Use Of Cell-free Extracts Research Articles

Xenopus cell-free extracts and their applications in cell biology study.

Xenopus has proven to be a remarkably versatile model organism in the realm of biological research for numerous years, owing to its straightforward maintenance in laboratory settings and its abundant provision of ample-sized oocytes, eggs, and embryos. The cell cycle of these oocytes, eggs, and early embryos exhibits synchrony, and extracts derived from these cells serve various research purposes. Many fundamental concepts in biochemistry, cell biology, and development have been elucidated through the use of cell-free extracts derived from Xenopus cells. Over the past few decades, a wide array of cell-free extracts has been prepared from oocytes, eggs, and early embryos of different Xenopus species at varying cell cycle stages. Each of these extracts possesses distinct characteristics. This review provides a concise overview of the Xenopus species employed in laboratory research, the diverse types of cell-free extracts available, and their respective properties. Furthermore, this review delves into the extensive investigation of spindle assembly in Xenopus egg extracts, underscoring the versatility and potency of these cell-free systems in the realm of cell biology.

Isolation and Demembranation of Xenopus Sperm Nuclei.

The inherent experimental advantages of intact amphibian eggs have been exploited for several decades to advance our understanding of fundamental developmental processes and the cell cycle. Characterization of these processes at the molecular level has been greatly advanced by the use of cell-free extracts, which permit the development of biochemically tractable approaches. Demembranated Xenopus laevis sperm nuclei have been used with cell-free extracts to recapitulate cell cycle progression and to control the cell cycle state of the egg extract. This system has become an invaluable and widely used tool for studies of cell cycle regulation and many downstream events. Here, we describe a protocol, derived in part from other published protocols and modified over time, for the preparation of Xenopus sperm nuclei that can be used in a variety of in vitro assays.

Analyzing the micromechanics of the cell division apparatus.

Cell division involves mechanical processes, such as chromosome transport and centrosome separation. Quantitative micromanipulation-based approaches have been central to dissecting the forces driving these processes. We highlight two biophysical assays that can be employed for such analyses. First, an in vitro "mini-spindle" assay is described that can be used to examine the collective mechanics of mitotic motor proteins cross-linking two microtubules. In the spindle, motor proteins (e.g., kinesin-5, kinesin-14, and dynein) can localize to overlapping microtubules that slide relative to each other, work as an ensemble, and equilibrate between cytoplasm and the microtubules. The "mini-spindle" assay can recapitulate these features and allows measurements of forces generated between adjacent microtubules and their dependence on filament orientation, sliding speed, overlap length, and motor protein density. Second, we describe a force-calibrated microneedle-based "whole-spindle" micromechanics assay. Microneedle-based micromanipulation can be a useful technique to examine cellular scale mechanics, but its use has been restricted by the difficulty in getting probes to penetrate the plasma membrane without disrupting cell physiology. As detailed here, the use of cell-free extracts prepared from metaphase-arrested Xenopus eggs can address this limitation. These micromanipulation studies also benefit from the use of frozen stocks of Xenopus egg extract. Together, these approaches can be used to decipher how micromechanics and biochemical activities ensure successful cell division.

Living Xenopus oocytes, eggs, and embryos as models for cell division.

Xenopus laevis has long been a popular model for studies of development and, based on the use of cell-free extracts derived from its eggs, as a model for reconstitution of cell cycle regulation and other basic cellular processes. However, work over the last several years has shown that intact Xenopus eggs and embryos are also powerful models for visualization and characterization of cell cycle-regulated cytoskeletal dynamics. These findings were something of a surprise, given that the relatively low opacity of Xenopus eggs and embryos was assumed to make them poor subjects for live-cell imaging. In fact, however, the high tolerance for light exposure, the development of new imaging approaches, new probes for cytoskeletal components and cytoskeletal regulators, and the ease of microinjection make the Xenopus oocytes, eggs, and embryos one of the most useful live-cell imaging models among the vertebrates. In this review, we describe the basics of using X. laevis as a model organism for studying cell division and outline experimental approaches for imaging cytoskeletal components in vivo in X. laevis embryos and eggs.

Growth Inhibition of Some Phytopathogenic Bacteria by Cell-Free Extracts from Enterococcus sp

Aims: To inhibit of bacterial growth of three important phyto-pathogenic bacteria: Erwinia carotovora, Clavibacter michiganensis sp. michiganensis and Xanthomonas axonopodis by cell-free extracts from submerged cultures of two strains of Enterococcus sp. was tested. Study Design: A complete randomized experimental design with factorial fix was used to evaluate the efficiency of growth inhibition against the phytopathogenic bacteria. Place and Duration of Study: Laboratory of Bioprocesses, Department of Food Science and Technology, School of Chemistry, Universidad Autonoma de Coahuila, Mexico, between December 2011 and July 2012. Methodology: Enterococci strains were isolated from goat milk, buttermilk and whey by typical microbiological procedures and primarily identified based on biochemical tests. Short Communication British Biotechnology Journal, 3(3): 359-366, 2013 360 Strains were subsequently activated in MRS broth and cells were separated by centrifugation and filtration. Cell-free extracts were tested against plant pathogenic bacteria to determine their growth inhibition potential. Results: Strains of Enterococcus MII-1 and MIV-2 were able to inhibit the growth of three pathogenic bacteria, demonstrating to be an attractive alternative for biological control assays. Conclusion: The cell-free extracts of Enterococcus spp. show inhibition potential to inhibit phytopathogenic bacteria that cause diseases in horticultural crops. Further studies are needed to completely evidence the high potential of use of cell-free extracts from Enterococcus MII-1 and MIV-2.

Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins

The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication.

The Snf1 kinase of the filamentous fungus Hypocrea jecorina phosphorylates regulation-relevant serine residues in the yeast carbon catabolite repressor Mig1 but not in the filamentous fungal counterpart Cre1

In Saccharomyces cerevisiae, the SNF1 gene product phosphorylates the carbon catabolite repressor protein Mig1 under conditions when glucose is limiting, thereby relieving the fungus from catabolite repression. We have investigated whether the corresponding counterpart of filamentous fungi—the Cre1 protein—is also phosphorylated by Snf1. To this end, snf1, an ortholog of SNF1, was isolated from the ascomycete Hypocrea jecorina. The gene encodes a protein with high similarity to Snf1 kinases from other eukaryotes in its N-terminal catalytic domain, but little similarity in the C-terminal half of the protein, albeit some short aa-areas were detected, however, which are conserved in filamentous fungi and in yeast. Expression of snf1 is independent of the carbon source. An overexpressed catalytic domain of H. jecorina Snf1 readily phosphorylated yeast Mig1, but not a Mig1 mutant form, in which all four identified Snf1 phosphorylation sites ( Φ X RXX SXXX Φ ) had been mutated. The enzyme did neither phosphorylate H. jecorina Cre1 nor histone H3, another substrate of Snf1 kinase in yeast. H. jecorina Snf1 also phosphorylated peptides comprising the strict Snf1 consensus, but notably did not phosphorylate peptides containing the regulatory serine residue in Cre1 (=Ser 241 in H. jecorina Cre1 and Ser 266 in Sclerotinia sclerotiorum CRE1). The use of cell-free extracts of H. jecorina as protein source for Snf1 showed phosphorylation of an unknown 36 kDa protein, which was present only in extracts from glucose-grown mycelia. We conclude that the Snf1 kinase from H. jecorina is not involved in the phosphorylation of Cre1.

Serodiagnosis of Leishmaniasis

Leishmaniasis is a spectrum of diseases ranging in severity from cutaneous (CL), post-kala-azar dermal (PKDL), and diffuse cutaneous (DCL) to mucocutaneous (MCL) and visceral (VL) infections that are endemic in 86 tropical and subtropical countries around the world, accounting for 75,000 deaths per year. Different forms of leishmaniases are generally caused by different distinct species of Leishmania having a digenetic life cycle alternating between an aflagellated amastigote form replicative within the macrophages of the host and a flagellated promastigote form that multiplies within the gut of the sandfly. VL, MCL, PKDL, DCL, and CL forms of the disease can be arranged on a priority basis in accordance with the humoral immune responses of host. Generally, the cell-mediated immunity, particularly the delayed-type hypersensitivity to leishmanial antigens, is associated with CL, MCL, PKDL, and cured VL cases. The serodiagnosis of leishmaniasis appears to be an alternative to parasite detection in biopsy samples either by the staining of amastigotes or by culturing the amastigotes, which transform to a promastigote form and replicate. A battery of immunological procedures have been developed or adapted to demonstrate either humoral or cell-mediated immune responses against Leishmania for diagnosis and epidemiological survey. The sensitivity and specificity of such diagnostic methods depend on the type, source, and purity of antigen employed, as some of the leishmanial antigens have common cross-reactive epitopes shared with other microorganisms, particularly Trypanosoma, Mycobacteria, Plasmodia, and Schistosoma. Serodiagnostic techniques for the detection of antileishmanial antibodies have been employed with about 72 to 100, 23 to 90, 83, and 33 to 100% success in VL, CL, MCL, and PKDL patients, respectively. The Leishmanin skin test (LST) is useful to detect MCL and CL, with about 100 and 84% success, respectively. In PKDL, the gradual fall of antileishmanial antibody titer to some extent and the rise of delayed hypersensitivity to the parasite antigen are the characteristic features associated with the chronicity of the disease. The use of whole promastigote as the source of antigens in the direct agglutination test (DAT) and immunofluorescent test (IFAT) gave cross-reactions with the sera of leprosy, tuberculosis, and African trypanosomiasis patients. Again, the use of cell-free extracts of promastigotes generally gave false positive results with the sera of normal human and Chagas' disease, leprosy, tuberculosis, and malaria patients in enzyme-linked immunosorbent assay (ELISA), dot ELISA, immunodiffusion, immunoelectrophoresis, and counter-current immunoelectrophoresis tests.(ABSTRACT TRUNCATED AT 400 WORDS)

The use of cell free extracts derived from fungal protoplasts in the study of aflatoxin biosynthesis.

A supernatant fraction derived from protoplasts of Aspergillus flavus was shown to be capable of converting both sterigmatocystin and versiconal hemiacetal acetate to aflatoxin B1. Versicolorin A was not converted under the same conditions.

Effect of interferon pretreatment on coupled transcription and translation in cell-free extracts of primary chick embryo cells

The mechanism of action of chick interferon was studied by the use of cell-free extracts of interferon-treated and mock-interferon-treated primary chick embryo cells. These cell-free extracts differed from those of chick origin which have been used previously in that they did not require supplementation with heterologous factor preparations. In extracts of cells which had been pretreated with mock preparations of interferon, purified vesicular stomatitis virus (VSV) directed the coupled transcription and translation of the viral genes. In extracts of interferon-treated cells, viral transcription was not inhibited, but the rate of translation was reduced by up to 70%, the extent of inhibition being dependent upon the interferon dose. The translation inhibition was not consistently reversed by the addition of chick cell transfer RNA, but was consistently accentuated in the presence of low concentrations of double-stranded RNA. To explore the possibility that this inhibition of protein synthesis was due to an effect on viral messenger RNA synthesis or processing, the products of VSV transcription in the two systems were compared for: (i) their size distribution, by polyacrylamide-gel electrophoresis under denaturing conditions; (ii) their stability, by UTP pulse-chase methods; (iii) their degree of polyadenylation, by oligo(dT)-cellulose column chromatography; (iv) their degree of methylation, by S-adenosyl[ methyl- 3H]methionine/[ 14C]UTP double-labeling; and (v) their ability to be translated upon reintroduction into cell-free systems from mock- or interferon-treated chick cells. These tests showed no difference between the preparations of viral mRNA made in the two systems, which indicated that translation itself was the site of inhibition under these conditions. Consistent with this conclusion was the fact that the translation of other messenger RNAs of both viral and animal cell origin was inhibited to a similar extent.

Isolation and role of nonheme iron protein in Clostridium botulinum.

A type of iron-bound protein was isolated from Clostridium botulinum by a modification of the method used for isolating ferredoxin from C. pasteurianum. This method involved acetone and diethylaminoethyl cellulose treatments followed by ammonium sulfate fractionation. The protein exhibited maximal absorption in the ultraviolet region near 260 mmu. Portions of the isolated iron protein were separated by disc electrophoresis and, following specific iron-bound protein staining, showed a positive reaction in the same position on the gel column as was first demonstrated by use of cell-free extract. Evidence accumulated by use of a cell-free extract of C. botulinum suggests that pyruvate is metabolized through a phosphoroclastic system as demonstrated in other clostridia. It is probable that ferredoxin is an electron mediator between pyruvic oxidase and hydrogenase for hydrogen evolution and acetyl phosphate formation.

Isolation and Role of Nonheme Iron Protein in Clostridium botulinum1

A type of iron-bound protein was isolated from Clostridium botulinum by a modification of the method used for isolating ferredoxin from C. pasteurianum. This method involved acetone and diethylaminoethyl cellulose treatments followed by ammonium sulfate fractionation. The protein exhibited maximal absorption in the ultraviolet region near 260 mmu. Portions of the isolated iron protein were separated by disc electrophoresis and, following specific iron-bound protein staining, showed a positive reaction in the same position on the gel column as was first demonstrated by use of cell-free extract. Evidence accumulated by use of a cell-free extract of C. botulinum suggests that pyruvate is metabolized through a phosphoroclastic system as demonstrated in other clostridia. It is probable that ferredoxin is an electron mediator between pyruvic oxidase and hydrogenase for hydrogen evolution and acetyl phosphate formation.

Characteristics of TCA Cycle of Cycloheximide-resistant Mutant ofZygosaccharomyces soja

Glucose catabolism inZygosaccharomyces sojawas carried out through fermentation, whereas riboflavin producing mutant obtained by cycloheximide treatment was found to utilize oxidative mechanisms. For instance, growth of mutant was sustained in minimal medium containing TCA cycle intermediates as the sole carbon source, whereas the mother strain could not grow on such culture media. Furthermore, malonate inhibited the oxidation of succinate with resting cells of mutant. As these results support the existence of TCA cycle in mutant, various enzyme activities relating to TCA cycle were investigated by the use of cell-free extracts of both strains. Aconitase, α-ketoglutaric decarboxylase, succinic dehydrogenase, fumarase, isocitritase and glyoxylic reductase were not detected in mother strain. These data indicated that both the TCA cycle and glyoxylate cycle did not operate in mother strain. On the other band, all enzyme activities relating to TCA cycle and glyoxylate cycle were verified in mutant. This finding indicates that both the TCA cycle and glyoxylate cycle performed a main role in carbohydrate catabolism in mutant. The evidence that alteration of the mode of glucose catabolism from fermentation in case of mother strain to respiration of mutant elicited by the action of cycloheximide was thus explained.

TRANSFORMATION IN RHIZOBIUM TRIFOLII

Transformation of streptomycin and chloramphenicol resistance and of crystal violet tolerance has been demonstrated in Rhizobium trifolii by the use of cell-free extracts prepared from a resistant homologue.

IN VITRO TRANSFORMATION OF PNEUMOCOCCAL TYPES

1. Further observations on the conditions under which transformation of pneumococcal types may be induced by in vitro procedures are presented. 2. R cultures possessing only slight degrees of R stability are most suitable for transformation purposes by in vitro procedures. 3. Vaccines prepared from cultures subjected to the action of bacterial enzymes liberated (a) in old broth cultures, (b) during mechanical disruption of young bacterial cells, are not effective in inducing transformation of type. 4. The property of an S vaccine responsible for transformation of type is not related to the specific soluble substance of pneumococcus. 5. Attempts to effect transformation of type by the use of cell-free extracts of pneumococcus have so far proved unsuccessful.