Evidence for a multiple innervation of purkinje cells by climbing fibers in the immature rat cerebellum

Climbing fiber (CF)–-Purkinje cell (PC) relationships were studied electrophysiologically on the cerebellum of 8 to 15 day old rats. Some animals were rendered agranular by x-irradiation from birth; some others were treated with 3-acetyl pyridine 3 days before study to selectively destroy the CF. Unitary extracellular recordings in 8–9 day old normal rats revealed that more than 50μ of the PC units each exhibited either two types of all-or-none climbing fiber responses (CFR) or stepwise graded CFRs. The other PC units only presented one type of all-or-none CFR. These activities were entirely mediated via CF since they persisted at the same age in x-irradiated rats, but were absent in animals treated with 3-acetyl pyridine. Interaction experiments were performed between juxtafastigial and Inferior Olive stimulations on 49 PC units in 8–9 day old normal rats. Collisions between impulses set up in CFs were disclosed in 21 out of the 24 PCs which exhibited only one type of CFR. In the three others and in each of the 25 PCs that displayed two types of all-or-none CFRs, or CFRs graded by steps, no collision was detected. Moreover intracellular recordings of epsp's mediated via CFs in PCs of 8–9 day old normal rats revealed that they frequently fluctuated in stepwise fashion. These results demonstrate that in the immature rat more than 50μ of PCs are each innervated by at least two distinct CFs; later on, the disappearance of the supernumerary synapses between CF and PC leads, as early as day 15, to the one-to-one relationship between CF and PC.     

Prostaglandins, cyclic AMP and the mechanism of opiate dependence.

Further evidence is given that dependence arises from the agonist action of opiates. From this and our previous propositions assigning a fundamental role to neuronal cyclic AMP in (i) the agonist action of opiates and (ii) the expression of the abstinence syndrome, it follows that opiate dependence is a state of heightened potential activity of a neuronal cyclic AMP mechanism, initiated and maintained by the blockade of an adenylate cyclase. Various possible mechanisms are discussed by which this potential is heightened. New evidence is given that morphine and naloxone stimulate prostaglandin biosynthesis without mutual antagonism. Preliminary evidence also is given that (i) the formation of cyclic AMP is enhanced in brain homogenates from heroin-dependent rats, and (ii) an acidified ethylacetate extract of brains of morphine-dependent rats induces quasi-abstinence effects when injected into a lateral cerebral ventricle of naive rats.     

MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea

Magnaporthe grisea produces an infection structure called an appressorium, which is used to breach the plant cuticle by mechanical force. Appressoria generate hydrostatic turgor by accumulating molar concentrations of glycerol. To investigate the genetic control and biochemical mechanism for turgor generation, we assayed glycerol biosynthetic enzymes during appressorium development, and the movement of storage reserves was monitored in developmental mutants. Enzymatic activities for glycerol generation from carbohydrate sources were present in appressoria but did not increase during development. In contrast, triacylglycerol lipase activity increased during appressorium maturation. Rapid glycogen degradation occurred during conidial germination, followed by accumulation in incipient appressoria and dissolution before turgor generation. Lipid droplets also moved to the incipient appressorium and coalesced into a central vacuole before degrading at the onset of turgor generation. Glycogen and lipid mobilization did not occur in a Deltapmk1 mutant, which lacked the mitogen-activated protein kinase (MAPK) required for appressorium differentiation, and was retarded markedly in a DeltacpkA mutant, which lacks the catalytic subunit of cAMP-dependent protein kinase A (PKA). Glycogen and lipid degradation were very rapid in a Deltamac1 sum1-99 mutant, which carries a mutation in the regulatory subunit of PKA, occurring before appressorium morphogenesis was complete. Mass transfer of storage carbohydrate and lipid reserves to the appressorium therefore occurs under control of the PMK1 MAPK pathway. Turgor generation then proceeds by compartmentalization and rapid degradation of lipid and glycogen reserves under control of the CPKA/SUM1-encoded PKA holoenzyme.     

Analysis of ribosomal DNA restriction patterns in the genusKluyveromyces

Riboprinting was used to determine the relationships among strains belonging to 15 species of the genusKluyveromyces. The small subunit ribosomal RNA gene (SSU rDNA) was amplified using the Polymerase Chain Reaction (PCR) and subjected to a battery of nine restriction enzymes. Similarity coefficients between strains were calculated based on shared and unique restriction fragments. Cluster analysis revealed three major groups that generally correlated with previously reported relationships based on other molecular data. Variations in SSU rDNA restriction fragments may be used for differentiation of theKluyveromyces strains included in this study.The U.S. Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.     

An interference-free fluorescent assay of telomerase for the high-throughput analysis of inhibitors

We have developed a telomerase assay that can quickly and accurately rank the ability of molecules to inhibit telomerase activity. It is based on the method of Orlando and co-workers which utilizes PicoGreen to detect dsDNA formed during the polymerase chain reaction (PCR) amplification of telomerase products. PCR cycles were optimized to give as linear a signal as possible relative to telomerase products; 96-well streptavidin-coated PCR plates were used to isolate the preamplification telomerase products and to wash inhibitors away before the amplification step. The inhibitor removal step is critical to prevent false positives potentially caused by inhibition of Taq polymerase during amplification. Use of the streptavidin-coated PCR plate allows this step to be done much more rapidly than use of the liquid/liquid extraction adopted by others. We have demonstrated that this assay can correctly order the ability of four inhibitors to inhibit telomerase and reproduce within a factor of two the absolute IC(50) values determined by the more time-consuming direct assay. We have shown that the difference in IC(50) values determined in this assay versus the direct assay can be corrected for by using the standard curve appropriately. Using this method 96 compounds can be assessed in 3-5h.     

An anatomical model to determine step height in step testing for estimating aerobic capacity

Physical fitness has been reported to be inversely related to coronary heart disease and other health related problems. One of the most valid means of assessing physical fitness is the test of aerobic capacity. Aerobic capacity is the greatest rate at which the body can consume oxygen and represents the most efficient integration of the various physiological processes which make up the oxygen transport system. However, direct measurement of aerobic capacity requires sophisticated laboratory equipment, and is adversive to subjects. Step tests are widely used to estimate aerobic capacity. Because the biomechanical efficiency and work rate is determined by step height, accommodation of step height to the subject's statute height should provide a better estimation of aerobic capacity. A hip angle of 73.3 degrees, when stepping, was found to give the best relationship of recovery heart rate of a step test to direct measurement of aerobic capacity. Using 73.3 degrees, the following equations were developed for determining the stepping height when using the step test: Hf = 0.189 Ih and Hf = 0.192 Ih for females and males respectively, where hf is the step height and Ih is the statute height of the subject. A correlation coefficient (r) of 0.93 was calculated between various hip angles and calculated foot height of 182 observations of 47 females while a correlation coefficient (r) of 0.96 was calculated from 208 observations of 53 males. Using these equations to determine step height, measurement of 30 females showed a mean hip angle of 73.3 degrees +/- 2.2 and measurement of 30 males showed a mean hip angle of 73.3 degrees +/- 2.1.(ABSTRACT TRUNCATED AT 250 WORDS)