Cleavage of the (1 goes to 3)-2-acetamido-2-deoxy-beta-D-glucopyranosyl linkage present in keratan sulfate. The A and B isoenzymes of human liver hexosaminidase (EC 3.2.1.30)

The disaccharide 2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 goes to 3)-D-[1-3H]-galactitol, prepared from keratan sulfate, was rapidly hydrolyzed by the A and B isoenzymes of normal human liver hexosaminidase (EC 3.2.1.30), and by the B isoenzyme prepared from the liver of a patient who had died of Tay-Sachs disease. The disaccharide substrate was also hydrolyzed by extracts of normal, cultured-skin fibroblasts, and fibroblasts of patients with Tay-Sachs disease, whereas it was not hydrolyzed by fibroblast extracts of patients with Sandhoff disease. Thus, effective degradation of keratan sulfate, secondary to a defect of the beta subunits present in the A and B isoenzymes of hexosaminidase, may contribute to the appearance of skeletal lesions in patients affected by Sandhoff disease.     

Analysis of side-chain organization on a refined model of charybdotoxin: structural and functional implications.

The spatial organization of side chains on a refined model of charybdotoxin is presented. First, the structural role of two groups of well-defined, low-accessible side chains (Thr3, Val5, Val16, Leu20, Cys33 and Leu20, His21, Thr23, Cys17, Cys35) is discussed. These side chains are conserved in three out of the five known scorpion toxins acting on K+ channels. Interestingly, they are not conserved in scyllatoxin which presents a slightly different secondary structure organization. Second, the spatial organization of all positively charged residues is analyzed. Comparison with the results presented by Park and Miller [(1992) Biochemistry (preceding paper in this issue)] shows that all functionally important positive residues are located on the beta-sheet side of the toxin. These results are different from those obtained by Auguste et al. [(1992) Biochemistry 31, 648-654] on scyllatoxin, which blocks a different type of K+ channel. This study shows, in fact, that functionally important positive residues are located on the helix side of the toxin. Thus, charybdotoxin and scyllatoxin, which present the same global fold, interact with two different classes of K+ channels by two different parts of the motif.     

Human ancestry indentification under resource constraints -- what can one chromosome tell us about human biogeographical ancestry?

Background

While continental level ancestry is relatively simple using genomic information, distinguishing between individuals from closely associated sub-populations (e.g., from the same continent) is still a difficult challenge.

Methods

We study the problem of predicting human biogeographical ancestry from genomic data under resource constraints. In particular, we focus on the case where the analysis is constrained to using single nucleotide polymorphisms (SNPs) from just one chromosome. We propose methods to construct such ancestry informative SNP panels using correlation-based and outlier-based methods.

Results

We accessed the performance of the proposed SNP panels derived from just one chromosome, using data from the 1000 Genome Project, Phase 3. For continental-level ancestry classification, we achieved an overall classification rate of 96.75% using 206 single nucleotide polymorphisms (SNPs). For sub-population level ancestry prediction, we achieved an average pairwise binary classification rates as follows: subpopulations in Europe: 76.6% (58 SNPs); Africa: 87.02% (87 SNPs); East Asia: 73.30% (68 SNPs); South Asia: 81.14% (75 SNPs); America: 85.85% (68 SNPs).

Conclusion

Our results demonstrate that one single chromosome (in particular, Chromosome 1), if carefully analyzed, could hold enough information for accurate prediction of human biogeographical ancestry. This has significant implications in terms of the computational resources required for analysis of ancestry, and in the applications of such analyses, such as in studies of genetic diseases, forensics, and soft biometrics.

     

Disentangling subpopulations in single-molecule FRET and ALEX experiments with photon distribution analysis

Among the advantages of the single-molecule approach when used to study biomolecular structural dynamics and interaction is its ability to distinguish between and independently observe minor subpopulations. In a single-molecule Förster resonance energy transfer (FRET) and alternating laser excitation diffusion experiment, the various populations are apparent in the resultant histograms. However, because histograms are calculated based on the per-burst mean FRET and stoichiometry ratio and not on the internal photon distribution, much of the acquired information is lost, thereby reducing the capabilities of the method. Here we suggest what to our knowledge is a novel statistical analysis tool that significantly enhances these capabilities, and we use it to identify and isolate static and dynamic subpopulations. Based on a kernel density estimator and a proper photon distribution analysis, for each individual burst, we calculate scores that reflect properties of interest. Specifically, we determine the FRET efficiency and brightness ratio distributions and use them to reveal 1), the underlying structure of a two-state DNA-hairpin and a DNA hairpin that is bound to DNA origami; 2), a minor doubly labeled dsDNA subpopulation concealed in a larger singly labeled dsDNA; and 3), functioning DNA origami motors concealed within a larger subpopulation of defective motors. Altogether, these findings demonstrate the usefulness of the proposed approach. The method was developed and tested using simulations, its rationality is described, and a computer algorithm is provided.     

Inflammasome activation via intracellular NLRs triggered by bacterial infection

Members of the nucleotide-binding, oligomerization domain (NOD)-like receptor (NLR) proteins assemble into a multiprotein platform, known as the inflammasome, to induce caspase-1 activation followed by the subsequent secretion of IL-1β and IL-18. In this review, we focus on the role of NLRs in inflammasome activation as part of the host defence against bacterial pathogens. One of activators of the NLRC4 inflammasome is bacterial flagellin secreted through type III or IV secretion systems, which are important for the pathogenicity of many Gram-negative bacteria. The NLRP3 inflammasome is mainly activated by a large number of bacterial pore-forming toxins. Despite our knowledge of inflammasome activation upon bacterial infection, the function of antibacterial defence under in vivo conditions remains to be elucidated. Further understanding of NLR function should provide new insights into the mechanisms of host pro-inflammatory responses and the pathogenesis of bacterial infections.     

Ischemic preconditioning phosphorylates mitogen-activated kinases and heat shock protein 27 in the diabetic rat heart

Diabetes mellitus blocks protection by ischemic preconditioning (IPC), but the mechanism is not known. We investigated the effect of ischemic preconditioning on mitogen-activated protein kinases (extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinases, p38 mitogen-activated kinase) and heat shock protein 27 phosphorylation in diabetic and nondiabetic rat hearts in vivo. Two groups of anaesthetized nondiabetic and diabetic rats underwent a preconditioning protocol (3 cycles of 3 min coronary artery occlusion and 5 min of reperfusion). Two further groups served as untreated controls. Hearts were excised for protein measurements by Western blot. Four additional groups underwent 25 min of coronary occlusion followed by 2 h of reperfusion to induce myocardial infarction. In these animals, infarct size was measured. IPC reduced infarct size in the nondiabetic rats but not in the diabetic animals. In diabetic rats, IPC induced phosphorylation of the mitogen-activated protein kinases and of heat shock protein 27. We conclude that protection by IPC is blocked by diabetes mellitus in the rat heart in vivo without affecting phosphorylation of mitogen-activated protein kinases or heat shock protein 27. Therefore, the blockade mechanism of diabetes mellitus is downstream of mitogen-activated kinases and heat shock protein 27.     

Vibrio parahaemolyticus Effector Proteins Suppress Inflammasome Activation by Interfering with Host Autophagy Signaling

Bacterial pathogens utilize pore-forming toxins or sophisticated secretion systems to establish infection in hosts. Recognition of these toxins or secretion system by nucleotide-binding oligomerization domain leucine-rich repeat proteins (NLRs) triggers the assembly of inflammasomes, the multiprotein complexes necessary for caspase-1 activation and the maturation of inflammatory cytokines such as IL-1β or IL-18. Here we demonstrate that both the NLRP3 and NLRC4 inflammasomes are activated by thermostable direct hemolysins (TDHs) and type III secretion system 1 (T3SS1) in response to V. parahaemolyticus infection. Furthermore, we identify T3SS1 secreted effector proteins, VopQ and VopS, which induce autophagy and the inactivation of Cdc42, respectively, to prevent mainly NLRC4 inflammasome activation. VopQ and VopS interfere with the assembly of specks in infected macrophages. These data suggest that bacterial effectors interfere with inflammasome activation and contribute to bacterial evasion from the host inflammatory responses.     

The effect of tosyl lysine chloromethyl ketone on the activity of uridine diphosphoglucose pyrophosphorylase of the cellular slime mold Dictyostelium discoideum

We report here that the specific activity of UDPG pyrophosphorylase in extracts of $\text{D. discoideum}$ amoebae and preculmination-stage cells increases as a function of the length of their exposure to tosyl lysine chloromethyl ketone, an irreversible inhibitor of a number of serine and sulfhydryl proteases. This compound also stabilizes the activity of the enzyme in crude extracts of amoebae. These results can be interpreted, with some assumptions, as evidence in support of the hypothesis that the levels of the enzyme are maintained in $\text{D. discoideum}$ by a balance of synthesis and degradation.