The perinuclear matrix as a structural element of the mouse sperm nucleus.

The mouse sperm nucleus, after the removal of protamines and DNA, consisted of a skeletal structure that conformed to the original nuclear shape. Sperm were extracted with 1% SDS, and the isolated nuclei, along with the enveloping perinuclear theca, were incubated in 25 mM dithiothreitol, and exposed to different reagents in an effort to displace the protamines, P1 and P2. Protamines, labeled with [3H]arginine, were displaced from the nucleus by CaCl2.MgCl2, but only partially by anionic detergents, monovalent cations, and polyvalent anions. Displacement of P1 and P2 was achieved by digesting the nuclei with DNase I and simultaneously extracting with CaCl2.MgCl2 (3:2; mol:mol) in stepwise increments of 125, 150, 175, 200, and 250 mM. Protamine displacement was concentration-dependent, occurring with an EC50 of approximately 205 mM and with maximal displacement at approximately 250 mM CaCl2.MgCl2. The nucleus was reduced to a skeletal structure consisting of the perinuclear theca and an internal network of transverse fibers. The evidence was consistent with the former being derived from the perforatorium and postacrosomal nuclear sheath (both cytoplasmic structures), whereas the fibers were most likely of nuclear origin. By SDS-PAGE and isoelectric focusing (IEF), perinuclear matrices consisted of greater than or equal to 230 protein spots, with M(r)s in the range of 70,000 to 8000 and pIs of greater than or equal to 7.5 to approximately 4.7, respectively. Monoclonal antibodies prepared against perinuclear matrices bound to specific proteins on IEF immunoblots and, based on light and electron microscopic observations, to discrete domains of the sperm perinuclear theca and nucleus.     

Changes in carbon,nitrogen and phosphorus levels due to deforestation and cultivation: A case study in Simlipal National Park,India

The study area, within the Simlipal National Park, India, provides a rare variety of soil sampling sites. These include virgin forests in the proximity of several cultivated areas (where no chemical fertilizers or any modern technology has been used and where periods of cultivation vary from 5 to a little over 100 yr); samples from evergreen forests, deciduous forests and natural grasslands could also be obtained. The availability of numerous such samples made it possible to use statistical methods to evaluate the changes. This study showed that deforestation and cultivation result in statistically significant (P_0.05) reduction in organic C, total N and C:N ratios but no significant changes in total and available P levels; C:P and N:P ratios are also reduced. Loss of organic C and N occurs rapidly in the first 15 yr of cultivation and reaches quasi-steady state values around 1–2% organic C and 0.1–0.2% total N; extent of reduction is not related to initial levels. Significant reduction in C:N, C:P ratios following cultivation suggest that mineralisation losses of C are higher than loss of N whereas loss of P is lowest. Lack of significant correlation between organic C and P levels in all types of soils, suggests that the bulk of the P is in the inorganic form. Highest levels of organic C and N were observed in evergreen forests followed by deciduous forests, grasslands and cultivated areas in that order; total and available P levels, however, showed no significant differences. Evergreen vegetative cover appears to provide the ideal environment for organic matter accumulation.     

Chondrocyte cluster formation in agarose cultures as a functional assay to identify genes expressed in osteoarthritis

Understanding altered gene expression in osteoarthritic cartilage can lead to new targets for drug intervention. We established a functional assay based on chondrocyte cluster formation, a phenotype associated with osteoarthritis (OA), to screen an OA cartilage gene library. Previous reports have demonstrated that normal chondrocytes grown in suspension culture maintain their chondrocytic phenotype, however, certain growth factors such as basic fibroblast growth factor (bFGF) will induce the cells to proliferate in tight clusters similar to those seen in osteoarthritic cartilage. In this study we validate that overexpression of bFGF by retrovirally transduced normal chondrocytes would similarly induce the proliferation of tight cell clusters. We then used this approach as a basis to set up a functional screen where an entire OA cartilage cDNA library was tranduced into normal chondrocytes to search for other genes that would also induce cluster formation. Seven potential genes were isolated from the OA gene library, including BPOZ, IL-17 receptor C, NADH ubiquinone oxidoreductase, COMP, Soluble carrier 16 (MCT 3), C1r, and bFGF itself. None of the identified genes were upregulated by bFGF, however, all of them upregulated the expression of bFGF suggesting a common pathway. Although cluster formation is not considered to be destructive in OA cartilage, it is consistent with the disease and could yield answers to the altered phenotype. Further studies are needed to elucidate how these genes are linked to the disease state.     

(TG:CA)(n) repeats in human housekeeping genes

The unravelling of human genome sequence gives a new opportunity to investigate the role of repetitive sequences in gene regulation. Among the various types of repetitive sequences, the dinucleotide (TG:CA)(n) repeats are one of the most abundant in human genome and exhibit polymorphism. Early on, it was observed that the (TG:CA)(n) repeats could modulate gene expression and has the propensity to undergo conformational transitions in in vivo conditions. Recent reports describe the role of polymorphic (TG:CA)(n) repeats in gene regulation in several genes. In this work, we have analysed the distribution of (TG:CA)(n) (n >or= 6) repeats in human 'housekeeping genes' on which recently released Gene Chip data is available. Our results indicate that (i). The number of short intragenic (TG:CA)(n) repeats is significantly higher than the number of long repeats (ii). the proportion of genes with (TG:CA)(n) repeats (n >or= 12 units) had lower mean expression levels compared to those without these repeats, (iii). the genes belonging to the functional class of 'signalling and communication' had a positive association with repeats in contrast to the genes belonging to the 'information' class that were negatively associated with repeats.     

Effect of season and artificial diet on amino acids and nucleic acids in gonads of green sea urchin Strongylocentrotus droebachiensis

The content of total and free amino acids (FAA) in green sea urchin (Strongylocentrotus droebachiensis) gonads varied with the season and feeding on an artificial diet. Glycine was the dominant amino acid in each season contributing 12.9-16.6% to the total amino acid (TAA) content, peaking in the spring. In the FAA profile, glycine accounted for 30.3-61.4% in different seasons. A grain-based artificial diet had noticeable effects on the total and FAA compositions of S. droebachiensis. Although, glycine was the dominant amino acid in the TAA profile during early harvesting, tyrosine in gonads became more dominant on week 9 of feeding. Furthermore, glycine was the dominant amino acid in the FAA pool after feeding the artificial diet. The total FAA content in the gonads increased significantly (P<0.05) from 20.6 on week 0 to 180.6 mg/g dry mass tissue on week 3. There were no significant (P<0.05) changes between week 6 and week 9. Deoxyribonucleic acid (DNA) content exceeded that of ribonucleic acid (RNA) in each season, while in cultured urchins, RNA content exceeded that of DNA only on week 6. The RNA/DNA ratio was significantly increased in the summer, whereas this ratio was increased up to week 6 followed by a decrease on week 9 in cultured counterparts.     

Antioxidant activity of wheat extracts as affected by in vitro digestion

Phenolic compounds from soft wheat and its milling fractions were extracted and their in vitro antioxidant activity evaluated. Non-hydrolyzed extracts were prepared by extracting phenolics into distilled deionized water. To make hydrolyzed extracts samples were first subjected to pH adjustments in order to simulate gastrointestinal pH conditions. Total phenolic content (TPC) was determined using Folin-Ciocalteu's procedure. Total antioxidant activity (TAA) was determined using Trolox equivalents antioxidant capacity (TEAC) assay and expressed as Trolox equivalents (TE). Antioxidant activity of wheat extracts was determined using the procedures of inhibition of beta-carotene bleaching, scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and inhibition of oxidation of human low density lipoprotein (LDL) cholesterol. The TPC and TAA of wheat extracts tested were significantly increased following hydrolysis. Similarly, the antioxidant activity, as determined by different procedures, was considerably increased when the samples were subjected to simulated digestion conditions.     

A GTPase Chimera Illustrates an Uncoupled Nucleotide Affinity and Release Rate,Providing Insight into the Activation Mechanism

The release of GDP from GTPases signals the initiation of a GTPase cycle, where the association of GTP triggers conformational changes promoting binding of downstream effector molecules. Studies have implicated the nucleotide-binding G5 loop to be involved in the GDP release mechanism. For example, biophysical studies on both the eukaryotic Gα proteins and the GTPase domain (NFeoB) of prokaryotic FeoB proteins have revealed conformational changes in the G5 loop that accompany nucleotide binding and release. However, it is unclear whether this conformational change in the G5 loop is a prerequisite for GDP release, or, alternatively, the movement is a consequence of release. To gain additional insight into the sequence of events leading to GDP release, we have created a chimeric protein comprised of Escherichia coli NFeoB and the G5 loop from the human Giα1 protein. The protein chimera retains GTPase activity at a similar level to wild-type NFeoB, and structural analyses of the nucleotide-free and GDP-bound proteins show that the G5 loop adopts conformations analogous to that of the human nucleotide-bound Giα1 protein in both states. Interestingly, isothermal titration calorimetry and stopped-flow kinetic analyses reveal uncoupled nucleotide affinity and release rates, supporting a model where G5 loop movement promotes nucleotide release.The hydrolysis of guanosine triphosphate (GTP) by GTPases, such as the oncoprotein p21 Ras and heterotrimeric Gα proteins, is a critical regulatory activity for cell growth and proliferation (1). Aberrant GTPases are consequently often implicated in tumorigenesis, developmental disorders, and metabolic diseases (2). Critical for the initiation of a GTPase cycle is the release of guanosine diphosphate (GDP), which allows GTP to bind and switch the protein from an inactive to an active conformation. The GTP is subsequently hydrolyzed to GDP and inorganic phosphate, returning the GTPase to an inactive conformation (3).Given that the release of GDP is the fundamental step in the initiation of a GTPase cycle, the detailed mechanism by which it is released has been under intense scrutiny. Studies using double electron-electron resonance, deuterium-exchange, Rosetta energy analysis, and electron paramagnetic resonance, have shown that the mechanism involves conformational changes in the nucleotide-coordinating G5 loop, one of five nucleotide recognition motifs (4, 5, 6, 7, 8, 9, 10, 11). Structural studies of eukaryotic Gα proteins and the intracellular TEES-type GTPase domain of the prokaryotic iron transporter FeoB (NFeoB) have also illustrated distinct conformations of the G5 loop, depending on the nucleotide-bound state (9, 12).Recently, we reported mutational studies of the G5 loop of Escherichia coli NFeoB, which illustrated a correlation between the sequence composition of the loop and the intrinsic GDP release rate (13). However, despite these observations, it is unclear whether the observed conformational changes in the G5 loop are a prerequisite for GDP release, or if the movement is a consequence of GDP release. To address this fundamental question, in this study we have used a combination of protein engineering and biophysical methods.Initially, to assess the relevance of conformational flexibility in the G5 loop, we aimed to create a protein chimera combining sequence and structural characteristics of both fast and slow GDP-releasing GTPases. We thus engineered a protein chimera using E. coli NFeoB as the scaffold (a protein with fast intrinsic GDP release) and substituted the G5 loop with that of a slow GDP-releasing protein (the human Giα1 protein; Gene ID 2770; Fig. 1 A (5)). GTP hydrolysis assays comparing wild-type (wt) NFeoB (wtNFeoB) and the protein chimera (ChiNFeoB) validated the integrity of the GTPase activities of both proteins (k_cat = 0.46 and 0.36 min⁻¹, respectively). To further assess the ChiNFeoB protein, we determined its crystal structure at 2.2 Å resolution (see Table S1 in the Supporting Material). The ChiNFeoB structure contains two molecules in the asymmetric unit, with molecule A bound to GDP. They are essentially identical to the nucleotide-bound wtNFeoB structure (root-mean-square deviation of 1.2 Å over 226 Cα atoms; Fig. 2).Open in a separate windowFigure 1Chimera model and structural comparison. (A) Illustration highlighting the chimera sequence change. (Orange) Sequence of the extended G5 loop from Giα1, which replaced the NFeoB sequence (gray). (B–F) Structural comparison of the G5 loop between (B) WT apo (PDB:3HYR) and nucleotide-bound (PDB:3HYT) NFeoB structures. (C) NFeoB nucleotide-bound and Giα1 (PDB:2ZJZ). (D) Nucleotide-bound NFeoB and chimera (Chi_GDP). (E) Nucleotide-bound chimera and Giα1. (F) Nucleotide-free (Chi_apo) and bound chimera protein. (G) Overview of the nucleotide binding site and structural overlay of chimera and Giα1 structures. To see this figure in color, go online.Open in a separate windowFigure 2Superimposition of nucleotide-bound NFeoB and chimera protein, with thermodynamic parameters. To see this figure in color, go online.However, the ChiNFeoB structure, when compared to the wtNFeoB structure, revealed an alteration in the conformation of the G5 loop, showing an extra turn on the N-terminal end of the α6 helix. This is structurally distinct from the wtFeoB protein, but with a conformation similar to that of the Giα1 protein (PDB:2ZJZ; Fig. 1, B–F). As in the crystal structures of wtNFeoB and Giα1, ChiNFeoB residues implicated in coordination of the nucleotide base maintain their positions in the G5 loop relative to GDP. In particular, residues Ala^∗150 and Thr^∗151 (NFeoB numbering, the asterisk indicates Giα1 chimera residue) are involved in electrostatic interactions with the nucleotide base moiety, analogous to the structures of both wtNFeoB and Giα1 (Fig. 1 G). Serendipitously, the second molecule in the asymmetric unit of ChiNFeoB (molecule B) was present in the nucleotide-free state. The two molecules (GDP-bound and nucleotide-free) are nearly identical (the superposition of molecules A and B yields a root-mean-square deviation of 0.36 Å over 229 Cα atoms), with the G5 loop adopting a nearly indistinguishable conformation compared to that of the GDP-bound molecule A (Fig. 1 F).Importantly, this conformation is independent of the crystallographic packing, inasmuch as the loop is not involved in any crystal contacts. In contrast, the structures of nucleotide-bound and nucleotide-free wtNFeoB illustrated a large conformational change in the G5 loop (Fig. 1 B). Hence, the substitution in the chimera extends the secondary structure of the α6 helix, and as hypothesized, the engineered ChiNFeoB protein has a G5 loop structure that is more conformationally stable than that of wtNFeoB.We subsequently measured the affinity of the ChiNFeoB protein for GDP using isothermal titration calorimetry (ITC). Nonlinear regression was used to attain the thermodynamic parameters (including the GDP binding affinity, K_a; the corresponding dissociation constant (K_d) was calculated from the equation K_d = 1/K_a). Interestingly, these measurements revealed the ChiNFeoB protein to have an almost 10-fold reduced affinity for GDP (82 vs. 9 μM measured for the WT protein; Fig. 2). In contrast, in a recent alanine scanning mutagenesis study of the G5 loop we observed a fivefold increase in affinity for GDP in a Ser¹⁵⁰Ala mutant (2 μM) (14). This mutant protein has a coordination environment for the GDP base analogous to that of the ChiNFeoB protein (Fig. 1 A), indicating that it is not the presence of an alanine at position 150 that causes the reduced GDP affinity observed for the chimera protein. Instead, the analysis by ITC and comparison with previous mutagenesis studies indicates that the GDP binding site is less accessible in the ChiNFeoB protein, likely due to the introduction of conformational rigidity that accompanies the extension of secondary structural elements within the loop (Fig. 1 D).To further evaluate the functional characteristics of the chimera protein, we used stopped-flow fluorescence assays to determine the rate of nucleotide dissociation (k_off) and association (k_on) for the ChiNFeoB protein. The association rate for the GTP analog mant-GMPPNP was determined from the slope of a linear plot of protein concentration versus the observed association constant (k_obs). The k_on for the chimera was determined to be 3.20 μM⁻¹ min⁻¹ (Supporting Material), the dissociation rate (k_off) of GDP for the chimera was determined to be 16.6 s⁻¹ (vs. 144 s⁻¹ for wtNFeoB;

Tissue specificity of 3′-untranslated sequence of myosin light chain gene: Unexpected interspecies homology with repetitive DNA

Using the 3′ noncoding and coding sequences of chick heart myosin light chain mRNA cloned into Escherichia coli as probes, it was observed that, while the coding sequence shared homology with myosin light-chain mRNAs from other sources, the 3′ noncoding sequence was specific for chick heart muscle. This property was used to detect chick heart-specific myosin light-chain gene activity in chick blastoderms of very early developmental stages where cells of different muscle origins cannot be distinguished morphologically. However, in spite of the tissue-specific divergence of the 3′ noncoding sequence of myosin light-chain gene, which is present in a single copy in the chick genome, a surprising homology with DNA from such a diverse source like Dictyostelium discoideum was noted. The sequence homologous to chick myosin light-chain DNA was apparently present in a high repetition frequency in the Dictyostelium genome.