RepD-mediated recruitment of PcrA helicase at the Staphylococcus aureus pC221 plasmid replication origin, oriD.

Plasmid encoded replication initiation (Rep) proteins recruit host helicases to plasmid replication origins. Previously, we showed that RepD recruits directionally the PcrA helicase to the pC221 oriD, remains associated with it, and increases its processivity during plasmid unwinding. Here we show that RepD forms a complex extending upstream and downstream of the core oriD. Binding of RepD causes remodelling of a region upstream from the core oriD forming a 'landing pad' for the PcrA. PcrA is recruited by this extended RepD-DNA complex via an interaction with RepD at this upstream site. PcrA appears to have weak affinity for this region even in the absence of RepD. Upon binding of ADPNP (non-hydrolysable analogue of ATP), by PcrA, a conformational rearrangement of the RepD-PcrA-ATP initiation complex confines it strictly within the boundaries of the core oriD. We conclude that RepD-mediated recruitment of PcrA at oriD is a three step process. First, an extended RepD-oriD complex includes a region upstream from the core oriD; second, the PcrA is recruited to this upstream region and thirdly upon ATP-binding PcrA relocates within the core oriD.

The synthesis and antibacterial activity of totarol derivatives. Part 1: modifications of ring-C and pro-drugs.

A series of analogues of, and potential pro-drugs derived from, the potent antibacterial diterpene totarol (1) were synthesized in order to elucidate the minimum structural requirements for antibacterial activity and to seek compounds with good bioavailability in vivo. These analogues varied in the structural features of their aromatic rings and the prodrugs were O-glycosylated derivatives. They were tested in vitro against three gram-positive bacteria: beta-lactamase-positive and high level gentamycin-resistant Enterococcus faecalis, penicillin-resistant Streptococcus pneumoniae, and methicillin-resistant Staphylococcus aureus (MRSA); and against the gram-negative multi-drug-resistant Klebsiella pneumoniae. None of the analogues was more potent than totarol itself, which is effective against these gram-positive bacteria at MIC values of 7 microM. The results were evaluated in terms of a structure-activity relationship and this showed that a phenolic moiety was essential for potent antibacterial activity. Amongst the pro-drugs, totaryl alpha-D-mannopyranoside (22) proved the most active in vitro (MIC 18 microM). The in vivo antibacterial activities of compounds 1, 22 and totarol beta-lactoside (23) were assessed in a mouse model of infection, but they were found to be ineffective. Compounds 1 and 22 were shown to be cytotoxic towards proliferating human cell cultures, CH 2983, HeLa, and MG 63, but only at concentrations of > 30 microM.

Synthesis of allosamidin analogues.

Two new isomers of allosamidin and allosamizoline, and two dimeric allosamidin analogues have been synthesized. The new compounds have been tested for inhibition of insect chitinase and for toxicity to insect larvae.

Influence of dietary palm oil supplementation on serum lipid metabolites, carcass characteristics, and lipid composition of carcass tissues of growing ram and ewe lambs.

The objective of this research was to determine the influence of dietary palm oil supplementation on carcass characteristics and lipid composition of tissues from growing lambs. Twenty-eight Suffolk x Hampshire lambs were weaned at 60 d of age (average 36 kg BW) and assigned to a 2 x 2 factorial arrangement consisting of diet (control [NPO] or 10.6% added dietary palm oil [PO]) and sex (ram vs ewe). The NPO diet (77% forage and 23% concentrate) contained 11.2% CP and 2.66 Mcal of ME/kg. Palm oil replaced molasses in the PO diet. Lambs were individually given ad libitum access to feed for 60 d to a final BW average of 50.1 kg. Lipid composition of the longissimus muscle and corresponding subcutaneous (s.c.) adipose tissue was determined by gas-liquid chromatography (GLC). Lambs fed PO were fatter (P less than .01) than lambs fed NPO (.77 vs .56 cm, s.c. fat). Diet had no effect on cholesterol content of lean tissue; however, feeding PO increased the saturated fatty acids of lean tissue. The s.c. fat from lambs fed PO had less (P less than .01) cholesterol (64.79 vs 89.67 mg/100 g) and more saturated fatty acids than that from lambs fed NPO. Ewes were fatter (P less than .01) than rams, yet they had less cholesterol content in the s.c. adipose tissue (68.71 vs 85.74 mg/100 g). High amounts of dietary palm oil fed to growing lambs caused changes in fatty acid deposition and cholesterol metabolism and may be a useful investigative tool to study lipid metabolism in growing ruminants.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dietary canola seed and soy lecithin in high-forage diets on cholesterol content and fatty acid composition of carcass tissues of growing ram lambs.

Phospholipids (soy lecithin) are important in the emulsification of lipids and may escape the rumen and influence the absorption of fatty acids in the small intestine. Our objectives were to determine the influence of dietary canola seed (high in unsaturated fatty acids) and soy lecithin in high-forage diets on total lipid content, cholesterol content, and fatty acid composition of carcass tissues. Forty-three Hampshire or Suffolk-sired ram lambs were weaned at 60 d of age (average 23.6 kg of BW) and assigned to a 2 x 2 factorial arrangement of treatments consisting of 1) basal diet (control = BAS), 2) BAS with 6% whole canola seed (CS), 3) BAS with 4.9% deoiled soy lecithin (SL), and 4) BAS with 6% CS and 4.8% SL (CSSL). The BAS diet consisted of 70% forage and 30% concentrate and contained 15% CP and 2.2 Mcal of ME/kg. Lambs were individually fed and given ad libitum access to feed to an average final BW of 52.1 kg. Longissimus muscle (LM) from the left side of each carcass posterior to the 13th rib (12 to 15 cm in length) was excised and the lean (LM) and corresponding subcutaneous (s.c.) adipose tissue were separated, frozen, and later used for lipid analysis by gas-liquid chromatography. In lean tissue, feeding lambs CS reduced (P less than .01) the proportion of total polyunsaturated fatty acids (PUFA) and feeding SL increased (P less than .01) the proportion of total PUFA. In s.c. adipose tissue, lambs fed CS had lower (P less than .01) saturated fatty acids (SFA) and lambs fed SL had increased (P less than .03) PUFA.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of rapeseed meal, whole rapeseed, and soybean meal on fatty acid composition and cholesterol content of muscle and adipose tissue from ram lambs.

Twenty-four Suffolk x Hampshire ram lambs (average 46 kg) were assigned to one of three diets containing rapeseed meal (RM), soybean meal (SBM), or whole rapeseed-soybean meal (RSSBM) as the protein source. Diets contained 75% roughage, 14% CP and 2.0 Mcal of ME/kg and lambs were allowed ad libitum access to diets for 35 d. Lipid composition of the longissimus, semimembranosus, and triceps brachii muscles and their corresponding s.c. adipose tissue was determined by gas-liquid chromatography (GLC). The total lipid content in either muscle or subcutaneous fat was not different (P greater than .01) by diet. In lean tissue, palmitic and palmitoleic acids were higher and stearic acid was lower (P less than .01) in rams fed RM than in rams fed RSSBM or SBM, regardless of anatomical location. In the s.c. adipose tissue, the amounts of myristoleic, pentadecylic, and palmitoleic acids were lower and the amount of stearic acid was higher (P less than .01) in rams fed RSSM than in those fed RM or SBM, regardless of anatomical location. The semimembranosus and triceps brachii muscles from all treatments contained 12 to 19% more polyunsaturated fatty acids (PUFA) than the longissimus muscle. The cholesterol content of the three muscles was highest in SBM-fed lambs, lowest in RM-fed lambs, and intermediate in RSSBM-fed lambs. These results demonstrate that dietary treatments of the types used in the present study elicit changes in fatty acid composition of both adipose and muscle tissue without affecting the quantity of total lipid.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dietary canola seed and soy lecithin in high-forage diets on performance, serum lipids, and carcass characteristics of growing ram lambs.

Phospholipids (soy lecithin) are important in the emulsification of lipids and may be able to escape the rumen and influence the absorption of fatty acids in the small intestine. The objectives of this study were to determine the influence of dietary canola seed (high in unsaturated fatty acids) and soy lecithin in high-forage diets on performance, serum lipid metabolites, and carcass characteristics of growing ram lambs. Forty-three Hampshire- or Suffolk-sired ram lambs were weaned at 60 d of age (average 23.6 kg BW) and assigned to a 2 x 2 factorial arrangement of the following treatments (% of DM): 1) basal diet (control = BAS); 2) BAS with 6% whole canola seed (CS); 3) BAS with 4.9% deoiled soy lecithin (SL); and 4) BAS with 6% CS and 4.8% SL (CSSL). The BAS diet consisted of 70% forage and 30% concentrate and contained 15% CP and 2.2 Mcal of ME/kg. Lambs had ad libitum access to their diet to an average final BW of 52.1 kg. Lambs were bled by jugular venipuncture on d 0, at 4-wk intervals, and 1 d before slaughter. Dry matter intake was not affected by treatment; however, energy intake (Mcal of ME/d) was greater (P less than .01) for lambs fed SL. Feeding SL and(or) CS to growing ram lambs increased (P less than .02) serum lipid concentrations. Carcasses from lambs fed SL were fatter, as evidenced by greater dressing percentage and subcutaneous fat thickness. Feeding CS to growing lambs had little effect on performance and carcass characteristics compared with feeding SL, which resulted in greater energy intake and fatter carcasses.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrogen metabolism of lactating ewes fed rumen-protected methionine and lysine.

Twenty multiparous, crossbred, black-faced ewes and their newborn twin lambs were assigned to one of four dietary treatments in a 2 x 2 factorial experiment to examine the effects of increased supply of CP or a mixture of encapsulated methionine and lysine or both on the performance of ewes and their nursing twin lambs. Ewes were fed ad libitum amounts of either a 10.2% low CP diet or a 16.2% moderate CP diet with or without additional encapsulated amino acids. Nitrogen metabolism trials were conducted simultaneously on both ewes and lambs at wk 2, 4, and 8 of lactation. Analyses were conducted for blood urea N, plasma 3-hydroxybutyrate, lactate, NEFA, insulin, and amino acids (plasma, feed, and milk). Ewe DMI, BW, BW gain, and milk yield were not changed by dietary treatments. Balance of N and N digested were increased by moderate CP treatment. The portion of retained N used for milk synthesis was increased by low CP treatment. Methionine and total branched-chain amino acids were increased by encapsulated amino acids and by protein treatment. Gains in BW and N balance were increased in lambs nursing ewes fed protected amino acids. Increased growth of nursing lambs would be an important beneficial effect of supplementing diets of ewes with encapsulated methionine and lysine.

Lipid composition of muscle and adipose tissue from crossbred ram, wether and cryptorchid lambs.

Lambs from rams (R), cryptorchids (C) and wethers (W) were examined to compare with the dietary guidelines for humans on fat and cholesterol intake. Twenty-four lambs were assigned to treatments as R, C or W at weaning (60 d). Each lamb had ad libitum access to a 77:23% forage:concentrate diet containing 11.7% crude protein and 1.84 Mcal ME/kg. The experiment was terminated when the lambs reached 50 kg body weight. Lipid composition of the longissimus muscle (LM) and s.c. adipose tissue was determined by gas-liquid chromatography. Rams has less (P less than .01) s.c. fat (cm) and i.m. lipid (%) (.26 cm; 3.*%) than W (.56 cm; 4.9%). Total polyunsaturated fatty acids were greater (P less than .01) in the LM of R (7.06%) than in LM of W (5.21%). Rams had higher percentages of C18:2, C18:3, C20:4 and C18:1 in their LM than W did. On the basis of a 100-g serving of LM, R had 1.73, C had 2.15 and W had 2.21 g saturated fatty acids. There were no (P less than .01) differences among sex types for cholesterol content of LM, which averaged 66.6 mg/100 g wet tissue. The only major difference of the s.c. fat lipid composition was in saturated fatty acids. Wethers had higher (P less than .01) saturated fatty acids (51.4%) than C 46.8%) and R (43.3%). Based on the lipid composition of these three types of lambs, the LM muscle from young R lambs approached dietary fat guidelines for humans more closely than did that from castrates.