World-wide surveillance of influenza.

Influenza epidemics and pandemics resulting in excess mortality are due to various influenza viruses, in which through the accumulation of mutations the structure changes. A world-wide surveillance has been set up for early detection of new influenza virus strains and of epidemics or pandemics resulting thereof. Basing on such data the World Health Organisation (WHO) issues recommendations to Public Health authorities on the most efficient means for prevention.

Immunocytochemical localization of cytoskeletal proteins and histone 2B in isolated membrane-depleted nuclei, metaphase chromatin, and whole Chinese hamster ovary cells.

Immunocytochemical techniques employing protein A-gold labeling were used to locate actin, tubulin, and histone 2B in thin sections of embedded, isolated membrane-depleted nuclei, metaphase chromosomes, and whole Chinese hamster ovary (CHO) cells. Actin and tubulin were detected in significant amounts in the cytoplasm and the nucleus of whole cells. The isolated membrane-depleted nuclei and metaphase chromosomes showed levels of actin and tubulin labeling either comparable to or sometimes lower than the levels of labeling in whole interphase cells. The results suggest that actin and tubulin are normal components of nuclei throughout the cell cycle. A mouse monoclonal antibody to histone 2B gave relatively weak labeling of nuclei and chromosomes in whole cells but intense labeling of isolated nucleoids and chromosomes. An increased concentration of antibody at the periphery of interphase nucleoids was noted.

Comparative studies on the structural organization of membrane-depleted nuclei and metaphase chromosomes.

Interphase membrane-depleted nuclei and metaphase chromosomes were prepared in parallel with a nonionic detergent lysis procedure at low ionic strength. By flow microfluorometry we showed for the first time that cell lysates contain all stages of the cell cycle in the same proportions as the starting cell population. Morphologically intact membrane-depleted nuclei and metaphase chromosomes were isolated as non-aggregated structures on sucrose gradients. When analysed in the electron microscope, membrane-depleted nuclei that had been treated with 2M NaCl appeared as residual structures containing the pore complex-lamina layer attached to a halo of DNA filaments. In contrast, no distinct high salt-resistant structure was found with metaphase chromosomes. They formed a highly fragile network which disintegrated easily into small complexes connected with DNA filaments. High salt-resistant DNA-protein complexes were purified by Metrizamide density gradient centrifugation. The main difference in the protein composition of interphase and metaphase residual complexes was the presence in interphase of a protein triplet in the 60-75 kilodalton molecular weight range and its absence in metaphase. This protein triplet most likely corresponds to the lamins A, B, and C of the nuclear lamina. The combined results suggest that the main difference in the structural organization of interphase nuclei and metaphase chromosomes is the presence or absence of the pore complex-lamina layer.

Involvement of higher order chromatin structures in metaphase chromosome organization.

Using electron microscopy we show that the metaphase chromatin fibers of Chinese hamster ovary cells form the same ionic strength-dependent higher order structures as the corresponding interphase chromatin fibers. We present evidence that such intact chromatin fibers are a prerequisite for the maintenance of the characteristic shape of metaphase chromosomes. The evidence is based on the finding that treatment of chromosomes with 0.5 M NaCl, a condition which is known to remove histone H1 and which destroys the higher order structure of chromatin fibers, also leads to a disintegration of the metaphase chromosome structure, whereas treatment with 0.3 M (or less) NaCl has no effect on the integrity of the chromosomes and their chromatin fibers. These data support a model in which the metaphase chromosome is maintained by a tight assembly of the 25-30 nm thick chromatin fibers containing all the histones.

In vitro studies of the fixation of DNA, nucleoprotamine, nucleohistone and proteins.

In view of improving fixation and embedding for thin sections of nuclear material we investigated the crosslinking of selected proteins, nucleic acid and nucleoproteins by observing the eventual gelation of solutions of these substances, which might be produced by current cytological fixatives. For those combinations where gelation occurs, we give thresholds of minimum concentrations required. We discuss the consequences of these findings for the preservation of thin sectioned nuclear structures within cells.

C-type virus particles in human urogenital tumours after heterotransplantation into nude mice.

C-type viruses were formed in heterotransplants of 5/14 human urogenital tumours which had been serially transferred in nude mice of NIH(S) background. Except for one case in which C-type particles were present in the epithelial cells as well as the connective tissue, the viruses were only found within the stroma of the heterotransplanted tumours. Peroxidase labelling with anti-mouse serum demonstrated that the connective tissue supporting the transplanted human tumours was of mouse origin. Competition radioimmunoassays demonstrated that MuLV interspecies viral protein was present in high titre in the transplanted tumour extracts and also in extracts of 2 spontaneous mouse-tumour extracts. These data suggest that endogenous viruses of the nude mice are activated by the graft, and only subsequently infect the human tumour cells and form particles.

Isolation of a human prostate carcinoma cell line (DU 145).

A long-term tissue culture cell line has been derived from a human prostate adenocarcinoma metastatic to the brain. The cell line, DU 145, has been passaged 90 times in vitro over a period of 2 years. The cells are epithelial, grow in isolated islands on plastic Petri dishes, and form colonies in soft agar suspension culture. Karyotypic analysis demonstrates an aneuploid human karyotype with a modal chromosome number of 64. Distinctive marker chromosomes (a translocation Y chromosome, metacentric minute chromosomes and three large acrocentic chromosomes) have been identified. Electron microscopy of the original tumor tissue and of the tissue culture cell line show a remarkable similarity in cell organelle structure.

Heterotransplantation of a human prostatic adenocarcinoma cell line in nude mice.

Nude mice of NIH/Swiss background were utilized for the heterotransplantation of a tissue culture cell line derived from a human prostate adenocarcinoma metastatic to the brain. These cells, which had been grown in vitro for 13 passages, formed solid tumors when injected s.c. into nude mice. The cell line DU 145 has been passaged 60 times in vitro over a period of 18 months. Tumors removed from the mice were serially transplanted to additional mice and reestablished in vitro. Light-microscopic analysis of the tumor grown in nude mice revealed a strong similarity to the patient's metastatic tumor. The ultrastructure of the tumor cells propagated in nude mice was compared to that of the original human tumor cells and to the tissue culture cells, both before and after passage in nude mouse. No major differences were detected. Karyotypic analysis of the tumor cells grown in vitro before mouse passage, grown in nude mouse, and grown in vitro after mouse passage indicated chromosomal identity and consistent marker chromosomes: three large acrocentric chromosomes and metacentric minute chromosomes.

Development and application of basic research techniques in bladder cancer research.

The growth of transitional epithelial cells with different growth media and growth supports was examined. Sephadex G-10, Bio-Gel P-20, Bio-Glas-1000, DEAE-Sephadex A-50, DEAE-cellulose, CM-Sephadex C-50, acid-soluble collagen, and immobilized collagen fibers were used to enhance plating efficiency. Acid-soluble collagen layers optimally increased the plating efficiency of primary cultures of bladder carcinoma. Media alterations with serial combinations of fetal calf, newborn calf, calf, bovine, and bull serum with minimum essential medium, Roswell Park Memorial Institute Tissue Culture Medium 1640, Connaught Medical Research Laboratories Medium 1066, Medium 199, Grand Island Biological, National Cancer Tissue Culture 135, 1415, McCoy's 5A, and National Cancer Institute medium were established. No promotion of cell division was noted with any one of these basic medium formulations.

Studies related to the head-maturation pathway of bacteriophages T4 and T2:I. morphology and kinetics of intracellular particles produced by mutants in the maturation genes.

Mutants in the genes governing the maturation of the head of bacteriophage T4 and in gene 24 were studied by electron microscopy of thin sections. We define morphologically: black particles, comprising mature, stable heads and immature, fragile heads, which break down upon lysis; grizzled particles, which apparently are partially filled or partially emptied; empty large particles without DNA or core which are all the same size as normal heads; empty small particles without DNA and without core which are of the size of the tau particle, which is the prehead of phage T4. The study of single and double mutants of the maturation genes demonstrates that the phenotypes are only different by the proportions of the different particles made except for 17- where only empty small and empty large particles accumulate. The mutants in gene 24 are epistatic on all other mutants. Mutants in gene 17 are epistatic on the remaining ones. The results are consistent with the hypothesis that the products of several of the maturation genes act on DNA to render it competent for packaging while the others act directly on the particle. By this uncoupling, bypasses and abortive pathways can result.

Studies related to the head-maturation pathway of bacteriophages T4 And T2:II. nuclear disruption, protein synthesis and particle formation with the mutant 43-.30-.46-.

We describe the aberrant phage multiplication of the triple conditional lethal mutant 43-(polymerase).30-(ligase).46-(exonuclease) of bacteriophage T4D in which phage DNA replication is arrested but some late protein synthesis occurs (33). The nuclear disruption is indistinguishable from wild type. Forty-five empty small and empty large particles are assembled per cell when the multiplicity of infection (m.o.i.) is 100. This number corresponds closely to the 38 phage equivalents of cleaved major head protein determined biochemically. By reducing the m.o.i. the number of observable particles decreases, reaching 1-5 per cell at an m.o.i. of 5(+5). The total synthesis of phage related proteins is not significantly dependant on the m.o.i. The synthesis of late proteins is about 10% of that of wild type at high m.o.i. and decreases with the m.o.i. The different early and late proteins do not show the same relative proportions as in wild type and respond differently to an increased m.o.i. These and other results are discussed with respect to the role of phage DNA in prehead assembly and head maturation.