Further Studies on Antigenic Changes in Trypanosoma lewisi*

SYNOPSIS. An investigation of day-to-day changes in adult form antigens of T. lewisi in the rat shows that adult antigens, 2 of which are present in trace amounts in young forms, suddenly appear in large amounts in 9-day-old trypanosomes. An additional antigen is formed on the 13th day. In addition, 2-day-old trypanosomes contain some antigens not present in 6-day-old forms. The combined results of the present paper and a previous one indicate that there are antigenically 3 distinct stages of T. lewisi in the rat: 1- to 3-day-old, 4- to 8-day-old, and 9- to 14-day-old forms. From 9- to 11-day-old trypanosomes, there is a form containing both young and adult antigens.     

Chlorophyll accumulation and breakdown in light-grown barley transferred to darkness: effect of seedling age

Diurnally grown barley (Hordeum vulgare L. cv. Clipper) seedlings of various ages (3–4, 5–6 and 10–11-days-old) were transferred to darkness for 17 h and changes in leaf fresh weight, chlorophyll a, chlorophyll b and protochlorophyllide measured. The results were consistent with previous evidence of a light-independent chlorophyll biosynthetic pathway in light-grown barley. There was a net gain in chlorophyll (μg leaf^-1) in 5–6- and 10–11-day-old plants after 17 h dark treatment. The amounts of chlorophyll that accumulated were similar (5.9 and 4.3 μg Chl leaf^-1), despite a twofold difference in leaf size at T₀. The rate of leaf expansion in 5–6-day-old plants greatly exceeded the rate of chlorophyll accumulation and leaves were noticeably paler after dark treatment i.e. there was a reduction in chlorophyll concentration (μg g fresh weight^-1) in spite of an increase in chlorophyll content (μg leaf^-1). The ability of light-grown barley to accumulate chlorophyll in darkness was a function of seedling age. Very young seedlings (3–4-day-old) generally lost chlorophyll in darkness. The decrease in chlorophyll per leaf resulted mainly from loss of chlorophyll b. Preferential loss of chlorophyll b resulted in dramatic increases in the chlorophyll a:b ratio. Since 3–4-day-old seedlings (1) accumulated 5-aminolevulinic acid in the presence of levulinic acid at a rate comparable to older seedlings, and (2) converted exogenous 5-aminolevulinic acid to chlorophyll in the absence of light, it is unlikely that failure of the youngest plants to accumulate chlorophyll in darkness was due to blocks at these steps in the pathway. Net loss of chlorophyll (μg leaf^-1) in 3–4-day-old seedlings in darkness was eliminated by the addition of chloramphenicol, which occasionally produced a small, but significant, gain in total chlorophyll. These results imply that chlorophyll degradation in young barley leaves is strongly influenced by the chloroplast genome, and is a major factor influencing changes in chlorophyll levels in darkness. The present findings are consistent with the suggestion that the failure of 3–4-day-old barley seedlings to accumulate chlorophyll in darkness may be due to chlorophyll turnover in which the rate of degradation exceeds the rate of synthesis.     

Effects of Extremely Low-Frequency Electromagnetic Fields on Helicobacter pylori Biofilm

The aim of this work was to investigate the effects of exposure to extremely low-frequency electromagnetic fields (ELF-EMF) both on biofilm formation and on mature biofilm of Helicobacter pylori. Bacterial cultures and 2-day-old biofilm of H. pylori ATCC 43629 were exposed to ELF-EMF (50 Hz frequency–1 mT intensity) for 2 days to assess their effect on the cell adhesion and on the mature biofilm detachment, respectively. All the exposed cultures and the respective sham exposed controls were studied for: the cell viability status, the cell morphological analysis, the biofilm mass measurement, the genotypic profile, and the luxS and amiA gene expression. The ELF-EMF acted on the bacterial population during the biofilm formation displaying significant differences in cell viability, as well as, in morphotypes measured by the prevalence of spiral forms (58.41%) in respect to the controls (33.14%), whereas, on mature biofilm, no significant differences were found when compared to the controls. The measurement of biofilm cell mass was significantly reduced in exposed cultures in both examined experimental conditions. No changes in DNA patterns were recorded, whereas a modulation in amiA gene expression was detected. An exposure to ELF-EMF of H. pylori biofilm induces phenotypic changes on adhering bacteria and decreases the cell adhesion unbalancing the bacterial population therefore reducing the H. pylori capability to protect itself.     

ACETYLCHOLINESTERASE IN DEVELOPING CHICK EMBRYO BRAIN

–Acetylcholinesterase has been assayed at different stages of development to see whether changes in the activity of this enzyme are correlated in any way with the ontogenesis of electrical activity in the brain of growing chick embryo. The specific activity of the enzyme was highest in the synaptosomal fraction of the brain. The activity of the enzyme increased progressively with the age of the embryo. There were three isozymic forms of the enzyme in the 6-day-old embryo brain. A new isozyme appeared around the 9th day. The K_m values of the enzyme for acetylthiocholine from 6- and 20-day-old embryo brains were 6.5 ± 10^-5m and 3.3 ± 10^-5m respectively. Enzyme preparations from 6-day-old embryos were found to lose 50 per cent of their activity when heated at 50°C for 10 min. Under similar conditions the loss in activity in 18-day-old embryo brain enzyme was 22 per cent.     

Polyamine content in Arabidopsis thaliana (L.) Heynh during recovery after low and high temperature treatments

Comparative studies on the effect of temperature treatment on the endogenous polyamine content in wild type and the ethylene insensitive mutant eti5 of Arabidopsis thaliana (L.) Heynh were performed. The levels of free and conjugated putrescine, spermidine and spermine were measured in rosette leaves of 38-day-old plants subjected to low and high temperature for 24 h in darkness. Data for fractions measured in treated wild type plants during recovery suggest that alterations in polyamine levels may be a consequence of the conversion of the supernatant-bound into free form and vice versa, while in treated eti5 plants de novo synthesis of spermidine and spermine could not be excluded. It was found that high temperature provoked more significant changes in polyamine levels than low temperature. The results suggest that the eti5 mutant showed a better ability to recover after the temperature treatments than wild type partly as a consequence of changes in polyamine content.     

MITOTIC ACTIVITY OF THE PARS INTERMEDIA IN THE FEMALE MOUSE: AGE-ASSOCIATED VARIATIONS IN PROLIFERATION RATE AND CIRCADIAN PERIODICITY

We previously reported daily variations in the mitotic activity of the endocrine cells in the pars intermedia of 21- and 28-day-old male mice. Since cellular proliferation might be affected by factors such as sex and age, we undertook the present experiments to study the mitotic activity of the pars intermedia from 14-, 28-, and 150-day-old female mice. Inbred C3H/S mice, grouped according to age, were housed under standard conditions (12h each of light and dark [LD 12:12]) for periodicity analysis and were killed in lots of 5–11 animals every 4h over a single 24h cycle, with each mouse receiving 2 μg/g of colchicine 4h before decapitation. Pituitaries were excised, extracted, fixed in buffered formaldehyde, embedded in celloidin-paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. We counted the total number of nuclei to estimate the total number of cells monitored and then calculated the mitotic index (metaphases/1000 nuclei). Differences were analyzed for statistical significance by the Student t test. While the 14-day-old animals manifested no significant changes in mitotic activity during the 24h cycle, the 28- and 150-day-old mice showed higher mitotic indices during the period of darkness. The average mitotic activity over the entire cycle, however, was higher in the two groups of younger animals than in the 150-day-old mice. Moreover, the averages for the 28-day-old females were higher than the corresponding values previously reported by us for male mice of the same age. (Chronobiology International, 17(6), 751–756, 2000)     

Abiotic factors affecting the infectivity of Steinernema carpocapsae (Rhabditida: Steinernematidae) on larvae of Anastrepha obliqua (Diptera: Tephritidae)

The effects of soil depth, soil type and temperature on the activity of the nematode Steinernema carpocapsae (Filipjev) were examined using larvae of the West Indian fruit fly, Anastrepha obliqua (Macquart). Bioassays involved applying infective juveniles (IJs) to the surface of sterilized sand in PVC tubes previously inoculated with fly larvae of two ages. The 50% lethal concentration (LC₅₀) values estimated for 6-day-old larvae were 9, 20 and 102 IJs/cm² in tubes containing 2, 5 and 8 cm depth of sand, respectively, whereas for 8-day-old larvae, LC₅₀ values were 16, 40 and 157 IJs/cm², respectively. The effect of soil texture on the activity of S. carpocapsae was tested by applying the corresponding LC₅₀ concentrations of nematodes to sand, sand–clay and loamy–sand soils. For 6-day-old larvae, soil type had a highly significant effect on infection with the highest percentages of infection observed in the sand–clay mixture (60–82% depending on depth) compared to 45–64% infection in sand and 23–30% infection in loamy–sand soil. A very similar pattern was observed in 8-day-old larvae except that infection rates were significantly lower than in younger larvae. There was a significant interaction between soil type and soil depth. The effect of three temperatures (19, 25 and 30°C) on infection was examined in sand–clay soil. The infectivity of S. carpocapsae was affected by temperature and soil depth and by the interaction of these two factors. Response surface analysis applied to second order multiple linear regression models indicated that the optimal temperature for infection of larvae of both ages was ~26°C, at a depth of 7.9 cm for 6-day-old larvae and <2 cm for 8-day-old larvae, resulting in a predicted 91.4% infection of 6-day-old larvae and 61.2% infection of 8-day-old larvae. These results suggest that S. carpocapsae may have the potential to control fruit fly pests in tropical ecosystems with warm temperatures and high soil moisture levels, although this assertion requires field testing.     

Effect of Adult Experience on the Host-Location Behavior of the Aphid ParasitoidAphidius colemaniViereck (Hymenoptera: Aphidiidae)

We investigated the relationship between age and learning ability of femaleAscogaster reticulatusWatanabe, an egg-larval parasitoid of the smaller tea tortrixAdoxophyessp. (Lepidoptera: Tortricidae). Females 1, 3, 5, 7, 9, 11, and 13 days old had higher fecundity than 0-day-old females and could associate tea leaf extract with the host egg mass through oviposition. Experienced females 1 ≈ 13 days old walked more than 10 cm along an extract-treated line while exhibiting the antennating response. However, the learned responses of 0-day-old females was significantly lower [mean = 1.4 ± 0.8 (S. E.) cm] than that of older females (1 ≈ 13 days). In considering the relation between fecundity and the learned response, a low response in 0-day-old females could be due to a low propensity by them to learn cues associated with host eggs. Average longevity was 12.0 ± 1.5 days, but even 13-day-old females could oviposit a large number of eggs and learn to associate the tea leaf extract with host presence. This suggests that females can learn as long as they are able to oviposit. This learning ability would be advantageous forA. reticulatusfemales throughout their lives.     

Heart-type fatty acid binding proteins are upregulated during terminal differentiation of mouse cardiomyocytes,as revealed by proteomic analysis

At birth, the cardiomyocytes in the mouse neonatal heart still retain their ability to proliferate. However, this lasts only a few days and then the cardiomyocytes irreversibly lose their potential to divide. It is still not fully understood what factors are involved in the cessation of cardiomyocyte proliferation. Using proliferating cell nuclear antigen (PCNA) antibodies, we established that cardiomyocytes could divide extensively in 2-day-old mouse neonatal hearts and to a lesser extent in 6-day-old hearts. By 13 days, the cardiomyocytes have mostly stopped dividing. Comparative two-dimensional gel electrophoresis (2-DE) was performed on total proteins extracted from the 2-day- and 13-day-old hearts, in order to identify peptides that might be involved in the inhibition of cardiomyocyte proliferation. Using matrix-assisted laser desorption ionization mass spectroscopy (MALDI-TOF), we identified two protein spots that have the same molecular weight (approximately 14 kDa) but different pIs (5.9 and 6.1). Mass spectra analysis determined the proteins to be isoforms of the heart-type fatty acid binding protein (H-FABP). The pI 6.1 H-FABP is also known as mammary-derived growth inhibitor (MDGI; Specht et al. 1996). MGDI is a breast tumour growth suppressor gene capable of inhibiting tumour cell proliferation (Huynh et al. 1995). Both H-FABP isoforms were expressed in 2-day-old hearts but became strongly upregulated in 13-day-old hearts. We examined whether H-FABPs and PCNA were coexpressed in 2-, 6- and 13-day-old heart histological sections, using MDGI antibodies. The antibody could detect both forms of H-FABPs. It was established that there was a correlation between an increase in H-FABP expression and a decrease in PCNA expression. Hence, we tentatively propose that H-FABP isoforms are involved in regulating cardiomyocyte growth and differentiation in mouse neonatal hearts.This project was supported by a grant from the National Natural Science Foundation of China (Project No. 30340038).     

Cecal Propionic Acid as a Biological Indicator of the Early Establishment of a Microbial Ecosystem Inhibitory toSalmonellain Chicks

Experiments were conducted to evaluate the effects of different competitive exclusion (CE) cultures on the concentration of cecal propionic acid in 3-day-old broiler chicks, and the correlation between cecal propionic acid concentration and protection againstSalmonellacolonization. CE cultures that significantly (P < 0.05) increased cecal propionic acid in 3-day-old chicks decreased (P < 0.05) cecalSalmonellacolonization in 10-day-old chicks compared with the untreated controls. CE cultures that failed to significantly (P > 0.05) increase cecal propionic acid concentrations in 3-day-old chicks failed to protect (P > 0.05) against cecalSalmonellacolonization in 10-day-old chicks compared with untreated controls. A significant (P < .05) correlation (−.88) was found between cecal propionic acid concentration in 3-day-old chicks and cecalSalmonellacolonization in 10-day-old chicks.     

Protective effect of bone marrow and spleen suspensions on radiation-induced leukemogenesis in C57BL/6 mice

Summary The present experiments are an attempt to precise the type and localization of the cells involved in the protective effect of hemopoietic suspensions against the radiation-induced thymic lymphosarcoma (TLS) of C57BL/6 mice. Inocula containing variable numbers of BM or spleen CFUs from 60-day-old and 360-day-old donors were tested. According to their origin, the suspensions differed with respect to the CFU replication rate, the CFU ability to differentiate towards the T lineage and the content of the suspensions in thymic precursors. Two levels of inhibition were observed: BM suspensions from 60-day-old donors containing 1,500 CFUs had the best protective effect: 14.5% of TLS; 1,500 CFUs from 360-day-old donors were slightly but not significantly less efficient (28.5%). The second level of inhibition (36–46% of TLS) was obtained with all the following inocula:a) 1,200 and 300 spleen CFUs or 300 and 95 BM CFUs from 60-day-old donors,b) 1,500 spleen CFUs from aged donors. Seventy-six spleen CFUs from 60-day-old donors, 120 BM or 175 spleen CFUs from aged donors had no effect. These results suggest that in addition to the high replication rate of the BM CFUs as compared with spleen CFUs, cells endowed with an optimal protective effect are present in BM suspensions and are either absent or present in very small amount in spleen suspensions. These cells which induce an early repopulation of the thymus might correspond to thymic precursors.     

Scanning-Beam Electron Microscopy of Mycoplasma pneumoniae

The morphology and the existence of a growth cycle of Mycoplasma pneumoniae have not been clearly established. There is disagreement as to whether this organism exists as a spherical or filamentous form, and whether it progresses from filamentous to spherical forms as the organism ages. A scanning-beam electron microscope (SEM) was utilized to provide detailed observations of the cycle of morphological changes during growth phases of M. pneumoniae. Cultures of cells grown and fixed in liquid suspension displayed morphological changes from spherical to filamentous and then to larger round forms. After 8 hr to 2 days of growth (phase I), spherical forms and aggregates were revealed. Two- to 6-day-old growth (phase II) was composed of both straight and branching filaments with bulbous elements situated at intervals along their lengths, and microcolonies composed predominantly of intertwined filaments. Six- to 10-day-old growth (phase III) was characterized by flattened, spherical organisms larger than those observed in phase I, occasional membranes or ghosts, and a paucity of aggregates or microcolonies. Thus, stereo-scan electron microscopic studies suggest that M. pneumoniae undergoes an orderly and sequential metamorphosis during its life cycle.     

Acquisition of symbiotic dinoflagellates (Symbiodinium) by juveniles of the coral Acropora longicyathus

Scleractinian corals may acquire Symbiodinium from their parents (vertically) or from the environment (horizontally). In the present study, adult colonies of the coral Acropora longicyathus from One Tree Island (OTI) on the southern Great Barrier Reef (Australia) acquired two distinct varieties of symbiotic dinoflagellates (Symbiodinium) from the environment. Adult colonies had either Symbiodinium from clade C (86.7%) or clade A (5.3%), or a mixture of both clades A and C (8.0% of all colonies). In contrast, all 10-day-old juveniles were associated with Symbiodinium from clade A, while 83-day-old colonies contained clades A, C and D even though they were growing at the same location. Symbiodinium from clade A were dominant in both 10- and 83-day-old juveniles (99 and 97% of all recruits, respectively), while clade D was also found in 31% of 83-day-old juveniles. Experimental manipulation also revealed that parental association (with clade A or C), or the location within the OTI reef, did not influence which clade of symbiont was acquired by juvenile corals. The differences between the genetic identity of populations of Symbiodinium resident in juveniles and adult A. longicyathus suggest that ontogenetic changes in the symbiosis may occur during the development of scleractinian corals. Whether or not these changes are due to host selective processes or differences in the physical environment associated with juvenile versus adult colonies remains to be determined.     

Fine Structure of the A Cells of the Pars Intercerebralis of Normal and Gamma-irradiated Female Milkweed Bug,Oncopeltus fasciatus (Heteroptera: Lygaeidae)

Neurosecretory A cells in the pars intercerebralis of O. fasciatus were identified at the ultrastructural level. Fine structural study of the A cells of female insects during the period of production of the first batch of eggs revealed that these cells undergo qualitative and quantitative changes during the first eight days of adult life. The A cells appear to be inactive in the newly emerged females. There is a significant depletion of neurosecretory granules (NSG) in the perikarya in the 2-day-old females followed by an increase and decrease in the 4- and 6-day-old females, respectively. The A cells in the 8-day-old females showed an accumulation of NSG. The mitochondrial population increases after adult emergence reaching a peak in the 4-day-old females, and then declines gradually in the subsequent age groups. The effect of sublethal dose of gamma radiation on the ultrastructure of the A cells was also investigated. Radiation damage is evident as early as four hours after irradiation with 10 kR. There is considerable swelling of the lysosomes and the membrane system in the A cells of irradiated insects. Synthetic activity of the A cells, based on the distribution of the endoplasmic reticulum, Golgi complex and mitochondria, as well as release of the NSG are apparently inhibited after irradiation. Radiation-induced damage becomes more conspicuous as post-irradiation interval increases. It is surmised that the damage to the A cells might have contributed at least in part, to the failure of yolk deposition in the irradiated insects.     

Studies on fungi in the root region

Summary Data are given on the fungi isolated from 30-day-old adventitious roots and seedling roots of severalAllium species by means of the root-washing technique. These fungi were typical root-surface forms includingCylindrocarpon, Fusarium, and sterile forms. The amount of root colonization observed, however, was not so great as with other higher plant roots.The nature and role of bulb surface contaminants is discussed in relation to the colonization of young roots, and is compared with the role of seed-coat contaminants in such colonization.     

Prostaglandin F2a inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F_2a (PGF_2a. The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF₂. A dose-dependent inhibition by PGF_2a (0.5–50 μM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165–165 μM). The stimulation by epinephrine on progesterone production was inhibited by PGF_2a (5 μM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF_2a can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF_2a shows in this respect the same agedependent inhibitory pattern as in relation to LH stimulation.     

Flight activity in the parasitoid waspNasonia vitripennis (Hymenoptera: Pteromalidae)

Flight activity in females of the parasitoid wasp Nasonia vitripennis(Walker) was examined by measuring still-air tethered flight. There was a large amount of variation among females in flight duration. The longest single flight (with no pauses of more than 5 s) was more than 2 h long. Mating status had a significant and large effect on flight: mated females flew twice as long as virgin females. There also was a slight but significant effect of age on flight, with 3-day-old females being less likely to fly than 1-day-old females. Flight duration was not affected by prior exposure to other females, to honey, or to a low or a high host density.     

Neural influence on the expression of acetylcholinesterase molecular forms in fast and slow rabbit skeletal muscles

With the aim of investigating the roles of motor innervation and activity on muscle characteristics, we studied the molecular forms of acetylcholinesterase (AChE) in fast-twitch (semimembranosus accessorius; SMa) and slow-twitch (semimembranosus proprius; SMp) muscles of the rabbit. We have shown that SMa and SMp express different patterns and tissue distribution of AChE forms and that the effect of long denervation varies with age. Three principal findings concerning expression of AChE molecular forms emerge from these studies. (1) The activity of AChE and the pattern of its molecular forms are particularly altered in adult denervated SMa and SMp muscles. AChE activity increases by 10-fold in both muscles, but asymmetric forms disappear in SMa and increase by 20-fold in SMp muscles. A similar alteration of AChE is found after tenotomy of these muscles, showing that the effect of denervation may be partly due to suppression of muscle activity. (2) The different changes occurring in the composition of AChE molecular forms in adult denervated SMa and SMp muscles are consistent with fluorescent staining with anti-AChE monoclonal antibodies and with DBA or VVA lectins, which bind to AChE asymmetric, collagen-tailed forms. These lectins poorly stain denervated SMa muscle surfaces but intensely stain neuromuscular junctions and extrasynaptic areas in denervated SMp muscle. (3) In contrast with the adult, denervation of 1-day-old muscles does not markedly modify the total amount of AChE or the proportions of its molecular forms, despite dramatic effects on muscle structure. These results are supported by studies of labeling with fluorescent DBA: the lectin only slightly stains the muscle fiber surface of denervated 15-day-old SMp muscle. Taken together, these data show that denervated muscles escape physiological regulation, producing increased levels of AChE with highly variable cellular distribution and patterns of molecular forms, depending on the age of operation and on the type of muscle.