Characterization of photosynthetically active duckweed (<Emphasis Type="Italic">Wolffia australiana</Emphasis>) in vitro culture by Respiration Activity Monitoring System (RAMOS)

The feasibility of oxygen transfer rate (OTR) measurement to non-destructively monitor plant propagation and vitality of photosynthetically active plant in vitro culture of duckweed (Wolffia australiana, Lemnaceae) was tested using Respiration Activity Monitoring System (RAMOS). As a result, OTR proofed to be a sensitive indicator for plant vitality. The culture characterization under day/night light conditions, however, revealed a complex interaction between oxygen production and consumption, rendering OTR measurement an unsuitable tool to track plant propagation. However, RAMOS was found to be a useful tool in preliminary studies for process development of photosynthetically active plant in vitro cultures.     

Intercellular pectic protuberances in Asplenium: new data on their composition and origin

BACKGROUND AND AIMS: Projections of cell wall material into the intercellular spaces between parenchymatic cells have been observed since the mid-19th century. Histochemical staining suggested that these intercellular protuberances are probably pectic in nature, but uncertainties about their origin, composition and biological function(s) have remained. METHODS: Using electron and light microscopy, including immunohistochemical methods, the structure and the presence of some major cell wall macromolecules in the intercellular pectic protuberances (IPPs) of the cortical parenchyma have been studied in a specimen of the Asplenium aethiopicum complex. KEY RESULTS: IPPs contained pectic homogalacturonan, but no evidence for pectic rhamnogalacturonan-I or xylogalacturonan epitopes was obtained. Arabinogalactan-proteins and xylan were not detected in cell walls, middle lamellae or IPPs of the cortical parenchyma, whereas xyloglucan was only found in its cell walls. Extensin (hydroxyproline-rich glycoproteins) LM1 and JIM11 and JIM20 epitopes were detected specifically in IPPs but not in their adjacent cell walls or middle lamellae. CONCLUSIONS: It is postulated that IPPs do not originate exclusively from the middle lamellae because extensins were only found in IPPs and not in surrounding cell walls, intercellular space linings or middle lamellae, and because IPPs and their adjacent cell walls are discontinuous.     

Melanocyte differentiation antigen RAB38/NY-MEL-1 induces frequent antibody responses exclusively in melanoma patients

Expression pattern and immunogenicity are critical issues that define tumor antigens as diagnostic markers and potential targets for immunotherapy. The development of SEREX (serological analysis of recombinant expression libraries) has provided substantial progress in the identification of tumor antigens eliciting both cellular and humoral immune responses in cancer patients. By SEREX, we have previously identified RAB38/NY-MEL-1 as a melanocyte differentiation antigen that is highly expressed in normal melanocytes and melanoma tissues but not in other normal tissues or cancer types. In this study, we further demonstrate that RAB38/NY-MEL-1 is strongly immunogenic, leading to spontaneous antibody responses in a significant proportion of melanoma patients. The immune response occurs solely in malignant melanoma patients and was not detected in patients with other diseases, such as vitiligo, affecting melanocytes. Fine analysis of the spontaneous anti-RAB38/NY-MEL-1 antibody response reveals a polyclonal B cell recognition targeting various epitopes, although a dominant immunogenic region was preferentially recognized. Interestingly, our data indicate that this recognition is not rigid in the course of a patient’s response, as the dominant epitope changes during the disease evolution. Implications for the understanding of spontaneous humoral immune responses are discussed.Alfred Zippelius and Asma Gati contributed equally to this work.     

Scanning behavior by larvae of the predacious diving beetle, <Emphasis Type="Italic">Thermonectus marmoratus</Emphasis> (Coleoptera: Dytiscidae) enlarges visual field prior to prey capture

Larvae of the predaceous diving beetle Thermonectus marmoratus bear six stemmata on each side of their head, two of which form relatively long tubes with linear retinas at their proximal ends. The physical organization of these eyes results in extremely narrow visual fields that extend only laterally in the horizontal body plane. There are other examples of animals possessing eyes with predominantly linear retinas, or with linear arrangements of specific receptor types. In these animals, the eyes, or parts of the eyes, are movable and perform scanning movements to increase the visual field. Based on anatomical data and observations of relatively transparent, immobilized young larvae, we report here that T. marmoratus larvae are incapable of moving their eyes or any part of their eyes within the head capsule. However, they do perform a series of bodily dorso-ventral pivots prior to prey capture, behaviorally extending the vertical visual field from 2° to up to 50°. Frame-by-frame analysis shows that such behavior is performed within a characteristic distance to the prey. These data provide first insights into the function of the very peculiar anatomical eye organization of T. marmoratus larvae.     

Early detection and treatment of vesicular adenitis in bulls

The objective of this study was to determine whether early detection and treatment with tilmicosin would reduce the number of yearling bulls culled due to vesicular gland disease at the time of their first semen test. Bulls (n=2207) of 15 breeds at 17 performance test stations were examined by transrectal palpation at 9-12 months of age and assigned to three treatment groups: (1) positive treated group, receiving subcutaneous injections of tilmicosin every second day for three treatments (2) positive untreated control group, (3) negative untreated control group. Transrectal palpation of the glands was done again at a pre-sale semen test 28-70 d (mean=42.8 d) after the first examination. Semen was evaluated for the presence of pus and/or leukocytes by light microscopy. The proportion of bulls with vesiculitis was 4.4% (97/2207). At the pre-sale semen test the number of bulls with vesiculitis had decreased to 1.3% (29/2207); however, seven of these were new cases that developed after the first examination. Therefore, there was a recovery rate of 75/97 (77.3%) in the original group of bulls positive for vesiculitis. At the pre-sale test, there was no difference in the proportion of bulls with enlarged glands in the positive treated group (15/66) versus the positive untreated group (7/31) and no difference in the proportion of bulls with leukocytes or pus in their semen samples. The results of this experiment did not indicate any advantage in detection and treatment of vesiculitis before a year on age due to a high rate of spontaneous recovery.     

Sperm accumulation in the ampullae and cauda epididymides of bulls

Bulls that appear to have an abnormality of sperm transport accumulate large numbers of senescent sperm in the excurrent ducts of the reproductive tract. Six bulls that accumulated sperm were used to determine the number of physiological ejaculations required to deplete accumulated senescent sperm, semen traits during depletion, the period of time to re-accumulate senescent sperm after depletion, the sites of sperm accumulation, and the effect of sperm accumulation on fertility during natural service. Semen was collected from three bulls (HH, PH1, and CH1) three times daily using internal artificial vaginas placed in cows in estrus. These bulls started to produce semen with >or=70% morphologically normal sperm on the third, fifth, and seventh day, respectively. The percentage of live sperm increased from 5% to 68%, 5% to 63%, and 18% to 68% in HH, PH1, and CH1, respectively. Two weeks later the same bulls were electroejaculated every second day for five electroejaculations to deplete stores of senescent sperm. Each time, electroejaculation was continued until the semen produced had a dilute appearance. The three bulls re-accumulated senescent sperm after 1 month of sexual rest. After re-accumulation of senescent sperm, the total volumes of semen in both ampullae recovered at slaughter from HH, PH1, and CH1 were 5.0, 5.0, and 9.5 ml, respectively. The volumes of semen in ampullae recovered at slaughter from two control bulls (RA and CHC) were 1.7 and 1.9 ml, respectively. The number of sperm recovered from both cauda epididymides of HH, PH1, CH1, RA and CHC was 37.3x10(9), 23.3x10(9), 15.0x10(9), 6.9x10(9), and 7.4x10(9), respectively. Bull CH1 and a fourth bull (LM) that also accumulated sperm, started to produce semen with >or=70% morphologically normal sperm, and >or=60% progressively motile sperm on the third electroejaculation after depletion of senescent sperm by repeated electroejaculations. Pregnancy rates achieved by two bulls that accumulated senescent sperm (CH2 and PH2) were less (P<0.05) during the first week of a 21-day breeding period and there was a tendency for lesser pregnancy rates at the end of the breeding period when compared with two normal control bulls. The present study indicates that bulls that accumulate senescent sperm may achieve greater pregnancy rates approximately 1 week after beginning a period of frequent ejaculation. Re-accumulation of an increased percentage of senescent sperm would likely occur after 1 month of sexual rest.     

<Emphasis Type="Italic">pspA</Emphasis> overexpression in <Emphasis Type="Italic">Streptomyces lividans</Emphasis> improves both Sec- and Tat-dependent protein secretion

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its innate capacity to secrete proteins at high level in the culture medium. In this report, we evaluated the importance of the phage-shock protein A (PspA) homologue on the protein secretion yield in Streptomyces lividans. The PspA protein is supposed to play a role in the maintenance of the proton motive force (PMF). As the PMF is an energy source for both Sec- and Tat-dependent secretion, we evaluated the influence of the PspA protein on both pathways by modulating the pspA expression. Results indicated that pspA overexpression can improve the Tat-dependent protein secretion as illustrated for the Tat-dependent xylanase C and enhanced green fluorescent protein (EGFP). The effect on Sec-dependent secretion was less pronounced and appeared to be protein dependent as evidenced by the increase in subtilisin inhibitor (Sti-1) secretion but the lack of increase in human tumour necrosis factor (hTNFα) secretion in a pspA-overexpressing strain.     

A statistical light scattering approach to separating fast and slow dynamics

Light scattering is a powerful technique to study the structural and dynamical properties of biomolecular systems or other soft materials such as polymeric solutions and blends or gels. An important application of this technique is the study of the kinetics of formation of supramolecular structures. However, in such cases, the system under study is rapidly changing, and consequently the integration time for each measurement is limited. In order to overcome this difficulty, a statistical approach has been developed based on the analysis of the scattered light intensity distribution (Manno et al. 2006, 2004). Indeed the intensity distribution depends upon the ratio between the integration time of each measurement and the coherence time of scattered radiation. This method has been applied to protein aggregation (Manno et al. 2006) and to sol-gel transition (Manno et al. 2004), to obtain information on the heterogeneity of morphological and dynamical features during such processes. In the present work, we accurately test the validity of this approach by analyzing the statistical properties of the light scattered by a model system: a solution of polystyrene spherical macromolecules of different sizes. Each molecular size is related to a given diffusion coefficient and to a given coherence time of the scattered intensity. The effect of changing the experimental integration time is systematically investigated. A mixture of particles of two different sizes is also analyzed to test the validity and robustness of the model based on the convolution of a gaussian with an exponential distribution. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.