Two distinct domains of protein 4.1 critical for assembly of functional nuclei in vitro

Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4.1 is essential for nuclear assembly and identify two distinct 4.1 domains, initially characterized in cytoskeletal interactions, that have crucial and versatile functions in nuclear assembly.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) destabilizes erythrocyte membrane skeleton

Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) is a parasite-derived protein that appears on the cytoplasmic surface of the host cell membrane in the later stages of the parasite's development where it associates with membrane skeleton. We have recently demonstrated that a 60-residue fragment (FIa1, residues 38-97) of PfEMP3 bound to spectrin. Here we show that this polypeptide binds specifically to a site near the C terminus of alpha-spectrin at the point that spectrin attaches to actin and protein 4.1R in forming the junctions of the membrane skeletal network. We further show that this polypeptide disrupts formation of the ternary spectrin-actin-4.1R complex in solution. Importantly, when incorporated into the cell, the PfEMP3 fragment causes extensive reduction in shear resistance of the cell. We conjecture that the loss of mechanical cohesion of the membrane may facilitate the exit of the mature merozoites from the cell.     

Aerobic training increases the expression of adiponectin receptor genes in the peripheral blood mononuclear cells of young men

Little is known about the effect of exercise training on the expression of adiponectin receptor genes in peripheral blood mononuclear cells (PBMCs). In this study, we investigated the effects of aerobic training on the expression of AdipoR1 and AidpoR2 mRNAs in PBMCs, whole body insulin sensitivity, and circulating adiponectins in men. Thirty young men were randomly assigned to either a control (n=15) or an exercise (n=15) group. Subjects assigned to the exercise group underwent a 12-week jogging and/or running programme on a motor-driven treadmill at an intensity of 60%-75% of the age-based maximum heart rate with duration of 40 minutes per session and a frequency of 5 days per week. Two-way mixed ANOVA with repeated measures was used to test any significant time-by-group interaction effects for the measured variables at p=0.05. We found significant time-by-group interaction effects for waist circumference (p=0.001), VO_2max (p<0.001), fasting insulin (p=0.016), homeostasis model assessment for insulin resistance (HOMA-IR) (p=0.010), area under the curve (AUC) for insulin response during the 75-g oral glucose tolerance test (p=0.002), high-molecular weight (HMW) adiponectin (p=0.016), and the PBMC mRNA levels of AdipoR1 (p<0.001) and AdipoR2 (p=0.001). The exercise group had significantly increased mRNA levels of AdipoR1 and AdipoR2 in PBMCs, along with increased whole body insulin sensitivity and HMW adiponectin, decreased waist circumference, and increased VO_2max compared with the control group. In summary, the current findings suggest that exercise training modulates the expression of AdipoR1 and AdipoR2 mRNAs in PBMCs, implying that manipulation of the expression of these genes could be a potential surrogate for lifestyle intervention-mediated improvements of whole body insulin sensitivity and glucose homeostasis.     

Structural and functional studies of interaction between Plasmodium falciparum knob-associated histidine-rich protein (KAHRP) and erythrocyte spectrin

Plasmodium falciparum dramatically modifies the structure and function of the membrane of the parasitized host erythrocyte. Altered membrane properties are the consequence of the interaction of a group of exported malaria proteins with host cell membrane proteins. KAHRP (the knob-associated histidine-rich protein), a member of this group, has been shown to interact with erythrocyte membrane skeletal protein spectrin. However, the molecular basis for this interaction has yet to be defined. In the present study, we defined the binding motifs in both KAHRP and spectrin and identified a functional role for this interaction. We showed that spectrin bound to a 72-amino-acid KAHRP fragment (residues 370-441). Among nine-spectrin fragments, which encompass the entire alpha and beta spectrin molecules (four alpha spectrin and five beta spectrin fragments), KAHRP bound only to one, the alpha N-5 fragment. The KAHRP-binding site within the alpha N-5 fragment was localized uniquely to repeat 4. The interaction of full-length spectrin dimer to KAHRP was inhibited by repeat 4 of alpha spectrin. Importantly, resealing of this repeat peptide into erythrocytes mislocalized KAHRP in the parasitized cells. We concluded that the interaction of KAHRP with spectrin is critical for appropriate membrane localization of KAHRP in parasitized erythrocytes. As the presence of KAHRP at the erythrocyte membrane is necessary for cytoadherence in vivo, our findings have implications for the development of new therapies for mitigating the severity of malaria infection.     

Identification and functional characterization of protein 4.1R and actin-binding sites in erythrocyte beta spectrin: regulation of the interactions by phosphatidylinositol-4,5-bisphosphate

The ternary complex of spectrin, F-actin, and protein 4.1R defines the erythrocyte membrane skeletal network, which governs the stability and elasticity of the membrane. It has been shown that both 4.1R and actin bind to the N-terminal region (residues 1-301) of the spectrin beta chain, which contains two calponin homology domains, designated CH1 and CH2. Here, we show that 4.1R also binds to the separate CH1 and CH2 domains. Unexpectedly, truncation of the CH2 domain by its 20 amino acids, corresponding to its N-terminal alpha helix, was found to greatly enhance its binding to 4.1R. The intact N terminus and the CH1 but not the CH2 domain bind to F-actin, but again, deletion of the first 20 amino acids of the latter exposes an actin-binding activity. As expected, the polypeptide 1-301 inhibits the binding of spectrin dimer to actin and formation of the spectrin-actin-4.1R ternary complex in vitro. Furthermore, the binding of 4.1R to 1-301 is greatly enhanced by PIP(2), implying the existence of a regulatory switch in the cell.     

Molecular genetic approach for identifying markers associated with yield traits in the silkworm, Bombyx mori using RFLP-STS primers

Bombyx mori, the mulberry silkworm, exhibits wide variability in yield and developmental attributes. The genetics of yield expression, shown to be of polygenic nature, is poorly studied in silkworm. To identify markers associated with 10 selected yield traits, multiple regression analysis (MRA) and discriminant function analysis (DFA) were applied on 64 markers generated with eight RFLP-derived sequence-tagged-site (STS) primers on the genomic DNA of 20 silkworm stocks of different origin and diverse yield potential. The analyses led to the identification of ten markers showing significant association with the different yield traits. The markers could classify the stocks according to yield potential, irrespective of their origin and status of diapause. Trait means were significantly different for stocks with and with out the associated marker. The inheritance of a marker G2(1300bp), selected at the first step of MRA for five yield traits was shown to segregate in 1:1 ratio in the F2 progeny from a cross between two divergent stocks. The relevance of the STS primers is discussed in the context of applying multiple regression model for identifying markers associated with yield expression and suitability for molecular breeding work in B. mori for yield improvement.     

Development of a CrN/Cu nanocomposite coating on titanium-modified stainless steel for antibacterial activity against Pseudomonas aeruginosa

A relatively simple method was developed to fabricate CrN/Cu nanocomposite coatings using pulsed DC magnetron sputtering for application in antibacterial activity. These nanocomposite coatings were applied on titanium (Ti)-modified stainless steel substrata (D-9 alloy) and the antibacterial activity of these coating with respect to the Gram-negative bacterium Pseudomonas aeruginosa was investigated qualitatively and quantitatively. Scanning electron microscopy, epifluorescence microscope analyses, and total viable counts confirmed that inclusion of copper in the CrN/Cu nanocomposite coatings provided antibacterial activity against P. aeruginosa. The quantitative examination of the bacterial activity of P. aeruginosa was estimated by the survival ratio as calculated from the number of viable cells which formed colonies on nutrient agar plates.