O-mannosylation of the Mycobacterium tuberculosis Adhesin Apa Is Crucial for T Cell Antigenicity during Infection but Is Expendable for Protection

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.     

Effect of Guanine Nucleotides on [3H]Glutamate Binding and on Adenylate Cyclase Activity in Rat Brain Membranes

GMP-PNP, a non-hydrolyzable analog of GTP binds tightly to G-protein in the presence of Mg²⁺, so that the binding is stable even after exhaustive washings. This property was exploited to prepare membrane samples of rat brain where G-protein GTP-binding sites were saturated with GMP-PNP. Experiments carried out with these membranes showed that GTP, GMP-PNP, GDP-S and GMP (1 mM) inhibit the sodium-independent [³H]glutamate binding by 30–40% [F(4,40) = 5.9; p < .001], whereas only GMP-PNP activates adenylate cyclase activity [F(6,42) = 3.56; p < .01]. The inhibition of sodium-independent [³H]glutamate binding occurred in the absence of Mg²⁺. These findings suggest that guanine nucleotides may inhibit glutamate binding and activate adenylate cyclase through distinct mechanisms by acting on different sites.     

The GABAA receptor complex in the developing chick optic tectum: characterization of benzodiazepine and ionophore-linked convulsant/barbiturate recognition sites

By use of membrane preparations and incubation conditions optimized for each binding site, we have characterized the benzodiazepine and ionophore-linked-convulsant/barbiturate modulatory sites within the chick tectal GABA_A receptor complex. Using [³H]flunitrazepam (FNZ) and [³⁵S]t-butylbicyclophosphorothionate (TBPS), respectively, as specific radioligand probes for the two sites, we have found in each case one single population of high-affinity, saturable, specific binding sites. The apparent dissociation constants (K_d) show no change during tectal development (9 nM for [³H]FNZ, and 25–28 nM for [³⁵S]TBPS) while the respective densities of binding sites at saturation (B_max) experience in both cases a twofold increase between embryonic day 16 and postnatal day 10. Ligand-specific pharmacological profiles and allosteric interactions between the transmitter and modulatory sites appear to be well preserved in the chick tectal membrane preparations employed in this study.     

Mitochondrial and nuclear genetic variation across calving lagoons in Eastern North Pacific gray whales (Eschrichtius robustus)

Accurate knowledge of population structure in cetaceans is critical for preserving and managing breeding habitat, particularly when habitat is not uniformly protected. Most eastern gray whales return to their major breeding range each winter along the Pacific coast of Baja California, Mexico, concentrating in 3 major calving lagoons, but it is unknown whether genetic differences exist between lagoons. Previous photo-identification studies and genetic studies suggest that gray whales may return to their natal lagoons to breed, potentially resulting in the buildup of genetic differences. However, an earlier genetic study used only one genetic marker and did not include samples from Bahia Magdalena, a major calving lagoon not currently designated as a wildlife refuge. To expand on this previous study, we collected genetic data from the mitochondrial control region (442 bp) and 9 microsatellite markers from 112 individuals across all 3 major calving lagoons. Our data suggest that migration rates between calving lagoons are high but that a small but significant departure from panmixia exists between Bahia Magdalena and Laguna San Ignacio (Fisher's Exact test, P < 0.0001; F(ST) = 0.006, P = 0.025). Coalescent simulations show that the lack of extensive population structure may result from the disruption of structure due to whaling. Another possibility is that rates of migration have always been high (>10% per generation). In addition, microsatellite data showed evidence of a severe population bottleneck. Eastern gray whales are still recovering from the impacts of whaling on their breeding grounds, and these populations should be protected and monitored for future genetic changes.     

Blunting of circadian rhythms and increased acrophase variability in sleep-time hypertensive subjects

24 h and ultradian rhythms of blood pressure (BP) have been previously shown to be disorganized in nocturnal hypertensive subjects. The present study was undertaken to further analyze the ultradian and circadian BP rhythm structure in sleep-time hypertensive subjects with normal or elevated awake-time BP levels. Fourier analysis was used to fit 24, 12, 8, and 6 h curves to mean BP as well as heart rate (HR) time series data derived from 24 h ambulatory blood pressure monitoring. Awake and sleep periods were defined according to individual sleep diaries. Awake-time hypertension was defined as diurnal systolic (SBP) and/or diastolic BP (DBP) means ≥135/85 mmHg. Sleep-time hypertension was defined as nocturnal SBP and/or DBP means ≥120/70 mmHg. The sample included 240 awake-time normotensive subjects (180 sleep-time normotensives and 60 sleep-time hypertensives) and 138 untreated awake-time hypertensive subjects (31 sleep-time normotensives and 107 sleep-time hypertensives). The amplitude and integrity (i.e., percent rhythm) of the 24 and 12 h BP rhythms were lower in the sleep-time hypertensive subjects and higher in the awake-time hypertensive subjects. However, no differences were detected when the integrity and amplitude of the 6 and 8 h mean BP rhythms were analyzed. The sleep-time hypertensive group showed significantly higher 24 h BP rhythm acrophase variability. No differences could be found in any of the HR rhythm parameters. Altogether, the findings suggest a disorganization of the BP circadian rhythm in sleep-time hypertensives that results in reduced 24 h rhythm amplitude and integrity that could be related to cardiovascular risk.