DNA binding properties of a 110 kDa nucleolar protein.

A single strand specific DNA binding protein was purified to homogeneity from calf thymus nucleoprotein. The monomeric protein is elongated in shape and has a molecular mass of 110 kDa. Since immunocytochemistry revealed that the protein is predominantly located in the nucleolus we refer to it as the 110 kDa nucleolar protein. The protein binds not only to single stranded DNA but also to single stranded RNA, including homopolymeric synthetic RNA. We have used the single stranded DNA binding properties of the 110 kDa protein in model studies to investigate its effects on the configuration of nucleic acid. Our results are: only 50-55 protein molecules are sufficient to saturate all binding sites on the 6408 nucleotides of phage fd DNA; protein binding cause a compaction of single stranded DNA; large nucleoprotein aggregates are formed in the presence of divalent cations; this is due to protein-protein interactions which occur at moderately high concentrations of magnesium-, calcium or manganese ions; the protein induces the reassociation of complementary nucleic acid sequences. We speculate that the 110 kDa protein performs similar reactions in vivo and may have a function related to the processing and packaging of preribosomal RNA.     

Generation and neutralization of pseudovirions of human papillomavirus type 33.

Since human papillomaviruses (HPV) cannot be propagated in cell culture, the generation of infectious virions in vitro is a highly desirable goal. Here we report that pseudovirions can be generated by the assembly of virus-like particles (VLPs) in COS-7 cells containing multiple copies of a marker plasmid. Using recombinant vaccinia viruses, we have obtained spherical VLPs of HPV type 33 (HPV-33) which fractionate into heavy and light VLPs in cesium chloride density gradients. VLPs in the heavy fraction (1.31 g/cm3) carry the plasmid in DNase-resistant form and are capable of transferring the genetic marker located on the plasmid to COS-7 cells in a DNase-resistant way (pseudoinfection). The minor capsid protein L2 is not required for encapsidation but is essential for efficient pseudoinfection. Antiserum to HPV-33 VLPs inhibits VLP-mediated DNA transfer with high efficiency. Antisera to VLPs of HPV-18 and HPV-16 are not neutralizing, although the HPV-16 antiserum exhibited some cross-reactivity with HPV-33 VLPs in an enzyme-linked immunosorbent assay. In a cell binding assay, the titer of the HPV-33 VLP antiserum was 1:200 compared to the neutralization titer of 1:10(5). This indicates that neutralization is essentially due to the inhibition of cellular processes after VLP binding to cells. The encapsidation of marker plasmids into VLPs provides a sensitive and fast assay for the evaluation of neutralizing potentials of antisera against papillomavirus infections.     

Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.     

Alterations in osteoclast morphology following osteoprotegerin administration in the magnesium-deficient mouse

In the present study, we used osteoprotegerin (OPG), which blocks osteoclastogenesis, to correct and thus explain the hypercalcemia that is seen during dietary Mg deficiency in the mouse. Control and Mg-deficient mice received injections for 12 days of either OPG or vehicle only. Serum Ca was similar in Mg-deficient mice treated with OPG and in control mice receiving OPG (9.2±0.3 mg/dl vs. 9.2±0.5). Both groups had significantly higher serum Ca than controls or Mg-deficient animals receiving vehicle alone. Surprisingly, Mg-depleted mice that received OPG in doses that inhibit osteoclastic bone resorption remained hypercalcemic. Because mature osteoclasts still present in the marrow might be hyperactive, we examined osteoclast morphology at the light microscopic and ultrastructural level. Light microscopic examination of trabecular bone showed few osteoclasts in OPG-treated mice. Ultrastructural examination revealed that osteoclasts in OPG-treated mice have decreased contact with the endosteal bone surface and absence of a ruffled border. Because the morphology of the existing pool of mature osteoclasts did not enhance resorption, another mechanism, such as increased intestinal absorption of Ca in Mg-deficient mice, likely contributes to the hypercalcemia observed during Mg deficiency.     

Methylation: a regulator of HIV-1 replication?

Pakistan, the second most populous Muslim nation in the world, has started to finally experience and confront the HIV/AIDS epidemic. The country had been relatively safe from any indigenous HIV cases for around two decades, with most of the infections being attributable to deported HIV positive migrants from the Gulf States. However, the virus finally seems to have found a home-base, as evidenced by the recent HIV outbreaks among the injection drug user community. Extremely high-risk behavior has also been documented among Hijras (sex workers) and long-distance truck drivers. The weak government response coupled with the extremely distressing social demographics of this South-Asian republic also helps to compound the problem. The time is ripe now to prepare in advance, to take the appropriate measures to curtail further spread of the disease. If this opportunity is not utilized right now, little if at all could be done later.     

Human papillomavirus infection requires cell surface heparan sulfate

Using pseudoinfection of cell lines, we demonstrate that cell surface heparan sulfate is required for infection by human papillomavirus type 16 (HPV-16) and HPV-33 pseudovirions. Pseudoinfection was inhibited by heparin but not dermatan or chondroitin sulfate, reduced by reducing the level of surface sulfation, and abolished by heparinase treatment. Carboxy-terminally deleted HPV-33 virus-like particles still bound efficiently to heparin. The kinetics of postattachment neutralization by antiserum or heparin indicated that pseudovirions were shifted on the cell surface from a heparin-sensitive into a heparin-resistant mode of binding, possibly involving a secondary receptor. Alpha-6 integrin is not a receptor for HPV-33 pseudoinfection.     

Viral contamination of embryos cryopreserved in liquid nitrogen

Despite the worldwide application of embryo-freezing technology as the means of preserving germplasm of mammalian species, there is no information available on the possible transmission of infectious agents to cryopreserved embryos via contaminated liquid nitrogen (LN). Recently, it has been reported that new methods of cryopreservation which employ ultrarapid freezing or vitrification require direct contact between the freezing medium containing oocytes or embryos and liquid phase nitrogen (LPN). As models for human and animal viral pathogens three bovine viruses, bovine viral diarrhea virus (BVDV), bovine herpesvirus-1 (BHV), and bovine immunodeficiency virus (BIV), were employed to study the potential for their transmission by experimentally contaminated LN to embryos frozen and stored in open freezing containers. Bovine embryos in a mixture of 20% ethylene glycol, 20% ME(2)SO, and 0.6% sucrose were vitrified in either unsealed standard 0.25 ml or modified open pulled straws or in plastic cryovials and then plunged into contaminated LPN. After 3-5 weeks of storage in LN, embryos were thawed and sequentially washed and only those with intact ZP were pooled together and tested in batches of three for viral contamination. From this pool of 83 batches, 13 of 61 (21.3%) batches exposed to BVDV and BHV-1 tested positive for viral association while all 22 batches exposed to BIV in unsealed containers tested negative. All control embryos vitrified in sealed cryovials and straws were free from viral contamination.