. In contrast, the cytoplasmic tail of mIgG is extended by 25 residues (gamma ct). We have tested the possibility that mIgG can perform antigen capture and presentation functions independently of the Ig(alpha)/beta dimer. We show that CD4/(gamma)ct chimeras are efficiently endocytosed partially dependent on a tyrosine residue in (gamma)ct. In addition, human mIgG was expressed on the surface of Ig(alpha)/Ig(beta)-negative non-lymphoid cells and mediated antigen capture and endocytosis. Antigen-specific human mIgG targeted antigen to MIIC-type vesicles in the Ig(alpha)/beta negative melanoma Mel JuSo and augmented antigen presentation 1000-fold, identical to the augmentation seen in Ig(alpha)/beta-positive B-cells expressing the same transfected mIgG. Thus, unlike mIgM, mIgG has autonomous antigen capture and presentation capacity, which may have evolved to reduce or eliminate the BCR's dependence on additional accessory molecules.

Evidence for fusion between multilamellar endosomes and autophagosomes in HeLa cells.

The organelles of the endocytic and autophagic pathways were studied in HeLa cells using immunoelectron microscopy and stereological techniques. In the absence of autophagic stimulation, characteristic particulate structures containing closely packed layers of membrane received the fluid-phase marker horseradish peroxidase after 25 min uptake. These multilamellar endosomes contained the majority of the cellular immunogold labeling obtained by using a monoclonal antibody (1B5) directed against a lysosomal glycoprotein. Lysosomes with homogeneous dense content were only poorly labeled. After stimulation of autophagy, two classes of autophagosome profile appeared. One had a double limiting membrane and content that resembled the surrounding cytoplasm. The other most abundant type possessed a single limiting membrane and contained multilamellar structures which were strikingly similar to multilamellar endosomes, not only in form, but also in volume and membrane packing density. Immunogold labeling showed that now the majority of 1B5 labeling was located in the class of autophagosomes which contained multilamellar structures. Stereological methods showed that, after autophagic stimulation, multilamellar endosomes had become depleted, while multilamellar structures had appeared within the autophagosomes. Taken together, these data provide evidence that autophagosomes of HeLa cells fuse with preexisting multilamellar endosomes.

Distinct compartmentalization of TGN46 and beta 1,4-galactosyltransferase in HeLa cells.

The Golgi proteins, TGN46 and GalT, were characterized in human HeLa cells using specific polyclonal and monoclonal antibodies. A bacterially expressed soluble recombinant TGN46 protein was used to raise rabbit polyclonal antibodies and used to probe HeLa cell extracts. Human TGN46 had an apparent molecular mass of 100 to 120 kDa which reflects extensive glycosylation. Epifluorescence light microscopy indicated substantial colocalization of TGN46 and GalT. However, confocal laser microscopy and three-dimensional reconstruction of double-labeled HeLa cells revealed large areas of colocalization but also specific differences in the distribution of these two proteins within the Golgi apparatus. Importantly, quantitative immunoelectron microscopy showed that there was little overlap between the distribution of GalT and TGN46. Approximately 75% of GalT was in the Golgi stack, whereas 80% of TGN46 was detected in tubules. Distinct GalT-positive regions within the Golgi cisternal stack were not labeled for TGN46.

Carbonic anhydrase isoenzymes I, II, III, and IV are present in human esophageal epithelium.

Carbonic anhydrase (CA) isoenzymes have been widely studied in the gastrointestinal tract, where they mediate membrane transport events and pH regulation. However, the esophagus has generally received scant attention. In an immunohistochemical study confirmed by Western blotting, we have detected for CA isoenzymes (CAI, II, III, and IV) in the epithelium of human esophagus. Isoenzymes I, III, and sometimes IV (< 10%) were present in the cytoplasm of basal cells and II and IV in the cytoplasm and cell surface membranes, respectively, of suprabasal cells (prickle cells). The localization of CAIV to the plasma membranes was confirmed by electron microscopic immunocytochemistry. CA was effectively divided at the basal-suprabasal interface between low-activity CAI and III (basal) and high-activity CAII and IV (suprabasal). Carbonic anhydrase in esophageal epithelial cells may have several functions: elimination of CO2 and metabolites, participation in membrane transport events during active cell growth, and pH regulation as a protective mechanism against acidic gastric reflux.