Isolation of differentially expressed genes in human heart tissues

We applied RNA arbitrarily primed-PCR (RAP-PCR) to screen the genes differentially expressed between common congenital heart defects (CHD) [atrial septal defect, ventricular septal defect, Tetrology of Fallot (TOF)] and normal human heart samples. Three of these differentially amplified fragments matched cDNA sequences coding for proteins of unknown function in humans: hCALO (human homologue of calossin), NP79 (coding for a nuclear protein of 79KD) and SUN2 (Sad-1 unc-84 domain protein 2). The other four fragments were from known human genes: apolipoprotein J, titin, dystrophin and protein kinase C-delta. Northern blot analysis confirmed that all of these genes are expressed in the human heart. The results of RAP-PCR were reconfirmed by quantitative RT-PCR in TOF and control heart samples. Both techniques showed the levels of expression of hCALO, NP79 and SUN2 to be comparable in TOF and control samples and the level of expression of dystrophin and titin, both coding for cytoskeletal proteins, to be significantly upregulated in TOF samples. In summary, we have shown that the RAP-PCR technique is useful in the identification of differentially expressed gene from biopsy samples of human CHD tissues. In this manner, we have identified three novel genes implicated in the normal function of the human heart and two known genes upregulated in TOF samples.     

Fasting and postprandial overproduction of intestinally derived lipoproteins in an animal model of insulin resistance. Evidence that chronic fructose feeding in the hamster is accompanied by enhanced intestinal de novo lipogenesis and ApoB48-containing lipoprotein overproduction

Insulin-resistant states are characterized by hypertriglyceridemia, predominantly because of overproduction of hepatic very low density lipoprotein particles. The additional contribution of intestinal lipoprotein overproduction to the dyslipidemia of insulin-resistant states has not been previously appreciated. Here, we have investigated intestinal lipoprotein production in a fructose-fed hamster model of insulin resistance previously documented to have whole body and hepatic insulin resistance, and hepatic very low density lipoprotein overproduction. Chronic fructose feeding for 3 weeks induced significant oversecretion of apolipoprotein B48 (apoB48)-containing lipoproteins in the fasting state and during steady state fat feeding, based on (a) in vivo Triton WR1339 studies of apoB48 production as well as (b) ex vivo pulse-chase labeling of intestinal enterocytes from fasted and fed hamsters. ApoB48 particle overproduction was accompanied by increased intracellular apoB48 stability, enhanced lipid synthesis, higher abundance of microsomal triglyceride transfer protein mass, and a significant shift toward the secretion of larger chylomicron-like particles. ApoB48 particle overproduction was not observed with short-term fructose feeding or in vitro incubation of enterocytes with fructose. Secretion of intestinal apoB48 and triglyceride was closely linked to intestinal enterocyte de novo lipogenesis, which was up-regulated in fructose-fed hamsters. Inhibition of fatty acid synthesis by cerulenin, a fatty acid synthase inhibitor, resulted in a dose-dependent decrease in intestinal apoB48 secretion. Overall, these findings further suggest that intestinal overproduction of apoB48 lipoproteins should also be considered as a major contributor to the fasting and postprandial dyslipidemia observed in response to chronic fructose feeding and development of an insulin-resistant state.     

Mating system of the rockfish, <Emphasis Type="Italic">Sebastiscus marmoratus</Emphasis> as revealed by DNA fingerprinting

The mating system of the viviparous rockfish, Sebastiscus marmoratus was studied using aquarium mating experiments and paternity testing by DNA fingerprinting. Individually specific DNA fingerprints were produced and the paternal relationships determined with the use of the restriction enzyme HinfI and the DNA probe (CAC)₅. Two types of mating experiments, multiple-male and single-male, were set up to investigate the effect of male sizes on their mating success, as determined by number of females fertilized. Male size was positively and significantly related to mating success in the multiple-male situation in which male–male interaction was present, but not in the single-male situation in which no male–male interaction was involved. Females produced one to seven batches of larvae, and all batches from individual females had identical paternity. Most females were fertilized by a single male but one case of multiple paternity was detected. The laboratory study suggested the principal mating system of S. marmoratus to be promiscuity, although multiple mating by females was uncommon.     

Immunization with attenuated Salmonella typhimurium producing catalase in protection against gastric Helicobacter pylori infection in mice

Aim. To evaluate the protective effect of live attenuated Salmonella typhimurium expressing catalase against gastric Helicobacter pylori infection in mice, and to explore the underlying mechanisms of the protective immune reaction. Materials and Methods The H. pylori catalase gene was introduced into attenuated S. typhimurium strain SL3261. C₅₇BL/6 mice were orally immunized with the SL3261 vaccine strain expressing catalase or with SL3261 alone or phosphate‐buffered saline (PBS). Mice were sacrificed 4 weeks after immunization and 5 weeks after H. pylori challenge, respectively. Results. All PBS control mice were infected. Eight of 13 (61.5%) mice immunized with the SL3261 vaccine strain and three of 14 (21%) mice immunized with SL3261 alone showed protection against H. pylori infection. Serum anti‐H. pylori IgG2a levels of S. typhimurium‐immunized mice were higher than those of PBS controls, both before and after H. pylori challenge, while there were no differences for IgG1 and IgA. Similarly, mRNA expression of interleukin (IL)‐2, IL‐12 and interferon‐γ in the gastric mucosa of S. typhimurium‐immunized mice was significantly higher than that of PBS controls both before and after challenge. Moreover, S. typhimurium‐immunized mice were characterized by marked infiltration of lymphocyte and mononuclear cells in the gastric mucosa after challenge. IL‐4 and IL‐10 were not detected in any of the three groups. IL‐6 expression was increased in the PBS group compared with the S. typhimurium‐immunized groups after challenge. Conclusions. This study demonstrates that oral immunization of mice with catalase delivered by an attenuated S. typhimurium strain offers protection against H. pylori infection. This protective immunity was mediated through a predominantly Th1‐type response and was associated with post‐immunization gastritis.     

TIP,a T-cell factor identified using high-throughput screening increases survival in a graft-versus-host disease model

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.     

A study of 110mAg in aquatic and terrestrial ecosystems

Experiments on a simulated terrestrial agricultural ecosystem were carried out using the pot culture approach. The most representative plants in local vegetable gardens were selected to investigate the root uptake of (110m)Ag. The results show that carrot, kale and flowering cabbage have the largest transfer factor values among the vegetables. Flowering cabbage, as the most popular leafy vegetable in Hong Kong and the South China area, can be used as a biomonitor for radioisotope contamination in vegetables. Soil column and adsorption tests were also carried out to study the leaching ability of the silver isotope in soil and (110m)Ag was mainly adsorbed in the top 1 cm of soil regardless of the pH value. Experiments on a simulated aquatic ecosystem for freshwater fish and marine organisms were carried out in glass aquaria. The freshwater fish Cyprinus carpio, the marine fish Cuvier and some local abundant seashore molluscs were selected to investigate the kinetic metabolism of (110m)Ag in the compartmental system. The results show that molluscs absorb (110m)Ag much more than fish. Clibanarius infraspinatus has the largest concentration factor among the marine organisms selected. Fish liver, although representing a minor portion of the total body mass, shows the highest (110m)Ag concentration factor, whereas muscle, although representing a major portion of the total body mass, is characterized by an absence of (110m)Ag.     

Development of a marine subtidal epibiotic community in Hong Kong: implications for deployment of artificial reefs

A 2-year study was conducted in Hong Kong to examine the effects of substratum, season and length of submersion on the development of a subtidal epibiotic community using four types of settlement panels (concrete, steel, wood and tyre). The season and length of submersion had a strong influence on the total biomass and on community structure while the type of substratum had very little impact on the total biomass or the structure of the epibiotic community. The season of submersion determined the species composition of the newly submerged surfaces. In the spring and summer, tubeworms were the most abundant. In the autumn and winter, barnacles and tunicates dominated. Community succession was not obvious in the first year of submersion as it was intermingled with strong seasonal settlement, growth and death of barnacles and tunicates. In the second year of submersion, green mussels and tunicates settled and grew to occupy most of the panel surfaces, forming an assemblage that was characteristic of climax communities in the local subtidal waters. The results suggest that the type of construction material has limited impact on the development of epibiotic communities on artificial reefs deployed in Hong Kong; the season of submersion may affect community structure in the early successional stage, but not the characteristics of the climax communities. This study indicates that the type of substratum should not be of concern when deploying artificial reefs in the subtidal waters in this region. The design of artificial reefs should focus more on other physical and economical aspects such as durability, flow dynamics, stability, cost, and effects on the ambient environment.