DNA condensation by high-affinity interaction with avidin

Avidin, the basic biotin-binding glycoprotein from chicken egg white, is known to interact with DNA, whereas streptavidin, its neutral non-glycosylated bacterial analog, does not. In the present study we investigated the DNA-binding properties of avidin. Its affinity for DNA in the presence and absence of biotin was compared with that of other positively charged molecules, namely the protein lysozyme, the cationic polymers polylysine and polyarginine and an avidin derivative with higher isoelectric point (pI approximately 11) in which most of the lysine residues were converted to homoarginines. Gel-shift assays, transmission electron microscopy and dynamic light scattering experiments demonstrated an unexpectedly strong interaction between avidin and DNA. The most pronounced gel-shift retardation occurred with the avidin-biotin complex, followed by avidin alone and then guanidylated avidin. Furthermore, ultrastructural and light-scattering studies showed that avidin assembles on the DNA molecule in an organized manner. The assembly leads to the formation of nanoparticles that are about 50-100 nm in size (DNA approximately 5 kb) and have a rod-like or toroidal shape. In these particles the DNA is highly condensed and one avidin is bound to each 18 +/- 4 DNA base pairs. The complexes are very stable even at high dilution ([DNA] =10 pM) and are not disrupted in the presence of buffers or salt (up to 200 mM NaCl). The other positively charged molecules also condense DNA and form particles with a globular shape. However, in this case, these particles disassemble by dilution or in the presence of low salt concentration. The results indicate that the interaction of avidin with DNA may also occur under physiological conditions, further enhanced by the presence of biotin. This DNA-binding property of avidin may thus shed light on a potentially new physiological role for the protein in its natural environment.     

Synthesis and preliminary pharmacological evaluation of new (+/-) 1,4-naphthoquinones structurally related to lapachol

Seven new 1,4-naphthoquinones structurally related to lapachol were synthesized from lawsone and oxygenated arylmercurials. These compounds can also be seen as pterocarpan derivatives where the A-ring was substituted by the 1,4-naphthoquinone nucleus. Pharmacological screening provided evidence of significant biological activities, including effects against proliferation of the MCF-7 human breast cancer cell line, against Herpes Simplex Virus type 2 infection, and against snake poison-induced myotoxicity. One derivative displaced flunitrazepam binding and showed benzodiazepine-like activity, suggesting novel neuroactive structural motifs.     

Prevalence of serum antibodies to LA-1 oncoprotein, herpes simplex virus type-2 glycoprotein and human papillomavirus type 16 transactivator (E2) protein among Indian women with cervical neoplasia.

Prevalence of serum antibodies to synthetic peptide to oncoprotein of LA-1 known as oncogene of herpes simplex virus type-2, herpes simplex virus type-2 glycoprotein-D as an determinant of viral pathogenicity and human papillomavirus type 16 transactivator E2 protein was studied among 46 Indian women with cervical neoplasia using immunoblot assay for HSV-2 gD glycoprotein and LA-1 antibodies as well as peptide ELISA assay to detect HPV16 E2 antibodies. The seropositivity to LA-1 oncoprotein was found to be high (61%) among patients with invasive cervical carcinoma as compared to 35% in various grades of cervical intraepithelial neoplasia (CIN) and 36% in normal control women. In contrast to this, a uniformly high frequency of antibody to HPV 16 E2 was observed among women with CIN (68%), normal healthy controls (50%) and invasive cervical carcinoma (43%). However, a low frequency of seropositivity (13%) to recombinant vaccinia virus HSV-2 gD protein was found among 15 tested sera each from group of women with various grades of CIN as well as invasive cervical carcinoma as compared to 28% among seven normal healthy control. A negative correlation of LA-1 and HPV16 E2 seropositivity on patient by patient comparison among CIN and invasive cervical carcinoma group was observed which is statistically significant (P = 0.019 for CIN; P = 0.038 for invasive cervical carcinoma). However, a positive correlation (P = 0.144) was found among normal control women. The study has shown a desirable serological marker of cervical neoplasia. This serological marker could be employed as a screening tool in conjunction with cytopathological screening to diagnose women harbouring LA-1 oncogene associated cervical lesions.     

Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

The large subunit of the herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (RR1) is demonstrated to possess serine/threonine-specific kinase activity. Computer-assisted sequence analysis identified regions within the amino terminus of ICP10 that are homologous to the catalytic domain of known protein kinases (PKs). An in vitro kinase assay confirmed the ability of ICP10, immunoprecipitated from either HSV-2-infected cells or from cells transfected with an ICP10 expression vector, to autophosphorylate and transphosphorylate exogenous substrates in the presence of ATP and Mg2+. The HSV-1 RR1 was shown to be negative for PK activity under these conditions. PK activity was localized to a 57-kDa amino-terminal region within ICP10 that lacked RR activity.     

Replication of canine herpesvirus: I. Synthesis of viral deoxyribonucleic Acid

This paper reports the results of two series of experiments. The first series indicated that deoxyribonucleic acid (DNA) extracted from partially purified canine herpesvirus virions is characterized by a high guanine plus cytosine molar base ratio (65 to 67 mole%), similar to the DNA of herpes simplex virus. In the second series of experiments it was estimated, on the basis of uptake of tritiated thymidine, that in dog kidney cells canine herpesvirus-DNA synthesis starts at 4 hr and continues until 16 hr after infection. Treatment of infected cells with puromycin during the first 4 hr of infection blocks the onset of viral DNA synthesis, whereas, after this time the uptake of thymidine is unaffected.     

Inducible expression of phospholipid transfer protein (PLTP) in transgenic mice: acute effects of PLTP on lipoprotein metabolism

One main determinant in high-density lipoprotein (HDL) metabolism is phospholipid transfer protein (PLTP), a plasma protein that is associated with HDL. In transgenic mice overexpressing human PLTP we found that elevated plasma PLTP levels dose-dependently increased the susceptibility to diet-induced atherosclerosis. This could be mainly due to the fact that most functions of PLTP are potentially atherogenic, such as decreasing plasma HDL levels. To further elucidate the role of PLTP in lipoprotein metabolism and atherosclerosis we generated a novel transgenic mouse model that allows conditional expression of human PLTP. In this mouse model a human PLTP encoding sequence is controlled by a Tet-On system. Upon induction of PLTP expression, our mouse model showed a strongly increased PLTP activity (from 3.0 ± 0.6 to 11.4 ± 2.8 AU, p < 0.001). The increase in PLTP activity resulted in an acute decrease in plasma cholesterol of 33% and a comparable decrease in phospholipids. The decrease in total plasma cholesterol and phospholipids was caused by a 35% decrease in HDL-cholesterol level and a 41% decrease in HDL-phospholipid level. These results demonstrate the feasibility of our mouse model to induce an acute elevation of PLTP activity, which is easily reversible. As a direct consequence of an increase in PLTP activity, HDL-cholesterol and HDL-phospholipid levels strongly decrease. Using this mouse model, it will be possible to study the effects of acute elevation of PLTP activity on lipoprotein metabolism and pre-existing atherosclerosis.     

Endothelial cell plasma membrane obtained by chemically induced vesiculation

A method using low concentrations of formaldehyde and dithiothreitol was applied to obtain 'right-side out' luminal plasmalemma-derived vesicles from bovine aortic endothelial cells (EC) in culture, and from human umbilical vein and bovine or porcine aortas perfused ex vivo with the vesiculation solution. Vesicle formation and shedding were examined by phase-contrast microscopy and by transmission (TEM) and scanning electron microscopy (SEM). Vesicles showed the characteristic trilaminar pattern of the unit membrane and did not contain cellular organelles. As detected in freeze-fracture preparations, vesicle membrane displayed intramembrane particles and filipin-detectable cholesterol. Like EC plasmalemma, vesicle surface was heavily stained by Ruthenium Red and bound under a normal pattern cationized ferritin and ferritin hydrazide. As indicated by lectin agglutination assays and by ultrastructural cytochemistry, vesicles maintained on their ectodomains glycoconjugates bearing monosaccharides such as N-acetyl-neuraminic acid, beta-N-acetylglucosamine and beta-D-galactose, and expressed 5'-nucleotidase activity. The electrophoretic profiles of externally disposed 125I-labelled polypeptides of vesicles were found to be similar to those of intact EC. Chemically-induced vesiculation appears as a suitable method to obtain EC plasmalemma for studying its composition and functions in various vascular beds.