Safety aspects of switched gradient fields

Examination of the first crystal structures of proteins from a halophilic organism suggests that an abundance of acidic residues distributed over the protein surface is a key determinant of adaptation to high-salt conditions. Although one extant theory suggests that acidic residues are favored because of their superior water-binding capacity, it is clear that extensive repulsive electrostatic interactions will also be present in such proteins at physiological pH. To investigate the magnitude and importance of such electrostatic interactions, we conducted a theoretical analysis of their contributions to the salt and pH-dependence of stability of two halophilic proteins. Our approach centers on use of the Poisson-Boltzmann equation of classical electrostatics, applied at an atomic level of detail to crystal structures of the proteins. We first show that in using the method, it is important to account for the fact that the dielectric constant of water decreases at high salt concentrations, in order to reproduce experimental changes in pKa values of small acids and bases. We then conduct a comparison of salt and pH effects on the stability of 2Fe-2S ferredoxins from the halophile Haloarcula marismortui and the non-halophile anabaena. In both proteins, substantial upward shifts in pKa accompany protein folding, though shifts are considerably larger, on average, in the halophile. Upward shifts for basic residues occur because of favorable salt-bridge interactions, whilst upward shifts for acidic residues result from unfavorable electrostatic interactions with other acidic groups. Our calculations suggest that at pH 7 the stability of the halophilic protein is decreased by 18.2 kcal/mol on lowering the salt concentration from 5 M to 100 mM, a result that is in line with the fact that halophilic proteins generally unfold at low salt concentrations. For comparison, the non-halophilic ferredoxin is calculated to be destabilized by only 5.1 kcal/mol over the same range. Analysis of the pH stability curve suggests that lowering the pH should increase the intrinsic stability of the halophilic protein at low salt concentrations, although in practice this is not observed because of aggregation effects. We report the results of a similar analysis carried out on the tetrameric malate dehydrogenase from H. marismortui. In this case, we investigated the salt and pH dependence of the various monomer-monomer interactions present in the tetramer. All monomer-monomer interactions are found to make substantial contributions to the salt-dependence of stability of the tetramer. Excellent agreement is obtained between our calculated results for the stability of the tetramer and experimental results. In particular, the finding that at 4 M NaCl, the tetramer is stable only between pH 4.8 and 10 is accurately reproduced. Taken together, our results suggest that repulsive electrostatic interactions between acidic residues are a major factor in the destabilization of halophilic proteins in low-salt conditions, and that these interactions remain destabilizing even at high salt concentrations. As a consequence, the role of acidic residues in halophilic proteins may be more to prevent aggregation than to make a positive contribution to intrinsic protein stability.     

The dorsal mesocardium and development of the pulmonary veins in human embryos

Pulmonary vein development was studied using serial histologic sections of normal human embryos of Carnegie stages 11 to 15. Three-dimensional models were created in the program Swivel 3D on a Macintosh IIfx computer. The position of the mesocardium was found to be an important factor in the placement of the vein. Since the vein grows through a gap in the myoepicardium of the dorsal atrial wall created by the mesocardium, the vein can only grow where the mesocardium is positioned. Displacement of the initially median pulmonary vein ostium into the left atrium appeared to be caused by the formation of the left valve of the sinus venosus. This latter structure displaces the mesocardium to the left from stage 14 and later, carrying the vein to the left as well. The subsequent development of several pulmonary veins from the original single pulmonary vein occurred later, as the apex of the heart rotated to the left and brought the left atrium into a dorsal midline position. The study shows that correct placement of the pulmonary vein in the left atrium is the consequence of the successful execution of a sequence of developmental events in cardiogenesis.     

The role of quenched-ln embryos in solid-state nucleation processes

Consideration is given the possibility that embryos retained from a higher temperature may assist in subsequent nucleation processes. Quantitative thermodynamic and kinetic constraints are found to exist for formation and retention of “precipitation-type” embryos. It is shown for dilute solutions that the spectrum of concentration fluctuations is independent of temperature, so that quenching rate is not a consideration. Various suggestions for the martensite nucleus are considered. This paper is based on a presentation made at a symposium on Altering the Time Cycle of Heat Treatment, held at the Philadelphia meeting of The Metallurgical Society of AIME, October 14, 1969, under the sponsorship of the IMD Heat Treatment Committee.     

The effects of lithium on neurulation stage mouse embryos

The aim of this study was to evaluate the maternal toxicity and teratogenicity of lithium following intraperitoneal injection (i.p.) with lithium carbonate (Li2CO3) in pregnant CD-1 mice at the developmental stage of neurulation (E8; day of vaginal plug, E0). Light (LM) and electron (TEM) microscopic studies were also done to document the tissue and cellular changes occurring in embryonic tissues during the 48 h following treatment with 300 mg/kg body wt. Li2CO3. Controls were untreated or given equimolar amounts of NaCl or Na2CO3. A pharmacokinetic study showed that lithium was rapidly absorbed from the peritoneal cavity after the above-stated dose, achieved peak serum levels of 9.8 mmol/l within 1 h, had a half-life in the blood of 5 h and was completely cleared by 16 to 24 h after injection. Doses of Li2CO3 > 300 mg/kg body wt. were toxic to adult CD-1 mice. The latter dose had no detectable maternal toxicity but caused a 19% resorption rate and 2% incidence of open cranial neural tube defect in gestations terminated on E18. The malformation and resorption rates in gestations terminated on E11, E12 and E14 were not significantly different from those of E18. A strong litter effect was seen both for the resorption and malformation rates at all stages examined. At 3 h after treatment cell death became evident in the neuroepithelium. Cells continued to die for approximately 17 h and all necrotic debris had been cleared by 48 h. Also at 3 h after treatment small densely stained inclusions began to appear in mesodermal cells. TEM showed these to be non-membrane bound with an irregular shape and variable size; the lack of staining for acid phosphatase indicated a non-lysosomal structure; the ultrastructural features suggested a lipoid basis. At 24 h after treatment vascular ruptures and surface ectodermal ruptures were seen in the cranial mesoderm. These ruptures with extravascated blood were also seen at 48 h after treatment. A litter effect was also noted with respect to the tissue and cellular changes. These experiments suggest that the developing vascular system may be a target for lithium. In addition, the possibility is discussed that lithium induced cell death in the neuroepithelium may lead to neural tube defects.     

The effect of propanediol on the morphology of fresh and frozen equine embryos

Seventeen horse embryos recovered on the sixth day after spontaneous ovulation were; 1) washed in PBS (n = 6), 2) treated with 1.5 M 1-2 propanediol (n = 6) or, 3) frozen and thawed using 1.5 M propanediol as the cryoprotectant (n = 5). After treatment, the embryos were incubated for 6 h in medium before they were fixed, serially sectioned and examined microscopically to count the total numbers of interphase, mitotic and pycnotic nuclei. Significant differences were measured only in the mean proportions of pycnotic cells (+/- s.d.), both between the control (9.2 +/- 7.3%) and frozen-thawed embryos (52.8 +/- 37.1%; P<0.05) and between the propanediol-treated (10.8 +/- 4.6%) and the frozen-thawed embryos (P<0.01). Propanediol appears to be minimally toxic to equine embryos but it is a poor cryoprotectant.     

The effects of cytochalasins on the ultrastructure of neurulating hamster embryos in vivo

Single intraperitoneal injections of cytochalasin B (CB) in dimethylsulfoxide were given to gravid Syrian hamsters on the eighth day of pregnancy at various dose levels. Exencephaly and encephalocele, the only defects which were seen in the term litters, occurred in dose-response patterns reaching peak frequencies of 14.9% and 53.2%, respectively, at the highest dose level, while accompanied by a mortality of 27.7% of implantations. Although these abnormalities were the same as those resulting from cytochalasin D (CD) treatment at this time, the frequencies were lower and the distribution of defects somewhat different. Morphological comparison of embryos fixed at various times after maternal treatment with 7.0 mg/kg CB or 1.5 mg/kg CD demonstrated qualitatively similar changes in response to either teratogen, leading to failure of the cranial neural folds to approximate and close. The principal ultrastructural changes involved alterations in the topography of the apical membranes of neuroectoderm cells. At doses which produced high frequencies of gross defects in the term litters, no changes were seen in the apical bundles of microfilaments in these cells, although much higher dose levels did disrupt these structures. The results support the hypothesis that the cell membrane is the primary target of these teratogens in vivo.     

The expression of beta-glucuronidase during preimplantation development of mouse embryos

The globin mRNAs containing between 30 and 40 polyadenylate residues can be separated from thos mRNAs containing longer poly(A) regions by Millipore filter binding. The molecular weights of the alpha-and beta-globin mRNAs containing this size class of poly(A) have beed determined by lectrophoresis on 3.7% polyacrylamide gels in the presence of 99% formamide. Because the number of adenylic acid residues in these mRNAs is known, the number of non-poly(A) nucleotides can be accurately calculated. The molecular weight of the beta-globin mRNA is 235 000 +/- 28 000 (736 +/- 88 nucleotides) and that of the alpha-globin mRNA is 208 900 +/- 43 870 (653 +/- 78 nucleotides). By subtracting the number of nucleotides in the coding and poly(A) regions, the number of non-coding nucleotides in the beta-globin mRNA were calculated to be 261, 69 more than the 193 present in the alpha-globin mRNA. Comparison of size estimates of newly synthesized globin mRNAs containing longer average lengths of poly(A) shhowed that there is no comparable processin of the 5' termini of the alpha-and beta-globin mRNAs concomitant with the stepwise degradation of the poly(A) regions which occur as the mRNAs mature.     

The ultrastructure of the cell center in the enterocytes of mouse embryos and newborn mice

The centrosome structure was studied in differentiating small intestine enterocytes of mouse embryos (day 16 to 17 of gestation) and newborn mice. It is shown that fine structure of the centrosome in embryonic enterocytes differs from that found in cells of the adult mice. The centrosome of the crypt cells is more active: a greater number of cytoplasmic microtubules terminates there; mother centriole has two types of satellites; both centrioles are surrounded by fine fibrillar material. In 20% of crypt cells, replication of centrioles is observed. In the upper part of the villus of embryonic intestine, there still are two centrioles per cell, but they are located 3-7 um apart and devoid of any cytoplasmic microtubules attached to or directed toward them. In newborn mice, centrosomes of cells located at the bottom of the crypt are less active, as compared with centrosomes of embryonic cells. There are 1-3 satellites on mother centriole and few cytoplasmic microtubules closing to the centrosome. In 20% of cells from the bottom of the crypt centrioles are replicating. In the lateral part of the crypt, centrosome is inactive: centrioles are located 1-3 um apart and do not replicate. In the villus, centrioles undergo changes similar to those observed in the villus of adult mice. Centrioles loose portions of microtubule triplets. Near the top of the villus, only one centriole was found in two of 25 studied cells. In the enterocytes of the murine small intestine, centrioles are always located far from nuclei and close to the apical cell surface (1-3 um from the brush border). Centrioles never form a primary cilium and are not attached to the plasma membrane.     

The chromosome constitution of human preimplantation embryos fertilized in vitro

The chromosome constitution of five haploid, 178 diploid and 11 triploid embryos fertilized in vitro was determined after fixation on day 2 or day 3 of development. Karyotype analysis of 178 diploid embryos revealed abnormalities in 40 (22.5%) cases: 34 (19.1%) aneuploids, four (2.2%) mosaic embryos and two (1.1%) structural anomalies were identified. The majority of aneuploid karyotypes (28/34) involved a single chromosome but six embryos had aneuploidy of two or three chromosomes. The E group was most frequently involved in aneuploid karyotypes (10/23 hyperdiploid embryos) and trisomy 16, the most common single anomaly in diploid embryos, was detected in 2.2% (4/178) of cases. Only one case of sex chromosome monosomy was identified. An excess of female karyotypes was detected in abnormal cases (sex ratio 0.48); this ratio was significantly (P < 0.05) different from that observed in normal cases (74:64, XY:XX). The incidence of aneuploidy increased with maternal age but this did not reach statistical significance. Embryo morphology and growth rate, assessed by embryo development rating (EDR), did not distinguish between normal (mean score 7.9; mean EDR 96.1) and aneuploid (mean score 8.1; mean EDR, 92.1) embryos. Numbers of hyperploid (n = 17) and hypoploid (n = 11) embryos (non-mosaic cases involving single chromosomes) were not statistically different. The relative proportions of chromosomes involved in trisomic karyotypes showed a remarkable similarity to the pattern in spontaneous abortions. Pronuclear status was an unreliable predictor of ploidy. Small numbers of karyotyped triploid embryos revealed equal proportions of XXX, XXY and XYY embryos.     

The ovarian hyperstimulation syndrome in extracorporeal oocyte fertilization and the transfer of cleavage-stage embryos

Major clinical characteristics of ovarian hyperstimulation syndrome are described and criteria of predicting the course of this disease and measures to prevent it presented. Statistical data on the presence of this syndrome in various forms during various schemes of superovulation stimulation are offered. Optimal schemes for the treatment of the condition are suggested.     

Cryopreservation of equine embryos

Principles and procedures for cryopreservation of equine embryos are described. Embryos less than 250 microM in diameter can be cryopreserved successfully if glycerol is used as the cryoprotectant. Cooling is takes place in such a way that most of the water leaves the cells before intracellular ice forms, and glycerol is removed after thawing without undue osmotic swelling of cells. Vitrification procedures also show promise for small embryos. Satisfactory procedures for cryopreserving embryos of more than 250 microM in diameter are not yet available.     

The influence of in-vitro culture and cooling on the survival and development of cow embryos

Cow morulae cultured in a phosphate-buffered medium containing serum developed normally and retained viability when transferred to recipients. Unlike earlier cleavage stages, cow blastocysts tolerated cooling to 0 degrees C and retained viability after storage for 48 hr at 0 degrees C when transferred to recipients.     

The effect of concanavalin-A on the reaggregation of cells dissociated from Xenopus laevis early embryos

The effects of concanavalin-A on the reaggregation and sorting of cells from Xenopus laevis early embryos have been studied. The results suggest that at high concentrations, concanavalin-A can prevent reaggregation.     

The deleterious effects of fungicides and herbicides on Xenopus laevis embryos

Groups of 30 Xenopus laevis embryos, at "tail-bud" stage (Nieukoop-Faber stages 22-24) were exposed to 0.1-2 ppm concentrations of various pesticides for 1 to 10 days. The pesticides used were chloranil and dichlone (both are fungicidal and herbicidal); diquat (herbicide); and nabam (fungicide). The parameters examined were mortality, gross morphology, histology, and behavior. Chloranil (1.25 to 1.75 ppm) treated embryos showed abnormalities of the otolith, optic cup, and general pigmentation. Their movement was sporadically convulsive and they were unable to maintain proper balance. Dichlone (0.1 to 0.15 ppm) disrupted the development of the cephalic end of the embryo. Many of these embryos developed a slightly retarded trunk and tail only. These headless embryos lived for a time and were relatively lethargic. Diquat (0.75 to 2.0 ppm) administration reduced body size and pigmentation, and altered body shape. When embryos were treated with both 1.0 ppm of diquat and 2.0 ppm of nabam the integrity of myomeres and myocommata of the musculature was disrupted. The histological bases of these morphological and behavioral changes are discussed.     

The strange case of the misplaced suppository

In a controversial decision, the General Medical Council has found a consultant anesthetist guilty of serious professional misconduct for giving a painkilling suppository without forewarning the patient and obtaining consent. Even though the suppository was misplaced in the patient's vagina, this was accepted as a mistake, at issue was whether a specific separate consent was required for insertion of the suppository.