Chemical Stimulators Research Articles

Inactivation of MPF and MAP kinase by single electrical stimulus for parthenogenetic development of porcine oocytes

This study was conducted to examine the activities of maturation-promoting factor (MPF) and mitogen-activated protein (MAP) kinase in the porcine oocytes after artificial activation. To determine optimal electrical activation condition, oocytes were exposed to single DC pulse in a variety of electric field strengths (120, 150, 180, and 210 V/mm) and pulse durations (15, 30, 45, and 60 microsec). After the artificial activation, 40-50 oocytes were cultured in a 50 microl drop of NCSU23 medium supplemented with 0.4% BSA at 39 degrees C, 5% CO2 in air for 6 days. No difference was detected in the preimplantation development of pocine oocytes and the mean nuclei number of blastocysts between electric field strengths. Under the 180 V/mm electric field strength, short pulse durations (15 and 30 microsec) showed a higher preimplantation developmental rate of the oocytes and mean nuclei number of blastocysts than an extended electric pulse (60 microsec) (P < 0.05). Single electrical stimulus (180 V/mm, 15 microsec) resulted in higher preimplantation development of porcine oocytes as compared to other chemical stimulators (P < 0.01). Western blot analyses showed the decrease of MPF and MAP kinase in the electrically-activated oocytes. After single electrical stimulus, the amounts of both cdc2 and ERK in porcine oocytes were remarkably reduced by 4 hr and then further decreased by 8 hr. However, the chemically-stimulated oocytes did not show any significant change at the levels of MPF and MAP kinase. Our results indicate that the optimal single electrical pulse is effective on the inactivation of MPF and MAP kinase, eventually leading to the parthenogenetic development of porcine oocytes.

269 INACTIVATION OF MATURATION PROMOTING FACTOR AND MITOGEN-ACTIVATED PROTEIN KINASE IN PORCINE OOCYTES BY A SINGLE ELECTRICAL PULSE

Activities of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) of mature oocytes should be decreased to begin subsequent development. In this study, activities of MPF and MAPK were investigated in porcine oocytes after artificial activation. To determine optimal electrical activation, porcine oocytes were exposed to 3 V AC pulse for 5 s followed by a single DC pulse of various electric field strengths (120, 150, 180, and 210 V/mm) and pulse durations (15, 30, 45, and 60 μs). For chemical activation, oocytes were exposed to 5 μM ionomycin for 5 min followed by 2 mM 6-dimethylaminopurine (6-DMAP) or 5 μg/mL cycloheximide for 4 h or 6 h. After activation, 40 to 50 oocytes were cultured in 50-μL drops of NCSU23 medium supplemented with 4 mg/mL BSA at 39°C, and 5% CO2 in air. After 6 days of culture, blastocyst formation was observed and then numbers of blastocyst nuclei were counted after staining with Hoechst 33342. in vitro development rates and numbers of blastocyst nuclei by the field strengths were not significantly different among experimental groups (P &gt; 0.05). However, development rates to the blastocyst stage of porcine oocytes exposed to 15 and 30 μ­s were 27.4 and 24.4%, respectively, which were significantly higher than that (12.5%) of 60 μs (P &lt; 0.05). Mean numbers of blastocyst nuclei in 15- and 30-μs groups (38.6 ± 13.4 and 37.9 ± 11.4, respectively) were significantly higher than that (25.8 ± 10.5) of the 60-μs group (P &lt; 0.05). Blastocyst development after optimal electrical pulse exposure was compared with that after different chemical treatments. Electrical stimulation induced 22.9% of blastocyst formation, which was significantly higher (P &lt; 0.01) than those induced by the chemical stimulators (3.4 and 2.7%). Based on these results, changes of constituent proteins (cdc2 and ERK) of MPF and MAPK after artificial activation were analyzed by immunoblotting using anti-PSTAIRE monoclonal antibody and anti-MAP kinase polyclonal antibody. Activities of both cdc2 and ERK in pig oocytes were reduced 4 h after electrical stimulus, but were maintained at optimal levels after treatment with ionomycin + 6-DMAP. Our results indicate that an optimal single electrical pulse is effective on the inactivation of MPF and MAPK in porcine oocytes, eventually resulting in activation of porcine oocytes produced in vitro.

Testing of Growth Promoting and Protective Activity of the Probiotic Lactiferm in Weaned Piglets

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Development of a model for cytomegalovirus infection of oligodendrocytes.

The well-characterized human oligodendroglioma (HOG) cell line, cells of which resemble immature oligodendrocytes, was used to investigate the level of permissiveness to human cytomegalovirus (CMV) infection. Expression of CMV genes was incomplete following exposure of HOG cells to CMV, in contrast with results observed with the astroglioma cell line U373-MG (used as a positive control). However, treatment with phorbol 12-myristate 13-acetate (PMA) or with dibutryl cAMP (dbcAMP) plus the phosphatase inhibitor 1-isobutyl-3,3-methyl xanthine (IBMX) rendered the HOG cells fully permissive to CMV; down-regulation of HLA class I and production of virions were only observed under these conditions. In contrast to the findings seen with the HOG cell line, treatment of U373-MG cells with dbcAMP/IBMX or PMA did not interfere with CMV-induced down-regulation of HLA class I. However, these chemical stimulators reduced virus production in U373-MG cells by 30 and 70%, respectively.

Volatile Chemical Germination Stimulators of Rust and Other Fungal Spores

Volatile Chemical Germination Stimulators of Rust and Other Fungal Spores

Volatile Chemical Germination Stimulators of Rust and Other Fungal Spores

(1992). Volatile Chemical Germination Stimulators of Rust and Other Fungal Spores. Mycologia: Vol. 84, No. 3, pp. 277-288.

Short-term efficient expression of transfected DNA in human hematopoietic cells by electroporation: definition of parameters and use of chemical stimulators.

The efficiency of DNA transfer into human hematopoietic cells by electroporation was investigated and compared to conventional transfection procedures. Important parameters of electroporation were optimized in human erythroleukemia cells using the chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) gene linked to the cytomegaloviral enhancer-promoter. In addition, selected chemicals with different modes of action were studied for their ability to aid DNA entry and gene expression in this system, and several were found to enhance gene transfection by electroporation in a significant manner. Using these chemical stimulators, many but not all human and mouse suspension cultures tested were successfully electroporated by the Baekon 2000 instrument. From these studies, it appears that electroporation can be enhanced by chemical additives. Because of its efficiency, reproductivity, and convenience electroporation is an attractive method of gene transfer in human hematopoietic cells.

Principles of Applied Biomedical Instrumentation.

Biomedical instruments and the measurement of physiological events resistive transducers inductive transducers capacitive transducers photoelectric transducers piezoelectric devices thermoelectric devices chemical transducers electrodes stimulation and stimulators detection of physiological events by impedance the bioelectric events radiant energy devices ventilation and ventilators criteria for the faithful reproduction of an event.

Studies on galactosyl ceramide and lactosyl ceramide β-galactosidase

Galactosyl ceramide and lactosyl ceramide β-galactosidase activity was measured in extracts of previously frozen human brain. Using the assay methods described here, both galactosyl ceramide and lactosyl ceramide gave Michaelis constants of about 20 μM. Evidence from these studies indicates that at least a major portion of human brain lactosyl ceramide β-galactosidase activity is identical to galactosyl ceramide β-galactosidase activity. The evidence includes the following: co-purification by all methods tried, identical heat denaturation pattern under various conditions, same response to chemical inhibitors or stimulators, competitive inhibition with either substrate by the other, and severe deficiency of both galactosyl ceramide and lactosyl ceramide β-galactosidase activity in all tissues tested from patients with Krabbe's disease. Liver, brain, fibroblast, leukocyte and spleen extracts from patients with G M1 gangliosidosis had normal ability to degrade both galactosyl ceramide and lactosyl ceramide.

Patterns of cellular organization and cell division in the epithelium of the cultured lens

Crystalline lenses (rabbit) were cultured for periods ranging from 1 hr to 46 days. A dialysis system which facilitates the culture of large numbers of lenses under virtually identical conditions was used. The lens was enclosed in a dialysis bag containing 2–3 ml of synthetic culture medium (inside medium), and immersed in a 125 ml flask containing 50 ml of the same medium (outside medium). Several lenses were incubated in one flask and the outside medium was changed at regular intervals of 1–3 days. Some lenses were injured after 8 days of culture and incubated for 2 further days. In most cases, tritiated thymidine was introduced into the medium just prior to termination of culture. Whole-mount preparations and autoradiographs of the epithelia were examined and the general and nuclear morphology, number of mitotic figures and number of radioactive nuclei noted. Lenses remained clear and refractile as long as 30 days. Except for small areas probably related to injuries accidentally inflicted during isolation of the lens, the epithelia of these lens showed little morphological alteration. These preparations indicated that cell density in the germinative zone approaches that of the central area as culture continues, and that the epithelium tends to extend on to the posterior capsule. Cell division in the epithelium of such lenses does not cease during culture, but the distribution of labeled nuclei and mitotic figures is altered; this alteration appears to follow a well defined sequence. The lenses injured during culture showed a great burst of cell division around the site of injury 48 hr later. Lenses that became cataractous did so after the appearance of microbial infection in the outside culture medium. The epithelia of these lenses showed great alterations in general morphology, very extensive nuclear labeling, and nearly complete population of the posterior capsule by epithelial cells. The various patterns of cell proliferation observed in the epithelia of cultured lenses, and comparison of these with that found in vivo, seem to indicate that physical forces exerted on the lens (such as those due to the varying tension of the suspensory ligaments) and the resulting wear and tear on the epithelium may be causally related to the rate of cell proliferation and the distribution of dividing cells in this tissue. This hypothesis, as opposed to that involving diffusible chemical inhibitors or stimulators, is discussed.

The Influence of Nucleic Acid on Dehydrogenase Systems a Contribution to the Problem of Gene Mechanism

The cellular components comprise a vast number of different compounds, salts, proteins, carbohydrates, fats, nucleates, etc., each in a state of metabolic flux, and each interacting with others to provide the energy for specific function, the supply of structural materials, and the disposal of breakdown products. This complicated and highly diverse mechanism of the cell is regulated by a mosaic of enzyme systems which are not independent but interrelated, and which are themselves regulated and affected by extrinsic and intrinsic factors and conditions. Thus, it is possible to affect the level of particular enzymes by nutritive deprivation of the cell, by the application of chemical inhibitors or stimulators, and by cellular mutations whether spontaneous or induced. Any attempt to elucidate these phenomena in toto is quite obviously a formidable task. The biochemist at present can study only a few systems at a time, under conditions often considerably removed from the physiological, and, as yet, with tools and concepts alike available only for first approximations. Biological chemistry owes its beginnings to the insight of men like Miescher, Kossel, and others, who believed that problems in tissue function might be at least partially explicable in terms of the chemical properties of isolated components and systems. It is with this hope undimmed, and with due recognition of the limitations inherent in the approach, that an attempt at a study of a chemical basis of gene action may be begun (Greenstein and Chalkley, '45). Nucleic acid combined with specific proteins appears to yield conjugated compounds often with remarkable biological properties. Among these compounds are the viruses and the components of the chromosomes. Considerable information exists suggestive of a linking of nucleic acids with gene mechanisms, but in the absence of evidence for the isolated gene any attempt to identify the latter as a nucleoprotein must be treated with some reserve. Nevertheless, there is evidence that growth and reduplication are associated with the presence of nucleic acid, and the relation of the latter type of substance with the gene may thus be strongly inferred. Since the genic material exerts a controlling influence over the multitudinous functional processes of the cell, it may be assumed that it accomplishes this not by a remote form of control but by specific kinds of chemical interaction with the cellular components involved in individual reactions, i. e., enzymes (cf. Tatum and Beadle, '45). It is for this reason that the study of the chemical interaction of nucleic acid with various kinds of cellular components should yield sig-

Experimental Zinc Teratomas of the Testis and their Transplantation: Preliminary Communication

Michalowsky in 1925 was the first to produce tumors of the testes in fowls by injecting a 5 per cent solution of zinc chloride. Histological examination of the tumors showed them to be composed of the most diverse tissues, including cartilage, developing bone, glandular formations, cysts, and ducts lined with stratified polymorphous epithelium, smooth and striated muscle, and nerve cells. On account of this variety of tissues the tumors were classified as teratomas. Of particular interest to Michalowsky were certain peculiar cells with a basophilic protoplasm and a clear nucleus, which were scattered throughout the tumor in separate foci or strands. Unfortunately, for a long time these experiments were not duplicated. Thus, some years after Michalowsky9s work had been published, Petrov wrote: “Among the tumors produced by chemical stimulators an isolated place is occupied by the complex teratomas described by Professor Michalowsky. Until his experiments are verified we do not find it possible to pass any judgment upon their significance.” The experiments were repeated in 1936 by an American investigator, Bagg, whose results fully confirmed the data originally obtained. Bagg considered the foci of dark cells, referred to above, as carcinomatous zones, but numerous attempts made to transplant his teratomas were not successful. At the suggestion of Professor Michalowsky experiments on the transplantation of the tumors were undertaken in this laboratory. In March 1937, 19 adult fowls were injected with a 5 per cent solution of zinc chloride, receiving 0.3 c.c. in each testis. Three fowls died soon afterward. In 2 of the 16 survivors teratomas of the testes developed. The first fowl was killed on June 14, 1937, and autopsy revealed a large tuberous tumor arising from the middle of the left testis, weighing 204 gm. and surrounded by a thin capsule through which small cysts were seen. At the site of the injection cyst cavities and small areas of cartilage were observed.