Antimicrobial activity in the skin of the channel catfish Ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins

Three antibacterial proteins were isolated from acid extracts of channel catfish (Ictalurus punctatus) skin by cation exchange chromatography and reverse-phase high-pressure liquid chromatography. The molecular masses of the proteins were 15.5, 15.5 and 30 kD as determined by SDS-polyacrylamide gel electrophoresis. Mass spectrometry, amino acid composition and amino acid sequence data suggest that the most abundant protein is closely related to histone H2B. The H2B-like protein was inhibitory to Aeromonas hydrophila and Saprolegnia spp., which are important bacterial and fungal pathogens of fish. These findings suggest that histones may be important defensive molecules in fish. Received 22 December 1997; received after revision 5 March 1998; accepted 5 March 1998     

Roles of the DNA mismatch repair and nucleotide excision repair proteins during meiosis

Numerous proteins are involved in the nucleotide excision repair (NER) and DNA mismatch repair (MMR) pathways. The function and specificity of these proteins during the mitotic cell cycle has been actively investigated, in large part due to the involvement of these systems in human diseases. In contrast, comparatively little is known about their functioning during meiosis. At least three repair pathways operate during meiosis in the yeast Saccharomyces cerevisiae to repair mismatches that occur as a consequence of heteroduplex formation in recombination. The first pathway is similar to the one acting during postreplicative mismatch repair in mitotically dividing cells, while two pathways are responsible for the repair of large loops during meiosis, using proteins from MMR and NER systems. Some MMR proteins also help prevent recombination between diverged sequences during meiosis, and act late in recombination to affect the resolution of crossovers. This review will discuss the current status of DNA mismatch repair and nucleotide excision repair proteins during meiosis, especially in the yeast S. cerevisiae. Received 21 September 1998; received after revision 23 November 1998; accepted 23 November 1998     

Chromatin assembly during S phase: contributions from histone deposition, DNA replication and the cell division cycle

During S phase of the eukaryotic cell division cycle, newly replicated DNA is rapidly assembled into chromatin. Newly synthesised histones form complexes with chromatin assembly factors, mediating their deposition onto nascent DNA and their assembly into nucleosomes. Chromatin assembly factor 1, CAF-1, is a specialised assembly factor that targets these histones to replicating DNA by association with the replication fork associated protein, proliferating cell nuclear antigen, PCNA. Nucleosomes are further organised into ordered arrays along the DNA by the activity of ATP-dependent chromatin assembly and spacing factors such as ATP-utilising chromatin assembly and remodelling factor ACF. An additional level of controlling chromatin assembly pathways has become apparent by the observation of functional requirements for cyclin-dependent protein kinases, casein kinase II and protein phosphatases. In this review, we will discuss replication-associated histone deposition and nucleosome assembly pathways, and we will focus in particular on how nucleosome assembly is linked to DNA replication and how it may be regulated by the cell cycle control machinery.     

Effect of histones from brain on DNA-synthesis in vitro

The results of these experiments demonstrate that histones from brain inhibit the replication of DNA in vitro. A similar effect is observed with polylysine or polyarginine. The reversion of inhibition by polyglutamic acid or acidic proteins is completed in all cases except when the DNA is previously complexed with histones, polyarginine or polylysine. This suggest that histones masking of DNA towards the polymerases involves electrostatic forces.     

Menadione-induced apoptosis and the degradation of lamin-like proteins in tobacco protoplasts

Detection of stereotypic hallmarks of apoptosis during cell death induced by menadione, including DNA laddering and the formation of apoptotic bodies, is reported. Comet assay and the TdT-mediated dUTP nick end labelling (TUNEL) procedure were also performed to detect DNA fragmentation. Inhibition of DNA fragmentation by Ac-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and phenylmethylsulfosyl (PMSF) implicated the involvement of caspase-like proteases in menadione-induced apoptosis in plants. We further studied the cleavage of lamin-like proteins during apoptosis in menadione-treated tobacco protoplasts. In animals, it has been reported that the solubilization of nuclear lamina and lamin degradation occurs during apoptotic cell death. However, little is known about the fate of lamins in apoptotic plant cells. Our study provided evidence that lamin-like proteins degraded into 35-kDa fragments in tobacco protoplasts induced by menadione, and this preceded DNA fragmentation. The results thus indicated that proteolytic cleavage of nuclear lamins was also conserved in programmed cell death in plants. Received 16 November 1998; received after revision 21 December 1998; accepted 23 December 1998     

Structure, function and evolution of antifreeze proteins

Antifreeze proteins bind to ice crystals and modify their growth. These proteins show great diversity in structure, and they have been found in a variety of organisms. The ice-binding mechanisms of antifreeze proteins are not completely understood. Recent findings on the evolution of antifreeze proteins and on their structures and mechanisms of action have provided new understanding of these proteins in different contexts. The purpose of this review is to present the developments in contrasting research areas and unite them in order to gain further insight into the structure and function of the antifreeze proteins. Received 2 September 1998; received after revision 21 October 1998; accepted 2 November 1998     

Immune responses to DNA vaccines

DNA vaccines, based on plasmid vectors expressing an antigen under the control of a strong promoter, have been shown to induce protective immune responses to a number of pathogens, including viruses, bacteria and parasites. They have also displayed efficacy in treatment or prevention of cancer, allergic diseases and autoimmunity. Immunologically, DNA vaccines induce a full spectrum of immune responses that include cytolytic T cells, T helper cells and antibodies. The immune response to DNA vaccines can be enhanced by genetic engineering of the antigen to facilitate its presentation to B and T cells. Furthermore, the immune response can be modulated by genetic adjuvants in the form of vectors expressing biologically active determinants or by more traditional adjuvants that facilitate uptake of DNA into cells. The ease of genetic manipulation of DNA vaccines invites their use not only as vaccines but also as research tools for immunologists and microbiologists. Received 26 October 1998; received after revision 3 December 1998; accepted 3 December 1998     

Effect of histones from brain on DNA-synthesis in vitro

Summary The results of these experiments demonstrate that histones from brain inhibit the replication of DNA in vitro. A similar effect is observed with polylysine or polyarginine. The reversion of inhibition by polyglutamic acid or acidic proteins is completed in all cases except when the DNA is previously complexed with histones, polyarginine or polylysine. This suggest that histones masking of DNA towards the polymerases involves electrostatic forces.These studies were supported by the Consejo Nacional de Investigaciones Científicas y Técnicas and the Instituto Nacional de Farmacología y Bromatología, Argentina.Abbreviations. DNA-polymerase: deoxynucleoside triphosphate: DNA deoxynucleotidyl transferase (E.C. 2.7.7.7.).     

Age-related changes of the pattern of non-histone proteins in active and condensed fractions of mouse liver chromatin and hepatocarcinoma

Summary Electrophoretic analysis of histones and non-histone acid-soluble proteins in active (nuclease sensitive) and inactive chromatin from liver of young and old CBA mice and in age-related hepatocarcinomas showed a higher ratio of NHP: histones in active chromatin in old cells. Some liver- and hepatoma-specific fractions of non-histone proteins have been identified as chromatin matrix proteins.     

Long-term changes in tyrosine phosphorylation of the abundant nuclear proteins during granulocytic differentiation of HL-60 cells

The two-dimensional electrophoretic patterns of nuclear proteins and their tyrosine phosphorylation were compared for HL-60 cells before and after differentiation induction to granulocytes by dimethyl sulfoxide, all-trans retinoic acid and N ⁶,O ²-dibutyryl adenosine 3′5′-cyclic monophosphate. Regardless of the inducer used, some nuclear proteins, which are tyrosine-phosphorylated in proliferating HL-60 cells, undergo gradual dephosphorylation 12–72 h after induction of differentiation, followed by drastic dephosphorylation during maturation to granulocytes. At least 13 nuclear proteins with a molecular mass of 35–110 kDa are dephosphorylated, and 6 nuclear proteins undergo tyrosine phosphorylation. Analysis of the nuclear proteins differentially extracted by salt and detergents indicates that changes in their tyrosine phosphorylation during the maturation stage of differentiating granulocytes occur mainly in proteins which are abundant in nucleoplasm, chromatin and residual nuclear structures. The abundance of these proteins, residing in the nuclear structures, and their long-term modification in phosphorylation during the maturation stages of differentiation strongly suggest that tyrosine phosphorylation of these proteins is involved in reorganization of the differentiating cell nucleus. Received 21 September 1998; received after revision 24 November 1998; accepted 3 December 1998     

Age-related changes of the pattern of non-histone proteins in active and condensed fractions of mouse liver chromatin and hepatocarcinoma

Electrophoretic analysis of histones and non-histone acid-soluble proteins in active (nuclease sensitive) and inactive chromatin from liver of young and old CBA mice and in age-related hepatocarcinomas showed a higher ratio of NHP:histones in active chromatin in old cells. Some liver- and hepatoma-specific fractions of non-histone proteins have been identified as chromatin matrix proteins.     

Gonadotrophin-releasing activity of histones H2A and H2B

We report that histones H2A and H2B possess gonadotrophin-releasing activity in vitro and assess the signal transduction pathways involved in these effects. Perifused and incubated rat anterior pituitary (AP) cells were used, and luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured by RIA. Perifusion of cells with histone H2A (30 μM) or histone H2B (30 μM), markedly stimulated LH release but failed to elicit any FSH response. Cells incubated with 6 or 30 μM histone H2A showed a dose- and time-dependent stimulatory effect on both LH and FSH release which was blocked by 1 μM peptide MB35, an 86–120 amino acid fragment of histone H2A. Incubation of pituitary cells with gonadotrophin-releasing hormone (GnRH) and histones H2A or H2B showed a stimulatory effect on LH and FSH release which was similar to the sum of the separate effects. Trifluoperazine, as well as ethylene glycol bis(b-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), alone or in the presence of the calcium ionophore A23187, significantly reduced the response of AP cells to histones. Various cyclic adenosine monophosphate (cAMP) enhancers had no effect on histone-stimulated release of gonadotrophins in incubated AP cells. Our results confirm previous evidence that histones may act as hypophysiotrophic signals. Calcium- and diacylglycerol-associated pathways, but not cAMP, appear to participate in these effects. Received 11 August 1997; received after revision 20 January 1998; accepted 26 January 1998     

Microtubule associated protein tau binds to double-stranded but not single-stranded DNA

Tau, a major microtubule-associated protein of the neuron, which is known to promote the assembly of and to stabilize microtubules, has also been seen associated with chromatin in neuronal cell lines, but its role in this subcellular compartment is still unknown. In this study, the binding of tau to DNA was investigated using the electrophoretic mobility shift assay. Using polynucleotide as probe, we found that tau bound to double-stranded but not to single-stranded DNA. Formation of tau-polynucleotide complex was disrupted by alkaline pH and a high concentration of NaCl, but was not affected by dithiothreitol. Electron microscopy revealed that the protein associated with the nucleic acid in a necklacelike manner. DNA-cellulose chromatography and radioimmunodot-blot analyses showed that calf thymus histones VI-S, VII-S and VIII-S could replace both recombinant human brain tau₃₅₂ (tau-23) and tau₄₄₁ (tau-40) from DNA. Thus, tau appears to bind to DNA reversibly in the presence of histones. Received 24 November 2002; received after revision 28 December 2002; accepted 30 December 2002 RID="*" ID="*"Corresponding author.