LeRoy Walters was at the center of public debate about emerging biological technologies, even as “biotechnology” began to take root. He chaired advisory panels on human gene therapy, the human genome project, and patenting DNA for the congressional Office of Technology Assessment. He chaired the subcommittee on Human Gene Therapy for NIH’s Recombinant DNA Advisory Committee. He was also a regular advisor to Congress, the executive branch, and academics concerned about policy governing emerging biotechnologies. In large part due to Prof. Walters, the Kennedy Institute of Ethics was one of the primary sources of talent in bioethics, including staff who populated policy and science agencies dealing with reproductive and genetic technologies, such as NIH and OTA. His legacy lies not only in his writings, but in those people, documents, and discussions that guided biotechnology policy in the United States for three decades.

The only causative treatment for IgE-mediated allergies is allergen-specific immunotherapy. However, fewer than 5% of allergy patients receive immunotherapy because of its long duration and risk of allergic side effects. We aimed at enhancing s.c. immunotherapy by direct administration of allergen into s.c. lymph nodes. The objective was to evaluate safety and efficacy compared with conventional s.c. immunotherapy. In a monocentric open-label trial, 165 patients with grass pollen-induced rhinoconjunctivitis were randomized to receive either 54 s.c. injections with pollen extract over 3 years [cumulative allergen dose 4,031,540 standardized quality units (SQ-U)] or 3 intralymphatic injections over 2 months (cumulative allergen dose 3,000 SQ-U). Patients were evaluated after 4 months, 1 year, and 3 years by nasal provocation, skin prick testing, IgE measurements, and symptom scores. Three low-dose intralymphatic allergen administrations increased tolerance to nasal provocation with pollen already within 4 months ( P < 0.001). Tolerance was long lasting and equivalent to that achievable after standard s.c. immunotherapy ( P = 0.291 after 3 years). Intralymphatic immunotherapy ameliorated hay fever symptoms ( P < 0.001), reduced skin prick test reactivity ( P < 0.001), decreased specific serum IgE ( P < 0.001), caused fewer adverse events than s.c. immunotherapy ( P = 0.001), enhanced compliance ( P < 0.001), and was less painful than venous puncture ( P = 0.018). In conclusion, intralymphatic allergen administration enhanced safety and efficacy of immunotherapy and reduced treatment time from 3 years to 8 weeks.

A survey of technology transfer of DNA inventions at 19 top US research universities reveals consensus, diversity and flexibility in intellectual property management. Patent filing and license terms are influenced by intended uses of inventions, outside market interest and NIH guidelines.

Genomic technologies are best defined as technologies used to manipulate and analyze genomic information. The evolution of this collective power began in earnest with the invention of DNA cloning in the 1970's and most of the technology derives from the last quarter of the 20th century. The historical impact of these technologies is clearly immense. With the genome sequence becoming available for many organisms, including humans, another new view of biology has recently emerged. This review examines the shape and texture of this recent evolution, with a particular emphasis on new technology: DNA cloning, macromolecular structure analysis (X-ray crystallography and NMR), DNA sequencing, DNA synthesis, amplification by the polymerase chain reaction, and transgenic animals (bacteria through mammals).

The chapters in this book reflect the range of technical advances and their applications that are currently transforming biology. Topics covered include the application of computational methods, the robotics of modern automation methodologies, the new chemical and imaging methods that reveal chromosome structure and elucidate gene function, and the emergence of evolutionary and functional information by comparative genomics. Eminent international scientists describe in detail the new technology necessary to study the entire genome in a holistic manner and all the high throughput and large-scale experimental methodologies currently being used in genomic science. In addition the authors describe the progress of the newest technologies that are currently being developed. Written by experts in the field, this concise yet informative volume covers all aspects of technology pertaining to genomic studies. An essential book for anyone involved in genomic science. Readership: bioscientists, molecular biologists, biotechnologists, geneticists, human, animal and plant geneticists, microbiologists, genome scientists. An essential book for anyone involved in genomic science.

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.

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 (), 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.

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; pp125(FAK)), 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.

Heparin can substitute for double-stranded (ds) RNA in the autophosphorylation and activation of the interferon-inducible, RNA-dependent eIF-2α 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 adenovirusVARNA, was competed by poly(rI):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, eIF-2α 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.