Cloned calves from chromatin remodeled in vitro

We have developed a novel system for remodeling mammalian somatic nuclei in vitro prior to cloning by nuclear transplantation. The system involves permeabilization of the donor cell and chromatin condensation in a mitotic cell extract to promote removal of nuclear factors solubilized during chromosome condensation. The condensed chromosomes are transferred into enucleated oocytes prior to activation. Unlike nuclei of nuclear transplant embryos, nuclei of chromatin transplant embryos exhibit a pattern of markers closely resembling that of normal embryos. Healthy calves were produced by chromatin transfer. Compared with nuclear transfer, chromatin transfer shows a trend toward greater survival of cloned calves up to at least 1 mo after birth. This is the first successful demonstration of a method for directly manipulating the somatic donor chromatin prior to transplantation. This procedure should be useful for investigating mechanisms of nuclear reprogramming and for making improvements in the efficiency of mammalian cloning.     

Phosphatidyl ethanolamine is essential for targeting the arginine transporter Can1p to the plasma membrane of yeast

In continuation of our previous study, we show that phosphatidyl ethanolamine (PE) depletion affects, in addition to amino acid transporters, activities of at least two other proton motive force (pmf)-driven transporters (Ura4p and Mal6p). For Can1p, we demonstrate that the lack of PE results in a failure of the permease targeting to plasma membrane. Despite the pleiotropic effect of PE depletion, a specific role of PE in secretion of a defined group of permeases can be distinguished. Pmf-driven transporters are more sensitive to the lack of PE than other plasma membrane proteins.     

Monitoring brevetoxins during a Gymnodinium breve red tide: comparison of sodium channel specific cytotoxicity assay and mouse bioassay for determination of neurotoxic shellfish toxins in shellfish extracts

In October of 1996, a Gymnodinium breve bloom occurred in shellfish harvesting waters of Alabama, Mississippi and Louisiana, Gulf of Mexico, USA. Bloom densities reached 5.6x10(5) cells liter(-1) and bloom residence at shellfish sampling stations ranged from 3 to 28 days. Brevetoxin-2 dominated G. breve toxin profiles in bloom seawater extracts. Shellfish toxicity, assessed by mouse bioassay, exceeded the guidance level for up to 75 days after the bloom had dissipated. Cytotoxicity assays and mouse bioassays showed similar temporal patterns of shellfish toxicity, but the two methods differed in estimations of brevetoxin-3 equivalent toxicity by a factor of 93 to 1. LC-ESI-MS showed the temporal patterns in shellfish toxicity reflected metabolism of G. breve toxins. The molecular ions m/z 1004, 1017 and 1033 dominated LC-ESI-MS spectra of toxic chromatographic fractions from the extracts and were identified as brevetoxin metabolites on the basis of LC-APCI-MS-MS. The discrepancy between cytotoxicity and mouse bioassay estimates of brevetoxin-3 equivalent toxicity resulted from the difference in extraction efficiency of solvents used in the respective methods and the relative sensitivity of the assays to toxin metabolite mixtures present in the extracts. The normalized cytotoxicity assay showed 75% agreement with mouse bioassay positive test samples and 64% agreement with mouse bioassay negative test samples. Published in 1999 by John Wiley & Sons, Ltd.     

Initiation and organization of events during the first cell cycle in mammals: applications in cloning

The technology of cloning involves transplanting a diploid nucleus into a mature oocyte cytoplast. The cytoplast is then activated to initiate the first cell cycle of development as a nuclear transplant embryo. Initiation and regulation of events during the first cell cycle are, therefore, critical for proper reprogramming of the donor nucleus and development as a cloned embryo. Activation is normally induced by the sperm and is mediated by a series of intracellular free calcium ([Ca(2+)](i)) oscillations that last for several hours. Although it is not known precisely how the sperm induces activation, current evidence favors the delivery, by the sperm, of a soluble protein factor that causes the production of IP3. IP3 acts to open a Ca(2+) channel in the endoplasmic reticulum and release Ca(2+) into the cytosol. A variety of methods have been used to duplicate or replace the sperm-induced [Ca(2+)](i) increase to cause activation in nuclear transplant embryos. It has been found that treatments that cause a single transient [Ca(2+)](i) activate some oocytes with the level of activation increasing as the oocyte ages. Attempts have been made to extend the period of time over which [Ca(2+)](i) oscillations occur. This has been successful in increasing activation rates of less mature oocytes but the techniques are still cumbersome. An alternative method, that has been very successful, is the combination of a treatment that elevates [Ca(2+)](i) and a treatment that maintains low levels of maturation promoting factor for several hours after the initial [Ca(2+)](i) elevation. The sperm also contributes the centrosome that organizes microtubules during the first cell cycle. One current hypothesis for regulation of sperm centrosomal activity consists of a dephosphorylation of sperm connecting piece proteins following sperm entry into the oocyte and activation of the oocyte. Dephosphorylation of these proteins results in the disassembly of the connecting piece and assembly of a functional centrosome. In nuclear transfer, centrosomal components are contributed by the donor cell. If the cell is fused to the cytoplast before centriole replication then a single aster forms. If the cell is fused after centriole replication then two asters form. In either case and even in parthenogenetic oocytes, which do not have centrioles, the first cell cycle progresses to metaphase. However, progress is slow and some defects are observed in the assembly of chromosomes into a metaphase plate.     

Use of Ascomycete Extracts in Enzymatic Cocktail Formulations Increases Sugar Cane Bagasse Hydrolysis

Hemicellulases and accessory enzymes are essential for supplementation of cellulolytic enzyme extracts, and combinations of these enzymes can lead to high performance in plant biomass hydrolysis. In this work, enzyme extracts rich in hemicellulases and β-glucosidase, produced by the unique ascomycete strains Annulohypoxylon stygium DR47 and Aspergillus niger DR02, were tested for use in formulations with Celluclast 1.5 L. Statistical analysis showed that a mixture based on these enzymes was able to increase the hydrolysis of hydrothermally pretreated sugar cane bagasse. The two A. stygium extracts only effectively increased glucose release when they were combined. These extracts had no positive effect when used together with the A. niger extract, and the findings suggested that a blend based on the commercial cellulose preparation and the xylanase-rich extract from A. niger provided the best carbohydrate solubilization. Supplementation at low cellulolytic loading resulted in 120 and 238 % increases in cellulose and hemicellulose hydrolysis yields.     

Molecular characteristics of diverse populations are consistent with the hypothesis of a recent invasion of Drosophila melanogaster by mobile P elements

Approximately 100 strains derived from natural populations of Drosophila melanogaster were tested for the presence or absence of P- element sequences by using two molecular probes derived from internal regions of a full-sized P element. Strains that had been collected from several continents at varying times during the past 60 years were examined. The oldest available strains, representing most major geographical regions of the world, exhibited no detectable hybridization to the P-element probes. In contrast, all recently collected natural populations that were tested carried P-element sequences. The earliest appearance of P elements occurred in collections made during the 1950s and early 1960s in the Americas and during the late 1960s on other continents. The youngest strains that were completely devoid of P elements originated in populations sampled during the mid-1960s in America, but as late as 1974 in populations from the USSR. There are differences in the patterns of hybridization to the two P-element probes between populations from different geographical regions. These differences are consistent with the varying P-M phenotypic properties of these populations. Taken together with the results of phenotypic tests reported in earlier studies, the available evidence is consistent with the hypothesis of a worldwide P-element invasion of D. melanogaster during the past 30 years and suggests that the putative invasion of the Americas possibly preceded by approximately a decade that in Europe, Africa, and the rest of the world.      

Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nuclear reprogramming in nuclear transplant rabbit embryos

The first six genetically verified nuclear transplant rabbits have been produced in this study. Individual eight-cell stage embryo blastomeres were transferred and fused with enucleated mature oocytes of which six full-term offspring were produced out of 164 manipulated eggs. The following efficiency rates were determined for the nuclear transplantation procedure: chromosomal removal from oocytes, 92%; fusion rate, 84%; activation rate, 46%; embryo transfer rate, 27%. Additional reasons for the low efficiency rate of nuclear transplant embryos may include limited development due to aging in recipient oocytes and asynchronous transfers of manipulated embryos to recipient females. The successful development to term may have been due to the ability of the mature oocyte to reprogram the eight-cell stage nuclei. The number of cells in blastocysts derived from isolated eight-cell blastomeres (18 +/- .08) was lower than that of nonmanipulated pronuclear (106 +/- 5.1) and nuclear transplant embryos derived from eight-cell stage nuclei (91 +/- 10.2) (p less than 0.001). This evidence along with the significant amount of nuclear swelling in nuclear transplant embryos and a delay in the time of blastocyst formation indicate that nuclear reprogramming had taken place in these embryos. Successful nuclear reprogramming indicates that serial transfers could result in the expanded multiplication of mammalian embryos.     

Enhancement of <Emphasis Type="Italic">Penicillium echinulatum</Emphasis> glycoside hydrolase enzyme complex

The enhancement of enzyme complex produced by Penicillium echinulatum grown in several culture media components (bagasse sugarcane pretreated by various methods, soybean meal, wheat bran, sucrose, and yeast extract) was studied to increment FPase, xylanase, pectinase, and β-glucosidase enzyme activities. The present results indicated that culture media composed with 10 g/L of the various bagasse pretreatment methods did not have any substantial influence with respect to the FPase, xylanase, and β-glucosidase attained maximum values of, respectively, 2.68 FPU/mL, 2.04, and 115.4 IU/mL. On the other hand, proposed culture media to enhance β-glucosidase production composed of 10 g/L steam-exploded bagasse supplemented with soybean flour 5.0 g/L, yeast extract 1.0 g/L, and sucrose 10.0 g/L attained, respectively, 3.19 FPU/mL and 3.06 IU/mL while xylanase was maintained at the same level. The proteomes obtained from the optimized culture media for enhanced FPase, xylanase, pectinase, and β-glucosidase production were analyzed using mass spectrometry and a panel of GH enzyme activities against 16 different substrates. Culture medium designed to enhance β-glucosidase activity achieved higher enzymatic activities values (13 measured activities), compared to the culture media for FPase/pectinase (9 measured activities) and xylanase (7 measured activities), when tested against the 16 substrates. Mass spectrometry analyses of secretome showed a consistent result and the greatest number of spectral counts of Cazy family enzymes was found in designed β-glucosidase culture medium, followed by FPase/pectinase and xylanase. Most of the Cazy identified protein was cellobiohydrolase (GH6 and GH7), endoglucanase (GH5), and endo-1,4-β-xylanase (GH10). Enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse performed with β-glucosidase enhanced cocktail achieved 51.4 % glucose yield with 10 % w/v insoluble solids at enzyme load of 15 FPU/g material. Collectively the results demonstrated that it was possible to rationally modulate the GH activity of the enzymatic complex secreted by P. echinulatum using adjustment of the culture medium composition. The proposed strategy may contribute to increase enzymatic hydrolysis of lignocellulosic materials.