Heterogeneous nuclear transfer embryos reconstructed by bovine oocytes and camel (Camelus bactrianus) skin fibroblasts and their subsequent development

Summary This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplasts to investigate the reprogramming of camel somatic cell nuclei in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Serum-starved skin fibroblast cells, obtained from adult camel, were electrically fused into enucleated bovine metaphase II (MII) oocytes that were matured in vitro. The fused eggs were activated by Inomycin with 2 mM/ml 6-dimethylaminopurine. The activated reconstructed embryos were cocultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum for 168 h. Results showed that 53% of the injected oocytes were successfully fused, 34% of the fused eggs underwent the first egg cleavage, and 100% of them developed to four- or 16-cell embryo stages. The first completed cleavage of xenonuclear transfer camel embryos occurred between 22 and 48 h following activation. This study demonstrated that the reconstructed embryos underwent the first embryonic division and that the reprogramming of camel fibroblast nuclei can be initiated in enucleated bovine MII oocytes.     

Purification and partial characterisation of camel milk xanthine oxidoreductase

Xanthine oxidoreductase (XOR) was purified in the presence of dithiothrietol from camel milk with yields of up to 22.2mg/l that were comparable to those obtained from bovine and human milk sources. On SDS-PAGE, the freshly purified camel milk XOR had a protein flavin (A280/A450) ratio of 5.3 +/- 0.4 and appeared homogenous with a single major band of approximately Mr 145.3 KDa. Surprisingly, in all the batches (n = 8) purified camel milk XOR showed no detectable activity towards xanthine or NADH. The molybdenum content of camel XOR was comparable to human and goat milk enzymes. After resulphuration, camel milk XOR gave a specific activity of 1.1 nmol/min/mg and 13.0 nmol/min/mg enzyme towards pterin (fluorimetric assay) and xanthine (spectrophotometric assay) respectively. This activity was markedly lower than that of human, bovine and goat enzymes obtained under the same conditions. These findings suggest that the molybdo-form of camel enzyme is totally under desulpho inactive form. It is possible that camel neonates are equipped with an enzymic system that reactivates XOR in their gut and consequently generates antibacterial reactive oxygen species.     

Characterisation of whey proteins of camel (Camelus dromedarius) milk and colostrum

Variation in the composition of whey proteins from camel (Camelus dromedarius) colostrum and milk was recorded over a 192 h period following parturition. Whey proteins were separated by cation-exchange fast protein liquid chromatography and identified by polyacrylamide gel electrophoresis. The main components of whey proteins in camel milk and colostrum were similar to that in bovine, except for the lack in β-lactoglobulin. Serum albumin was the major whey protein present in camel milk, with an average concentration of 10.8 g/l. Camel colostrum was rich in immunoglobulins G, which consist of IgG1, and the enzyme inhibitory antibodies IgG2 and IgG3. The concentration of these proteins decreased rapidly 48 h post partum. Lactophorin (proteose peptone-component 3) and basic whey protein were detected only within 48 h after parturition, reaching a level of 4.9 and 3.1 g/l at 192 h post partum, respectively. The maximum level of lactoferrin (2.3 g/l) was observed at 48 h after parturition. Camel milk and colostrum were shown to be rich in protective proteins, especially IgG2 and IgG3, which revealed to be a potential source of inhibitory antibodies.     

The response of small vessel endothelial cells from fetal rat lung to growth factors

Small vessel pulmonary endothelial cells were obtained from rat fetal lung at day 20 of gestation, and were maintained in culture to passage three for study. Endothelial cells grown on a collagen matrix with Dulbecco's minimal essential medium: Ham's F12 medium (1:1, v/v) supplemented with 20 ml/l fetal bovine serum, bovine pituitary extract (50 mg/l), endothelial cell growth supplement (100 mg/l), hydrocortisone (1 mg/l) and an increased (10 mmol/l) magnesium concentration retained the characteristic endothelial cell marker factor VIII antigen during the third passage in culture. The factors responsible for small vessel growth in the developing fetal lung are unknown. To test the hypothesis that small vessel pulmonary endothelial cells would respond to autocrine or paracrine growth factors the effects of conditioned media from fetal lung endothelial cells, fibroblasts and pneumocytes from lungs of the same gestational age were studied in vitro. None of the tested conditioned media had any effect on endothelial cell DNA synthesis in the presence of 20 ml/l fetal bovine serum. Since no paracrine or autocrine effects of conditioned media were observed, the effect of other growth factors that could be derived from the circulation, or from storage sites in subcellular matrix, were studied for effect. When endothelial cells were studied in the presence of 20 ml/l fetal bovine serum and 100 mg/l endothelial cell growth supplement they had enhanced DNA synthesis in response to the progression-type growth factors insulin (5 mg/l), insulin-like growth factor-I and insulin-like growth factor-II (20 micrograms/l) and epidermal growth factor (10 micrograms/l). In the absence of serum or endothelial growth supplement endothelial cell DNA synthesis was enhanced by the competence-type growth factors acidic and basic fibroblastic growth factors at 100 micrograms/l and platelet derived growth factor at 10 micrograms/l. In the absence of exogenous competence-type growth factors neutralizing antibodies to basic fibroblast growth factor reduce DNA synthesis. Of various cytokines tested only interleukin-1 (1 x 10(3) U/l) and tumor necrosis factor (25 x 10(4) U/l) had an effect on endothelial cell DNA synthesis. Endothelial cell division during fetal lung development may be controlled by progression growth factors present in serum, and by either autocrine release of the competence factor basic fibroblast growth factor or paracrine release of platelet-derived growth factor by other cell types.     

Chaperone activities of bovine and camel beta-caseins: Importance of their surface hydrophobicity in protection against alcohol dehydrogenase aggregation

β-Casein (β-CN) showing properties of intrinsically unstructured proteins (IUP) displays many similarities with molecular chaperones and shows anti-aggregation activity in vitro. Chaperone activities of bovine and camel β-CN were studied using alcohol dehydrogenase (ADH) as a substrate. To obtain an adequate relevant information about the chaperone capacities of studied caseins, three different physical parameters including chaperone constant (k_c, μM⁻¹), thermal aggregation constant (k_T, °C⁻¹) and aggregation rate constant (k_t, min⁻¹) were measured. Bovine β-CN displays greater chaperone activity than camel β-CN. Fluorescence studies of 8-anilino-1-naphthalenesulfonic acid (ANS) binding demonstrated that bovine β-CN is doted with larger effective hydrophobic surfaces at all studied temperatures than camel β-CN. Greater relative hydrophobicity of bovine β-CN than camel β-CN may be a factor responsible for stronger interactions of bovine β-CN with the aggregation-prone pre denatured molecular species of the substrate ADH, which resulted in greater chaperone activity of bovine β-CN.     

An investigation of the antibacterial effect of carrot on Listeria monocytogenes.

Carrot slices immersed in a potassium phosphate buffer (0.1 mol/l, pH 7.0) or carrot tissue macerated in the buffer had a lethal effect on Listeria monocytogenes. This antilisterial activity was suppressed by anaerobiosis, thiol compounds (1 mmol/l) and bovine serum albumin (0.05%) but was not affected by sodium ascorbate (200 mmol/l), propyl gallate (25 mmol/l), catalase (1100 U/ml), superoxide dismutase (357 U/ml), or chelating agents (10 mmol/l). Free-radical scavengers had no effect at 10 mmol or 50 mmol/l but histidine and diazabenzocyclooctane at 100 mmol/l reduced the antilisterial activity. The addition of Tween 20, 0.05% (v/v), to carrot macerates improved the recovery of the activity in the supernatant liquid after centrifugation at 10,000 g for 2 min. The addition of higher concentrations of the detergent to the macerate reduced the antilisterial activity.     

An investigation of the antibacterial effect of carrot on Listeria monocytogenes

Carrot slices immersed in a potassium phosphate buffer (0.1 mol/l, pH 7.0) or carrot tissue macerated in the buffer had a lethal effect on Listeria monocytogenes. This antilisterial activity was suppressed by anaerobiosis, thiol compounds (1 mmol/l) and bovine serum albumin (0.05%) but was not affected by sodium ascorbate (200 mmol/l), propyl gallate (25 mmol/l), catalase (1100 U/ml), superoxide dismutase (357 U/ml), or chelating agents (10 mmol/l). Free-radical scavengers had no effect at 10 mmol or 50 mmol/l but histidine and diazabenzocyclooctane at 100 mmol/l reduced the antilisterial activity. The addition of Tween 20, 0.05% (v/v), to carrot macerates improved the recovery of the activity in the supernatant liquid after centrifugation at 10 000 g for 2 min. The addition of higher concentrations of the detergent to the macerate reduced the antilisterial activity.     

Macro CK type 1 as a major component of serum creatine kinase activity in pregnant sheep

Electrophoretic separation of creatine kinase (ATP: creatine phosphotransferase, E.C. 2.7.3.2) isoenzymes in agarose gel was made in the serum of 93 sheep of three Slovenian domestic breeds (Solcavska, Pramenka and Bovska breeds). 44 sheep were pregnant between 112 and 135 days at the time of the blood collection. The median value of serum total CK activity for all animals investigated was 82 U/l. After direct immunoinhibition of CK with anti-M-CK monoclonal antibodies the total CK activity remained the same (88 U/l, P = 0.354). There were significant differences among breeds in CK activity for the Solcavska (101 U/l), Bovska (89 U/l) and Pramenka breeds (73 U/l), respectively (Kruskal–Wallis one way analysis of variance, P < 0.01), and between pregnant (105 U/l) and non-pregnant animals (76 U/l), irrespective of the breed (Mann–Whitney rank sum test, P < 0.05). According to electrophoresis, all non-pregnant sheep had exclusively free CK-BB serum bands activity. In all pregnant sheep coupled dimeric BB variant appeared as macro-CK type 1 in the range between 80% and 100% of total CK activity. The present study confirms the existence of an elimination mechanism for CK from the plasma abundance free CK-BB enzyme.     

Purification and functional characterization of pancreatic insulin from camel (Camelus dromedarius)

Large-scale production of insulin still represents the key step in helping diabetic patients throughout the world. Many species and approaches have been used for the production of insulin. In this study, we purified and characterized for the first time pancreatic insulin from the Arabian camel (Camelus dromedarius) using a modified acid-alcohol extraction method. After extraction insulin was purified using a one-step gel filtration on a Sephadex G-50 column leading to a purification yield of 80 mg/kg (20%) of camel pancreas. The purity of camel insulin was assessed by SDS–PAGE and HPLC using insulin from human, bovine and porcine as standards. Molecular weight was determined for purified camel insulin as 5800 Daltons and its amino acid composition is similar to that known for other species. The functional characterization of purified crude camel insulin was demonstrated in vitro by positive competition by radioimmunoassay and in vivo showing camel insulin inducing acute hypoglycaemia in mice. Together, our study reports for the first time the successful purification of functional insulin from camel pancreas with similar properties compared to other insulin species. This is of great interest given that the camel represents considerable economic worth in many countries.     

Lack of induction of serum gamma-glutamyltransferase in diabetes mellitus

Some authors have suggested that carbohydrates can induce hepatic microsomal enzymes, resulting in increased serum gamma-glutamyltransferase (GGT) in diabetes mellitus. Previously we demonstrated the lack of serum GGT increases in patients with acute diabetic crises. In this work we studied serum GGT activity, blood glucose levels and glycosylated hemoglobin levels (HB A1) in 35 patients with diabetes mellitus and 27 healthy volunteers. We did not see differences in the serum GGT activity among controlled (25.05 +/- 2.72 U/l) and uncontrolled (26.44 +/- 4.05 U/l) diabetics and the control group (22.51 +/- 2.95 U/l). Also, there was no significant correlation between serum GGT and HB A1 levels in diabetic patients (r = 0.279). We think that our observations may be relevant because they support the hypothesis that hyperglycemia does not act as an enzyme-inducing agent in chronically uncontrolled diabetics and, furthermore, they indicate that in the presence of abnormal serum GGT levels in diabetics it is necessary to investigate other associated diseases.     

Characterization of recombinant camel chymosin reveals superior properties for the coagulation of bovine and camel milk

Enzymatic milk coagulation for cheese manufacturing involves the cleavage of the scissile bond in kappa-casein by an aspartic acid protease. Bovine chymosin is the preferred enzyme, combining a strong clotting activity with a low general proteolytic activity. In the present study, we report expression and enzymatic properties of recombinant camel chymosin expressed in Aspergillus niger. Camel chymosin was shown to have different characteristics than bovine chymosin. Camel chymosin exhibits a 70% higher clotting activity for bovine milk and has only 20% of the unspecific protease activity for bovine chymosin. This results in a sevenfold higher ratio of clotting to general proteolytic activity. The enzyme is more thermostable than bovine chymosin. Kinetic analysis showed that half-saturation is achieved with less than 50% of the substrate required for bovine chymosin and turnover rates are lower. While raw camel milk cannot be clotted with bovine chymosin, a high clotting activity was found with camel chymosin.     

Irisin/FNDC5: A participant in camel metabolism

The quantification, localization, production, function, and regulation of irisin/FNDC5 in camel species have not been previously studied. The objective of this study was to detect the irisin content in Arabian camel blood and tissues and study the gene expression of FNDC5 and PGC-1α in camel skeletal muscles and white adipose tissue depots under basal conditions. To monitor if exercise influences blood and tissue irisin protein levels as well as FNDC5 and PGC-1α gene expression levels, we analyzed irisin concentrations in the serum, skeletal muscles (soleus and gastrocnemius), and white adipose tissues (hump, subcutaneous, visceral, epididymal, and perirenal) in both control (n = 6) and exercised group (n = 6) using ELISA and determined the cellular localization of irisin/FNDC5 and the mRNA levels of FNDC5 and PGC-1α in skeletal muscles and adipose tissues via immunohistochemistry and real-time PCR, respectively. The possible regulatory roles of exercise on some hormones and metabolites as well as the detection of links between serum irisin and other circulating hormones (insulin, leptin, and cortisol) and metabolites (glucose, free fatty acids, triglycerides, and ATP) were explored for the first time in camels. Our results indicated that exercise induces tissue-specific regulation of the camel irisin, FNDC5, and PGC-1α levels, which subsequently regulates the circulating irisin level. Significant associations were detected between the levels of irisin/FNDC5/PGC-1α in camels and the metabolic and hormonal responses to exercise. Our study suggested that irisin regulates, or is regulated by, glucose, FFA, insulin, leptin, and cortisol in camels. The novel results of the present study will serve as baseline data for camels.     

Thyroid-stimulatory effects of human chorionic gonadotrophin in early pregnancy. In vivo and in vitro studies

Human chorionic gonadotrophin (hCG) shares structural similarity with pituitary thyrotrophin (TSH) and may act as a thyroid stimulator. We have studied serum hCG levels, thyroid function tests and the ability of serum to stimulate cultured thyroid cells in 40 subjects between 6 and 12 weeks of pregnancy. Serum free tri-iodothyronine was increased and serum TSH reduced in pregnancy samples (both p less than 0.05). hCG was detectable in all pregnancy sera with a mean level of 105.6 X 10(3) U/l. Serum from 24 of the 40 (60%) patients stimulated iodide uptake into cultured FRTL-5 thyroid cells. The potency of sera in stimulating cells correlated with the hCG level (r = 0.710, p less than 0.01). The stimulatory activity in some, but not all, sera could be specifically neutralized with antiserum to hCG. Partially purified hCG stimulated iodide uptake and growth of thyroid cells at concentrations of 50 X 10(3) U/l and above. In these experiments, 25 X 10(3) U/l of hCG produced equivalent stimulation to 1 mU/l of TSH. In 8 patients tested before and after termination of pregnancy, the thyroid-cell-stimulatory activity of serum declined rapidly in parallel with serum hCG. hCG may stimulate the thyroid gland at concentrations which prevail in normal pregnancy. Its potential as a physiological regulator of the thyroid gland is not widely appreciated and requires further study.     

Studies with bovine parainfluenza - 3 virus in u.a.R. (Egypt)

A bovine strain of myxovirus parainfluenza-3 (MP3) virus, designated S virus, was isolated from lung tissue collected from cattle with respiratory illness in 1963. The virus agglutinates mammalian and avian erythrocytes, and is sensitive to ether, sodium desoxycholate and trypsin. It grows in primary calf kidney, buffalo kidney, dog kidney, camel kidney and MS cell cultures. The S virus forms well-defined plaques in buffalo and calf kidney cells on the 5th or 6th day after inoculation. Examination of cell cultures following inoculation with S virus revealed giant cell formation, and introcytoplasmic and intranuclear inclusions. At 37 degrees C the virus titer dropped from 10(10.4) to 10(2.6) in 3 days. Virus was completely inactivated at 56 degrees C within 15 minutes. Growth-curve studies in tissue culture monolayer cells revealed a latent period of 10 hours. The intracellular virus titer was slightly lower than that of extracellular virus. The isolate was identified as MP3 virus by serum neutralization and hemagglutination-inhibition tests. Antibodies (HI) to S virus were shown to be present in a significant proportion of Egyptian cattle. The epidemiological significance of MP3 (bovine strain) virus in U.A.R. is discussed.     

On the effect of mutations in bovine or camel chymosin on the thermodynamics of binding κ‐caseins

Bovine and camel chymosins are aspartic proteases that are used in dairy food manufacturing. Both enzymes catalyze proteolysis of a milk protein, κ‐casein, which helps to initiate milk coagulation. Surprisingly, camel chymosin shows a 70% higher clotting activity than bovine chymosin for bovine milk, while exhibiting only 20% of the unspecific proteolytic activity. By contrast, bovine chymosin is a poor coagulant for camel milk. Although both enzymes are marketed commercially, the disparity in their catalytic activity is not yet well understood at a molecular level, due in part to a lack of atomistic resolution data about the chymosin—κ‐casein complexes. Here, we report computational alanine scanning calculations of all four chymosin—κ‐casein complexes, allowing us to elucidate the influence that individual residues have on binding thermodynamics. Of the 12 sequence differences in the binding sites of bovine and camel chymosin, eight are shown to be particularly important for understanding differences in the binding thermodynamics (Asp112Glu, Lys221Val, Gln242Arg, Gln278Lys. Glu290Asp, His292Asn, Gln294Glu, and Lys295Leu. Residue in bovine chymosin written first). The relative binding free energies of single‐point mutants of chymosin are calculated using the molecular mechanics three dimensional reference interaction site model (MM‐3DRISM). Visualization of the solvent density functions calculated by 3DRISM reveals the difference in solvation of the binding sites of chymosin mutants.     

Serum zinc and angiotensin-converting enzyme levels in patients with lung cancer

Forty-two patients with lung cancer and 72 healthy subjects were studied in order to determine a possible relationship between serum zinc and angiotensin-converting enzyme (ACE), a peptidyl dipeptide hydrolase. Serum zinc levels were 105 +/- 21 micrograms/dl in control subjects and 50 +/- 19 micrograms/dl in patients, and angiotensin-converting enzyme activity was 296 +/- 28 U/l in the former and 240 +/- 55 U/l in the latter using hippurylglycylglycine as a substrate. The findings obtained show that the decreased levels of angiotensin-converting enzyme may be related to decreased serum zinc levels and that the primary defect may be the zinc deficiency in these patients.     

Blood protein polymorphism in the one-humped camel (Camelus dromedarius) in the Sudan

The blood protein polymorphism of serum albumin, haptoglobin, transferrin, ceruloplasmin and haemoglobin have been studied in 135 samples from one-humped Arabian camel (Camelus dromedarius) of the Sudan by starch gel electrophoresis. Only the serum albumin and haptoglobin systems exhibited polymorphism with the estimated frequencies of 0.0222, 0.2227 and 0.7773 for Alb^v, Hp¹Hp⁰respectively. The frequency of Hp was 0.0325. No electrophoretic variant was observed at transferrin, ceruloplasmin and haemoglobin loci in the camel. The activity of the ceruloplasmin of the camel sera was weak.     

Metabolomic and elemental analysis of camel and bovine urine by GC–MS and ICP–MS

Recent studies from the author’s laboratory indicated that camel urine possesses antiplatelet activity and anti-cancer activity which is not present in bovine urine. The objective of this study is to compare the volatile and elemental components of bovine and camel urine using GC–MS and ICP–MS analysis. We are interested to know the component that performs these biological activities. The freeze dried urine was dissolved in dichloromethane and then derivatization process followed by using BSTFA for GC–MS analysis. Thirty different compounds were analyzed by the derivatization process in full scan mode. For ICP–MS analysis twenty eight important elements were analyzed in both bovine and camel urine. The results of GC–MS and ICP–MS analysis showed marked difference in the urinary metabolites. GC–MS evaluation of camel urine finds a lot of products of metabolism like benzene propanoic acid derivatives, fatty acid derivatives, amino acid derivatives, sugars, prostaglandins and canavanine. Several research reports reveal the metabolomics studies on camel urine but none of them completely reported the pharmacology related metabolomics. The present data of GC–MS suggest and support the previous studies and activities related to camel urine.     

On the contemporaneous, reversible interaction of different ligands with serum albumins in dilute aqueous solutions: Fluorescein and phenylbutazone

The binding affinity of fluorescein and of phenylbutazone to human serum albumin (HSA) and to bovine serum albumin (BSA), respectively, as well as of the two drugs together to each protein in dilute aqueous solution has been studied by means of gel permeation chromatography, circular dichroism, U.V. absorption and fluorescence spectroscopy. Identity of and/or interdependence between primary binding sites for the two ligands considered on HSA and BSA are evidenced and correlated with a simple theoretical approach to mixed drugs binding.     

Oxidase activity of ceruloplasmin and concentrations of copper and zinc in serum in chronic arterial occlusion of the lower limbs.

The presence of ischaemic tissue excites an inflammatory reaction and synthesis of acute phase proteins (APhPs). Ceruloplasmin (Cp) protein binds 90% of the copper in plasma and it is one of the positive APhPs, and its concentration increases in infection, inflammation or necrosis. The study presents the relationship of the oxidase activity of Cp and concentrations of Cu and Zn in serum of men with different degrees of ischaemia of the lower limbs. The subjects were 32 men with chronic arterial occlusion (AO) of the lower limbs. The oxidase activity of Cp was measured in serum with o-dianisidine as a substrate. Concentrations of Cu and Zn were determined by using atomic absorption spectrometry. The mean activity of Cp in serum in AO (173 +/- 69.2 U/l) was higher as compared with the control group (123.7 +/- 28.6 U/l), and in men with critical ischaemia (> or = 194.8 U/l) than in men with a moderate level of ischaemia (109.3 +/- 31.6 U/l). The mean concentrations of Cu and Zn in serum were found to be higher in AO (22.2 +/- 4.2 and 19.1 +/- 6.9 mumol/l, respectively) than in the control group (16.3 +/- 1.8 and 15.2 +/- 2.3 mumol/l), and in men with critical ischaemia (> or = 22.2 and 19.1 mumol/l) than in men with a moderate level of ischaemia (18.5 +/- 3.3 and 14.5 +/- 4.3 mumol/l). Significant positive correlation coefficients were calculated for the activities of Cp and concentrations of Cu in the control group (r = 0.86) and the AO group (r = 0.76), and low, but significant, correlations for Cp and Zn in the AO group (r = 0.66). The increase in the oxidase activity of Cp and concentration of Cu in serum in ischaemia is caused by the acute phase response. The relationship of Zn concentration and Cp activity in ischaemia is indirect and needs further study.