Labeling of Retina and Optic Tectum Phospholipids in Chickens Exposed to Light or Dark

The labeling of retina ganglion cell and optic tectum phospholipids was determined in chickens given an intraocular injection of 32P and then either exposed to light or maintained in the dark. Significantly higher labeling was found in the optic tectum phospholipids of light-exposed compared with dark-maintained animals after 3-24 h of labeling. In the ganglion cells, the labeling of phospholipids increased in dark with respect to light at 15 and 30 min of labeling; from 60 min to 24 h, the labeling of phospholipids was significantly higher in light with respect to dark, even if the precursor pool showed a higher labeling in dark at all times studied. When labeling was allowed to proceed in the dark for 30 min and then half of the animals were exposed to light for 15 min, the labeling of ganglion cell phospholipids of light-exposed animals was significantly higher than those of animals kept in the dark. No individual phospholipid accounted for the differences observed in the labeling of the total phospholipid pool. These results are interpreted as an increase in the biosynthesis of phospholipids in the ganglion cell somas in light with respect to dark.     

Lambda repressor mutants that are better substrates for RecA-mediated cleavage

RecA-mediated cleavage of the bacteriophage lambda repressor results in inactivation of the protein and leads to induction of the lambda prophage. Here, we report the identification of three mutations in lambda repressor that significantly increase the rate of RecA-mediated cleavage. These mutations were isolated as intragenic second-site suppressors of a mutation (ind-) which prevents cleavage. Purified repressor proteins that contain both the ind- mutation and one of the second-site mutations undergo cleavage at near wild-type rates. Purified repressors that contain the second-site mutations in otherwise wild-type backgrounds undergo RecA-mediated cleavage at significantly faster rates than wild-type, and form dimers more poorly than the wild-type protein. In related experiments, we found that other repressor mutants that dimerize poorly are also better substrates for RecA-mediated cleavage. Conversely, we show that a covalent disulfide-bonded repressor dimer is resistant to cleavage. These results support a model in which repressor monomers are the only substrate in the cleavage reaction.     

Induction of mineral deposition by primary cultures of chicken growth plate chondrocytes in ascorbate-containing media. Evidence of an association between matrix vesicles and collagen

A serum-free primary culture system for chicken growth plate chondrocytes has been developed which consistently undergoes mineral deposition. Upon attainment of confluency, the chondrocytes develop locally into multilayer cellular nodules leading to matrix calcification. Mineralization first occurs in matrix vesicles (MV) that are abundant in the extraterritorial matrix between the hypertrophic cells. Studies with 45Ca reveal that significant accumulation of Ca2+ occurs as early as day 12, continuing progressively throughout the culture period. By day 24, the nodules become densely calcified. Fourier transform infrared spectroscopy reveals the mineral to be similar to apatite, with features essentially identical to those of mineral formed by MV in vitro. The presence of ascorbate is critical to the culture system; in its absence, calcification is rarely observed. Ascorbate stimulates MV formation and synthesis of cellular protein, alkaline phosphatase, and especially types II and X collagens. In addition, there is strong evidence that the types II and X collagens are associated with MV. 1) Electron microscopy reveals MV embedded in a type II collagenous network; 2) Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of MV using monospecific antibodies to types X and II collagen indicate that both collagens are present in specific MV fractions; 3) sucrose gradient purification of MV does not remove associated collagens; 4) graded salt extraction selectively releases type II collagen from MV; and 5) incubation of radiolabeled types II and X collagens with MV leads to their cosedimentation upon subsequent centrifugation. Taken together, the data suggest that coordinated synthesis of the collagens, alkaline phosphatase, MV formation, and Ca2+ accumulation by the cultures combine to induce mineral deposition in the multilayer nodules.     

The major 45-kDa protein of the yeast mitochondrial outer membrane is not essential for cell growth or mitochondrial function

As part of an analysis of the function and assembly of the mitochondrial outer membrane, we have cloned and characterized the yeast gene encoding a 45-kDa polypeptide (OM45) which is a major constituent of this membrane. The nuclear gene was isolated by immunological screening of plaques of recombinant phage lambda gt11 containing fragments of yeast genomic DNA using an antibody against OM45. Determination of the nucleotide sequence of the DNA fragment isolated by this approach revealed a single open reading frame of 1179 base pairs which encodes a protein having a predicted molecular mass of 44.6-kDa. Disruption of the OM45 gene in haploid yeast cells eliminated the expression of OM45. The mutant strain showed no apparent defect in cell viability, growth, mitochondrial function, or mitochondrial protein import.     

Structural and energetic consequences of disruptive mutations in a protein core.

We have characterized the properties of a set of variants of the N-terminal domain of lambda repressor bearing disruptive mutations in the hydrophobic core. These mutations include some that dramatically alter the total core residue volume (by up to six methylene groups) and some that place a single polar residue into the otherwise hydrophobic core. The structural properties of the purified proteins have been studied by CD spectroscopy, biological activity, recognition by conformation-specific monoclonal antibodies, and 1H NMR spectroscopy. The stabilities of the proteins have been measured by thermal and guanidine hydrochloride denaturation. Proteins with disruptive core mutations are found to display a continuum of increasingly nonnative properties. Large internal volume changes cause both significant conformational rearrangements and destabilization by up to 5 kcal/mol. Variants with polar substitutions at core positions no longer behave like well-folded proteins but rather display characteristics of molten globules. However, even proteins bearing some of the most disruptive mutations retain many of the crude secondary and tertiary structural features of the wild-type protein. These results indicate that primitive elements of native structure can form in the absence of normal core packing.     

Time-resolved spectroscopy of the blue fluorescence of spinach leaves

When excited by ultraviolet radiation, leaves of a great number of species of higher plants exhibit emission of blue fluorescence, comparable in intensity to the red emission of chlorophyll. The fluorescence decay of the blue emission of spinach leaves recorded by single photon counting techniques is decomposed into exponential components and it is shown that at least three different components are present. The lifetime of the three components does not show significant variations with the excitation or emission wavelengths. The excitation and emission spectra of each component were determined. The nature of the chemical compounds which cause this emission is discussed in relation to these spectra.     

Transients of perforin pore formation observed by fluorescence microscopic single channel recording.

A new type of single channel recording is described. Large pores were generated in the membranes of resealed human erythrocyte ghosts by incubation with perforin (cytolysin). The flux of the polar fluorescent probe Lucifer Yellow was measured in single ghosts by the fluorescence microphotolysis (photobleaching) technique. The distribution of flux rates for ghosts treated with a limiting perforin concentration showed equidistantly spaced peaks suggesting that subpopulations of ghosts with 0, 1 and 2 pores were resolved. Furthermore, distributions obtained for very different perforin concentrations could be well simulated by using one common value for the flux rate of the single pore (k = 4.65 x 10(-3) s) and assuming a Poisson distribution of pores among ghosts. The flux rate of the single pore corresponds to a pore radius of approximately 50 A, a value which is much smaller than that obtained previously by electron microscopic studies but which agrees well with recent electrical single channel recordings. Mature perforin pores were observed to be very stable. No closing events were detected at a time resolution of 0.2 s for a wide range of temperatures and Ca2+ concentrations. However, the formation of new pores was an unexpectedly slow process. Fluorescence microscopic single channel recording as introduced by this study is applicable to a variety of cellular systems and fluorescent probes and thus may complement the information obtainable by electrical single channel recording of anorganic ion fluxes.     

Duplication of the PMP22 gene in 17p partial trisomy patients with Charcot-Marie-Tooth type-1A neuropathy

Autosomal dominant Charcot-Marie-Tooth type-1A neuropathy (CMT1A) is a demyelinating peripheral nerve disorder that is commonly associated with a submicroscopic tandem DNA duplication of a 1.5-Mb region of 17p11.2p12 that contains the peripheral myelin gene PMP22. Clinical features of CMT1A include progressive distal muscle atrophy and weakness, foot and hand deformities, gait abnormalities, absent reflexes, and the completely penetrant electrophysiologic phenotype of symmetric reductions in motor nerve conduction velocities (NCVs). Molecular and fluorescence in situ hybridization (FISH) analyses were performed to determine the duplication status of the PMP22 gene in four patients with rare cytogenetic duplications of 17p. Neuropathologic features of CMT1A were seen in two of these four patients, in addition to the complex phenotype associated with 17p partial trisomy. Our findings show that the CMT1A phenotype of reduced NCV is specifically associated with PMP22 gene duplication, thus providing further support for the PMP22 gene dosage mechanism for CMT1A. Received: 3 May 1995 / Revised: 1 August 1995     

“Bacillus thermoantarcticus” sp. nov., from Mount Melbourne,Antarctica: a novel thermophilic species

 A novel thermophilic Gram-positive bacillus, “Bacillus thermoantarcticus”, isolated from geothermal soil near the crater of Mount Melbourne, is described. The organism grows at an optimal temperature of 63^°C at pH 6.0, is oxidase-positive, catalase-negative and produces an exopolysaccharide, an exocellular xylanase, an intracellular alcohol dehydrogenase and exo- and endocellular α-glucosidase(s). The sequence of 16S rDNA is very similar to that of “Bacillus thermoglucosidasius”; however, the guanine-plus-cytosine (G+C) content is 8 mol% higher. The type strain is “Bacillus thermoantarcticus” (DSM 9572). Received : 3 February 1995/Accepted : 12 May 1995