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Video-assisted thoracoscopic surgery is emerging as a valuable tool in the investigation and management of thoracic neoplasms, including those involving the pleura, lung, and mediastinum with the e...

Biotinylated peptides Biot-Gln-Gln-Ile-Val and Biot-epsilon-Aca-Gln-Gln-Ile-Val were shown to act as acceptor substrates for amines in reactions catalyzed by both tissue transglutaminase and coagulation factor XIIIa. Moreover, the peptides could be employed for specifically blocking the potential amine donor sites of protein substrates participating in biological cross-linking with these enzymes. The presence of the biotin label allowed for ready detectability of the marked donor substrates during the cross-linking of crystallins in lens homogenate by the intrinsic transglutaminase and that of the alpha chains of human fibrin by factor XIIIa.

High resolution localized surface plasmon resonance (HR-LSPR) sensors were combined with matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) for the first time. LSPR sensors provide real-time label-free detection of molecular adsorption. Subsequent MALDI-MS analysis enables identification of the adsorbed molecules. This synergistic LSPR-MS approach was applied to the detection and identification of amyloid beta oligomers which play an important role in the molecular pathogenesis of Alzheimer’s Disease.

Individuals with early Alzheimer's disease (AD) suffer from a selective and profound failure to form new memories. A novel molecular mechanism with implications for therapeutics and diagnostics is now emerging in which the specificity of AD for memory derives from disruption of plasticity at synapses targeted by toxic Abeta oligomers (also known as ADDLs). ADDLs accumulate in AD brain and constitute long-lived alternatives to the disease-defining Abeta fibrils deposited in amyloid plaques. The AD-like cellular pathologies induced by ADDLs suggest their impact could provide a unifying mechanism for AD pathogenesis, explaining why early stage disease is specific for memory and accounting for major facets of AD neuropathology. Discovery of these new toxins has provided an appealing target for disease-modifying immunotherapy. For optimal protection against these toxins, antibodies should bind to the pathological oligomers without being depleted by their monomeric subunits, which are rapidly generated by membrane protein turnover. A solution to this problem is likely to come from the continued development of conformation-specific antibodies, as described here. Prototype conformation-specific antibodies, not yet in the clinic, have been introduced and utilized in multiple applications for their ability to bind with high specificity and affinity to ADDLs. It can be anticipated that further development of such antibodies for use in clinical trials will come in the near future.

The basis for memory loss in early Alzheimer's disease (AD) seems likely to involve synaptic damage caused by soluble Aβ-derived oligomers (ADDLs). ADDLs have been shown to build up in the brain and CSF of AD patients and are known to interfere with mechanisms of synaptic plasticity, acting as gain-of-function ligands that attach to synapses. Because of the correlation between AD dementia and synaptic degeneration, we investigated here the ability of ADDLs to affect synapse composition, structure, and abundance. Using highly differentiated cultures of hippocampal neurons, a preferred model for studies of synapse cell biology, we found that ADDLs bound to neurons with specificity, attaching to presumed excitatory pyramidal neurons but not GABAergic neurons. Fractionation of ADDLs bound to forebrain synaptosomes showed association with postsynaptic density complexes containing NMDA receptors, consistent with observed attachment of ADDLs to dendritic spines. During binding to hippocampal neurons, ADDLs promoted a rapid decrease in membrane expression of memory-related receptors (NMDA and EphB2). Continued exposure resulted in abnormal spine morphology, with induction of long thin spines reminiscent of the morphology found in mental retardation, deafferentation, and prionoses. Ultimately, ADDLs caused a significant decrease in spine density. Synaptic deterioration, which was accompanied by decreased levels of the spine cytoskeletal protein drebrin, was blocked by the Alzheimer's therapeutic drug Namenda. The observed disruption of dendritic spines links ADDLs to a major facet of AD pathology, providing strong evidence that ADDLs in AD brain cause neuropil damage believed to underlie dementia.

Background Rheumatoid arthritis (RA) is a chronic disease that leads to inflammation of the joints and other tissues. Rituximab (Rtx) is a therapeutic monoclonal antibody directed against CD20 that induces an important depletion of B cells. Although little is known about how it modifies the homeostasis of lymphoid subpopulations, it has been successfully used in RA. Objectives The objective of this study was to know whether Rtx modifies the ratio of the different lymphocyte subpopulations from peripheral blood in RA patients. Methods We studied 62 RA patients that underwent treatment with Rtx because of active RA and failure to at least one previous anti-rheumatic drug. The patients were divided into those naive for Rtx treatment (RtxN n=21) and those who had received at least one previous cycle of treatment with Rtx (RtxS n=41). Peripheral blood samples for lymphoid subpopulation analysis were obtained at different times in both RtxN and RtxS patients: before the first infusion of Rtx (baseline) and at 3, 6 and 8 months after it (T3, T6 and T8 respectively). We studied different combinations of molecules that allowed us to calculate the percentage of T cell subpopulations, including memory and naive T cells, effector and central memory T cells, follicular T helper cells and regulatory T cells. In the case of B cells, we analyzed memory and naive B cells, Marginal Zone B cells, follicular B cells and plasmablasts B cells, at each visit. Differences between groups were analyzed by ANOVA test. Results At baseline, there was a significant difference in the percentage of a considerable number of B cell subsets. In particular, we observed an important decrease in memory B cells and an increase in follicular B cells. We also detected an increase in CD38+CD24+CD10+ B cells in RtxS. Regarding T cell subpopulations, we only observed a significant decrease in Th17 cells and in follicular T helper cells in RtxS. During the follow up period, the differences in T cells observed at baseline became much smaller presumably because of the effects of Rtx. At T3 only CD4 + T cells and memory T cells, defined by the absence of CD62L, were significantly lower in RtxS. At T6, we observed an increase in the CD8+CCR6- CXCR6- subpopulation in RtxS. At T8, as some patients had already repopulated B cell subsets, we found differences in B cells, and we detected a decrease in the CD38- B cell population. Taking into account the T cell population, we noted an increase in total T cells in RtxS with a significant decrease in effector-memory T cells. Conclusions Depletion of B cells through Rtx treatment leads to a profound change in the subpopulations of peripheral blood B cells, due to repopulation, and also of T cells. This suggests that mature B cells play a relevant role in the homeostasis of T cells. Acknowledgements * L. Merino and J. Lόpez have contributed equally to this work. Disclosure of Interest None declared

Two siblings with hypofibrinogenemia have lifelong trauma-related bleeding. Recently, the brother experienced recurrent thrombosis after cryoprecipitate infusions following surgery. The sister had 6 miscarriages. Plasma clots in each were resistant to compression and fibrinolysis and were soluble in 5 M urea. Examination by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) revealed only the presence of crosslinked γ–γ fibrin chain dimers without high polymers of αn. Fibrin clots contained an abnormal 35-kDa constituent recognized by an antibody to the mature fibrinogen Aα–chain residues 241-476 but not by antibodies to Aα219-348 or Aα349-406. DNA analysis revealed a heterozygous CAA → TAA mutation at the codon for amino acid 328 of the Aα gene in these siblings and 2 asymptomatic family members. The Gln328stop mutation (fibrinogen Keokuk) predicted a 46% truncation and the production of a 35-kDa Aα chain. Analysis of purified fibrinogen revealed expression of the abnormal Aα chain in 4 family members but found no normal fibrinogen in the 2 hypofibrinogenemic patients. This paradox was resolved when they and their asymptomatic mother were found to be heterozygous for a second Aα mutation, a GT → TT splice site mutation in intron 4 (IVS4 + 1 G> T). However, compound heterozygosity for both mutations was required for the expression of severe hypodysfibrinogenemia and for clinical symptoms.

Intracranial hemorrhage in a young woman with systemic lupus erythematosus necessitated two surgical evacuations. In the absence of a family history of bleeding, clot solubility in urea suggested a factor XIII (FXIII) inhibitor. The patient's IgG bound well to the virgin and the thrombin-modified zymogen ensemble (A(2)B(2) and A(2)'B(2)) and to the free rA(2) but reacted poorly with the thrombin-modified rA(2)'. Since the IgG did not block the thrombin-catalyzed proteolysis of A subunits nor the dissociation of the A(2)'B(2), its action might be to interfere with the release of activation peptides from the thrombin-cleaved zymogen, hindering the conformational change necessary for generating FXIIIa. Treatment with cryoprecipitate and cyclophosphamide arrested the hemorrhage and almost neutralized the antibody so that the patient's clot became insoluble in urea and showed a close to normally crosslinked gamma-gamma and alpha(n) fibrin chain profile. Nevertheless, she still has detectable anti-FXIII antibody and may be at risk for hemorrhage.

Background The recommended therapeutic regime for Rituximab (RTX) in Rheumatoid Arthritis (RA), according to prescribing information, includes two 1000-milligram infusions given two weeks apart, every 6 months. However, this is often not the case in clinical practice, since both consensus documents and information from clinical trials consider other alternatives. Objectives Our objective in this study was to analyze the regime of use of RTX in RA in daily clinical practice. Methods This is a retrospective study that includes patients treated with RTX between 1998 and 2013 in a single university hospital. We reviewed medical records and collected demographic data, number of cycles, doses and intervals of RTX administered to the patients, response duration, as well as frequency and reasons of treatment discontinuation. Descriptive analysis was performed using the statistical package Stata v. 12. Results Ninety-three patients were studied, of which 83% were women. Median age at disease onset was 51 years with an interquartile range (IQR) of 39 to 60 years. Median age at the start of treatment with RTX was 60 [IQR: 51-70] years. Out of the 93 patients, 11 had negative rheumatoid factor. The number of cycles of RTX administered to each patient ranged from one to nine. Treatment was discontinued in 33% of the patients. The reasons for discontinuation were inefficacy (16%), adverse effects (7%) and others (10%). RTX was most commonly withdrawn during the first two cycles. The main data related to use of RTX in our study are summarized in the following table. Response duration in males tended to be longer [12 months; IQR: 8-13] than in females [10 months; IQR: 7-12], but this didn9t reach statistical significance (p=0911, Mann-Whitney9s test). Longer response duration was observed in patients with a longer RA history (r =0.24, P=0.001, Pearson9s test). RTX dose per cycle did not modify the response duration (1 vs 2 grams, 9.5 and 10 months respectively). Conclusions Our data show that, in daily clinical practice, RTX is more frequently used on demand, tending to abandon the fixed regime of 2 grams every six months. In addition, we observe a tendency to an increased use of 1 gram cycles with time. This results in cost savings without apparent decrease in healthcare quality. References Martin Mola et al. Grupo de Expertos en Rituximab. Reumatol Clin. 2011;7:30-44 Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4562

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

Without a prior history of hemorrhagic disease, a 62-year-old man suffered recurrent episodes of bleeding. Solubility of the patient’s clot in 5 mol/L urea indicated a problem with fibrin stabilization. The transamidase activity potential of factor XIII, measured by the incorporation of radioactive putrescine into N,N-dimethylcasein as test substrate, was 62% of control, close to the normal range of values. Examination of the patient’s clot from recalcified plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that essentially none of the  chains and only about two thirds of the γ chains of fibrin became cross-linked under conditions where both were fully cross-linked in the controls. An antibody to factor XIII was isolated which, although recognizing the recombinant rA2subunits, as well as the virgin A2B2 plasma ensemble, showed a 100-fold greater affinity for the thrombin-activated rA2′ and A2′B2 forms of the zymogen, suggesting that the latter would be its main target during coagulation. Furthermore, the patient’s IgG has an ability, never seen before, for inducing an enzymatically active configuration in the thrombin-activated zymogen in the absence of Ca2+.

Mere addition of Ca2+ to a lens cortical homogenate (bovine) generates a series of products composed of a variety of high molecular weight vimentin species. The Ca2+-induced cross-linking of this cytoskeletal element seems to be mediated by the intrinsic transglutaminase of lens, because the reaction could be blocked at the monomeric state of vimentin by the inclusion of small synthetic substrates of the enzyme dansylcadaverine or dansyl-epsilon-aminocaproyl-Gln-Gln-Ile-Val. These compounds are known to compete against the Gln or Lys functionalities of proteins that would participate in forming the Nepsilon(gamma-glutamyl)lysine protein-to-protein cross-links. The cytosolic transglutaminase-catalyzed reactions could be reproduced with purified bovine lens vimentin and also with recombinant human vimentin preparations. Employing the latter system, we have titrated the transglutaminase-reactive sites of vimentin and, by sequencing the dansyl-tracer-labeled segments of the protein, we have shown that residues Gln453 and Gln460 served as acceptor functionalities and Lys97, Lys104, Lys294, and Lys439 as electron donor functionalities in vimentin. The transglutaminase-dependent reaction of this intermediate filament protein might influence the shape and plasticity of the fiber cells, and the enzyme-catalyzed cross-linking of vimentin, in conjunction with other lens constituents, may contribute to the process of cataract formation.

Gel filtration of the water-soluble extract from bovine lens yields a group of proteins, emerging between the peaks of beta H and beta L crystallins, which show a considerably greater sensitivity to heat-induced aggregation/precipitation than the far more abundant beta and gamma crystallins. However, the small heat shock protein: alpha crystallin was effective in protecting these trace constituents of the lens from precipitating out of solution at 55 degrees C (measured under the standard conditions in a pH 7.5 buffer containing 50 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA and 0.05% NaN3). Prominent components of the precipitate, formed in the absence of a recombinant alpha B crystallin chaperone could be resolved by one- and two-dimensional electrophoresis. Identification by amino acid sequencing revealed that the heat-sensitive group of lens proteins comprised glyceraldehyde-3-phosphate dehydrogenase (M(r) approximately 39 kDa), enolase (approximately 48 kDa), leucine aminopeptidase (approximately 52 kDa) and aldehyde dehydrogenase (approximately 53 kDa). These findings indicate for the first time that the aggregation of such minor lens constituents could possibly contribute to initiating the process of opacification in the development of cataracts.

A mouse monoclonal IgG, G82, directed against guinea pig liver transglutaminase recognizes a transglutaminase-related antigen that is associated with the keratin intermediate filament network in some primary mouse keratinocytes. The association can be seen at the resolution of individual keratin tonofibrils following fixation and staining for double-label indirect immunofluorescence. Western blots indicate that G82 reacts with two proteins of 95 kDa and 280 kDa, respectively, in extracts of these cells. The 95-kDa band is also recognized by a polyclonal antibody against purified guinea pig liver transglutaminase, and the 280-kDa protein seems to correspond to a similar protein that was shown to be recognized by G92.1.2 in the intermediate filament fraction of primary mouse fibroblasts. The transglutaminase-related antigen was shown by confocal microscopy to co-localize only with nonbasal cell specific keratin intermediate filaments.

A multidisciplinary project was undertaken at The New York Hospital-Cornell Medical Center to develop critical pathways for open-heart surgery to help reduce cost, shorten hospital length of stay (LOS), and streamline patient care.A critical pathway for elective coronary artery bypass grafting instituted on March 1, 1995, was developed through a cooperative effort involving surgeons, anesthesiologists, nurses, social workers, physical therapists, nutritionists, and patient case managers. Prospective data collected on consecutive patients forming a critical pathway group (n = 114) over a 6-month period were compared with retrospective data on consecutive patients forming a cohort group (n = 382) who underwent elective coronary artery bypass grafting in 1994.The critical pathway group of patients experienced a significantly shorter total hospital LOS (7.7 +/- 2.3 days vs 11.1 +/- 6 days, p0.0001) and shorter intensive care unit LOS (1.5 +/- 0.9 days vs 2.0 +/- 2.8 days, p0.0001). Direct costs were computed by use of hospital charges multiplied by the Medicare cost-to-charge ratio. Mean hospital direct cost (ancillary resources) was $1181 lower in the critical pathway group when compared with the control group (p0.0001). The postoperative mortality and readmission rates were similar for the two groups of patients.The ongoing analysis of cost, LOSs, and outcomes has made possible a process of continuous quality improvement on the cardiothoracic service in which further areas for improvement are identified and studied. The use of a critical pathway for elective coronary artery bypass grafting at our institution significantly reduced hospital LOS and direct costs while maintaining the overall quality of patient care.

A mouse monoclonal antibody, G92.1.2, raised against guinea pig liver transglutaminase (TGase) recognizes an antigen present in primary mouse dermal fibroblasts. A filamentous pattern, bearing remarkable similarity to the vimentin intermediate filament (IF) network, is seen when these cells are fixed and processed for indirect immunofluorescence with the antibody. Double-label immunofluorescence reveals that the antigen reacting with the antibody colocalizes precisely with vimentin IF and that this colocalization is retained after the treatment of fibroblasts with colchicine, which induces a redistribution of the majority of IFs into perinuclear aggregates. These morphological observations are further supported by the finding that the protein reacting with G92.1.2 is retained in IF-enriched cytoskeletal preparations made by using nonionic detergent-containing high ionic strength solutions. Western blots of the IF fraction show that G92.1.2 recognizes a major band of approximately 280 kDa and does not cross react with vimentin. Furthermore, when the antibody is microinjected into live dermal fibroblasts, it causes a collapse of the vimentin IF network in the majority of injected cells. The results suggest that a form of TGase, or a TGase-related antigen, is closely associated with the vimentin IF network of primary cultures of mouse dermal fibroblasts.

The size and shape of Alzheimer’s β-amyloid structures, as well as the kinetics of their self-assembly, exhibits a very pronounced dependence on concentration and environment. In the present study, we are reporting the direct observation of Aβ oligomers and fibrils assemblies using atomic force microscopy imaging in fluid environment. These results demonstrate that in the Aβ preparations at lower concentrations, predominant are the globular, smaller oligomers, while for higher concentrations, globular oligomers co-exist with higher molecular weight fibrillar structures. At higher concentrations, the fibril formation is a dynamic and continuous process, yielding amyloid fibrils with multiple structures and diameters.

To study the early hemodynamic effects of the rapid infusion of 7.5g/dl NaCl/ 6g/dl dextran-70 solution in dogs submitted to hemorrhagic shock.Mongrel dogs were anesthetized with pentobarbital and a electromagnetic flowmeter probe was placed around the ascending aorta or the portal vein. By external bleeding the arterial pressure was lowered to 40mmHg and held for 30min. The animals received a 4ml/kg infusion of the hypertonic solution in 90s. Arterial blood pressure and flow were registered continuously during 3min and the derived hemodynamic variables were calculated at regular time intervals.The total plasma protein concentration decreased and the cardiac output showed a continuous elevation during the infusion. The arterial blood pressure showed two oscillations and then decreased during a short period of time. This moment was coincident with the initial increase of the portal flow and preceded the elevation of the systemic vascular resistance and the arterial pressure.The rapid infusion of hypertonic NaCl/dextran solution to dogs in hemorrhagic shock determines immediate and intense hemodynamic effects. During the infusion period there is volemic expansion and the cardiac output increases rapidly. The arterial pressure shows oscillations and decreases as a consequence of visceral arterial dilation before starting its final elevation that occurs as the vascular resistance increases.

The transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13)-catalyzed cross-linking of proteins in biological systems can often be inhibited by inclusion of small primary amines or glutamine-containing peptides, which act as site-specific blockers of the relevant acceptor (i.e., glutamine) and donor (i.e., lysine) functionalities of the natural substrates. Compounds such as dansylcadaverine and dansyl-epsilon-aminocaproyl-Gln-Gln-Ile-Val are particularly useful in sorting out acceptor-donor relationships among lens crystallins. Apart from its fluorescent properties, the dansyl hapten offered special advantages as a "handle" for the rapid isolation of transglutaminase targets even in the complex system of lens cortical homogenate. The dansylated peptide was incorporated into bovine lens proteins under the influence of the Ca(2+)-activated intrinsic transglutaminase and, after digestion by endoproteinase Glu-C, the tracer-containing fragments were isolated by affinity chromatography on an anti-dansyl antibody column. The major fluorescent peak was isolated by HPLC and sequenced by Edman degradation, which yielded phenylthiohydantoin amino acid derivatives for the first 10 cycles, EKPAVTAAPK, and none for the next 2. The sequence, corresponding to residues 165-174 of alpha B-crystallin, unambiguously identifies the known carboxyl-terminal domain, EK-PAVTAAPKK, as the prominent lysine-donating fragment in bovine lens.

The transglutaminase-catalyzed incorporation of the fluorescent amine, dansylcadaverine, into casein derivatives, such as N,N-dimethylcasein, is accompanied by a large increase in intensity of emission (Lorand et al., Anal. Biochem. 44, 221-231, 1971). We have sought to make use of this sensitive detection device for the continuous, on-line monitoring of an amide-splitting reaction in which dansylcadaverine served as the leaving group. The transglutaminase-coupled test system comprised gamma-glutamyldansylcadaverine as the first substrate and gamma-glutamylamine cyclotransferase as the enzyme responsible for releasing dansylcadaverine from the gamma-amide. At close to saturating levels of transglutaminase, the measured rate of increase of fluorescence, i.e. the steady-state rate of dansylcadaverine incorporation into N,N-dimethylcasein, showed a near-linear relationship with the concentration of gamma-glutamylamine cyclotransferase present in the assay mixture. The general approach developed may be applicable to the assay of other amide cleaving enzymes.

The hemodynamic effects, after infusion of 4 ml/kg of hypertonic (7.5%) saline solution (HS), were evaluated in six patients (mean age = 56.6 years) with cardiogenic shock (CS) due to right ventricular infarction (RVI). Basal condition data (mean +/- SEM) were as follows: cardiac index (CI) = 1.9 +/- 0.1 1/min/m2, arterial pressure (AP) = 66.5 +/- 0.9 mmHg, and systemic vascular resistance (SVR) = 31.3 +/- 1.0 mmHg/1/min/m2. Five- and 240-minute post-HS infusion data (respectively) revealed: CI = 3.3 +/- 0.1* and 2.9 +/- 0.1* 1/min/m2, AP = 87.7 +/- 1.6* and 80.7 +/- 2.2* mmHg, and SVR = 22.5 +/- 0.6* and 24.5 +/- 1.1* mmHg/1/min/m2 (*P less than 0.05 compared to baseline values). These data suggest that small-volume infusion of HS induced an important acute and sustained hemodynamic improvement in these patients with CS due to RVI.

Twenty six patients at different stages of pregnancy and diagnosis of rubella were studied prospectively in order to evaluate the perinatal outcome. Diagnosis was based in the detection of specific IgM or on a four-fold rise in antibody titre (IHA) with an interval of three weeks between one and other specimen blood. The stage of pregnancy was defined as the interval between the last menstrual period and onset of the rash. The pregnancies surveillance was performed with biophysics test periodically. The pregnancies were terminated in base of the obstetrical conditions. The analyzed variables were: Maternal age, stage of pregnancy at the attack, duration of the rash, parity, clinical diagnosis of the newborn. Pregnancy continued in 16 patients and were followed up to the birth of the infants. The frequency of congenital infection after maternal rubella was more than 80% during the 4-8 weeks. Congenital rubella syndrome occurred in 2 newborn infected at the organogenesis period (4-8 weeks). Nevertheless there are 12 children identified serologically which do it interesting the following of these infants for to identify in an opportune form possible structural alterations as late manifestations of rubella.

The effects of various hypertonic solutions on the intraventricular conduction disturbances and on the cardiac arrhythmias caused by the intravenous (i.v.) injection of bupivacaine were studied in sodium pentobarbital anesthetized mongrel dogs. Bupivacaine was injected in 2 doses: 3.0 mg/kg and 6.5 mg/kg. Hypertonic solutions, given intravenously 5 minutes before bupivacaine, were 7.5% NaCl, 5.4% LiCl or 50% glucose (2,400 mOsm/l, 5 ml/kg), or 20% mannitol (1,200 mOsm/l, 10 ml/kg). The highest dose of bupivacaine induced severe cardiac arrhythmias and intraventricular conduction disturbances, as reflected by significant increases in QRS complex duration, HV interval and IV interval, as well as a severe hemodynamic impairment. Significant prevention against intraventricular conduction disturbances and ventricular arrhythmias was observed with 7.5% NaCl (QRS complex duration percent increase: 164 +/- 21% in the non pretreated group vs. 75 +/- 14% in the pretreated group, P less than .01; HV interval percent increase: 131 +/- 16% in the non pretreated group vs. 58 +/- 7% in the pretreated group, P less than .01; cardiac index percent decrease: 46 +/- 6% in the non pretreated group vs. 28 +/- 5% in the pretreated group, P less than .025). The three other hypertonic solutions were ineffective. These findings suggest an involvement of sodium ions in the mechanism of hypertonic protection.

Fifty pentobarbital anesthetized dogs were subjected to pressure driven hemorrhage (PDH) in which (a) an initial bleeding rate (25 ml/min) was set, and (b) reset min-to-min in proportion to prevailing mean arterial pressure (MAP). When blood loss reached 40 ml/kg, experimental time was set to zero and dogs were divided into five groups: (1) CTR (untreated controls); (2) HSD (NaCl 7.5%-Dextran70 6%, 6 ml/kg, at zero time); (3) LR (lactated Ringers, 25 ml/min from 0-60 min); (4) HSD-LR (combines HSD and LR); (5) DBL-HSD-LR (as HSD-LR, plus second HSD injection, 4 ml/kg, at 30 min). PDH was continued throughout the postresuscitation period. CTR dogs bled 55.5 +/- 2.1 ml/kg and survived to 34.7 +/- 5.0 min postzero; HSD dogs bled 78.6 +/- 2.0 ml/kg, and survived to 51.2 +/- 2.9 min with transient recovery of MAP, cardiac output (CO), and O2 availability (O2A); LR dogs bled 94.5 +/- 3.4 ml/kg and survived for over 60 min, with sustained, partial recovery of MAP, CO, and O2A. HSD-LR dogs bled 111.5 +/- 3.7 ml/kg and survived for over 60 min with improved hemodynamic and metabolic response. In DBL-HSD-LR dogs, the second HSD produced higher MAP, CO, and O2A, but hematocrit was lowered to a critical level. Thus, standard LR resuscitation is effective in PDH, in spite of increased blood loss; a single HSD lengthens survival when used alone and improves recovery when added to LR.

We report here the direct observation of high resolution structures of assemblies of Alzheimer beta-amyloid oligomers and monomers using liquid atomic force microscopy (AFM). Visualization of nanoscale features of Abeta oligomers (also known as ADDLs) was carried out in tapping mode AFM in F12 solution. Our results indicate that ADDL preparations exist in solution primarily as a mixture of monomeric peptides and higher molecular mass oligomers. Our study clearly reveals that the size and shape of these oligomer aggregates exhibit a pronounced dependence on concentration. These studies show that wet AFM enables direct assessment of oligomers in physiological fluids and suggests that this method may be developed to visualize Abeta oligomers from human fluids.

Peptides patterned on the N-terminal sequence of fibronectin were synthesized and tested for amine acceptor qualities in reactions with dansylcadaverine catalyzed either by coagulation factor XIIIa or intracellular transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13). On the basis of inverse half-saturations of the enzymes, the order of acceptor substrate affinity for factor XIIIa was pEAQQIV much greater than Boc-AQQIV greater than Boc-QQIV, and for transglutaminase, Boc-QQIV greater than Boc-AQQIV greater than pEAQQIV (amino acid residues are shown in one-letter code; pE, pyroglutamic acid; Boc, tert-butyloxycarbonyl). Sequence analysis of dansylcadaverine-substituted pEAQQIV indicated that the first of the two adjacent glutamine residues was the target of enzymatic modification. Boc-QIV showed no substrate activity with either enzyme. Crosslinking of crystallins in Ca2(+)-treated rabbit lens homogenate was readily inhibited by Boc-QQIV, Boc-AQQIV, and pEAQQIV, as was the formation of alpha-chain polymers in human fibrin by pEAQQIV in the presence of human factor XIIIa. SDS/PAGE analysis suggested that the inhibitory peptides selectively blocked the electron donor functionalities in these enzymatic crosslinking reactions.

Hypertonic saline, or saline-dextran resuscitation is normally achieved with an Na+ load of 4.8 to 7.2 mEq/kg given in a small volume (typically 4 to 6 ml/kg NaCl 7.5%). Na+ can also be administered saturated in a smaller volume, e.g., 1 to 1.5 ml/kg NaCl 25%, with similar results. Such reduction in administered volume would be an asset in prehospital trauma management. In the present experiments, severely bled (45 ml/kg) dogs were treated with one of three NaCl/dextran-70 solutions: S1, 25% NaCl + 24% dextran (1.5 ml/kg); S2, 15% NaCl + 14.4% dextran (2.5 ml/kg); S3, 7.5% NaCl + 6% dextran (5 ml/kg). S1, S2, and S3 were pump-infused in 10 min into a peripheral vein; S1 and S2 were also given into the right atrium. S1, S2, or S3 produced a number of similar responses irrespective of the route of administration; arterial pressure, cardiac index, and base excess reverted to near control levels, plasma Na+ was raised to 155-158 mEq/L, and 5-day survival was high and comparable. Plasma volume, and total and mean red cell volumes were similarly affected in all groups; however, peripheral injections of S1 and S2 induced severe hemolysis (plasma Hgb: 53 +/- 6 and 34 +/- 4 mg/dl, respectively), while right atrial S1 and S2 caused mild hemolysis (22 +/- 3 and 14 +/- 3 mg/dl, respectively). In contrast, S3 never induced hemolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

The dansyl-conjugated (Dns) peptides Dns-Pro-Gly-Gly-Gln-Gln-Ile-Val and Dns-Ala-Gln-Gln-Ile-Val, patterned on the N-terminal sequence of fibronectin, were synthesized and used for the transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13)-directed selective blocking of lens proteins that otherwise might participate in donating lysyl side chains in forming N epsilon-(gamma-glutamyl)-lysine cross-linked oligomers and polymers. Labeling profiles with these peptides could be readily visualized by fluorescence as well as by immunoblotting with anti-dansyl antibody. The labeling patterns in rabbit lens homogenates were quite different with the dansylated peptides than those obtained with dansylcadaverine. Use of such glutamine-containing dansylated peptides should clearly aid in identifying, isolating, and sequencing potential donor substrates of transglutaminases in many biological systems.

To identify the incidence, risk factors, and treatment of diarrhea and Clostridium difficile-associated diarrhea (CDAD) in surgery patients.Prospective and historical retrospective analysis.Major urban tertiary care referral hospital.Consecutive patients (N = 475) admitted to the vascular, trauma, and general surgical surgery services, prospectively evaluated during a 10-week period. A retrospective historical control of the same surgical services was used for comparison.None.Incidence of diarrhea and CDAD, use of bowel preparations, surgical procedure, use of C difficile toxin assay, white blood cell count, symptoms, treatment, and delay in hospital discharge.The incidence of diarrhea in surgery patients analyzed prospectively was 6.1%; the incidence of CDAD during the prospective and retrospective periods was 2%. Preoperative bowel preparations were associated with an increased risk of diarrhea (relative risk, 4.2; 95% confidence interval, 2.6-6.8; P.001) and CDAD (relative risk, 3.2; 95% confidence interval, 1.5-7.2; P.03). Leukocytosis (white blood cell count11 x 10(9)/L) was significantly higher in the CDAD group compared with the diarrhea group only on the day of diagnosis (P.05). By subjective analysis, diarrhea was directly responsible for a delay in discharge in 7 of 29 patients for a mean (+/-SEM) of 4.0 +/- 1.0 days.Patients undergoing preoperative bowel preparations are at increased risk of experiencing diarrhea and CDAD. Among patients with diarrhea, an elevated white blood cell count may help identify those with C difficile. Early treatment of diarrhea with oral metronidazole while awaiting the results of the stool toxin assay is recommended for treating diarrhea in surgery patients. Prophylactic treatment of surgery patients undergoing bowel preparations should be considered.

The amine specificity of guinea pig liver transglutaminase, a model enzyme for endo-gamma-glutamine:epsilon-lysin transferases, was explored with the aid of synthetic substrates of high apparent affinities. As exemplified by dansyl- (5-dimethylamino-1-naphthalenesulfonyl), (2,4-dinitrobenzenesulfonyl)-, and (2,4,6-triisopropylbenzenesulfonyl)-cadaverines--each of which showed affinities of approximately 4 x 10(7) M-1--the best amine substrates carried a large hydrophobic substituent attached to an alkylamine side chain of about 7.2 A in length. Altogether, our results point to the importance of a hydrophobic binding region in the enzyme from where the alkyl side chain reaches into a narrow crevice toward the active center and positions the primary amine of the substrate for attacking the carbonyl group of the acyl enzyme intermediate.

An immunoblotting procedure, using anti-dansyl antibody, was employed to demonstrate that band 3 protein was the predominant substrate in inside-out vesicles from human erythrocytes reacting with transglutaminase.

Incubation of lens in Ca2+-containing media, considered by several investigators to be a useful model of cataract formation, gave rise to significant alterations in the covalent structures of various proteins. In rabbit lens, when sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used after reduction of disulfides in urea, the most readily observable changes were (i) disappearance of 210K, 95K, and 60K proteins, (ii) modifications of alpha crystallin subunits, (iii) alterations of beta H crystallins, and (iv) de novo production of 55K and higher molecular weight polymers. The addition of leupeptin inhibited the disappearances of 210K, 95K, and 60K proteins and the alteration of alpha crystallins, suggesting that all these were caused by a Ca2+-activated protease. The proteolytically sensitive 60K species was identified as vimentin, a component of intermediate filaments. Formation of the 55K material and of higher molecular weight polymers during Ca2+ treatment of the lens could be prevented by histamine, a compound known to inhibit the transglutaminase-mediated cross-linking of proteins by epsilon-(gamma-glutamyl)lysine peptide bonds in other biological systems. It could also be shown by immunoblotting that an antibody raised against the 55K material reacted selectively with beta crystallins of normal lens. This indicates that the 55K product is in all likelihood an essential intermediate toward higher polymers and that the 55K product is a cross-linked dimer of certain polypeptides of beta crystallin.(ABSTRACT TRUNCATED AT 250 WORDS)

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Severe hemorrhage in pentobarbital-anesthetized dogs (25 mg/kg) is reversed by intravenous NaCl (4 ml/kg, 2,400 mosmol/l, 98% long-term survival). This paper compares survival rates and hemodynamic and metabolic effects of hypertonic NaCl with sodium salts (acetate, bicarbonate, and nitrate), chlorides [lithium and tris(hydroxymethyl)aminomethane (Tris)], and nonelectrolytes (glucose, mannitol, and urea) after severe hemorrhage (44.5 +/- 2.3 ml/kg blood loss). Sodium salts had higher survival rates (chloride, 100%; acetate, 72%; bicarbonate, 61%; nitrate, 55%) with normal stable arterial pressure after chloride and nitrate; near normal cardiac output after sodium chloride; normal acid-base equilibrium after all sodium salts; and normal mean circulatory filling pressure after chloride, acetate, and bicarbonate. Chlorides and nonelectrolytes produced low survival rates (glucose and lithium, 5%; mannitol, 11%; Tris, 22%; urea, 33%) with low cardiac output, low mean circulatory filling pressure, and severe metabolic acidosis. Plasma sodium, plasma bicarbonate, mean circulatory filling pressure, cardiac output, and arterial pressure correlated significantly with survival; other parameters, including plasma volume expansion or plasma osmolarity, did not. It is proposed that high plasma sodium is essential for survival.

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The accumulation of Ca2+ ions in intact human erythrocytes leads to the production of membrane protein polymers larger than spectrin. The polymer has a heterogeneous size distribution and is rich in gamma-glutamyl-epsilon-lysine cross-links. Isolation of this isodipeptide, in amounts as high as 6 mol/10(5) g of protein, confirms the idea [Lorand L., Weissmann, L.B., Epel, D.L., and Bruner-Lorand, J. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 4479] that the Ca2+-induced membrane protein polymerization is mediated by transglutaminase. Formation of the polymer in the intact cells is inhibited by the addition of small, water-soluble primary amines. Inasmuch as these amines are known to prevent the Ca2+-dependent loss of deformability of the membrane, it is suggested that transglutaminase-catalyzed cross-linking may be a biochemical cause of irreversible membrane stiffening.

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The dansyl hapten in dansylcadaverine offers unique possibilities for measuring the incorporation of the monoamine into proteins (e.g. N,N′-dimethylcasein) by transamidating enzymes such as factor XIIIa and the transglutaminases. The protein-bound dansylcadaverine was assayed by an ELISA procedure based on a monoclonal antibody to the dansyl moiety.