In vitro production of Sudanese camel (Camelus dromedarius) embryos from epididymal spermatozoa and follicular oocytes of slaughtered animals.

Application of assisted reproductive technology in camelidea, such as artificial insemination (AI) and embryo transfer, has been slow in comparison to that for other livestock species. In Egypt, there are few attempts to establish in vitro maturation (IVM) and fertilization (IVF) techniques in dromedary camel. The present study was carried out to produce Sudanese camel embryos using in vitro matured oocytes and epididymal spermatozoa. Dromedary camel ovaries were collected from abattoirs and then, the oocytes were aspirated from all the visible follicles on the ovarian surface (~2-8 mm in a diameter). Meanwhile, Fetal Dromedary Camel Serum (FDCS) was obtained from camel fetuses after slaughtering. Thereafter, only Cumulus Oocyte Complexes (COCs) were matured in vitro in the Tissue Culture Medium (TCM-199) complemented with 10% FDCS. Spermatozoa required for in vitro fertilization were collected from testes (epididymal cauda) of the slaughtered camel bulls. The results clearly showed that the maturation rate of oocytes at metaphase II was about 59.5% while the fertilization rate was around 70.4%. Intriguingly, the embryo rates determined were 13.1%, in 2-cell; 0.0%, in 4-cell; 34.7%, in 8-16% cell; 39.1%, in morula and 13.1% in a blastocyst stage. This study represented a successful in vitro production of Sudanese dromedary camel embryos from epididymal sperm cells and in vitro matured oocytes recovered from slaughtered camels.

Biocompatibility of BioAggregate and mineral trioxide aggregate on the liver and kidney.

AIM: To investigate and compare the systemic toxic effect of DiaRoot BioAggregate and grey ProRoot Mineral trioxide aggregate (MTA) on the liver and kidney after 7 and 30 days. METHODOLOGY: Forty-two white albino rats were divided into two main groups. Group (1), considered the control group (n = 18), was further divided into two subgroups. The negative control subgroup (n = 6) received no treatment. The empty tube subgroup (n = 12) received empty sterile Teflon tubes. In Group (2), considered the experimental group (n = 24), the rats were divided equally into two subgroups. One subgroup received MTA, whilst the other received BioAggregate. The materials in the Teflon tubes were implanted subcutaneously in the dorsal side of the rats. Blood samples were taken to investigate the change of kidney and liver functions on day 7 and day 30. The liver and kidney organs were subjected to histopathological examination and calculation of the number of inflammatory cells. Data analysis was performed using one-way anova with post hoc multiple comparisons with the Tukey's test. Student's t-test was used to compare the changes in liver and kidney functions amongst the groups. RESULTS: On day 7, a significantly more severe inflammatory reaction was observed in both experimental subgroups compared with the control (P < 0.05); the severity decreased after 30 days. The kidney functions were not affected after 7 days but had subsequently increased after 30 days (P < 0.001). Liver functions increased after 7 days and had decreased in the BioAggregate subgroup after 30 days, whilst in the MTA subgroup, a continuous increase in the level of liver function was observed. CONCLUSIONS: Mineral trioxide aggregate had adverse effects on the liver and kidney that were significantly more severe than BioAggregate but with no permanent damage.

Efficiency of cytoplasmic delivery by non-cationic liposomes to cells in vitro: a confocal laser scanning microscopy study.

It is necessary to understand liposomal uptake mechanisms and intracellular distribution in order to design more efficient gene (drug) carrier systems. Until now, a few studies have been carried out using confocal laser scanning microscopy (CLSM) to investigate the cellular uptake and transfection mediated with liposomes. So, by CLSM, we demonstrated that artificial virus-like envelope (AVE) vesicles labeled with rhodamine-PE (Rh-PE), carbocyanine (DiI) and carboxyfluorescein (CF) were investigated into the cytoplasm of two human cell lines, Mewo (human melanoma cell line) and HepG2 (human hepatoma cell line) cells grown in DMEM medium supplemented with different percentages (0%, 30%, and 100%) fetal calf serum (FCS). The liposome uptake was dependent on the cell line, in view that the whole process of liposomes associated with cells (uptake) is a two-step process involving binding and endocytosis. Based upon the various assays used to measure cellular uptake of liposomes, we conclude the efficacy of cytoplasmic delivery by AVE-liposomes to cells in culture.

Interaction of type-I collagen with phospholipid monolayer.

The effects of type-I collagen on dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) monolayer films with different compositions were studied using monolayer technique. The addition of collagen in the subphase of different monolayer films induced a considerable shift towards larger area/molecule in the compression-isotherm curves. This is either referred to the insertion of collagen into the monolayer by its hydrophobic residues or to an adsorption process causing a protein layer to be located parallel to the lipid monolayer [1]. The variation of collagen interaction with different lipid compositions was also verified through the penetration-kinetics experiment. Comparing our results to the results of Pajean et al. [2] and Pajean and Herbage [3] on the effect of collagen on the stability of lipid vesicles implies that the collagen induced stability could be explained on the basis of collagen-lipid monolayer interaction.

Interaction of Doxorubicin with phospholipid monolayer and liposomes.

The effect of Doxorubicin which is (an anthracycline antibiotic with a broad spectrum of antitumor activity) on the monolayer and bilayer in the form of large Multilamellar Vesicles (MLV's) of Dipalmitoyl phosphatidylcholine (DPPC) were studied by means of monolayer techniques (surface pressure, penetration kinetics, and association constant) and light scattering technique. The monolayer technique showed that addition of DXR to a lipid film composed of (DPPC/CHOL/PEG-PE) at a molar ratio of (100:0:0) produced a less condensed Monolayer. In the (pie-A) curves, DXR induced shift towards larger area/molecule, where the area/molecule was shifted from 61 to 89 A2, and 116 A2 in the presence of 20 and 40 nM DXR, respectively. The three curves collapsed at a pressure pi = 45 mN/m. In penetration kinetics experiment (delta pi-t), the change in pressure with time was 8 and 14 mN/m for a DXR concentration of 20 and 40 nM, respectively, and the increase in surface pressure presented a plateau over a period of 30 min. The measured association constant (K) was found to be 5 x 10(5)/M. In the light scattering experiment, there was a shift of the transition temperature (Tm) of (MLV's) of the same composition of the monolayer towards a smaller value from 40.5 degrees to 34.5 degrees C. Incorporation of CHOL and PEG-PE as DPPC/CHOL/PEG-PE at a molar ratio of (100:20:0), (100:20:4) and (100:20:4) greatly counteracted the effect of DXR and made the lipid membrane more condense and rigid. Moreover, the penetration of DXR into the membrane was greatly reduced. There was a very small shift for the (pi-A) and (delta pi-t) curves, and the association constant of the drug for these different lipid compositions was greatly reduced down to 2.5 x 10(5)/M and the transition temperature (Tm) was increased up to (42.5 degrees C) in the presence of 40 nM DXR. Our results suggest that DXR has a great effect on the phospholipid membrane, and that addition of CHOL or PEG-PE to the phospholipid membrane causes stabilization for the membrane, and reduces the interaction with Doxorubicin.

Light scattering study of irradiated lipid bilayer.

Vesicular phospholipid bilayer membranes in the form of giant unilamellar vesicles (GUVs) of dipalmitoylphosphatidylcholine (DPPC) were irradiated with fast neutron fluences ranging from 10(4) to 10(7) n cm-2. The phase behaviour of both non-irradiated and irradiated GUVs was investigated using an angular light scattering technique. A model independent size distribution of the samples and their optical anisotropy (delta) were determined using a maximum entropy technique and the theory of light scattering from spherical shells composed of anisotropic cylindrical molecules arranged radially in the shells. The structural changes in the lipid bilayer exposed to fission neutrons are discussed on the basis of the damaging mechanisms of fast neutrons to both the hydrophobic and hydrophilic regions of the lipid bilayer.

Efficient gene delivery with serum into human cancer cells using targeted anionic liposomes.

Success of human gene therapy depends upon the development of delivery vehicles or vectors, which can selectively deliver therapeutic genes to target cells with efficiency and safety. Previous studies have shown an efficient, systemic trans-gene expression in many cell lines (in vitro) by using an anionic liposomal vector, based on the composition of retroviral envelopes (artificial viral envelopes, AVEs). The AVE-liposomes and their complexes with plasmid (DNA) were characterized according to zeta potential measurements and transmission electron microscopy (TEM). We successfully demonstrated that AVE liposomes, dispersed in 10% serum-containing growth medium, efficiently delivered plasmid DNA to HuH-7 (human hepatoma cell line) cells. We assessed the utility of liver-targeted vesicles as a drug/gene delivery system for the treatment of liver diseases. We found that small unilamellar AVE vesicles containing 15 mol% digalactosyl diglyceride (DGDG) are efficiently targeted to the liver via the hepatic asialoglycoprotein receptor.

[Suckling-induced changes in oxytocin and alpha-melanocyte-stimulating hormone contents of the median eminence and various lobes of the pituitary gland]. / Az alpha-melanocyta-stimuláló hormon és oxytocin elválasztásának és lokális transzportjának vizsgálata szopási inger hatására a hypophysis különbözó régióiban és az eminencia medianaban.

Previous reports have implicated that pituitary-derived prolactin (PRL) is secreted from two distinct zones of mammotropes within the anterior lobe (AL). The inner zone (AL-IZ), located adjacent to the NIL, is supposed to be involved in the rapid and massive discharge of PRL from the pituitary gland due to suckling stimulus. Anatomically, the AL-IZ has an intimate contact with the NIL because the blood arriving from the posterior pituitary through the short portal vessels (SPV) baths it first. Based on these facts it would be hypothesized that the locally released and/or produced important compounds, like oxytocin (OXT) and alpha-melanocyte-stimulating hormone (alpha-MSH), can be delivered to the AL-IZ. Therefore, the purpose of this study was to examine the possible local transportation of these hormones into various regions of the pituitary gland (adenohypophysis inner-zone (AL-IZ), outer zone (AL-OZ), intermediate lobe (IL), neural lobe (NL)) and median eminence of lactating rats. We have measured the concentration of OXT and alpha-MSH from tissue samples of nonsuckled and suckled rats using specific RIA-s. There were no changes in the concentration of OXY and alpha-MSH in the AL-IZ and AL-OZ due to suckling stimulus. In contrast, our data provide compelling evidence that OXT is transported into the IL, which can be further increased by suckling stimulus. Our data have shown a lack of local delivery of either alpha-MSH or OXY into the AL that raises serious doubt about their possible role in PRL secretion during suckling stimulus.

Spectroscopic study of the experimental parameters controlling the structural properties of chitosan-Ag nanoparticles composite.

Chitosan as reducing, stabilizing and capping agent was used to synthesize chitosan-silver nanoparticles composite under different experimental conditions of temperature or time. The UV-Vis spectra exhibited a single peak at 430nm which provided strong evidence for the formation of surface plasmon resonance (SPR) band of Ag nanoparticles. The rate of the increase of this absorbance with temperature increases with increasing the time of reduction. It was found that the variation of the temperature from 60°C to 100°C and the time of reduction from 6h to 16h resulted in no significant changes in the intensities and positions of the FTIR absorption bands of the composite. The TEM micrographs showed distinct typical spherical silver nanoparticles separated from each other quite well at reduction temperature range (60-80°C) and displayed some of accumulations at high temperature range (90-100°C). The TEM micrographs investigation indicated various shapes with different reduction time. The SEM images of the prepared samples were discussed.

Effect of ovary preservation period on recovery rate and categories of dromedary camel oocytes.

The aim of this study was to evaluate the effect of different periods of ovary preservation at 25-30 °C for 5, 6, 7, 9, 12 and 24 h on recovery rate and oocyte categories of dromedary camel oocytes. Camel ovaries were collected from El-Bassatein slaughterhouse, Cairo. The collected ovaries were placed immediately after slaughtering into thermos in saline solution (0.9% NaCl) supplemented with antibiotics (100 IU penicillin and 100 µg streptomycin/ml) at 25-30 °C and transported to the laboratory within 4-5 h. Ovaries were washed three times with warmed (30 °C) phosphate buffer solution (PBS) and one time with ethanol (70%). All visible follicles on the ovarian surface (2-8 mm in diameter) were counted. Oocytes were aspirated using a 20-gauge hypodermic needle. Oocyte yield was recorded and the number of oocytes/ovary was calculated. Oocytes were classified into five categories (compact, partial denuded, denuded, shrunken and cleaved oocytes). Results show that average number of follicles on each ovary was not significantly affected by preservation period, although tended to reduce only after 5 h of ovary preservation. However, this number was insignificantly reduced by increasing period of ovary preservation more than 5 up to 24 h. Average number of oocytes on each ovary was significantly (P < 0.05) reduced only between 5 and 6 h of ovary preservation. Average number of oocytes showed higher reduction rate between 5 and 6 h from 12.4 to 9.3/ovary as well as between 9 and 12 h. Oocyte recovery rate showed insignificant decrease from 88.1% at 5 h to 78.6% at 9 h of preservation. However, it showed significant (P < 0.05) reduction to 62.0% between 9 and 12 h, then insignificantly decreased to 58.6 at 24 h of preservation of the ovaries. Frequency distribution and recovery rate of each category was the highest for compact oocytes and the lowest for cleaved oocytes at all periods of preservation. Increasing preservation period significantly (P < 0.05) decreased frequency distribution of compact and cleaved oocytes, while increased frequency distribution of partial denuded, denude and shrunken oocytes. It might be concluded from the present results that the preservation of dromedary camel ovaries at 25-30 °C for 5-6 h was effective for maintaining the oocytes quality and recovery rate compared with the other preservation periods.

Effect of microwave radiation on the biophysical properties of liposomes.

Steadily growing use of electromagnetic fields, especially in conjunction with wireless communication systems, has led to increasing public concern about possible health effects of electromagnetic radiation. However, besides the well-known thermal effect of electromagnetic fields on biological tissue, there is no clear evidence of further athermal interaction mechanisms with biological systems. The present study was designed to determine the changes in bilayer permeability in egg lecithin multilamellar vesicles after exposure to 900 MHz microwave radiation for a period of 5 h. Specific absorption rate (SAR) of the radiation for the investigated liposome sample was found to be 12 +/- 1 W/kg. Liposomal changes in permeability were monitored using a light scattering technique. Optical anisotropy of the liposome sample decreased dramatically upon exposure to microwave radiation, indicating structural changes in acyl chain packing. IR and NMR ((1)H NMR) studies, which have been employed to reveal structural alterations in microwave, exposed vesicles showed an increased damage upon exposure to microwave. The changes observed in the (1)H NMR spectrum of the microwave exposed sample indicated hydrolysis of carboxylic and phosphoric esters. IR study showed conformational changes in the acyl chains of the lipids upon microwave exposure. However, both IR and (31)P NMR did not show any appreciable changes in the head group part of the lipids.