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Background The rise of nature-based ecotourism in the past decade has introduced unprecedented challenges in managing the increasing interaction between humans and animals. The potential transmission of antibiotic resistant microbes between humans and non-human primate populations is a concern due to their genetic similarity. Malaysia is well known for hotspots of wildlife diversity where non-human primates like monkeys and orangutans have become popular tourist attractions. In this study, we assessed the prevalence of antimicrobial resistant Staphylococcus aureus, Enterococcus species, and other Enterobacteriaceae in the faeces of human (HS) and two non-human primates (NHP) in Malaysia, the Long-tailed macaque (Macaca fascicularis, MF) and Silvered leaf monkey (Trachypithecus cristatus, TC). In addition, the faecal bacterial composition was profiled to evaluate the potential association between antibiotic resistant profiles and composition of gut microbiota. Results We tested the isolated bacteria using a selection of antibiotics. The results showed that both the number of antibiotic resistant strains and resistance level were higher in humans than NHPs. Overall, the composition of gut microbiome and pattern of antibiotic resistance showed that there was higher similarity between MF and TC, the two NHPs, than with HS. In addition, samples with higher levels of antibiotic resistance showed lower bacterial richness. Homo sapiens had the lowest bacterial diversity and yet it had higher abundance of Bacteroides. In contrast, NHPs displayed higher bacterial richness and greater prevalence of Firmicutes such as Ruminococceae and Oscillospira. Conclusion Higher antibiotic susceptibility in NHPs is likely related to low direct exposure to antibiotics. The lack of resistance may also suggest limited antimicrobial resistance transmission between humans and NHP. Nonetheless, continued monitoring over a long period will help mitigate the risk of anthropozoonosis and zooanthroponosis.

Embryonic diapause is a maternally controlled phenomenon. The molecule controlling the onset of the phenomenon is unknown. We demonstrated that overexpression of microRNA let-7a or incubation with let-7g-enriched extracellular vesicles from endometrial epithelial cells prolonged the in vitro survival of mouse blastocysts, which developed into live pups after having been transferred to foster mothers. Similar to in vivo dormant blastocysts, let-7-induced dormant blastocysts exhibited low level of proliferation, apoptosis, and nutrient metabolism. Let-7 suppressed c-myc/mTORC1 and mTORC2 signaling to induce embryonic diapause. It also inhibited ODC1 expression reducing biosynthesis of polyamines, which are known to reactivate dormant embryos. Furthermore, the overexpression of let-7 blocked trophoblast differentiation and implantation potential of human embryo surrogates, and prolonged survival of human blastocysts in vitro, supporting the idea that embryonic diapause was an evolutionary conserved phenomenon. In conclusion, let-7 is the main factor inducing embryonic diapause.

The entity 'B cell lymphoma, unclassifiable, with features intermediate between diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL)' refers to B cell neoplasms that share overlapping characteristics of BL and DLBCL. A subset of these 'grey-zone lymphomas' possesses C-MYC and IGH translocations but, in addition, contains additional rearrangements of BCL2 and/or BCL6 genes. The aim of this study was to investigate if the proliferation fraction by Ki67 immunostaining can be used to identify such double-/triple-hit lymphomas. We studied 492 cases of mature aggressive B cell neoplasms by histology, immunohistochemistry and interphase fluorescence in-situ hybridization (FISH) using break-apart probes against C-MYC, BCL2, BCL6, IGH, MALT1, PAX5 and CCND1. Forty Burkitt lymphomas and 28 cases of MYC(+) double-/triple-hit lymphomas were identified. Of the latter, 77% and 54% displayed proliferation fractions exceeding 75% and 90%, respectively. With a cut-off of >75% by Ki67 immunostaining, the sensitivity and specificity for detection of MYC(+) double/triple translocations was 0.77 and 0.36. Raising the proliferation fraction criterion to >90% improved the specificity to 0.62 at the expense of a low sensitivity of 0.54. Immunostaining for Ki67 is not a useful approach to prescreen B cell lymphomas for MYC(+) double/triple translocations.

International audience; Salmonella genomic island I (variant SGII-J3) has been previously identified in multi-drug resistant (MDR) Salmonella enterica serovar Virchow isolated from humans in 1994. In this study, antimicrobial resistance, genotypes and genetic relationship were investigated in 96 S. Virchow isolates collected from humans in 2004-2006. XbaI-PFGE analysis separated 96 isolates into two main related-clusters, I and II, which consisted of four major pulsotypes differing in prevalence by year. The majority of isolates were MDR to chloramphenicol, sulfonamide, trimethoprim and tetracyclines associated with antimicrobial resistance genes dfrA1, floR2, sull and tet(G) of variant SGII-J3. Among nine variants, we determined two novel variants SGII-J4 and -J5, which have undergone different homologous recombinational events resulting in partial deletions of the MDR region. The first one contained an empty integron structure and the second presented a deletion extending from the IS6100 element to the adjacent SGII backbone. SGII-J3 is largely encountered in clonally related MDR S. Virchow isolates collected from humans which spread vertically. The genomic island SGII appears to be largely responsible for the diversity of MDR phenotypes among S. Virchow isolates in Taiwan.

The risk of acquiring variant Creutzfeldt–Jakob disease is closely related to polymorphism at codon 129 of the human prion gene, because almost all variant Creutzfeldt–Jakob disease patients are Met/Met homozygotes. Although animal transmission experiments corroborated this seeding discrimination, the origin of the differential seeding efficiency of the bovine prion seed for human codon 129 polymorphism remained elusive. Here, we used a short prion protein (PrP) peptide as a model system to test whether seeding discrimination can be found in this simple system. We used a previously developed ‘seed-titration method’ and time-resolved CD spectroscopy to compare sequence-dependent seeding efficiency regarding codon 129 polymorphism. Our results showed that the MetVal substitution on the human PrP (huPrP) peptide decreased seeding efficiency by 10 times when fibrils formed from bovine PrP (bPrP) peptide were used as the seed. To explore whether the different seeding barrier is due to the chemical and structural properties of Met and Val or whether another residue is involved in this peptide model, we constructed three bPrP mutants, V112M, L138I and N143S, in each of which one residue was replaced by the corresponding human residue. Our data showed that Leu138 in the bPrP seed might be the key residue causing the different seeding efficiencies related to 129M/V polymorphism and the interference effect of huPrP129V in the huPrP129M/V mixture. We propose a ‘surface competition hypothesis’ to explain the big seeding barrier caused by 129V in the PrP peptide seeding experiment. Structured digital abstract • huPrP aggregates with bPrP by circular dichroism (View Interaction 1, 2) • bPrP aggregates with bPrP by circular dichroism (View interaction) • bPrP aggregates with bPrP by electron microscopy (View interaction) • bPrP aggregates with bPrP by fluorescence technology (View interaction) • huPrP aggregates with huPrP by electron microscopy (View interaction) • huPrP aggregates with huPrP by fluorescence technology (View interaction) • huPrP aggregates with huPrP by circular dichroism (View interaction)

More than 20 unrelated proteins can form amyloid fibrils in vivo which are related to various diseases, such as Alzheimer's disease, prion disease, and systematic amyloidosis. Amyloid fibrils are an ordered protein aggregate with a lamellar cross-beta structure. Enhancing amyloid clearance is one of the targets of the therapy of these amyloid-related diseases. Although there is debate on whether the toxicity is due to amyloids or their precursors, research on the degradation of amyloids may help prevent or alleviate these diseases. In this study, we explored the amyloid-degrading ability of nattokinase, a fibrinolytic subtilisin-like serine protease, and determined the optimal conditions for amyloid hydrolysis. This ability is shared by proteinase K and subtilisin Carlsberg, but not by trypsin or plasmin.

Efficient incorporation of novel DNA sequences into a specific site in the genome of living human cells remains a challenge despite its potential utility to genetic medicine, biotechnology, and basic research. We find that a precisely placed double-strand break induced by engineered zinc finger nucleases (ZFNs) can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in high-efficiency site-specific gene addition. Using an extrachromosomal DNA donor carrying a 12-bp tag, a 900-bp ORF, or a 1.5-kb promoter-transcription unit flanked by locus-specific homology arms, we find targeted integration frequencies of 15%, 6%, and 5%, respectively, within 72 h of treatment, and with no selection for the desired event. Importantly, we find that the integration event occurs in a homology-directed manner and leads to the accurate reconstruction of the donor-specified genotype at the endogenous chromosomal locus, and hence presumably results from synthesis-dependent strand annealing repair of the break using the donor DNA as a template. This site-specific gene addition occurs with no measurable increase in the rate of random integration. Remarkably, we also find that ZFNs can drive the addition of an 8-kb sequence carrying three distinct promoter-transcription units into an endogenous locus at a frequency of 6%, also in the absence of any selection. These data reveal the surprising versatility of the specialized polymerase machinery involved in double-strand break repair, illuminate a powerful approach to mammalian cell engineering, and open the possibility of ZFN-driven gene addition therapy for human genetic disease.

The secreted Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) is a virulence factor that binds to both the complement component C3b and fibrinogen. Our laboratory previously reported that by binding to C3b, Efb inhibited complement activation and blocked opsonophagocytosis. We have now located the Efb binding domain in C3b to the C3d fragment and determined a disassociation constant (Kd) of 0.24 μm for the Efb-C3d binding using intrinsic fluorescence quenching assays. Using truncated, recombinant forms of Efb, we also demonstrate that the C3b binding region of Efb is located within the C terminus, in contrast to the fibrinogen binding domains that are located at the N-terminal end of the protein. Enzyme-linked immunosorbent assay-type binding assays demonstrated that recombinant Efb could bind to both C3b and fibrinogen simultaneously, forming a trimolecular complex and that the C-terminal region of Efb could inhibit complement activity in vitro. In addition, secondary structure analysis using circular dichroism spectroscopy revealed that the C-terminal, C3b binding region of Efb is composed primarily of α-helices, suggesting that this domain of Efb represents a novel type of C3b-binding protein.

The receptor for macrophage colony-stimulating factor (M-CSFR; c-fms) is expressed at increased levels by microglia in Alzheimer's disease (AD) and in mouse models for AD. Increased expression of M-CSFR on cultured microglia results in a strong proinflammatory response, but the relevance of this cell culture finding to intact brain is unknown. To determine the effects of increased microglial expression of M-CSFR in a complex organotypic environment, we developed a system for biolistic transfection of microglia in hippocampal slice cultures. The promoter for the Mac-1 integrin alpha subunit CD11b is active in cells of myeloid origin. In the brain, CD11b expression is restricted to microglia. Constructs consisting of the promoter for CD11b and a c-fms cDNA or an enhanced green fluorescent protein (EGFP) cDNA were introduced into monotypic cultures of microglia, neurons, and astrocytes. Strong CD11b promoter activity was observed in microglia, whereas little activity was observed in other cell types. Biolistic transfection of organotypic hippocampal cultures with the CD11b/c-fms construct resulted in expression of the c-fms mRNA and protein that was localized to microglia. Furthermore, biolistic overexpression of M-CSFR on microglia resulted in significantly increased production by the hippocampal cultures of the proinflammatory cytokines interleukin (IL)-1alpha macrophage inflammatory protein (MIP-1alpha), and trends toward increased production of IL-6 and M-CSF. These findings demonstrate that microglial overexpression of M-CSFR in an organotypic environment induces an inflammatory response, and suggest that increased microglial expression of M-CSFR could contribute to the inflammatory response observed in AD brain.

Staphylococcus aureus can cause a variety of acute and chronic diseases. The ability of S. aureus to cause persistent infections has been linked to its ability to evade or inactivate host immune responses. We have identified a secreted 19-kDa protein produced by S. aureus that binds to the complement protein C3. N-terminal sequencing of this protein identified it as the extracellular fibrinogen-binding protein (Efb). In this study, we demonstrate that Efb can bind to the alpha -chain of C3 and inhibit both the classical and alternative pathways of complement activation. In addition, we show that Efb can inhibit complement-mediated opsonophagocytosis in a dose-dependent manner and that Efb inhibits complement activity by blocking deposition of C3 or by preventing further complement activation beyond C3b. These data suggest that Efb is a virulence factor involved in facilitating persistent S. aureus infections by interfering with complement activity in vivo.

An efficient method has been developed for regeneration of complete plants via somatic embryogenesis in Corydalis yanhusuo (Fumariaceae), an important medicinal plant, using tuber-derived callus. Primary callus was induced by culturing mature tuber pieces on Murashige and Skoog's (MS) medium supplemented with 2.0 mg l(-1) N(6)-benzyladenine (BA) and 0.5 mg l(-1) alpha-naphthaleneacetic acid (NAA) in darkness. Somatic embryos were induced by subculturing the primary callus on MS medium supplemented with 0.5-4.0 mg l(-1) BA, kinetin, or zeatin, within 2 weeks of culture in light. Embryos with well-developed cotyledonary leaves were transferred in half-strength liquid MS medium supplemented with 1.0 mg l(-1) zeatin riboside for the development of roots. Converted somatic embryos were cultured on half-strength MS medium supplemented with 6% sucrose, and with 0.5-10.0 mg l(-1) abscisic acid (ABA), paclobutrazol, or ancymidol, 0.5-5.0 mg l(-1) GA(3) and 15-100 mg l(-1) polyethylene glycol (PEG) 4000 for further development of plantlets and in vitro tuber formation. The development of somatic embryos over the surface of tuber and/or cotyledonary leaf base region of the converted primary somatic embryo was observed. Before ex vitro establishment of somatic embryo-derived plants, plants with well-developed tubers were cultured on half-strength MS medium with 2% sucrose and 0.1 mg l(-1) GA(3) for 3 weeks.

Spinal cord injury is accompanied by an initial inflammatory reaction followed by secondary injury that is caused, in part, by apoptosis. Recruitment of leukocytes from the blood compartment to the site of inflammation in the injured spinal cord has been attributed to locally generated chemotactic agents (cytokines and chemokines). In addition to upregulation in the message levels of a number of chemokines, we have found up-regulation in the message levels of several chemokine receptors following spinal cord contusion injury. To reduce the inflammatory response after spinal cord injury, we have blocked the interaction of chemokine receptors with their ligands using the vMIPII chemokine antagonist. Using a rat model of spinal cord contusion injury, we show that continuous infusion of the antagonist for up to 7 days results in a decrease in infiltrating hematogenous cells at the site of injury. Histological evaluation ofthe tissue showed fewer activated macrophages at the site of injury. Concomitantly, reduced neuronal loss and gliosis were observed in the antagonist infused spinal cord. In addition, increased expression of Bcl-2 gene, an endogenous inhibitor of apoptosis, was seen in the antagonist infused spinal cord at 7 days post injury. Morphologically, staining with the bisbenzamide dye Hoechst 33342 showed significantly more apoptotic bodies in the vehicle compared to antagonist infused spinal cord. Our data suggest that chemokine antagonist infusion post-injury results in limiting the inflammatory response following spinal cord contusion injury, thereby attenuating neuronal loss, possibly due to decreased apoptosis. These findings support the contention that disrupting chemokine interactions with their receptors may be an effective approach in reducing the secondary damage after spinal cord injury.

The present study evaluates the physiological effects of magnetic stimulation on astrocyte cultures. Cell cultures were exposed to pulsed magnetic stimulation (10 Hz, 10 sec) at the following levels: 0.10 tesla (T; Group A); 0.21 T (Group B); 0.42 T (Group C); and 0.63 T (Group D). Glial fibrillary acidic protein (GFAP) levels from immunoblots, total protein concentrations, and cellular morphology were analyzed at 0, 1, 3, 5, 7, 13, and 20 days poststimulation. Significantly higher GFAP levels were observed in Group D at day 3 (P = 0.0114). The change was transient as the GFAP levels quickly returned to control levels by day 5. No other significant changes in GFAP levels were observed. In comparison to control protein levels at day 0, concentrations from Groups B, C, and D were significantly lower (P < 0.006), whereas at day 3, Groups C and D were significantly higher (P < 0.02). Differences in astrocyte morphology due to magnetic stimulation were not observed. This study demonstrated that high intensity magnetic stimulation for only 10 sec induced a transient biological response.

Traumatic injury to the central nervous system (CNS) results in the breakdown of the blood-brain barrier and recruitment of hematogenous cells at the site of injury. The role of chemokines in this process has been well recognized and they have been regarded as promising targets for development of anti-inflammatory therapies. The expression of monocyte chemoattractant protein (MCP-1), in particular, has been closely linked to macrophage infiltration following trauma in rat brain. In this study we determined whether inhibition of MCP-1 following stab wound injury would reduce macrophage infiltration. Stab wound injured Sprague-Dawley rats were infused with MCP-1 sense or antisense oligonucleotides using an Alzet miniosmotic pump (1 microl/h for 3 days). Three days following injury, widespread gliosis was observed in both groups of rats as judged by glial fibrillary acidic protein (GFAP) immunoreactivity. Immunohistochemistry showed significantly less staining for MCP-1 in antisense treated animals. In addition, the number of macrophages were reduced by 30% in the antisense compared to the sense treated animals (P0.05). These results demonstrate that modulation of MCP-1 expression in stab wound injury directly affects monocytic infiltration and provide a basis for MCP-1 inhibition as a therapeutic strategy for controlling inflammatory events of traumatic brain injury.

Lon protease is a multifunction protein and operates in protein quality control and stress response pathways in mitochondria. Human Lon is upregulated under oxidative and hypoxic stresses that represent the stress phenotypes of cancer. However, little literature undertakes comprehensive and detailed investigations on the tumorigenic role of Lon. Overexpression of Lon promotes cell proliferation, apoptotic resistance to stresses, and transformation. Furthermore, Lon overexpression induces the production of mitochondrial reactive oxygen species (ROS) that result from Lon-mediated upregulation of NDUFS8, a mitochondrial Fe-S protein in complex I of electron transport chain. Increased level of mitochondrial ROS promotes cell proliferation, cell survival, cell migration, and epithelial–mesenchymal transition through mitogen-activated protein kinase (MAPK) and Ras-ERK activation. Overall, the present report for the first time demonstrates the role of Lon overexpression in tumorigenesis. Lon overexpression gives an apoptotic resistance to stresses and induces mitochondrial ROS production through Complex I as signaling molecules to activate Ras and MAPK signaling, giving the survival advantages and adaptation to cancer cells. Finally, in silico and immunohistochemistry analysis showed that Lon is overexpressed specifically in various types of cancer tissue including oral cancer.

A traumatic injury to the adult mammalian central nervous system (CNS) results in reactive astrogliosis and the migration of hematogenous cells into the damaged neural tissue. Chemokines, a novel class of chemoattractant cytokines, are now being recognized as mediators of the inflammatory changes that occur following injury. The expression of MCP-1 (macrophage chemotactic peptide-1), a member of the beta family of chemokines, has recently been demonstrated in trauma in the rat brain (Berman et al.: J Immunol 156:3017-3023, 1996). Using a stab wound model for mechanical injury, we studied the expression of two other beta chemokines: RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) and MIP-1 beta (macrophage inflammatory protein-1 beta) in the rat brain. The stab wound injury was characterized by widespread gliosis and infiltration of hematogenous cells. Immunohistochemical staining revealed the presence of RANTES and MIP-1 beta in the injured brain. RANTES and MIP-1 beta were both diffusely expressed in the necrotic tissue and were detected as early as 1 day post-injury (dpi). Double-labeling studies showed that MIP-1 beta, but not RANTES, was expressed by reactive astrocytes near the lesion site. In addition, MIP-1 beta staining was also detected on macrophages at the site of injury. The initial expression of the chemokines closely correlated with the appearance of inflammatory cells in the injured CNS, suggesting that RANTES and MIP-1 beta may play a role in the inflammatory events of traumatic brain injury. This study also demonstrates for the first time MIP-1 beta expression in reactive astrocytes following trauma to the rat CNS.

Animals representing different trophic levels were collected from two estuarine environments; the subarctic Strait of Georgia ( c. 50°N, 124°W) and the tropical coast of Kaohsiung ( c. 22°N, 121°E). The animals were analysed for their heavy isotope composition of carbon and nitrogen in order to determine the trophic structure of the two environments. The tropical environment showed a greater enrichment in the 13 C isotope which could be attributed to previously reported latitudinal differences in the source of carbon for photosynthesis. In both environments there was a further enrichment of 13 C at higher trophic levels which was believed to be the result of significant grazing on benthic organisms in both systems. The much higher variation in the data encountered in the tropical environment was attributed to the sharp contrast between offshore oligotrophic waters from the South China Sea and the Kuroshio current that mix with estuarine waters, within a short distance of the coast.

Previous studies have shown that the mechanical wounding of 3-week-old cultured rat astrocytes results in cell proliferation and hypertrophy resembling astrocyte responses to a brain injury in vivo. We now report the effects of basic fibroblast growth factor (bFGF) and an anti-bFGF antibody on astrocyte morphology, proliferation, and migration following in vitro wounding of confluent secondary cultures. Addition of bFGF (20 ng/ml) to wounded cultures induced morphological changes characteristic of differentiation in wounded and nonwounded areas of the culture. Combined treatment with bFGF and an anti-bFGF antibody (100 micrograms/ml) prevented this effect. Astrocyte proliferation along the edges of a scratch wound was at maximum 24 hr after wounding in cells growing in Eagle's minimum essential medium (EMEM) containing 10% serum. Low serum concentration and treatment with dibutyryl cyclic adenosine monophosphate (dbc-AMP) reduced injury-associated astrocyte proliferation. Addition of bFGF to cultures in EMEM with serum increased astrocyte proliferation at 18 and 24 hr after wounding. This effect was reduced considerably by treatment of cultures with bFGF in combination with an anti-bFGF antibody. The combined treatment and the antibody alone reduced cell division to a level lower than in control cultures. Twenty-four hr following wounding, astrocytes along the edges of the wound exhibited extension of thick, flat processes into the wound area. At 3 and 5 days after wounding, a bodily migration of astrocytes into the wounded area was observed. Addition of bFGF significantly increased astrocyte migration 1 day after wounding, with maximum effect on day 3 and no subsequent increase on day 5. A combination of bFGF and anti-bFGF antibody as well as the antibody alone reduced astrocyte migration to a level lower than in controls. Immunohistochemical localization and isoform pattern of bFGF in astrocytes did not change with dbc-AMP treatment or wounding. We conclude that mechanically wounded confluent astrocytes respond to bFGF added to the culture medium by enhancing cell division, differentiation, and migration. In addition, the results of the antibody treatment also suggest a role for endogenous bFGF in astrocyte proliferation and migration elicited by wounding in vitro. These results support the notion that in vivo, both bFGF released by injury and endogenous bFGF synthesized by astrocytes, contribute to the cellular responses that lead to astrogliosis.

The survival of colony-forming cells in pig epidermis after irradiation was measured using different electron field sizes. The sensitivity of the colony-forming cells was characterized by D(o) = 2.5-3.0 Gy. There was an effect of field size, described approximately by: Dose (to give five colonies/cm2) (Gy) = (31 +/- 2) x Area-(0.048 +/- 0.015). The effect of field size was less than found previously using tolerance to skin reactions (area exponent = 0.16). This work indicates for the first time that the effects of different large field sizes in skin can be detected at the level of colony-forming cell survival.

In conscious rats, we have previously shown that immunoneutralization of circulating insulin with a rabbit anti-insulin serum abolished the pancreatic exocrine secretion stimulated by a meal or a combination of exogenous secretin and cholecystokinin octapeptide (CCK-8). To investigate the mechanism of endogenous insulin action on the exocrine pancreas, isolated rat pancreata were perfused with intra-arterial infusion of Krebs-Henseleit solution (37 degrees C) at 1.2 ml/min, whereas both pancreatic juice and portal venous effluent were collected separately in 15-min samples. Simultaneous intra-arterial infusion of secretin and CCK-8 in doses of 0.75 and 4.2 pmol/h; respectively, significantly increased volume, bicarbonate, and protein output in 7 rat pancreata (P < 0.01). When a rabbit anti-insulin serum was administered intra-arterially (0.1-ml bolus followed by 0.1 ml for 10 min), pancreatic secretion of volume, bicarbonate, and protein output was profoundly suppressed (n = 7, P < 0.01), whereas a normal rabbit serum failed to influence pancreatic secretion. The decrease in pancreatic secretion by the antiserum coincided with a significant increase in somatostatin in portal venous effluent from 1.4 +/- 0.2 to 4.1 +/- 0.8 pM (n = 6, P < 0.05). The combined administration of a rabbit antisomatostatin serum (0.4 ml) and the anti-insulin serum partially reversed the effect of the anti-insulin serum alone. Thus the pancreatic secretion was significantly greater than that achieved by the anti-insulin serum alone (P < 0.05). These observations strongly suggest that the action of insulin on exocrine pancreas is mediated by its local or paracrine action.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present investigation, we have studied the effect of atropine on the pancreatic secretion stimulated by intraduodenal administration of either sodium oleate or exogenous cholecystokinin (CCK). In four dogs prepared with gastric and Thomas duodenal cannulas, pancreatic juice was collected for measurement of volume, bicarbonate, and protein output, and peripheral venous blood samples were obtained for radioimmunoassay of both secretin and CCK. Volume, bicarbonate, and protein output of the pancreatic juice increased significantly in response to sodium oleate (1-4 mmol/h) in a dose-dependent manner. The increase in pancreatic secretion paralleled the increments in both plasma CCK and secretin. Atropine given intravenously suppressed completely both pancreatic secretion and release of CCK stimulated by sodium oleate, whereas the release of secretin was not affected. Pancreatic secretion was significantly increased in a dose-dependent manner by exogenous CCK octapeptide (CCK-8) at 16, 32, and 64 micrograms (14, 28, and 56 pmol).kg-1.h-1. Atropine inhibited protein output only partially, but it did not influence bicarbonate output. In five additional dogs, the effect of atropine on L-tryptophan-stimulated pancreatic secretion was studied. Interestingly, atropine failed to influence the CCK release and pancreatic secretion of volume and bicarbonate, except for protein secretion, which was significantly inhibited. It was shown previously that atropine inhibited significantly the pancreatic secretion of bicarbonate stimulated by secretin in physiological doses. Thus we conclude that the inhibition by atropine of the pancreatic exocrine secretion stimulated by sodium oleate is mediated by both suppression of CCK release and inhibition of action of secretin on the exocrine pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosteres arinsanus (Sonan) (=B. oophilus Fullaway) showed a strong preference forDacus dorsalis Hendel overCeratitis capitata (Wiedemann) in choice tests using papaya fruits infested with eggs of both hosts in laboratory studies. In laboratory and field studiesD. dorsalis was the most suitable host. A total of 13,871 parasitoids was produced fromD. dorsalis infested fruits compared with 2,772 fromC. capitata infested fruits.

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann), was reared at three larval densities in ripe mock orange, Murraya paniculata (L.), obtained from five locations in the field. Larvae and preadult survival decreased with increase in larval density. C. capitata field infestation rates varied between locations from 0.5 to 148.4 larvae/kg of fruit. More Dacus dorsalis Hendel were reared from field collected fruits than C. capitata . The weight of mock orange fruit varied between locations from 0.31 to 4.0 g/fruit. Survival and reproduction parameters are discussed in relation to availability of fruit in the field. We conclude that the availability of ripe mock orange fruit in the field ultimately influences the number of fruit flies that survive to the next generation.

Revertant cell lines were established from cisplatin (CP) resistant HeLa cells. Expression of CP damaged plasmid DNA carrying bacterial chloramphenicol acetyltransferase (CAT) gene after transfection into cells was measured. Revertant cells showed reduced host cell reactivation of damaged plasmid, as compared to resistant cells. Addition of aphidicolin, an inhibitor for DNA polymerase alpha, to resistant cells effectively blocked enhanced plasmid reactivation and acquired resistance. The results are consistent with the notion that cellular ability in repair CP-damaged DNA is a mechanism for CP resistance.

Human Lon protease is a mitochondrial matrix protein with several functions, including protein degradation, mitochondrial DNA (mtDNA) binding, and chaperone activity. Lon is currently emerging as an important regulator of mitochondria-contributed tumorigenesis due to its overexpression in cancer cells. To understand the mechanism of increased Lon in tumor cells, we studied the interactome to identify the chaperone Lon-associated proteins by proteomics approaches using the cells overexpressing Lon. In the present study, we designed a method connecting co-immunoprecipitation (Co-IP) to in-solution digestion for the shotgun mass spectrometry. We identified 76 proteins that were putative Lon-associated proteins that participated in mitochondrial chaperone system, cellular metabolism and energy, cell death and survival, and mtDNA stability. The association between Lon and NDUFS8 or Hsp60–mtHsp70 complex was confirmed by Co-IP and immunofluorescence co-localization assay. We then found that the protein stability/level of Hsp60–mtHsp70 complex depends on the level of Lon under oxidative stress. Most importantly, the ability of increased Lon-inhibited apoptosis is dependent on Hsp60 that binds p53 to inhibit apoptosis. These results suggest that the mechanism underlying cell survival regulated by Lon is mediated by the maintenance of the protein stability of Hsp60–mtHsp70 complex. This new knowledge of chaperone Lon interactome will allow us to better understand the cellular mechanism of Lon in mitochondrial function and of its overexpression in enhancing cell survival and tumorigenesis.

An efficient method for regeneration of entire plants via somatic embryogenesis in Corydalis yanhusuo using tuber-derived callus has been developed. Primary callus was obtained by culturing tuber explants on Murashige and Skoog’s (MS) (1962) medium supplemented with 2.0 mg l−1 N6-benzyladenine (BA) and 0.5 mg l−1 α-naphthaleneacetic acid (NAA) in darkness for one month. Somatic embryos were induced by subculturing the primary callus on MS medium supplemented with 0.5–4.0 mg l−1 BA, kinetin, or zeatin, within 2 weeks of culture in light. Rooting in the embryos with well developed cotyledonary leaves was achieved by transferring them to half-strength liquid MS medium supplemented with 1.0 mg l−1 zeatin riboside for three weeks. Converted somatic embryos were cultured on half-strength MS medium supplemented with 6% (w/v) sucrose, and with 0.5–10.0 mg l−1 abscisic acid (ABA), paclobutrazol, or ancymidol, 0.5–5.0 mg l−1 gibberellic acid (GA3) and 15–100 mg l−1 polyethylene glycol (PEG) 4000 for further development of plantlets and tuber formation in vitro. Recurrent somatic embryogenesis has been observed in the region at the junction of cotyledon and root when the converted somatic embryos were cultured on MS basal medium supplemented with 0–10 mg l−1 ABA. The use of liquid MS basal medium with 0.1 mg l−1 GA3 enhanced embryo conversion within fifteen days of culture. Phenotypically normal plants were recovered from converted somatic embryos and the plants showed well-developed tuber formation and in vitro flowering after 4 months of culture. Also, the effects of 0.5–5 mg l−1 ABA, paclobutrazol and 0.5–2 mg l−1 ancymidol, 0.5–5 mg l−1 GA3 and 15–100 mg l−1 PEG 4000 supplemented in half-strength MS medium on the production of the two major protoberberine-type alkaloids (D,L-tetrahydropalmatine and D-corydaline) by the tubers of somatic embryo-derived plants of C. yanhusuo were examined. The high performance liquid chromatography (HPLC) analysis revealed that the contents of D,L- tetrahydropalmatine and D-corydaline in the tubers of somatic embryo-derived plants were greater than the marketed crude drug and varied with growth regulator / PEG-4000 treatment and the age of the plant.

Microglia are important in the inflammatory response in Alzheimer's disease (AD). We showed previously that macrophage colony-stimulating factor receptor (M-CSFR), encoded by the c-fms protooncogene, is overexpressed on microglia surrounding amyloid beta (Abeta) deposits in the APP(V717F) mouse model for AD. The M-CSFR is also increased on microglia after experimental brain injury and in AD. To determine the relevance of these findings, we transiently expressed M-CSFR on murine BV-2 and human SV-A3 microglial cell lines using an SV40-promoted c-fms construct. M-CSFR overexpression resulted in microglial proliferation and increased expression of inducible nitric-oxide synthase, the proinflammatory cytokines interleukin-1alpha, macrophage inflammatory protein 1-alpha, and interleukin-6 and of macrophage colony-stimulating factor (M-CSF) itself. Antibody neutralization of M-CSF showed that the M-CSFR-induced proinflammatory response was dependent on M-CSF in the culture media. By using a co-culture of c-fms-transfected murine microglia and rat organotypic hippocampal slices and a species-specific real time reverse transcriptase-polymerase chain reaction assay and enzyme-linked immunosorbent assay, we showed that M-CSFR overexpression on exogenous microglia induced expression of interleukin-1alpha by the organotypic culture. These results show that increased M-CSFR expression induces microglial proliferation, cytokine expression, and a paracrine inflammatory response, suggesting that in APP(V717F) mice increased M-CSFR on microglia could be an important factor in Abeta-induced inflammatory response.

Tumor necrosis factor-alpha is a pluripotent cytokine that is reportedly mitogenic to astrocytes. We examined expression of the astrocyte intermediate filament component glial fibrillary acidic protein in astrocyte cultures and the U373 glioblastoma cell line after treatment with tumor necrosis factor-alpha. Treatment with tumor necrosis factor-alpha for 72 h resulted in a decrease in content of glial fibrillary acidic protein and its encoding mRNA. At the same time, tumor necrosis factor-alpha treatment increased the expression of the cytokine interleukin-6 by astrocytes. The decrease in glial fibrillary acidic protein expression was greater when cells were subconfluent than when they were confluent. Thymidine uptake studies demonstrated that U373 cells proliferated in response to tumor necrosis factor-alpha, but primary neonatal astrocytes did not. However, in both U373 cells and primary astrocytes tumor necrosis factor-alpha induced an increase in total cellular protein content. Treatment of astrocytes and U373 cells for 72 h with the mitogenic cytokine basic fibroblast growth factor also induced a decrease in glial fibrillary acidic protein content and an increase in total protein level, demonstrating that this effect is not specific for tumor necrosis factor-alpha. The decrease in content of glial fibrillary acidic protein detected after tumor necrosis factor-alpha treatment is most likely due to dilution by other proteins that are synthesized rapidly in response to cytokine stimulation.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine synthesized by a variety of cell types. In the nervous system LIF affects neuronal differentiation, and may be important during cerebral infection and inflammation. To clarify the cellular source of LIF in the brain, we examined the expression of LIF mRNA by primary cortical astrocyte cultures and an immortalized microglial cell line. The microglial cell line did not express LIF mRNA in response to pro-inflammatory agents such as lipopolysaccharide (LPS) that induced expression of other cytokine mRNAs. In contrast, primary astrocyte cultures grown in serum-containing medium expressed LIF mRNA constitutively, and this expression was regulated by pro-inflammatory and anti-inflammatory stimuli. Agents which activate the cAMP and protein kinase C second messenger systems also increased LIF mRNA in astrocyte cultures. These results suggest that astrocytes, but not microglia, may be an important source of LIF during cerebral inflammation and infection.

Publisher Summary This chapter discusses the reverse transcriptase polymerase chain reaction (RT-PCR) method to study cytokine expression in astrocytes in response to exogenous agents and mechanical injury. By quantitative RT-PCR, it has been recently shown that TNFα/ interleukin (IL)-1β induced IL-6 in mouse results in astrocyte cultures. IL-6 belongs to a family of neuroactive cytokines including leukemia inhibitor factor (LIF) and ciliary neurotrophic factor (CNTF). RT-PCR has been used to demonstrate that the cholinergic differentiation factor/leukemia inhibitory factor (CDFLIF) and CNTF induce mRNAs for choline acetyltransferase, somatostatin, substance P, vasoactive intestinal peptide, cholecystokinin, and enkephalin in cultured sympathetic neurons. These data suggest that CDFLIF and CNTF may share receptor subunits and signal transduction pathways. Lipopolysaccharide, PMA, tumor necrosis factor (TNFα), and IL-lβ have also been found to induce LIF in cultured mouse astrocytes but not in the immortalized microglial cell line (BV-2) provided by Bocchini.

Publisher Summary Ischemia is a combination of substrate deprivation, severe hypoxia, and a failure to remove toxic metabolic products. This chapter discusses the expression of some specific genes in astrocytes during and after ischemia. The expression of the late response genes that produce long-term changes in cells is regulated by the immediate early genes (IEGs) and is greatly influenced by cerebral ischemia. Involvement of the immediate early genes (IEGs) in brain injury and ischemia is under intensive investigation. There are several families of the IEGs. They include the fos, jun, and zinc finger genes that encode transcription factors. c-fos induction by ischemia has been found to be rapid and transient. Ischemia induces c-fos in nerve cells is believed to be related to calcium entry into the cells. The measurement of c-fos gene expression in ischemic astrocytes provides significant evidence demonstrating the induction of response to the injury. The detection of hsp-70 gene would reflect the late responses in cellular protection. A change in the expression of cytoskeleton protein genes in the ischemic astrocytes may reflect the activation of astrocytes to ischemia. Understanding the chronology of these gene expressions would provide some early markers for detecting the injury and reactivation of astrocytes under these pathologic conditions.

The reverse transcription and polymerase chain reaction technique (RT-PCR) was assessed for the quantification of changes in mRNA levels from primary astrocyte cultures. The effects of dibutyryl cyclic AMP (dBcAMP) on glial fibrillary acidic protein (GFAP) mRNA and the effects of tumor necrosis factor-alpha (TNF-α), interlekin-1 beta (IL-1β), and lipopolysaccharide (LPS) on interleukin-6 (IL-6) mRNA were examined. Two quantitative PCR methods were used: one involved carrying out the reaction in the exponential phase and the other involved the coamplification of a competitive target sequence. Increased GFAP mRNA in response to chronic dBcAMP treatment and increased IL-6 mRNA in response to TNF-α/IL-1β were readily detected. Both RT-PCR techniques were found to be suitable for the detection of large as well as smaller (twofold) changes in mRNA levels. The advantages and limitations of RT-PCR for mRNA quantification are discussed. © 1993 Wiley-Liss, Inc.

Glial fibrillary acidic protein (GFAP), the major component of the intermediate filament in differentiated astrocytes (Eng et al., 1971; Eng, 1985), is extensively synthesized within and adjacent to the site of injury (Eng, 1988a; Condorelli et al., 1990; Hozumi et al., 1990; Vijayan et al., 1990). Other than GFAP accumulation, astrogliosis is also characterized by astrocyte proliferation (hyperplasia) and extensive hypertrophy of the cell body, nucleus as well as cytoplasmic processes (Eng, 1988a). Astrogliosis may participate in the healing phase following CNS injury by actively monitoring and controlling the molecular and ionic contents of the extracellular space of the CNS. They can wall off areas of the CNS that are exposed to non-CNS tissue environments following trauma. On the other hand, such responses may interfere with the function of residual neuronal circuits, by preventing remyelination, or by inhibiting axonal regeneration (Eng et al., 1987; Stensaas et al., 1987; Reier and Houle, 1988). Although astrogliosis has received considerable attention in term of its proposed inhibitory effect on CNS repair, there is still very little specific information available concerning the properties of reactive astrocytes, what triggers glial reactivity, and many of the cellular dynamics associated with scar formation. Control of astrocyte proliferation, differentiation, and astrogliosis may be linked to GFAP synthesis. Our aim was to transfect astrocytes with exogenous synthetic oligo- or polynucleotides, which would allow the manipulation of a transient suppression of GFAP synthesis which might delay the gliotic reaction and the scar formation, thus allowing neurons and oligodendrocytes to re-establish a functional environment.

Brucella abortus may be useful as a component of vaccines. This is because it possesses several unique properties as a carrier that enable it to stimulate human B cells even in the relative absence of T cells. Human immunodeficiency virus type 1 proteins conjugated to B. abortus could induce neutralizing antibodies against human immunodeficiency virus type 1. Recently we showed that the characteristics of lipopolysaccharide (LPS) derived from B. abortus are similar to those of the whole bacterium in that the LPS acts as a T-independent type 1 carrier in mice. In this study we wanted to determine whether LPS derived from B. abortus is associated with the adverse effects seen with other bacterial endotoxins. LPS purified from B. abortus by butanol extraction was shown to have less than 2% (wt/wt) contamination by protein and less than 1% (wt/wt) contamination by nucleic acids and to contain 1% (wt/wt) ketodeoxyoctanic acid. Compared with LPS derived from Escherichia coli, B. abortus LPS was 10,000-fold less potent in eliciting fever in rabbits, 268-fold less potent in killing D-galactosamine-sensitized mice, and 1,400-fold and 400-fold less potent in inducing interleukin-1 beta and tumor necrosis factor alpha production, respectively. These results suggest that B. abortus LPS is much less likely than the LPS from E. coli to evoke endotoxic shock; therefore, it may be feasible to incorporate B. abortus as a component of vaccines.

Publisher Summary The chapter discusses the role of the astrocyte in the central nervous system (CNS) injury and disease. Astrocytes comprise as much as 25% of the cells and 35% of the total mass of the CNS. Astrocytes form barriers around blood vessels and connections between nerve cells. Numerous functions have been assigned to the astrocyte depending on its stage of maturation, location in the CNS, and response to CNS insult. Some signals that regulate gene expression in development and response to astrocyte injury are: growth factors, prion protein from Scrapies, neural and immunological adhesion molecules, such as NCAM, LFA-1, gangliosides, low density lipoproteins, cytokines from T-cells, macrophages and other glia, neurotransmitters and neuropeptides, such as catecholamines, monoamines, glutamate, ATP, substance P, and antigen-antibody complexes. Astrocytic responses to these signals include: (1) proliferation, movement and differentiation; (2) changes in shape, cell volume, cytoskeletal organization, endocytic activity, lysosomal fragility, and enzyme content; (3) buffering capacity for K + , glutamate and GABA; (4) expression of nerve growth factor, tumor necrosis factor, interferon α and β , interleukin 1 and 6, colony stimulating factor-1, fibroblast growth factor, neurotropic factors, neurite promoting agents, MHC class I and I1 histocompatibility antigens, amyloid protein, GD3 ganglioside, ICAM- 1, Na + channel protein, GFAP, crystallin, vimentin and heat shock proteins.

Publisher Summary This chapter discusses the role of glutamate as a metabolic substrate. Glutamate is the most plentiful amino acid and the major excitatory neurotransmitter in adult central nervous system (CNS). Glutamate participates in the synthesis of proteins, peptides and fatty acids, and in the control of osmotic or anionic balance. It is a constituent of at least two important co-factors, glutathione and folic acid; it contributes along with glutamine to the regulation of ammonia levels, and it serves as precursor for GABA and various tricarboxylic acid (TCA) cycle intermediates. Glutamate is released from neurons in large amounts. Uptake studies demonstrates that both neurons and astrocytes take up glutamate. The uptake of glutamate into astrocytes represents a net transfer of carbon skeleton from the neurons to astrocytes. There are three probable roles for this uptake process: (1) to remove the glutamate from extracellular space and synaptic clefts as a means of termination of the transmitter activity; (2) to form glutamine during the detoxification of ammonia and (3) to serve as a metabolic substrate for astrocytes. Metabolic studies have shown that, a part of the glutamate taken up by astrocytes is metabolized to CO2 and another part to glutamine, the latter of which then can be returned to neurons as a precursor for glutamate and GABA. Glutamate exerts a regulatory effect on glycogen metabolism in astrocytes and also affects glucose utilization in astrocytes. Glutamate also plays an important role in the pathogenesis of various neurologic diseases and insult.

Glial fibrillary acidic protein (GFAP) accumulation is a prominent feature of astrocytic gliosis. The inhibition or delay in GFAP synthesis might delay scar formation resulting from an insult such as spinal cord injury or central nervous system (CNS) demyelination. The delay in the formation of a physical barrier might allow the neurons and oligodendrocytes to reestablish a functional environment. We delivered antisense GFAP RNA complexed with LipofectinTM (LF), a cationic liposome, into cerebral astrocytes in culture and tested the feasibility of inhibiting GFAP synthesis. Our results demonstrate that LF facilitated antisense RNA uptake into astrocytes. Astrocytes took up 3H-antisense GFAP RNA alone and reached an equilibrium of 7–8.8 ηg per mg protein after 2.5 hr. When complexed with LF, astrocytes could increase the uptake to 14 ηg per mg protein and the time for reaching this quantity was shortened to 10 min. This uptake level was further enhanced if experiments were carried out in HEPES buffered saline (HBS). All uptake studies were dose- and time-dependent. Dibutyryl cyclic AMP (dBcAMP) is known to induce an increase of GFAP content in cultured astrocytes. We studied the effect of LF/antisense GFAP RNA on the GFAP content in dBcAMP (0.25 mM)-treated astrocytes. Cultures of astrocytes treated with dBcAMP contained almost twice as much GFAP as untreated cultures after 2 days. Similar cultures treated with LF/antisense RNA in HBS did not show an increase but a 30–40% decrease in GFAP content 2 days after treatment. A similar decrease in GFAP content was obtained in cultures grown in a chemically defined medium, another condition known to induce an increase in GFAP content in cultures. This study has demonstrated that antisense GFAP RNA can inhibit GFAP synthesis in astrocytes and may be useful for regulating astrogliosis immediately following CNS injury.

Glial fibrillary acidic protein (GFAP) is the major constituent of glial filaments and is restricted within the CNS to astrocytes. As with other classes of intermediate filament proteins, the regulation of GFAP expression is poorly understood. Utilizing highly purified cultures of astrocytes and a chemically defined (CD) medium, we have demonstrated that the expression of GFAP is subject to regulation by hormones and growth factors. The concentration of GFAP/mg protein was induced 2-4-fold in the presence of hydrocortisone, putrescine, prostaglandin F-2 alpha (PGF2 alpha), and pituitary fibroblast growth factor (FGF). Augmentation of the levels of GFAP continued for up to 3 weeks after conversion to CD medium and paralleled the morphological maturation of astrocytes. The accumulation of GFAP resulted from an increase in its specific rate of synthesis. Conversion of astrocytes from serum-supplemented (SS) to CD medium did not alter its rate of degradation. GFAP appeared quite stable under both sets of conditions, exhibiting a half-life of approximately 7.5 days. The data demonstrate that GFAP expression in astrocytes is subject to hormonal regulation, which may have implications for gliosis.

Infestations (1- to 3-day-old eggs and larvae) of the oriental fruit fly, Dacus dorsalis Hendel, or the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), were eliminated from packaged papayas, Carica papaya L., by low-temperature (12.8°C) fumigation with phosphine generated from ½ of a FUMI-CEL®. There were no survivors from 103,880 oriental fruit flies after an exposure of 4940 ppm-h. Where fumigations were done at 12.8° or 18.5°–26.1°C and 80% RH (ambient), mortality varied both with the natural logarithm of the concentration and treatment time because of the variable rates of phosphine generated. Time influenced efficacy more than concentration. The residues of phosphine in the fumigated papayas were 0.08 ppb after low-temperature fumigation and an exposure of 7600 ppm-h. These residues dissipated to trace levels after 2 days of storage at 12.8°C. Residues were 1.0–1.4 ppb after fumigation at ambient and an exposure of 7700–9100 ppm-h. These residues decreased to 0.18–0.3 ppb after 1 day of storage. Residues of 1.3 ppb on the carton and the polyethylene foam sheet dissipated to levels ranging from trace to 0.4 ppb. Papayas and 10 Hawaiian varieties of avocado, Persea Americana Mill., were not injured by low-temperature fumigation, but fumigated avocados ripened faster than unfumigated avocados. ‘California Wonder’ bell peppers, Capsicum annuum L. var. annuum, ‘apple’ and ‘Williams Hybrid’ bananas, Musa paradisiaca ssp. sapientum (L.) O. Ktze., ‘N-52’ and ‘Anahu’ tomatoes, Lycopersicon esculentum Mill., and 5 varieties of avocado were uninjured after fumigation for 12–63 h and exposures of 5500–20,000 ppm-h at ambient. ‘Black Beauty’ eggplant, Solanum melongena L., tolerated 5500 ppm-h.

Neonatal rat brain astrocyte secondary cultures were established on nitrocellulose membrane filters (13-mm diameter Millipore disk) and on plastic coverslips in serum-supplemented medium. On these substrata, cultured astrocytes changed their shape from flat and polygonal to stellate in the absence of hormones or growth factor supplements. Cultures became confluent after 4 days, and astrocytes on nitrocellulose filters continued to differentiate morphologically and biochemically, as evidenced by extensive cytoplasmic process formation and glial fibrillary acid protein (GFAP) accumulation. Cultures were immunostained for GFAP and vimentin. mRNAs to GFAP, vimentin, alpha and beta tubulin, and actin also were detected by in situ hybridization with biotinylated cDNA probes. The astrocyte culture method on nitrocellulose provides a simple, versatile means of comparing undifferentiated, morphologically mature, reactive, and neoplastic astrocytes in vitro.

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann), showed a distinct preference for a dry habitat ( 180 cm/yr rainfall) on the island of Oahu, but they were persistent in dry areas, particularly Makaha Valley. The mean infestation rate was 0.21 and 33.5 flies per kilogram of coffee in wet and dry areas. Dissection of coffee collected in the field showed that larval recoveries per 100 fruits were equal in wet and dry areas, but larval mortality was statistically higher in wet areas; the larvae apparently die from decay of the fruit, especially if they crack open. We conclude that C. capitata and Dacus dorsalis Hendel distribution and abundance are directly affected by the elimination of preferred host fruits and indirectly by climate causing differential effects on their survival in coffee and other fruits in wet versus dry areas.

The geographical, seasonal, and annual distributions of Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann), populations were monitored for 7 yr (1977–83) by means of traps. containing a male lure to catch flies, and host fruits periodically collected to detect infested fruits in Honolulu and leeward and windward residential areas of the Koolau Mountains of Oahu. Medflies often coexisted in fruit samples with the oriental fruit fly, Dacus dorsalis Hendel, with more oriental fruit flies than medflies reared. Medflies were virtually absent from wet leeward and windward areas of Kaneohe and Kahaluu. The results indicated that medfly host plants were highly variable in distribution, abundance, and fruiting cycle among seasons and years because of climatic factors, especially rainfall. Distribution, dispersal, and abundance of the flies followed the same trends.

Cells cultured from the caudalmost area of regenerating teleost spinal cord differ both morphologically and in terms of molecular architecture from those of more rostral (more fully differentiated) areas of the cord. The caudalmost regenerating cord consists of an ependymal tube. Cells from this region have flattened or partially flattened cell somas and short spike projections in vitro; they do not exhibit the rounded cell somas nor the long, thin, branching neurites typical of differentiated neurons. A series of cultures taken from different areas along the length of regenerating spinal cord were examined for molecular differentiation by staining with a monoclonal antibody against non-phosphorylated neurofilament protein (SMI 32). None of the cells from the caudalmost culture of the regenerating spinal cord stained with antibody SMI 32. In cultures of more rostral regenerated cord, the cells with neuronal morphology do stain positively with the anti-neurofilament antibody. This result suggests that the cells in the cultures of caudalmost cord represent relatively undifferentiated ependymal cells, or ependyma-derived cells, which later differentiate into neurons and glia in the regenerating spinal cord.

The minimal dose of ethylene dibromide (EOB) needed to obtain 99.997% mortality of eggs and larvae of Dacus dorsalis Hendel in papayas, Carica papaya L., varied with the concentration of EDB in the fumigation chamber, the type and number of containers, and the fumigation temperature. The effect of container type on the EDB concentration after 0.5 h of fumigation was increased adsorption of EDB in the following order: perforated polyethylene tray < painted wooden field box < wooden field box ≤ fiberboard carton. Hot water treatments to control anthracnose of papaya improved the efficacy of EDB fumigation and reduced EDB residues more as a postfumigation treatment than prefumigation treatment. The quarantine treatment developed for oriental fruit fly-infested papayas in perforated polyethylene trays was fumigation with 7 g EDB/m3 for 2 h at 21.1°C and loading of 46% (by volume). The EDB residues in such fumigated papayas after 1-, 3-, and 5-day storage in cartons at 12.8°C were 2.1±0.12, 0.6±0.05, and 0.4±0.07 ppm, respectively. When similarly treated fruits were hot water-treated for 20 min at 46.1°C after the fumigation, the residues, at similar times and storage, were 1.8±0.2, 0.5±0.05, and 0.1 ±0.02 ppm. The quarantine treatment developed for fruits in wooden field boxes was fumigation with 8 g EDB/m3 for 2 h at 21.1°C plus postfumigation hot water treatment at 60.6°c for 20 sec. For such fruits stored 1 and 3 days in cartons at 20.0°-25.6°C (ambient temperature), the EDB residues were 0.4±0.04 and 0.05 ppm to trace, respectively; after 1, 3, and 5 days of storage at 7.2°C, the residues were 2.1±0.13, 0.7±0.06, and 0.3±0.02 ppm, respectively.

The minimum doses of methyl bromide (administered at the optimum temperature after a period of low-temperature storage), the minimum period of low-temperature storage, and the most effective combination of the 2 treatments were established for quarantine treatment of papayas, Carica papaya L., and avocados, Persea americana Mill., infested with the 3 Hawaiian tephritids, the Mediterranean fruit fly, Ceratitis capitata (Wiedemann); the oriental fruit fly, Dacus dorsalis Hendel; and the melon fly, D. cucurbitae Coquillett. Equations were used to approximate the separate effects of each of the subtreatments and of the combined treatments. The effective treatments projected about 32 survivors from a million eggs and larvae of the 3 species. Also, residues were determined in papayas and avocados after fumigation with methyl bromide, and tests were made with the minimum treatments to establish tolerance levels for residues in these commodities.

4-Methoxygentisic acid was an intermediate formed when Arthrobacter degraded, 2,4,5-trimethoxybenzoic acid. Isolates of Pseudomonas and Arthrobacter from soil grew at the expense of 3-methoxycrotonic acid. Evidence is presented that enzymatic hydration, with elimination of methanol, accounted for replacement of the methoxyl group of 3-methoxycrotonic acid and also of one methoxyl group of 2,4,5-trimethoxybenzoic acid.

Heating of 4-h-old eggs and nearly mature 3rd-instars of the oriental fruit fly in water in the laboratory indicated that minimum periods for 100% kill were 60, 14, 3, 1, or ¼ min at constant 43.3, 46.1, 48.9, 51.7 or 54.4°C, respectively, for 4-h-old eggs and 65, 18, 5, or ¼ min at constant 43.3, 46.1, 48.9, and 54.4°C, respectively, for 3rd instars. None of 262,555 eggs, 1st, 2nd, and 3rd-instars located in 1095 heavily infested papayas at depths of 0–2.0 cm survived commercial-type vapor heat treatment. Treatment consisted of an average 11-h approach period to raise the mean temperature of the fruit from 23.3° to 44.4°C, and then holding the fruit at a constant mean temperature of 44.4°C for 8-¾h.