Types and Distribution of Anaerobic Bacteria in the Large Intestine of Pigs

An examination was made of various sites along the length of the swine large intestine, using strictly anaerobic culture methods. Sites were separated by differential washing into fractions described as lumenal content, lumenal surface layer, and intestinal wall tissue. Direct microscopic clump counts averaged 13.3 × 10¹⁰ organisms per g (dry weight) of material in the lumenal content, 14.0 × 10¹⁰ in the surface layer, and 5.1 × 10¹⁰ in the intestinal wall tissue. Both direct microscopic counts and viable culture counts were higher from the lumenal content and surface layer than from the intestinal tissue at all sites sampled in the intestine. Cultural counts averaged 56.2% of the direct microscopic counts in lumenal content and surface layer and 20.2% in intestinal tissue. Over 90% of the bacteria isolated were gram positive and consisted mainly of gram-positive cocci, lactobacilli, eubacteria, and clostridia. Of 192 isolates recovered, only 124 could be assigned to recognized species.     

Light-Emitting Diode Phototherapy Reduces Nocifensive Behavior Induced by Thermal and Chemical Noxious Stimuli in Mice: Evidence for the Involvement of Capsaicin-Sensitive Central Afferent Fibers

Low-intensity phototherapy using light fonts, like light-emitting diode (LED), in the red to infrared spectrum is a promising alternative for the treatment of pain. However, the underlying mechanisms by which LED phototherapy reduces acute pain are not yet well understood. This study investigated the analgesic effect of multisource LED phototherapy on the acute nocifensive behavior of mice induced by thermal and chemical noxious stimuli. The involvement of central afferent C fibers sensitive to capsaicin in this effect was also investigated. Mice exposed to multisource LED (output power 234, 390, or 780 mW and power density 10.4, 17.3, and 34.6 mW/cm², respectively, from 10 to 30 min of stimulation with a wavelength of 890 nm) showed rapid and significant reductions in formalin- and acetic acid-induced nocifensive behavior. This effect gradually reduced but remained significant for up to 7 h after LED treatment in the last model used. Moreover, LED (390 mW, 17.3 mW/cm²/20 min) irradiation also reduced nocifensive behavior in mice due to chemical [endogenous (i.e., glutamate, prostaglandins, and bradykinin) or exogenous (i.e., formalin, acetic acid, TRPs and ASIC agonist, and protein kinase A and C activators)] and thermal (hot plate test) stimuli. Finally, ablating central afferent C fibers abolished LED analgesia. These experimental results indicate that LED phototherapy reduces the acute painful behavior of animals caused by chemical and thermal stimuli and that LED analgesia depends on the integrity of central afferent C fibers sensitive to capsaicin. These findings provide new information regarding the underlying mechanism by which LED phototherapy reduces acute pain. Thus, LED phototherapy may be an important tool for the management of acute pain.     

Bacterial Responses to Different Dietary Cereal Types and Xylanase Supplementation in the Intestine of Broiler Chicken

Several studies were carried out to investigate the influence of dietary cereals differing in soluble non starch polysaccharides (NSP) content and a xylanase preparation on selected bacterial parameters in the small intestine of broiler chicken. Compared to a maize diet colony forming units (CFU) of mucosa associated bacteria were higher in a wheat/rye diet, most notably for enterobacteria and enterococci. Xylanase supplementation to the wheat/rye diet generally led to lower CFU, especially in the first week of life. However, xylanase supplementation also displayed higher in vitro growth potentials for enterobacteria and enterococci. Bacterial growth of luminal samples in minimal media supplemented with selected NSP showed that the wheat/rye diet enhanced bacterial capacities to utilize NSP only in ileal samples. The xylanase application generally shifted respective maximum growth to the proximal part of the small intestine. The presence of soluble NSP from wheat or rye in the diet per se did not enhance bacterial NSP hydrolyzing enzyme activities in the small intestine, but xylanase supplementation resulted in higher 1,3-1,4- g - glucanase activity. Compared to a maize diet the activity of bacterial bile salt hydrolases in samples of the small intestine was not increased due to inclusion of wheat/rye or triticale to the diet. However, xylanase supplementation led to a reduction with a corresponding increase of lipase activity. It was concluded that dietary cereals producing high intestinal viscosities lead to increased overall bacterial activity in the small intestine. The supplementation of a xylanase to cereal based diets producing high intestinal viscosity, changes composition and metabolic potential of bacterial populations and may specifically influence fat absorption in young animals.     

The Presence of Two Types of Gene That Encode the Chlorophyll a/b-Binding Protein (LHCPII) and Their Light-Independent Expression in Pine (Pinus thunbergii)

A cDNA clone (pPDLHC3117) for LHCPII of pine (Pinus thunbergii)was isolated and sequenced. From a comparison of the deducedamino acid sequence with those of proteins from other seed plants,pPDLHC3117 was identified as encoding a type I LHCPII. Specificprobes for genes for type I and type II LHCPII were made frompPDLHC3117 and a type II cDNA (pPDLHC2176), respectively. Northernblotting with the specific probes showed that both types ofLHCPII were expressed in dark-grown seedlings of pine. ¹This work was supported by a Grant-in-Aid from the Ministryof Agriculture, Forestry and Fisheries of Japan (IntegratedResearch Program for the Use of Biotechnological Proceduresfor Plant Breeding).     

Identification of a Maize Locus That Modulates the Hypersensitive Defense Response,Using Mutant-Assisted Gene Identification and Characterization

Potentially useful naturally occurring genetic variation is often difficult to identify as the effects of individual genes are subtle and difficult to observe. In this study, a novel genetic technique called Mutant-Assisted Gene Identification and Characterization is used to identify naturally occurring loci modulating the hypersensitive defense response (HR) in maize. Mutant-Assisted Gene Identification and Characterization facilitates the identification of naturally occurring alleles underlying phenotypic variation from diverse germplasm, using a mutant phenotype as a “reporter.” In this study the reporter phenotype was caused by a partially dominant autoactive disease resistance gene, Rp1-D21, which caused HR lesions to form spontaneously all over the plant. Here it is demonstrated that the Rp1-D21 phenotype is profoundly affected by genetic background. By crossing the Rp1-D21 gene into the IBM mapping population, it was possible to map and identify Hrml1 on chromosome 10, a locus responsible for modulating the HR phenotype conferred by Rp1-D21. Other loci with smaller effects were identified on chromosomes 1 and 9. These results demonstrate that Mutant-Assisted Gene Identification and Characterization is a viable approach for identifying naturally occurring useful genetic variation.POTENTIALLY useful naturally occurring genetic variation is often difficult to identify as the effects of individual genes are subtle and difficult to observe. Furthermore, so many different alleles are available that it is a major challenge just to sift through the enormous diversity available. To this end, we recently conceptualized a simple yet effective method to discover and characterize variation present naturally in plant germplasm (Johal et al. 2008). This method, Mutant-Assisted Gene Identification and Characterization, makes use of a mutant phenotype for a gene affecting the trait of interest as a reporter to discover and analyze relevant, interacting genes present naturally in diverse germplasm. Mutant-Assisted Gene Identification and Characterization involves crossing a mutant to diverse germplasm and then evaluating the mutant progeny for transgressive changes (both suppressed and severe) in the mutant phenotype(s). If the mutation is recessive, the population needs to be advanced to the F₂ generation to be able to detect and analyze such variation. However, for a dominant or partially dominant mutant, evaluations can be made immediately in the F₁ to discover lines that contain suppressors or enhancers of the trait (mutation) under study. Mutant F₁ progenies from such crosses can then be propagated further to identify, map, and clone genes/QTL that affect the trait positively or negatively. In the case of maize and other species for which genetically characterized mapping populations are available, modifying loci can be rapidly mapped by crossing a mutant line to each member of a mapping population and evaluating the resulting F₁ families. In this study we provide a proof-of-concept for the Mutant-Assisted Gene Identification and Characterization technique, using it to identify loci involved in the defense response of maize.Plants are constantly exposed to numerous potential pathogens with diverse modes of attack. Nevertheless, it is rather rare to see plants succumbing to disease. One key reason for this is the presence of a highly effective and inducible defense system, a major component of which is the hypersensitive response (HR). HR is usually associated with a specific recognition event and is activated after other nonspecific resistance mechanisms have been overcome or evaded (see Bent and Mackey 2007). Although it was initially coined to refer to the rapid collapse of cells at the site of infection, over the years the term HR has been used to refer to both cell death and the associated induction of a number of other defense responses, including the accumulation of phytoalexins and pathogenesis-related (PR) proteins at the site of infection, to name a few (Mur et al. 2007). Reactive oxygen species such as superoxide and H₂O₂ appear to be causally involved in cell death underlying the HR response (Jones and Dangl 2006).HR is under the control of a subset of disease-resistance genes, commonly referred to as R genes. These R genes specifically recognize matching avirulence (Avr) effectors from the pathogen. Many R genes encode products containing a nucleotide-binding site (NBS) domain in the middle of the protein and a leucine-rich repeat (LRR) domain at the C-terminal end (Bent and Mackey 2007). R proteins are involved both in the recognition of the pathogen and the subsequent induction of the HR response. How R proteins remain in a quiescent but “vigilant” state remains to be established. Certain mutations in R genes have been found that abolish their dependence on AVR proteins for activation. Such aberrant R genes mostly behave as dominant or partially dominant alleles and trigger the HR constitutively in the absence of the pathogen (Hu et al. 1996; Zhang et al. 2003; Dodds et al. 2006). Two consequences of such “autoactive” or “ectopically active” R genes are a massive induction of cell death and the consequential stunting of the organism (Dodds et al. 2006). Although autoactive R genes have been found to exist in many plant species, the first few examples came from the maize Rp1 locus, which confers race-specific resistance to common rust, caused by Puccinia sorghi (Hu et al. 1996). Such autoactive R genes can be used to investigate HR genetics and etiology in the absence of confounding effects from the pathogen and constitute an excellent candidate for analysis using Mutant-Assisted Gene Identification and Characterization.The details of the HR cell death reaction as well as the pathway(s) that link R gene activation with the HR remain unclear (Mur et al. 2007). Despite considerable research over the past decade, only a few components have been found thus far. Some of these, Ndr1, Eds1, Pad4, Rar1, and Sgt1, were identified in mutagenesis screens conducted to identify mutants that failed to undergo an HR reaction in response to infection by an avirulent pathogen (reviewed in Bent and Mackey 2007). A few others, RIN4, for example, were identified in yeast two-hybrid assays using an NBS–LRR protein as bait (Mackey et al. 2003). Recently, an Arabidopsis gain-of-function mutant that carries a point mutation in an R gene analog (a gene with the structure of an R gene but not known to be involved in resistance to any pathogen) was used to isolate a few more potential genes in the HR pathway in a second site suppressor approach following mutagenesis with ethane methyl sulfonate (EMS) (Palma et al. 2005; Zhang and Li 2005; Goritschnig et al. 2007). A problem with approaches based on intentional mutagenesis is that they fail to uncover genes that have either redundant or essential functions. One way to avoid this problem would be to seek naturally occurring allelic variants affecting HR. Such natural variation is pervasive in all species, being generated and selected for over millions of years of evolution.Although natural variation has served as a constant provider of the R genes in all plant species, natural variability has not been tapped as a tool for understanding other aspects of the disease-resistance response (Holub 2007). The Rp1-D21 gene is an autoactive allele from the maize Rp1 disease-resistance locus that initiates HR randomly all over the plant (Pryor 1993; Collins et al. 1999; Sun et al. 2001). Our objective for this study was to use the Rp1-D21 gene phenotype as a test case for the Mutant-Assisted Gene Identification and Characterization approach. We show here that enormous variation exists in the maize germplasm that is capable of affecting the HR response positively or negatively and we identify loci that modulate expression of the HR phenotype segregating in the well-known Intermated B73 × Mo17 (IBM) advanced intercross line (AIL) population (Coe et al. 2002; Lee et al. 2002). This constitutes the first demonstration of the utility of the Mutant-Assisted Gene Identification and Characterization approach—an approach that is likely to prove widely applicable.     

Somatosensory Abnormalities for Painful and Innocuous Stimuli at the Back and at a Site Distinct from the Region of Pain in Chronic Back Pain Patients

Chronic low back pain (CLBP) was shown to be associated with pathophysiological changes at several levels of the sensorimotor system. Changes in sensory thresholds have been reported but complete profiles of Quantitative Sensory Testing (QST) were only rarely obtained in CLBP patients. The aim of the present study was to investigate comprehensive QST profiles in CLBP at the painful site (back) and at a site distinct from their painful region (hand) and to compare these data with similar data in healthy controls. We found increased detection thresholds in CLBP patients compared to healthy controls for all innocuous stimuli at the back and extraterritorial to the painful region at the hand. Additionally, CLBP patients showed decreased pain thresholds at both sites. Importantly, there was no interaction between the investigated site and group, i.e. thresholds were changed both at the affected body site and for the site distinct from the painful region (hand). Our results demonstrate severe, widespread changes in somatosensory sensitivity in CLBP patients. These widespread changes point to alterations at higher levels of the neuraxis or/and to a vulnerability to nociceptive plasticity in CLBP patients.     

Spinal Autofluorescent Flavoprotein Imaging in a Rat Model of Nerve Injury-Induced Pain and the Effect of Spinal Cord Stimulation

Nerve injury may cause neuropathic pain, which involves hyperexcitability of spinal dorsal horn neurons. The mechanisms of action of spinal cord stimulation (SCS), an established treatment for intractable neuropathic pain, are only partially understood. We used Autofluorescent Flavoprotein Imaging (AFI) to study changes in spinal dorsal horn metabolic activity. In the Seltzer model of nerve-injury induced pain, hypersensitivity was confirmed using the von Frey and hotplate test. 14 Days after nerve-injury, rats were anesthetized, a bipolar electrode was placed around the affected sciatic nerve and the spinal cord was exposed by a laminectomy at T13. AFI recordings were obtained in neuropathic rats and a control group of naïve rats following 10 seconds of electrical stimulation of the sciatic nerve at C-fiber strength, or following non-noxious palpation. Neuropathic rats were then treated with 30 minutes of SCS or sham stimulation and AFI recordings were obtained for up to 60 minutes after cessation of SCS/sham. Although AFI responses to noxious electrical stimulation were similar in neuropathic and naïve rats, only neuropathic rats demonstrated an AFI-response to palpation. Secondly, an immediate, short-lasting, but strong reduction in AFI intensity and area of excitation occurred following SCS, but not following sham stimulation. Our data confirm that AFI can be used to directly visualize changes in spinal metabolic activity following nerve injury and they imply that SCS acts through rapid modulation of nociceptive processing at the spinal level.     

Comparison of the Structure and Expression of Odd-Skipped and Two Related Genes That Encode a New Family of Zinc Finger Proteins in Drosophila

The odd-skipped (odd) gene, which was identified on the basis of a pair-rule segmentation phenotype in mutant embryos, is initially expressed in the Drosophila embryo in seven pair-rule stripes, but later exhibits a segment polarity-like pattern for which no phenotypic correlate is apparent. We have molecularly characterized two embryonically expressed odd-cognate genes, sob and bowel (bowl), that encode proteins with highly conserved C(2)H(2) zinc fingers. While the Sob and Bowl proteins each contain five tandem fingers, the Odd protein lacks a fifth (C-terminal) finger and is also less conserved among the four common fingers. Reminiscent of many segmentation gene paralogues, the closely linked odd and sob genes are expressed during embryogenesis in similar striped patterns; in contrast, the less-tightly linked bowl gene is expressed in a distinctly different pattern at the termini of the early embryo. Although our results indicate that odd and sob are more likely than bowl to share overlapping developmental roles, some functional divergence between the Odd and Sob proteins is suggested by the absence of homology outside the zinc fingers, and also by amino acid substitutions in the Odd zinc fingers at positions that appear to be constrained in Sob and Bowl.     

Identification of a Novel Drug Lead That Inhibits HCV Infection and Cell-to-Cell Transmission by Targeting the HCV E2 Glycoprotein

Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2’s interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421–645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50’s ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.     

Identification and Quantification of AKT Isoforms and Phosphoforms in Breast Cancer Using a Novel Nanofluidic Immunoassay

Breast cancer subtype-specific molecular variations can dramatically affect patient responses to existing therapies. It is thought that differentially phosphorylated protein isoforms might be a useful prognostic biomarker of drug response in the clinic. However, the accurate detection and quantitative analysis of cancer-related protein isoforms and phospho-isoforms in tumors are limited by current technologies. Using a novel, fully automated nanocapillary electrophoresis immunoassay (NanoPro^TM 1000) designed to separate protein molecules based on their isoelectric point, we developed a reliable and highly sensitive assay for the detection and quantitation of AKT isoforms and phosphoforms in breast cancer. This assay enabled the measurement of activated AKT1/2/3 in breast cancer cells using protein produced from as few as 56 cells. Importantly, we were able to assign an identity for the phosphorylated S473 phosphoform of AKT1, the major form of activated AKT involved in multiple cancers, including breast, and a current focus in clinical trials for targeted intervention. The ability of our AKT assay to detect and measure AKT phosphorylation from very low amounts of total protein will allow the accurate evaluation of patient response to drugs targeting activated PI3K-AKT using scarce clinical specimens. Moreover, the capacity of this assay to detect and measure all three AKT isoforms using one single pan-specific antibody enables the study of the multiple and variable roles that these isoforms play in AKT tumorigenesis.Activation of the PI3K-AKT signaling pathway is one of the most common events in cancer (1, 2). Pathway activation can confer a number of advantages to the cancer cells, including enhanced proliferation and survival (1, 2). Multiple mechanisms exist by which the pathway may become activated, including amplification or activation of receptor tyrosine kinases (e.g. ERBB2 in breast and EGFR in lung tumors), mutation of the catalytic or regulatory subunits of PI3K (e.g. PIK3CA in colorectal and breast tumors), loss of the negative regulator PTEN (e.g. mutation in prostate and melanoma), and gain of function of AKT (e.g. amplification or mutation in breast and pancreatic tumors) (reviewed in Refs. 1 and 2).AKT represents a central node in the PI3K signaling cascade (3). AKT is recruited to the cell membrane via its pleckstrin homology domain when PI3K phosphorylates PIP₂ to form PIP₃ (4, 5). Following recruitment, AKT is phosphorylated by PDK1 and the rictor-mTOR complex, resulting in conformational changes and activation of the protein (5–8). Multiple studies have shown that the phosphorylation of AKT leads to the phosphorylation and activation of downstream effectors of the signaling pathway, such as mTOR complex 1 and S6K (reviewed in Ref. 1). The central role of this pathway in cancer is further underscored by the efforts of multiple pharmaceutical companies that have developed inhibitors against AKT as potential anti-oncogenic therapeutics (9).Despite the importance of AKT in growth and survival signaling in cancer, there are surprisingly few data that address the specific roles played in growth and survival by the multiple AKT family members (AKT-1, -2, and -3) and different phosphorylation and putative phosphorylation sites that can potentially activate the protein. Western blot analysis has been the foundation of most AKT studies, but in many cases pan-AKT antibodies have been employed that fail to distinguish between the different AKT isoforms. Recent siRNA silencing studies have indicated distinct functions for different AKT family members within a cell (10, 11). Moreover, there is evidence in breast cancer that the three isoforms exhibit different localizations and therefore must have at least partially distinct functions (12). Similarly, evidence is mounting for multiple phosphorylation sites in AKT beyond the two most studied phosphorylation events (Thr-308 and Ser-473) (5–8). Phosphorylation at serine and threonine residues at Thr-72 and Ser-246 may be required for the activation or regulation of kinase activity (13). The functional significance of constitutive phosphorylation of Ser-124 and Thr-450 is still unknown (14). Finally, there is evidence that phosphorylation of tyrosine residues at Tyr-315 and Tyr-326 is required for full kinase activity (15).Analysis of such phospho- and isoform-specific activation often requires complicated in-depth analyses using large quantities of proteins, purified recombinant protein, immunoprecipitation, incorporation of ³²P isotopes, and/or mass spectroscopy, which makes such studies more difficult to perform and not easily adaptable to clinical specimens. Thus, better methods are required for the accurate assessment of both phosphoform and isoform usage in cells with an activated PI3K-AKT pathway and the effects of pathway inhibitors using relatively small amounts of starting material. We describe here the development of such an assay using nanocapillary-based isoelectric focusing (16). This approach allows the separation of AKT into distinct peaks that correspond to different iso- and phosphoforms using a small amount of starting material and a single pan-specific antibody. This approach should allow for more accurate determinations of isoform usage in different cell types, as well as of changes in phosphorylation states in response to pathway inhibition, including in clinical specimens.     

The Effects of Benzyladenine on the Accumulation of Messenger RNAs That Encode the Large and Small Subunits of Ribulose-l,5-bisphosphate Carboxylase/oxygenase and Light-Harvesting Chlorophyll a/b Protein in Excised Cucumber Cotyledons

Benzyladenine promoted rapid accumulation of mRNAs that encodedthe small subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase(RuBisCO) and light-harvesting chlorophyll a/b protein in etiolatedcucumber cotyledons, but only after prior incubation of thecotyledons in water. However, benzyladenine hardly affectedthe level of mRNA for the large subunit of RuBisCO. ¹ Present address: Shonan Junior College, Yokosuka, Kanagawa238, Japan (Received September 3, 1990; Accepted March 4, 1991)     

Isolation and Molecular Characterization of a Poliovirus Type 1 Mutant That Replicates in the Spinal Cords of Mice

The Mahoney strain of poliovirus type 1 (OM) is generally unable to cause paralysis in mice. We isolated a mouse-adapted mutant, PV1/OM-SA (SA), from the spinal cord of a mouse that had been intracerebrally inoculated with OM. SA showed mouse neurovirulence only with intraspinal inoculation, and the infected mice developed a flaccid paralysis, which was indistinguishable from that observed in poliovirus-sensitive transgenic mice inoculated with OM. SA antigens were detected in neurons of the spinal cords of the infected mice. Nucleotide (nt) sequence analysis revealed 9 nt changes on the SA genome, resulting in three amino acid (a.a.) substitutions, i.e., one each in the capsid proteins VP4 and VP1 and in the noncapsid protein 2C. To identify the key mutation site(s) for the mouse neurovirulence, virus recombinants between OM and SA were constructed by using infectious cDNA clones of these two viruses and tested for their mouse neurovirulence after inoculation via an intraspinal route. The results indicated that a mutation at nt 928 (replacement of A with G), resulting in a substitution of Met for Ile at a.a. 62 within VP4, was responsible for conferring the mouse neurovirulence phenotype of the mutant SA. The mutation in VP4 may render the virus accessible to a molecule that acts as a virus receptor and is located on the surfaces of neurons of the mouse spinal cord. This molecule appears not to be expressed in the mouse brain.     

The Identification of a Novel Gene,MAPO2, That Is Involved in the Induction of Apoptosis Triggered by O6-Methylguanine

O⁶-Methylguanine, one of alkylated DNA bases, is especially mutagenic. Cells containing this lesion are eliminated by induction of apoptosis, associated with the function of mismatch repair (MMR) proteins. A retrovirus-mediated gene-trap mutagenesis was used to isolate new genes related to the induction of apoptosis, triggered by the treatment with an alkylating agent, N-methyl-N-nitrosourea (MNU). This report describes the identification of a novel gene, MAPO2 (O⁶-methylguanine-induced apoptosis 2), which is originally annotated as C1orf201. The MAPO2 gene is conserved among a wide variety of multicellular organisms and encodes a protein containing characteristic PxPxxY repeats. To elucidate the function of the gene product in the apoptosis pathway, a human cell line derived from HeLa MR cells, in which the MAPO2 gene was stably knocked down by expressing specific miRNA, was constructed. The knockdown cells grew at the same rate as HeLa MR, thus indicating that MAPO2 played no role in the cellular growth. After exposure to MNU, HeLa MR cells and the knockdown cells underwent cell cycle arrest at G₂/M phase, however, the production of the sub-G₁ population in the knockdown cells was significantly suppressed in comparison to that in HeLa MR cells. Moreover, the activation of BAK and caspase-3, and depolarization of mitochondrial membrane, hallmarks for the induction of apoptosis, were also suppressed in the knockdown cells. These results suggest that the MAPO2 gene product might positively contribute to the induction of apoptosis triggered by O⁶-methylguanine.     

Ulex europaeus Agglutinin-I Binding to Dental Primary Afferent Projections in the Spinal Trigeminal Complex Combined with Double Immunolabeling of Substance P and GABA Elements Using Peroxidase and Colloidal Gold

Ulex europaeus agglutinin I (UEA-I) is a plant lectin with an affinity for L-fucosyl residues in the chains of lactoseries oligosaccharides associated with medium and smaller-diameter dorsal root ganglion neurons and their axonal processes. These enter Lissauer's tract and terminate within the superficial laminae of the spinal cord overlapping projections known to have a nociceptive function. This implies that the surface coatings of neuronal membranes may have a relationship with functional modalities. The present investigation further examined this concept by studying a neuronal projection with a nociceptive function to determine whether fucosyl-lactoseries residues were incorporated in its primary afferent terminals. Transganglionic transport of horseradish peroxidase (HRP) following injection into tooth pulp chambers was employed to demonstrate dental pulp terminals in the trigeminal spinal complex, while peroxidase and fluorescent tags were used concomitantly to stain for UEA-I. Double immunolabeling for substance P (SP) and γ-aminobutyric acid (GABA) using peroxidase and colloidal gold allowed a comparison of the distribution of a known excitatory nociceptive transmitter with that of UEA-I binding in specific subnuclei. Synaptic interrelationships between UEA-I positive dental pulp primary afferent inputs and specific inhibitory terminals were also examined.

SP immunoreactivity occurred in laminae I and outer lamina II (II_o) of subnucleus caudalis (Vc) and in the ventrolateral and lateral marginal region of the caudal half of subnucleus interpolaris (Vi), including the periobex area in which Vi is slightly overlapped on its lateral aspect by cellular elements of Vc. The adjacent interstitial nucleus (IN) also showed an intense immunoreactivity for this peptide antibody. UEA-I binding displayed a similar distribution pattern in both Vc and Vi, but extended into lamina II; and the superficial part of Lamina III in Vc. Dental pulp terminals were found to have a comparable distribution; however, many extended into the dorsal portion of the caudal half of Vi and the ventromedial quadrant of rostral Vi.

Electron-microscopic analysis showed that transganglionically labeled dental pulp terminals contained ovoid, complex membrane-bound vacuoles laden with transported HRP. The preterminal axon and synaptic membranes of those dental pulp terminals located in zones of Vc and Vi displaying an affinity for UEA-I were usually characterized by a patchy, electron-dense coating of the peroxidase tag. SP was demonstrated ultrastructurally with Protein-A colloidal gold (3-nm particles), whereas GABA immunoreactivity was revealed by the avidin—biotin—peroxidase method. This combined approach labeled a variety of simple axodendritic to large complex scalloped dental terminals which contained SP and were shown to have a UEA-I affinity. In addition, many of the larger terminals formed contacts with GABA-ergic dendrites and received inputs from GABA-ergic synapses. These complexes were most concentrated in lamina II_o of Vc and the ventrolateral zone of Vi. Many terminals in laminae II_i; and III with a UEA-I-positive surface coating failed to bind with the antiserum for SP, indicating that other transmitters may colocalize with UEA-I and suggesting that absolute correlations between specific oligosaccharide plasmalemmal coatings and functional modalities should be approached with caution. Further studies employing antisera to different transmitters are currently underway to better define the relationship between transmitter localization and anatomical substrates within this circuitry. These studies should eventually provide additional clues about relationships between functional properties and oligosaccharide coatings of primary afferent projections.     

Induction,Purification and Characterization of a Novel Manganese Peroxidase from Irpex lacteus CD2 and Its Application in the Decolorization of Different Types of Dye

Manganese peroxidase (MnP) is the one of the important ligninolytic enzymes produced by lignin-degrading fungi which has the great application value in the field of environmental biotechnology. Searching for new MnP with stronger tolerance to metal ions and organic solvents is important for the maximization of potential of MnP in the biodegradation of recalcitrant xenobiotics. In this study, it was found that oxalic acid, veratryl alcohol and 2,6-Dimehoxyphenol could stimulate the synthesis of MnP in the white-rot fungus Irpex lacteus CD2. A novel manganese peroxidase named as CD2-MnP was purified and characterized from this fungus. CD2-MnP had a strong capability for tolerating different metal ions such as Ca²⁺, Cd²⁺, Co²⁺, Mg²⁺, Ni²⁺ and Zn²⁺ as well as organic solvents such as methanol, ethanol, DMSO, ethylene glycol, isopropyl alcohol, butanediol and glycerin. The different types of dyes including the azo dye (Remazol Brilliant Violet 5R, Direct Red 5B), anthraquinone dye (Remazol Brilliant Blue R), indigo dye (Indigo Carmine) and triphenylmethane dye (Methyl Green) as well as simulated textile wastewater could be efficiently decolorized by CD2-MnP. CD2-MnP also had a strong ability of decolorizing different dyes with the coexistence of metal ions and organic solvents. In summary, CD2-MnP from Irpex lacteus CD2 could effectively degrade a broad range of synthetic dyes and exhibit a great potential for environmental biotechnology.     

用电子克隆新基因C17orf32和ZNF362对NCBI人类基因数据库模式参考序列5种错误类型的分析与纠正

采用生物信息学分析与实验确认相结合的技术路线,通过所识别的基因在非冗余数据库比对发现了网上公布的计算机注释人类基因组编码序列存在各种类型的多处错误。该策略既有助于发现更多的人类新基因,又有助于纠正美国国家生物技术信息中心(NCBI)基因组注释项目公布的参考序列(REFSEQs)中所存在的错误。比如他们采用基因预测方法通过自动计算分析从NCBIcontig NT_010808预测到两个模式参考序列LOC124919和LOC147007,本该都是C17orf32,但却都是C17orf32的不同错误形式,分别为第1和2类型错误;再如,他们采用基因预测方法通过自动计算分析从NCBIcontig NT_004511预测到3个模式参考序列LOC14907、LOC200084和LOC91126,实际上都是．ZNF362一种基因,却提交了ZNF362的3种不同错误形式,分别为第4、5和7类型错误。本研究利用计算机识别并结合实验验证能够纠正或避免现有的人类基因组编码序列错误。以前公开发表的文献没有明确指出NCBI人类基因模式参考序列存在错误,因此直当慎重看待计算机注释的可能存在各种类型错误的人类基因组编码序列。人类新基因的正确识别和注释仍是一项长期而繁重的任务。