Formalin fixation strongly influences biomechanical properties of the spine

As fresh human cadaveric spine specimens for in vitro testing are hard to obtain and carry a potential risk of infection, the possibility of using embalmed spine specimens has been considered. The cross-linking effect of formalin fixation, however, raises uncertainties regarding the biomechanical likeness of preserved specimens. They have been reported to be stiffer, but no quantitative data exist.

The purpose of this study was to determine the biomechanical differences between fresh and formalin-fixed spine specimens, using L1–2 motion segments from six 16-week-old calf spines. The range of motion and neutral zone were determined in flexion-/extension, left/right axial rotation, and right/left lateral bending.

The range of motion decreased in the formalin fixed specimens by as much as 80%, and the neutral zone by as much as 96%. The results of this study therefore imply that, for biomechanical testing, formalin-fixed specimens are not representative of the in vivo conditions.     

Properties of the adenovirus IVa2 gene product, an effector of late-phase-dependent activation of the major late promoter.

The adenovirus major late promoter is strongly activated after the onset of viral DNA replication. Sequence elements located downstream of the major later promoter start site have previously been shown to be essential for this activation. Two proteins (DEF-A and DEF-B) bind to these elements in a late-phase-dependent manner. DEF-B has been identified as the product of adenovirus intermediate gene IVa2 (pIVa2) (C. Tribouley, P. Lutz, A. Staub, and C. Kedinger, J. Virol. 68:4450-4457, 1994). Here we show that pIVa2, while monomeric in solution, binds to its recognition sequence as a dimer and that two 20-residue amphipathic alpha helices play an essential role in this DNA-binding activity. Attempts to purify DEF-A have failed, but its chromatographic behavior, together with its immunological properties, established that pIVa2 is also a component of this heteromeric protein. In addition, the time course of pIVa2 synthesis during infection correlated with simultaneous detection of the binding of both DEF-A and DEF-B complexes to the downstream elements. Finally, as revealed by immunomicroscopy, pIVa2 is targeted to the nucleus, where it distributes to restricted locations in the nucleoplasm, as well as to the nucleoli. Altogether, these results demonstrate that pIVa2 plays a critical role in the transition from the early to the late phase of the lytic cycle. Furthermore, pIVa2 may serve additional functions yet to be uncovered, as suggested by its presence within the cell nucleolus.     

Site-specific integration of the phage ΦCTX genome into the Pseudomonas aeruginosa chromosome: characterization of the functional integrase gene located close to and upstream of attP

The site-specific integration of the phage ?CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 by integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage ?CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates.     

A Nonradioactive Fluorescent Gel-Shift Assay for the Analysis of Protein Phosphatase and Kinase Activities toward Protein-Specific Peptide Substrates

Synthetic peptides are important tools with which to study the activities of protein kinases and phosphatases toward specific substrate sequences which are present within selected regions of a protein. Most existing assays for the phosphorylation or dephosphorylation of such peptides utilize ³²P and either affinity chromatography or HPLC separation and require extensive characterization and validation. Here, we describe a method for monitoring the phosphorylation or dephosphorylation of almost any peptide of interest which does not require the use of radioactivity, making its reagents stable for a prolonged period, and which can be performed in any standard laboratory. For this, after performance of kinase or phosphatase reactions with the peptide of interest, products are derivatized with fluorescamine and are separated according to charge by agarose gel electrophoresis. Phosphorylated and nonphosphorylated peptides are readily separated and can be both identified and quantified by uv detection. The lower limit for detection of peptide in the agarose gel was 0.02 nmol using the gel-shift kinase assay with cAMP-dependent kinase and Kemptide as substrate. This had sensitivity and reproducibility similar to those of a standard assay using [γ-³²P]ATP with this substrate. Dephosphorylation of a synthetic phosphopeptide corresponding to a segment of the cholecystokinin receptor was tested in an analogous assay with known amounts of protein phosphatase 2A. Phosphopeptide and dephosphopeptide were easily detected and quantified with as little as 0.03 mU/mI protein phosphatase 2A activity. Therefore, with this assay, most synthetic peptides and phosphopeptides can be used as substrates without further modification. This will be of particular interest for monitoring the purification of highly specific protein kinase and phosphatase activities.     

Pigment interactions in chlorosomes of various green bacteria

Resonance Raman experiments were performed on different green bacteria. With blue excitation, i.e. under Soret resonance or preresonance conditions, resonance Raman contributions were essentially arising from the chlorosome pigments. By comparing these spectra and those of isolated chlorosomes, it is possible to evaluate how the latter retain their native structure during the isolation procedures. The structure of bacteriochlorophyll oligomers in chlorosomes was interspecifically compared, in bacteriochlorophyllc- and bacteriochlorophylle- synthesising bacteria. It appears that interactions assumed by the 9-keto carbonyl group are identical inChlorobium limicola, Chlorobium tepidum, andChlorobium phaeobacteroides. In the latter strain, the 3-formyl carbonyl group of bacteriochlorophylle is kept free from intermolecular interactions. By contrast, resonance Raman spectra unambiguously indicate that the structure of bacteriochlorophyll oligomers is slightly different in chlorosomes fromChloroflexus auranticus, either isolated or in the whole bacteria.     

A site accessible to extracellular TEA+ and K+ influences intracellular Mg2+ block of cloned potassium channels

The members of the RCK family of cloned voltage-dependent K⁺ channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K⁺ ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg²⁺ ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K⁺ solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg²⁺ block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K⁺ ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K⁺ and thereby indirectly affects internal Mg²⁺ block and channel closing.Abbreviations TEA tetraethylammonium - EGTA Ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid - 2S3B model 2-site 3-barrier model Correspondence to: S. H. Heinemann     

Characterization of a class Ib gene of the rat major histocompatibility complex

The cDNA and a partial genomic sequence of a rat class I major histocompatibility (RT1) gene, 11/3R, is reported here. The sequence contains several unique amino acid residues at certain positions, mutations in exon 7 (which is not expressed), a mutation of the canonical exon 8 stop codon to a sense codon, and includes a long 3 unstranslated region (utr). The structure of exon 7 differs from that found in most rat class I genes and resembles exon 7 of most H-2K,D,L.Q genes. Parts of the 3 noncoding region are homologous to the RT1.A-4 and certain H-2 genes. Expression is detectable by northern blot analysis in mitogen-stimulated lymphocytes only, by polymerase chain reaction (PCR) in each tissue tested. After transfection into L cells 11/3R can be shown to be expressible at the cell surface. Probes derived from the 3 noncoding part crosshybridize with a number of restriction fragments which map to the RT1.C region, thus defining a subfamily of RT1.C region genes. Several members of this subfamily are deleted in the M1 RT1 mutant. The 11/3R gene presents typical features of a class Ib gene. Aspects of evolution and the potential of the gene are discussed.The nucleotide sequence data reported in this paper have been submitted to the GenBank molecule sequence data base and have been assigned the accession numbers X67503 ande X67504.