Purification and characterization of the rat spermatid basic nuclear protein TP4.

Following elongation of spermatids in mammals, the histones are replaced by a set of basic nuclear transition proteins; in the rat there are four, named TP1-TP4. Of these, TP1 and TP2 are well characterized. Here we report the purification to homogeneity of TP4 from rat spermatids. It is a low molecular mass (about 13-20 kDa) basic protein with arginine and lysine constituting 24 mol % and histidine 2.2 mol %. Its 25 N-terminal amino acids were sequenced, and no sequence homologies with any known protein were found. Polyclonal antibodies raised against it in rabbit did not cross-react with other transition proteins, protamines, or histones. The presence of TP4 during sperm development was monitored by cell separation studies. No TP4 was detected in round spermatids, and along with TP1 and TP2, it is present in step 13-15 spermatids and its amount decreased in steps 16-19. Trace amounts of TP4 were also detected in epididymal sperm. A possible role for TP4 in spermatid and sperm chromatin structure is discussed.     

Stabilization of type I collagen against collagenases (type I) and thermal degradation using iron complex

The widespread application of collagen as a biomaterial warrants research in understanding the stabilization of the same. In this study, interaction of iron-tetrakis (hydroxymethyl) phosphonium (THP) complex with type I collagen has been investigated. DSC and hydrothermal measurement studies reveal that the shrinkage temperature of iron-THP treated rat tail tendon (RTT) collagen is 33 degrees C higher than that of native RTT collagen. Fe-THP complex also brings about high degree of enzymatic stability to type I collagen. The effect of Fe-THP on the conformation of collagen was studied using circular dichroism and it was found that no major alterations in the triple helical structure of collagen occur on treatment with Fe-THP. It is observed from viscosity experiment results that though Fe-THP complex is able to bring about long range ordering of collagen, as evident from the thermal and enzymatic stability imparted to collagen, this ordering does not lead to any aggregation of collagen. Since THPS is reducing in nature, it is expected to keep iron in the +2 state and if THP chelates to Fe(II), the hydrolytic behavior of iron can also be controlled.     

Long-term serotonin administration leads to higher bone mineral density, affects bone architecture, and leads to higher femoral bone stiffness in rats

New evidence suggests a control of bone mass by the central nervous system. We have previously shown that functional serotonin receptors are present in bone cells and that serotonin stimulates proliferation of osteoblast precursor cells in vitro. In the present study we investigated the effects of serotonin on bone tissue in vivo. Ten, 2-month-old female Sprague-Dawley rats were injected with serotonin subcutaneously (s.c.) (5 mg/kg) once daily for 3 months, controls received saline. Using microdialysis and HPLC, free circulating serotonin levels were measured. DXA scans were made after 3 months of serotonin administration. Bone architecture and mechanical properties were investigated by micro-computed tomography (microCT), histomorphometry, and mechanical testing. A long-lasting hyperserotoninemia with a >10-fold increase in serotonin appeared. Total body BMD was significantly higher (0.1976+/-0.0015 vs. 0.1913+/-0.0012 g/cm2) in rats receiving serotonin. Cortical thickness (Ct.Th) measured by microCT analysis was also higher, whereas trabecular bone volume (BV) was lower. Interestingly, the perimeter and cross-sectional moment of inertia (MOI), a proxy for geometrical bone strength, were the same in both groups. These data suggest that serotonin reduces resorption or/and increases apposition of endosteal bone. Mechanical testing showed that femoral stiffness was higher in serotonin-dosed animals. The energy absorption also seemed slightly, but not significantly higher. In conclusion, hyperserotoninemia led to a higher BMD, altered bone architecture and higher femural bone stiffness in growing rats, demonstrating that serotonin may have important effects on bone in vivo.     

Bioaccumulation and biosorption of chromium by Aspergillus niger MTCC 2594

Chromium toxicity is of prime concern due to chrome tanning processes in the leather sector. Chrome tanning results in the discharge of toxic levels of chromium causing pollution hazards. Chromium levels of Cr(III) and Cr(VI) were high above permissible limits in chrome samples after chrome tanning. The potential of Aspergillus niger MTCC 2594 to accumulate chromium as well as its biosorption capacity is investigated in this study. Bioaccumulation of Cr(III) and Cr(VI) in the spent chrome liquor has resulted in a 75-78% reduction of the initial Cr content in 24-36 h. A. niger biomass is found to be very effective in the biosorption of Cr(III) and Cr(VI) in spent chrome liquor. Maximum adsorption of 83% for biosorption of Cr(III) at 48 h and 79% of Cr(VI) at 36 h in spent chrome liquor is observed. The biosorption characteristics fit well with Langmuir and Freundlich isotherms and the adsorption parameters are evaluated. The biosorption of Cr also follows Lagergren kinetics. A. niger biomass is effectively used for the biosorption of chromium with 79-83% Cr removal in 36-48 h.     

Use of hunters in wildlife surveys: does hunter and forest grouse habitat selection coincide?

Wildlife monitoring performed by volunteer personnel may suffer from bias with regard to their habitat use. Such errors can lead to erroneous population estimates, evidently influencing both management programmes and research that are based on the monitoring. We used a dataset on hunters’ habitat use in forest while searching for black grouse (Tetrao tetrix) and capercaillie (Tetrao urogallus) to test whether hunters’ utilisation of the habitat was independent of that of grouse or if it corresponded to the grouse habitat preference. Twenty volunteer hunters with dogs registered their tracks and all observations of capercaillie and black grouse in Østfold County, Norway, during August 2003 and 2004. We performed an ecological niche factor analysis (ENFA) and a K-select analysis with respect to hunters’ selection for habitat, described with ecogeographical variables related to forest stand characteristics, as well as the habitat preference of the observed grouse, conditional on the habitat utilisation of the hunters. Individual ENFA on the hunter's tracks revealed large variation in the habitat preferences of the hunters. The K-select indicated few overall patterns in the habitat characteristics of grouse observations, conditional on the hunters selected habitat. Accordingly, the results indicate that hunters’ observation of grouse prior to the hunt may give indicators of changes in grouse density unbiased by habitat preference due to the large between-hunter variation in habitat preference, given that a sufficient number of hunters is used. This suggests that such monitoring programmes can provide information about fluctuations in grouse population sizes valuable for both the management and research of forest grouse species.     

Identification of Bilirubin Binding Site in Type I Collagen

The interaction of bilirubin with collagen in the significance of jaundice incidence have been previously reported and investigated. The novel peptide sequences containing bilirubin binding domain was identified and located to develop a basis for further studies investigating the interactions of collagen with bilirubin in the present study. In this study an intricate interaction between bilirubin and collagen was characterized and their binding domain has been established using in-gel digestion and LC–MS/MS analysis based on the collagen sequencing and peptide mass fingerprinting. The biotinylated bilirubin derivatives bind to α₁(I) chain but not to α₂(I) chains which clearly designates that bilirubin shows greater affinity to α₁ chains of collagen. The intact proteins collected after analyzing the resulting complex mixture of peptides was used for peptide mapping. Using the electrospray method, among the other peptide sequence information obtained, the molecular weight of collagen alpha-2(I) chain was obtained by locating a 130 kDa weight peptide sequences with greater pi value (9.14) with 1,364 amino acid residues and collagen alpha-1(I) chain with 1,463 amino acid residues with 138.9 kDa molecular weight. This information leads to locate the exact sequence of these helices focussing on the domain identification. The total charge of the peptide domain sequences infers that the bilirubin participates in the electrostatic mode of interaction with collagen peptide. Moreover, other modes of interactions such as hydrogen bonding, covalent interactions and hydrophobic interactions are possible.     

Rational discovery of a SOD1 tryptophan oxidation inhibitor with therapeutic potential for amyotrophic lateral sclerosis

Abstract

Formation of Cu, Zn superoxide dismutase 1 (SOD1) protein inclusions within motor neurons is one of the principal characteristics of SOD1-related amyotrophic lateral sclerosis (ALS). A hypothesis as to the nature of SOD1 aggregation implicates oxidative damage to a solvent-exposed tryptophan as causative. Here, we chart the discovery of a phenanthridinone based compound (Lig9) from the NCI Diversity Set III by rational methods by in silico screening and crystallographic validation. The crystal structure of the complex with SOD1, refined to 2.5 Å, revealed that Lig9 binds the SOD1 β-barrel in the β-strand 2 and 3 region which is known to scaffold SOD1 fibrillation. The phenanthridinone moiety makes a substantial π–π interaction with Trp32 of SOD1. The compound possesses a significant binding affinity for SOD1 and inhibits oxidation of Trp32; a critical residue for SOD1 aggregation. Thus, Lig9 is a good candidate from which to develop a new library of SOD1 aggregation inhibitors through protection of Trp32 oxidation.

Communicated by Ramaswamy H. Sarma     

Novel dual cyclooxygenase and lipoxygenase inhibitors targeting hyaluronan–CD44v6 pathway and inducing cytotoxicity in colon cancer cells

Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzyme have been found to play a role in promoting growth in colon cancer cell lines. The di-tert-butyl phenol class of compounds has been found to inhibit both COX-2 and 5-LOX enzymes with proven effectiveness in arresting tumor growth. In the present study, the structural analogs of 2,6 di-tert-butyl-p-benzoquinone (BQ) appended with hydrazide side chain were found to inhibit COX-2 and 5-LOX enzymes at micromolar concentrations. Molecular docking of the compounds into COX-2 and 5-LOX protein cavities indicated strong binding interactions supporting the observed cytototoxicities. The signaling interaction between endogenous hyaluronan and CD44 has been shown to regulate COX-2 activities through ErbB2 receptor tyrosine kinase (RTK) activation. In the present studies it has been observed for the first time, that three of our COX/5-LOX dual inhibitors inhibit proliferation upon hydrazide substitution and prevent the activity of pro-angiogenic factors in HCA-7, HT-29, Apc10.1 cells as well as the hyaluronan synthase-2 (Has2) enzyme over-expressed in colon cancer cells, through inhibition of the hyaluronan/CD44v6 cell survival pathway. Since there is a substantial enhancement in the antiproliferative activities of these compounds upon hydrazide substitution, the present work opens up new opportunities for evolving novel active compounds of BQ series for inhibiting colon cancer.     

Enhancement of anti-tumor CD8 immunity by IgG1-mediated targeting of Fc receptors

Dendritic cells (DCs) function as professional antigen presenting cells and are critical for linking innate immune responses to the induction of adaptive immunity. Many current cancer DC vaccine strategies rely on differentiating DCs, feeding them tumor antigens ex vivo, and infusing them into patients. Importantly, this strategy relies on prior knowledge of suitable “tumor-specific” antigens to prime an effective anti-tumor response. DCs express a variety of receptors specific for the Fc region of immunoglobulins, and antigen uptake via Fc receptors is highly efficient and facilitates antigen presentation to T cells. Therefore, we hypothesized that expression of the mouse IgG1 Fc region on the surface of tumors would enhance tumor cell uptake by DCs and other myeloid cells and promote the induction of anti-tumor T cell responses. To test this, we engineered a murine lymphoma cell line expressing surface IgG1 Fc and discovered that such tumor cells were taken up rapidly by DCs, leading to enhanced cross-presentation of tumor-derived antigen to CD8+ T cells. IgG1-Fc tumors failed to grow in vivo and prophylactic vaccination of mice with IgG1-Fc tumors resulted in rejection of unmanipulated tumor cells. Furthermore, IgG1-Fc tumor cells were able to slow the growth of an unmanipulated primary tumor when used as a therapeutic tumor vaccine. Our data demonstrate that engagement of Fc receptors by tumors expressing the Fc region of IgG1 is a viable strategy to induce efficient and protective anti-tumor CD8+ T cell responses without prior knowledge of tumor-specific antigens.     

Monocyte gene expression signature of patients with early onset coronary artery disease

The burden of cardiovascular disease (CVD) cannot be fully addressed by therapy targeting known pathophysiological pathways. Even with stringent control of all risk factors CVD events are only diminished by half. A number of additional pathways probably play a role in the development of CVD and might serve as novel therapeutic targets. Genome wide expression studies represent a powerful tool to identify such novel pathways. We compared the expression profiles in monocytes from twenty two young male patients with premature familial CAD with those from controls matched for age, sex and smoking status, without a family history of CVD. Since all patients were on statins and aspirin treatment, potentially affecting the expression of genes in monocytes, twelve controls were subsequently treated with simvastatin and aspirin for 6 and 2 weeks, respectively. By whole genome expression arrays six genes were identified to have differential expression in the monocytes of patients versus controls; ABCA1, ABCG1 and RGS1 were downregulated in patients, whereas ADRB2, FOLR3 and GSTM1 were upregulated. Differential expression of all genes, apart from GSTM1, was confirmed by qPCR. Aspirin and statins altered gene expression of ABCG1 and ADBR2. All finding were validated in a second group of twenty four patients and controls. Differential expression of ABCA1, RSG1 and ADBR2 was replicated. In conclusion, we identified these 3 genes to be expressed differently in CAD cases which might play a role in the pathogenesis of atherosclerotic vascular disease.