Visualization of a 21-nm axial periodicity in shadowed keratin filaments and neurofilaments.

Unidirectional and rotary shadowing techniques have been applied in studying the surface structure of two types of intermediate filaments. Keratin filaments and neurofilaments demonstrate a approximately 21-nm axial periodicity which probably indicates the helical pitch of the outer shell of the filament. Analysis of unidirectionally shadowed keratin showed that the helix is left-handed. The observation of a left-handed helix of 21-nm pitch supports the three-stranded protofilament model of Fraser, Macrae, and Suzuki (1976, J. Mol. Biol. 108:435-452), and indicates that keratin filaments probably consist of 10 three-stranded protofilaments surrounding a core of three such protofilaments, as predicted by models based on x-ray diffraction of hard keratin filaments. Neurofilaments do not demonstrate an easily identifiable hand, so their consistency with the model is, as yet, uncertain.

Genital Findings in Cases of Child Sexual Abuse: Genital vs Vaginal Penetration.

STUDY OBJECTIVE: To (1) examine the prevalence of abnormal genital findings in a large cohort of female children presenting with concerns of sexual abuse; and (2) explore how children use language when describing genital contact and genital anatomy. DESIGN: In this prospective study we documented medical histories and genital findings in all children who met inclusion criteria. Findings were categorized as normal, indeterminate, and diagnostic of trauma. Logistic regression analysis was used to determine the effects of key covariates on predicting diagnostic findings. Children older than 4 years of age were asked questions related to genital anatomy to assess their use of language. SETTING: A regional, university-affiliated sexual abuse clinic. PARTICIPANTS: Female children (N = 1500) aged from birth to 17 years (inclusive) who received an anogenital examination with digital images. INTERVENTIONS AND MAIN OUTCOME MEASURES: Physical exam findings, medical history, and the child's use of language were recorded. RESULTS: Physical findings were determined in 99% (n = 1491) of patients. Diagnostic findings were present in 7% (99 of 1491). After adjusting for age, acuity, and type of sexual contact reported by the adult, the estimated odds of diagnostic findings were 12.5 times higher for children reporting genital penetration compared with those who reported only contact (95% confidence interval, 3.46-45.34). Finally, children used the word "inside" to describe contact other than penetration of the vaginal canal (ie, labial penetration). CONCLUSION: A history of penetration by the child was the primary predictor of diagnostic findings. Interpretation of children's use of "inside" might explain the low prevalence of diagnostic findings and warrants further study.

Malignant transformation of an infinite life span human fibroblast cell strain by transfection with v-Ki-ras.

To determine if human fibroblasts can be transformed into malignant cells by transfection of a K-ras oncogene, we transfected the provirus of Kirsten murine sarcoma virus (v-Ki-ras) into an infinite life span human cell strain, MSU-1.1, which has a normal morphology, is not anchorage independent, and has a stable, near-diploid karyotype. The transfected populations gave rise to distinct foci composed of morphologically-altered cells. The cells from several independent foci were isolated, propagated, and assayed for anchorage independence and/or tumorigenicity. They formed large-sized colonies in soft agar at a high frequency. Cell strains derived from colonies isolated from agar as well as focus-derived cell strains were injected subcutaneously into athymic mice to test for tumorigenicity. One cell strain yielded myxoid fibromas, the rest produced well-differentiated, progressively-growing, invasive, myxoid or spindle cell sarcomas. The karyotype of each of the cell strains tested, including cell strains derived from tumors, was identical to that of non-transfected MSU-1.1 cells. Two focus-derived strains, and two cell strains derived from sarcomas produced from them, were tested and shown by DNA and RNA hybridization to contain and express the v-Ki-ras oncogene. Radioimmunoprecipitation analysis showed that these strains expressed ras-specific p21 products not found in non-transfected MSU.1.1 cells. When injected intraperitoneally, a cell strain derived from a myxoid tumor gave rise to invasive myxoid tumors at various sites in the body. The same cell strain gave rise to invasive spindle cell sarcomas when injected into the tail vein of the animals.

Electron microscopy of rotary shadowed vinculin and vinculin complexes.

Chicken gizzard smooth muscle vinculin, purified according to the method of Feramisco & Burridge (1980), was examined by rotary shadowing and electron microscopy. Individual vinculin molecules have two domains: a globular head with a diameter of 8.0 nm, and a tail 20 nm long. In high salt, vinculin self-associates into multimers containing two to six individual molecules. These molecules associate head to head and tail to tail, but the tail to tail association appears to be favored. Electron microscopy of the approximately 100,000 Mr major fragment of vinculin was performed. The tail region appeared to be cleaved off, making the head region less compact.

Malignant transformation of human fibroblasts by ionizing radiation.

As one step in developing an assay for quantifying the induction of malignant transformation of human cells by ionizing radiation, we exposed cells from a non-tumorigenic, infinite life span, near-diploid fibroblast strain MSU-1.1 to 4.35 Gy 60Co radiation and assayed them for focus formation. The mean frequency of foci in the irradiated population was 6 x 10(-7) cells assayed. No foci were found in the control cells. Of four focus-derived cell strains studied in detail, two produced malignant tumours within 3-7 weeks. The other two did not produce tumours during the 12-month period of study. The tumours from one strain were classified as sarcomas composed exclusively of spindle-shaped cells. Tumours from the other strain were sarcomas consisting of a mixed population of round and spindle cells. Immunoprecipitation analysis of the status of the p53 gene in the focus-derived strains, using a mutant-specific anti-body (Pab240) and an antibody that recognizes both mutant and wild-type p53 protein (Pab421), showed that the tumorigenic strains were completely devoid of p53 protein. One non-tumorigenic strain expressed wild-type p53 protein, and the other expressed a lower molecular weight form of the protein. Karyotypic analysis showed that the tumour-derived cells from one tumorigenic strain had lost one copy of chromosome 6, 14, 16 and 17. The tumour-derived cells from the second strain had lost one copy of chromosome 7, 13, 14 and 17 and part of chromosome 6, as well as part of the other copy of chromosome 7 and 17. These results suggest that the common loss of one copy of chromosome 14, 17 and part of 6 plays a causal role in the malignant transformation of these cells. Furthermore, the results indicate that it will be possible to develop a system that uses near-diploid human fibroblasts to quantify radiation-induced malignant transformation.

A structural comparison of tryptic fragments of three types of intermediate filaments.

We have compared tryptic fragments of three types of intermediate filaments, emphasizing structural characteristics as seen in the electron microscope. Variable, long alpha-helical rod fragments were found to be similar for keratin, neurofilaments and desmin filaments. Short rod fragments from keratin and neurofilaments appeared similar when observed by electron microscopy. Short rod fragments were not seen in desmin filament digests. In addition to these elongated particles, globular fragments, which have not been described previously, were obtained from all three types of intermediate filaments. These globular fragments were characterized by gel filtration and electron microscopy, and compared to globular proteins of known size using both methods. The diameter was about 6 nm and the molecular weight was estimated to be 50 000-60 000. These globular particles may comprise the short, nonhelical regions from several IF protein subunits, which are clustered into an interface in the intact filament or protofilaments.

Structural characteristics of the desmin protofilament.

Biochemical investigations of intermediate filaments in soluble or partially assembled forms are often difficult to perform due to the unusual insolubility of most types of intermediate filaments. However, desmin is soluble in 10 mM Tris. The structure of partially soluble native desmin was studied by gel-filtration chromatography and electron microscopy. The lowest molecular weight species of soluble desmin is a flexible rod averaging 53 nm in length. Calculations of f/fmin values from a previously published sedimentation value allowed comparisons with other elongated proteins. These values and the dimensions obtained from electron microscopy suggest that the desmin protofilament contains three or four protein subunits.

Transformation of diploid human fibroblasts by DNA transfection with the v-sis oncogene.

The simian sarcoma virus (SSV) oncogene (v-sis) has a high degree of homology to the cellular gene coding for the B peptide of human platelet-derived growth factor (PDGF), a potent fibroblast mitogen. The cellular homolog of v-sis is activated in some mesenchymal human tumors and cell lines derived from them. To determine the phenotype produced by v-sis in diploid human fibroblasts, we constructed plasmids containing the SSV provirus and drug-resistance markers and transfected them into early-passage human cells. Fibroblasts that had integrated the plasmid were selected for drug resistance and shown to contain and express the v-sis oncogene by DNA and RNA hybridization. The v-sis-expressing cells grew to higher saturation densities than control cells transfected with the vector plasmid alone and formed large, well defined foci. This allowed selection of transfectants directly for focus formation. The v-sis transformed cells continued to grow well in the absence of serum, whereas age-matched, vector-transfected control cells ceased replicating under these conditions so that the final difference in density between the two populations was tenfold. Incorporation of thymidine in serum-free medium by the v-sis-transformed cells was independent of exogenous PDGF. In contrast, PDGF increased thymidine incorporation in such medium by the control cells to the level found in the v-sis-transformed cells with or without added PDGF. These results suggest that expression of the v-sis oncogene in diploid human fibroblasts causes sufficient endogenous synthesis of the B chain of PDGF to allow transformants to grow to abnormally high cell densities. When individual v-sis-transformed cells were grown on a background of normal cells, this higher cell density at confluence could be visualized as a focus.

A major determinant of hnRNP C protein binding to RNA is a novel bZIP-like RNA binding domain.

The C protein tetramer of hnRNP complexes binds approximately 150-230 nt of RNA with high cooperativity (McAfee J et al., 1996, Biochemistry 35:1212-1222). Three contiguously bound tetramers fold 700-nt lengths of RNA into a 19S triangular intermediate that nucleates 40S hnRNP assembly in vitro (Huang M et al., 1994, Mol Cell Biol 14:518-533). Although it has been assumed that the consensus RNA recognition motif (RRM) of C protein (residues 8-87) is the primary determinant of RNA binding, we report here that a recombinant construct containing residues 1-115 has very low affinity for RNA at physiological ionic strength (100 mM NaCl). Moreover, we demonstrate that an N-terminal deletion construct lacking the consensus RRM but containing residues 140-290 binds RNA with an affinity sufficient to account for the total free energy change observed for the binding of intact protein. Like native C protein, the 140-290 construct is a tetramer in solution and binds RNA stoichiometrically in a salt-resistant manner in 100-300 mM NaCl. Residues 140-179 of the N-terminal truncated variant contain 11 basic and 2 acidic residues, whereas residues 180-207 specify a leucine zipper motif that directs dimer assembly. Elements within the 50-residue carboxy terminus of C protein are required for tetramer assembly. A basic region followed by a leucine zipper is identical to the domain organization of the basic-leucine zipper (bZIP) class of DNA binding proteins. Sequence homologies with other proteins containing RRMs and the bZIP motif suggest that residues 140-207 represent a conserved bZIP-like RNA binding motif (designated bZLM). The steric orientation of four high-affinity RNA binding sites about rigid leucine zipper domains may explain in part C protein's asymmetry, its large occluded site size, and its RNA folding activity.

The heterogeneous nuclear ribonucleoprotein C protein tetramer binds U1, U2, and U6 snRNAs through its high affinity RNA binding domain (the bZIP-like motif).

Based on UV cross-linking experiments, it has been reported that the C protein tetramer of 40 S heterogeneous nuclear ribonucleoprotein complexes specifically interacts with stem-loop I of U2 small nuclear RNA (snRNA) (Temsamani, J., and Pederson, T. (1996) J. Biol. Chem. 271, 24922-24926), that C protein disrupts U4:U6 snRNA complexes (Forne, T., Rossi, F., Labourier, E., Antoine, E., Cathala, G., Brunel, C., and Tazi, J. (1995) J. Biol. Chem. 270, 16476-16481), that U6 snRNA may modulate C protein phosphorylation (Mayrand, S. H., Fung, P. A., and Pederson, T. (1996) Mol. Cell. Biol. 16, 1241-1246), and that hyperphosphorylated C protein lacks pre-mRNA binding activity. These findings suggest that snRNA-C protein interactions may function to recruit snRNA to, or displace C protein from, splice junctions. In this study, both equilibrium and non-equilibrium RNA binding assays reveal that purified native C protein binds U1, U2, and U6 snRNA with significant affinity ( approximately 7.5-50 nM) although nonspecifically. Competition binding assays reveal that U2 snRNA (the highest affinity snRNA substrate) is ineffective in C protein displacement from branch-point/splice junctions or as a competitor of C protein's self-cooperative RNA binding mode. Additionally, C protein binds snRNA through its high affinity bZLM and mutations in the RNA recognition motif at suggested RNA binding sites primarily affect protein oligomerization.

Oligonucleotide binding specificities of the hnRNP C protein tetramer.

Through the use of various non-equilibrium RNA binding techniques, the C protein tetramer of mammalian40S hnRNP particles has been characterized previously as a poly(U) binding protein with specificity for the pyrimidine-rich sequences that often precede 3' intron-exon junctions. C protein has also been characterized as a sequence-independent RNA chaperonin that is distributed along nascent transcripts through cooperative binding and as a protein ruler that defines the length of RNA packaged in 40S monoparticles. In this study fluorescence spectroscopy was used to monitor C protein-oligonucleotide binding in a competition binding assay under equilibrium conditions. Twenty nucleotide substrates corresponding to polypyrimidine tracts from IVS1 of the adenovirus-2 major late transcript, the adenovirus-2 oncoprotein E1A 3' splice site, IVS2 of human alpha-tropomyosin, the consensus polypyrimidine tract for U2AF65, AUUUA repeats and r(U)20were used as competitors. A 20 nt beta-globin intronic sequence and a randomly generated oligo were used as competitor controls. These studies reveal that native C protein possesses no enhanced affinity for uridine-rich oligonucleotides, but they confirm the enhanced affinity of C protein for an oligonucleotide identified as a high affinity substrate through selection and amplification. Evidence that the affinity of C protein for the winner sequence is due primarily to its unique structure or to a unique context is seen in its retained substrate affinity when contiguous uridines are replaced with contiguous guanosines.