pp 835-843 Perspectives
Animal and human anatomy is among the most complex systems known, and suitable teaching methods have been of great importance in the progress of knowledge. Examining the human body is part of the process by which medical students come to understand living forms. However, the need to preserve cadavers has led to the development of various techniques to manufacture models for teaching purposes. A variety of materials, such as wax, wood, papier-mâché, or glass, have long been used to construct animal and plant models. In the case of the human body, the most innovative, yet controversial, method of preservation has been plastination, invented by the German physician Gunther von Hagens, by which actual human bodies are preserved as odourless and aesthetic models for teaching and exhibitions. We point out in our study that the ‘hands-on’ approach that some anatomical models allow, namely, the (clastic) disassembly and reassembly of the parts of complex systems and their models, is not only a crucial tool for learning, but is far superior to the simple passive observation that rigid, single-piece models allow. And what is valid for the learning of anatomy can be generalized to the acquisition of knowledge of other complex physical systems.
pp 845-848 Series
pp 849-852 Brief communication
In an attempt to see if frog blood vessels possess a plasma membrane electron transport system, the postcaval vein and aorta isolated from Rana tigrina were tested for their ability to reduce ferricyanide, methylene blue, and 2,6-dichloroindophenol. While the dyes remained unchanged, ferricyanide was reduced to ferrocyanide. This reduction was resistant to inhibition by cyanide and azide. Heptane extraction or formalin fixation of the tissues markedly reduced the capability to reduce ferricyanide. Denuded aortas retained only 30% of the activity of intact tissue. Our results indicate that the amphibian postcaval vein and aorta exhibit plasma membrane electron transport
pp 853-872 Articles
In a previous paper, we pointed out that the capability to synthesize glycine from serine is constrained by the stoichiometry of the glycine hydroxymethyltransferase reaction, which limits the amount of glycine produced to be no more than equimolar with the amount of C1 units produced. This constraint predicts a shortage of available glycine if there are no adequate compensating processes. Here, we test this prediction by comparing all reported fluxes for the production and consumption of glycine in a human adult. Detailed assessment of all possible sources of glycine shows that synthesis from serine accounts for more than 85% of the total, and that the amount of glycine available from synthesis, about 3 g/day, together with that available from the diet, in the range 1.5–3.0 g/day, may fall significantly short of the amount needed for all metabolic uses, including collagen synthesis by about 10 g per day for a 70 kg human. This result supports earlier suggestions in the literature that glycine is a semi-essential amino acid and that it should be taken as a nutritional supplement to guarantee a healthy metabolism.
pp 873-879 Articles
In this study, mouse mesoangioblasts were seeded onto bidimensional matrices within three-dimensional porous scaffolds of poly (L-lactic acid) (PLLA), in the presence or absence of a type I collagen coating. The cells were observed under a scanning electron microscope and tested for their adhesion, survival and proliferation. Immunolocalization of heat shock protein (Hsp) 70, an abundant and ubiquitous intracellular protein in these cells, was also performed in sectioned cell-containing scaffolds under a confocal fluorescence microscope to determine if in situ analysis of intracellular constituents was feasible. The data show that PLLA films allow direct cell adhesion and represent an optimal support for cell growth, and that the internal surfaces of PLLA polymeric sponges can be colonized by mesoangioblasts, which can be submitted for in situ confocal microscopic analyses for possible monitoring of timedependent expression of differentiation markers.
pp 881-890 Articles
Xylose reductase from the thermophilic fungus Talaromyces emersonii: cloning and heterologous expression of the native gene (Texr) and a double mutant (TexrK271R+N273D) with altered coenzyme specificity
Xylose reductase is involved in the first step of the fungal pentose catabolic pathway. The gene encoding xylose reductase (Texr) was isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli and purified to homogeneity. Texr encodes a 320 amino acid protein with a molecular weight of 36 kDa, which exhibited high sequence identity with other xylose reductase sequences and was shown to be a member of the aldoketoreductase (AKR) superfamily with a preference for reduced nicotinamide adenine dinucleotide phosphate (NADPH) as coenzyme. Given the potential application of xylose reductase enzymes that preferentially utilize the reduced form of nicotinamide adenine dinucleotide (NADH) rather than NADPH in the fermentation of five carbon sugars by genetically engineered microorganisms, the coenzyme selectivity of TeXR was altered by site-directed mutagenesis. The TeXRK271R+N273D double mutant displayed an altered coenzyme preference with a 16-fold improvement in NADH utilization relative to the wild type and therefore has the potential to reduce redox imbalance of xylose fermentation in recombinant S. cerevisiae strains. Expression of Texr was shown to be inducible by the same carbon sources responsible for the induction of genes encoding enzymes relevant to lignocellulose hydrolysis, suggesting a coordinated expression of intracellular and extracellular enzymes relevant to hydrolysis and metabolism of pentose sugars in T. emersonii in adaptation to its natural habitat. This indicates a potential advantage in survival and response to a nutrient-poor environment.
pp 891-898 Articles
Sticholysins I and II (St I/II) are cytolysins purified from the sea anemone Stichodactyla helianthus. In this study, we show their pharmacological action on guinea-pig and snail models in native and pH-denatured conditions in order to correlate the pharmacological findings with the pore-forming activity of both isoforms. In guinea-pig erythrocytes (𝑁 = 3), St II possessed higher haemolytic activity in comparison with St I and this activity was lost at an alkaline pH. In molluscan central neurons (𝑁 = 30), they irreversibly decreased the amplitude of the cholinergic response; St I (EC50 0.6 𝜇molL–1) was more potent than St II (EC50 > 6.6 𝜇molL–1) and they both increased the duration of the action potential; these effects were absent at an alkaline pH. In guinea-pig isolated atrium (𝑁 = 25), both increased the amplitude of the contraction force, but St II was more potent than St I (EC50 0.03 𝜇molL–1 and 0.3 𝜇molL–1, respectively) and this effect persisted at an alkaline pH. In summary, both cytolysins have neuroactive and cardioactive properties. The main mechanism in molluscan neurons seems to be associated with the cytolytic activity of these molecules, whereas in guinea-pig atrium, the existence of an additional pharmacological mechanism might be contributing to the observed effect.
pp 899-908 Articles
Glycosaminoglycans, especially heparin, are involved in various cell processes such as apoptosis, cell cycle control, platelet activation, capacitation, acrosome reaction and sperm decondensation. Heparin-binding proteins (HBPs) are essential constituents of human seminal fluid, which bind to sperm lipids containing the phosphorylcholine group and mediate the fertilization process. We utilized a proteomic set-up consisting of affinity chromatography, isoelectric focusing (IEF) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI TOF/MS) for protein analysis of human HBPs. We resolved 70 different spots on two-dimensional (2-D) gel and subsequently identified these proteins. Forty different types of proteins were identified. Functional analysis revealed that 38% of the proteins belonged to the enzyme category, 20% were involved in RNA processing and transcription, 18% in structure and transport function, and 16% in cell recognition and signal transduction. We also identified 8% of proteins with unknown functions, although their expression in seminal fluid has been documented. Proteins of seminal fluid that bind heparin may be directly involved in sperm capacitation and acrosome reaction (AR), which are the two critical steps for fertilization. This information on HBPs would be useful for identifying potential biomarkers of fertility in the near future.
pp 909-916 Articles
A number of therapeutic options are available for patients with prostate carcinoma till the time that the tumour is hormone dependent. However, no fully effective therapy is available for the treatment of androgen-independent prostate carcinomas. Antibodies directed at epitopes unique to or overexpressed on the cancer cells could be of therapeutic utility. A monoclonal antibody (Moab) 2C4 has been generated, which binds with cells of two androgenindependent prostate cancers, DU145 and PC3, and does not bind to peripheral blood leukocytes (PBLs) of healthy donors. This antibody, along with the previously developed Moab 730, kills 100% of both DU145 and PC3 cells in the presence of complement and does not have a deleterious effect on PBLs of healthy males. The anti-tumour action of the two antibodies prevents the establishment of DU145 cell tumour in nude mice in vivo. Moab 2C4 in combination with 730 has potential for use as therapy for androgen-independent cancers.
pp 917-925 Articles
Although cervical cancer is preventable with early detection, it remains the second most common malignancy among women. An understanding of how proteins change in their expression during a particular diseased state such as cervical cancer will contribute to an understanding of how the disease develops and progresses. Potentially, it may also lead to the ability to predict the occurrence of the disease. With this in mind, we aimed to identify differentially expressed proteins in the plasma of cervical cancer patients. Plasma from control, cervical intraepithelial neoplasia (CIN) grade 3 and squamous cell carcinoma (SCC) stage IV subjects was resolved by two-dimensional gel electrophoresis and the resulting proteome profiles compared. Differentially expressed protein spots were then identified by mass spectrometry. Eighteen proteins were found to be differentially expressed in the plasma of CIN 3 and SCC stage IV samples when compared with that of controls. Competitive ELISA further validated the expression of cytokeratin 19 and tetranectin. Functional analyses of these differentially expressed proteins will provide further insight into their potential role(s) in cervical cancer-specific monitoring and therapeutics.
pp 927-940 Articles
We studied the expression of inducible heat shock protein (HSP27, HSP72) and multidrug-resistance protein (MRP) in co-cultures of human colon carcinoma cell spheroids obtained from different grades of tumour with normal human colon epithelium, myofibroblast and endothelial cell monolayers. We also measured the influence of recombinant human transforming growth factor 𝛽1 (rhTGF-𝛽1) and camptothecin (CPT-11), added as single agents or in combination, on the levels of the HSPs, MRP, interleukin (IL)-6 and nitric oxide (NO).
An immunoblotting analysis with densitometry showed that rhTGF-𝛽1 and/or CPT-11 increased HSP27, HSP72 and MRP expression in tumour cells and myofibroblasts, as well as in co-cultures compared with appropriate controls. By contrast, in colonic epithelium, inhibition of HSPs and MRP was comparable with that of the control. In endothelial cells, HSP72 was undetectable.
Direct interaction of colon tumour spheroids with normal myofibroblasts caused a significant, tumour-grade dependent increase in IL-6 production. Production of IL-6 was significantly lowered by rhTGF-𝛽1 and/or CPT-11. Tumour cell spheroids cultivated alone produced larger amounts of NO than normal cells. In co-culture, the level of the radical decreased compared with the sum of NO produced by the monocultures of the two types of cells. rhTGF-𝛽1 and/or CPT-11 decreased NO production both in tumour and normal cell monocultures and their co-cultures. In conclusion, direct interactions between tumour and normal cells influence the expression of HSP27, HSP72 and MRP, and alter IL-6 and NO production. rhTGF-𝛽1 and/or CPT-11 may potentate resistance to chemotherapy by increasing HSP and MRP expression but, on the other hand, they may limit tumour cell spread by decreasing the level of some soluble mediators of inflammation (IL-6 and NO).
pp 941-951 Articles
An AGAMOUS (AG)-like gene, GbAGL2, was isolated from Gossypium barbadense and characterized. Alignment and phylogenetic analysis indicated that GbAGL2 shared high homology with AG-subfamily genes and belonged to a C-class gene family. DNA gel blot analysis showed that GbAGL2 belonged to a low-copy gene family. Reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) revealed that GbAGL2 was highly expressed in reproductive tissues including ovules and carpels, but barely expressed in vegetative tissues. In addition, GbAGL2 expression in a cotton cultivar XuZhou142 (wt) (XZ142, G. hirsutum L.) and its fibreless mutant XZ142 (fl) was examined. RNA in situ hybridization analysis indicated that GbAGL2 transcripts were preferentially restricted to outer ovule integuments, carpels and fibres. These expression patterns implied that GbAGL2 might participate in the development of the carpel and ovule. Furthermore, Arabidopsis transformation was performed and modifications occurred in flowers, and the silique length of transgenic plants also increased slightly, suggesting that the GbAGL2 gene may have a positive effect on the development of the ovary or ovule. Our findings suggest that GbAGL2 might not only specify the identity of floral organs but also play a potential key role in ovary or fibre development in cotton.
pp 953-962 Articles
Variations in transgene expression due to position effect and copy number are normalized when analysing and comparing the strengths of different promoters. In such experiments, the promoter to be tested is placed upstream to a reporter gene and a second expression cassette is introduced in a linked fashion in the same transfer DNA (T-DNA). Normalization in the activity of the test promoter is carried out by calculating the ratio of activities of the test and reference promoters. When an appropriate number of independent transgenic events are analysed, normalization facilitates assessment of the relative strengths of the test promoters being compared. In this study, using different modified versions of the Cauliflower Mosaic Virus (CaMV) 35S promoter expressing the reporter gene 𝛽-glucuronidase (gus) (test cassette) linked to a chloramphenicol acetyl transferase (cat) gene under the wild-type 35S promoter (reference cassette) in transgenic tobacco lines, we observed that cat gene expression varied depending upon the strength of the modified 35S promoter expressing the gus gene. The 35S promoter in the reference cassette was found to have been upregulated in cases where the modified 35S promoter was weaker than the wild-type 35S promoter. Many studies have been carried out in different organisms to study the phenomenon of transcriptional interference, which refers to the reduced expression of the downstream promoter by a closely linked upstream promoter. However, we observed a positive interaction wherein the weakened activity of a promoter led to upregulation of a contiguous promoter. These observations suggest that, in situations where the promoters of the test and reference gene share the same transcription factors, the activity of the test promoter can influence the activity of the reference promoter in a way that the test promoter’s strength is underestimated when normalized by the reference promoter.
pp 963-967 Articles
In most insect-pollinated flowers, pollinators cannot detect the presence of nectar without entering the flower. Therefore, flowers may cheat by not producing nectar and may still get pollinated. Earlier studies supported this ‘cheater flower’ hypothesis and suggested that the cost saving by cheater flowers could be the most predominant selective force in the evolution of nectarless flowers. Previous models as well as empirical studies have addressed the problem of optimizing the proportion of nectarless and nectarful flowers. However, there has been no attempt to optimize the investment in nectar production along with that in floral display. One of the key questions that arises is whether the floral display will evolve to be an honest indicator of nectar reward. We use a mathematical model to cooptimize the investments in nectar and floral display in order to achieve maximum reproductive success. The model assumes that pollinators rely on a relative rather than an absolute judgement of reward. A conspicuous floral display attracts naïve pollinators on the one hand and enhances pollinator learning on the other. We show that under these assumptions, plant–pollinator co-evolution leads to honest signalling, i.e. a positive correlation between display and reward.
pp 969-976 Articles
This study was conducted to test the hypothesis that spatial variations in soil microbial variables in a Thai rice paddy are accurately described by multivariate profiles of the soil bacterial communities. We found that community-level physiological profiles of soil bacterial communities could better describe the population density of Rhizoctonia solani in soil than the physicochemical profiles do. However, soil dehydrogenase levels were closely correlated with soil fertility (𝑃 < 0.05), and these were better described by the physicochemical profiles. Hence, the hypothesis was rejected, and we suspect that soil microbial variables react differently to the same physicochemical changes. The average population density of R. solani (35 colony-forming units/g dry soil) was relatively high in the soil we studied, and the soil fertility was found to be among the poorest in Thailand. The soil quality was comparable to the most degraded bare ground soil in an adjacent bioreserve in terms of Shannon diversity index based on the community-level physiological profile as well as values of soil fertility indices. Overall, the soil microbial and physicochemical indicators showed that the paddy soil needs to be supplemented with soil nutrients. Otherwise, R. solani may cause a significant reduction in rice production.
pp 977-990 Articles
This paper describes the results of a series of experiments conducted to unravel the patterns of sex expression and reproductive output in a fascinating species with high variation in sexuality. Commelina benghalensis L., an andromonoecious rainy season weed, bears male and bisexual flowers in axillary spathes of all the plants investigated. Bisexual flowers are of two types; chasmogamous (CH) and cleistogamous (CL). The former are borne on subaerial and the latter on subterranean shoots, in addition to those on aerial spathes. Three populations of the species, designated JU1, JU2 and JU3, were scanned for three consecutive years from 1996 to 1998, and the number and distribution of male, CH and CL flowers per plant were found to vary. The mere number of CH/CL flowers per plant is by itself not an accurate measure of mixed mating. It is necessary to confirm that CH flowers actually outcross and, if they do so, to what extent. Comparison of the pollen/ovule (P/O) ratio and percentage pollen germination on the stigmas of the CH and CL flowers have been used as indices of the pollination system. Confirmation of this was sought from the fruit and seed sets obtained after manual pollination of emasculated flowers with self- and cross-pollen. Results so obtained were compared with those of natural pollination. In the majority of CH flowers, the male and female reproductive phases (i.e. anther dehiscence and stigma receptivity) overlap, providing for self-pollination. However, two exceptions to this general behaviour were found in some plants of all the three populations. In some CH flowers, the female phase matures prior to anther dehiscence while in others, the anthers are sterile. Such plants, designated as variants 1 and 2, respectively, facilitate cross-pollination. While the CL flowers contribute to the production of selfed progeny, the variants of CH ones permit formation of outcrossed progeny, indicating a mixed mating strategy in C. benghalensis.
pp 991-994 Mini-review
pp 995-1003 Review
In the past 100 years, vaccination has contributed immensely to public health by preventing a number of infectious diseases. Attenuated, killed or part of the microorganism is employed to stimulate the immune system against it. Progress in biotechnology has provided protective immunity through DNA vaccines. In recent years, nanovaccine is a novel approach to the methodology of vaccination. Nanomaterials are delivered in the form of microspheres, nanobeads or micro-nanoprojections. Painless, effective and safe needle-free routes such as the intranasal or the oral route, or patches of microprojections to the skin are some of the approaches which are in the experimental stage at present but may have a great future ahead in nanovaccination.
Volume 42 | Issue 4