Dihedral angle and secondary structure database of short amino acid fragments

Dihedral angles of amino acids are of considerable importance in protein tertiary structure prediction as they define the backbone of a protein and hence almost define the protein's entire conformation. Most ab initio protein structure prediction methods predict the secondary structure of a protein before predicting the tertiary structure because three-dimensional fold consists of repeating units of secondary structures. Hence, both dihedral angles and secondary structures are important in tertiary structure prediction of proteins. Here we describe a database called DASSD (Dihedral Angle and Secondary Structure Database of Short Amino acid Fragments) that contains dihedral angle values and secondary structure details of short amino acid fragments of lengths 1, 3 and 5. Information stored in this database was extracted from a set of 5,227 non-redundant high resolution (less than 2-angstroms) protein structures. In total, DASSD stores details for about 733,000 fragments. This database finds application in the development of ab initio protein structure prediction methods using fragment libraries and fragment assembly techniques. It is also useful in protein secondary structure prediction.

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Comparative effects of insulin and insulin-like growth factor-1 on follicle-stimulating hormone-induced responses in porcine granulosa cells

The effect of insulin and insulin-like growth factor-1 (IGF-1) on progesterone secretion by porcine granulosa cells and their modulatory effect on follicle-stimulating hormone (FSH)-induced responses were examined. For comparative purposes, growth hormone (GH), previously shown to stimulate IGF-1 secretion, was also included. Granulosa cells from ovarian follicles (3 to 5 mm) were cultured in multiwell plates for the first 48 hours, either in the presence or absence of 1% fetal bovine serum (FBS). Following plating, all cultures were maintained in serum-free media. The addition of only insulin, but not IGF-1 or GH, enhanced progesterone secretion under both culture conditions. When low-density lipoprotein was provided as steroid substrate, a stimulatory effect of insulin on progesterone accumulation was observed with a minimum dose of 10 ng/ml. Granulosa cells cultured in serum-free media from the time of plating secreted less progesterone and were less responsive to FSH compared with cultures plated with 1% FBS. Only insulin, but not IGF-1, enhanced FSH responses to threefold in cells cultured with 1% FBS. However, when cells were cultured in serum-free media from the time of plating, both insulin and IGF-1, but not GH, potentiated the responses to FSH, but insulin was more potent than IGF-1. Insulin-like growth-factor-1 binding studies with granulosa cells indicate the presence of specific high-affinity binding sites (Kd 3.96 nM). A dose of 100 ng/ml of insulin had negligible cross-reactivity with IGF-1 receptors.     

Effect of 67Cu and 99Mo labeled tetrathiomolybdate on the distribution of 67Cu, Cu, and 99Mo in bile fractions in sheep

The effect of intravenous administration of 67Cu and 99Mo labeled tetrathiomolybdate (TTM) on the appearance of 67Cu, stable Cu, and 99Mo in gel chromatographic fractions of bile was examined in sheep fed either 5 or 35 mg Cu kg-1 DM. Peak excretory periods of biliary 67Cu, stable Cu, and 99Mo were observed at 30 min-1.25 hr, 2-3 hr, and 11-13 hr after 67Cu and after 99Mo labeled TTM. Sephadex G-75 gel filtration of bile samples collected at 1, 3, and 12 hr after 67Cu administration revealed two major protein peaks of molecular weights of greater than 80,000 (peak I) and 7,000 (peak II) containing both 67Cu and Cu. But the ratio of 67Cu in the two peaks varied with time of bile collection. The ratio of areas of peak I:II 1 hr after 67Cu administration was approximately 0.48; at 3 hr, 0.62, and at 12 hr 1.35. Tetrathiomolybdate administration increased both 67Cu and stable Cu in bile by severalfold and induced a major shift of Cu into the higher molecular weight protein fraction. The experiments confirm the effectiveness of TTM as a "decoppering" agent. Furthermore, TTM not only promoted bile Cu excretion, but it also increased the incorporation of Cu into the macromolecular fraction. This may limit enterohepatic circulation of biliary Cu and thereby cause an overall Cu depletion and a negative Cu balance.     

Distribution of Cu, Zn, and Fe in the soluble fraction of the kidney in normal, copper-poisoned, and thiomolybdate-treated sheep

Twenty-seven sheep were used in two experiments to study the distribution of copper (Cu), zinc (Zn), and iron (Fe) in the kidney cytosol of control, Cu-loaded, and thiomolybdate (TM)-treated sheep. A comparison was made of the patterns of distribution on Sephadex G-75 of Cu, Zn, and Fe from the cytosol of fresh and frozen kidneys and after thawing of frozen cytosol. In both Cu-dosed and TM-treated sheep, the absolute level of Cu increased in the cytosol, but the percent of Cu decreased. The percent recovery of Cu from the frozen kidney was comparable to that from fresh kidney, but the extraction of Cu from the supernatant of frozen cytosol was approximately 10% less. This was due to a loss of Cu in a precipitate that formed when the frozen cytosol was thawed. Most of the Cu in the cytosol from the kidney of Cu-loaded sheep was in a metallothionein (MT)-like protein fraction and was trichloroacetic acid (TCA) soluble. In contrast, that from the cytosol of TM-treated sheep was mostly in a high molecular weight fraction that was TCA insoluble. The chromatograms obtained from cytosol derived from frozen kidneys, or cytosol that had itself been frozen, contained a similar distribution of Cu, Zn, and Fe, but the peak heights were lower in the latter samples.     

The initiation of lactation in sows and the mastitis-metritis-agalactia syndrome

The initiation of lactation was studied in 46 sows farrowing normally in a commercial piggery practising intensive swine husbandry. Eleven of these sows showed typical clinical symptoms of mastitis-metritis-agalactia (MMA) syndrome, 12–48 h after parturition. Mammary secretion was collected from all sows at farrowing and analysed for lactose, protein, fat, Na⁺ and K⁺. The sows which subsequently developed MMA had significantly higher concentrations of lactose and significantly lower concentrations of protein and Na⁺ in colostrum, compared to normal sows.Analysis of milk from MMA sows showed an altered composition of milk (increased Na⁺ and decreased K⁺ and lactose concentrations). Results from this study suggest that an early initation of lactation resulting in the engorgement of the mammary glands predisposes the sows to MMA. Analysis of mammary secretion may be used to identify affected glands.     

Measuring Turnover of SIV DNA in Resting CD4+ T Cells Using Pyrosequencing: Implications for the Timing of HIV Eradication Therapies

Resting CD4+ T cells are a reservoir of latent HIV-1. Understanding the turnover of HIV DNA in these cells has implications for the development of eradication strategies. Most studies of viral latency focus on viral persistence under antiretroviral therapy (ART). We studied the turnover of SIV DNA resting CD4+ T cells during active infection in a cohort of 20 SIV-infected pigtail macaques. We compared SIV sequences at two Mane-A1*084:01-restricted CTL epitopes using serial plasma RNA and resting CD4+ T cell DNA samples by pyrosequencing, and used a mathematical modeling approach to estimate SIV DNA turnover. We found SIV DNA turnover in resting CD4+ T cells was slow in animals with low chronic viral loads, consistent with the long persistence of latency seen under ART. However, in animals with high levels of chronic viral replication, turnover was high. SIV DNA half-life within resting CD4 cells correleated with viral load (p = 0.0052) at the Gag KP9 CTL epitope. At a second CTL epitope in Tat (KVA10) there was a trend towards an association of SIV DNA half-life in resting CD4 cells and viral load (p = 0.0971). Further, we found that the turnover of resting CD4+ T cell SIV DNA was higher for escape during early infection than for escape later in infection (p = 0.0084). Our results suggest viral DNA within resting CD4 T cells is more labile and may be more susceptible to reactivation/eradication treatments when there are higher levels of virus replication and during early/acute infection.     

Subcellular distribution of copper in the kidneys of normal, copper-poisoned, and thiomolybdate-treated sheep

Twenty-seven sheep given either copper (Cu) and/or tetrathiomolybdate (TM) were used to study the subcellular distribution of Cu within the kidney and to monitor the location of lysosomes within the subcellular fractions using acid phosphatase (AP) as a marker enzyme. Copper dosing alone increased the Cu content in the liver and the kidneys. The administration of intravenous TM prevented the development of chronic copper poisoning (CCP) in sheep, reduced the rate of accumulation of Cu in the liver of Cu-dosed animals, but increased the Cu content of kidneys in both the control and Cu-dosed sheep. The total amount of Cu that accumulated in the kidneys of sheep given TM appears to depend on several factors: a) liver Cu concentration, b) Cu intake, and c) dosage of TM. Thus, the highest Cu concentration was found in the kidneys of sheep that continued to receive Cu orally at the same time as they were given TM. The intracellular distribution of Cu and AP in the kidneys showed that in the control sheep given neither Cu or TM, the highest proportion of Cu was in the cytosol fraction, and the highest specific activity of AP was in the light mitochondrial (lysosomal) fraction. Dosing with Cu markedly increased the Cu concentration and greatly elevated the total activity of AP in the heavier fractions, i.e., the nuclear (N) and heavy mitochondrial (MH). Thus, the increase in Cu observed in the N and MH fractions was not caused by an accumulation of Cu by nuclei and mitochondria, but was due to an accumulation of Cu by lysosomes that sedimented with the heavier fractions. The intracellular distribution of Cu in the kidneys of TM-treated sheep was similar to that seen in Cu-loaded sheep. Although Cu accumulated readily in the kidneys of animals receiving TM, kidney function tests showed neither glomerular nor tubular functional impairment.