Functional and physical interaction between p53 and BZLF1: implications for Epstein-Barr virus latency.

The p53 tumor suppressor protein, which is commonly mutated in human cancers, has been shown to interact directly with virally encoded from papillomavirus, adenovirus, and simian virus 40. The disruption of p53 function may be required for efficient replication of certain viruses and may also play a role in the development of virally induced malignancies. Infection with Epstein-Barr virus (EBV) has been associated with the development of B-cell lymphomas and nasopharyngeal carcinoma. Here we show that the EBV immediate-early protein, BZLF1 (Z), which is responsible for initiating the switch from latent to lytic infection, can interact directly in vitro and in vivo with the tumor suppressor protein, p53. This interaction requires the coiled-coil dimerization domain of the Z protein and the carboxy-terminal portion of p53. Overexpression of wild-type p53 inhibits the ability of Z to disrupt viral latency. Likewise, Z inhibits p53-dependent transactivation in lymphoid cells. The direct interaction between Z and p53 may play a role in regulating the switch from latent to lytic viral infection.     

Reductive methylation andpKa determination of the lysine side chains in calbindin D9k

The Lys residues in the 75-residue Ca²⁺-binding protein calbindin D_9k were reductively methylated with¹³C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca²⁺- and apo-forms of the protein, the¹³C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (¹H,¹³C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca²⁺-calbindin D_9k and apo-calbindin D_9k were obtained by combiningpH titration experiments and (¹H,¹³C)-HMQC NMR spectroscopy. Each Lys residue in the Ca²⁺- and apo-forms of calbindin D_9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D_9k has a very similar structure compared to Ca²⁺-calbindin D_9k, the removal of two Ca²⁺ ions from the protein leads to an increase of thepKa values of the Lys residues.     

Autophosphorylation-dependent protein kinase predominantly phosphorylates Ser115, thein vivo site in brain myelin basic protein

In a previous report [Yanget al., (1987a),J. Biol Chem. 262, 7034–7040], a cyclic-AMP- and calcium-independent brain kinase which requires autophosphorylation for activity was identified as a very potent myelin basic protein (MBP) kinase. In this report, the phosphorylation sites of MBP by this autophosphorylation-dependent protein kinase (autokinase) are further determined by two-dimensional electrophoresis/thin-layer chromatography, phosphoamino acid analysis, high-performance liquid chromatography, tryptic peptide mapping, sequential manual Edman degradation, and direct peptide sequencing. Autokinase phosphorylates MBP on both threonine and serine residues. Three major tryptic phosphopeptide peaks were resolved by C₁₈-reversed phase highper-formance liquid chromatography. Sequential manual Edman degradation together with direct sequence analysis revealed that FS(p)WGAEGQKPGFGYGGR is the phosphorylation site sequence (molar ratio 1.0) for the first major phosphopeptide peak. When mapping with bovine brain MBP sequence, we finally demonstrate Ser¹¹⁵, one of thein vivo phosphorylation sites in MBP, as the major site phosphorylated by autokinase, implicating a physiologically relevant role of autokinase in the regulation of brain myelin function. By using the same approach, we also identified HRDT(p)GILDSLGR (molar ratio 0.9) and TT(p)HYGSLPQK (molar ratio 0.8) as the major phosphorylation site sequences in³²P-MBP phosphorylated by autokinase, further indicating that -Arg-XSer/Thr-(neutral amino acid)₃-(amino acid-containing hydroxyl group such as Ser/Glu/Asp)-(neutral amino acid)₂-may represent a unique consensus sequence motif specifically recognized by this autophosphorylation-dependent multisubstrate/ multifunctional protein kinase in the brain.     

Mutation of the gene encoding protein kinase C 1 stimulates mitotic recombination in Saccharomyces cerevisiae.

We have isolated a recessive allele of the yeast protein kinase C gene (PKC1) which promotes an elevated rate of mitotic recombination and confers a temperature-sensitive growth defect. The rate of recombination was increased between genes in direct repeat and at a series of heteroalleles and was dependent upon the RAD52 gene product. The mutant pkc1 allele was sequenced and found to encode a single amino acid change within the catalytic domain. Osmotic stabilizing agents rescued the temperature-sensitive growth defect but not the hyperrecombination phenotype, indicating that the two traits are separable. This separability suggests that the PKC1 gene product (Pkc1p) regulates DNA metabolism by an alternate pathway to that used in the regulation of cell lysis. The regulation of recombination is a previously unidentified role for Pkc1p.     

Localisation of substance P-like immunoreactivity in the ciliary ganglia of monkey (Macaca fascicularis) and cat: a light- and electron-microscopic study

The present study describes substance P-like immunoreactivity in the ciliary ganglia of monkey (Macaca fascicularis) and cat. About 60% of neurons in the monkey ciliary ganglion and 40% in the cat ciliary ganglion were substance P-like immunoreactive, ranging from faint to moderate staining. Substance P-like immunoreactivity was located in cell bodies, dendritic profiles and axons. In the monkey, substance P-like immunoreactive pericellular arborisations were associated with about 0.5%–3% of the ganglion cells, which were either negatively, faintly or moderately stained. An electron-microscopic study demonstrated the presence of either substance P-like immunoreactive positive or negative axon terminals synapsing or closely associated with positive dendritic profiles in both the monkey and cat ciliary ganglia. The results suggest that substance P plays an important role in the ciliary ganglion, perhaps as a modulator or transmitter.     

Oligonucleotides with novel, cationic backbone substituents: aminoethylphosphonates.

Oligonucleotide (2-aminoethyl)phosphonates in which the backbone consisted of isomerically pure, alternating (2-aminoethyl)-phosphonate and phosphodiester linkages have been prepared and characterized. One of these single isomer oligonucleotides (Rp) formed a more stable duplex with DNA or RNA than its corresponding natural counterpart. Hybrid stability was more pH-dependent, but less salt-dependent than a natural duplex. The specificity of hybridization was examined by hybridization of an oligonucleotide containing one (2-aminoethyl)phosphonate to oligonucleotides possessing mismatches in the region opposite to the aminoethyl group. In contrast to oligonucleotides containing (aminomethyl)-phosphonate linkages, oligonucleotide (2-aminoethyl)phosphonates were completely stable to hydrolysis in aqueous solution. These oligonucleotides were resistant to nuclease activity but did not induce RNase H mediated cleavage of a complementary RNA strand. Incubation in a serum-containing medium resulted in minimal degradation over 24 hours. Studies of cell uptake by flow cytometry and confocal microscopy demonstrated temperature dependent uptake and intracellular localization. (2-Aminoethyl)phosphonates represent a novel approach to the introduction of positive charges into the backbone of oligonucleotides.