Analysis of simian immunodeficiency virus sequence variation in tissues of rhesus macaques with simian AIDS.

One rhesus macaque displayed severe encephalomyelitis and another displayed severe enterocolitis following infection with molecularly cloned simian immunodeficiency virus (SIV) strain SIVmac239. Little or no free anti-SIV antibody developed in these two macaques, and they died relatively quickly (4 to 6 months) after infection. Manifestation of the tissue-specific disease in these macaques was associated with the emergence of variants with high replicative capacity for macrophages and primary infection of tissue macrophages. The nature of sequence variation in the central region (vif, vpr, and vpx), the env gene, and the nef long terminal repeat (LTR) region in brain, colon, and other tissues was examined to see whether specific genetic changes were associated with SIV replication in brain or gut. Sequence analysis revealed strong conservation of the intergenic central region, nef, and the LTR. However, analysis of env sequences in these two macaques and one other revealed significant, interesting patterns of sequence variation. (i) Changes in env that were found previously to contribute to the replicative ability of SIVmac for macrophages in culture were present in the tissues of these animals. (ii) The greatest variability was located in the regions between V1 and V2 and from "V3" through C3 in gp120, which are different in location from the variable regions observed previously in animals with strong antibody responses and long-term persistent infection. (iii) The predominant sequence change of D-->N at position 385 in C3 is most surprising, since this change in both SIV and human immunodeficiency virus type 1 has been associated with dramatically diminished affinity for CD4 and replication in vitro. (iv) The nature of sequence changes at some positions (146, 178, 345, 385, and "V3") suggests that viral replication in brain and gut may be facilitated by specific sequence changes in env in addition to those that impart a general ability to replicate well in macrophages. These results demonstrate that complex selective pressures, including immune responses and varying cell and tissue specificity, can influence the nature of sequence changes in env.     

Establishment of a human anaplastic thyroid cancer cell line secreting granulocyte colony-stimulating factor in response to cytokines

Summary A human anaplastic thyroid cancer cell line K-119, derived from a 77-yr-old woman who had developed marked neutrophilia and underwent surgery for anaplastic thyroid cancer, has been established. The spindlelike and polygonal cells in shape are stably proliferating since the beginning of its culture 2 yr ago. The cells grow rapidly and the population doubling time is 26 h. The chromosomes show many abnormalities and many marker chromosomes have been observed. Heterotransplantation of the cells into nude mice has resulted in the formation of tumors that are histologically interpreted as anaplastic cancer. The most noteworthy characteristics of the cell line are the many Ki-67-positive cells (86.3%) and that the cell line spontaneously secretes granulocyte colony-stimulating factor (G-CSF) and releases increased amounts of G-CSF in response to the stimulation of tumor necrosis factor, interleukin 1α, and interleukin 1β. The conditioned medium obtained from K-119 cells contains an autocrine factor stimulating the proliferation of themselves.     

The Effect of Unheated Bovine Serum on the Complement-Fixing Activity of Heat-Inactivated Bovine Antiserum with Homologous Antigen. IV. Chromatographic Studies

Unheated, non-dialyzed, normal bovine sera were fractionated by column chromatography on the cross-linked dextran, Sephadex G-25, and the fraction tested for "supplementing" properties, that is for complement-fixation augmenting activities when added to mixtures of heated bovine antiserum and homologous antigen. Supplementing activity was shown by precipitated fractions from earlier eluates with pH values below 7.2 and also by both supernatant and precipitated fractions of the later eluates with pH values from 7.6 to 8.1. The possibility is briefly discussed that certain alkaline protein substances of relatively lower molecular weight may be involved in the supplementing activities of the later fractions. Heating at 56 degrees C. for 30 min. destroyed the supplementing activity of each of these fractions. Some of the supplementing fractions proved to be anti-complementary, others were not or only slightly so. First component of complement, C(1)1, was detected in the precipitated fractions of certain of the earlier eluates with pH values below 6.5; second component of complement, C(1)2, was found exclusively in supernatant fractions of earlier eluates with pH values less than 6.2. Conglutinin was not separated from C(1)1 by this method.     

Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product.

We attempted to clone an inosine kinase gene of Escherichia coli. A mutant strain which grows slowly with inosine as the sole purine source was used as a host for cloning. A cloned 2.8-kbp DNA fragment can accelerate the growth of the mutant with inosine. The fragment was sequenced, and one protein of 434 amino acids long was found. This protein was overexpressed. The overexpressed protein was purified and characterized. The enzyme had both inosine and guanosine kinase activity. The Vmaxs for guanosine and inosine were 2.9 and 4.9 mumol/min/mg of protein, respectively. The Kms for guanosine and inosine were 6.1 microM and 2.1 mM, respectively. This enzyme accepted ATP and dATP as a phosphate donor but not p-nitrophenyl phosphate. These results show clearly that this enzyme is not a phosphotransferase but a guanosine kinase having low (Vmax/Km) activity with inosine. The sequence of the gene we have cloned is almost identical to that of the gsk gene (K.W. Harlow, P. Nygaard, and B. Hove-Jensen, J. Bacteriol. 177:2236-2240, 1995).     

Post-ischemic administration of the acetylcholinesterase inhibitor ENA-713 prevents delayed neuronal death in the gerbil hippocampus

We examined by morphological methodology the effect of (S)-N-ethyl-3-[(1-dimethyl-amino)ethyl]-N-methyl-phenylcarbamate hydrogentartrate (ENA-713), an acetylcholinesterase (AChE) inhibitor, on ischemia-induced neuronal death in the gerbil hippocampus due to a 5-min ligation of bilateral common carotid arteries after light ether anesthesia. Pyramidal cells had been decreased to 27% of sham-operated controls and the number of hypertrophic astrocytes expressing glial fibrillary acidic protein (GFAP) markedly increased in the hippocampal CA1 subfield 14 days after ischemia. However, post-ischemic administration of ENA-713 (three times 0.2 mg/kg, i.p.) significantly ameliorated this ischemia-induced decrease in the number of pyramidal cells by 47% of sham-operated controls, furthermore, it reduced the ischemia-induced accumulation of GFAP-positive astrocyte in the CA1 region. Together with previous results showing that ENA-713 protected against the ischemia-induced cholinergic abnormalities in the gerbil brain and improved cholinergic dysfunctions in the senescent rat brain, our present findings suggest that ENA-713 prove to be useful for treatment with senile dementia such as cerebrovascular dementia.