Development of OPS vitrified pig blastocysts: effects of size of the collected blastocysts, cryoprotectant concentration used for vitrification and number of blastocysts transferred

Unhatched blastocysts from Large White hyperprolific gilts (n=103) were identified, measured and vitrified using the Open Pulled Straw (OPS) technique to evaluate the effects of the collected blastocyst size and cryoprotectant concentrations used for vitrification, and the number of embryos transferred per recipient. Vitrified/warmed blastocyst viability was estimated in vitro, as the percentage of embryos developing after 72h, and in vivo, on pregnancy Day 30. In the in vitro study, we compared the use of three cryoprotectant concentrations (16.5, 18, or 20% DMSO+16.5, 18, or 20% EG+0.4M sucrose). Survival rates differed significantly between the control (98.3%) and the three cryoprotectant concentrations (67, 62.3, and 57%, respectively). Blastocyst size at vitrification determined the further in vitro development of embryos (26% survival for blastocysts 126-144microm versus 100% for blastocysts >199microm). For the in vivo study, blastocysts were vitrified using cryoprotectant concentrations of 16.5 or 18% DMSO+EG and transferred surgically in groups of 20 or 30 per recipient (n=40). Recipients were slaughtered on pregnancy D30. No significant differences were detected in gestation rates (50-70%) and embryo survival rates (14.7-25%), although survival was higher (P=0.0003) when 20 blastocysts were transferred compared to 30 (24.7% versus 15.5%). Our findings indicate that best results, in terms of subsequent in vivo embryo survival, were achieved after transferring 20 embryos at the blastocyst or expanded blastocyst stage, previously vitrified using cryoprotectant concentrations of 16.5 or 18%.     

THE APPEARANCE OF ACETYLCHOLINESTERASE IN THE MYOTOME OF THE EMBRYONIC RABBIT : An Electron Microscope Cytochemical and Biochemical Study

Acetylcholinesterase (AChE) activity has been studied in the myoblast of skeletal muscle of the 9–13 day fetal rabbit. Cytochemical activity is present in the nuclear envelope and the endoplasmic reticulum, including its derivatives the subsurface reticulum and the sarcoplasmic reticulum. End product is also found in the Golgi complex of the more differentiated myoblasts. The formation of reticulum-bound acetylcholinesterase in the myoblast appears to be independent of nerve-muscle contact, since the enzyme is present before the outgrowth of the spinal nerve. The nerve lacks cytochemical end product until the myoblast is well differentiated. Possible mechanisms of spontaneous muscle contraction have been discussed. A second type of myotomal cell, which exhibits a poorly localized end product of AChE activity, has been described. The ready solubility of the enzyme or diffusibility of its end product suggests that the enzyme may be a lyoesterase. This cell may be the precursor of the morphologically undifferentiated cell which is closely apposed to the myotubes in later stages of skeletal muscle development. Biochemical studies show a significant increase in AChE activity in the dermomyotome by day 12, when many of the myoblasts are well differentiated and the second type of myotomal cell is prominent. Cytochemical studies have indicated that many of the cells in the sample lack reaction product of enzymic activity, whereas others are very active. Biochemical values, therefore, reflect the amount of enzyme in the dermomyotome as a whole, but give little information on the enzymic content of individual cells.     

Quantitative evaluation of the trapping reaction of copperthiocholine histochemical procedures for localization of cholinesterases

Summary The aim of the present study was to investigate whether the trapping reaction of the histochemical procedure for the localization of ChE of Koelle and Friedenwald (1949) and its modification by Brzin and Pucihar (1976) proceeds quantitatively. The weight of the precipitate formed in the tissue sample during the histochemical procedure was compared with enzyme activity of an equal sample. The differential magnetic microbalance was used for measurements of reduced weight and for previous determination of density of the precipitate. The evidence for the composition of the final product was drawn from the quantitative analysis of copper and iodine and from the infrared spectra.Tsuji's statement (1974) that cuprous copper thiocholine iodide is the final product of histochemical procedures investigated was confirmed. It was found that the trapping reaction of the original as well as of the modified procedure under our experimental conditions in tissue sections proceeds quantiatively which means that one of the basic conditions for reliable localization is fulfilled.This work was supported by the grant of Research Association of Slovenia and NIH Grant No 02-008-1, Z-ZF-6     

Syntheses of di-hydrocarbyl derivatives of carbonyl phosphine complexes of iron

Complexes cis,trans-Fe(CO)₂(PMe₃)₂RR′ (R = CH₃, R′ = Ph (2); R = CH₃, R′ = CHCH₂ (3); R = CHCH₂, R′ = Ph (4); R = R′ = CHCH₂ (5); R = R′ = CH₃ (6)) were prepared by reaction of cis,trans-Fe(CO)₂(PMe₃)₂RCl (1) with organolithium reagents LiR′. All complexes were characterized in solution by IR and ¹H, ³¹P and, in a few cases, ¹³C NMR mono- and bi-dimensional spectroscopies. Complexes 5 and 6 were structurally characterized by X-ray diffractometric methods. In solution complexes 2, 3 and 4 undergo slowly coupling of the σ-hydrocarbyl substituents leading to Fe(CO)₃(PMe₃)₂ and other decomposition products. Complex 6 was very stable in solution in the absence of nucleophiles and in the solid state. Complex 5 transformed through intramolecular coupling of the vinyl groups into Fe(CO)(PMe₃)₂(η⁴-butadiene) (7), which was characterized in solution by IR and NMR spectroscopies.     

Kinetics of charge translocation in the passive downhill uptake mode of the Na+/H+ antiporter NhaA of Escherichia coli

The Na+/H+ antiporter NhaA is the main Na+ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA's electrogenicity, its specificity for Li+ and Na+ and its pronounced pH dependence in the range pH 6.5-8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na+ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent Km values were 11 mM for Na+ and 7.3 mM for Li+ at basic pH and 180 mM for Na+ and 50 mM for Li+ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent Km as well as turnover (Vmax).     

Kinetics of charge translocation in the passive downhill uptake mode of the Na/H antiporter NhaA of Escherichia coli

The Na⁺/H⁺ antiporter NhaA is the main Na⁺ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA's electrogenicity, its specificity for Li⁺ and Na⁺ and its pronounced pH dependence in the range pH 6.5-8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na⁺ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent K_m values were 11 mM for Na⁺ and 7.3 mM for Li⁺ at basic pH and 180 mM for Na⁺ and 50 mM for Li⁺ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent K_m as well as turnover (V_max).     

Intranuclear arrangement of human chromosome 12 is reflected in metaphase chromosomes as non-random bending

We have found a high correlation of non-random bending of human metaphase chromosome 12 with the intranuclear arrangement deduced by Nogami et al. (Chromosoma 108 (2000) 514), providing further evidence of the relation of non-random bending and the interphase organization of the nucleus.     

War, mental disorder and suicide

War as a human disaster of major significance has led to an increase in the number of suicides committed by people suffering from mental disorders. Considering the results of similar research, we were particularly interested in the effect that war has on the incidence of suicide among of people with mental disorders. The research included 16,362 patients with mental disorders, treated at the Clinic for Psychiatry at the Clinical Hospital Split during the nine-year timeframe which were divided into pre-war (April 6th 1988- April 7rh 1991), wartime (April 6th 1991 -April 7rh 1994) and post-war (April 6th 1997 - April 7th 2000) periods. We studied the effects of how wartime events upon people with mental disorders in terms of their suicide rates, taking into account gender, age group, and the diagnosis under which they were treated. In our research, we found a statistically significant difference in suicide incidence between three observed periods (prewar April 6th 1988 April 7th 1991; wartime April 6th 1991 -April 7th 1994; and postwar April 6th 1997 -April 7th 2000) with the incidence being the highest during the wartime period (chi2 =9.98: p=0.007). Out of 16,362 patients treated at the clinic during the observed timeframe, a total of 78 people committed suicide. Twenty-two patients committed suicide during the first three year pre-war period; 36, during the three year wartime period; and 20, during the third three year post-war period. With this research we intended to offer a better understanding of the complexity of the suicide problem of mental patients as a phenomenon.     

Extracellular Levels of Amino Acids and Choline in Human High Grade Gliomas: An Intraoperative Microdialysis Study

The concentrations of endogenous amino acids and choline in the extracellular fluid of human cerebral gliomas have been measured, for the first time, by in vivo microdialysis. Glioblastoma growth was associated with increased concentrations of choline, GABA, isoleucine, leucine, lysine, phenylalanine, taurine, tyrosine, and valine. There was no difference between grade III and grade IV tumors in the concentrations of phenylalanine, isoleucine, tyrosine, valine, and lysine, whereas the concentrations of choline, aspartate, taurine, GABA, leucine, and glutamate were significantly different in the two tumor-grade subgroups. In contrast to the other compounds, the concentration of glutamate was decreased in glioma. The parenchyma adjacent to the tumor showed significant changes only in the extracellular concentration of glutamate, isoleucine, and valine. The concentrations of choline and the amino acids, glutamate, leucine, taurine, and tyrosine showed significant positive correlations with the degree of cell proliferation. Epilepsy, which is relatively common in subjects with gliomas, was shown to be a significant confounding variable when the extracellular concentrations of aspartate, glutamate and GABA were considered.