Impaired muscle glycogen storage after muscle biopsy

To assess the effects of repeated needle biopsies on the rate of muscle glycogen repletion, eight male subjects were studied immediately after and 2 days after an exhaustive cycling bout. A single biopsy was obtained from the right vastus lateralis muscle immediately after an exhaustive cycling bout. Two days later, a sample was taken 1 cm lateral or medial to sample A. In four of these subjects, additional biopsies were taken 3 cm distal and proximal. A control specimen was also taken from the left leg 2 days after the exercise. Ten days after the exercise, muscle was again sampled from each leg of these four subjects. Analysis of these samples revealed that the initial biopsy impaired glycogen storage in the muscle taken 1 cm medial or lateral to the previous site. This reduction in glycogen storage was most pronounced in the first 2 days after the exercise. Samples taken distal and proximal to the initial biopsy contained, on the average, less glycogen than the contralateral leg, but these differences were only significantly different in the distal muscle sample. Alteration in muscle glycogen storage was seen to persist for 10 days after the first biopsy, suggesting that care must be taken in selecting the site for repeated biopsies from the same muscle.     

Mutations Affecting Ty-Mediated Expression of the HIS4 Gene of SACCHAROMYCES CEREVISIAE

We have identified mutations in seven unlinked genes (SPT genes) that affect the phenotypes of Ty and δ insertion mutations in the 5' noncoding region of the HIS4 gene of S. cerevisiae. Spt mutants were selected for suppression of his4-912δ, a solo δ derivative of Ty912. Other Ty and δ insertions at HIS4 are suppressed by mutations in some but not all of the SPT genes. Only spt4 suppresses a non-Ty insertion at HIS4. In addition to their effects on Ty and δ insertions, mutations in several SPT genes show defects in general cellular functions—mating. DNA repair and growth.     

Protein evolution in different cellular environments: cytochrome b in sharks and mammals

DNA sequences for the mitochondrial cytochrome b gene were determined for 13 species of sharks. Rates and patterns of amino acid replacement are compared for sharks and mammals. Absolute rates of cytochrome b evolution are six times slower in sharks than in mammals. Bivariate plots of the number of nonsynonymous and silent transversions are indistinguishable in the two groups, however, suggesting that the differences in amino acid replacement rates are due primarily to differences in DNA substitution rates. Patterns of amino acid replacement are also similar in the two groups. Conserved and variable regions occur in the same parts of the cytochrome b gene, and there is little evidence that the types of amino acid changes are significantly different between the groups. Similarity in the relative rates and patterns of protein change between the two groups prevails despite dramatic differences in the cellular environments of sharks and mammals. Poor penetrance of physiological differences through to rates of protein evolution provides support for the neutral theory and suggests that, for cytochrome b, patterns of evolution have been relatively constant throughout much of vertebrate history.      

Distribution and relative proportions of neuropeptide Y- and proenkephalin-containing noradrenergic neurones in rat superior cervical ganglion: Separate projections to submaxillary lymph nodes

The distribution and relative proportions of neuropeptide Y (NPY)- and [Met]enkephalyl-Arg-Gly-Leu (ME-RGL)-containing sympathetic neurones in the rat superior cervical ganglion (SCG) and their projections to submaxillary lymph nodes (SLN) were determined by retrograde tracing and immunocytochemistry. Three subpopulations of neurones were detected in the SCG: 64% contained NPY, 30% contained ME-RGL, and 6% were immunonegative for both. Immunoreactive neurones were also present inside the external carotid nerve of the SCG. An injection of Fluoro-Gold (FG) into the left SLN retrogradely labeled a few neurones in the ipsilateral SCG. FG-labeled neurones contained tyrosine hydroxylase (TH) and were either positive for ME-RGL or for NPY. FG-labeled neurones immunostained for ME-RGL outnumbered by 4:1 FG-labeled neurones immunopositive for NPY. It is suggested that the sympathetic/peptidergic innervation to SLN may have distinct vasoregulatory and immunomodulatory functions.     

A role for autophosphorylation revealed by activated alleles of FUS3, the yeast MAP kinase homolog.

We have isolated dominant gain-of-function (gf) mutations in FUS3, a Saccharomyces cerevisiae mitogen-activated protein (MAP) kinase homolog, that constitutively activate the yeast mating signal transduction pathway and confer hypersensitivity to mating pheromone. Surprisingly, the phenotypes of dominant FUS3gf mutations require the two protein kinases, STE7 and STE11. FUS3gf kinases are hyperphosphorylated in yeast independently of STE7. Consistent with this, FUS3gf kinases expressed in Escherichia coli exhibit an increased ability to autophosphorylate on tyrosine in vivo. FUS3gf mutations suppress the signal transduction defect of a severely catalytically impaired allele of STE7. This finding suggests that the tyrosine-phosphorylated form of FUS3 is a better substrate for activation by STE7. Furthermore, these results imply that the degree of autophosphorylation of a MAP kinase determines its threshold of sensitivity to upstream signals.     

Multicellular Stalk-Like Structures in Saccharomyces cerevisiae

Stalk formation is a novel pattern of multicellular organization. Yeast cells which survive UV irradiation form colonies that grow vertically to form very long (0.5 to 3.0 cm) and thin (0.5 to 4 mm in diameter) multicellular structures. We describe the conditions required to obtain these stalk-like structures reproducibly in large numbers. Yeast mutants, mutated for control of cell polarity, developmental processes, UV response, and signal transduction cascades were tested and found capable of forming stalk-like structures. We suggest a model that explains the mechanism of stalk formation by mechanical environmental forces. We show that other microorganisms (Candida albicans, Schizosaccharomyces pombe, and Escherichia coli) also form stalks, suggesting that the ability to produce stalks may be a general property of microorganisms. Diploid yeast stalks sporulate at an elevated frequency, raising the possibility that the physiological role of stalks might be disseminating spores.     

Intracellular fluorescent probe concentrations by confocal microscopy.

A general method is described that takes advantage of the optical sectioning properties of a confocal microscope to enable measurement of both absolute and relative concentrations of fluorescent molecules inside cells. For compartments within cells that are substantially larger than the point spread function, the fluorescence intensity is simply proportional to the concentration of the fluorophore. For small compartments, the fluorescence intensity is diluted by contributions from regions outside the compartment. Corrections for this dilution can be estimated via calibrations that are based on the intensity distribution found in a computationally synthesized model for a cell or organelle that has been blurred by convolution with the microscope point spread function. The method is illustrated with four test cases: estimation of intracellular concentration of a fluorescent calcium indicator; estimation of the relative distribution between the neurite and soma of a neuronal cell of the InsP3 receptor on the endoplasmic reticulum; estimation of the distribution of the bradykinin receptor along the surface of a neuronal cell; and relative distribution of a potentiometric dye between the mitochondria and cytosol as a means of assaying mitochondrial membrane potential.     

Paroxysmal dystonic choreoathetosis: tight linkage to chromosome 2q.

Paroxysmal dystonic choreoathetosis (PDC) is characterized by attacks of involuntary movements that last up to several hours and occur at rest both spontaneously and following caffeine or alcohol consumption. We analyzed a Polish-American kindred with autosomal dominant PDC and identified tight linkage between the disorder and microsatellite markers on chromosome 2q (maximum two-point LOD score 4.77; recombination fraction 0). Our results clearly establish the existence of a locus for autosomal dominant PDC on distal chromosome 2q. The fact that three other paroxysmal neurological disorders (periodic ataxia with myokymia and hypo- and hyperkalemic periodic paralysis) are due to mutation in ion-channel genes raises the possibility that PDC is also due to an ion-channel gene mutation. It is noteworthy that a cluster of sodium-channel genes is located on distal chromosome 2q, near the PDC locus. Identifying the PDC locus on chromosome 2q will facilitate discovery of the PDC gene and enable investigators to determine whether PDC is genetically homogeneous and whether other paroxysmal movement disorders are also genetically linked to the PDC locus.