Two step continuous method to synthesize colloidal spheroid gold nanorods.

This research investigated a two-step continuous process to synthesize colloidal suspension of spheroid gold nanorods. In the first step; gold precursor was reduced to seed-like particles in the presence of polyvinylpyrrolidone and ascorbic acid. In continuous second step; silver nitrate and alkaline sodium hydroxide produced various shape and size Au nanoparticles. The shape was manipulated through weight ratio of ascorbic acid to silver nitrate by varying silver nitrate concentration. The specific weight ratio of 1.35-1.75 grew spheroid gold nanorods of aspect ratio ∼1.85 to ∼2.2. Lower weight ratio of 0.5-1.1 formed spherical nanoparticle. The alkaline medium increased the yield of gold nanorods and reduced reaction time at room temperature. The synthesized gold nanorods retained their shape and size in ethanol. The surface plasmon resonance was red shifted by ∼5 nm due to higher refractive index of ethanol than water.

A recurring pattern of chromosomal aberrations in mammary gland tumors of MMTV-cmyc transgenic mice.

Mice carrying the MMTV-cmyc transgene develop mammary tumors at 9 to 12 months of age. Little is known about karyotypic changes in this model of human breast cancer. We have developed and applied molecular cytogenetic techniques to study chromosomal aberrations that occur in these tumors, namely, comparative genomic hybridization and spectral karyotyping. Cell lines from eight tumors were established and analyzed, four of which carried a heterozygous p53 mutation. All of the tumor cell lines revealed increases in ploidy and/or multiple numerical and structural chromosomal aberrations. No consistent differences were observed between cmyc/p53+/+ and cmyc/p53+/- tumors, suggesting that cmyc induces karyotype instability independent of p53 status. Loss of whole chromosome (Chr) 4 was detected in five of the eight tumors. Parts of Chr 4 are syntenic to human 1p31-p36, a region that is also deleted in human breast carcinomas. Four tumors carried translocations involving the distal portion of Chr 11 (syntenic to human chromosome arm 17q), including two translocations T(X;11), with cytogenetically identical breakpoints. We compare the pattern of chromosomal aberrations with human breast cancers, find similarities in several syntenic regions, and discuss the potential of an interspecies cytogenetic map of chromosomal gains and losses.

Myc/p53 interactions in transgenic mouse mammary development, tumorigenesis and chromosomal instability.

We have examined defects in mammary development and tumorigenesis in a transgenic model expressing the c-myc gene under the MMTV-LTR promoter. The stochastic tumors which arise from hyperplastic ductal and lobular lesions in this model are characterized by high rates both of apoptosis and of chromosomal instability. Since the p53 gene product is thought to be central in the maintenance of genomic integrity, in part due to its ability to induce apoptosis in cells harboring DNA damage, we examined its expression and possible mutation. Initially, we observed that unmutated p53 is strongly expressed in premalignant mammary glands and in mammary tumors derived from the MMTV-c-myc strain. We then mated the MMTV-myc strain to a p53-deficient strain as a means of examining the effect of this lesion on mammary development and tumorigenesis in the context of c-myc overexpression. A lack of both p53 alleles in the presence of c-myc overexpression resulted in a dramatic hyerplastic alteration in mammary gland development. Specifically, in female bitransgenic MMTV-c-myc/p53 null mice (MMTV-myc/p53(-/-)), lobular hyperplasias were observed at almost every ductal end bud as early as 32 days of age. In contrast, only mild ductal and lobular hyperplasias were seen in MMTV-myc mice that contained both p53 alleles (MMTV-myc/p53(+/+)); an intermediate phenotype occurred in mice with a single intact (MMTV-myc/p53(+/-)) p53 allele. Mammary carcinomas arose with a high frequency in MMTV-myc/p53(+/-) mice; the tumors were comparable in frequency, histology and apoptotic index to the tumors in MMTV-myc/p53(+/+) mice. Also, as previously observed (Elson et al., 1995), lymphomas arose with extremely short latency in MMTV-myc/p53(-/-) mice, precluding study of the fate of their hyperplastic mammary lesions in situ. The frequency of p53 mutations in MMTV-myc/p53(+/+) and MMTV-myc/p53(+/-) mammary tumors and in cell lines derived from these tumors was examined by direct sequencing. No point mutations or deletions in p53 were observed in mammary tumors or cell lines from either genotype. Finally, a detailed chromosomal analysis using multicolor spectral karyotyping (SKY) revealed that there were multiple chromosomal alterations in the c-myc-overexpressing cells that contained either one or two unmutated p53 alleles. Variable ploidy changes, a common translocation of chromosome 11, and other chromosomal aberrations were observed. Our data thus support an interaction between c-Myc and p53 in mammary development, but suggest that loss of p53 is required neither for c-myc-dependent tumorigenesis nor for c-myc-dependent chromosomal instability.

Activation of the focal adhesion kinase signal transduction pathway in cervical carcinoma cell lines and human genital epithelial cells immortalized with human papillomavirus type 18.

The inappropriate activation of protein-tyrosine kinases (PTKs) has been associated with initiation and progression of several types of human cancers. We therefore postulated that immortalization by DNA tumor viruses results in the induction of PTKs fundamental to these processes. An RT-PCR-based screen was thus used to identify PTKs that were abundantly expressed in HPV-18-immortalized epithelial cells and HPV-containing carcinoma cell lines. One of the genes isolated in this screen was the focal adhesion kinase (FAK; pp125FAK), a cytoplasmic protein kinase that is activated in v-src transformed cells or by stimulation with mitogenic polypeptides. FAK also becomes catalytically active upon integrin engagement with extracellular matrix proteins, such as fibronectin. We found that FAK expression and activity were significantly elevated in HPV-18 E6/E7-immortalized human genital epithelial cells relative to their primary cell counterparts. Protein expression and tyrosine phosphorylation of the putative FAK substrate, paxillin, were also notably increased upon HPV-18 immortalization of genital epithelial cells and in HPV-containing cervical carcinoma cell lines. Most significantly, these cells expressed markedly higher levels of both intracellular and extracellular fibronectin, thus providing a mechanism for activation of FAK and increased tyrosine phosphorylation of paxillin. These findings suggest a role for the integrin/FAK-mediated signaling pathway in cervical carcinogenesis and represent one of the first demonstrations of a tyrosine kinase whose activity is elevated following viral immortalization.

Characterization of the heparin-mediated activation of PKR, the interferon-inducible RNA-dependent protein kinase.

Heparin can substitute for double-stranded (ds) RNA in the autophosphorylation and activation of the interferon-inducible, RNA-dependent elF-2 alpha protein kinase (PKR). We have used heparin oligosaccharides of defined lengths to examine the heparin-mediated activation of human PKR. Heparin oligosaccharide with 8 sugar residues was nearly as efficient as 16-residue heparin (Hep-16) in mediating the activation of PKR autophosphorylation, whereas 6-residue heparin was a poor activator. When examined in combination, Hep-16 and dsRNA did not act synergistically in activating PKR autophosphorylation. The RNA-binding activity of recombinant PKR, measured with adenovirus VA RNA, was competed by poly(rl):poly(rC) but not by Hep-16. When the catalytically inactive, histidine-tagged mutant PKR protein [His-PKR(K296R)] was examined as a substrate for purified wild-type PKR, the intermolecular phosphorylation of His-PKR(K296R) was efficiently catalyzed by dsRNA-activated PKR but not by heparin-activated PKR. However, elF-2 alpha phosphorylation was catalyzed by both heparin-and dsRNA-activated PKR. Preincubation of PKR with Hep-16 in the absence of ATP blocked subsequent autophosphorylation mediated either by Hep-16 or dsRNA, whereas preincubation with dsRNA either alone or in combination with Hep-16 did not impair subsequent autophosphorylation. Neither Hep-16 nor dsRNA caused a detectable degradation of PKR during preincubation or subsequent autophosphorylation of PKR. These results suggest that, while both dsRNA and heparin are capable of activating PKR autophosphorylation, the structural and functional basis of PKR activation differs for these two classes of polyanionic biomolecules.

Mechanism of interferon action. Biochemical and genetic evidence for the intermolecular association of the RNA-dependent protein kinase PKR from human cells.

The interferon-inducible protein kinase (PKR) is activated by an RNA-dependent autophosphorylation. Structure-function studies of the 551 amino acid PKR kinase from human cells have revealed that catalytic-deficient PKR mutants such as PKR(1-551)K296R display a dominant negative behavior when expressed in transfected cells. The potential for PKR to form protein multimers has therefore been examined. Three types of studies, including both genetic and biochemical analyses, demonstrated that PKR from human cells undergoes an intermolecular association that is not dependent upon RNA. First, the intermolecular association of PKR in vitro was demonstrated in the context of an enzyme-substrate interaction. Purified recombinant histidine-tagged PKR(1-551)K296R mutant protein was phosphorylated by purified wild-type PKR; this intermolecular phosphorylation of PKR was dependent on double-stranded RNA. At a fixed RNA concentration, high concentrations of the HIS-PKR(1-551)K296R mutant both impaired the autophosphorylation of wild-type PKR and blocked the trans-phosphorylation of itself. Second, the yeast two-hybrid system was used to probe the intermolecular association of PKR in vivo. Coexpression of the full-length catalytic-deficient phosphotransfer mutant PKR(1-551)K296R as a fusion protein with the Gal4 activation domain and the Gal4 DNA binding domain resulted in the expression of two Gal4-responsive reporter genes, HIS3 and lacZ. The full-length RNA-binding deficient PKR(1-551)K64E/K296R double mutant also interacted with PKR(1-551)K296R sufficiently to activate Gal4-responsive reporter genes; however, other PKR mutants including PKR(1-280)wt and PKR(281-551)K296R as well as p53, RAS, and BCL2 did not. Third, both PKR(1-551)K296R and PKR(1-551)K64E/K296R enhanced the expression of the reovirus S1 gene and S1/S4 chimeric gene in cotransfected COS cells. By contrast, the expression of the reovirus S4 gene was not enhanced by cotransfection with either PKR(1-551)K296R or PKR(1-551)K64E/K296R. These results indicate that PKR interacts with itself in an intermolecular manner both in vivo and in vitro, and that RNA binding is neither necessary nor sufficient for PKR multimerization.

Mechanism of interferon action: RNA-binding activity of full-length and R-domain forms of the RNA-dependent protein kinase PKR--determination of KD values for VAI and TAR RNAs.

The RNA-binding activity of the interferon-inducible, RNA-dependent protein kinase PKR, expressed from the human PKR cDNA, was quantitated using a gel mobility-shift assay. The N-terminal R-domain truncation Wt(1-243) and the full-length catalytic mutant K296R(21-551) were analyzed for their abilities to bind adenovirus VAI RNA, human immunodeficiency virus TAR RNA, and the synthetic homopolymer pI:pC RNA. The N-terminal 243 amino acid residue form of PKR [Wt(1-243)] bound VAI RNA with similar affinity as the 551 amino acid residue full-length catalytic mutant [K296R(1-551)]. The dissociation constant for VAI RNA was approximately 2 x 10(-9) M for both the K296R(1-551) and Wt(1-243) proteins. The K64E mutation significantly impaired the VAI RNA-binding activity as measured with the full-length double-point mutant PKR protein, K64E/K296R(1-551). Using a gel-shift competition assay, the dissociation constants of K296R(1-551) and Wt(1-243) for VAI(1-160) RNA and pI:pC RNA were comparable. By contrast, the dissociation constants of K296R(1-551) and Wt(1-243) for TAR(1-82) RNA were both about 1 x 10(-7) M. These results suggest that the RNA-binding affinity of PKR is approximately 100-fold lower for TAR RNA than for either VAI RNA or pI:pC RNA and that the full-length and N-terminal R-domain forms of PKR bind RNA with similar affinity.

Mechanism of interferon action. Translational control and the RNA-dependent protein kinase (PKR): antagonists of PKR enhance the translational activity of mRNAs that include a 161 nucleotide region from reovirus S1 mRNA.

The interferon-inducible, RNA-dependent protein kinase (PKR) is an important regulator of viral protein synthesis. Activated PKR inhibits protein synthesis by phosphorylating initiation factor eIF-2 alpha. The reovirus S4 gene, whose 1196 nucleotide mRNA transcript does not activate the PKR kinase, is efficiently expressed in vector-transfected monkey COS cells. By contrast, the 1463 nucleotide S1 gene of reovirus, which is a potent activator of PKR, is poorly expressed in COS cells. Virus genetic engineering was therefore used to examine the effect of the PKR activator sequence from the reovirus S1 gene on the expression of chimeric genes of reovirus in transfected COS cells. Chimeric S1/S4 and S4/S1/S4 reovirus constructions that included the PKR activator sequence from S1 in the sigma 3 ORF of S4 were expressed much less efficiently than wild-type S4. However, expression of sigma 3 from S4 (3'UTR/S1), which included the PKR activator sequence from S1 within the 3'-UTR of S4, was comparable to that from wild-type S4. Treatment of COS cells with 2-aminopurine, an inhibitor of PKR, increased the expression of the reovirus S1, S1/S4, and S4/S1/S4 chimeric genes but not the S4 gene or S4 (3'UTR/S1) chimera in transfected COS cells. Likewise, coexpression of the phosphotransfer-negative mutant PKR (K296R) increased the expression of reovirus S1, S1/S4 and S4/S1/S4 chimeric genes but not the S4 gene or S4 (3'UTR/S1) chimera in cotransfected COS cells. Truncated PKR(1-243) which includes the dsRNA binding domain but not the kinase catalytic subdomains was able to enhance the expression of reovirus S1, but did not affect S4 expression. The dsRNA binding protein E3L encoded by vaccinia virus also increased S1 expression similar to PKR (1-243) and PKR(K296R). These results suggest that the translational repression in vivo mediated by PKR is selective for mRNAs that possess the kinase activator region, and that the dominant negative effect of PKR on gene expression is likely mediated by the RNA binding activity of the PKR protein.

Mechanism of interferon action motif I of the interferon-induced, RNA-dependent protein kinase (PKR) is sufficient to mediate RNA-binding activity.

The interferon-induced P1/eIF-2 alpha protein kinase cDNA, designated PKR, was expressed both in Escherichia coli and in transfected monkey COS cells. TrpE-PKR fusion proteins and PKR nonfusion proteins were examined for their RNA-binding activity by Northwestern blot analysis. PKR is a RNA-binding protein that possesses two copies of a highly conserved motif, RI and RII, within the N-terminal region of the protein. Amino acid residues between 11 and 243 of PKR, which includes both copies of the R motif, displayed RNA-binding activity comparable to that of the full-length 551-amino-acid PKR protein. Analysis of substitution and deletion mutant PKR proteins revealed that motif RI was both necessary and sufficient for RNA-binding activity, whereas motif RII was not. Substitution of the highly conserved lysine at position 64 within the RI motif abolished RNA-binding activity, both of full-length PKR and the N-terminal 243-amino-acid truncated PKR. Finally, PKR substitution and deletion mutant cDNAs deficient for kinase function were expressed to much higher levels in transfected monkey cells than was the full-length wild-type PKR cDNA.

Mechanism of interferon action: identification of a RNA binding domain within the N-terminal region of the human RNA-dependent P1/eIF-2 alpha protein kinase.

A molecular cDNA clone of the human RNA-dependent P1/eIF-2 alpha protein kinase was expressed in Escherichia coli. Mutant P1 proteins were examined for RNA binding activity by Northwestern blot analysis using the reovirus s1 mRNA, an activator of the kinase; the adenovirus VAI RNA, an inhibitor of kinase activation; or human immunodeficiency virus (HIV) TAR RNA as probe. Analysis of TrpE-P1 deletion mutant fusion proteins revealed that the 11-kDa N-terminal region of the P1 protein bound reovirus s1 mRNA, adenovirus VAI RNA, and HIV TAR RNA. Neither s1 RNA, VAI RNA, nor TAR RNA was bound by truncated P1 proteins which lacked the N-terminal 98 amino acids. Computer analysis revealed that the human protein P1 sequence corresponding to amino acid residues within the N-terminal RNA binding domain displays high homology (greater than 54% identity; 61 to 94% similarity) with two animal virus proteins which possess RNA binding activity (vaccinia virus E3L; rotavirus VP2) and two proteins of unknown function (murine TIK; rotavirus NS34), but which are likely RNA binding proteins.