Characterization of the murine corticosteroid binding globulin: variations between mammalian forms

Corticosteroid binding globulin (CGB) from term-pregnant mouse serum was isolated and characterized by peptide analysis after treatment with CNBr and Lys-specific protease, respectively. Amino acid sequence analysis of six segments, covering 189 of 383 positions in different regions of the protein, showed unexpectedly low overall homology (60%) to the indirectly deduced human amino acid sequence previously reported. However, some segments displayed a greater resemblance to their human counterparts. Differences were observed in at least two of six potential glycosylation sites. The nature of electrophoretic CBG variants and their immunological properties are described.     

High mobility group proteins HMG1 and HMG2 do not decrease the melting temperature of DNA

High mobility group proteins 1 and 2 isolated in non-denaturing conditions cannot decrease the temperature of denaturation of DNA. When they are isolated or treated with tricloroacetic acid a hyperchromic peak below the melting temperature of free DNA appears in agreement with previous data ( Javaherian et al. (1979) Nucl . Acids Res. 6, 3569-3580). We show that this is due to light scattering of aggregated protein at submelting temperatures and not to melting of DNA.     

Residues in the 1A rod domain segment and the linker L2 are required for stabilizing the A11 molecular alignment mode in keratin intermediate filaments

Both analyses of x-ray diffraction patterns of well oriented specimens of trichocyte keratin intermediate filaments (IF) and in vitro cross-linking experiments on several types of IF have documented that there are three modes of alignment of pairs of antiparallel molecules in all IF: A11, A22 and A12, based on which parts of the major rod domain segments are overlapped. Here we have examined which residues may be important for stabilizing the A11 mode. Using the K5/K14 system, we have made point mutations of charged residues along the chains and examined the propensities of equimolar mixtures of wild type and mutant chains to reassemble using as criteria: the formation (or not) of IF in vitro or in vivo; and stabilities of one- and two-molecule assemblies. We identified that the conserved residue Arg10 of the 1A rod domain, and the conserved residues Glu4 and Glu6 of the linker L2, were essential for stability. Additionally, conserved residues Lys31 of 1A and Asp1 of 2A and non-conserved residues Asp/Asn9 of 1A, Asp/Asn3 of 2A, and Asp7 of L2 are important for stability. Notably, these groups of residues lie close to each other when two antiparallel molecules are aligned in the A11 mode, and are located toward the ends of the overlap region. Although other sets of residues might theoretically also contribute, we conclude that these residues in particular engage in favorable intermolecular ionic and/or H-bonding interactions and thereby may play a role in stabilizing the A11 mode of alignment in keratin IF.     

Involucrin cross-linking by transglutaminase 1. Binding to membranes directs residue specificity

The transglutaminase 1 (TGase 1) enzyme is essential for the assembly of the cell envelope barrier in stratified squamous epithelia. It is usually bound to membranes, but to date most studies with it have involved solution assays. Here we describe an in vitro model system for characterizing the function of TGase 1 on the surface of synthetic lipid vesicles (SLV) of composition similar to eukaryote plasma membranes. Recombinant baculovirus-expressed human TGase 1 readily binds to SLV and becomes active in cross-linking above 10 microM Ca2+, in comparison to above 100 microM in solution assays, suggesting that the membrane surface is important for enzyme function. Involucrin also binds to SLV containing 12-18% phosphatidylserine and at Ca2+ concentrations above 1 microM. In reactions of involucrin with TGase 1 enzyme in solution, 80 of its 150 glutamines serve as donor residues. However, on SLV carrying both involucrin and TGase 1, only five glutamines serve as donors, of which glutamine 496 was the most favored. As controls, there was no change in specificity toward the glutamines of other substrates used by free or SLV-bound TGase 1 enzyme. We propose a model in which involucrin and TGase 1 bind to membranes shortly after expression in differentiating keratinocytes, but cross-linking begins only later as intracellular Ca2+ levels increase. Furthermore, the data suggest that the membrane surface regulates the steric interaction of TGase 1 with substrates such as involucrin to permit specific cross-linking for initiation of cell envelope barrier formation.     

Effect of quercetin on daunorubicin-induced heart mitochondria changes in rats

Cancer therapy with daunorubicin is limited by its cardiotoxicity. It has been suggested that daunorubicin-induced free radical generation can be involved. The precise molecular mechanism of daunorubicin-induced cardiotoxicity is still not well understood but it is believed that mitochondria play an important role in this process. It has been reported that flavonoids with antioxidant properties may prevent anthracycline-induced cardiotoxicity. In this work, we investigated the effects of daunorubicin and quercetin on mitochondrial enzyme activities such as ATPase, glutathione peroxidase (GPx) and glutathione reductase (GR). Moreover, we also studied the changes of outer mitochondrial membrane using synchronous fluorescence spectra. The activity of ATPase and GR were significantly increased after daunorubicin application. Pretreatment with quercetin significantly alleviated this increase. On the other hand, GPx activity was significantly decreased and quercetin prevented this decrease. Treatment with quercetin alone had no significant effect on the enzyme activity studied. Quercetin also completely prevented daunorubicin-induced changes in fluorescence of the outer mitochondrial membrane. In conclusion, our data indicate that quercetin may be useful in mitigating daunorubicin-induced cardiotoxicity.     

Caffeine induces a second wave of apoptosis after low dose-rate gamma radiation of HL-60 cells

Most cell lines that lack functional p53 protein are arrested in the G₂ phase of the cell cycle due to DNA damage. It was previously found that the human promyelocyte leukemia cells HL-60 (TP53 negative) that had been exposed to ionizing radiation at doses up to 10 Gy were arrested in the G₂ phase for a period of 24 h. The radioresistance of HL-60 cells that were exposed to low dose-rate gamma irradiation of 3.9 mGy/min, which resulted in a pronounced accumulation of the cells in the G₂ phase during the exposure period, increased compared with the radioresistance of cells that were exposed to a high dose-rate gamma irradiation of 0.6 Gy/min. The D₀ value (i.e. the radiation dose leading to 37% cell survival) for low dose-rate radiation was 3.7 Gy and for high dose-rate radiation 2.2 Gy. In this study, prevention of G₂ phase arrest by caffeine (2 mM) and irradiation of cells with low dose-rate irradiation in all phases of the cell cycle proved to cause radiosensitization (D₀=2.2 Gy). The irradiation in the presence of caffeine resulted in a second wave of apoptosis on days 5–7post-irradiation. Caffeine-induced apoptosis occurring later than day 7 post-irradiation is postulated to be a result of unscheduled DNA replication and cell cycle progress.     

In vitro assembly and structure of trichocyte keratin intermediate filaments: a novel role for stabilization by disulfide bonding

Intermediate filaments (IF) have been recognized as ubiquitous components of the cytoskeletons of eukaryotic cells for 25 yr. Historically, the first IF proteins to be characterized were those from wool in the 1960s, when they were defined as low sulfur keratins derived from "microfibrils." These proteins are now known as the type Ia/type IIa trichocyte keratins that constitute keratin IF of several hardened epithelial cell types. However, to date, of the entire class of >40 IF proteins, the trichocyte keratins remain the only ones for which efficient in vitro assembly remains unavailable. In this paper, we describe the assembly of expressed mouse type Ia and type IIa trichocyte keratins into IF in high yield. In cross-linking experiments, we document that the alignments of molecules within reduced trichocyte IF are the same as in type Ib/IIb cytokeratins. However, when oxidized in vitro, several intermolecular disulfide bonds form and the molecular alignments rearrange into the pattern shown earlier by x-ray diffraction analyses of intact wool. We suggest the realignments occur because the disulfide bonds confer substantially increased stability to trichocyte keratin IF. Our data suggest a novel role for disulfide bond cross linking in stabilization of these IF and the tissues containing them.     

Coiled-coil trigger motifs in the 1B and 2B rod domain segments are required for the stability of keratin intermediate filaments

Many alpha-helical proteins that form two-chain coiled coils possess a 13-residue trigger motif that seems to be required for the stability of the coiled coil. However, as currently defined, the motif is absent from intermediate filament (IF) protein chains, which nevertheless form segmented two-chain coiled coils. In the present work, we have searched for and identified two regions in IF chains that are essential for the stability necessary for the formation of coiled-coil molecules and thus may function as trigger motifs. We made a series of point substitutions with the keratin 5/keratin 14 IF system. Combinations of the wild-type and mutant chains were assembled in vitro and in vivo, and the stabilities of two-chain (one-molecule) and two-molecule assemblies were examined with use of a urea disassembly assay. Our new data document that there is a region located between residues 100 and 113 of the 2B rod domain segment that is absolutely required for molecular stability and IF assembly. This potential trigger motif differs slightly from the consensus in having an Asp residue at position 4 (instead of a Glu) and a Thr residue at position 9 (instead of a charged residue), but there is an absolute requirement for a Glu residue at position 6. Because these 13 residues are highly conserved, it seems possible that this motif functions in all IF chains. Likewise, by testing keratin IF with substitutions in both chains, we identified a second potential trigger motif between residues 79 and 91 of the 1B rod domain segment, which may also be conserved in all IF chains. However, we were unable to find a trigger motif in the 1A rod domain segment. In addition, many other point substitutions had little detectable effect on IF assembly, except for the conserved Lys-23 residue of the 2B rod domain segment. Cross-linking and modeling studies revealed that Lys-23 may lie very close to Glu-106 when two molecules are aligned in the A(22) mode. Thus, the Glu-106 residue may have a dual role in IF structure: it may participate in trigger formation to afford special stability to the two-chain coiled-coil molecule, and it may participate in stabilization of the two-molecule hierarchical stage of IF structure.     

Allosteric signaling and a nuclear exit strategy: binding of UL25/UL17 heterodimers to DNA-Filled HSV-1 capsids

UL25 and UL17 are two essential minor capsid proteins of HSV-1, implicated in DNA packaging and capsid maturation. We used cryo-electron microscopy to examine their binding to capsids, whose architecture observes T = 16 icosahedral geometry. C-capsids (mature DNA-filled capsids) have an elongated two-domain molecule present at a unique, vertex-adjacent site that is not seen at other quasiequivalent sites or on unfilled capsids. Using SDS-PAGE and mass spectrometry to analyze wild-type capsids, UL25 null capsids, and denaturant-extracted capsids, we conclude that (1) the C-capsid-specific component is a heterodimer of UL25 and UL17, and (2) capsids have additional populations of UL25 and UL17 that are invisible in reconstructions because of sparsity and/or disorder. We infer that binding of the ordered population reflects structural changes induced on the outer surface as pressure builds up inside the capsid during DNA packaging. Its binding may signal that the C-capsid is ready to exit the nucleus.     

Terpenes with antimicrobial activity from Cretan propolis

Five terpenes, the diterpenes: 14,15-dinor-13-oxo-8(17)-labden-19-oic acid and a mixture of labda-8(17),13E-dien-19-carboxy-15-yl oleate and palmitate as well as the triterpenes, 3,4-seco-cycloart-12-hydroxy-4(28),24-dien-3-oic acid and cycloart-3,7-dihydroxy-24-en-28-oic acid were isolated from Cretan propolis. Moreover, 18 known compounds were also isolated, seven of them for the first time as propolis components. All structures were established on the basis of spectroscopic analysis and chemical evidence. All isolated compounds were tested for antimicrobial activity against some Gram-positive and Gram-negative bacteria as well as against some human pathogenic fungi showing a broad spectrum of antimicrobial activity.