Structure-based development of a canine TNF-α-specific antibody using adalimumab as a template.

The canine anti-tumor necrosis factor-alpha (TNF-α) monoclonal antibody is a potential therapeutic option for treating canine arthritis. The current treatments for arthritis in dogs have limitations due to side effects, emphasizing the need for safer and more effective therapies. The crystal structure of canine TNF-α (cTNF-α) was successfully determined at a resolution of 1.85 Å, and the protein was shown to assemble as a trimer, with high similarity to the functional quaternary structure of human TNF-α (hTNF-α). Adalimumab (Humira), a known TNF-α inhibitor, effectively targets and neutralizes TNF-α to reduce inflammation and has been used to manage autoimmune conditions such as rheumatoid arthritis. By comparing the structure of cTNF-α with the complex structure of hTNF-α and adalimumab-Fab, the epitope of adalimumab on cTNF-α was identified. The significant structural similarities of epitopes in cTNF-α and hTNF-α indicate the potential of using adalimumab to target cTNF-α. Therefore, a canine/human chimeric antibody, Humivet-R1, was created by grafting the variable domain of adalimumab onto a canine antibody framework derived from ranevetmab. Humivet-R1 exhibits potent neutralizing ability (IC50 = 0.05 nM) and high binding affinity (EC50 = 0.416 nM) to cTNF-α, comparable to that of adalimumab for both hTNF-α and cTNF-α. These results strongly suggest that Humivet-R1 has the potential to provide effective treatment for canine arthritis with reduced side effects. Here, we propose a structure-guided antibody design for the use of a chimeric antibody to treat canine inflammatory disease. Our successful development strategy can speed up therapeutic antibody discovery for animals and has the potential to revolutionize veterinary medicine.

Mechanistic effects of microwave radiation on pupal emergence in the leafminer fly, Liriomyza trifolii.

Liriomyza trifolii is a significant pest of vegetable and ornamental crops across the globe. Microwave radiation has been used for controlling pests in stored products; however, there are few reports on the use of microwaves for eradicating agricultural pests such as L. trifolii, and its effects on pests at the molecular level is unclear. In this study, we show that microwave radiation inhibited the emergence of L. trifolii pupae. Transcriptomic studies of L. trifolii indicated significant enrichment of differentially expressed genes (DEGs) in 'post-translational modification, protein turnover, chaperones', 'sensory perception of pain/transcription repressor complex/zinc ion binding' and 'insulin signaling pathway' when analyzed with the Clusters of Orthologous Groups, Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes databases, respectively. The top DEGs were related to reproduction, immunity and development and were significantly expressed after microwave radiation. Interestingly, there was no significant difference in the expression of genes encoding heat shock proteins or antioxidant enzymes in L. trifolii treated with microwave radiation as compared to the untreated control. The expression of DEGs encoding cuticular protein and protein takeout were silenced by RNA interference, and the results showed that knockdown of these two DEGs reduced the survival of L. trifolii exposed to microwave radiation. The results of this study help elucidate the molecular response of L. trifolii exposed to microwave radiation and provide novel ideas for control.

Associations between ALDH Genetic Variants, Alcohol Consumption, and the Risk of Nasopharyngeal Carcinoma in an East Asian Population.

Nasopharyngeal carcinoma (NPC) and alcohol flush syndrome are thought to be strongly influenced by genetic factors and are highly prevalent amongst East Asians. Diminished activity of aldehyde dehydrogenase (ALDH), a major enzyme in the alcohol-metabolizing pathway, causes the flushing syndrome associated with alcoholic consumption. The genetic effect of ALDH isoforms on NPC is unknown. We therefore investigated the association between the genetic polymorphisms of all 19 ALDH isoforms and NPC among 458 patients with NPC and 1672 age- and gender-matched healthy controls in Taiwan. Single-nucleotide polymorphisms (SNPs) located between the 40,000 base pairs upstream and downstream of the 19 ALDH isoform coding regions were collected from two genome-wise association studies conducted in Taiwan and from the Taiwan Biobank. Thirteen SNPs located on ALDH4A1, ALDH18A1, ALDH3B2, ALDH1L2, ALDH1A2, and ALDH2 Glu487Lys (rs671) were associated with NPC susceptibility. Stratification by alcohol status revealed a cumulative risk effect for NPC amongst drinkers and non-drinkers, with odds ratios of 4.89 (95% confidence interval 2.15-11.08) and 3.57 (1.97-6.47), respectively. A synergistic effect was observed between SNPs and alcohol. This study is the first to report associations between genetic variants in 19 ALDH isoforms, their interaction with alcohol consumption and NPC in an East Asian population.

Neuroprotective effects of intraoperative dexmedetomidine versus saline infusion combined with goal-directed haemodynamic therapy for patients undergoing cranial surgery: A randomised controlled trial.

BACKGROUND: By inhibiting neuroinflammation dexmedetomidine may be neuroprotective in patients undergoing cranial surgery, but it reduces cardiac output and cerebral blood flow. OBJECTIVE: To investigate whether intra-operative dexmedetomidine combined with goal-directed haemodynamic therapy (GDHT) has neuroprotective effects in cranial surgery. DESIGN: A double-blind, single-institution, randomised controlled trial. SETTING: A single university hospital, from April 2017 to April 2020. PATIENTS: A total of 160 adults undergoing elective cranial surgery. INTERVENTION: Infusion of dexmedetomidine (0.5 µg kg-1 h-1) or saline combined with GDHT to optimise stroke volume during surgery. MAIN OUTCOME MEASURES: The proportion who developed postoperative neurological complications was compared. Postoperative disability was assessed using the Barthel Index at time points between admission and discharge, and also the 30-day modified Rankin Scale (mRS). Postoperative delirium was assessed. The concentration of a peri-operative serum neuroinflammatory mediator, high-mobility group box 1 protein (HMGB1), was compared. RESULTS: Fewer patients in the dexmedetomidine group developed new postoperative neurological complications (26.3% vs. 43.8%; P = 0.031), but the number of patients developing severe neurological complications was comparable between the two groups (11.3% vs. 20.0%; P = 0.191). In the dexmedetomidine group the Barthel Index reduction [0 (-10 to 0)] was less than that in the control group [-5 (-15 to 0)]; P = 0.023, and there was a more favourable 30-day mRS (P = 0.013) with more patients without postoperative delirium (84.6% vs. 64.2%; P = 0.012). Furthermore, dexmedetomidine induced a significant reduction in peri-operative serum HMGB1 level from the baseline (222.5 ±â€Š408.3 pg ml-1) to the first postoperative day (152.2 ±â€Š280.0 pg ml-1) P = 0.0033. There was no significant change in the control group. The dexmedetomidine group had a lower cardiac index than did the control group (3.0 ±â€Š0.8 vs. 3.4 ±â€Š1.8 l min-1 m-2; P = 0.0482) without lactate accumulation. CONCLUSIONS: Dexmedetomidine infusion combined with GDHT may mitigate neuroinflammation without undesirable haemodynamic effects during cranial surgery and therefore be neuroprotective. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT02878707.

Structure-based Development of Human Interleukin-1ß-Specific Antibody That Simultaneously Inhibits Binding to Both IL-1RI and IL-1RAcP.

Interleukin-1ß (IL-1ß) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1ß regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1ß to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1ß initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-1ß neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human IL-1ß. We validated IgG26AW-neutralizing antibodies specific for IL-1ß in vivo to prevent human IL-1ß-driving IL-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to IL-1ß also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1ß complex structure. Meanwhile, SPR experiments showed that IL-1ß bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1ß/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1ß, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1ß is critical to pathogenesis.

Revalidation of morphological characteristics and multiplex PCR for the identification of three congener invasive Liriomyza species (Diptera: Agromyzidae) in China.

Due to varietal differences, diminutive size, and similar morphological characters, it is difficult to classify and identify Liriomyza spp., a genus comprised of economically-important, highly-polyphagous insect pests. In this study, we reconfirmed the morphological characteristics of three closely-related invasive leafminers, L. trifolii, L. sativae, and L. huidobrensis. Morphological results showed that characteristics imparted by the male genitalia were the most reliable morphological features for identification. The colors exhibited by vertical setae were variable among species, and the ratio of the length of the ultimate section of vein CuA1 divided by penultimate section also varied within species. Although the patterns of abdominal tergites were diverse among Liriomyza spp., L. trifolii exhibited a unique pattern with a yellow patch at the 5th black visible tergite; this pattern can be profiled as a prominent characteristic for morphological identification. In order to identify the three Liriomyza spp. quickly and accurately, we developed an improved molecular identification method using multiplex PCR based on the gene encoding mitochondrial cytochrome oxidase I (COI); this method enabled direct identification based on the size of amplified products. The results of this study provide a valuable reference for the identification of Liriomyza spp., which will ultimately improve our ability to control individual species.

Comparative transcriptome analysis of three invasive leafminer flies provides insights into interspecific competition.

Liriomyza spp. (Diptera: Agromyzidae) represent a group of economically-significant highly polyphagous pests of plants grown in field and greenhouse conditions. Liriomyza spp. share similar biological and morphological characteristics, and complex interspecific interactions have been documented among these species in various geographical regions. Where the displacement of one of these species by the other has been studied, no unique mechanisms have been identified as causing it. The impact of competitive factors (such as, insecticide tolerance, thermotolerance, and adaptability to cropping systems) may be unique to specific geographic regions of Liriomyza spp., but more research is needed to confirm these hypotheses. In this study, RNA-seq was used to determine the transcriptomes of three closely-related leafminers, e.g. L. sativae, L. trifolii, and L. huidobrensis. Over 20 Gb of clean reads were generated and assembled into unique transcriptomes, and 38,747 unigenes were annotated in different databases. In pairwise comparisons, L. trifolii and L. sativae had more up-regulated genes than L. huidobrensis. With respect to common differentially-expressed genes (Co-DEGs), the three leafminers exhibited distinct groups of highly-expressed gene clusters. When genes related to competitive factors were compared, expression patterns in L. trifolii and L. sativae were more closely related to each other than to L. huidobrensis. The data suggest that DEGs involved in competitive factors may play a key role in competition and displacement of leafminers. The divergent genes identified in this study will be valuable in revealing possible mechanisms of invasion, displacement and interspecific competition in Liriomyza spp.

Development of a Versatile and Modular Linker for Antibody-Drug Conjugates Based on Oligonucleotide Strand Pairing.

Linker design is crucial to the success of antibody-drug conjugates (ADCs). In this work, we developed a modular linker format for attaching molecular cargos to antibodies based on strand pairing between complementary oligonucleotides. We prepared antibody-oligonucleotide conjugates (AOCs) by attaching 18-mer oligonucleotides to an anti-HER2 antibody through thiol-maleimide chemistry, a method generally applicable to any immunoglobulin with interchain disulfide bridges. The hybridization of drug-bearing complementary oligonucleotides to our AOCs was rapid, stoichiometric, and sequence-specific. AOCs loaded with cytotoxic payloads were able to selectively target HER2-overexpressing cell lines such as SK-BR-3 and N87, with in vitro potencies similar to that of the marketed ADC Kadcyla (T-DM1). Our results demonstrated the potential of utilizing AOCs as a highly versatile and modular platform, where a panel of well-characterized AOCs bearing DNA, RNA, or various nucleic acid analogs, such as peptide nucleic acids, could be easily paired with any cargo of choice for a wide range of diagnostic or therapeutic applications.

Population genetics of Liriomyza trifolii (Diptera: Agromyzidae) and comparison with four Liriomyza species in China based on COI, EF-1a and microsatellites loci.

Liriomyza is a large genus that includes polyphagous and invasive species (L. trifolii, L. sativae, and L. huidobrensis), and oligophagous species such as L. Chinensis in China. Effective control of these invasive and oligophagous species is not easy due to the fast invasion rate, interspecific competition, and pesticide resistance. In this study, we investigated population genetics of five Liriomyza species L. trifolii, L. sativae, L. huidobrensis, L. bryoniae, and L. chinensis based on COI and EF-1a genes, and microsatellite DNA. These five Liriomyza species revealed highly conservative characteristics in the COI gene among populations collected from different geographical regions and host plants. By contrast, the mutation rate of the EF-1a gene was higher than COI, and phylogenetic tree based on EF-1a showed that haplotypes of L. trifolii and L. sativae were not distinguished well. Genetic differentiation in microsatellite loci was obvious among the five species. Our results also indicated that geographic isolation had a greater impact on genetic differentiation in L. trifolii than the host plant. Populations of L. trifolii in China showed a high to moderate level of genetic differentiation and they had divided into two groups representing the coastal areas of southern China and northern regions. The genetic diversity of the southern group was higher than the northern group. We speculated that the invasion of L. trifolii likely occurred in southern regions of China and then spread northward. Bottleneck analyses revealed that the L. trifolii population in China was in a steady growth period.

Lifestyle in Visually Impaired or Blind Massage Therapists: A Preliminary Study.

BACKGROUND: Lifestyle is among the most important factors affecting individual health status. Limited access to health information may limit the ability of people with visual impairment or blindness to practice healthy lifestyles. However, no studies have investigated how lifestyle practices affect health specifically in visually impaired and blind populations. PURPOSE: The aim of this study was to investigate the lifestyle behaviors of visually impaired and blind massage therapists (VIBMTs) in Taiwan. METHODS: This exploratory study used a purposive sampling technique to recruit 50 VIBMTs who were employed at massage stations in southern Taiwan. All of the participants completed the Health-Promoting Lifestyle Profile II (HPLP-II) and a survey of demographic characteristics. Descriptive and inferential statistical tests, including the Mann-Whitney U test and the Kruskal-Wallis H test, were used. Statistical significance was defined as p < .05 in two-tailed tests. RESULTS: Fifty participants completed both the HPLP-II and the demographic survey. The mean subscale score for the HPLP-II was 2.52 ± 0.37. The lowest scores were on the physical activity (2.09 ± 0.67) and nutrition (2.35 ± 0.39) subscales, and the highest scores were on the spiritual growth (2.89 ± 0.56) and interpersonal relations (2.79 ± 0.46) subscales. Scores on the stress management and physical activity subscales were significantly higher in men than in women (p < .05). In addition, mean HPLP-II scores were significantly higher in VIBMTs who exercised regularly compared with those who did not (p < .05). Compared with nonsmokers, current smokers had significantly higher scores on the stress management subscale (p < .05). CONCLUSIONS/IMPLICATIONS FOR PRACTICE: The low physical activity scores in this population may be improved by developing physical activity programs for the home and workplace and by establishing community recreational and exercise facilities for visually impaired populations. The low scores for nutrition may be improved by establishing nutrition education programs that are designed specifically for VIBMTs to increase their consumption of fresh produce and other healthy foods and by requiring food manufacturers to use labels that may be easily read or understood by visually impaired populations.

Cloning and expression of genes encoding heat shock proteins in Liriomyza trifolii and comparison with two congener leafminer species.

The polyphagous agromyzid fly, Liriomyza trifolii, is a significant and important insect pest of ornamental and vegetable crops worldwide. The adaptation of insects to different environments is facilitated by heat shock proteins (HSPs), which play an important role in acclimation to thermal stress. In this study, we cloned and characterized five HSP-encoding genes of L. trifolii (Lthsp20, Lthsp40, Lthsp60, Lthsp70, and Lthsp90) and monitored their expression under different thermal stresses using real-time quantitative PCR. Pupae of L. trifolii were exposed to 19 different temperatures ranging from -20 to 45°C. The results revealed that Lthsp20, Lthsp40, Lthsp70 and Lthsp90 were significantly upregulated in response to both heat and cold stress, while Lthsp60 was induced only by heat temperatures. The temperatures of the onset (Ton) and maximal (Tmax) expression of the five Lthsps were also determined and compared with published Ton and Tmax values of homologous genes in L. sativae and L. huidobrensis. Although L. trifolii occurs primarily in southern China, it has cold tolerance comparable with the other two Liriomyza species. Based on the heat shock proteins expression patterns, L. trifolii has the capacity to tolerate extreme temperatures and the potential to disseminate to northern regions of China.

Bipolar disorder moderates associations between linoleic acid and markers of inflammation.

Dietary polyunsaturated fatty acids (PUFA) and inflammatory proteins associate with immune activation and have been implicated in the pathophysiology of mood disorders. We have previously reported that individuals with bipolar disorder (BPD) have decreased PUFA intake, including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA); and decreased PUFA concentration of plasma EPA and linoleic acid (LA). We have also reported an association between plasma LA and its metabolites and burden of disease measures in BPD. In the current cross-sectional study we collected blood samples and diet records from both bipolar (n = 91) and control subjects (n = 75) to quantify plasma cytokine concentrations and dietary LA intake, respectively. Using multiple linear regression techniques, we tested for case control differences in plasma cytokine levels and associations between cytokines and dietary LA intake, adjusting for sex, age, BMI, and total energy intake. We found significantly higher plasma levels of interleukin 18 (IL-18) (p = 0.036), IL-18 binding protein (IL-18BP) (p = 0.001), soluble tumor necrosis factor receptor (sTNFR) 1 (p = 0.006), and sTNFR2 (p = 0.007) in BPD compared with controls. Moreover, BPD significantly moderated the associations of dietary LA intake with plasma levels of IL-18, sTNFR1 and sTNFR2, which were inverse associations in bipolar individuals and positive associations in controls (p for dietary LA x BPD diagnosis interaction < 0.05 for all three). These findings suggest potential dysregulation of LA metabolism in BPD, which may extend to a modified influence of dietary LA on specific inflammatory pathways in individuals with BPD compared to healthy controls.

Colossolactone H, a new Ganoderma triterpenoid exhibits cytotoxicity and potentiates drug efficacy of gefitinib in lung cancer.

Three pentacyclic triterpene dilactones were isolated from the fruiting bodies of Ganoderma colossum, a medicinal mushroom. Colossolactone H (colo H) as a new compound and the most cytotoxic among the isolates was studied for its anticancer mechanism and the potential use in cancer therapy. Gene expression profiling analysis indicated that treatment of lung cancer cells with colo H caused upregulation of 252 genes and downregulation of 398 genes. Gene ontology enrichment analysis indicated that the downregulated genes were the most significantly enriched in cell cycle progression, and the upregulated genes were significantly enriched in metabolic process, cellular response to stimulus, and oxidation reduction. Accordingly, colo H was found to halt cell growth and induce cell apoptosis via the elevation of cellular reactive oxygen species to cause DNA damage and the increase of tumor suppressor p53 protein. These events facilitate additive cytotoxicity of colo H and gefitinib for gefitinib-resistant H1650 lung cancer cells. Furthermore, combination of colo H and gefitinib effectively inhibited the growth of tumor xenografts in athymic mice. In addition to the efficacy in adjunctive cancer therapy, we have also demonstrated the isolation of colo H from cultivated G. colossum. Thus it is feasible to use colo H or Ganoderma colossum for cancer therapy.

Characterization of Cell Wall Proteins in Saccharomyces cerevisiae Clinical Isolates Elucidates Hsp150p in Virulence.

The budding yeast Saccharomyces cerevisiae has recently been described as an emerging opportunistic fungal pathogen. Fungal cell wall mannoproteins have been demonstrated to be involved in adhesion to inert surfaces and might be engaged in virulence. In this study, we observed four clinical isolates of S. cerevisiae with relatively hydrophobic cell surfaces. Yeast cell wall subproteome was evaluated quantitatively by liquid chromatography/tandem mass spectrometry. We identified totally 25 cell wall proteins (CWPs) from log-phase cells, within which 15 CWPs were quantified. The abundance of Scw10p, Pst1p, and Hsp150p/Pir2p were at least 2 folds higher in the clinical isolates than in S288c lab strain. Hsp150p is one of the members in Pir family conserved in pathogenic fungi Candida glabrata and Candida albicans. Overexpression of Hsp150p in lab strain increased cell wall integrity and potentially enhanced the virulence of yeast. Altogether, these results demonstrated that quantitative cell wall subproteome was analyzed in clinical isolates of S. cerevisiae, and several CWPs, especially Hsp150p, were found to be expressed at higher levels which presumably contribute to strain virulence and fungal pathogenicity.

Biomechanical effects of screw fixation in second mandibular reconstruction plate.

The purpose of this study was to investigate biomechanical effects of second reconstruction plate with different combinations of fixed screws in patient with mandibular tumor resection by three-dimensional finite element analysis (FEA). The FEA models were consisted of defected mandible, reconstruction plate with different screw holes, and bone screws. The results indicated that application of the second reconstruction plate appeared to increase stability and decrease stress magnitude on the plates and screws accompanying with increasing screw number. For clinical cost consideration in usage of the second reconstruction plate, the conclusion showed that the second reconstruction plate could offer a better mechanical efficacy accompanying with increase of screw quantity, but single screw applied for the second plate fixation to defected mandible of tumor resection was enough to stabilize without increase of screw quantities.

A cysteine-reactive alkyl hydroquinone modifies topoisomerase IIα, enhances DNA breakage, and induces apoptosis in cancer cells.

We previously reported that the anticancer activity of a botanical compound 10'(Z),13'(E),15'(E)-heptadecatrienylhydroquinone [HQ17(3)] was attributed to topoisomerase (Topo) IIα poisoning and the induction of oxidative damage. HQ17(3) irreversibly inhibits Topo IIα activity in vitro and is more cytotoxic in leukemia HL-60 cells than in Topo IIα-deficient variant HL-60/MX2 cells, which suggests that Topo IIα is a cellular target of HQ17(3). This study further characterizes the molecular mechanisms of the anticancer activity of HQ17(3). Proteomic analyses indicated that HQ17(3) reacted with Cys-427, Cys-733, and Cys-997 of recombinant Topo IIα in vitro, whereas it reacted with Cys-427 of cellular Topo IIα in Huh7 hepatoma cells. The modification of HQ17(3) inhibited Topo IIα catalytic activity, increased the Topo IIα-DNA cleavage complex, and caused the accumulation of DNA breakage. In Huh7 cells, HQ17(3) treatment caused prompt inhibition of DNA synthesis and consequently induced the expression of DNA damage-related genes DDIT3, GADD45A, and GADD45G. Topo IIα inhibition, apoptosis, and oxidative stress were found to account for cytotoxicity caused by HQ17(3). Pretreatment of Huh7 cells with N-acetylcysteine (NAC) partially attenuated mitochondrial membrane damage, DNA breakage, and caspase activation. However, NAC pretreatment did not diminish HQ17(3)-induced cell death. These results suggest that the anticancer activity of HQ17(3) is attributed significantly to Topo IIα poisoning. The structural feature of HQ17(3) can be used as a model for the design of Topo IIα inhibitors and anticancer drugs.

YC-1 potentiates cAMP-induced CREB activation and nitric oxide production in alveolar macrophages.

Alveolar macrophages play significant roles in the pathogenesis of several inflammatory lung diseases. Increases in exhaled nitric oxide (NO) are well documented to reflect disease severity in the airway. In this study, we investigated the effect of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on prostaglandin (PG)E1 (a stable PGE2 analogue) and forskolin (a adenylate cyclase activator) induced NO production and inducible NO synthase (iNOS) expression in rat alveolar macrophages (NR8383). YC-1 did not directly cause NO production or iNOS expression, but drastically potentiated PGE1- or forskolin-induced NO production and iNOS expression in NR8383 alveolar macrophages. Combination treatment with YC-1 and PGE1 significantly increased phosphorylation of the cAMP response element-binding protein (CREB), but not nuclear factor (NF)-κB activation. The combined effect on NO production, iNOS expression, and CREB phosphorylation was reversed by a protein kinase (PK)A inhibitor (H89), suggesting that the potentiating functions were mediated through a cAMP/PKA signaling pathway. Consistent with this, cAMP analogues, but not the cGMP analogue, caused NO release, iNOS expression, and CREB activation. YC-1 treatment induced an increase in PGE1-induced cAMP formation, which occurred through the inhibition of cAMP-specific phosphodiesterase (PDE) activity. Furthermore, the combination of rolipram (an inhibitor of PDE4), but not milronone (an inhibitor of PDE3), and PGE1 also triggered NO production and iNOS expression. In summary, YC-1 potentiates PGE1-induced NO production and iNOS expression in alveolar macrophages through inhibition of cAMP PDE activity and activation of the cAMP/PKA/CREB signaling pathway.

Inhibitory effects of terpenoids from the fermented broth of the ascomycete Stilbohypoxylon elaeicola YMJ173 on nitric oxide production in RAW264.7 macrophages.

A series of six isopimarane-type diterpene glycosides, along with an eremophilane-type sesquiterpene, i.e., elaeicolasides A-C (1-3, resp.), 16-(α-D-mannopyranosyloxy)isopimar-7-en-19-oic acid (4), hymatoxin K (5), hymatoxin L (6), and elaeicolalactone (7), were isolated from the AcOEt extract of the fermented broth of Stilbohypoxylon elaeicola YMJ173. Among these, 1-3 and 7 are new compounds based on their spectroscopic data and sugar composition analysis. The effects of 1-7 on the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells were evaluated. All these compounds inhibited NO production, detected as nitrite in the culture medium, in activated macrophages without any cytotoxicity at a concentration of 100 µM. Among these compounds, 2 showed a significant activity with the average maximum inhibition (E(max)) and median inhibitory concentration (IC(50)) values of 93.3±0.5% and 79.3±0.4 µM, respectively.

[The relationship between oral feeding and cardiorespiratory regulation of premature infants].

Levels of maturity and diseases of premature infants are factors that produce individual differences in cardiorespiratory responses to oral feeding and feeding performances. Recognition of mechanisms and cardiorespiratory regulation of oral feeding may increase sensitivity of clinical observation of nurses and facilitate safe and comfort feeding experiences for premature infants. Four major concepts of this integrated literature review are: (a) The maturation of sucking skills and coordination of sucking, swallowing, and breathing play an important role in successful oral feeding. (b) There are significant relationships between the feeding performances and cardiorespiratory stability of premature infants in the early period of oral feeding. (c) During the oral feeding period, the reduction of vagal tone will increase the heart rate of premature infants in order to balance physiological metastasis. (d) During the early period of oral feeding, the SaO2 of premature infants might decrease continually. This condition can be improved gradually with experience of oral feeding. These concepts provide knowledge for assessing feeding readiness and facilitate protocols for safe feeding by reducing the cardiorespiratory workload of premature infants.

The Ras/PKA signaling pathway directly targets the Srb9 protein, a component of the general RNA polymerase II transcription apparatus.

RNA polymerase II transcription is a complex process that is controlled at multiple levels. The data presented here add to this repertoire by showing that signal transduction pathways can directly regulate gene expression by targeting components of the general RNA polymerase II apparatus. In particular, this study shows that the Ras/PKA signaling pathway in Saccharomyces cerevisiae regulates the activity of the Srb complex, a regulatory group of proteins that is part of the RNA polymerase II holoenzyme. Genetic and biochemical data indicate that Srb9p is a substrate for PKA and that this phosphorylation modulates the activity of the Srb complex. The Srb complex, like many components of the RNA II polymerase machinery, is responsible for regulating the expression of a relatively large number of genes. Thus, this type of a transcriptional control mechanism would provide the cell with an efficient way of bringing about broad changes in gene expression.