Formation of crystalloid inclusions in the small intestine of neonatal pigs: an immunocytochemical study using colloidal gold

Summary Enterocytes of the small intestine in 1-day-old suckling piglets contain numerous vesicles in the apical cytoplasm and a large granule located beneath the nucleus. Within the next 3 days, these granules transform into electron-dense crystalloid inclusions. These membrane-bound inclusions are up to 10 m in length and 1–2 m in diameter, and they are composed of electron-dense lamellae 3.9 nm apart. Postembedding immunocytochemistry, using rabbit anti-porcine IgG and goat anti-rabbit IgG conjugated to 10 nm colloidal gold, revealed that both the granules and the crystalloid inclusions contained a high concentration of maternal IgG. Although the IgG content of the crystalloid inclusions was detected on epoxy-embedded sections, the use of LR White resin resulted in a much higher density of labelling. Quantification of the labelling density showed that the concentration of IgG in the crystalloid inclusions was approximately ten times higher than that in the lumenal colostrum and approximately three times higher than that in the granules. These observations showed that there are at least three compartments involved in the accretion of IgG in the small intestine of neonatal piglets: smaller apical endocytotic vesicles, large subnuclear granules and crystalloid inclusions. The role of these compartments in maternal immunoglobulin absorption and in the acquisition of passive immunity has yet to be explored.     

Lymphatic and Angiogenic Candidate Genes Predict the Development of Secondary Lymphedema following Breast Cancer Surgery

The purposes of this study were to evaluate for differences in phenotypic and genotypic characteristics in women who did and did not develop lymphedema (LE) following breast cancer treatment. Breast cancer patients completed a number of self-report questionnaires. LE was evaluated using bioimpedance spectroscopy. Genotyping was done using a custom genotyping array. No differences were found between patients with (n = 155) and without LE (n = 387) for the majority of the demographic and clinical characteristics. Patients with LE had a significantly higher body mass index, more advanced disease and a higher number of lymph nodes removed. Genetic associations were identified for four genes (i.e., lymphocyte cytosolic protein 2 (rs315721), neuropilin-2 (rs849530), protein tyrosine kinase (rs158689), vascular cell adhesion molecule 1 (rs3176861)) and three haplotypes (i.e., Forkhead box protein C2 (haplotype A03), neuropilin-2 (haplotype F03), vascular endothelial growth factor-C (haplotype B03)) involved in lymphangiogensis and angiogenesis. These genetic associations suggest a role for a number of lymphatic and angiogenic genes in the development of LE following breast cancer treatment.     

Hepatitis B surface antigen immunopurification using a plant-derived specific antibody produced in large scale

This paper provides an evaluation of a plant-derived HBsAg-specific antibody in the immunopurification of the recombinant HBsAg for vaccine purposes. This plant-derived antibody was obtained from different batches of 100-200kg of tobacco leaves and coupled to Sepharose CL-4B with high efficiency. The plant-derived antibody immunoaffinity matrix purification behavior (elution capacity, antigen purity, purification cycles, and ligand leakage) was comparable to that of its mouse-derived monoclonal antibody homolog. This result supports the feasibility of using this plant-derived antibody for the immunopurification of the Hepatitis B surface antigen for human use, opening a new possibility to overcome the constrain of monoclonal antibody production in mice.     

Microgravity culture reduces apoptosis and increases the differentiation of a human colorectal carcinoma cell line

Our hypothesis is that rotation increases apoptosis in standard tissue culture medium at shear stresses of greater than approximately 0.3 dyn/cm2. Human MIP-101 poorly differentiated colorectal carcinoma cells were cultured for 6 d in complete medium in monolayers, on Teflon-coated nonadherent surfaces (static three-dimensional [3D]) or in rotating 3D cultures either in microgravity in low-earth orbit (3D microg) or in unit gravity on the ground (3D 1g). Apoptosis (determined morphologically), proliferation (by MIB1 staining), and the expression of epidermal growth-factor receptor (EGF-R), TGF-alpha, or TGF-beta were assessed by immunohistochemistry, while the expression of the differentiation marker carcinoembryonic antigen (CEA) was assessed on Western blots. Over the course of 6 d, static 3D cultures displayed the highest rates of proliferation and lowest apoptosis. This was associated with high EGF-R, TGF-alpha, and TGF-beta expression which was greater than that of a monolayer culture. Both rotated 3D lg and 3D microg cultures displayed lower expression of EGF-R, TGF-alpha, or TGF-beta and proliferation than that of monolayer or static 3D cultures. However, rotated 3D microg displayed significantly less apoptosis and greater CEA expression than rotated 3D 1g cultures. When rotated cultures of MIP-101 cells were grown uncler static conditions for another 3 d, proliferation increased and apoptosis decreased. Thus, rotation appears to increase apoptosis and decrease proliferation, whereas static 3D cultures in either unit or microgravity have less apoptosis, and reduced rotation in microgravity increases CEA expression.     

Cell cycle regulation of the microtubular cytoskeleton

The microtubular element of the plant cytoskeleton undergoes dramatic architectural changes in the course of the cell cycle, specifically at the entry into and exit from mitosis. These changes underlie the acquisition of specialized properties and functions involved, for example, in the equal segregation of chromosomes and the correct positioning and formation of the new cell wall. Here we review some of the molecular mechanisms by which the dynamics and the organization of microtubules are regulated and suggest how these mechanisms may be under the control of cell cycle events.     

Stable individual differences in separation calls during early development in cats and mice

Background

The development of ethologically meaningful test paradigms in young animals is an essential step in the study of the ontogeny of animal personality. Here we explore the possibility to integrate offspring separation (distress) calls into the study of consistent individual differences in behaviour in two species of mammals, the domestic cat (Felis silvestris catus) and the mound-building mouse (Mus spicilegus). Such vocal responses in young mammals are a potentially useful test option as they represent an important element of mother-offspring communication with strong implications for offspring survival. In addition, the neural control of vocalisation is closely associated with emotional state.

Results

We found marked similarities in the pattern of individual responses of the young of both species to separation from their mother and littermates. In the domestic cat as well as in the mound-building mouse, individual differences in the frequency of calls and to a lesser extent in locomotor activity were repeatable across age, indicating the existence of personality types. Such consistencies across age were also apparent when only considering relative individual differences among litter siblings. In both species, however, individual patterns of vocalisation and locomotor activity were unrelated. This suggests that these two forms of behavioural responses to isolation represent different domains of personality, presumably based on different underlying neurophysiological mechanisms.

Conclusions

Brief separation experiments in young mammals, and particularly the measurement of separation calls, provide a promising approach to study the ontogeny of personality traits. Future long-term studies are needed to investigate the association of these traits with biologically meaningful and potentially repeatable elements of behaviour during later life.

     

The Wound Healing and Antibacterial Activity of Five Ethnomedical Calophyllum inophyllum Oils: An Alternative Therapeutic Strategy to Treat Infected Wounds

Background

Calophyllum inophyllum L. (Calophyllaceae) is an evergreen tree ethno-medically used along the seashores and islands of the Indian and Pacific Oceans, especially in Polynesia. Oil extracted from the seeds is traditionally used topically to treat a wide range of skin injuries from burn, scar and infected wounds to skin diseases such as dermatosis, urticaria and eczema. However, very few scientific studies reported and quantified the therapeutic properties of Calophyllum inophyllum oil (CIO). In this work, five CIO from Indonesia (CIO1), Tahiti (CIO2, 3), Fiji islands (CIO4) and New Caledonia (CIO5) were studied and their cytotoxic, wound healing, and antibacterial properties were presented in order to provide a scientific support to their traditional use and verify their safety.

Methods

The safety of the five CIO was ascertained using the Alamar blue assay on human keratinocyte cells. CIO wound healing properties were determined using the scratch test assay on human keratinocyte cells. CIO-stimulated antibacterial innate immune response was evaluated using ELISA by measuring β defensin-2 release in human derivative macrophage cells. CIO antibacterial activity was tested using oilogramme against twenty aerobic Gram- bacteria species, twenty aerobic Gram+ bacteria species, including a multi-drug resistant Staphylococcus aureus strain and two anaerobic Gram+ bacteria species e.g. Propionibacterium acnes and Propionibacterium granulosum. To detect polarity profile of the components responsible of the antibacterial activity, we performed bioautography against a Staphylococcus aureus strain.

Results

Based on Alamar Blue assay, we showed that CIO can be safely used on keratinocyte cells between 2.7% and 11.2% depending on CIO origin. Concerning the healing activity, all the CIO tested accelerated in vitro wound closure, the healing factor being 1.3 to 2.1 higher compared to control when keratinocytes were incubated after scratch with CIO at 0.1%. Furthermore, our results showed that CIO exhibit two distinct antibacterial effects: one against Gram+ bacteria by direct inhibition of mitotic growth and another potent effect against Gram- bacteria due to increased release of β-defensin 2 peptide by macrophages. Interestingly, the needed concentrations of CIO to inhibit bacteria growth and to promote wound healing are lower than concentrations exhibiting cytotoxic effects on keratinocyte cells. Finally, we performed bioautography assay against Staphylococcus aureus to determine polarity profile of the components responsible for CIO antibacterial activity. Our results showed for the five tested CIO that components responsible of the bacterial growth inhibition are the more polar one on the TLC chromatographic profile and are contained in the resinous fraction of the oil.

Conclusions

This study was conducted to evaluate cytotoxicity, wound healing and antibacterial properties of five CIO traditionally used to treat infected wounds. Using cell and bacteria cultures, we confirmed the pharmacological effects of CIO as wound healing and antimicrobial agent. Moreover, we showed that concentration of CIO needed to exhibit therapeutic effects are lower than concentrations exhibiting cytotoxic effects in vitro. For the first time, this study provides support for traditional uses of CIO. These wound healing and antibiotic properties make CIO a valuable candidate to treat infected wounds especially in tropical areas.     

Arabidopsis kinetochore fiber-associated MAP65-4 cross-links microtubules and promotes microtubule bundle elongation

The acentrosomal plant mitotic spindle is uniquely structured in that it lacks opposing centrosomes at its poles and is equipped with a connective preprophase band that regulates the spatial framework for spindle orientation and mobility. These features are supported by specialized microtubule-associated proteins and motors. Here, we show that Arabidopsis thaliana MAP65-4, a non-motor microtubule associated protein (MAP) that belongs to the evolutionarily conserved MAP65 family, specifically associates with the forming mitotic spindle during prophase and with the kinetochore fibers from prometaphase to the end of anaphase. In vitro, MAP65-4 induces microtubule (MT) bundling through the formation of cross-bridges between adjacent MTs both in polar and antipolar orientations. The association of MAP65-4 with an MT bundle is concomitant with its elongation. Furthermore, MAP65-4 modulates the MT dynamic instability parameters of individual MTs within a bundle, mainly by decreasing the frequency of catastrophes and increasing the frequency of rescue events, and thereby supports the progressive lengthening of MT bundles over time. These properties are in line with its role of initiating kinetochore fibers during prospindle formation.     

A comparison of periodization models during nine weeks with equated volume and intensity for strength

The purpose of the present investigation was to determine if significant differences exist among 3 different periodization programs in eliciting changes in strength. Twenty-eight recreationally trained college-aged volunteers (mean +/- SD; 22.29 +/- 3.98) of both genders were tested for bench press, leg press, body fat percentage, chest circumference, and thigh circumference during initial testing. After initial testing, subjects were randomly assigned to 1 of 3 training groups: (a) linear periodization (n = 9), (b) daily undulating periodization (n = 10), or (c) weekly undulating periodization (n = 9). The training regimen for each group consisted of a 9-week, 3-day-per-week program. Training loads were assigned as heavy (90%, 4 repetition maximum [4RM]), medium (85%, 6RM), or light (80%, 8RM) for bench press and leg press exercises. Subjects were familiarized with the CR-10 rated perceived exertion scale and instructed to achieve an 8 or 9 on the final repetition of each set for all other exercises. Subjects were then retested after 4 weeks of training. Training loads were then adjusted according to the new 1RM. Subjects were then retested after 5 more weeks of exercise. For all subjects, significant (p < 0.05) increases in bench press and leg press strength were demonstrated at all time points (T1-T3). No significant differences (p > 0.05) were observed between groups for bench press, leg press, body fat percentage, chest circumference, or thigh circumference at all time points. These results indicate that no separation based on periodization model is seen in early-phase training.