Therapeutic potential of amniotic fluid stem cells to treat bilateral ovarian dystrophy in dairy cows in a subtropical region.

Amniotic fluid is a rich source of multipotent mesenchymal stem cells (MSCs). Amniotic fluid stem cells (AFSCs) have become a new source of stem cells; they have low immunogenicity and are easily harvested. For this reason, they may be useful in clinical tissue engineering. Moreover, AFSCs have anti-inflammatory properties and can repair tissues. This study evaluated the utility of AFSC injection to treat bilateral ovarian dystrophy in Holstein-Friesian cows. Bovine AFSCs (BAFSCs) were collected at slaughter from Holstein-Friesian cows during the third or fourth month of pregnancy and cultured in vitro. The BAFSCs began to show a fibroblast-like morphology. They were positive for ß-integrin, CD44, CD73, CD106 and Oct4 and negative for CD34 and CD45. After induction, the cells differentiated into mesodermal lineages. Bilateral ovarian dystrophy was confirmed by ultrasonography in 16 lactating cows. The subsequent experiment lasted 15 weeks. Serum was collected weekly to analyse progesterone concentrations, and weekly ultrasonography recorded ovarian changes. Each cow was equipped with an automatic heat detection system to facilitate oestrus observation and breeding records. The progesterone concentration of two cows in the treatment group (25%) significantly increased during weeks 10-15. On ultrasonography, the treatment group demonstrated mature follicles after BAFSCs injection, and foetuses were visualized approximately 40 days after artificial insemination (AI). Oestrus rates in the control and treatment groups were 0% (0/8) and 50% (4/8), respectively; pregnancy rates were 0% (0/8) and 25% (2/8), respectively. Calves were successfully delivered in both cases of pregnancy. These results show that BAFSCs can alleviate bovine ovarian dystrophy and restore fertility.

[Protection of Verapamil against AGE induced apoptosis of human lens epithelial cell].

OBJECTIVE: To investigate the protection of Verapamil against advanced glycation end products (AGE) induced human lens epithelial cells (HLEC) apoptosis. METHODS: Experiment study. SRA01/04 (HLEC line) was cultivated and passaged to the third generation and then divided into four groups. A group was named as control group, and B group was named as AGE group (LEC was treated by 20 µmol/L AGE). C group was AGE+SB202190 group (LEC was treated 2 hours by SB2012190 and then treated by 15 µmol/L AGE). D group was AGE+ Verapamil group (LEC was treated 2 hours by 50 µmol/L Verapamil and then treated by AGE). MTT was used to evaluate the cell viability. Flow cytometry with Annexin V-FITC apoptosis detection was used to assess cell apoptosis.The expression of p-p38 and caspase3 was detected by Western blot between groups. One way Chi-square analysis was used for data analysis. LSD-t test was used as comparison between every two groups. RESULTS: After 24 hours, LEC viability (A570) was (0.28±0.08) in B group, which was significantly lower than A group (0.97±0.05) (LSD-t test, P=0.008). LEC viability in C and D group was (0.79±0.06) and (0.62±0.07) separately, which can partly higher than it was in B group (F=34.52, P=0.001). The apoptosis cells were (19.9±1.1)% in B group, which were significantly higher than they were in A group (2.5±0.6)% (P=0.003). The apoptosis cells in C and D group were (4.23±1.20) and (5.79±1.75) separately, which were significant lower than they were in B group (F=371.61, P<0.01). In additional, expressions of p-p38, Caspase3 proteins in the cells of group B were (223.35±20.15) and (256.77±19.88) separately, which were higher than it were in A group,which were (106.44±10.74) and (100.26±18.65) separately. However, they were (139.17±19.10) and (142.75±23.36) in group C and (154.79±21.87) and (139.79±25.73) in group D (F=248.01, F=76.68; P<0.01), which were lower than they were in A group. CONCLUSION: p38 pathway is involved in the apoptotic procedure of LEC induced by AGE. Verapamil can interdict the p38 signal pathway and protect LEC apoptosis. (Chin J Ophthalmol, 2016, 52: 444-448).

Effects of injecting exogenous lipid transfer protein into rats.

Rats were injected intravenously with preparations of partially purified lipid transfer protein isolated from human plasma. Cholesteryl ester transfer activity disappeared from the plasma of recipient rats with a t1/2 of about 10 h and after 24 h had fallen to a level comparable to that in human plasma. By contrast there was no measurable cholesteryl ester transfer activity in the plasma of control rats. Plasma collected from rats 24 h after the injection was subjected to ultracentrifugation at 1.225 g/ml; lipoproteins in the 1.225 g/ml supernatant were subsequently separated by both gel filtration chromatography and gradient gel electrophoresis. The major change in the treated animals was a total loss of the large, cholesteryl ester-rich, apolipoprotein E-rich high-density lipoproteins, HDL1, which are prominent in the plasma of control rats. This loss of HDL1 unmasked an obvious peak of low-density lipoproteins that had been obscured in the control rats. Other changes in the treated rats included an increase in the relative cholesteryl ester content of very-low-density lipoproteins and the emergence of a peak of triacylglycerol in the high-density lipoproteins.

Sodium oleate dissociates the heteroexchange of cholesteryl esters and triacylglycerol between HDL and triacylglycerol-rich lipoproteins.

Mixtures of human high-density lipoproteins (HDL) and triacylglycerol-rich lipoproteins (TGRL) have been incubated in the presence of partially pure cholesteryl ester transfer protein (CETP). There were net mass transfers of cholesteryl ester from HDL to TGRL and of triacylglycerol from TGRL to HDL which were accompanied by the formation of minor subpopulations of small HDL particles. When the mixture of HDL, TGRL and CETP was supplemented with fatty acid-poor bovine serum albumin (40 mg/ml) there was a 7% reduction in the transfer of cholesteryl esters out of HDL (P less than 0.05) and a 14% increase in the transfer of triacylglycerol into HDL (P less than 0.05); there was also a reduction in the formation of very small HDL particles. In contrast, when the mixture of HDL, TGRL and CETP was supplemented with 0.16 mM sodium oleate the transfer of cholesteryl esters out of HDL was increased by 31% (P less than 0.001) and the transfer of triacylglycerol into HDL was decreased by 25% (P less than 0.01); under these conditions the formation of very small HDL particles was enhanced. It has been concluded that in the presence of sodium oleate, there is a dissociation of the CETP-mediated heteroexchange of cholesteryl esters and triacylglycerol between HDL and TGRL.

The interaction of cholesteryl ester transfer protein and unesterified fatty acids promotes a reduction in the particle size of high-density lipoproteins.

Purified human cholesteryl ester transfer protein (CETP) has been found, under certain conditions, to promote changes to the particle size distribution of high-density lipoproteins (HDL) which are comparable to those attributed to a putative HDL conversion factor. When preparations of either the conversion factor or CETP are incubated with HDL3 in the presence of very-low-density lipoproteins (VLDL) or low-density lipoproteins (LDL), the HDL3 are converted to very small particles. The possibility that the conversion factor may be identical to CETP was supported by two observations: (1) CETP was found to be the main protein constituent of preparations of the conversion factor and (2) an antibody to CETP not only abolished the cholesteryl ester transfer activity of the conversion factor preparations but also inhibited changes to HDL particle size. In additional studies, the changes to HDL particle size promoted by purified CETP were inhibited by the presence of fatty-acid-free bovine serum albumin; by contrast, albumin had no effect on the cholesteryl ester transfer activity of the CETP. The possibility that albumin may inhibit changes to HDL particle size by removing unesterified fatty acids from either the lipoproteins or CETP was tested by adding exogenous unesterified fatty acids to the incubations. In incubations of HDL with either VLDL or LDL, sodium oleate had no effect on HDL particle size. However, when CETP was also present in the incubation mixtures the capacity of CETP to reduce the particle size of HDL was greatly enhanced by the addition of sodium oleate. It is concluded that the changes in HDL particle size which were previously attributed to an HDL conversion factor can be explained in terms of the interacting effects of CETP and unesterified fatty acids.

Particle size distribution of high density lipoproteins as a function of plasma triglyceride concentration in human subjects.

Polyacrylamide gradient gel electrophoresis has been used to examine the particle size distribution of high density lipoproteins (HDL) in human subjects with a wide range of plasma triglyceride concentrations. In studies of groups of both male and female subjects, it was confirmed that the concentration of HDL cholesterol decreases with increasing plasma triglyceride concentration. The HDL fraction from subjects with elevated concentrations of plasma triglyceride was depleted of cholesteryl ester and enriched in triglyceride. It was also confirmed that the proportion of HDL subfraction 2 (HDL2) declines as the plasma triglyceride increases. A new finding was that there were also significant changes in the size of particles in HDL subfraction 3 (HDL3). At low concentrations of plasma triglyceride the predominant subpopulation of HDL3 comprised particles of mean radius 4.3 nm. As the triglyceride concentration increased, however, there was a progressive appearance of HDL3 particles of radius 3.9 nm; in plasma samples with the highest concentrations of triglyceride there was an almost complete disappearance of the 4.3-nm particles, with the population of 3.9-nm particles now predominant.

Sodium oleate promotes a redistribution of cholesteryl esters from high to low density lipoproteins.

Cholesteryl esters readily exchanges between the low density lipoproteins (LDL) and high density lipoproteins (HDL) in human plasma in a process of equilibration catalysed by the cholesteryl ester transfer protein (CETP). In the present studies, in which mixtures of human LDL and HDL have been incubated in vitro with partially pure CETP, it has been found that Na oleate disrupts the CETP-mediated equilibrium between LDL and HDL and promotes a concentration dependent redistribution of cholesteryl esters from HDL to LDL. The end result of the redistribution is the appearance of a cholesteryl ester enriched LDL fraction and an HDL fraction which is protein-rich, lipid-depleted and markedly reduced in particle size.

Sequential MRI studies of a patient with complex partial status--a case report.

Complex partial status (CPS), the status epilepticus of complex partial seizure, is rarely seen in clinical practice. The clinical presentations of CPS are characterized by confusion, slowness in response, together with stereotypic or complex automatisms and occasional secondary generalization. The electroencephalographic findings of CPS reveal characteristic focal epileptiform activities of mesial temporal region. Magnetic resonance image (MRI) is the imaging method of choice for studying epilepsy, particularly when focus is in the temporal lobe. We report a 49-year-old female with diagnosis of viral encephalitis and clinical presentation of CPS. We present the sequential brain MRI findings at acute, subacute and chronic stages of this patient.

Gate length scaling effect on high-electron mobility transistors devices using AlGaN/GaN and AlInN/AlN/GaN heterostructures.

Compared to AlGaN/GaN HEMT with 0.15 µm T-gate length, the AlInN/AlN/GaN one exhibits much higher current density and transconductance of 1558 mA/mm at Vd = 2 V and 330 mS/mm, respectively. The high extrinsic ft and fmax of 82 GHz and 70 GHz are extracted from AlInN/AlN/GaN HEMT. Besides, we find that the transconductance roll-off is significant in AlGaN/GaN, but largely improved in AlInN/AlN/GaN HEMT, suggesting that the high carrier density and lattice-matched epitaxial heterostructure is important to reach both large RF output power and high operation frequency, especially for an aggressively gate length scaling.

Incubation of human lipid transfer protein with plasma from normal-fed and cholesterol-fed rats: effect on the distribution of constituents between lipoprotein fractions.

Samples of rat plasma, in which activity of lecithin-cholesterol acyltransferase was inhibited, were incubated in vitro at 37 degrees C for 6 hr in the presence and absence of a partially purified preparation of human lipid transfer protein (LTP). After the incubation gel filtration chromatography was used to separate plasma lipoproteins into multiple fractions which were individually assayed to obtain a complete profile of all constituents across the whole lipoprotein spectrum. When plasma from both normal and cholesterol-fed rats was incubated in the absence of LTP there were no changes in the distribution of any of the lipoprotein constituents between different fractions. When, however, normal plasma was incubated in the presence of LTP there was an obvious transfer of cholesteryl esters from high density lipoproteins (HDL) to very low density lipoproteins (VLDL) and of triglyceride from VLDL to HDL. The presence of LTP in incubations of plasma from cholesterol-fed rats, by contrast, resulted in only minimal redistributions of constituents.

Role of lipid transfers in the formation of a subpopulation of small high density lipoproteins.

The effect of lipid transfers on the structure and composition of high density lipoproteins (HDL) has been studied in vitro in incubations that contained the lipoprotein-free fraction of human plasma as a source of lipid transfer protein. These incubations did not contain lecithin:cholesterol acyltransferase activity and were not supplemented with lipoprotein lipase. Incubations were performed at 37 degrees C for 6 hr in both the presence and absence of either added very low density lipoproteins (VLDL) or the artificial triglyceride emulsion, Intralipid. Incubation in the absence of added VLDL or Intralipid had little or no effect on the HDL. By contrast, incubation in the presence of either VLDL or Intralipid resulted in marked changes in the HDL. The effect of incubation with VLDL was qualitatively similar to that of Intralipid; both resulted in obvious transfers of lipid and changes in the density, particle size, and composition of HDL. Incubation of the plasma fraction of density 1.006-1.21 g/ml, total HDL, or HDL3 with either VLDL or Intralipid resulted in the following: 1) a depletion of the cholesteryl ester and free cholesterol content and an increase in the triglyceride content of both HDL2 and HDL3; 2) a decrease in density and an increase in particle size of the HDL3 to form a population of HDL2-like particles; and 3) the formation of a discrete population of very small lipoproteins with a density greater than that of the parent HDL3. The newly formed lipoproteins had a mean particle radius of 3.7-3.8 nm and consisted mainly of protein, predominantly apolipoprotein A-I and phospholipid.

Evidence that glucagon acts on the liver to decrease mitochondrial calcium stores.

1. Mitochondria isolated from rats treated with glucagon for 60 min or lives perfused in the presence of glucagon for 10 min exhibited lower rates of 45Ca2+ exchange than did control mitochondria when this was measured under steady-state conditions in the presence of Mg2+, ATP, Pi and 0.13 microM- or 0.16 microM-free Ca2+ at pH 7.4 and at 25 degrees C or 37 degrees C. Under these conditions no significant difference in the rates of Ruthenium Red-induced 45Ca2+ efflux was observed. These results contrast with earlier work in which mitochondria isolated from glucagon-treated livers were shown to exhibit faster rates of Ca2+ uptake [Yamazaki (1975) J. Biol. Chem. 250, 7924-7930] and slower rates of spontaneous Ca2+ efflux [Hughes & Barritt (1978) Biochem. J. 176, 295-304] when these parameters were measured under different incubation conditions, including supra-physiological concentrations of free Ca2+ and the absence of added Mg2+ and ATP. 2. Perfusion of livers with glucagon before the addition of adrenaline or the Ca2+-selective ionophore A23187, to release Ca2+ from intracellular stores, decreased the amount of Ca2+ released by these agents. 3. Incubation of isolated hepatocytes in the presence of glucagon at 1.3 mM extracellular Ca2+ induced a small decrease in the plateau of the 45Ca2+-exchange curve obtained under steady-state conditions. 4. It is concluded that the actions of glucagon on liver mitochondrial Ca2+ transporters lead to a decrease, rather than an increase, in mitochondrial Ca2+ stores in the intact cell.

Glycosaminoglycans of brain during development.

The concentration of hyaluronic acid, chondroitin sulfate, and heparan sulfate was measured in rat brain at 2-day intervals from birth to 1 month of age, and in 40-day-old and adult animals. The levels of all three glycosaminoglycans increased after birth to reach a peak at 7 days after which they declined steadily, attaining by 30 days concentrations within 10% of those present in adult brain. The greatest change was seen in hyaluronic acid, which decreased by 50% in 3 days, and declined to adult levels (28% of the peak concentration) by 18 days of age. Only heparan sulfate showed a significant change in metabolic activity during development (a fourfold increase in the relative specific activity of glucosamine), most of which occurred after 1 week of age. In 7-day-old rats almost 90% of the hyaluronic acid in brain is extractable by water alone, as compared to only 15% in adult animals, and this large amount of soluble hyaluronic acid in young rat brain is relatively inactive metabolically. On the basis of our data we propose that the higher amounts of hyaluronic acid found in very young brain may be responsible for the higher water content of brain at these ages, and that the hydrated hyaluronic acid serves as a matrix through which neuronal migration and differentiation may take place during early brain development.

Isolation of a high-density-lipoprotein conversion factor from human plasma. A possible role of apolipoprotein A-IV as its activator.

1. A high-density-lipoprotein (HDL) conversion factor was partially purified from human plasma by precipitation with (NH4)2SO4, ultracentrifugation, cation-exchange chromatography, anion-exchange chromatography and chromatography on a column of hydroxyapatite. 2. This factor modulates the particle size of HDL by converting a homogeneous population into new populations of particles, some of which are smaller and others larger than those in the original population. 3. The isolated HDL conversion factor appeared as one major band and at least three minor bands on SDS/polyacrylamide-gel electrophoresis; attempts to purify this factor further resulted in loss of conversion activity. 4. Preparations of the HDL conversion factor were stable after heating to 58 degrees C for 1 h, and were shown not to possess proteolytic activity. 5. The conversion factor was distinct from the known apolipoproteins, none of which had HDL conversion activity. 6. Addition of apolipoprotein A-IV had a dose-dependent potentiating effect on the process promoted by the HDL conversion factor.

Use of botulinum toxin A in the treatment of hemifacial spasm and blepharospasm.

BACKGROUND: Hemifacial spasm and blepharospasm are both dystonic disorders. They may seriously affect individuals' lifestyle and social activities. In 1990, the Food and Drug Administration of the USA approved botulinum toxin A as a therapeutic agent in the treatment of hemifacial spasm and blepharospasm. We present a therapeutic review of botulinum toxin A in 80 patients in Taiwan. METHODS: Fifty-eight patients with hemifacial spasm and 22 with blepharospasm. Botulinum toxin A was prepared and injected into the facial and eyelid muscles. Patients were monitored every two weeks and classified into four groups (excellent, moderate, mild and no improvement) according to the clinical improvement scale. Complications were also recorded. RESULTS: A total of 86.2% of hemifacial spasm patients and 81.8% of blepharospasm patients had excellent improvement on the spasm intensity scale, while 6.8% of hemifacial spasm and 9.0% of blepharospasm patients had moderate improvement. The complication rate was low and included transient mild facial weakness (5%), ptosis (3.8%), eyelid swelling and/or ecchymosis (3.8%), nausea/vomiting (2.5%) and transient severe facial weakness (1.3%). CONCLUSION: Botulinum toxin A is an excellent therapeutic agent to improve spasm intensity and has a low complication rate.