Probing the oligomeric assemblies of pea porphobilinogen synthase by analytical ultracentrifugation

The enzyme porphobilinogen synthase (PBGS) can exist in different nonadditive homooligomeric assemblies, and under appropriate conditions, the distribution of these assemblies can respond to ligands such as metals or substrate. PBGS from most organisms was believed to be octameric until work on a rare allele of human PBGS revealed an alternate hexameric assembly, which is also available to the wild-type enzyme at elevated pH [Breinig, S., et al. (2003) Nat. Struct. Biol. 10, 757-763]. Herein, we establish that the distribution of pea PBGS quaternary structures also contains octamers and hexamers, using both sedimentation velocity and sedimentation equilibrium experiments. We report results in which the octamer dominates under purification conditions and discuss conditions that influence the octamer:hexamer ratio. As predicted by PBGS crystal structures from related organisms, in the absence of magnesium, the octameric assembly is significantly destabilized, and the oligomeric distribution is dominated largely by the hexameric assembly. Although the PBGS hexamer-to-octamer oligomeric rearrangement is well documented under some conditions, both assemblies are very stable (under AU conditions) in the time frame of our ultracentrifuge experiments.     

Late quaternary sediments,minerals, and inferred geochemical history of Didwana Lake,Thar Desert,India

Variations in clastic sediment texture, mineralogy of both evaporites formed at the surface and precipitates formed below the lake floor, and the relative chemical activities of the major dissolved components of the chemical precipitates, have allowed reconstruction of the history of salinity and water-level changes in Didwana Lake, Thar Desert, India. Hypersaline conditions prevailed at about the Last Glacial Maximum, with little evidence of clastic sediments entering the lake. Between ca. 13,000 and 6000 B.P. the lake level fluctuated widely, the lake alternately hypersaline and fresh, and clastic sediments were delivered to the lake at a low rate. Deep-water conditions occurred ca. 6000 B.P. and clastic influx increased abruptly. The water level dropped towards 4000 B.P. when the lake dried briefly. Since 4000 B.P. the lake has been ephemeral with a lowered rate of sedimentation and mildly saline conditions rather like those of today. This sequence of changes documented in the lake parallels changes in vegetation recorded in published pollen diagrams from both the Thar and the Arabian Sea. Correlation of the various lines of evidence suggests that the climate of the Last Glacial Maximum at Didwana was dry and windy with a weak monsson circulation. The monsson was re-established between ca. 13,000 and a little before 6000 B.P., and, when winter rainfall increased ca. 6000 B.P., the lake filled to its maximum depth.     

Blood pressure in a hypertensive mouse model of SLE is not salt-sensitive

Systemic lupus erythematosus (SLE) is a risk factor for hypertension. Previously, we demonstrated that an established mouse model of SLE (female NZBWF1 mice) develops hypertension with renal inflammation and oxidative stress, both characteristics known as contributing mechanisms to the development of salt-sensitive hypertension. On the basis of this model, we hypothesized that blood pressure in SLE mice would be salt-sensitive. Thirty-week-old female SLE and control mice (NZW/LacJ) were fed 8% high-salt (HS) diet or normal diet (0.4% salt) for 4 wk. Plasma levels of double-stranded DNA (dsDNA) autoantibodies, a marker of SLE disease activity, were increased in SLE mice compared with controls (472 ± 148 vs. 57 ± 17 U/ml × 1,000, P < 0.001). HS did not alter dsDNA autoantibody levels in SLE or control mice. Mean arterial pressure was increased in SLE mice compared with controls (132 ± 3 vs. 118 ± 2 mmHg, P < 0.001) and was not significantly altered by the HS diet in either group. Similarly, albuminuria was higher in SLE mice compared with controls (10.7 ± 9.0 vs. 0.3 ± 0.1 mg/day) but was not significantly increased in SLE or control mice fed a HS diet. In summary, blood pressure during SLE is not salt-sensitive, and the HS diet did not adversely affect SLE disease activity or significantly augment albuminuria. These data suggest that renal inflammation and oxidative stress, characteristics common to both SLE and models of salt-sensitive hypertension, may have diverging mechanistic roles in the development of hypertension.     

Molecular characterization of Encephalitozoon intestinalis (Microspora) replication kinetics in a murine intestinal cell line

Microsporidia are obligate intracellular pathogens of invertebrate and vertebrate animals. Most human infections are caused by Enterocytozoon bieneusi or Encephalitozoon intestinalis, and result in chronic diarrhea. In order to determine the signals involved in microsporidial spore activation and invasion, kinetics of in vitro E. intestinalis replication were defined using real-time quantitative PCR. Segments of small subunit ribosomal RNA and polar tube protein 2 genes of E. intestinalis were used to quantify parasite gene copy number following infection in murine colon carcinoma cells. Parasite DNA was detectable in small but significant amounts within host cells as early as 4 h postinoculation, genome replication was completed by 36 h, and parasite progeny were released into the supernatant beginning 72 h postinoculation. Heat-treating spores did not prevent transfer of parasite DNA into cells, but did inhibit parasite replication. Treating cell cultures with albendazole suppressed but did not completely inhibit parasite replication. These results confirm observations that E. intestinalis completes its life cycle within the turnover time of its target host cells; invasion into susceptible host cells occurs independently of spore viability; and real-time quantitative PCR is a sensitive and reproducible method with which to monitor microsporidial infection under varying treatments or conditions.     

Altered T cell surface glycosylation in HIV-1 infection results in increased susceptibility to galectin-1-induced cell death

The massive T cell death that occurs in HIV type 1 (HIV-1) infection contributes profoundly to the pathophysiology associated with AIDS. The mechanisms controlling cell death of both infected and uninfected T cells ("bystander" death) are not completely understood. We have shown that HIV-1 infection of T cells results in altered glycosylation of cell surface glycoproteins; specifically, it decreased sialylation and increased expression of core 2 O-glycans. Galectin-1 is an endogenous human lectin that recognizes these types of glycosylation changes and induces cell death of activated lymphocytes. Therefore we studied the possible contribution of galectin-1 in the pathophysiology of AIDS. O-glycan modifications were investigated on peripheral lymphocytes from AIDS patients. Oligosaccharides from CD43 and CD45 of CEM cells latently infected with HIV-1 were chemically analyzed. Consistent with our previous results, we show that HIV-1 infection results in accumulation of exposed lactosamine residues, oligosaccharides recognized by galectin-1 on cell surface glycoproteins. Both latently HIV-1-infected T cell lines and peripheral CD4 and CD8 T cells from AIDS patients exhibited exposed lactosamine residues and demonstrated marked susceptibility to galectin-1-induced cell death, in contrast to control cultures or cells from uninfected donors. The fraction of cells that died in response to galectin-1 exceeded the fraction of infected cells, indicating that death of uninfected cells occurred. Altered cell surface glycosylation of T cells during HIV-1 infection increases the susceptibility to galectin-1-induced cell death, and this death pathway can contribute to loss of both infected and uninfected T cells in AIDS.     

Characterization of human poly(ADP-ribose) polymerase with autoantibodies

The addition of poly(ADP-ribose) chains to nuclear proteins has been reported to affect DNA repair and DNA synthesis in mammalian cells. The enzyme that mediates this reaction, poly(ADP-ribose) polymerase, requires DNA for catalytic activity and is activated by DNA with strand breaks. Because the catalytic activity of poly(ADP-ribose) polymerase does not necessarily reflect enzyme quantity, little is known about the total cellular poly(ADP-ribose) polymerase content and the rate of its synthesis and degradation. In the present experiments, specific human autoantibodies to poly(ADP-ribose) polymerase and a sensitive immunoblotting technique were used to determine the cellular content of poly(ADP-ribose) polymerase in human lymphocytes. Resting peripheral blood lymphocytes contained 0.5 X 10(6) enzyme copies per cell. After stimulation of the cells by phytohemagglutinin, the poly(ADP-ribose) polymerase content increased before DNA synthesis. During balanced growth, the T lymphoblastoid cell line CEM contained approximately 2 X 10(6) poly(ADP-ribose) polymerase molecules per cell. This value did not vary by more than 2-fold during the cell growth cycle. Similarly, mRNA encoding poly(ADP-ribose) polymerase was detectable throughout S phase. Poly(ADP-ribose) polymerase turned over at a rate equivalent to the average of total cellular proteins. Neither the cellular content nor the turnover rate of poly(ADP-ribose) polymerase changed after the introduction of DNA strand breaks by gamma irradiation. These results show that in lymphoblasts poly(ADP-ribose) polymerase is an abundant nuclear protein that turns over relatively slowly and suggest that most of the enzyme may exist in a catalytically inactive state.