The dynamics of infectious diseases that are spread through direct contact have been proven to depend on the strength of community structure or modularity within the underlying network. It has been recently shown that weighted networks with similar modularity values may exhibit different mixing styles regarding the number of connections among communities and their respective weights. However, the effect of mixing style on epidemic behavior was still unclear. In this paper, we simulate the spread of disease within networks with different mixing styles: a dense-weak style (i.e., many edges among the communities with small weights) and a sparse-strong style (i.e., a few edges among the communities with large weights). Simulation results show that, with the same modularity: 1) the mixing style significantly influences the epidemic size, speed, pattern and immunization strategy; 2) the increase of the number of communities amplifies the effect of the mixing style; 3) when the mixing style changes from sparse-strong to dense-weak, there is a ‘saturation point’, after which the epidemic size and pattern become stable. We also provide a mean-field solution of the epidemic threshold and size on weighted community networks with arbitrary external and internal degree distribution. The solution explains the effect of the second moment of the degree distribution, and a symmetric effect of internal and external connections (incl. degree distribution and weight). Our study has both potential significance for designing more accurate metrics for the community structure and exploring diffusion dynamics on metapopulation networks.

Mass spectrometry (MS)-based proteomics is playing an increasingly important role in cardiovascular research. Proteomics includes not only identification and quantification of proteins, but also the characterization of protein modifications such as post-translational modifications and sequence variants. The conventional bottom-up approach, involving proteolytic digestion of proteins into small peptides prior to MS analysis, is routinely used for protein identification and quantification with high throughput and automation. Nevertheless, it has limitations in the analysis of protein modifications mainly due to the partial sequence coverage and loss of connections among modifications on disparate portions of a protein. An alternative approach, top-down MS, has emerged as a powerful tool for the analysis of protein modifications. The top-down approach analyzes whole proteins directly, providing a “bird’s eye” view of all existing modifications. Subsequently, each modified protein form can be isolated and fragmented in the mass spectrometer to locate the modification site. The incorporation of the non-ergodic dissociation methods such as electron capture dissociation (ECD) greatly enhances the top-down capabilities. ECD is especially useful for mapping labile post-translational modifications which are well-preserved during the ECD fragmentation process. Top-down MS with ECD has been successfully applied to cardiovascular research with the unique advantages in unraveling the molecular complexity, quantifying modified protein forms, complete mapping of modifications with full sequence coverage, discovering unexpected modifications, and identifying and quantifying positional isomers and determining the order of multiple modifications. Nevertheless, top-down MS still needs to overcome some technical challenges to realize its full potential. Herein, we reviewed the advantages and challenges of top-down methodology with a focus on its application in cardiovascular research.

Background

To compare the characteristics between 22-channel water-perfusion manometry (WPM) and solid-state manometry (SSM) with 36 sensors of the pressure measurements, as well as patients’ discomfort indices in nose and pharynx, the preparation and operation time of the manometry.

Methods

12 volunteers were included in the study. Each of the volunteers underwent esophageal manometry by both 22-channel water-perfusion catheter (WPC) and solid-state catheter (SSC) with 36 sensors in random order, and separated by 30 min. The subjects gave a VAS score soon after each test. Non-parametric tests were used to analyze the differences and Bland-Altman plots were used to assess the consistency of the two systems.

Results

During the wet swallows, there were significant differences between the two systems in three measurements of location of lower esophageal sphincter (LES) upper margin (Z = -2.11, P = 0.035), LES relax ratio (Z = -2.20, P = 0.028) and IRP4s (Z = -2.05, P = 0.041). During the jelly pocket swallows, LES relax ratio measurements of the two systems showed significant differences (Z = -2.805, P = 0.005). Further Bland–Altman plots analysis presented good agreement between the two systems measurements of location of LES upper margin, LES relax ratio and IRP4s. The discomfort indices of subjects’ nasal sensation were higher when inserting the solid-state catheter [5(3.75-5)] than water-perfusion one (2.5(2-4)) (Z = -2.471, P = 0.013), as well as the discomfort indices of pharyngeal sensation (7.5(4.75-9) vs. 4.5(3.75-6.5)), (Z = -2.354, P = 0.019). The preparation time for WPC was 40(39-41) minutes, which was much longer than that for SSC 32.5(31.75-33) minutes, (Z = -3.087, P = 0.002). And the nurses reported it’s much easier to insert WPC (Z = -3.126, P = 0.002).

Conclusions

In conclusion, most pressure measurements were consistent between WPM and SSM. Patients tolerated better with WPC, while for operators, the SSC presented more convenient.

The rapid increase in the prevalence of chronic heart failure (CHF) worldwide underscores an urgent need to identify biomarkers for the early detection of CHF. Post-translational modifications (PTMs) are associated with many critical signaling events during disease progression and thus offer a plethora of candidate biomarkers. We have employed top-down quantitative proteomics methodology for comprehensive assessment of PTMs in whole proteins extracted from normal and diseased tissues. We have systematically analyzed thirty-six clinical human heart tissue samples and identified phosphorylation of cardiac troponin I (cTnI) as a candidate biomarker for CHF. The relative percentages of the total phosphorylated cTnI forms over the entire cTnI populations (%Ptotal) were 56.4±3.5%, 36.9±1.6%, 6.1±2.4%, and 1.0±0.6% for postmortem hearts with normal cardiac function (n=7), early-stage of mild hypertrophy (n=5), severe hypertrophy/dilation (n=4), and end-stage CHF (n=6), respectively. In fresh transplant samples, the %Ptotal of cTnI from non-failing donor (n=4), and end-stage failing hearts (n=10) were 49.5±5.9% and 18.8±2.9%, respectively. Top-down MS with electron capture dissociation unequivocally localized the altered phosphorylation sites to Ser22/23 and determined the order of phosphorylation/dephosphorylation. This study represents the first clinical application of top-down MS-based quantitative proteomics for biomarker discovery from tissues, highlighting the potential of PTM as disease biomarkers.

Cardiac troponin I (cTnI) is the inhibitory subunit of cardiac troponin, a key myofilament regulatory protein complex located on the thin filaments of the contractile apparatus. cTnI is uniquely specific for the heart and is widely used in clinics as a serum biomarker for cardiac injury. Phosphorylation of cTnI plays a critical role in modulating cardiac function. cTnI is known to be regulated by protein kinase A and protein kinase C at five sites, Ser22/Ser23, Ser42/44, and Thr143, primarily based on results from in vitro phosphorylation assays by the specific kinase(s). However, a comprehensive characterization of phosphorylation of mouse cTnI occurring in vivo has been lacking. Herein, we have employed top-down mass spectrometry (MS) methodology with electron capture dissociation for precise mapping of in vivo phosphorylation sites of cTnI affinity purified from wild-type and transgenic mouse hearts. As demonstrated, top-down MS (analysis of intact proteins) is an extremely valuable technology for global characterization of labile phosphorylation occurring in vivo without a priori knowledge. Our top-down MS data unambiguously identified Ser22/23 as the only two sites basally phosphorylated in wild-type mouse cTnI with full sequence coverage, which was confirmed by the lack of phosphorylation in cTnI-Ala2 transgenic mice where Ser22/23 in cTnI have been rendered nonphosphorylatable by mutation to alanine.

Objective

GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin biosynthesis and has been shown to be a promising therapeutic target in ischemic heart disease, hypertension, atherosclerosis and diabetes. The endogenous GCH1-interacting partners have not been identified. Here, we determined endogenous GCH1-interacting proteins in rat.

Methods and Results

A pulldown and proteomics approach were used to identify GCH1 interacting proteins in rat liver, brain, heart and kidney. We demonstrated that GCH1 interacts with at least 17 proteins including GTP cyclohydrolase I feedback regulatory protein (GFRP) in rat liver by affinity purification followed by proteomics and validated six protein partners in liver, brain, heart and kidney by immunoblotting. GCH1 interacts with GFRP and very long-chain specific acyl-CoA dehydrogenase in the liver, tubulin beta-2A chain in the liver and brain, DnaJ homolog subfamily A member 1 and fatty aldehyde dehydrogenase in the liver, heart and kidney and eukaryotic translation initiation factor 3 subunit I (EIF3I) in all organs tested. Furthermore, GCH1 associates with mitochondrial proteins and GCH1 itself locates in mitochondria.

Conclusion

GCH1 interacts with proteins in an organ dependant manner and EIF3I might be a general regulator of GCH1. Our finding indicates GCH1 might have broader functions beyond tetrahydrobiopterin biosynthesis.

Cardiac troponin T (cTnT), the tropomyosin binding subunit of the troponin complex, plays a pivotal regulatory role in the Ca2+-mediated interaction between actin thin filament and myosin thick filament. The post-translational modifications (PTMs) and alternative splicing of cTnT may represent important regulatory mechanisms of cardiac contractility. However, a complete characterization of PTMs and alternatively spliced isoforms in cTnT present in vivo is lacking. Top-down protein mass spectrometry (MS) analyzes whole proteins, thus providing a global view of all types of modifications, including PTMs and sequence variants, simultaneously in one spectrum without a priori knowledge. In this study, we applied an integrated immunoaffinity chromatography and top-down MS approach to comprehensively characterize PTMs and alternatively spliced isoforms of cTnT purified from healthy human and wild-type mouse heart tissue. High-resolution Fourier transform MS revealed that human cTnT (hcTnT) and mouse cTnT (mcTnT) have similar phosphorylation patterns, whereas higher molecular heterogeneity was observed for mcTnT than hcTnT. Further MS/MS fragmentation of monophosphorylated hcTnT and mcTnT by electron capture dissociation and collisionally activated dissociation unambiguously identified Ser1 as the conserved in vivo phosphorylation site. In contrast, we identified a single spliced isoform for hcTnT but three alternatively spliced isoforms for mcTnT. Moreover, we observed distinct proteolytic degradation products for hcTnT and mcTnT. This study also demonstrates the advantage of top-down MS/MS with complementary fragmentation techniques for the identification of modification sites in the highly acidic N-terminal region of cTnT.

Heterotrimeric cardiac troponin (cTn) is a critical component of the thin filament regulatory complex in cardiac muscle. Two of the three subunits, cTnI and cTnT, are subject to post-translational modifications such as proteolysis and phosphorylation, but linking modification patterns to function remains a major challenge. To obtain a global view of the biochemical state of cTn in native tissue, we performed high resolution top-down mass spectrometry of cTn heterotrimers from healthy adult rat hearts. cTn heterotrimers were affinity purified, desalted and then directly subjected to mass spectrometry using a 7 Tesla Thermo LTQ-FT-ICR instrument equipped with an ESI source. Molecular ions for N-terminally processed and acetylated cTnI and cTnT were readily detected as were other post-translationally modified forms of these proteins. cTnI was phosphorylated with a distribution of un-, mono- and bisphosphorylated forms of 41 ± 3%, 46 ± 1%, 13 ± 3%, respectively. cTnT was predominantly mono-phosphorylated and partially proteolyzed at the Glu29-Pro30 peptide bond. Also observed in high resolution spectra were ‘shadow’ peaks of similar intensity to ‘parent’ peaks exhibiting masses of cTnI+16 Da and cTnT+128 Da, subsequently shown by tandem mass spectrometry (MS/MS) to be single amino acid polymorphisms. Intact and protease-digested cTn subunits were fragmented by electron capture dissociation or collision activated dissociation to localize an Ala/Ser polymorphism at residue 7 of cTnI. Similar analysis of cTnT localized an additional Gln within a three residue alternative splice site beginning at residue 192. Besides being able to provide unique insights into the global state of post-translational modification of cTn subunits, high resolution top-down mass spectrometry readily revealed naturally occurring single amino acid sequence variants including a genetic polymorphism at residue 7 in cTnI, and an alternative splice isoform that affects a putative hinge region around residue 192 of cTnT, all of which co-exist within a single rat heart.

Mass spectrometry (MS)-based phosphoproteomics remains challenging due to the low abundance of phosphoproteins and substoichiometric phosphorylation. This demands better methods to effectively enrich phosphoproteins/peptides prior to MS analysis. We have previously communicated the first use of mesoporous zirconium oxide (ZrO2) nanomaterials for effective phosphopeptide enrichment. Here we present the full report including the synthesis, characterization, and application of mesoporous titanium dioxide (TiO2), ZrO2, and hafnium oxide (HfO2) in phosphopeptide enrichment and MS analysis. Mesoporous ZrO2 and HfO2 are demonstrated to be superior to TiO2 for phosphopeptide enrichment from a complex mixture with high specificity (>99%), which could almost be considered as “a purification”, mainly because of the extremely large active surface area of mesoporous nanomaterials. A single enrichment and Fourier transform MS analysis of phosphopeptides digested from a complex mixture containing 7% of α-casein identified 21 out of 22 phosphorylation sites for α-casein. Moreover, the mesoporous ZrO2 and HfO2 can be reused after a simple solution regeneration procedure with comparable enrichment performance to that of fresh materials. Mesoporous ZrO2 and HfO2 nanomaterials hold great promise for applications in MS-based phosphoproteomics.

AIM: To investigate the role of α-fetoprotein (AFP), a cancer-associated fetal glycoprotein, in glucocorticoid-induced precocious maturation in rat colon.

METHODS: Colons from suckling Sprague-Dawley rats were used in this study. Corticosterone acetate at a dose of 100 μg/g body weight was given to normal pups on days 7, 9 and 11 after birth to induce hypercorticoidism. Control animals were injected with identical volumes of normal saline. Some rats receiving corticosterone 7 d after birth were also treated with mifepristone (RU38486), a glucocorticoid cytoplasm receptor antagonist to investigate the effects of glucocorticoids (GCs). The morphological changes of the crypt depth and villous height of the villous zone in colon were observed as indices of colon maturation. Expression levels of AFP in colons were detected by reverse transcriptase polymerase chain reaction and Western blotting. To identify the cellular localization of AFP in developing rat colons, double-immunofluorescent staining was performed using antibodies to specific mesenchymal cell marker and AFP.

RESULTS: Corticosterone increased the crypt depth and villous height in the colon of 8- and 10-d-old rats with hypercorticoidism compared with that in the control animals (120% in 8-d-old rats and 118% in 10-d-old rats in villous height, P = 0.021; 145% in 8-d-old rats and 124% in 10-d-old rats in crypt depth, P = 0.017). These increases were accompanied by an increase of AFP expression in both mRNA and protein (2.5-folds in 8-d-old and 2.5-folds in 10-d-old rats higher than in control animals, P = 0.035; 1.8-folds in 8-d-old and 1.3-folds in 10-d-old rats higher than in control animals, P = 0.023). Increased crypt depth and villous height and increased expression of AFP in the colon of rats with hypercorticoidism were blocked by mifepristone. Both had positive staining for AFP or vimentin, and overlapped in mesenchymal cells at each tested colon.

CONCLUSION: GCs promote the development of rat colon. AFP appears to be involved, in part, in mediating the effects of GCs in the developmental colon.

Cardiac troponin I (cTnI) is an important regulatory protein in cardiac muscle and its modification represents a key mechanism in the regulation of cardiac muscle contraction and relaxation. cTnI is often referred to as the “gold-standard” serum biomarker for diagnosing patients with acute cardiac injury since it is unique to the heart and released into the circulation following necrotic death of cardiac tissue. The swine (Sus scrofa) heart model is extremely valuable for cardiovascular research since the heart anatomy and coronary artery distribution of swine are almost identical to those of humans. Herein we report a comprehensive characterization of the modifications in swine cTnI using top-down high-resolution tandem mass spectrometry in conjugation with immunoaffinity chromatography purification. High-resolution high accuracy mass spectrometry revealed that swine cTnI affinity purified from domestic pig hearts was N-terminally acetylated and phosphorylated. Electron capture disassociation is uniquely suited for localization of labile phosphorylations, which unambiguously identified Ser22/Ser23 as the only basally phosphorylation sites that are well-known to be regulated by protein kinase A and protein kinase C. Moreover, a combination of tandem mass spectrometry with sequence homology alignment effectively localized a single amino acid polymorphism, V116A, representing a novel genetic variant of swine cTnI. Overall, our studies demonstrated the unique power of top-down high-resolution tandem mass spectrometry in the characterization of protein modifications including labile phosphorylation and unexpected sequence variants.

Coactivator-associated arginine methyltransferase 1 (CARM1), the histone arginine methyltransferase and coactivator for many transcription factors, is subject to multiple post-translational modifications (PTMs). To unbiasedly investigate novel CARM1 PTMs we employed high-resolution top-down mass spectrometry. Surprisingly, mouse CARM1 expressed in insect and mammalian expression systems was completely dimethylated at a single site in the C-terminal domain (CTD). We demonstrate that dimethylation of CARM1 occurs both in vivo and in vitro and proceeds via an automethylation mechanism. To probe function of automethylation, we mutated arginine 551 to lysine to create an automethylation-deficient CARM1. Although mutation of CARM1's automethylation site did not affect its enzymatic activity, it did impair both CARM1-activated transcription and pre-mRNA splicing. These results strongly imply that automethylation of CARM1 provides a direct link to couple transcription and pre-mRNA splicing in a manner differing from the other steroid receptor coactivators. Furthermore, our study identifies a self-regulatory signaling mechanism from CARM1's catalytic domain to its CTD.

Objective

The post-translational regulation of GTP cyclohydrolase I (GCH-1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, remains elusive. Here, we identified specific phosphorylation sites on GCH-1 and characterized the function of these sites.

Methods and Results

Mass spectrometry studies showed overexpressed rat GCH-1 was phosphorylated at serine (S) 51, S167 and threonine (T) 231 in HEK293 cells whereas a computational analysis of GCH-1 revealed 8 potential phosphorylation sites [S51, S72, T85, T91, T103, S130, S167 and T231]. GCH-1 activity and BH4 were significantly decreased in cells transfected with the phospho-defective mutants (S72A, T85A, T91A, T103A or S130A) and increased in cells transfected with the T231A mutant. BH4 and BH2 were increased in cells transfected with S51E, S72E, T85E, T91E, T103D or T130D mutants, but decreased in cells transfected with the T231D mutant, while cells transfected with the S167A or the S167E mutant had increased BH2. Additionally, cells transfected with the T231A mutant had reduced GCH-1 nuclear localization and nuclear GCH-1 activity.

Conclusion

Our data suggest GCH-1 activity is regulated either positively by phosphorylation S51, S72, T85, T91, T103 and S130, or negatively at T231. Such information might be useful in designing new therapies aiming at improving BH4 bioavailability.

This work represents the first use of mesoporous zirconium oxide nanomaterials for highly effective and selective enrichment of phosphorylated peptides.

Tg2576 mice produce high levels of beta-amyloid (Aβ) and develop amyloid deposits, but lack neurofibrillary tangles and do not suffer the extensive neuronal cell loss characteristic of Alzheimer's disease. Protection from Aβ toxicity has been attributed to up-regulation of transthyretin (TTR), a normal component of plasma and cerebrospinal fluid. We compared the effect of TTR purified from human plasma (pTTR) with that produced recombinantly (rTTR) on Aβ aggregation and toxicity. pTTR slowed Aβ aggregation but failed to protect primary cortical neurons from Aβ toxicity. In contrast, rTTR accelerated aggregation, while effectively protecting neurons. This inverse correlation between Aβ aggregation kinetics and toxicity is consistent with the hypothesis that soluble intermediates rather than insoluble fibrils are the most toxic Aβ species. We carried out a detailed comparison of pTTR with rTTR to ascertain the probable cause of these different effects. No differences in secondary, tertiary or quaternary structure were detected. However, pTTR differed from rTTR in the extent and nature of modification at Cys10. We hypothesize that differential modification at Cys10 regulates TTR's effect on Aβ aggregation and toxicity.

Background

Plant roots are the primary site of perception and injury for saline-alkaline stress. The current knowledge of saline-alkaline stress transcriptome is mostly focused on saline (NaCl) stress and only limited information on alkaline (NaHCO3) stress is available.

Results

Using Affymetrix® Soybean GeneChip®, we conducted transcriptional profiling on Glycine soja roots subjected to 50 mmol/L NaHCO3 treatment. In a total of 7088 probe sets, 3307 were up-regulated and 5720 were down-regulated at various time points. The number of significantly stress regulated genes increased dramatically after 3 h stress treatment and peaked at 6 h. GO enrichment test revealed that most of the differentially expressed genes were involved in signal transduction, energy, transcription, secondary metabolism, transporter, disease and defence response. We also detected 11 microRNAs regulated by NaHCO3 stress.

Conclusions

This is the first comprehensive wild soybean root transcriptome analysis under alkaline stress. These analyses have identified an inventory of genes with altered expression regulated by alkaline stress. The data extend the current understanding of wild soybean alkali stress response by providing a set of robustly selected, differentially expressed genes for further investigation.

The crustacean hyperglycemic hormone (CHH) is a 72-amino acid residue polypeptide with multiple physiological effects. The X-organ/sinus gland is the primary source for CHH and its family members. However, the amino acid sequence of CHH in Cancer borealis, a premier model system for neuromodulation, has not been characterized. In this study, a novel hybrid strategy combining “bottom-up” and “top-down” methodologies enabled direct sequencing of CHH peptide in the sinus gland of C. borealis. Multiple mass spectrometry (MS)-based techniques were employed to characterize the CHH peptide, including direct tissue analysis by MALDI FT-ICR MS, de novo sequencing of tryptic digested CHH by nano-LC-ESI Q-TOF MS and intact CHH analysis by LC FT-ICR MS. In-trap cleaning removed the extensive matrix adducts of CHH in the direct tissue analysis by MALDI FT-ICR MS. Fragmentation efficiency of the intact CHH was drastically improved after the reduction-alkylation of the disulfide bonds. The sequence coverage was further enhanced by employing multiple complementary fragmentation techniques. Overall, this example is the largest neuropeptide de novo sequenced in C. borealis by mass spectrometric methods.

Oncolytic adenoviral vectors that express immunostimulatory transgenes are currently being evaluated in clinic. Preclinical testing of these vectors has thus far been limited to immunodeficient xenograft tumor models since human adenoviruses do not replicate effectively in murine tumor cells. The effect of the immunostimulatory transgene on overall virus potency can therefore not be readily assessed in these models. Here, a model is described that allows the effective testing of mouse armed oncolytic adenovirus (MAV) vectors in immunocompetent syngeneic tumor models. These studies demonstrate that the MAV vectors have a high level of cytotoxicity in a wide range of murine tumor cells. The murine oncolytic viruses were successfully armed with murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) by a novel method which resulted in vectors with a high level of tumor-specific transgene expression. The mGM-CSF-armed MAV vectors showed an improved level of antitumor potency and induced a systemic antitumor immune response that was greater than that induced by unarmed parental vectors in immunocompetent syngeneic tumor models. Thus, the oncolytic MAV-1 system described here provides a murine homolog model for the testing of murine armed oncolytic adenovirus vectors in immunocompetent animals. The model allows evaluation of the impact of virus replication and the host immune response on overall virus potency and enables the generation of translational data that will be important for guiding the clinical development of these viruses.

The increasingly serious problem of acid rain is leading to increased potassium (K) loss from soils, and in our field investigation, we found that even congenerically relative Mosla species show different tolerance to K-deficiency. A hydroponic study was conducted on the growth of two Mosla species and their morphological, physiological and stoichiometric traits in response to limited (0.35 mmol K/L), normal (3.25 mmol K/L) and excessive (6.50 mmol K/L) K concentrations. Mosla hangchowensis is an endangered plant, whereas Mosla dianthera a widespread weed. In the case of M. hangchowensis, in comparison with normal K concentration, K-limitation induced a significant reduction in net photosynthetic rate (P n), soluble protein content, and superoxide dismutase (SOD) activity, but an increase in malondialdehyde (MDA) concentration. However, leaf mass ratio (LMR) and root mass ratio (RMR) were changed little by K-limitation. In contrast, for M. dianthera, K-limitation had little effect on P n, soluble protein content, SOD activity, and MDA concentration, but increased LMR and RMR. Critical values of N (nitrogen):K and K:P (phosphorus) ratios in the shoots indicated that limitation in acquiring K occurred under K-limited conditions for M. hangchowensis but not for M. dianthera. We found that low K content in natural habitats was a restrictive factor in the growth and distribution of M. hangchowensis, and soil K-deficiency caused by acid rain worsened the situation of M. hangchowensis, while M. dianthera could well acclimate to the increasing K-deficiency. We suggest that controlling the acid rain and applying K fertilizers may be an effective way to rescue the endangered M. hangchowensis.

AIM: To investigate the expression of α-fetoprotein (AFP), a cancer-associated fetal glycoprotein, and its involvement during rat colon development.

METHODS: Colons from Sprague-Dawley rat fetuses, young and adult (8 wk old) animals were used in this study. Expression levels of AFP in colons of different development stage were detected by reverse-transcriptase PCR (RT-PCR) and Western blotting. To identify the cell location of AFP in the developing rat colons, double-immunofluorescent staining was performed using antibodies to specific cell markers and AFP, respectively.

RESULTS: The highest levels of AFP mRNA were detected in colons of rats at embryonic day 18.5 (e18.5). Compared to e18.5 d, the AFP expression was significantly decreased during rat development [85% for e20.5, P < 0.05, 58% for postnatal day 0.5 (P0.5), P < 0.05, 37% for P7, P < 0.05, 24% for P14, P < 0.05, and 11% for P21, P < 0.05] and undetected in adult rats. Only the 72-kDa isoform of AFP was detected by Western blotting, the expression pattern was similar to AFP mRNA and conformed to the results of mRNA expression. The AFP positive staining was identical to different distribution patterns in fetuses, young and adult animals and positive staining for both AFP and vimentin was overlapped in mesenchymal cells at each stage tested.

CONCLUSION: This study has for the first time demonstrated that AFP is localized in the mesenchyme of rat colon from the embryo to the weaning stage by immunofluorescence and presents 72-kDa isoform in the developing rat colons by Western blotting. The dynamic expression of AFP in the various developmental stages of the colon indicates that AFP might be involved in many aspects of colon development.

AIM: To investigate the effect of quercetin (3,3’,4’,5,7-pentahydroxy flavone), a major flavonoid in human diet, on hyper-proliferation of gastric mucosal cells in rats treated with chronic oral ethanol.

METHODS: Forty male Sprague-Dawley rats, weighing 200-250 g, were randomly divided into control group (tap water ad libitum), ethanol treatment group (6 mL/L ethanol), quercetin treatment group (intragastric gavage with 100 mg/kg of quercetin per day), and ethanol plus quercetin treatment group (quercetin and 6 mL/L ethanol). Expression levels of proliferating cell nuclear antigen (PCNA) and Cyclin D1 were detected by Western blot to assay gastric mucosal cell proliferation in rats. To demonstrate the influence of quercetin on the production of extra-cellular reactive oxygen species/nitrogen species (ROS/RNS) in rats, changes in levels of thiobarbituric acid reactive substance (TBARS), protein carbonyl, nitrite and nitrate (NOx) and nitrotyrosine (NT) were determined. The activity of inducible nitric oxide synthase (NOS) including iNOS and nNOS was also detected by Western blot.

RESULTS: Compared to control animals, cell proliferation in the gastric mucosa of animals subjected to ethanol treatment for 7 days was significant increased (increased to 290% for PCNA density P < 0.05, increased to 150 for Cyclin D1 density P < 0.05 and 21.6 ± 0.8 vs 42.3 ± 0.7 for PCNA positive cells per view field), accompanied by an increase in ROS generation (1.298 ± 0.135 μmol vs 1.772 ± 0.078 μmol for TBARS P < 0.05; 4.36 ± 0.39 mmol vs 7.48 ± 0.40 mmol for carbonyl contents P < 0.05) and decrease in NO generation (11.334 ± 0.467 μmol vs 7.978 ± 0.334 μmol P < 0.01 for NOx; 8.986 ± 1.351 μmol vs 6.854 ± 0.460 μmol for nitrotyrosine P < 0.01) and nNOS activity (decreased to 43% P < 0.05). This function was abolished by the co-administration of quercetin.

CONCLUSION: The antioxidant action of quercetin relies, in part, on its ability to stimulate nNOS and enhance production of NO that would interact with endogenously produced reactive oxygen to inhibit hyper-proliferation of gastric mucosal cells in rats treated with chronic oral ethanol.

The aim of this study was to determine which human immunodeficiency virus type 1 (HIV-1) subtypes were circulating in Australia and to correlate the subtypes with risk factors associated with the acquisition of HIV-1 infection. DNA was extracted from peripheral blood mononuclear cells, and HIV-1 env genes were amplified and subtyped using heteroduplex mobility analysis, with selected samples sequenced and phylogenetic analysis performed. The HIV-1 env subtypes were determined for 141 samples, of which 40 were from female patients and 101 were from male patients; 13 samples were from children. Forty-seven patients were infected by homosexual or bisexual contact, 46 were infected through heterosexual contact, 21 were infected from injecting drug use (IDU), 13 were infected by vertical transmission, 8 were infected from nosocomial exposure, and 6 were infected by other modes of transmission, including exposure to blood products, ritualistic practices, and two cases of intrafamilial transmission. Five subtypes were detected; B (n = 104), A (n = 5), C (n = 17), E (CRF01_AE; n = 13), and G (n = 2). Subtype B predominated in HIV-1 acquired homosexually (94% of cases) and by IDU (100%), whereas non-subtype B infections were mostly seen in heterosexually (57%) or vertically (22%) acquired HIV-1 infections and were usually imported from Africa and Asia. Subtype B strains of group M viruses predominate in Australia in HIV-1 transmitted by homosexual or bisexual contact and IDU. However, non-B subtypes have been introduced, mostly acquired via heterosexual contact.