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Innate lymphoid cells (ILCs) are key players during an immune response at the mucosal surfaces, such as lung, skin, and gastrointestinal tract. Giardia lamblia is an extracellular protozoan pathogen that inhabits the human small intestine. In this study, ILCs prepared from the lamina propria of mouse small intestine were incubated with G. lamblia trophozoites. Transcriptional changes in G. lamblia-exposed ILCs resulted in identification of activation of several immune pathways. Secretion of interleukin (IL)-17A, IL-17F, IL-1β, and interferon-γ was increased, whereas levels of IL-13, IL-5, and IL-22, was maintained or reduced upon exposure to G. lamblia. Goup 3 ILC (ILC3) was found to be dominant amongst the ILCs, and increased significantly upon co-cultivation with G. lamblia trophozoites. Oral inoculation of G. lamblia trophozoites into mice resulted in their presence in the small intestine, of which, the highest number of parasites was detected at the 5 days-post infection. Increased ILC3 was observed amongst the ILC population at the 5 days-post infection. These findings indicate that ILC3 from the lamina propria secretes IL-17 in response to G. lamblia, leading to the intestinal pathology observed in giardiasis.

Background A foodborne pathogen, Vibrio vulnificus, encounters normal microflora inhabiting the gut environments prior to causing fatal septicemia or gastroenteritis and should overcome the barriers derived from the gut commensals for successful infection. Its interactions with gut commensals during the infection process, however, have not yet been understood. In the present study, the effect of V. vulnificus on the community structures of gut microbiota in mice was examined. Results Analyses of microbiota in the fecal samples of mice that died due to V. vulnificus infection revealed the decreased abundance of bacteria belonged to Bacteroidetes, notably, the species Bacteroides vulgatus. In vitro coculturing of the two bacterial species resulted in the decreased survival of B. vulgatus. The antagonistic effect of V. vulnificus against B. vulgatus was found to be mediated by cyclo-Phe-Pro (cFP), one of the major compounds secreted by V. vulnificus. cFP-treated B. vulgatus showed collapsed cellular morphology with an undulated cell surface, enlarged periplasmic space, and lysed membranes, suggesting the occurrence of membrane disruption. The degree of membrane disruption caused by cFP was dependent upon the cellular levels of ObgE in B. vulgatus. Recombinant ObgE exhibited a high affinity to cFP at a 1:1 ratio. When mice were orally injected with cFP, their feces contained significantly reduced B. vulgatus levels, and their susceptibility to V. vulnificus infection was considerably increased. Conclusions This study demonstrates that V. vulnificus-derived cFP modulates the abundance of the predominant species among gut commensals, which made V. vulnificus increase its pathogenicity in the hosts.

The cardiac homeobox transcription factor Nkx2-5 is a major determinant of cardiac identity and cardiac morphogenesis. Nkx2-5 operates as part of a complex and mutually reinforcing network of early transcription factors of the homeobox, GATA zinc finger and MADS domain families to initiate the program of cardiac development and differentiation, particularly in outflow tract precursor cells in the second heart field (SHF). We have now found evidence for another aspect of cardiac transcription factor cooperativity between Nkx2-5 and the cardiac enriched MADS domain transcription factor Srf. Specifically, Srf interaction with an evolutionarily conserved binding site in the Nkx2-5 CpG island-like proximal promoter is required for cardiac specific expression mediated by an SHF enhancer, and for combinatorial activation of these elements by cardiac transcription factors. These results provide further insight into cooperative gene regulation during cardiogenesis at the level of promoter-enhancer interactions.

The cardiac transcription factor Nkx2-5 is essential for normal outflow tract (OFT) and right ventricle (RV) development. Nkx2-5−/− null mouse embryos display severe OFT and RV hypoplasia and a single ventricle phenotype due to decreased proliferation of Second Heart Field (SHF) cells, a pool of cardiac progenitors present in anterior pharyngeal arch mesoderm at mid-gestation. However, definition of the precise role of Nkx2-5 in facilitating SHF expansion is incomplete. We have found that Nkx2-5 positively and directly regulates a novel target gene, Ccdc117, in cells of the SHF at these stages. The nuclear/mitotic spindle associated protein Ccdc117 interacts with the MIP18/MMS19 cytoplasmic iron-sulfur (FeS) cluster assembly (CIA) complex, which transfers critical FeS clusters to several key enzymes with functions in DNA repair and replication. Loss of cellular Ccdc117 expression results in reduced proliferation rates associated with a delay at the G1-S transition, decreased rates of DNA synthesis, and unresolved DNA damage. These results implicate a novel role for Nkx2-5 in the regulation of cell cycle events in the developing heart, through Ccdc117′s interaction with elements of the CIA pathway and the facilitation of DNA replication during SHF expansion.

An inflammatory form of phagocyte death evoked by the Gram-negative bacterium Vibrio (V.) vulnificus (WT) is one of hallmarks to promote their colonization, but the virulence factor and infectious mechanism involved in this process remain largely unknown. Here, we identified extracellular metalloprotease VvpM as a new virulence factor and investigated the molecular mechanism of VvpM which acts during the regulation of the inflammatory form of macrophage death and bacterial colonization. Mutation of the vvpM gene appeared to play major role in the prevention of IL-1β production due to V. vulnificus infection in macrophage. However, the recombinant protein (r) VvpM caused IL-1β production coupled with necrotic cell death, which is highly susceptible to the knockdown of annexin A2 (ANXA2) located in both membrane lipid and non-lipid rafts. In lipid rafts, rVvpM recruited NOX enzymes coupled with ANXA2 to facilitate the production of ROS responsible for the epigenetic and transcriptional regulation of NF-κB in the IL-1β promoter. rVvpM acting on non-lipid rafts increased LC3 puncta formation and autophagic flux, which are required for the mRNA expression of Atg5 involved in the autophagosome formation process. The autophagy activation caused by rVvpM induced NLRP3 inflammasome-dependent caspase-1 activation in the promoting of IL-1β production. In mouse models of V. vulnificus infection, the VvpM mutant failed to elevate the level of pro-inflammatory responses closely related to IL-1β production and prevented bacterial colonization. These findings delineate VvpM efficiently regulates two pathogenic pathways that stimulate NF-κB-dependent IL-1β production and autophagy-mediated NLRP3 inflammasome via distinct spatial targeting by ANXA2.

Giardia lamblia is a protozoan pathogen with distinct cytoskeletal structures, including median bodies and eight flagella. In this study, we examined components comprising G. lamblia flagella. Crude flagellar extracts were prepared from G. lamblia trophozoites, and analyzed by two-dimensional (2-D) gel electrophoresis. The 19 protein spots were analyzed by MALDI–TOF mass spectrometry, identifying ten metabolic enzymes, six distinct giardins, Giardia trophozoite antigen 1, translational initiation factor eIF-4A, and an extracellular signal-regulated kinase 2. Among the identified proteins, we studied α-11 giardin which belongs to a group of cytoskeletal proteins specific to Giardia . Western blot analysis and real-time PCR indicated that expression of α-11 giardin is not significantly increased during encystation of G. lamblia . Immunofluorescence assays using anti-α-11 giardin antibodies revealed that α-11 giardin protein mainly localized to the plasma membranes and basal bodies of the anterior flagella of G. lamblia trophozoites, suggesting that α-11 giardin is a genuine component of the G. lamblia cytoskeleton.

The cardiac homeobox gene Nkx2.5 plays a key and dosage-sensitive role in the differentiation of outflow tract and right ventricle from progenitors of the second heart field (SHF) and Nkx2.5 mutation is strongly associated with human outflow tract congenital heart disease (OFT CHD). Therefore defining the regulatory mechanisms controlling Nkx2.5 expression in SHF populations serves an important function in understanding the etiology of complex CHD. Through a comparative analysis of regulatory elements controlling SHF expression of Nkx2.5 in the chicken and mouse, we have found evidence that Nkx2.5 autoregulation is important for maintaining Nkx2.5 expression during SHF differentiation in both species. However the mechanism of Nkx2.5 maintenance differs between placental mammals and non-mammalian vertebrates: in chick Nkx2.5 binds directly to a genomic enhancer element that is required to maintain Nkx2.5 expression in the SHF. In addition, it is likely that this is true in other non-mammalian vertebrates given that they possess a similar genomic organization. By contrast, in placental mammals, Nkx2.5 autoregulation in the SHF functions indirectly through Mef2c. These data underscore a tight relationship in mammals between Nkx2.5 and Mef2c in SHF transcriptional regulation, and highlight the potential for evolutionary cis-regulatory analysis to identify core, conserved components of the gene networks controlling heart development.

Vibrio parahaemolyticusis a Gram-negative halophilic bacterium that causes food-borne gastroenteritis in humans who consumeV. parahaemolyticus-contaminated seafood.The FORC_023 strain was isolated from raw fish storage water, containing live fish at a sashimi restaurant. Here, we aimed to sequence and characterize the genome of the FORC_023 strain. The genome of the FORC_023 strain showed two circular chromosomes, which contained 4227 open reading frames (ORFs), 131 tRNA genes and 37 rRNA genes. Although the genome of FORC_023 did not include major virulence genes, such as genes encoding thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), it contained genes encoding other hemolysins, secretion systems, iron uptake-related proteins and severalV. parahaemolyticusislands. The highest average nucleotide identity value was obtained between the FORC_023 strain and UCM-V493 (CP007004-6). Comparative genomic analysis of FORC_023 with UCM-V493 revealed that FORC_023 carried an additional genomic region encoding virulence factors, such as repeats-in-toxin and type II secretion factors. Furthermore,in vitrocytotoxicity testing showed that FORC_023 exhibited a high level of cytotoxicity toward INT-407 human epithelial cells. These results suggested that the FORC_023 strain may be a food-borne pathogen.

EpsC, one of the components comprising the type II secretion system (T2SS), was isolated from a human-pathogenic bacterium, Vibrio vulnificus , to evaluate its role in eliciting virulence. An espC -deleted mutant of V. vulnificus displayed a reduced cytotoxicity to the human cell line HEp-2 and an attenuated virulence in a mouse model. This mutant exhibited dramatic defects in the secretion of diverse extracellular proteins, such as outer membrane proteins, transporters, and the known secreted factors, notably, a hemolysin (VvhA) and an elastase (VvpE). A defect in its secretion of proteins was restored by in trans complementation of the intact epsC gene. Analyses of cellular fractions revealed that VvhA and VvpE of the ΔepsC mutant were not excreted outside the cell but were present mainly in the periplasmic space. Examination of a V. vulnificus mutant deficient in TolC, a component of the T1SS, showed that it is not involved in the secretion of VvhA and VvpE but that it is necessary for the secretion of another major toxin of V. vulnificus , RtxA. Therefore, the T2SS is required for V. vulnificus pathogenicity, which is mediated by at least two secreted factors, VvhA and VvpE, via facilitating the secretion and exposure of these factors to host cells.

Vibrio vulnificus is an opportunistic human pathogen that causes severe infections in susceptible individuals. While the components of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) have been shown to regulate numerous targets, little such information is available for the V. vulnificus PTS. Here we show that enzyme IIAGlc of the PTS regulates the peptidase activity of a mammalian insulysin homolog in V. vulnificus. While interaction of IIAGlc with the insulysin homolog is independent of the phosphorylation state of IIAGlc, only unphosphorylated IIAGlc activates the insulysin homolog. Taken together, our results suggest that the V. vulnificus insulysin-IIAGlc complex plays a role in survival in the host by sensing glucose. Structured summary MINT- 8045996 : IIA glu (uniprotkb:Q7MBY2) binds (MI: 0407 ) to vIDE (uniprotkb: Q7MIS6 ) by pull down (MI: 0096 ) MINT- 8045817 , MINT- 8045967 : IIA glu (uniprotkb:Q7MBY2) physically interacts (MI: 0915 ) with vIDE (uniprotkb: Q7MIS6 ) by pull down (MI: 0096 )

Nkx2.5, a transcription factor implicated in human congenital heart disease, is required for regulation of second heart field (SHF) progenitors contributing to outflow tract (OFT). Here, we define a set of genes (Lrrn1, Elovl2, Safb, Slc39a6, Khdrbs1, Hoxb4, Fez1, Ccdc117, Jarid2, Nrcam, and Enpp3) expressed in SHF containing pharyngeal arch tissue whose regulation is dependent on Nkx2.5. Further investigation shows that Jarid2, which has been implicated in OFT morphogenesis, is a direct target of Nkx2.5 regulation. Jarid2 expression was up-regulated in SHF mesoderm of Nkx2.5-deficient embryos. Chromatin immunoprecipitation analysis showed Nkx2.5 interaction with consensus binding sites in the Jarid2 promoter in pharyngeal arch cells. Finally, Jarid2 promoter activity and mRNA expression levels were down-regulated by Nkx2.5 overexpression. Given the role of Jarid2 as a regulator of early cardiac proliferation, these findings highlight Jarid2 as one of several potential mediators of the critical role played by Nkx2.5 during OFT morphogenesis.

Calcium signaling is a central regulator of cardiomyocyte growth and function. Calmodulin is a critical mediator of calcium signals. Because the amount of calmodulin within cardiomyocytes is limiting, the precise control of calmodulin expression is important for the regulation of calcium signaling. In this study, we show for the first time that calmodulin levels are regulated posttranscriptionally in heart failure. The cardiomyocyterestricted microRNA miR-1 inhibited the translation of calmodulin-encoding mRNAs via highly conserved target sites within their 3 untranslated regions. In keeping with its effect on calmodulin expression, miR-1 downregulated calcium-calmodulin signaling through calcineurin to NFAT. miR-1 also negatively regulated the expression of Mef2a and Gata4, key transcription factors that mediate calcium-dependent changes in gene expression. Consistent with the downregulation of these hypertrophy-associated genes, miR-1 attenuated cardiomyocyte hypertrophy in cultured neonatal rat cardiomyocytes and in the intact adult heart. Our data indicate that miR-1 regulates cardiomyocyte growth responses by negatively regulating the calcium signaling components calmodulin, Mef2a, and Gata4. In the face of biomechanical stress, the heart develops compensatory hypertrophy, the hallmark of which is an increase in the size of cardiomyocytes. Over time, these hypertrophic changes become maladaptive, resulting in decreased contractile performance, cardiomyocyte loss, and, ultimately, heart failure (16). These changes in cardiomyocyte phenotype are linked to pathological changes in cardiomyocyte gene expression (24). Calcium, in conjunction with the calcium-binding protein calmodulin (CaM), is a critical mediator of hypertrophy signaling. Ca/CaM activates the phosphatase calcineurin (CN), leading to nuclear accumulation and the activation of

The proteome of Giardia lamblia at its cyst stage was compared with that of trophozoites by using two-dimensional SDS-PAGE gel electrophoresis. Protein spots that increased in the extracts of cysts compared to trophozoites were identified by MALDI-TOF mass spectroscopy and categorized as cytoskeletal proteins, metabolic enzymes, a cell-cycle-specific kinase, stress resistance proteins, and a protein involved in translation. Expression patterns of five of the identified proteins were examined during encystation by real-time PCR. Expression of cwp1 (encoding cyst wall protein 1), a marker for encystation, was increased 11-fold. In contrast, tim (encoding triose-1-phosphate isomerase) was expressed constitutively during encystation, and its transcription level was therefore used as a mRNA loading control. Expression of three genes encoding beta-tubulin, vacuolar ATPase, and never-in-mitosis-A-related protein kinase did not vary significantly during encystation. Interestingly, genes encoding two heat shock proteins (Hsp70 and Hsp90) showed increased expression during encystation, suggesting that this differentiation process accompanies a cellular response to stress in G. lamblia.

A cDNA clone encoding a putative Bop1 homologous protein was identified in Giardia lamblia. Since Bop1 is a nucleolar protein involved in rRNA processing, thereby controlling the cell cycle, we investigated components of cell cycle control in G. lamblia by identifying the protein(s) that interact with Bop 1. Through an immunoaffinity column made with polyclonal antibodies specific to the recombinant Bop1 of G. lamblia, a pool of proteins was obtained from the crude extracts of Giardia and then used as antigens to immunize rats. By employing the resultant sera for cDNA library immunoscreening, we isolated cDNA clones encoding an immunopurified protein, which turned out to contain the gene for beta-giardin, a Giardia-specific cytoskeletal protein. The interaction between Bop1 and beta-giardin was confirmed via two different methods, yeast two-hybrid assay and coimmunoprecipitation.

The interaction between fermentation-respiration switch (FrsA) protein and glucose-specific enzyme IIA(Glc) increases glucose fermentation under oxygen-limited conditions. We show that FrsA converts pyruvate to acetaldehyde and carbon dioxide in a cofactor-independent manner and that its pyruvate decarboxylation activity is enhanced by the dephosphorylated form of IIA(Glc) (d-IIA(Glc)). Crystal structures of FrsA and its complex with d-IIA(Glc) revealed residues required for catalysis as well as the structural basis for the activation by d-IIA(Glc).

Hypertrophic cardiomyopathy is a common cause of mortality in congenital heart disease (CHD). Many gene abnormalities are associated with cardiac hypertrophy, but their function in cardiac development is not well understood. Loss-of-function mutations in PTPN11, which encodes the protein tyrosine phosphatase (PTP) SHP2, are implicated in CHD and cause Noonan syndrome with multiple lentigines (NSML), a condition that often presents with cardiac hypertrophic defects. Here, we found that NSML-associated hypertrophy stems from aberrant signaling mechanisms originating in developing endocardium. Trabeculation and valvular hyperplasia were diminished in hearts of embryonic mice expressing a human NSML-associated variant of SHP2, and these defects were recapitulated in mice expressing NSML-associated SHP2 specifically in endothelial, but not myocardial or neural crest, cells. In contrast, mice with myocardial- but not endothelial-specific NSML SHP2 expression developed ventricular septal defects, suggesting that NSML-associated mutations have both cell-autonomous and nonautonomous functions in cardiac development. However, only endothelial-specific expression of NSML-associated SHP2 induced adult-onset cardiac hypertrophy. Further, embryos expressing the NSML-associated SHP2 mutation exhibited aberrant AKT activity and decreased downstream forkhead box P1 (FOXP1)/FGF and NOTCH1/EPHB2 signaling, indicating that SHP2 is required for regulating reciprocal crosstalk between developing endocardium and myocardium. Together, our data provide functional and disease-based evidence that aberrant SHP2 signaling during cardiac development leads to CHD and adult-onset heart hypertrophy.

Mutation of sucker (suc) disrupts development of the lower jaw and other ventral cartilages in pharyngeal segments of the zebrafish head. Our sequencing, cosegregation and rescue results indicate that suc encodes an Endothelin-1 (Et-1). Like mouse and chick Et-1, suc/et-1 is expressed in a central core of arch paraxial mesoderm and in arch epithelia, both surface ectoderm and pharyngeal endoderm, but not in skeletogenic neural crest. Long before chondrogenesis, suc/et-1 mutant embryos have severe defects in ventral arch neural crest expression of dHAND, dlx2, msxE, gsc, dlx3 and EphA3 in the anterior arches. Dorsal expression patterns are unaffected. Later in development, suc/et-1 mutant embryos display defects in mesodermal and endodermal tissues of the pharynx. Ventral premyogenic condensations fail to express myoD, which correlates with a ventral muscle defect. Further, expression of shh in endoderm of the first pharyngeal pouch fails to extend as far laterally as in wild types. We use mosaic analyses to show that suc/et-1 functions nonautonomously in neural crest cells, and is thus required in the environment of postmigratory neural crest cells to specify ventral arch fates. Our mosaic analyses further show that suc/et-1 nonautonomously functions in mesendoderm for ventral arch muscle formation. Collectively our results support a model for dorsoventral patterning of the gnathostome pharyngeal arches in which Et-1 in the environment of the postmigratory cranial neural crest specifies the lower jaw and other ventral arch fates.

Early dorsoventral pattern formation in vertebrate embryos is regulated by opposing activities of ventralizing bone morphogenetic proteins (BMPs) and dorsal-specific BMP antagonists such as Chordin, Noggin and Follistatin. Specific defects in early dorsoventral patterning have been recently found in a number of zebrafish mutants, which exhibit either a ventralized or dorsalized phenotype. One of these, the ventralized mutant chordino (originally called dino) is caused by a mutation in the zebrafish chordin homologue and interacts genetically with the dorsalized mutant swirl. In swirl mutant embryos, dorsal structures such as notochord and somites are expanded while ventral structures such as blood and nephros are missing. Here we demonstrate that the swirl phenotype is caused by mutations in the zebrafish bmp2 gene (zbmp2). While injection of mRNAs encoded by the mutant alleles has no ventralizing effect, injection of wild-type zbmp2 mRNA leads to a complete rescue of the swirl mutant phenotype. Fertile adult mutant fish were obtained, showing that development after gastrulation is not dependent on zbmp2 function. In addition zBMP2 has no maternal role in mesoderm induction. Our analysis shows that swirl/BMP2, unlike mouse BMP2 but like mouse BMP4, is required for early dorsoventral patterning of the zebrafish embryo.

Objectives In the present study, we aimed to examine whether fractions from an edible sea weed, Hizikia fusiformis, had immunomodulatory effects, particularly an anti-atopic effect, by attenuating the expression of T cell-dependent cytokines using in-vitro and in-vivo animal atopic dermatitis-like models. Methods The anti-atopic activities were examined in in vitro, and a 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis-like mouse model using quantitative real-time polymerase chain reaction, electrophoretic-mobility shift and histophathological analysis. Key findings Our results showed that the final fraction (F2′) of H. fusiformis contained a higher amount of butanoic acid which was not found in the other fractions, and effectively inhibited T cell activation by inhibiting dephosphorylation of nuclear factor of activated T cells in electrophoretic-mobility shift assay. As a consequence, helper T cell-dependent cytokines, such as interleukin-2, -4 and interferon-γ, were significantly inhibited while activated with an anti-CD3 antibody. We also showed that skin challenged with DNCB successfully recovered when treated with 2.5 mg/kg, comparable to that by 0.25% prednicarbate. These results indicate that F2′ may contribute to inhibit T cell activation by eliminating Th cell-dependent cytokines. Conclusions Taken together, we concluded that F2′ containing butanoic acid may be a new functional anti-atopic candidate, which probably acts through nuclear factor of activated T cell inactivation mechanisms.

Cardiac gene expression is precisely regulated and its perturbation causes developmental defects and heart disease. Leucine-rich repeat containing 10 (Lrrc10) is a cardiac-specific factor that is crucial for proper cardiac development and deletion of Lrrc10 in mice results in dilated cardiomyopathy. However, the mechanisms regulating Lrrc10 expression in cardiomyocytes remain unknown. Therefore, we set out to determine trans-acting factors and cis-elements critical for mediating Lrrc10 expression. We identify Lrrc10 as a transcriptional target of Nkx2-5 and GATA4. The Lrrc10 promoter region contains two highly conserved cardiac regulatory elements, which are functional in cardiomyocytes but not in fibroblasts. In vivo, Nkx2-5 and GATA4 endogenously occupy the proximal and distal cardiac regulatory elements of Lrrc10 in the heart. Moreover, embryonic hearts of Nkx2-5 knockout mice have dramatically reduced expression of Lrrc10. These data demonstrate the importance of Nkx2-5 and GATA4 in regulation of Lrrc10 expression in vivo. The proximal cardiac regulatory element located at around −200 bp is synergistically activated by Nkx2-5 and GATA4 while the distal cardiac regulatory element present around −3 Kb requires SRF in addition to Nkx2-5 and GATA4 for synergistic activation. Mutational analyses identify a pair of adjacent Nkx2-5 and GATA binding sites within the proximal cardiac regulatory element that are necessary to induce expression of Lrrc10. In contrast, only the GATA site is functional in the distal regulatory element. Taken together, our data demonstrate that the transcription factors Nkx2-5 and GATA4 cooperatively regulate cardiac-specific expression of Lrrc10.

The TME, true amino acid availability (TAAA), AME, and apparent amino acid availability (AAAA) were determined in full-fat seeds, meals, oils, and meal-oil mixtures of flax and canola using Single Comb White Leghorn roosters by the force feeding and total excreta collection assay. The metabolizable energy values were corrected to zero nitrogen balance to obtain AMEn and TMEn. The AMEn and TMEn contents of canola seed (4.46 and 4.56 kcal/g) were respectively higher (P < .05) than those of flax seed (3.75 and 3.75 kcal/g). Flax meal contains contents of AMEn (2.07 kcal/g) and TMEn (2.07 kcal/g) similar to those (1.98 and 2.09 kcal/g) of canola meal. There were no differences in AMEn and TMEn values between flax oil (8.10 and 8.28 kcal/g) and canola oil (8.25 and 8.46 kcal/g). No differences were observed among AME, AMEn, TME, and TMEn values within each test ingredient except canola meal: its TME value was higher (P < .05) than its AMEn value. The TAAA values were higher for full-fat seeds than for the meals and also higher for canola seed and meal than for flax seed and meal. The AAAA values, although lower than TAAA values, followed similar patterns as TAAA values. Both AAAA and TAAA values were higher when the ingredients were fed with a basal diet than when fed alone.

This study examined whether steam-dried ginseng berries fermented with Lactobacillus plantarum (FSGB) could improve the indices of type 2 diabetes mellitus (T2DM) in obese db/db mice. FSGB was shown to have an effect on body weight and blood glucose/serum parameters when administered at a dose of 0.5 g/kg. In the intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT), FSGB was clearly shown to improve insulin tolerance and glucose tolerance. Moreover, FSGB was shown to enhance immune activities by increasing the immune cell population, and glucose transpoter 1 (GLUT1) mRNA expression in L6 cells was up-regulated, suggesting that FSGB can increase glucose transport activity in target cells. These results indicate that steam- and dry-processed ginseng berries fermented with L. plantarum can be used to effectively control blood sugar metabolism via improving insulin and glucose tolerance and body weight gain in db/db mice.

Colonization of the light-emitting organ of the Hawaiian squid Euprymna scolopes is initiated when the nascent organ of a newly hatched squid becomes inoculated with Vibrio fischeri cells present in the ambient seawater. Although they are induced for luminescence in the light organ, these symbiotic strains are characteristically non-visibly luminous (NVL) when grown in laboratory culture. The more typical visibly luminous (VL) type of V. fischeri co-occurs in Hawaiian seawater with these NVL strains; thus, two phenotypically distinct groups of this species potentially have access to the symbiotic niche, yet only the NVL ones are found there. In laboratory inoculation experiments, VL strains, when presented in pure culture, showed the same capability for colonizing the light organ as NVL strains. However, in experiments with mixed cultures composed of both VL and NVL strains, the VL ones were unable to compete with the NVL ones and did not persist within the light organ as the symbiosis became established. In addition, NVL strains entered light organs that had already been colonized by VL strains and displaced them. The mechanism underlying the symbiotic competitiveness exhibited by NVL strains remains unknown; however, it does not appear to be due to a higher potential for siderophore activity. While a difference in luminescence phenotype between VL and NVL strains in culture is not likely to be significant in the symbiosis, it has helped identify two distinct groups of V. fischeri that express different colonization capabilities in the squid light organ. This competitive difference provides a useful indication of important traits in light organ colonization.

Little is known about the molecular mechanism for autolysis of Gram-negative bacteria. In the present study, we identified the vvpS gene encoding a serine protease, VvpS, from Vibrio vulnificus , a Gram-negative food-borne pathogen. The amino acid sequence predicted that VvpS consists of two functional domains, an N-terminal protease catalytic domain (PCD) and a C-terminal carbohydrate binding domain (CBD). A null mutation of vvpS significantly enhanced viability during stationary phase, as measured by enumerating CFU and differentially staining viable cells. The vvpS mutant reduced the release of cytoplasmic β-galactosidase and high-molecular-weight extracellular chromosomal DNA into the culture supernatants, indicating that VvpS contributes to the autolysis of V. vulnificus during stationary phase. VvpS is secreted via a type II secretion system (T2SS), and it exerts its effects on autolysis through intracellular accumulation during stationary phase. Consistent with this, a disruption of the T2SS accelerated intracellular accumulation of VvpS and thereby the autolysis of V. vulnificus . VvpS also showed peptidoglycan-hydrolyzing activity, indicating that the autolysis of V. vulnificus is attributed to the self-digestion of the cell wall by VvpS. The functions of the VvpS domains were assessed by C-terminal deletion analysis and demonstrated that the PCD indeed possesses a proteolytic activity and that the CBD is required for hydrolyzing peptidoglycan effectively. Finally, the vvpS mutant exhibited reduced virulence in the infection of mice. In conclusion, VvpS is a serine protease with a modular structure and plays an essential role in the autolysis and pathogenesis of V. vulnificus .

Vibrio vulnificus is a pathogenic bacterium causing primary septicemia, which is followed by a classical septic shock pathway including an overwhelming inflammatory cytokine response. V. vulnificus IlpA is a potent immunogenic lipoprotein that triggers cytokine production in human monocytes by activating the toll-like receptor 2 (TLR2). In this study, we further defined the IlpA signaling pathways involved in cytokine production in the human monocytic cell line, THP-1. TLR2 was involved in cytokine production by complexing with TLR1, but not with TLR6. MyD88 was necessary for IlpA-induced cytokine expression through TLR1/TLR2. Three mitogen activated protein kinases (MAPK), p38, ERK1/2, and JNK, were activated in THP-1 cells stimulated with recombinant IlpA (rIlpA). Selective inhibition of each MAPK resulted in significant decrease of rIlpA-induced cytokine production. Especially, functional TLR2 was necessary for IlpA-induced activation of p38 and JNK. IlpA augmented the DNA-binding activity of nuclear factor-kappaB (NF-κB) and activator protein-1 (AP-1) transcriptional factors to their recognition sites in THP-1 cells. These results suggest that serial activation of TLR1/TLR2, MyD88, the three MAPKs, and NF-κB/AP-1 comprises the signaling pathway responsible for proinflammatory cytokine production by V. vulnificus IlpA.

Vibrio vulnificus has been shown to require a global transcription factor, NtrC for mature biofilm development via controlling the biosyntheses of lipopolysaccharide and exopolysaccharide (EPS). Biofilm formation and EPS production were dramatically increased in a medium including a tricarboxylic acid cycle-intermediate as a carbon source. These phenotypes required functional NtrC and were abolished by the addition of ammonium chloride. During the initial stage of biofilm formation, both expression of the ntrC gene and the cellular content of NtrC protein increased. Thus, the regulatory roles of NtrC in EPS biosynthesis were studied with three gene clusters for EPS biosyntheses. Transcriptions of the three clusters were positively controlled by NtrC and showed maximal expression at the early stage of biofilm development. Mutants deficient in one of the genes (VV1_2661, VV2_1579 and VV1_2305) in each cluster showed decreased production of EPS, attenuated ability to form biofilm and lowered cytoadherence to human epithelial cells. However, mutations in VV2_1579 and VV1_2305 resulted in lower cytotoxicity to human cells and mortality to mice than the mutation in VV1_2661. These results demonstrate that NtrC-regulated EPS are crucial in biofilm formation of V. vulnificus, and some EPS components play important roles in interacting with hosts.

Vibrio vulnificus, a pathogenic bacterium causing primary septicemia, exhibited cytotoxicity towards Jurkat cells of T-lymphocytes through intracellular reactive oxygen species (ROS) production. Pretreatment of Jurkat T-cells with diphenyleneiodonium chloride (DPI) abolished V. vulnificus-induced ROS generation and bacterial ability to cause cell death. Jurkat T-cells expressing dominant-negative protein of Rac subunit of NADPH oxidase (NOX) did not show increased ROS production and cell death by V. vulnificus. Vibrio vulnificus also triggered phosphorylation of mitogen-activated protein kinases (MAPKs) including p38 and ERK1/2 in Jurkat T-cells. Experiments using inhibitors or small interfering RNAs for each MAPK showed that both MAPKs are involved in V. vulnificus-induced cell death. DPI only blocked the phosphorylation of p38 MAPK in Jurkat T-cells exposed by V. vulnificus. This study demonstrates that V. vulnificus induces death of Jurkat T-cells via ROS-dependent activation of p38 MAPK, and that NOX plays a major role in ROS generation in V. vulnificus-exposed cells.

A monoclonal antibody (mab) against the antimicrobial sulfamethazine was prepared and characterized by an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). Sulfamethazine in the range of 0.2 and 45 ng/ml could be determined with the mab by IC-ELISA. cDNAs encoding a variable heavy chain and variable light chain of the mab were cloned to produce recombinant antibodies using phage display technology. Following phage rescue and three rounds of panning, a single-chain variable fragment (scFv) antibody with high sulfamethazine-binding affinity was obtained. ELISA analysis revealed that scFv antibody and parent mab showed similar, but not identical, characteristics. The IC50 value by IC-ELISA with scFv antibody was 4.8 ng/ml, compared with 1.6 ng/ml with the parent mab. Performances of the assays in the presence of milk matrix were compared; the mab-based assay was less affected than the scFv-based assay. Sixty milk samples were analyzed by mab-based IC-ELISA, and four samples were sulfamethazine positive; these results were favorably correlated with those obtained by HPLC.

The adhesion of Campylobacter jejuni to chicken skin, along with the associated morphological changes under aerobic conditions at 4, 25, and 37 degrees C and microaerobic (O2 5%, CO2 10%, N2 85%) conditions, were investigated using confocal laser scanning microscopy (CLSM), flow cytometry, and plate counting. The morphological change of C. jejuni from a spiral shape to a coccoid form or VBNC form (viable but nonculturable form) progressed rapidly under aerobic conditions at 25, 37, and 4 degrees C. As regards adhesion, the C. jejuni cells were mostly located in the crevices and feather follicles of the chicken skin, where the cells in the feather follicles floated freely in the entrapped water, even after the skin was rinsed quite thoroughly. CLSM also revealed the penetration of some spiral-shaped C. jejuni cells into the chicken skin. Even after changing their shape at various temperatures, coccoid-form C. jejuni cells were still found in the crevices and feather follicles of the chicken skin.

Vibrio vulnificus is a pathogenic bacterium causing primary septicemia, which follows a classical septic shock pathway, including an overwhelming inflammatory cytokine response. In this study, we identified a putative lipoprotein of V. vulnificus, encoded by the ilpA gene, as one of the surface proteins that specifically reacted with the antibodies raised against outer membrane proteins of V. vulnificus. Using a mutant V. vulnificus in which its ilpA gene was knocked out, we found that IlpA is important in the production of interferon-gamma in human peripheral blood mononuclear cells. Production of tumor necrosis factor-alpha and interleukin-6 is also induced by the recombinant IlpA (rIlpA) in human monocytes. Lipidation of the rIlpA was observed by in vivo labeling in Escherichia coli. Experiments using the mutant IlpA, which is unable to be modified by lipidation, indicate that the lipid moiety of this protein has an essential property for cytokine production in human cells. Pretreatment of monocytes with antibodies against Toll-like receptor 2 (TLR2) inhibited production of both tumor necrosis factor-alpha and interleukin-6. The role of TLR2 in IlpA-induced cytokine production was confirmed by an in vitro assay, in which only the TLR2-expressing cells showed a dramatic induction of nuclear factor-kappaB activity by rIlpA. In addition, rIlpA treatment resulted in induction of TLR2 transcription in human cells. In comparison with the wild type V. vulnificus, the ilpA mutant showed a reduced mortality in mice. These results demonstrate that IlpA of V. vulnificus functions as an immunostimulant to human cells via TLR2.

Vibrio vulnificus is a pathogenic bacterium that causes gastroenteritis and primary septicemia. To identify factors involved in microbial adherence to the host cells, we investigated bacterial proteins capable of binding to fibronectin, one of the main components comprised of the extracellular matrix of mammalian cells. A protein of ∼35 kDa was purified from the extracts of V. vulnificus by its property to bind to immobilized fibronectin. This protein was identified as OmpU, one of the major outer membrane proteins of V. vulnificus . In binding assays using immobilized fibronectin, the number of ompU mutant cells bound to fibronectin was only 4% of that of wild-type cells bound to fibronectin. In addition, the exogenous addition of antibodies against OmpU resulted in a decreased ability of wild-type V. vulnificus to adhere to fibronectin. The ompU mutant was also defective in its adherence to RGD tripeptide (5% of the adherence of the wild type to RGD), cytoadherence to HEp-2 cells (7% of the adherence of the wild type to HEp-2), cytotoxicity to cell cultures (39% of the cytotoxicity of the wild type), and mortality in mice (10-fold increase in the 50% lethal dose). The ompU mutant complemented with the intact ompU gene restored its abilities for adherence to fibronectin, RGD tripeptide, and HEp-2 cells; cytotoxicity to HEp-2 cells; and mouse lethality. This study indicates that OmpU is an important virulence factor involved in the adherence of V. vulnificus to the host cells.

Cardiac malformations due to aberrant development of the atrioventricular(AV) valves are among the most common forms of congenital heart disease. At localized swellings of extracellular matrix known as the endocardial cushions,the endothelial lining of the heart undergoes an epithelial to mesenchymal transition (EMT) to form the mesenchymal progenitors of the AV valves. Further growth and differentiation of these mesenchymal precursors results in the formation of portions of the atrial and ventricular septae, and the generation of thin, pliable valves. Gata4, which encodes a zinc finger transcription factor, is expressed in the endothelium and mesenchyme of the AV valves. Using a Tie2-Cre transgene, we selectively inactivated Gata4 within endothelial-derived cells. Mutant endothelium failed to undergo EMT, resulting in hypocellular cushions. Mutant cushions had decreased levels of Erbb3, an EGF-family receptor essential for EMT in the atrioventricular cushions. In Gata4 mutant embryos, Erbb3downregulation was associated with impaired activation of Erk, which is also required for EMT. Expression of a Gata4 mutant protein defective in interaction with Friend of Gata (FOG) cofactors rescued the EMT defect, but resulted in a decreased proliferation of mesenchyme and hypoplastic cushions that failed to septate the ventricular inlet. We demonstrate two novel functions of Gata4 in development of the AV valves. First, Gata4 functions as an upstream regulator of an Erbb3-Erk pathway necessary for EMT, and second, Gata4 acts to promote cushion mesenchyme growth and remodeling.

In a previous paper, the ogdH gene that encodes 2-oxoglutarate dehydrogenase was isolated from Salmonella typhimurium. The catalytic N-terminal region in the enzyme was found to be very specific for the Salmonella species. Therefore, the aim of the present study was to detect S. typhimurium in food sources using primers designed for OGDH-1 and OGDH-2 which were based on the salmonella-specific region of the ogdH gene. A simple polymerase chain reaction (PCR) detection method was developed to detect low numbers of S. typhimurium in a chicken meat microbial consortium. Using the ogdH-specific primers under stringent amplification conditions and for gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the PCR assay was run on S. typhimurium-contaminated meat contents, only the positive meat samples containing as few as 200 CFUs reacted to the assay. The method employed for sample processing is simple and it was determined to provide a sensitive means of detecting trace amounts of S. typhimurium-specific sequences in the presence of mixed meat microbial populations. When compared with six representative intestinal gram-negative bacterial strains in foods, including Vibrio parahaemolyticus, V. vulnificus, Enterobacter cloacae, E. coli O157:H7, Pseudomonas aeruginosa, and Proteus sp., S. typhimurium had a unique and distinct PCR product (796 bp). In conclusion, the two OGDH primers were found to be rapid and sensitive detectors of Salmonella spp for the PCR method.

Coherent x-rays from synchrotron sources are increasingly used in non-conventional radiological techniques ('phase-contrast' radiology). Our experiments demonstrate that by using white (unmonochromatic) radiation and a time-resolving system, it is possible to image microscopic details of moving blood vessels in different live animals without using any contrast agent. The images have excellent contrast plus unprecedented spatial resolution for microangiography (10 microm). This result is likely to impact many different areas of biological and medical research and of diagnostic radiology.

Ionizing radiation has long been used in medicine since the discovery of X-rays. Diagnostic imaging using synchrotron radiation has been under investigation since Rubenstein et al. reported dual-energy iodine-K-edge subtraction coronary angiography. Recently, computed tomography (CT) and magnetic resonance imaging (MRI) have provided better quality results than conventional radiology, providing important information on human internal structures. However, such techniques are unable to detect fine micron sized structures for the early diagnosis of tumors, vascular diseases and other medical objectives. Third generation synchrotron X-rays are well known for their superiority in coherence and energy tunability with respect to conventional X-rays. Consequently, new contrast mechanisms with a superior spatial resolution are becoming available. Here we present the extremely fine details of live animal internal structures using unmonochromatized synchrotron X-rays (white beam) and a simple detector system. Natural movements of the internal organs are also shown. The results indicate that this imaging technique can be applied to investigating microstructures and evaluating the function of the internal organs. Furthermore, this imaging system may be applied to humans as the next tool beyond CT and MRI.

The Drosophila homeobox gene tinman and its vertebrate homologs Nkx-2.5 and Nkx-2.3 are critical determinants of cardiac development. We report here the identification of a new tinman-related gene, nkx2.7, as well as orthologs of Nkx-2.5 and Nkx-2.3 in the zebrafish. Analysis of their expression in the developing zebrafish embryo reveals that nkx2.7 transcripts are the first to appear in cardiac mesodermal and pharyngeal endodermal precursors of the anterior hypoblast, anticipating both temporally and spatially the later expression of nkx2.5 and nkx2.3 in these lineages. The preeminence of nkx2.7 in these embryonic lineages is consistent with a key role in cell fate determination, perhaps in part through the induction of nkx2.5 and nkx2.3. The findings provide the first molecular clues as to the spatial organization of endodermal and cardiac mesodermal precursors in the zebrafish hypoblast immediately following gastrulation. They suggest a coordinate role for these three tinman-related genes in the development of the heart and pharyngeal arches, and reinforce the paradigm of gene duplication and subspecialization between Drosophila and vertebrate species. The results provide a framework in which to analyze potential changes in tinman-related gene expression during abnormal zebrafish development.

We have cloned and characterized a new member of the receptor tyrosine kinase family. The cDNA clone, isolated from a rat olfactory cDNA library, has considerable homology to the family of receptors that includes the colony-stimulating factor 1 receptor, the c-kit proto-oncogene, and the platelet-derived growth factor (PDGF) receptors. Analysis of DNA sequence homology, ligand-binding, and ligand-stimulated phosphorylation data suggests that this clone encodes the rat PDGF-A/B or alpha-receptor. Comparison of its sequence to those of other receptors allows us to postulate a mechanism for receptor dimerization and activation. The expression of the rat alpha-PDGF receptor in nonneuronal cells of the olfactory epithelium and in the olfactory bulb is consistent with a role for PDGF in glial cell generation.

Responses of dorsal horn cells to ventral root stimulation were determined for the L7 and S1 levels of the spinal cord of 14 anesthetized cats. Forty-six dorsal horn cells were found that were excited by stimulation of the distal stump of the cut ventral root. For maximum excitation it was necessary to use a train of stimuli. For the 34 dorsal horn cells whose peripheral receptive-field properties could be characterized, 14 were wide dynamic range cells and 19 were high-threshold cells. The other cell responded exclusively to stimulation of deep tissue. None of the cells responded exclusively to innocuous stimuli, and all responded more vigorously to noxious than to innocuous stimuli. Some cells also responded to noxious heat applied to the skin of the receptive field. Locations of 10 of the activated dorsal horn cells were identified. They were distributed throughout the dorsal horn, but most were found in laminae V and VI. In four animals, both the proximal and distal stumps of the cut S1 ventral root were stimulated while searching for dorsal horn cells. Ten dorsal horn cells were found that were excited by stimulation of the distal stump of the ventral root. No cells were found that responded to proximal stump stimulation. To prevent current spread by stimulation of the ventral root, an extra ground electrode was placed distal to the stimulating electrodes. When the ground electrode was removed, distinctive signs of current spread appeared in that a cord dorsum potential could be recorded and the dorsal horn neuronal responses changed. Dorsal horn neurons could also be excited by nonelectrical stimuli such as crushing the ventral root. If the ventral root was crushed distal to the stimulating electrodes, however, the initially excited cell could no longer be activated by ventral root stimulation. Activation of dorsal horn cells by stimulation of the distal stump of a cut ventral root was abolished when the dorsal root of the same segment was sectioned. Conduction velocities of the fibers in the ventral root that excited dorsal horn cells ranged between 0.25 and 1.78 m/s with a mean of 0.91 +/- 0.47 (SD) m/s. These results show that there are unmyelinated afferent fibers in the ventral root that enter the spinal cord through the dorsal root and excite dorsal horn cells.(ABSTRACT TRUNCATED AT 400 WORDS)

✓ A late component of the cortical evoked potential elicited by somatosensory afferent input was studied in cats anesthetized with α-chloralose. Cortical evoked potentials were recorded from the somatosensory-motor cortex during stimulation of the sural nerve with graded intensities. The stimulus intensity was adjusted to activate Aαβ fibers only, then both Aαβ and Aδ fibers, and both A and C fibers, as judged by afferent volleys monitored from the sural nerve proximal to the stimulating site. In addition to early components reported previously, a very late component was identified at a latency of 400 to 600 msec following stimulation of the sural nerve with intensities above threshold for Aδ fibers. A further increase in stimulation intensity to include activation of C fibers did not reveal any more components. This late component was depressed by a systemic intravenous injection of morphine (2 mg/kg), and intravenous naloxone (0.1 mg/kg) reversed the effect of morphine. The late component of the evoked potential could also be recorded from subcortical tissue after decortication of the sensorimotor cortex. From these results, it appears that a very late component of the cortical evoked potential can be recorded from cats anesthetized with α-chloralose. The late component is evoked by activation of peripheral Aδ fibers. Furthermore, its morphine sensitivity suggests that this component may be elicited by nociceptive afferent fibers. If further investigations prove this, the late component, which is analogous to human long-latency potentials, could be used in an experimental model for pain research.

The segmental distribution of visceral afferent neurons with axons in the cardiac nerve was studied in cats with the horseradish peroxidase (HRP) technique. After HRP was introduced into the left inferior cardiac nerve, labeled dorsal root ganglion cells were found in the ganglia from the C7 to T7 spinal levels, with a wide variation in distribution among the individual animals. An average of 93 cells was labeled per animal. The data indicate that the afferent nerve fibers in the inferior cardiac nerve project to a much wider range of segments of the spinal cord than previously believed.

The activity of 132 neurons in the caudal part of the ventral posterior lateral nucleus (VPLc) of the thalamus was recorded from 23 anesthetized monkeys. All single thalamic units that could be excited by electrical search stimuli applied to the contralateral sciatic nerve were investigated. Responses of these cells to mechanical, thermal, and electrical stimuli applied in the periphery indicated that at least half of the sampled cells were nociceptive. Based on responses to graded mechanical stimuli applied to the periphery, 110 of the sampled cells that received a predominant input from cutaneous receptive fields were classified. There were 56 low-threshold, 39 wide dynamic range, and 15 high-threshold cells. The same neurons were also classified into five mechanical types based on a cluster analysis: types 1-5 contained 25, 34, 17, 10, and 24 cells, respectively. The fact that about half the population of cells belonged to either the wide dynamic or the high threshold group (or mechanical types 3-5) suggested that a large population of VPLc neurons respond to mechanical nociceptive stimuli either exclusively or preferentially. Responses of 63 thalamic neurons were tested to noxious heat pulses applied to their cutaneous receptive fields with a contact thermostimulator. Of these, 47 cells were excited, whereas only 16 cells did not respond. The peripheral nerve that innervated the receptive field of each of 82 thalamic neurons was stimulated with graded strengths to activate A fibers only or both A and C fibers. All tested cells responded to peripheral A fiber volleys. In addition, 42 of these cells responded to peripheral C fiber volleys. The C fiber responses could be either short lasting (a few hundreds of milliseconds) or long lasting (up to several seconds). The recording sites of 80 cells were reconstructed. Of these, 78 were in the VPLc nucleus and the remaining two were in the reticular nucleus of the thalamus. No obvious relationship between the response characteristics and the locations of the cells within the VPLc nucleus was found. Sampled thalamic units had a variety of sources of input from the periphery, including both cutaneous and/or deep tissue receptive fields. The majority of the cells, however, had exclusively cutaneous receptive fields. The sizes of the cutaneous receptive fields were often very small, so that nearly half (41%) of the receptive fields of cells sampled occupied an area of skin smaller than half the foot.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular cloning techniques were used to isolate and characterize a protein possibly involved in the signal transducing system in olfactory tissue of the frog Rana pipiens. A complementary DNA library was constructed with messenger RNA obtained from frog olfactory neuroepithelium. A 700-base pair complementary DNA clone encoding a protein with a molecular weight of 20,300 was identified by differential hybridization analysis with polyadenylated RNA from olfactory epithelium and nonsensory respiratory epithelium. The messenger RNA corresponding to this clone was abundant in the cells of Bowman's glands in olfactory tissue but not in respiratory epithelium nor in several other tissues. The predicted sequence of this protein is homologous to members of a family of proteins that bind and transport small molecules in serum, suggesting that this protein may also bind and transport odorants in the mucus secreted by Bowman's glands.

Measurements of cell body and peripheral and central axon sizes were made for primary sensory neurons outlined by the intracellular injection of HRP. Conduction velocities were also measured on the outlined processes. The sensory neurons were then subdivided into A and C cells on the basis of the conduction velocity of the impulses carried by the processes of these cells. Central processes of both A and C cells are smaller than the peripheral processes, but the size differential is greater for the C cells. For A cells there is a linear relation between the size of the peripheral axon and the conduction velocity of the impulses carried by these axons, but the confidence limits are wide. For C cells there is a linear relation between the size of the central process and conduction velocity of the impulses carried by the processes, but for the peripheral processes two aberrant processes resulted in no correlation between process size and conduction velocity. For A cells, the size of the central and peripheral processes and the conduction velocity of the impulses carried by the peripheral processes are linearly correlated with cell body size. By contrast no such correlations can be demonstrated for C cells. This presumably implies an important difference in that the size of the cell body is correlated with axon size and impulse conduction velocity for A cells but not for C cells. A widely accepted generalization is that large sensory cells give rise to myelinated axons and small sensory cells to unmyelinated axons. In this study, myelinated and unmyelinated are defined on the basis of impulse conduction velocity. For those cells that are clearly large (greater than 50 microns in diameter), the conduction velocity of the impulses carried by their processes is always greater than 2.5 m/s, and for those cells that are clearly small (less than 35 microns in diameter), the conduction velocity is always less than 2.5 m/s. Thus for these cells the above generalization holds. For the intermediate-sized cells (35-50 microns), however, the size of the cell body bears no predictable relation to the conduction velocity of the impulses carried by those processes, and thus to whether the axons are myelinated or unmyelinated. Thus the above generalization does not hold for this intermediate group of cells, and since there are many cells in this size range, we feel that the generalization that large cells give rise to myelinated axons and small cells to unmyelinated axons is an oversimplification.

✓ Transcutaneous electrical nerve stimulation (TENS) was applied in an experimental animal model to investigate the underlying mechanisms of this treatment. Recordings were made from identified spinothalamic tract (STT) neurons in the lumbosacral spinal cords of seven anesthetized monkeys. The STT cells were activated by stimulating the common peroneal nerve at a suprathreshold intensity for C-fibers. Evoked responses of C-fibers were compared before, during, and after application of TENS for 5 minutes from a commercially available TENS unit. The current delivered by the TENS unit was monitored. In 14 STT cells, some degree of inhibition of C-fiber evoked responses occurred only when the intensity of TENS exceeded the threshold of Aδ fibers. At a given stimulus intensity, bursts of pulses repeated at a low rate were more effective than high-rate pulses. When TENS was applied to an area of the skin within a cell's receptive field, it was more effective than when it was applied outside the receptive field. The C-fiber volley recorded from a peripheral nerve was not reduced in size, and there were no substantial changes in its latency due to TENS. The inhibition of the activity of STT cells was not altered appreciably after intravenous injection of naloxone hydrochloride. These results suggest that TENS produces central nervous system inhibition by activating Aδ afferent fibers. The inhibitory effects of TENS on STT cells appear to be due to a mechanism that does not involve release of endogenous opioid substances.

Data analyzed in this study were derived from the responses of 128 spinothalamic tract (STT) cells and 110 thalamic neurons recorded in 75 anesthetized monkeys. A k-means cluster analysis, a nonhierarchical clustering technique, was performed using the relative magnitudes of responses to a graded series of innocuous and noxious mechanical stimuli applied to the receptive field. For comparison, a parallel analysis was performed based on definitions of low-threshold (LT), wide dynamic range (WDR), and high-threshold (HT) cells used by our laboratory. For 128 STT cells, a classification scheme with three clusters was found statistically to be the best. This yielded groups of 22, 57, and 49 cells in clusters 1, 2, and 3, respectively. Cluster 1 cells were activated best by low-intensity mechanical stimuli, whereas cluster 3 cells were activated primarily by nociceptive stimuli. Cluster 2 cells had intermediate characteristics. When the classification scheme based on the cluster analysis was compared with the classification of the same neurons as LT, WDR, and HT cells, cluster 1 cells were divided into LT and WDR cells, whereas cluster 2 and 3 cells included WDR and HT cells. For 110 thalamic neurons, a classification scheme with five clusters was found statistically to be the best. Clusters 1-5 contained 25, 34, 17, 10, and 24 cells, respectively. Response characteristics of cells in each group indicated a gradual change in sensitivity to higher intensities of peripheral input from cluster 1 to 5. When this classification scheme was compared with the classification scheme previously used by our laboratory, cluster 1 cells belonged to the LT group, clusters 2 and 3 split into LT and WDR cells, and clusters 4 and 5 included WDR and HT cells. It is concluded that a classification scheme based on a cluster analysis of the responses of neurons to standardized stimuli may provide an objective and functionally meaningful way to categorize somatosensory neurons.