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ABSTRACTBackgroundThe systemic immune-inflammation index (SII) is a novel parameter and its role in the prognosis of sepsis has never been explored previously.MethodsWe retrospectively assessed 267 patients with blood-culture confirmed sepsis. Clinical and laboratory data recorded at intensive care unit (ICU) admission were analyzed. Outcomes of interest included in-hospital mortality and length-of-stay (LOS) in the ICU. Sequential Organ Failure Assessment (SOFA) scores, SII, neutrophil-lymphocyte ratio (NLR), and platelet-lymphocyte ratio (PLR) were calculated. Multivariable regression analysis was used to identify independent predictors of LOS and mortality. Area under receiver operator characteristic (AUROC) curves were used to determine optimum cutoffs, and the incremental effect of SII on the SOFA score was assessed using model discrimination and calibration properties.ResultsThere were 76 (28.5%) non-survivors. SII, NLR, and PLR were independent predictors of sepsis mortality, with adjusted odds ratios of 1.51 (1.24–1.84), 1.67 (1.30–2.13) and 1.24 (1.11–1.39). SII and SOFA score were independent predictors of LOS. SII had an AUROC of 0.848, and the optimum cutoff was 564 with a sensitivity and specificity of 85.5% and 71.2%. The addition of SII to the model had a significant incremental effect on the predictive ability of SOFA score (Net Reclassification Index = 0.084, P= 0.025; Integrated Discrimination Index = 0.056, P= 0.001).ConclusionThe SII is an inexpensive parameter that can be used in addition to clinical sepsis scores to improve the accuracy of patient assessment.

Archaeosomes composed of sulfated lactosyl archaeol (SLA) glycolipids from stereoisomerically pure archaeol (1) are vaccine adjuvants that can boost immunogenicity and vaccine efficacy in preclinical models. Herein, we report a new synthesis of 2,3-bis((3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol (3) by treating (±)-3-benzyloxy-1,2-propanediol with a mesylated phytol derivative through a double nucleophilic substitution reaction, followed by reductive debenzylation. Three SLA archaeosomes from archaeols of different chiral purities were prepared, and the effect of stereochemistry on their adjuvanticity toward ovalbumin was investigated. It was found that all SLA archaeosomes induced strong humoral and cell-mediated antigen-specific immune responses following immunization of C57BL/6NCrl mice, with no significant differences, irrespective of the chiral purities. The responses were comparable or better than those obtained using mimetics of approved adjuvants. The performance of SLA archaeosomes during immunization and their lack of dependence on the stereochemistry of archaeol points toward a promising, safe, scalable, and economically viable vaccine adjuvant system.

Metastatic renal cell carcinoma (mRCC) comprises a highly heterogeneous group of diseases with varied clinical outcomes. As a result, models to estimate prognosis were developed in an attempt to aid patient counseling, treatment selection, and clinical trial design. Contemporary prognostic models have been mostly generated based on clinical factors because of their ease of use. Recent advances in molecular techniques have allowed unprecedented molecular profiling of RCC and the discovery of genomic and proteotranscriptomic factors that may contribute to disease trajectory. With the advent of multiple systemic therapies in mRCC in recent years, predictive biomarkers have become increasingly relevant in treatment selection. In this review, we discuss the existing staging systems and prognostic models in mRCC. We also highlight various promising molecular biomarkers according to the subtypes of RCC and explore their integration into the traditional prognostic models. In addition, we discuss emerging predictive biomarkers in the era of immuno-oncology. Lastly, we explore future directions with a focus on liquid biopsies and composite biomarkers.

Ultrasmall gold nanoclusters (AuNCs) have emerged as agile probes for in vivo imaging, as they exhibit exceptional tumour accumulation and efficient renal clearance properties. However, their intrinsic catalytic activity, which can enable an increased detection sensitivity, has yet to be explored for in vivo sensing. By exploiting the peroxidase-mimicking activity of AuNCs and the precise nanometre-size filtration of the kidney, we designed multifunctional protease nanosensors that respond to disease microenvironments to produce a direct colorimetric urinary readout of the disease state in less than one hour. We monitored the catalytic activity of AuNCs in the collected urine of a mouse model of colorectal cancer in which tumour-bearing mice showed a 13-fold increase in colorimetric signal compared to healthy mice. The nanosensors were eliminated completely through hepatic and renal excretion within four weeks of injection with no evidence of toxicity. We envision that this modular approach will enable the rapid detection of a diverse range of diseases by exploiting their specific enzymatic signatures.

This paper examines the current state of cryptocurrency forensics focusing on digital forensics investigation supporting law enforcement activities. We begin with a brief overview of cryptocurrency and blockchain technologies, and with that understanding, we explore criminal and malicious activities enabled by these technologies which drive the need for cryptocurrency forensics. Followed by this, the paper will explore current cryptocurrency forensics techniques and challenges pertaining to mobile forensics, computer & memory forensics, and blockchain & network forensics. We will analyze the current body of research and identify gaps in the area. Also, current researchers and practicing investigators indicate that cryptocurrency forensics seems to be lagging and needs improved inter-agency coordination along with forensic techniques to counter the existing and emerging threats.

Proteases play diverse and important roles in physiology and disease, including influencing critical processes in development, immune responses, and malignancies. Both the abundance and activity of these enzymes are tightly regulated and highly contextual; thus, in order to elucidate their specific impact on disease progression, better tools are needed to precisely monitor in situprotease activity. Current strategies for detecting protease activity are focused on functionalizing synthetic peptide substrates with reporters that emit detection signals following peptide cleavage. However, these activity-based probes lack the capacity to be turned on at sites of interest and, therefore, are subject to off-target activation. Here we report a strategy that uses light to precisely control both the location and time of activity-based sensing. We develop photocaged activity-based sensors by conjugating photolabile molecules directly onto peptide substrates, thereby blocking protease cleavage by steric hindrance. At sites of disease, exposure to ultraviolet light unveils the nanosensors to allow proteases to cleave and release a reporter fragment that can be detected remotely. We apply this spatiotemporally controlled system to probe secreted protease activity in vitroand tumor protease activity in vivo. In vitro, we demonstrate the ability to dynamically and spatially measure metalloproteinase activity in a 3D model of colorectal cancer. In vivo, veiled nanosensors are selectively activated at the primary tumor site in colorectal cancer xenografts to capture the tumor microenvironment-enriched protease activity. The ability to remotely control activity-based sensors may offer a valuable complement to existing tools for measuring biological activity.

AbstractThis study was carried out along the altitudinal gradients of Uttara Kannada district of Karnataka, representing different agro-climatic zones – coast, hilly and plains. 10 different sectors were selected for the study. Transect based survey resulted in documenting a total of 134 species of trees and 146 species of shrubs. It was found that the sectors 6, 7 and 8, lying in the Western Ghats section, were having semi-evergreen to evergreen forests and had the highest species diversity, percentage evergreen and percentage endemism. The sectors 9 and 10 in eastern plains harbour dry deciduous forests dominated by teak. The coastal sectors have more moist deciduous forests affected by human disturbances except for a patch of sacred grove in sector 1. However, the regeneration of evergreens, especially in the Ghat section areas, can be considered as a good sign for the return of evergreen forests and need to be austerely protected.

Pathogens that reside in the phagosomes of infected cells persist despite the presence of potent T cell responses. We addressed the mechanism of immune evasion by using a mouse model of Salmonella typhimurium(ST). Recombinants of ST were generated that translocated antigen to the cytosol or phagosomes of infected cells. We find that the kinetics of antigen presentation and CD8+T cell priming is accelerated by cytosolic antigen delivery, although the magnitude of CD8+T cell response is not influenced by antigenic location. More importantly, only those targets that readily display antigen on the cell surface, owing to antigenic translocation to the cytosol, are recognized and killed by CD8+T cells. Thus, vaccination approaches developed to control phagosomal pathogens should incorporate methods for modulating antigen presentation such that infected target cells can be readily recognized by CD8+T cells.

Xenotransplantation with pig organs is being considered to alleviate donor organ shortages; however, the risk of introducing porcine viruses into humans is heightened in this setting. The goal of this study was to determine the infectious potential of porcine cytomegalovirus (PCMV), a xenozoonotic virus of interest, in human fibroblasts in vitro.

Human granulocyte-macrophage colony stimulating factor (GM-CSF), a cytokine with many applications in clinical medicine, was produced specifically in the seeds of transgenic tobacco plants. Two rice endosperm-specific glutelin promoters of different size and sequence, Gt1 and Gt3, were used to direct expression. Also in the Gt3 construct, the GM-CSF coding region was in fusion with the first 24 nucleotides of the mature rice glutelin sequence at its 5' end. With the Gt1 construct plants, seed extracts contained the recombinant human GM-CSF protein up to a level of 0.03% of total soluble protein. Transgenic seed extracts actively stimulated the growth of human TF-1 cells suggesting that the seed-produced GM-CSF alone and in fusion with the rice glutelin peptide was stable and biologically active. Furthermore, native tobacco seed extracts inhibited the activity of E. coli-derived GM-CSF in this cytokine-dependent cell line. The seeds of F1 generation plants retained the biological activity of human GM-CSF protein indicating that the human coding sequence was stably inherited. The feasibility of oral delivery of such stable seed-produced cytokines is discussed.

Type 1 T-cell responses against intracellular pathogens play a crucial role in mediating protection. We examined whether the induction of a strong type 1 T-cell response during a chronic bacterial infection influences responses to superantigens capable of inducing acute shock. Intravenous infection of mice with Mycobacterium bovis BCG appeared to induce a progressive anergy towards staphylococcal enterotoxin B (SEB) and towards antigen preparation of BCG (BCG-Ag) itself, based on diminished gamma interferon (IFN-gamma) production by SEB- and BCG-Ag-stimulated splenocytes from infected mice. In contrast to these in vitro results, injection of SEB into BCG-infected mice led to a dramatic increase in the serum IFN-gamma levels and the death of infected but not of control mice. In vitro hyporesponsiveness towards SEB and BCG-Ag occurred only with unfractionated splenocyte cultures, as purified T cells from infected mice produced higher levels of IFN-gamma. Hyporesponsiveness towards SEB and BCG-Ag in unfractionated splenocyte cultures was not due to suppressive antigen-presenting cells (APCs), as APCs from infected mice stimulated higher levels of IFN-gamma from purified T cells. The diminished IFN-gamma levels observed with bulk splenocytes appear to be due to changes in the T-cell-to-APC ratio that result in a decreased proportion of T cells, coupled to reduced proliferative responses and an increased susceptibility of effector T cells to activation-induced cell death in vitro. Our results indicate that the reported phenomena of T-cell anergy during mycobacterial infection may be an in vitro consequence of the development of a strong type 1 response in vivo.

ABSTRACTType 1 T-cell responses against intracellular pathogens play a crucial role in mediating protection. We examined whether the induction of a strong type 1 T-cell response during a chronic bacterial infection influences responses to superantigens capable of inducing acute shock. Intravenous infection of mice with Mycobacterium bovisBCG appeared to induce a progressive anergy towards staphylococcal enterotoxin B (SEB) and towards antigen preparation of BCG (BCG-Ag) itself, based on diminished gamma interferon (IFN-γ) production by SEB- and BCG-Ag-stimulated splenocytes from infected mice. In contrast to these in vitro results, injection of SEB into BCG-infected mice led to a dramatic increase in the serum IFN-γ levels and the death of infected but not of control mice. In vitro hyporesponsiveness towards SEB and BCG-Ag occurred only with unfractionated splenocyte cultures, as purified T cells from infected mice produced higher levels of IFN-γ. Hyporesponsiveness towards SEB and BCG-Ag in unfractionated splenocyte cultures was not due to suppressive antigen-presenting cells (APCs), as APCs from infected mice stimulated higher levels of IFN-γ from purified T cells. The diminished IFN-γ levels observed with bulk splenocytes appear to be due to changes in the T-cell-to-APC ratio that result in a decreased proportion of T cells, coupled to reduced proliferative responses and an increased susceptibility of effector T cells to activation-induced cell death in vitro. Our results indicate that the reported phenomena of T-cell anergy during mycobacterial infection may be an in vitro consequence of the development of a strong type 1 response in vivo.

ABSTRACTInduction of T-cell memory by vaccination ensures long-term protection against pathogens. We determined whether on-going inflammatory responses during vaccination influenced T-cell priming. A preexposure of mice to Mycobacterium bovisBCG impaired their subsequent ability to prime T cells against Listeria monocytogenes. This was characterized by a decrease in L. monocytogenes-specific gamma interferon (IFN-γ)-secreting CD4+and CD8+T cells. The intensity of T-cell priming towards L. monocytogenesdepended on the extent of L. monocytogenesexpansion, and a cessation of this expansion caused by M. bovisBCG-induced inflammation resulted in impairment in T-cell priming. A challenge of M. bovisBCG-infected mice with a higher L. monocytogenesdose increased L. monocytogenessurvival and restored T-cell priming towards L. monocytogenes. Impairment in T-cell priming towards L. monocytogenesdue to M. bovisBCG-induced inflammation resulted in a compromised protective efficacy in the long term after mice were rechallenged with L. monocytogenes. Preexisting inflammation selectively impaired T-cell priming for replicating immunogens as CD8+T-cell response to ovalbumin administered as an inert antigen (ovalbumin-archaeosomes) was enhanced by M. bovisBCG preimmunization, whereas priming towards ovalbumin administered as a live immunogen (L. monocytogenes-ovalbumin) was impaired. Thus, depending on the nature of the immunogen, the presence of prior inflammatory responses may either impede or boost vaccine efficacy.

Induction of T-cell memory by vaccination ensures long-term protection against pathogens. We determined whether on-going inflammatory responses during vaccination influenced T-cell priming. A preexposure of mice to Mycobacterium bovis BCG impaired their subsequent ability to prime T cells against Listeria monocytogenes. This was characterized by a decrease in L. monocytogenes-specific gamma interferon (IFN-gamma)-secreting CD4(+) and CD8(+) T cells. The intensity of T-cell priming towards L. monocytogenes depended on the extent of L. monocytogenes expansion, and a cessation of this expansion caused by M. bovis BCG-induced inflammation resulted in impairment in T-cell priming. A challenge of M. bovis BCG-infected mice with a higher L. monocytogenes dose increased L. monocytogenes survival and restored T-cell priming towards L. monocytogenes. Impairment in T-cell priming towards L. monocytogenes due to M. bovis BCG-induced inflammation resulted in a compromised protective efficacy in the long term after mice were rechallenged with L. monocytogenes. Preexisting inflammation selectively impaired T-cell priming for replicating immunogens as CD8(+) T-cell response to ovalbumin administered as an inert antigen (ovalbumin-archaeosomes) was enhanced by M. bovis BCG preimmunization, whereas priming towards ovalbumin administered as a live immunogen (L. monocytogenes-ovalbumin) was impaired. Thus, depending on the nature of the immunogen, the presence of prior inflammatory responses may either impede or boost vaccine efficacy.

A plant based high fidelity vaccine production system is being developed with emphasis on producing antigens capable of being orally delivered in multivalent or subunit plant packets. Plant-based edible vaccines may provide an attractive, safe and inexpensive alternative to conventional vaccine production. Edible plant tissues are not normally antigenic in nature. However, foreign antigens from common infectious organisms like hepatitis-B virus (HBV) can be produced along with naturally occurring storage proteins in DNA-transformed plants. Upon administration via the oral route, these transgenic plant tissues may mobilize the protective humoral and mucosal immune responses to challenge the natural infectious agent. When tobacco, carrot and rice plants were transformed with the truncated version of the HBV nucleocapsid gene expression construct, non-infective hepatitis B viral core particles were observed via electron microscopy. A second plant codon-optimised HBV expression construct was designed that included the extensin signal sequence for augmented HBV particle accumulation. Upon transformation of tobacco plants with the codon-optimised construct, over 4 times more transgenic plants with high levels of expression of the HBV nucleocapsid protein were generated in comparison with a similar vector containing the unmodified wild-type HBV gene codon sequence. Further analysis via Western blotting confirmed the presence of the viral antigen in the total protein extracts from transgenic tobacco leaves and seeds. Electron microscopy showed that the expressed protein self-assembled into viral-like particles of 25–30 nm in diameter. To develop an edible subunit vaccine in plant seeds, a third plant transformation construct was used for the synthesis of the human cytomegalovirus glycoprotein B (HCMV gB) subunit. The gB protein derived from tobacco seeds retained critical structural features including epitopes for neutralizing antibodies and was targeted to the protein storage vesicles of tobacco seed endosperm. Two different strains of mice were orally immunized with tobacco seeds containing low concentrations of HCMV gB, with varying dosages, but without adjuvant. No anti-gB response was detected in intestinal or serum samples. However, a systemic immune response to normal tobacco seed proteins was observed in both strains of mice. While higher expression levels of antigens in seeds must be achieved, seeds may provide an effective and immunostimulatory vehicle for delivering edible vaccines to the intestinal mucosa. One of the outstanding challenges includes defining optimum conditions of antigen presentation, dosage and immunization schedules that will induce strong mucosal and/or systemic immune responses in heterogeneous populations. Here we review the different strategies being employed to produce specific oral antigens in plant tissues.

As part of ongoing studies into the use of plant expression systems for making human therapeutic proteins, we have successfully expressed the major glycoprotein, gB, of human cytomegalovirus (HCMV) in transgenic tobacco plants. Viral glycoprotein was detectable in the protein extracts of mature tobacco seeds using neutralizing and non-neutralizing monoclonal antibodies specific for gB. Although several mammalian proteins have been expressed in tobacco, localization of these proteins in transgenic tobacco tissue has not been extensively examined. The objective of this study was to identify the site(s) of recombinant gB deposition in mature tobacco seeds. Using immunogold labelling and electron microscopy, we found specific labelling for gB in the endosperm of transgenic seeds, with gB localized almost exclusively in protein storage vesicles (PSV). This occurred in seeds that were freshly harvested and in seeds that had been stored for several months. These data indicate that gB behaves like a plant storage protein when expressed in tobacco seeds, and provide further support for the suitability of plants for producing recombinant proteins of potential clinical relevance.

Translocation renal cell carcinoma (tRCC) is a poorly characterized subtype of kidney cancer driven by MiT/TFEgene fusions. Here, we define the landmarks of tRCC through an integrative analysis of 152 patients with tRCC identified across genomic, clinical trial, and retrospective cohorts. Most tRCCs harbor few somatic alterations apart from MiT/TFEfusions and homozygous deletions at chromosome 9p21.3 (19.2% of cases). Transcriptionally, tRCCs display a heightened NRF2-driven antioxidant response that is associated with resistance to targeted therapies. Consistently, we find that outcomes for patients with tRCC treated with vascular endothelial growth factor receptor inhibitors (VEGFR-TKIs) are worse than those treated with immune checkpoint inhibitors (ICI). Using multiparametric immunofluorescence, we find that the tumors are infiltrated with CD8+T cells, though the T cells harbor an exhaustion immunophenotype distinct from that of clear cell RCC. Our findings comprehensively define the clinical and molecular features of tRCC and may inspire new therapeutic hypotheses.

In HeLa cells, four different types of mutants resistant to cardiac glycosides viz. ouabain and SC4453, which differ from each other in cross resistance pattern, have been isolated after single-step selections [J Biol Chem 260 (1985) 6843–6850; J Biol Chem 261 (1986) 2034–2040]. Using cloned cDNA probes specific for the a and ß subunits of Na+/K+ ATPase, these mutants have been investigated for amplification and/or increased transcription of Na+/K+ ATPase genes. Results from dot blots, Southern and Northern hybridizations provide evidence that these mutants do not involve any amplification or increased transcription or gross structural alterations in Na+/K+ ATPase genes or their transcripts. Similar results were obtained with the mutant cells grown either in the absence or presence of cardiac glycosides, the latter conditions of which cause 3–4-fold increase in the resistant form of the enzyme within the mutant cells. These results are consistent with the inference that resistance to cardiac glycosides in these mutants may be due to specific point mutations within the structural gene(s) of Na+/K+ ATPase leading to an altered enzyme that is resistant to inhibition by different cardiac glycosides.

The status of glyoxalase-I was explored in exponentially growing and G1arrested temperature sensitive (ts) cell division cycle (cdc) mutants of Saccharomyces cerevisiae. It was observed that the specific activity of this enzyme was correlated with overall growth status. The activity was high in actively growing cells and was low in G1arrested cells. Specific activities of glyoxalase-I were also low in G1arrested prolonged stationary phase (PSP) cells of S. cerevisiaeand Candida albicans. The activity of glyoxalase-I recovered when G1arrested S. cerevisiae(ts) cells were allowed to regrow under permissive conditions. Results demonstrate that although glyoxalase-I activity is a good indicator of cell growth status, it is not involved in cell cycle regulation of this eukaryotic organism.

It was shown that the decrease in phosphatidylinositol (PI) content in cdc 28 G 1-cells was due to a defect in inositol transport. This decrease in inositol transport was linked to microtubular function which was evident by the effect of a microtubular disrupting agent (colcemid) on inositol transport in stationary phase A364A cells. The involvement of PI in yeast G 1phase was further substantiated by the observation that o-phenanthroline, which blocks yeast cells in G 1phase, could inhibit inositol transport and PI levels as well. It is proposed that the regulation of PI metabolism is mediated by the gene cdc 28 and that microtubules may play a major role in the mechanism of action of this gene product.

The toxicity of the protein synthesis inhibitor puromycin towards a number of human and Chinese hamster cell lines has been examined. In comparison to cells of human origin, Chinese hamster cells exhibited about 25-fold higher resistance towards puromycin. These differences appeared to be species related as all the cell lines from any one species showed similar sensitivity towards puromycin. The incorporation of [ 3H]leucine in the hamster cell lines was accordingly found to be more resistant to the inhibitory effects of puromycin as compared to human cells. Studies on the cellular uptake of [ 3H]puromycin showed that in comparison to human cells, the drug uptake/binding in the hamster cell lines was greatly reduced. However, protein synthesis in the extracts of hamster and human cells showed no significant differences in sensitivity towards puromycin. These results show that the observed species related differences in cellular toxicity to puromycin are due to differences in the cellular uptake/binding of the drug.

A total of 245 samples of Indian milk products comprising khoa, burfi, and perawere examined for chemical, microbiological, and organoleptic qualities, and samples were graded as ‘good,’ ‘fair,' and ‘poor'. The chemical composition of these products varied considerably. Higher bacterial and fungal counts were noted in khoa, as compared to burfiand pera. A variety of microorganisms such as micrococci, sarcinae, aerobic spore-formers, coliforms, staphylococci, streptococci, and lactobacilli were isolated from the samples.

The toxicity of the protein synthesis inhibitor puromycin towards a number of human and Chinese hamster cell lines has been examined. In comparison to cells of human origin, Chinese hamster cells exhibited about 25‐fold higher resistance towards puromycin. These differences appeared to be species related as all the cell lines from any one species showed similar sensitivity towards puromycin. The incorporation of [3H]leucine in the hamster cell lines was accordingly found to be more resistant to the inhibitory effects of puromycin as compared to human cells. Studies on the cellular uptake of [3H]puromycin showed that in comparison to human cells, the drug uptake/binding in the hamster cell lines was greatly reduced. However, protein synthesis in the extracts of hamster and human cells showed no significant differences in sensitivity towards puromycin. These results show that the observed species related differences in cellular toxicity to puromycin are due to differences in the cellular uptake/binding of the drug.

One of the mechanisms by which endothelial cells (ECs) regulate fibrinolysis is through the regulated assembly of proteins such as plasminogen, tissue plasminogen activator (tPA) and urokinase (uPA) on their membrane surface. Receptors for many of these fibrinolytic factors have been isolated and characterized. A unique 45 kD plasminogen receptor present on ECs derived from vein vasculature has been identified and resolved into two plasminogen binding components. One component consists of the unique 45 kD plasminogen receptor (pI = 6.3) whereas the other component (pI = 5.1) is identified as the cytoskeletal protein, actin. Immunofluorescent studies of isolated ECs confirm the presence of actin on their extracellular surface. This observation is consistent with a number of other recent reports of actin externally localized on other cell types. In vitrostudies using purified actin confirm that plasminogen binds to actin both saturably and with relatively high affinity. Competition studies with lysine indicated that the binding was largely kringle‐dependent, and when binding of tPA to actin was assessed, it also bound to actin with 70–80% of binding inhibited by lysine. Lipoprotein(a), which shows homology with plasminogen, also interacted with actin. Addition of plasminogen and low‐density lipoproteins inhibited Lp(a) binding to actin in a dose‐dependent fashion. Moreover, in competition with tPA, partial inhibition of plasminogen binding to actin was also observed. In experiments using anti‐actin antibodies added in excess to cultured ECs, binding of plasminogen was inhibited by 45%, tPA binding was inhibited by 46% and Lp(a) binding was reduced by 56%, confirming actin as a binding site for these various ligands whilst attesting to the presence of other EC receptors for these proteins. Collectively, the data presented are consistent with actin playing a major role in localizing binding not only of plasminogen, but also of tissue plasminogen activator and Lp(a) to the surface of human endothelial cells.

It was shown that the decrease in phosphatidylinositol (PI) content in cdc 28 G1‐cells was due to a defect in inositol transport. This decrease in inositol transport was linked to microtubular function which was evident by the effect of a microtubular disrupting agent (colcemid) on inositol transport in stationary phase A364A cells. The involvement of PI in yeast G1phase was further substantiated by the observation that o‐phenanthroline, which blocks yeast cells in G1phase, could inhibit inositol transport and PI levels as well. It is proposed that the regulation of PI metabolism is mediated by the gene cdc 28 and that microtubules may play a major role in the mechanism of action of this gene product.

Sodding of vascular grafts involves coating the biomaterial with cells prepared from collagenase-digested fat tissue after removal of the adipocytes by centrifugation. The goal of this study was to investigate the staining characteristics of the sodding cells as well as their ability to express the procoagulant protein tissue factor, and to compare these findings to those found with extensively purified microvascular endothelial cells (MEC) prepared from similar tissue. Sodding cells and MEC, isolated using immunomagnetic separation with anti-PECAM antibodies, were prepared from liposuction material and endothelial-specific staining was compared. The expression of tissue factor on these cells was examined using both an ELISA and a chromogenic assay to assess the rate of generation of factor Xa. Sodding cells expressed significantly more tissue factor than the unstimulated MEC in which the expression was undetectable (sodding cells 2466 ± 830 pg/mL,P< 0.05). There was no further increase in tissue factor expression in the sodding cells with stimulation with lipopolysaccharide (LPS); however, purified MEC expressed significantly more tissue factor after exposure to LPS (1247 ± 356 pg/mL,P< 0.05). These results were confirmed by the determination of procoagulant activity of the cells whereby the procoagulant activity on unstimulated MEC was significantly less than that found after stimulation of these cells, and it was also less than stimulated and unstimulated sodding cells (absorbance at 405 nm: 0.423 ± 0.125, unstimulated MEC; 1.000 ± 0.438, stimulated MEC; 1.129 ± 0.396, unstimulated sodding cells; 1.171 ± 0.254, stimulated sodding cells,P< 0.05). Staining of these two cell types also demonstrated significant uptake of acetylated LDL (Ac-LDL) in the purified MEC which was essentially absent in the sodding cells. Further, vWf staining was found to a greater degree in the purified MEC than in the sodding cells. These experiments demonstrated that the cells prepared for cell sodding express large amounts of tissue factor. The sodding cells do not stain for antigens known to be specific for endothelial cells, whereas MEC do and therefore the concentration of endothelial cells in the sodding cells is small. The significance of the tissue factor expression on the surface of sodded grafts is not yet known.

A human mitochondrial protein, designated P1 (63 kilodaltons [kDa], shows extensive sequence homology (47% identical residues and an additional approximately 20% conserved changes) to the 65-kDa mycobacterial antigen. To understand the relationship of these proteins, the cross-reactivity of several monoclonal antibodies directed against the 65-kDa Mycobacterium leprae antigen towards human, Chinese hamster, chicken, and bacterial cells has been examined. A number of antibodies (Y1-2, ML 30-A2, and F47-9-1) were found to cross-react with a 63-kDa antigen in vertebrate cell extracts and stained mitochondria in immunofluorescence studies. Some of these antibodies also reacted with a P1-beta-galactosidase fusion protein in recombinant Escherichia coli cells, expressing part of the human P1 protein. These results provide strong evidence that P1 is the mammalian homolog of the 65-kDa antigen. The human P1 protein also shows significant similarity (P less than 0.001) to a number of other bacterial and viral proteins including the pol polyprotein of human immunodeficiency viruses and the penicillin-binding protein of Neisseria gonorrhoeae. The observed similarity between human P1 protein and the major antigenic proteins of pathogenic organisms (e.g., 60- to 65-kDa mycobacterial antigen) suggests its possible involvement in autoimmune diseases (e.g., rheumatoid arthritis) by antigenic mimicry.