The human gene (RB) that determines susceptibility to hereditary retinoblastoma has been identified recently by molecular genetic techniques. Previous results indicate that complete inactivation of the RB gene is required for tumour formation. As a 'cancer suppressor' gene, RB thus functions in a manner opposite to that of most other oncogenes. Sequence analysis of RB complementary DNA clones demonstrated a long open reading frame encoding a hypothetical protein with features suggestive of a DNA-binding function. To further substantiate and identify the RB protein, we have prepared rabbit antisera against a trypE-RB fusion protein. The purified anti-RB IgG immunoprecipitates a protein doublet with apparent relative molecular mass (Mr) of 110,000-114,000. The specific protein(s) are present in all cell lines expressing normal RB mRNA, but are not detected in five retinoblastoma cell lines examined. The RB protein can be metabolically labelled with 32P-phosphoric acid, indicating that it is a phosphoprotein. Biochemical fractionation and immunofluorescence studies demonstrate that the majority of the protein is located within the nucleus. Furthermore, the protein can be retained by and eluted from DNA-cellulose columns, suggesting that it is associated with DNA binding activity. Taken together, these results imply that the RB gene product may function in regulating other genes within the cell.

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Abstract— Two easily prepared derivatives of chlorophyll,purpurin–18 and chlorin p6, are potent sensitizers of cell killing by low‐intensity red light. The internal anhydride group inpurpurin–18 provides the potential of covalently linking in one step the chlorin to cell targeting agents such as antibodies.

A gene in chromosome region 13q14 has been identified as the human retinoblastoma susceptibility (RB) gene on the basis of altered gene expression found in virtually all retinoblastomas. In order to further characterize the RB gene and its structural alterations, we examined genomic clones of the RB gene isolated from both a normal human genomic library and a library made from DNA of the retinoblastoma cell line Y79. First, a retiction and exon map of the RB gene was constructed by al overlapping genomic clones, yielding three contiguous regions ("contig') of 150 kilobases total length separated by two gaps. At least 20 exons were identified ip genomic clones, and these were provisionally numbered. Second, two overlapping genomic clones that demonstrated a DNA deletion of exons 2 through 6 from one RB allele were isolated from the Y79 library. To confirm and extend this result, a unique sequence probe from intron 1 was used to detect similar and possibly identical heterozygous deletions in genomic DNA from three retinoblastoma cell lines, thereby explaining the origs of their shortened RB mRNA htansripts. The same probe detecte genomic rearrangements in fibroblasts from two hereditary retinoblastoma patients, indicating that intron 1 includes a frequent site for mutations conferring predisposition to retinoblastona. Third, this probe also detete a polymorphic site for BamEil with allele frequencies near 0.5/0.5.Identification of commonly mutated regions will contribute significantly to genetic diagnosis in retinoblastoma patients and families.Retinoblastoma is an intraocular cancer of early childhood that arises from the developing retina. In a substantial proportion of cases, susceptibility to retinoblastoma can be inherited from a parent who was previously cured of the tumor (1,2 Recently, a gene in band 13q14 encoding an mRNA of 4.7 kilobases (kb) was identified as the RB gene based on tumor-specific alterations in expression and its apparent recessive nature at the cellular level (10). cDNA segments representing the RB gene transcript have been cloned (10-12) and sequenced (10). All hereditary and nonhereditary retinoblastomas examined to date have demonstrated altered RB gene expression: RB mRNA transcripts are either markedly reduced in quantity or are abnormal in length (10-12). About 40% of retinoblastomas show DNA deletions detectable by RB cDNA probes. Antibodies generated against the RB gene product, ppllORn, show complete absence of this nucleophosphoprotein in five out of five retinoblastomas, whereas it was easily detected in all normal or neoplastic cells containing a normal RB mRNA transcript (13). These data further strengthen the hypothesis that the absence of functional RB protein is potentially oncogenic.Given the different patterns of genetic alteration observed in retinoblastomas, it is evident that the RB gene is subject to a variety of mutational mechanisms, the details of which are as yet unknown. In order to further characterize the RB gene and some of its mutations, we construc...

Inflammation processes cause activation of phospholipase A in plasma membranes resulting in the production of various lysophospholipids. Treatment of mice with L-alpha-lysophosphatidyl-DL-glycerol (lyso-Pg) resulted in an enhanced ingestion activity of peritoneal macrophages as did other lysophospholipids. However, lyso-Pg is rather toxic as indicated by a rapid decrease in macrophage activity 3 days after treatment while macrophage activity of lysophosphatidylcholine-treated mice continued to increase at least up to the 6th day after treatment. Alkyl-lysophospholipid derivatives, racemic 1-0-octadecyl-2-methylglycero-3-phosphocholine and -phosphoethanolamine stimulated mouse macrophages for Fc-mediated ingestion. Decomposed products of alkyl-lysophospholipids, alkyl-glycerols, were also found to be excellent activators of macrophages not only for ingestion of IgG-coated target cells but also antibody-mediated tumoricidal activity. Macrophages from mice treated with alkylglycerols developed superoxide generating capacity. Furthermore, alkyl-glycerols were found to be tumoricidal by direct contact with retinoblastoma cells. Therefore, the advantage of the potential application of alkylglycerols as chemotherapeutic agents is that they have dual beneficial effects: potentiation of macrophage activity and cytotoxicity to malignant cells.

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1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.

Photoradiation therapy (PRT) against the Greene-Harvey amelanotic malignant melanoma on the rabbit iris was effectively used to cause tumor regression. A dose of 2.5 mg/kg of hematoporphyrin derivative (HPD) given intravenously, followed by photoradiation at a wavelength of 632 nm and a power density as low as 71 mW/cm2 for 24 minutes (102 J/cm2) was found to be lethal for tumors 4 mm in diameter with an acceptable level of reversible toxicity to the surrounding tissues. This was best accomplished with a dye laser as the source of light because of its very narrow expanding cone of light, as emitted from a fiber optic. A 1000 Watt xenon arc lamp was also effective but not as efficient. Because of this tumor's exceptionally rapid growth rate, it was necessary to compromise one important variable - the 3 to 4 day period between injection of HPD and photoradiation to allow for HPD depletion from normal tissues. Thus, the best tumor death responses were achieved when the light was given 1 to 16 hours after administering HPD. It is surmised that with this rapidly growing tumor, new cell progeny possess insufficient concentrations of HPD to be killed by the radiant energy. At such a short delay period, toxicity to normal tissues was observed mainly as conjunctivitis and conjunctival chemosis. A dose level of HPD at 5 mg/kg was very close to the threshold where minor increases in light intensity would cause strong inflammatory reactions. Higher doses, at 7.5 and 10 mg/kg were excessive. A dose of 23 mg/kg accompanied by mild light energy exposures, even after 30 days, caused massive damage to normal tissues.