Gabriel Corkidi

Biochemical Characterization of Rotavirus Receptors in MA104 Cells

ABSTRACT We have tested the effect of metabolic inhibitors, membrane cholesterol depletion, and detergent extraction of cell surface molecules on the susceptibility of MA104 cells to infection by rotaviruses. Treatment of cells with tunicamycin, an inhibitor of protein N glycosylation, blocked the infectivity of the SA-dependent rotavirus RRV and its SA-independent variant nar3 by about 50%, while the inhibition of O glycosylation had no effect. The inhibitor of glycolipid biosynthesisd,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) blocked the infectivity of RRV, nar3, and the human rotavirus strain Wa by about 70%. Sequestration of cholesterol from the cell membrane with β-cyclodextrin reduced the infectivity of the three viruses by more than 90%. The involvement of N-glycoproteins, glycolipids, and cholesterol in rotavirus infection suggests that the virus receptor(s) might be forming part of lipid microdomains in the cell membrane. MA104 cells incubated with the nonionic detergent octyl-β-glucoside (OG) showed a ca. 60% reduction in their ability to bind rotaviruses, the same degree to which they became refractory to infection, suggesting that OG extracts the potential virus receptor(s) from the cell surface. Accordingly, when preincubated with the viruses, the OG extract inhibited the virus infectivity by more than 95%. This inhibition was abolished when the extract was treated with either proteases or heat but not when it was treated with neuraminidase, indicating the protein nature of the inhibitor. Two protein fractions of around 57 and 75 kDa were isolated from the extract, and these fractions were shown to have rotavirus-blocking activity. Also, antibodies to these fractions efficiently inhibited the infectivity of the viruses in untreated as well as in neuraminidase-treated cells. Five individual protein bands of 30, 45, 57, 75, and 110 kDa, which exhibited virus-blocking activity, were finally isolated from the OG extract. These proteins are good candidates to function as rotavirus receptors.

A no hydrotropic response Root Mutant that Responds Positively to Gravitropism in Arabidopsis

For most plants survival depends upon the capacity of root tips to sense and move towards water and other nutrients in the soil. Because land plants cannot escape environmental stress they use developmental solutions to remodel themselves in order to better adapt to the new conditions. The primary site for perception of underground signals is the root cap (RC). Plant roots have positive hydrotropic response and modify their growth direction in search of water. Using a screening system with a water potential gradient, we isolated ano hydrotropic response (nhr) semi-dominant mutant of Arabidopsis that continued to grow downwardly into the medium with the lowest water potential contrary to the positive hydrotropic and negative gravitropic response seen in wild type-roots. The lack of hydrotropic response of nhr1roots was confirmed in a system with a gradient in air moisture. The root gravitropic response of nhr1 seedlings was significantly faster in comparison with those of wild type. The frequency of the waving pattern in nhr1 roots was increased compared to those of wild type. nhr1 seedlings had abnormal root cap morphogenesis and reduced root growth sensitivity to abscisic acid (ABA) and the polar auxin transport inhibitor N-(1-naphtyl)phtalamic acid (NPA). These results showed that hydrotropism is amenable to genetic analysis and that an ABA signaling pathway participates in sensing water potential gradients through the root cap.

Effects of prenatal malnutrition and postnatal nutritional rehabilitation on CA3 hippocampal pyramidal cells in rats of four ages.

The effects of prenatal protein malnutrition and postnatal nutritional rehabilitation on CA3 hippocampal pyramidal cells were investigated in rats of 15, 30, 90 and 220 days of age. Female rats were fed either 6% or 25% casein diet 5 weeks before conception. Following delivery, litters born the same day to 6% and 25% casein diet rats were randomly cross-fostered to 25% casein diet dams and maintained on that diet until sacrificed. In 288 rapid-Golgi impregnated cells, we measured somal size, length of the longest apical dendrite, number of apical and basal dendrites intersecting 10 concentric rings 38 microns apart, synaptic spine density in three 50 microns segments of the largest apical dendrite and the thorny excrescence area. Prenatal protein malnutrition produced differential morphological changes on CA3 pyramidal cells. We observed significant decreases of somal size (at 90 and 220 days of age), of length of apical dendrites (at 15 days old), of apical (in 15 day animals) and basal (in 15, 90 and 220 day animals) dendritic branching and of spine density (in 30, 90 and 220 day animals). We also found significant increases of apical dendritic branching in 90 and 220 day old rats. These results indicate that prenatal protein malnutrition affects normal development and produces long-term effects on CA3 pyramidal cells.

Differential responses of thyrotropin-releasing hormone (TRH) neurons to cold exposure or suckling indicate functional heterogeneity of the TRH system in the paraventricular nucleus of the rat hypothalamus

Thyrotropin-releasing hormone (TRH) is released from the median eminence upon neural stimulation such as cold or suckling exposure. Concomitant with the cold- or suckling-induced release of TRH is a rapid and transient increase in the expression of proTRH mRNA in the paraventricular nucleus (PVN) of the hypothalamus. We employed two strategies to determine whether TRH neurons responding to cold exposure are different from those responding to suckling. First, we attempted to identify a marker of cellular activation in TRH neurons of the PVN. Cold induced c-fos expression in about 25% of TRH neurons of the PVN, but no induction was observed by suckling. Moreover, we explored the expression of a variety of immediate early genes including NGFI-A, fra-1 and c-jun, or CREB phosphorylation but found none to be induced by suckling. The number of cells expressing high levels of proTRH mRNA was counted and compared to total expressing cells. An increased number of cells expressing high levels of proTRH mRNA was observed when both stimuli were applied to the same animal, suggesting that different cells respond separately to each stimulus. We therefore analyzed the distribution of responsive TRH neurons as defined by the cellular level of proTRH mRNA. The proTRH mRNA signal was analyzed within three rostrocaudal zones of the PVN and within six mediolateral columns. Results showed that in response to cold, all areas of the PVN of the lactating rat present increased proTRH mRNA levels, including the anterior zone where few hypophysiotropic TRHergic cells are believed to reside. The distribution of the proTRH mRNA expressing cells in response to cold was quite comparable in female and in male rats. In contrast, the response after suckling was confined to the middle and caudal zones. Our results provide evidence of a functional specialization of TRH cells in the PVN.

Tracking sperm in three-dimensions.

Sperm motility, crucial for fertilization, has been mostly studied in two dimensions (2D) by recording their swimming trajectories near a flat surface. However, spermatozoa swim in three-dimensions (3D) to find eggs, with their speed being the main impediment to track them under realistic conditions. Here, we describe a novel method allowing 3D tracking and analysis of the trajectories of multiple free-swimming sperm. The system uses a piezo-electric device displacing a large focal distance objective mounted on a microscope to acquire 70 image stacks per second, each stack composed of 60 images that span a depth of 100 microm. With this method, 3D paths of multiple sperm in the same field could be visualized simultaneously during 1 s. Within the same sample we found that surface-confined sperm swam 25% slower, produced 3-fold fewer circular revolutions per second, and had trajectories of 134% greater radius of curvature than those sperm swimming freely in 3D.

Effect of protein malnutrition on CA3 hippocampal pyramidal cells in rats of three ages

Prenatal and postnatal protein deprivation effects on CA3-hippocampal pyramidal cells were investigated in 30-, 90- and 220-day-old rats. Female rats were fed either a 6% or a 25% casein diet 5 wk before conception and the litters were maintained on their respective diet until sacrificed. In 216 rapid Golgi-impregnated cells, we measured somal size, length and diameter of apical dendrite, number of apical dendrites intersecting 10 concentric rings 38 microns apart, thorny excrescence area and length, head diameter and density of synaptic spines on 50-microns segments of apical dendrite. The present experiments showed that malnutrition produced significant reductions of somal size in animals at 220 days of age. There were significant reductions of apical dendrite diameters in animals of 30 and 90 days, and of density and head diameter of synaptic spines at the three ages studied, and significant decrease of the thorny excrescence area at 220 days of age. At this latter age, dendritic branching was significantly decreased in the last four rings representing the area into which the perforant pathway projects. In 30-day malnourished rats, dendritic branching showed a significant increase in rings 4-6 representing the area in which the Schaffer collaterals synapse. The location of the deficit in dendritic spines corresponds to the sites where mossy fibers synapse on the apical dendrites of CA3 neurons. Age-related changes normally observed in control rats (e.g., the 30-day-old control group showed the smallest somal size and 220-day-old controls the largest size) failed to occur in the malnourished rats. The deficits in spine density and dendritic branching (in animals of 220 days old) were similar to those found in our previous studies on fascia dentata.

Strategies for locating the female gamete: the importance of measuring sperm trajectories in three spatial dimensions.

The spermatozoon must find its female gamete partner and deliver its genetic material to generate a new individual. This requires that the spermatozoon be motile and endowed with sophisticated swimming strategies to locate the oocyte. A common strategy is chemotaxis, in which spermatozoa detect and follow a gradient of chemical signals released by the egg and its associated structures. Decoding the female gamete’s positional information is a process that spermatozoa undergo in a three-dimensional (3D) space; however, due to their speed and small size, this process has been studied almost exclusively in spermatozoa restricted to swimming in two dimensions (2D). This review examines the relationship between the mechanics of sperm propulsion and the physiological function of these cells in 3D. It also considers whether it is possible to derive all the 3D sperm swimming characteristics by extrapolating from 2D measurements. It is concluded that full insight into flagellar beat dynamics, swimming paths and chemotaxis under physiological conditions will eventually require quantitative imaging of flagellar form, ion flux changes, cell trajectories and modelling of free-swimming spermatozoa in 3D.

Comparison between Low Frequency Magnetic Field Stimulation and Nerve Growth Factor Treatment of Cultured Chromaffin Cells, on Neurite Growth, Noradrenaline Release, Excitable Properties, and Grafting in Nigrostriatal Lesioned Rats

Adrenal chromaffin cells in vitro respond to nerve growth factor (NGF) by expressing neuronal traits. Low frequency magnetic (LFM) field stimulation, while inducing a variety of effects on several cell types, has never been studies as to its effects on chromaffin cell cultures. The purpose of this study was to compare the effects of LFM field stimulation with that of NGF on the morphological phenotype, on noradrenaline (NA) release, and on membrane excitability of cultured chromaffin cells. We also tested the effects of grafting LFM and NGF-treated chromaffin cells into the caudate nucleus of rats with 6-hydroxydopamine lesions of the nigrostriatal pathway. The results of this study showed that LFM field stimulation produced neurite growth of cultured chromaffin cells in a manner similar to that of NGF exposure. The combination of the two procedures did not induce changes above those observed by NGF alone. Both NGF- and LFM-treated chromaffin cells released [3H]NA equally in response to a depolarizing concentration of KCl. On the other, Na+ current density of LFM field stimulation increased, but to a lesser extent than that seen in NGF-treated cells. In addition both types of cells when transplanted into nigrostriatal-lesioned animals induced a similar decrease in the motor asymmetries produced by the lesion. When NGF- or LFM-treated chromaffin cells where compared to untreated control cells, no significant differences were observed in [3H]NA release, on Na+ current densities, or on postgraft motor asymmetries. The results are discussed in terms of the fact that LFM-stimulated cells can be differentiated in a manner similar to NGF-treated cells, by acquiring sympathetic like traits which in turn can diminish motor asymmetries when grafted into nigrostriatal-lesioned rats.

Oil and air dispersion in a simulated fermentation broth as a function of mycelial morphology.

The culture conditions of a multiphase fermentation involving morphologically complex mycelia were simulated in order to investigate the influence of mycelial morphology ( Trichoderma harzianum) on castor oil and air dispersion. Measurements of oil drops and air bubbles were obtained using an image analysis system coupled to a mixing tank. Complex interactions of the phases involved could be clearly observed. The Sauter diameter and the size distributions of drops and bubbles were affected by the morphological type of biomass (pellets or dispersed mycelia) added to the system. Larger oil drop sizes were obtained with dispersed mycelia than with pellets, as a result of the high apparent viscosity of the broth, which caused a drop in the power drawn, reducing oil drop break‐up. Unexpectedly, bubble sizes observed with dispersed mycelia were smaller than with pellets, a phenomenon which can be explained by the segregation occurring at high biomass concentrations with the dispersed mycelia. Very complex oil drops were produced, containing air bubbles and a high number of structures likely consisting of small water droplets. Bubble location was influenced by biomass morphology. The percentage (in volume) of oil‐trapped bubbles increased (from 32 to 80%) as dispersed mycelia concentration increased. A practically constant (32%) percentage of oil‐trapped bubbles was observed with pelleted morphology at all biomass concentrations. The results evidenced the high complexity of phases interactions and the importance of mycelial morphology in such processes.

Expression of the proprotein convertases PC1 and PC2 mRNAs in thyrotropin releasing hormone neurons of the rat paraventricular nucleus of hypothalamus.

PC1 and PC2 are subtilisin-like processing enzymes capable of cleaving thyrotropin releasing hormone (TRH) precursor (pro-TRH) at paired basic residues in vitro. In the paraventricular nucleus of the hypothalamus (PVN), pro-TRH is synthesized to control adenohypophysial thyrotropin and prolactin release. Biochemical and immunological approaches have shown that in the hypothalamus, pro-TRH is extensively cleaved at pairs of basic amino acids. We quantified, by two different approaches, in situ hybridization (ISH) on consecutive cryostat sections or double label ISH, the proportion of PVN TRH neurons containing either PC1 or PC2 mRNAs. Both techniques gave similar results: PC2 mRNA was present in 60-70% of TRH neurons, and PC1 mRNA in 37-46%. Values were similar in the anterior and medial parts of the parvocellular PVN. TRH neurons containing either PC1 or PC2 mRNA were found throughout the areas containing TRH cells without any evidence of anatomical segregation. These results suggest a biochemical heterogeneity in PVN TRH biosynthetic machinery.