Fine structure of early human embryos frozen with 1,2 propanediol

Previous studies by a French group (Fertil Steril 44:645–651, 1985) have shown that two-to eight-cell human embryos can survive slow freeze-thawing with propanediol in a biological freezer. These embryos were assessed for morphological appearance by phase-contrast microscopy. We assessed the structure of 25 frozen-thawed one- to 12-cell embryos, obtained from our in vitro fertilization (IVF) and GIFT programmes, by phase-contrast and electron microscopy, using the same method of cryopreservation. One-fourth of the embryos examined had all cells intact, and more than one-half the embryos had over 50% of their cells well preserved. Some of these embryos had unequal blastomeres and cytoplasmic fragments. Ultrastructural assessment revealed good preservation of fine structure in the intact blastomeres of all embryos and maintenance of cell-to-cell contacts. Most cytoplasmic organelles, cell membranes, and nuclei were well preserved compared to nonfrozen controls. The cells that were cryoinjured showed varying degrees of disorganization of the cell membrane, cytosol, and cellular membranes, including swelling and disruption of the nuclear envelope. Disruption of the zona was somewhat rare. Small cytoplasmic fragments were less prone to cryoinjury than blastomeres. The use of propanediol for embryo cryopreservation seems to be feasible; frozen embryos with more than 50% cells intact have produced 10 pregnancies after embryo transfer (Fertil Steril 46:268–272, 1986). Replacement of 17 frozen embryos in seven patients has resulted in a twin pregnancy in Singapore. However, the effects of freezing on the mitotic spindles of embryonic cells need to be investigated further.     

Structural heterogeneity of the alpha subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding

The structural basis for the heterogeneity of the two agonist binding sites of the Torpedo californica acetylcholine receptor with respect to antagonist binding and reactivity toward affinity alkylating reagents was investigated. There is one agonist binding site on each of the two alpha subunits in a receptor monomer. One of these sites is easily affinity labeled with bromoacetylcholine, while more extreme conditions are required to label the other. Evidence is presented that the site which is easily labeled with bromoacetylcholine is the site with higher affinity for the antagonist d-tubocurarine. Digestion of purified alpha subunits with staphylococcal V8 protease gave two limit fragments with apparent molecular weights of 17K and 19K. Both of these fragments began at residue 46 of the alpha sequence, and both reacted with monoclonal antibodies specific for the sequence alpha 152-159 but not with antibodies specific for alpha 235-242. Their tryptic peptide maps and reactivity with a number of monoclonal antibodies were virtually identical. Only the 17-kilodalton (17-kDa) fragments stained heavily for sugars with Schiff's reagent. However, both fragments bound 125I-labeled concanavalin A. Complete removal of carbohydrate detectable with concanavalin A from V8 protease digests of alpha subunits resulted in two fragments of lower apparent molecular weights, indicating that these fragments differed not only in carbohydrate content but also in their C-termini or by another covalent modification. Covalent labeling of one of the two agonist sites of the intact receptor with bromo[3H]acetylcholine followed by digestion with V8 protease resulted in labeling of only the 19-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laboratory studies on salmonella-contaminated cheese involved in a major outbreak of gastroenteritis

A major outbreak of gastroenteritis was traced to Cheddar cheese contaminated with Salmonella typhimurium. There were no significant differences in pH values of the contaminated (mean pH 5.31) and non-contaminated (mean pH 5.39) cheese. The isolation rates of Salm. typhimurium were about the same when cheese samples were homogenized in lactose broth, lactose broth containing 1% Tween 80, or in aqueous 2% sodium citrate. Salmonella typhimurium was isolated regardless of preenrichment in lactose broth, but required selective enrichment in selenite cystine or tetrathionate brilliant green broth. There were no marked differences in the isolation rates obtained with different selective enrichment media, or after incubation at 36 degrees and 43 degrees C for 24 or 48 h. Contaminated samples of cheese failed to yield Salm. typhimurium consistently despite large and multiple samplings; samples from the interior of cheese blocks yielded positive results more frequently than the samples from the exterior. The number of Salm. typhimurium in factory sealed blocks as well as in samples obtained from the homes of known cases of salmonellosis was found to range from less than 3/100 g to 9/100 g of cheese. The infective dose of Salm. typhimurium in contaminated cheese was probably no greater than 10(4) organisms, and a rapid decline in numbers of Salm. typhimurium must have occurred subsequent to the outbreak.     

Laboratory studies on salmonella-contaminated cheese involved in a major outbreak of gastroenteritis

Dried biomass of Streptomyces cyaneus NCIB 9616 harvested at 1, 3, 7, 10 and 14 d from a liquid culture was degraded by alkaline methanolysis and the fatty acid methyl esters released examined quantitatively by gas chromatography. Isoprenoid quinones were extracted with petroleum ether and methanolic saline, purified by thin-layer chromatography and quantified by high performance liquid chromatography. Data for each component were plotted against time. The results showed that at the beginning of logarithmic growth there was a small quantitative shift in the fatty acid composition but, the fatty acid profiles remained relatively constant during both the logarithmic and stationary phases of growth. In contrast, the isoprenoid quinone composition depended markedly on the stage of growth. These results indicate that valid comparison of isoprenoid quinone profiles requires that sampling take place at defined stages of the growth cycle.     

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

A vigorous mutant sugarcane (Saccharum sp.) clone Co 527

Summary A vigorous fast growing mutant which ends vegetative growth sixty days earlier than the parent variety Co 527 was isolated from gamma irradiated vegetative buds. The mutant initially segregated but stabilized in the vM₄ generation. Its growth rate was almost fifty per cent higher than Co 527 beginning in the early stages of growth. It produced a significantly higher early shoot population which enabled it to yield a higher number of millable canes at maturity. Economic characters like sucrose content and juice purity remained unaffected. This mutant had a chromosomal basis in that the number was 2 to 3 lower than in the parent variety.Research carried out under the FAO/IAEA Co-ordinated Research Programme on the Improvement of Vegetatively Propagated Plants and Tree Crops through Induced mutations (Research Contract No. 1680)     

Concentration of plasminogen activators and inhibitor in the human endometrium at different phases of the menstrual cycle.

The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor (PAI-1) have been determined in endometrial curettings obtained from 46 subfertile women during proliferative, early or late secretory phases of the menstrual cycle. t-PA activity and antigen concentrations was significantly higher (P < 0.001) in late secretory endometrium than in proliferative or early secretory endometrium. Higher concentrations of PAI-1 antigen (P < 0.05) were also noted in late secretory phase than in proliferative and early secretory endometrium. However, u-PA concentration was not significantly different and no PAI activity could be demonstrated in the menstrual phases studied. Zymography studies confirmed the presence of both t-PA and u-PA in the endometrium. Ovarian hormonal patterns may therefore influence the activity of plasminogen activators especially of t-PA in the endometrium during various phases of the menstrual cycle.     

Mapping the antigenic epitopes of human dihydrofolate reductase by systematic synthesis of peptides on solid supports.

All of the 181 possible overlapping hexapeptides as well as 179 octapeptides covering the amino acid sequence of human dihydrofolate reductase (hDHFR) were synthesized on polyethylene supports. The synthetic procedure of Geysen et al. (Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G., and Schoofs, P. G. (1987) J. Immunol. Methods 102, 259-274) was modified to obtain up to 100 nmol of peptide on each pin. Peptides constituting antigenic epitopes on hDHFR were identified by examining the binding of antibodies raised against both native and denatured hDHFR to these peptides by enzyme-linked immunosorbent assay. The peptides bound in a similar pattern to polyclonal antibodies against both native and denatured dihydrofolate reductase (DHFR). Six major epitopes were located corresponding to residues 27-33, 45-51, 67-74, 133-139, 153-158, and 176-181 using both hexapeptides and octapeptides. An additional epitope, constituting residues 14-21, was found by the use of octapeptides. Most of the epitopes are hydrophilic and reside largely in "loop" regions at the boundaries of secondary structural elements of hDHFR. This observation is consistent with our previous results which suggested that ligand binding at the active site of the enzyme can cause a dramatic reduction in antibody binding to DHFR due to conformational constraints in flexible loop regions in various parts of the molecule. The similarity of the immunogenic profiles of native versus denatured hDHFR indicates that the two forms of the antigen share the same amino acid sequence-specific epitopes. Competitive enzyme-linked immunosorbent assay showed that the binding of anti-hDHFR antiserum to both native and denatured hDHFR was inhibited by approximately 30% by the seven antigenic peptides, indicating that a significant proportion of the antibodies elicited by this enzyme is specific for short peptides. Besides revealing the antigenic structure of DHFR our results provide a rational basis for the design of mutant DHFRs to study the importance of loop residues in the conformational dynamics of the enzyme.     

Effect of imipramine hydrochloride on the activity of gamma-aminobutyric acid transaminase in regions of the rat brain

The effect of intraperitoneal injection of imipramine hydrochloride on the activity of gamma-aminobutyric acid transaminase was determined in three regions of the rat brain.The cerebral hemispheres did not show a significant change in the activity of gamma-aminobutyric acid transaminase. Cerebellum and brain stem, both, however, showed a very significant decrease in the activity of the enzyme at 15 and 30 minutes after drug administration. At 90 minutes after drug administration, the activity of gamma-aminobutyric acid transaminase had returned to nearly control values.