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.

Nitrogen metabolism by lactating ewes and their lambs.

Twenty multiparous ewes and their newborn lambs were assigned to a 2 x 2 factorial experiment in which ewes were fed ad libitum either a moderate (MP, 15%) or a low (LP, 10%) CP diet and nursed either twin (T) or single (S) lambs. Nitrogen (N) balance trials were conducted on both the ewes and lambs during wk 2, 4 and 8 of lactation. Nitrogen balance, N digested and the portion of digested N retained were greater (P less than .01) with the MP diet. Nitrogen retention and serum urea-N values were lower for the ewes nursing T lambs. Plasma beta-hydroxybutyrate and serum glucose values were lower (P less than .01) for ewes fed the LP diet, and certain plasma amino acids and the ketogenic amino acids were lower with the LP diet. Insulin increased (P less than .01) with time throughout lactation. Triiodothyronine (T3) concentrations were higher (P less than .01) in ewes nursing T lambs. Thyroxine (T4) was greater (P less than .04) with the LP diet and greater (P less than .01) for ewes nursing T lambs. The T4:T3 ratio was lower (P less than .02) in the ewes consuming LP. The portion of the ewe's retained N used for milk synthesis was lower (P less than .01) with the MP diet. Ewes fed LP and nursing T lambs utilized all of their retained N plus a portion of their body protein reserves for milk production by the 2nd wk of lactation. Body weights of creep-fed lambs were not changed by protein content of the ewe's diet.

Serum amino acids as indicators of protein status in gestating ewes.

Changes in the serum amino acids of ewes obtained with regulated protein intakes may be of value for indicating the status of protein metabolism during gestation. Thirty crossbred ewes in midgestation were assigned in equal numbers to three treatments of 12%, 9% and 7% crude protein, isocaloric diets fed al libitum. Serum amino acids were determined by reverse phase, high performance liquid chromatography. Several consistent amino acid changes were related to protein intake over the last half of gestation. Glutamine plus alanine were increased and the branched chain amino acids (isoleucine, valine and leucine) were reduced. These responses suggest that tissues, such as muscle, undergo catabolism during late gestation as a result of low protein intake to release glutamine and alanine for gluconeogenesis. Serum Leu was reduced by a decreased level of dietary protein indicating inadequate protein status. Lowering protein intake also induced a consistent increase in serine through the last half of gestation. Increasing serine values may be the result of induced synthesis by liver in response to the low protein intake. This possibility suggests that the protein status of gestating ewes can be evaluated by following serum amino acid changes and the release of amino acids from specific tissues such as liver and muscle can be monitored through changes in the circulating amino acid values.

Relationship of lactose to nitrogen metabolism of artificially reared beef calves.

The purpose was to study the relationship of lactose to nitrogen metabolism of artificially-reared beef calves. Calves from 35 market heifers were fed whole milk at 12% of body weight daily to 14 days and then at 8% of body weight to 28 days. An 18.3% crude protein dry diet was fed for ad libitum consumption on days 1 to 28. For days 29 to 84, nine calves were assigned to each of three treatments: A) 60:40 grain:hay dry diet, B) 60:40 grain diet with liquid lactose fed separately, and C) 60:40 dry diet containing dried lactose. During days 1 to 28, body weights were not reduced. Calves compensated for reduction of whole milk intake by increasing their intakes of dry diet. gamma-Glutamyl transferase and urea nitrogen in blood serum were reduced when milk intake was decreased. Beef calves can be adapted to early weaning and artificial rearing if started soon after birth. Lactose treatments decreased dry matter intakes and vitamin E in blood, but body weights were not different. Feed conversion was improved; nitrogen balance and urinary nitrogen excretion were decreased by liquid lactose. Urea nitrogen in blood was related to nitrogen balance. Liquid lactose increased serum alkaline phosphatase and serum glucose. The role of liquid lactose was to supply adequate energy for improved utilization of retained nitrogen.

Changes of tocopherols in blood serum of cows fed hay or silage.

The purpose was to determine if cows fed corn silage during gestation have calves with low serum tocopherol and high erythrocyte hemolytic rates. Ten cows were fed hay, six fed corn silage with one vitamin E injection, and six fed corn silage with no injection beginning with the 6th mo of gestation. Hay intakes were adjusted to equalize intakes of dry matter. Analysis of tocopherol isomers was by reverse phase, high performance liquid chromatography. Alpha-tocopherol and potassium were lower for hay-fed cows than for cows fed predominantly corn silage, but creatinine phosphokinase, glutamic-oxaloacetic transaminase, and glucose were higher. Calves from hay-fed cows had lower alpha-tocopherol and higher glutamic-oxaloacetic transaminase. No consistent histopathological changes of muscle tissue were seen. Erythrocyte hemolytic rates were low. Serum vitamin E of cows may change during gestation because of type of forage fed. Specific enzyme changes may give an early indication of a change of tocopherol status of cattle.

Effects of sarcocystosis on milk production of dairy cows.

Sixteen multiparous Holstein cows were allotted randomly to four groups of four cows each. Cows in 1 and 2 were uninfected. Those in 3 received 60,000 and those in 4 received 120,000 Sarcocystis bovicanis sporocysts per os approximately 30 days before the expected onset of lactation to produce nonclinical and clinical infections in 3 and 4, respectively. Combined stresses of infection, parturition, lactation, and high ambient temperatures caused all infected cows to develop clinical illness. Clinical signs included fever, anemia, glossitis, myositis, nasal discharge, hypersalivation, anorexia, and hind limb weakness; two cows died and two others were killed in extremis. Six cows in 3 and 4 developed high Sarcocystis-specific immunoglobulin G1 antibody. Uninfected control cows had no clinical signs and no rising concentrations of antibody against Sarcocystis antigen. When lactation began, cows were milked twice daily, and milk production was recorded for 70 consecutive days. All sarcocystis-infected cows (3 and 4) decreased feed intake and milk production compared with uninfected controls. The Wisconsin Mastitis Test on milk production compared with uninfected controls. The Wisconsin Mastitis Test on milk samples at 1, 2, 4, 8, and 12 wk of lactation did not differ among groups.

Attempted transmission of Sarcocystis bovicanis from cows to calves via colostrum.

Sixteen pregnant cows were divided into four equal groups. Groups 1 and 2 were uninoculated controls; Groups 3 and 4 received 60,000 and 120,000 S. bovicanis sporocysts per os, respectively, about 30 days before expected parturition. Stained smears of colostrum from each quarter of the udder of each cow were examined microscopically for zoites. As a bioassay for infectious zoites in colostrum of acutely infected cows, colostrum from infected Group 4 cows was fed to calves from control Group 1 cows. Calves in Groups 2 and 3 received colostrum from their dams. Calves in Group 1 received colostrum from uninoculated normal cows. All calves were killed at 61 to 68 days of age and their tissues examined microscopically for Sarcocystis cysts. Because zoites were not found in colostrum smears and cysts were not found in calf tissues, it was concluded that lactogenic transmission of S. bovicanis did not occur.

Pathophysiological changes in urine and blood from calves experimentally infected with Sarcocystit cruzi.

Two 8-week experiments were conducted to determine the relationships between nutritional stress and pathophysiological changes in male Holstein calves infected with Sarcocystis cruzi. Calves were infected by oral inoculation with 200 000 S. cruzi sporocysts. In the first experiment weight gain reduction was greatest in inoculated calves during weeks 4 and 5 after inoculation. Feed intake was reduced during the 5th week. Erythrocyte count was reduced during week 5 and haemoglobin was reduced during week 6. The 24-h excretion of urinary and urea nitrogen from the inoculated calves was increased by treatment. In the 2nd experiments, both the feed-restricted and inoculated calves lost weight during weeks 4 and 5; feed intake was lower from week 5 to 8 inclusive. Urine volume from inoculated calves was lower during week 8. Lower urine excretions of sodium and potassium resulted from S. cruzi inoculation. There was a non-significant trend for higher urinary zinc excretion in the inoculated group during week 4. Urine nitrogen excretion from inoculated calves was higher during week 6. The urinary excretion of 3-methylhistidine from the inoculated calves was higher during week 4 and excretion of guanine was higher during weeks 4, 5 and 8. S. cruzi has several specific pathophysiological effects on calves beyond those induced by nutritional stress.

Heinz body formation in calf erythrocytes.

Heinz body formation was induced in calf erythrocytes by a daily dose of 8 mg phenylhydrazine/kg body weight. Body weights and feed intakes were not affected, the glutathione content of erythrocytes was reduced, but the hemolysis rate of erythrocytes was not changed. Morphological changes in erythrocytes were shown by light and high resolution scanning microscopy. The appearance of Heinz bodies may have value as an indicator of oxidative stress under conditions such as copper toxicity or deficiency of selenium-vitamin E.

Responses of young calves to low doses of lead.

Twenty calves, week-old male Holsteins, 42.8 kg body weight, were assigned to four treatments by oral dose 0, 1.5, 3.0, and 6.0 mg lead carbonate/kg body weight given three times weekly until calf fetal hemoglobin had been converted to 100% adult hemoglobin for 2 consecutive wk. Analyses were for the first 7 wk when data were complete. Variables included lead in blood, body weight changes, erythrocyte delta-aminolevulinic acid dehydrase activity, packed cell volume, hemoglobin, conversion of fetal to adult hemoglobin, and erythrocyte 2,3-diphosphoglyceric acid. Early changes due to lead treatment were inhibition of delta-aminolevulinic acid dehydrase at all doses and increased loss of hemoglobin at 3.0 and 6.0 mg doses. Growth rate was not impaired seriously by lead treatment. The 2,3-diphosphoglyceric acid in erythrocytes reached peak value at 3 to 4 wk of age and then decreased with age, but 2,3-diphosphoglyceric acid of the treated calves was not changed by the low lead dosing. Low lead dosing will induce early metabolic changes in the young calf prior to inhibition of growth.