Whole-cell recording of neuroblastoma x glioma cells during downregulation of a major substrate, 80K/MARCKS,of protein kinase C

Summary Differentiated neuroblastoma cells exhibit both the delayed rectifier potassium current (I _K) and the M-current (I _M). The present study was designed to determine the roles of protein kinase C (PKC) and of the calmodulin-binding protein 80K/MARCKS, a prominent substrate for PKC and possible regulator of these currents. Neuroblastoma x glioma (NG108-15) hybrid cells transfected with m1 muscarinic receptors were grown with 1% fetal bovine serum (FBS) without the prostaglandin E₁ (PGE₁) and isobutylmethylxanthine (IBMX) usually added in preparation for electrophysiological studies. Under these conditions, the usual pleomorphism was largely abolished, leaving two populations of small cells with stellate and spherically symmetrical geometries. Whole-cell patch clamping indicated that the two cell types had identical electrophysiological properties, displaying: I _k, a small current through a T-like Ca²⁺ channel, and no M-current.Stimulation with carbachol shifted the distribution of cells to a more stellate morphology within 24 hr and later (after 48 hr) reduced the PKC substrate 80K/MARCKS by 22±7%. In contrast to the stimulation of I _k observed with cardiac cells, PKC activation produced only a small inhibition of I _k, which was independent of carbachol pretreatment. Thus, PKC and 80K/MARCKS can be dissociated from the regulation of I _k in neuroblastoma cells.Supported in part by research grants from the National Institutes of Health (DK-40145 and EY-08343) and from the U.K. Medical Research Council.We thank Dr. Peter J. Parker for his generous gift of PKC, and Yvonne Vallis for her skillful assistance with the cultures and harvesting of the NG108-15 transfected cells.     

A NEW METHOD OF POLARIZATION MICROSCOPIC ANALYSIS : I. Scanning with a Birefringence Detection System

A new method of polarized light analysis is described in which a highly sensitive electronic detector specific for birefringence is used to identify the crystalline axes of an object and then measure its phase retardation due to birefringence. The microscopic system employed in the method consists of an electronic birefringence detection system (BDS), a microscope with strain-free lenses, and a driven stage for passing the specimen at appropriate velocities across the image of an aperture placed at the field stop and imaged in the specimen plane by the condenser. The detector registers retardations directly as voltage at a constant deflection sensitivity of ca. 1.1 v per angstrom unit over a range of 120 angstrom units. The basal rms noise level is 0.002 A for a spot 36 µ in diameter formed by a 95 x, N. A. 1.25 objective pair, and increases in proportion to the reciprocal of the diameter of the scanning spot. The increase in noise with high resolution scanning can be offset by increasing the instrumental time constant, which is adjustable in decades between 0.004 and 0.4 seconds. A number of difficult problems in high extinction polarization microscopy are avoided by the use of modulated light and a rapid electronic detector. For example: (a) The measured distribution of birefringence is unaffected by the usual diffraction anomaly; therefore polarization rectifiers are not required. (b) The detector is selective for birefringence, so that there is no problem in separating contrast due to different optical properties (e.g. dichroism, light scattering). (c) The speed and sensitivity are both increased by between one and two orders of magnitude over that attainable by visual or photographic methods, thereby rendering a vast number of weakly birefringent, light-scattering, and motile objects readily analyzable for the first time with polarized light.     

GENETICS OF STERILITY IN HYBRIDS BETWEEN TWO SUBSPECIES OF DROSOPHILA

Hybrids between D. pseudoobscura bogotana and D. pseudoobscura pseudoobscura are fertile except for males produced in one of the two reciprocal crosses. As there is no premating isolation between these subspecies, nonreciprocal male sterility represents the first step in speciation. Genetic analysis reveals two causes of hybrid F₁ sterility: a maternal effect and incompatibilities between chromosomes within males. The maternal effect appears to play the greatest role in hybrid sterility. The X chromosome has the largest effect on fertility of any chromosome, a ubiquitous result in analyses of hybrid sterility and inviability in Drosophila. This effect is entirely attributable to a region comprising less than 30% of the X chromosome. These results are compared to those from a similar study of D. pseudoobscura-D. persimilis hybrids, an older and more reproductively isolated species pair in the same lineage. Such comparisons may allow one to identify the genetic changes characterizing the early versus late stages of speciation.     

Manipulating photosynthesis

The levels of individual photosynthetic proteins can be independently decreased by theAgrobacterium-mediated transformation of plants with antisens RNA constructs. Protocols for the introduction of such constructs intoAgrobacterium, theAgrobacterium-mediated transformation of tobacco leaf disks, and the screening and analysis of the transgenic plants produced are described.     

Inositol lipid-mediated signalling in response to endothelin and ATP in the mammalian testis

Molecular and Cellular Biochemistry - The testis is a complex organ in which local control is achieved by signalling between its constituent cells. Herein we describe the responses of cultured rat...