Analysis of the structure and expression of the c-myc oncogene in cervical tumor and in cervical tumor-derived cell lines

The structure of the c-myc oncogene in 17 cervical tumors and patient-matched nontumor tissues from Chinese patients residing in Taiwan was analysed. In contrast to recent reports on Mexican patients, none of the samples showed rearrangements and sequence amplification in the c-myc gene. The discrepancy may be explained by different carcinogenesis mechanisms being in operation in different geographic regions. Although no structural alterations in the c-myc gene were found in seven cervical carcinoma cell lines analysed, Northern blot analysis indicated different levels of c-myc gene expression which may be related to the presence of human papillomavirus (HPV) sequence in the cell and suggests a possible c-myc-hpv interaction in some stages of the transformation process.     

High-Throughput Nonlinear Optical Microscopy

High-resolution microscopy methods based on different nonlinear optical (NLO) contrast mechanisms are finding numerous applications in biology and medicine. While the basic implementations of these microscopy methods are relatively mature, an important direction of continuing technological innovation lies in improving the throughput of these systems. Throughput improvement is expected to be important for studying fast kinetic processes, for enabling clinical diagnosis and treatment, and for extending the field of image informatics. This review will provide an overview of the fundamental limitations on NLO microscopy throughput. We will further cover several important classes of high-throughput NLO microscope designs with discussions on their strengths and weaknesses and their key biomedical applications. Finally, this review will close with a perspective of potential future technological improvements in this field.     

Physiologic characterization of transformed and cloned rat granulosa cells

Properties of a clonal line of SV40-transformed rat granulosa cells (DC3 cells) were elucidated. DC3 cells were maintained in vitro in Iscove Modified Dulbecco Medium that contained 20% fetal bovine serum. The cells had a logarithmic growth phase doubling time of approximately 18 h and produced detectable quantities of estrone, estradiol, and progesterone. Steroidogenesis was increased by supplementation with either steroidogenic substrates or agents that stimulated activity of adenylate cyclase. Production of progesterone and estrogens was enhanced when medium was supplemented with 25-hydroxycholesterol, and production of estradiol was enhanced by medium supplementation with androstenedione. Treatments with forskolin and cholera toxin resulted in marked increases of cyclic adenosine 3',5'-monophosphate (cAMP) in medium and cells and enhanced steroidogenesis. Isoproterenol and vasoactive intestinal peptide, but not follicle-stimulating hormone (FSH), luteinizing hormone (LH), insulin or prolactin, stimulated cAMP secretion by suspended cells. DC3 cells had small but detectable levels of binding to FSH, but binding of LH and epidermal growth factor could not be detected. DC3 cells possess characteristics expected of granulosa cells arrested in an early stage of differentiation and may provide a useful model for studies of "immature" granulosa cell functions.     

The malmö polymorphism of coagulation factor IX, an immunologic polymorphism due to dimorphism of residue 148 that is in linkage disequilibrium with two other F.IX polymorphisms

A mouse monoclonal antibody (MAB 9.9) to coagulation factor IX (F.IX) detects a polymorphism in the plasma of normal people. Its epitope has been narrowed down to <6 amino acids in the activation peptide of the X-linked F.IX protein. The activation peptide contains a dimorphism—Thr:Ala—at position 148 of the protein. Using synthetic oligonucleotides, we have demonstrated that (1) the F.IX which reacts with 9.9 has Thr at position 148 and (2) that which does not has Ala. Positive reactors (148^thr) are designated Malmö A, and negative reactors (148^ala) are designated Malmö B. The plasma levels of AA women are indistinguishable from those of A men, and both B men and BB women are null against MAB 9.9. The plasma level of Malmö A in AB women is approximately half that of AA women, and “lyonization” is clearly operating in the heterozygotes. The dimorphism is in strong linkage disequilibrium with two other intragenic RFLPs, TaqI and XmnI. Furthermore, intragenic crossing-over—including double crossing-over—appears to have occurred between the three sites. Seven of the eight possible haplotypes have been identified, five in men and two others in women. The immunoassay that identifies ～50% of the AB women in the pool of Malmö A females with 95% confidence identifies men unambiguously as A or B. The assay would be very useful for population-genetic studies of the Malmö epitope if the studies were limited to men.     

Prophylactic immunization against experimental leishmaniasis. IV. Subcutaneous immunization prevents the induction of protective immunity against fatal Leishmania major infection

Durable immunity against fatal L. major infection in genetically susceptible mice can be induced by immunization with 150,000-rad irradiated or heat-killed promastigotes administered i.v. or to a lesser extent i.p. Conversely, subcutaneous (s.c.) and intramuscular (i.m.) injections are not only totally ineffective but generally increase susceptibility to and enhance the progression of the disease, leading to earlier mortality. This detrimental effect is particularly evident with lower infecting challenge doses. Disease exacerbation is apparent in mice given 4 X s.c. injections of as few as 2 X 10(4) irradiated promastigotes, but it appears most potent after doses of 2 X 10(7). When mice given 4 X s.c. injections were subsequently immunized i.v. with 2 X 10(7) irradiated promastigotes, they failed to develop any evidence of protection against infection with 2 X 10(5) promastigotes, whereas mice given i.v. immunization alone were strongly protected. Thus, s.c. injections are capable of blocking the prophylactic effect of i.v. immunization with irradiated parasites. This inhibitory effect can be achieved with a single s.c. injection, although rather less potently than with four, and is even effective against four repeated weekly i.v. immunizations. Once induced, the effect persists undiminished after 100 days. A weaker effect is also inducible by s.c. injection given after i.v. immunization. The blocking effect of s.c. injection is not dependent on continuing viability of the promastigotes, as it can be induced equally readily with heat-killed, formalin-fixed, or sonicated parasites. The phenomenon extends to mouse strains genetically resistant as well as susceptible to L. major infection and, in congenic mice of BALB background, is independent of the major histocompatibility (H-2) gene complex.     

Prophylactic immunization against experimental leishmaniasis. V. Mechanism of the anti-protective blocking effect induced by subcutaneous immunization against Leishmania major infection

The responsiveness of BALB/c mice to protective i.v. immunization with 150,000-rad irradiated or heat-killed Leishmania major promastigotes can be totally suppressed by prior subcutaneous (s.c.) injection of the same "vaccine." Induction of this effect is leishmania specific for although prevention of protection against L. major infection can be obtained with either homologous or Leishmania donovani promastigotes, it does not follow s.c. administration of an immunogenic Trypanosoma cruzi epimastigote preparation. Multiple s.c. injections of irradiated L. major promastigotes do not inhibit the subsequent antibody response of any major isotype to i.v. immunization, but rather induce some priming. The same s.c. injections induced delayed-type hypersensitivity (DTH) reactivity that could be transferred locally or systemically, although it was weaker than in mice with cured infections. Parallel cell-mediated immunity (CMI) responses were also reflected in vitro in specific lymphocyte transformation assays. Despite this evidence of a DTH/helper type of T cell response, transfer of 5 X 10(7) viable T cell-enriched spleen cells from 4 X s.c. immunized donors to normal recipients completely abrogated the protective response to i.v. immunization. Conversely, T cell-depleted (anti-Thy-1.2 + C treated) cells were without effect. The inhibitory T cells were defined by monoclonal antibody pretreatment as possessing an Lyt-1+2-,L3T4+ phenotype. T cells from s.c. immunized donors were also shown, by mixed transfer experiments, to counteract completely the protective effect of T cells from i.v. immunized donors in 550-rad irradiated recipients. They were as potent as suppressor T cells from donors with progressive disease both in this capacity and in abrogating the prophylactic effect of sublethal irradiation itself. The similarities and differences between suppressor and immune effector T cells induced by s.c. or i.v. immunization and those arising in response to leishmanial infection itself are discussed.     

Isolation of nuclear acidic proteins from rat tissues. Characterization of acetylated liver nuclear acidic proteins

Nuclear acidic proteins isolated from rat brain, heart, kidney and liver showed similar, complex patterns on electrophoresis in sodium dodecyl sulphate-polyacrylamide gels. The contamination of nuclear acidic proteins by nuclear-membrane acidic proteins was found to the extent of 11%. Incorporation of [(3)H]acetate into the various nuclear acidic proteins in vivo, which were fractionated by polyacrylamide-gel electrophoresis, differed from tissue to tissue. Hydrolysis of these acetylated nuclear acidic proteins with 6m-HCl at 110 degrees C released 70% of the radioactivity, which indicated that labile acetyl groups had been incorporated into these proteins. Analysis of [(3)H]acetate-labelled nuclear acidic proteins revealed two acetylated amino acid residues, N(2)-acetylserine and N(2)-acetyl-lysine. The significance of the role played by nuclear acidic proteins in relation to gene regulation is discussed.     

Tumor necrosis factor-alpha synergizes with IFN-gamma in mediating killing of Leishmania major through the induction of nitric oxide

CBA mice develop cutaneous lesions when infected with Leishmania major. The disease development was significantly reduced by injecting into the lesion a combination of rIFN-gamma and rTNF-alpha. The doses of IFN-gamma and TNF-alpha used were suboptimal in that either cytokine alone did not have any effect. The therapeutic effect of IFN-gamma and TNF-alpha in vivo is reflected in their ability to activate macrophages to kill the intracellular parasites in vitro. The macrophage leishmanicidal activity induced by TNF-alpha and IFN-gamma can be completely inhibited by a specific inhibitor (L-NG monomethyl arginine) of nitric oxide synthesis. There was a direct correlation between the intracellular killing of the parasites and the production of nitric oxide by the macrophages. In contrast, there was no correlation between leishmanicidal activity and superoxide production by macrophages.