Reliable exclusion of prognostically significant coronary disease in left ventricular dysfunction by cardiac MRI.

AIM: To assess the ability of cardiac magnetic resonance (CMR) to exclude prognostically significant coronary artery disease (CAD) in patients with left ventricular systolic dysfunction (LVSD). MATERIALS AND METHODS: A cohort of patients who underwent both X-ray angiography and CMR since 2006 was reviewed retrospectively. Records of those with European criteria for LVSD (left ventricular ejection fraction [LVEF] <50% or LV end-diastolic volume index [LVEDVI] ≥97 ml/m2) on CMR or transthoracic echo were analysed. The presence and extent of subendocardial late gadolinium enhancement (LGE) was recorded with the 17-segment model. The degree of coronary stenosis at X-ray angiography was assessed visually and significant disease defined as stenosis of the LMS ≥50%, or proximal left anterior descending ≥75%, or ≥70% in two main coronary vessels. RESULTS: One hundred and sixteen patients were included. The mean age was 64 years and 78% were male. The mean LVEF was 40%. The prevalence of prognostic CAD was 47%. The presence of subendocardial LGE detected prognostically significant CAD with a sensitivity of 100% (95% CI: 94-100%) with no false-negative results. CONCLUSIONS: The absence of subendocardial LGE on CMR reliably excludes prognostic CAD in patients with LVSD.

A model of nitrogen efficiency in contrasting grass-based dairy systems.

Nitrogen (N) efficiency is one of the key drivers of environmentally and economically sustainable agricultural production systems. An N balance model was developed, evaluated, and validated to assess N use efficiency and N surplus and to predict N losses from contrasting grass-based dairy production systems in Ireland. Data from a 5-yr study were used to evaluate and validate the model. Grass-based and high-concentrate production systems combined with 3 divergent strains of Holstein-Friesian (HF) dairy cows-high-production North American (HP), high-durability North American (HD), and New Zealand (NZ)-were evaluated. As concentrate input increased, N surplus per hectare increased and N use efficiency per hectare decreased (23 and 10%, respectively). When the N required to rear replacement animals to maintain the production system was considered, the N surplus of the HP genetic strain was greater (156 kg of N/cow) than that of the HD (140 kg of N/cow) or the NZ (128 kg of N/cow). The model estimated N leaching of 8.1mg of NO(3)-N/L, similar to that measured by others at the same site. The model creates awareness of methods and indicators available to assess the most suitable and environmentally sustainable grass based dairy production systems.

Extended lactations in a seasonal-calving pastoral system of production to modulate the effects of reproductive failure.

This study was conducted to determine whether extending the calving interval (CI) to 24 mo would be an alternative to culling and replacing cows that had failed to become pregnant. Forty-six nonpregnant lactating cows were assembled in November 2004 and assigned to receive either 3kg (low) or 6kg (high) of concentrate supplement and a basal diet of grass silage and maize silage over the winter period (13 wk). Cows returned to pasture in late March and received 1kg of concentrate/d until dry-off (milk yield <5 kg/d). Cumulative milk production was calculated from calving to the end of November 2004 (12-mo CI) and from the start of December 2004 until dry off in 2005 (extended lactation part of 24-mo CI). High winter feeding resulted in greater milk production over the winter confinement (20.0+/-0.3 vs. 17.8+/-0.3 kg/d for high and low winter feeding, respectively) and had a carryover effect during the remainder of the 24-mo CI period (5,177 vs. 4,686kg; SEM=173kg). At the end of the study, cows were ranked on cumulative milk solids and separated into 3 groups (R1, R2, and R3). During the 24-mo CI, milk yields were 7,287, 6,267, and 5,273kg (SEM=308kg) in yr 1, and 5,738, 4,836, and 4,266 (SEM=241kg) in yr 2 for R1, R2, and R3, respectively. Eighty-five percent of the cows became pregnant during the breeding season of yr 2, with a conception rate to first service of 52%. An economic analysis of different ranks with a 12-mo CI, a 24-mo CI, and an annualized herd effect, which compared an efficient spring calving system with 30% recycled cows in R1 and 10% recycled cows in R3, was carried out. Farm profit was reduced by 60% and 65% at a milk price of 22.3 euro-cents (c)/L with the corresponding values of 17% and 30% for a milk price of 30 c/L, respectively, when R1 and R3 systems were compared with an efficient spring milk (12-mo CI) production system. Within a spring system where 30% and 10% of R1 and R3 animals were subjected to extended lactations, the profit difference was reduced compared with an efficient spring system, The results indicated that lactations with a 24-mo CI may be a viable alternative to culling nonpregnant cows and be economically more suited to higher producing cows.

Short communication: the effect of dry period duration and dietary energy density in early lactation on the rennet gelation properties of milk.

This study was carried out to examine the effects of decreasing the dry period (DP) duration and altering the energy density of the diet during early lactation on the rheological characteristics of milk. Forty mature Holstein-Friesian cows were used in a completely randomized design with a 2 x 2 factorial arrangement of treatments. Cows were randomly assigned to 1 of 2 dry period treatments and 1 of 2 nutritional treatments. The DP treatments were continuous milking (CM) or an 8-wk standard dry period (SDP), and the nutritional treatments were a standard energy diet (SE) or a high energy diet (HE). Actual DP lengths were 6.3+/-1.7 d and 62.1+/-1.9 d for cows on the CM and SDP treatments, respectively. Milk samples were collected at 2, 6, and 10 wk postpartum. The concentrations of fat, protein, and lactose were determined in each sample. The rennet gelation properties were measured at 31 degrees C using dynamic low-amplitude strain oscillatory rheometry. The following parameters were obtained from the resultant elastic shear modulus (G'): gelation time, maximum curd firming rate, and gel strength. Reducing the DP duration from 62 to 6 d resulted in increases in milk protein concentration (31.8 vs. 34.7 g/kg), maximum curd firming rate (2.58 vs. 3.60 Pa/min), and gel strength (69.4 vs. 90.5Pa). Increasing the dietary energy density decreased percentage milk fat (43.1 vs. 37.7 g/kg) but otherwise had no effect. Gel strength was correlated with maximum curd firming rate (r=0.99), and both variables were correlated with milk protein concentration (r=0.71 and r=0.73, respectively). The results indicate that decreasing the duration of the DP increased milk protein concentration and improved the rennet gelation properties of milk, but that dietary energy density had little effect.

A comparison between Holstein-Friesian and Jersey dairy cows and their F(1) hybrid on milk fatty acid composition under grazing conditions.

The objective of this study was to investigate the effect of 2 breeds, Holstein and Jersey, and their F(1) hybrid (Jersey x Holstein) on milk fatty acid (FA) concentrations under grazing conditions, especially conjugated linoleic acid (CLA) and n-3 polyunsaturated fatty acids because of their importance to human health. Eighty-one cows (27 per breed grouping) were allocated a predominantly perennial ryegrass pasture. Samples were collected over 2 periods (June and July). Breed affected dry matter intake and milk production and composition. Holstein cows had the highest dry matter intake (18.4+/-0.40kg of DM/d) and milk production (21.1+/-0.53kg of DM/d). Holstein and Jersey x Holstein cows had similar 4% fat corrected milk, fat yield, and protein yield; with the exception of fat yield, these were all higher than for Jersey cows. Milk fat concentration was highest for Jersey cows and lowest for Holstein cows, with the hybrid cows intermediate. Total FA and linolenic acid intake (1.09+/-0.023 and 0.58+/-0.012 kg/d, respectively) were highest for Holstein cows. In terms of milk FA, Holstein cows had higher contents of C14:1, cis-9 C18:1 and linoleic acid. In turn, Jersey and Jersey x Holstein cows had higher content of C16:0. Milk concentrations of neither the cis-9,trans-11 isomer of CLA nor its precursor, vaccenic acid, were affected by breed. Nevertheless, large variation between individual animals within breed grouping was observed for CLA and estimated Delta(9)-desaturase activity. There was some evidence for a negative heterotic effect on milk concentration of CLA, with the F(1) hybrid cows having lower concentrations compared with the mid parent average. Plasma FA profile did not accurately reflect differences in milk FA composition. In conclusion, there was little evidence for either breed or beneficial heterotic effects on milk FA content with human health-promoting potential, though significant within-breed, interanimal variation was observed.

Pleiotropic effects of negative energy balance in the postpartum dairy cow on splenic gene expression: repercussions for innate and adaptive immunity.

Increased energy demands to support lactation, coupled with lowered feed intake capacity results in negative energy balance (NEB) and is typically characterized by extensive mobilization of body energy reserves in the early postpartum dairy cow. The catabolism of stored lipid leads to an increase in the systemic concentrations of nonesterified fatty acids (NEFA) and beta-hydroxy butyrate (BHB). Oxidation of NEFA in the liver result in the increased production of reactive oxygen species and the onset of oxidative stress and can lead to disruption of normal metabolism and physiology. The immune system is depressed in the peripartum period and early lactation and dairy cows are therefore more vulnerable to bacterial infections causing mastitis and or endometritis at this time. A bovine Affymetrix oligonucleotide array was used to determine global gene expression in the spleen of dairy cows in the early postpartum period. Spleen tissue was removed post mortem from five severe NEB (SNEB) and five medium NEB (MNEB) cows 15 days postpartum. SNEB increased systemic concentrations of NEFA and BHB, and white blood cell and lymphocyte numbers were decreased in SNEB animals. A total of 545 genes were altered by SNEB. Network analysis using Ingenuity Pathway Analysis revealed that SNEB was associated with NRF2-mediated oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, natural killer cell signaling, p53 signaling, downregulation of IL-15, BCL-2, and IFN-gamma; upregulation of BAX and CHOP and increased apoptosis with a potential negative impact on innate and adaptive immunity.

Fatty acid intake and milk fatty acid composition of Holstein dairy cows under different grazing strategies: herbage mass and daily herbage allowance.

The objective of this study was to investigate the effect of level of 1) pregrazing herbage mass (HM) and 2) level of daily herbage allowance (DHA) on the performance and fatty acid (FA) composition of milk from grazing dairy cows. Sixty-eight Holstein-Friesian dairy cows were allocated to either a high or low pregrazing HM (1,700 vs. 2,400 kg of DM/ha; >40 mm), and within HM treatment, cows were further allocated to either a high or low DHA (16 vs. 20 kg of DM/d per cow; >40 mm) in a 2 x 2 factorial design. Pregrazing HM did not affect dry matter intake (17.5 +/- 0.75 kg/d), milk production (22.1 +/- 0.99 kg/d), milk composition (milk fat, 3.88 +/- 0.114%; milk protein, 3.28 +/- 0.051%), body weight (525 +/- 16 kg), or body condition score (2.65 +/- 0.064). Increasing DHA increased dry matter intake (+1.5 kg/d) but did not affect any other variable measured. Cows grazing the low HM or high DHA had a higher daily intake of total FA (+0.12 and +0.09 kg/d, respectively, for the low HM and high DHA), alpha-linolenic acid (LNA; +0.08 and +0.05 kg/d, respectively, for the low HM and high DHA), and linoleic acid (+0.01 for both the low HM and high DHA) compared with either the high HM or low DHA. Milk conjugated linoleic acid (cis-9, trans-11 isomer) was not affected by treatment (13.0 +/- 0.77 g/kg of total FA); however, large variation was recorded between individual animals (range from 5.9 to 20.6 g/kg of total FA). Milk concentrations of LNA were higher for animals offered the low HM (5.3 g/kg of total FA), but across treatments, milk concentrations of LNA were low (4.9 +/- 0.33 g/kg of total FA). The present study indicates that changes in HM and DHA do not have a great effect on the milk FA composition of grazing dairy cows. Further enhancement of the beneficial FA content in milk purely from changes in grazing strategy may be difficult when pasture quality is already high.

Grazing cows are more efficient than zero-grazed and grass silage-fed cows in milk rumenic acid production.

Six rumen-cannulated Holstein cows in early lactation were assigned to 3 treatments: grazing (G), zero-grazing (ZG), and grass silage (GS) harvested from the same perennial rye grass sward in a 3 x 3 Latin square design with three 21-d periods. The objectives of this study were to investigate the underlying mechanisms for the reported elevation in milk rumenic acid (RA) concentration associated with G compared with ZG and GS, and to identify the important variables contributing to the milk RA response. Grazing animals were offered 20 kg of dry matter/cow per day; indoor animals were offered ad libitum grass or silage. A concentrate at a rate of 3 kg/d was also offered to all cows. Rumen, plasma, and milk samples were collected in the third week of each period. Data were analyzed by the MIXED procedure of SAS. Dry matter intakes were less for GS with no difference between G and ZG. Milk yield was greater for G than for ZG or GS. Milk fat and protein contents were less for GS with no difference between G and ZG. The combined intake (g/d) of linoleic and linolenic (18:3n-3) acids was different across the treatments (G: 433; ZG: 327; and GS: 164). Rumen pH was less for G with no difference between ZG and GS. Concentrations of volatile fatty acids and ammonia nitrogen in rumens were not different across the treatments. Wet rumen fill was less for G with no difference between ZG and GS. Vaccenic acid concentrations were different across the treatments in rumen (G: 12.30%, ZG: 9.31%, and GS: 4.21%); plasma (G: 2.18%, ZG: 1.47%, and GS: 0.66%) and milk (G: 4.73%, ZG: 3.49%, and GS: 0.99%). Milk RA concentrations were greater for G (2.07%) than for ZG (1.38%) and GS (0.54%). Milk desaturase index based on the ratio cis-9-14:1/14:0 was not different across the treatments. Milk RA yield per 100 g of linoleic acid and linolenic acid intake (efficiency) was 2.23, 1.50, and 0.62 g in G, ZG, and GS, respectively, suggesting that G cows were more efficient than ZG and GS cows in milk RA production. Stepwise regression analysis of a group of variables revealed that plasma vaccenic acid accounted for 95% of the variation in milk RA production. Milk desaturase index did not enter into the model. Overall findings suggest that substrate intake influenced milk RA production but it was not the only factor involved. There were differences in efficiency of milk RA production, which appears to depend on the factors regulating ruminal vaccenic acid production and its supply to the mammary tissue.

Cheese manufacture with milk with elevated conjugated linoleic acid levels caused by dietary manipulation.

The objective of this study was to assess the effect of dietary supplementation of cows on pasture with sunflower oil for conjugated linoleic acid (cis-9, trans-11 CLA) enrichment of milk, for the production of CLA-enriched cheese. A group of 40 autumn-calving dairy cows were assigned to either a control group (indoor feeding on grass silage ad libitum and 6 kg/d of a typical indoor concentrate) or an experimental group (on pasture, being fed 6 kg of a supplement containing 100 g/kg of sunflower oil per d). These diets were fed for 16 d, during which time milk was collected for pilot-scale hard cheese manufacture. The pasture-based diet with sunflower oil resulted in a significant effect on the milk fatty acid CLA content. The concentration of cis-9, trans-11 CLA in the milk produced from cows on this diet increased to 2.22 g/100 g of fatty acid methyl esters (FAME) after 14 d, compared with 0.46 g/100 g of FAME in milk produced on the control indoor diet. The content of cis-9, trans-11 CLA in the cheese manufactured from the indoor control milk was 0.78 g/100 g of FAME and that from the pasture-based sunflower oil milk was 1.93 g/100 g of FAME. The cheese was assessed during the ripening period and CLA concentrations were stable throughout the 6 mo of ripening. Other cheese variables (microbiology, composition, flavor, free AA) were monitored during the ripening period, and the cheese with the elevated CLA concentrations compared favorably with the control cheese. Thus, a pasture-based diet supplemented with an oil source rich in linoleic acid resulted in an enhanced CLA content of bovine milk fat, compared with an indoor grass silage-based diet.

Comparative evaluation of alternative forages to grass silage in the diet of early lactation dairy cows.

Fifty-six autumn-calving Holstein-Friesian cows, blocked on the basis of days in milk (27.6 +/- 10.65 d), lactation number (3.1 +/- 2.21), and preexperimental milk yield (28.4 +/- 6.69 kg) were used to examine the effects of replacing 330 g/kg of dry matter (DM) of first-cut perennial ryegrass silage with either fermented whole-crop wheat (WCW), urea-treated processed WCW, or corn silage on subsequent feed intake, milk production, and efficiency of nitrogen utilization. The DM (g/kg), crude protein (CP, g/kg of DM) and in vitro DM digestibility (g/kg) of the forages were 204, 179, and 762 for grass silage; 389, 90, and 711 for fermented WCW; 795, 141, and 768 for urea-treated processed WCW; and 346, 93, and 783 for corn silage, respectively. Four forage treatments were evaluated as follows: 1) grass silage as the sole forage (GS); 2) a mixture of grass silage and fermented WCW silage, (F-WCW); 3) a mixture of grass silage and urea-treated processed WCW, (UP-WCW); and 4) a mixture of grass silage and corn silage (CS). In all cases, the alternative forages comprised 67% of the forage mix on a DM basis. Isonitrogenous diets were formulated by offering all cows 8 kg of concentrate as fed, formulated to different CP concentrations. Cows were offered these diets from 28 to 104 d in milk. Total DM intake and milk yield were greater on UP-WCW (20.0 and 30.2 kg/d) and CS (18.3 and 33.2 kg/d) than on GS (13.5 and 26.5 kg/d). Although DM intake was greater on F-WCW (17.1 kg/d) than on GS, milk yield was not significantly greater (+2.7 kg/d). Milk protein concentration was greater on F-WCW (30.5 g/kg), UP-WCW (31.3 g/kg), and CS (30.7 g/kg) than on GS (28.5 g/kg). However, there was no difference between treatments in milk fat or lactose concentrations. Body weight change was greater for cows offered GS (-0.27 kg/d) than for those offered UP-WCW (-0.01 kg/d) and CS (+0.05 kg/d) but not compared with those offered F-WCW (-0.06 kg/d). There was no effect of treatment on plasma glucose, nonesterified fatty acids, beta-hydroxybutyrate, urea, or total protein at d 64 +/- 17.4 and d 92 +/- 17.4 postpartum. Efficiency of N utilization was greatest for CS with 0.36 of N intake being recovered in milk compared with 0.28, 0.32, and 0.26 for GS, F-WCW, and UP-WCW, respectively. There was no effect of treatment on milk urea N concentration or the urinary allantoin N to creatinine N ratio. The results of this experiment indicate that corn silage is a more suitable supplementary forage to grass silage than fermented or urea-treated processed WCW, with advantages realized in milk production and more efficient N utilization.

Relationships among milk production, energy balance, plasma analytes, and reproduction in Holstein-Friesian cows.

Associations were examined between components and indicators of early lactation energy balance (EB) and measures of fertility in Holstein cows. Milk production, dry matter intake (DMI), body condition score (BCS), and endocrine and metabolite data from 96 cows were analyzed using multivariate logistic regression and survival analysis. Fertility variables investigated were interval to commencement of luteal activity (C-LA), calving to conception interval (CCI), and conception rate to first service (CON1). Mean daily EB, milk protein content, and DMI during the first 28 d in milk were associated positively with CON1. Cows having poorer BCS (< or =2.25) at first service had a lower CON1. Positive associations were identified among EB, milk protein content, DMI, and the likelihood of a shorter interval to C-LA. Cows having greater DMI and a more positive EB had an increased likelihood of a shorter CCI, whereas a lower nadir BCS was associated with an increased likelihood of a longer CCI. Milk yield was not associated with any of the fertility variables investigated. A greater plasma concentration of insulin-like growth factor I (IGF-I) during the first 2 wk of lactation was associated with a greater CON1 and an increased likelihood of a shorter interval to C-LA. In conclusion, we identified DMI as the principal component of EB influencing subsequent fertility. Furthermore, results indicate that milk protein content and plasma IGF-I concentration in early lactation may be useful indicators of reproductive efficiency.

Embryo survival in dairy cows managed under pastoral conditions.

Efficient pasture-based milk production systems require a compact calving pattern aligned to the onset of the grazing season, a 365-day calving interval and low culling rates for infertility. Achievement of these targets requires high herd reproductive performance. While high genetic merit Holstein cows produce more milk in grass-based systems their fertility is compromised. Management of the modern high genetic merit Holstein dairy cow presents a major challenge in pasture-based systems of production. It appears that the extent of early embryo loss is greater (up to 20% points greater) in the modern high-producing dairy cow and that a much higher proportion of the embryos die before day 7 following insemination in contrast to heifers and lower yielding cows. About 7-8% of pregnancies are lost between days 30 and 90 of gestation with no evidence that loss rate is related to cow genetic merit, parity or level of production. Systemic concentrations of progesterone during both the cycle preceding and following insemination affect embryo survival rate with evidence that too low or indeed too high a concentration of progesterone been negatively associated with embryo survival rate. Peripheral concentrations of both progesterone and oestradiol are lowered by increased plane of feed intake due to increased metabolic clearance rate of the steroids, which is related to liver blood flow. It appears that high producing dairy cows have an increased risk of embryo death as a result of lowered peripheral concentrations of progesterone as a consequence of increased hepatic metabolism of progesterone. Uterine expression of mRNA for progesterone receptor, oestradiol receptor and retinol binding protein mRNA appears to be sensitive to changes in peripheral concentrations of progesterone during the first week after AI. It would appear that energy balance and dry matter intake during the 4 weeks, immediately after calving are critically important in determining conception rate when cows are inseminated at 70-100 days post-calving. Concentrate supplementation of cows at pasture during the breeding period has minimal affects on conception rates though sudden reduction in dietary intake should be avoided. For pasture-based systems of milk production more balanced breeding strategies, with greater emphasis on fertility and feed intake must be developed.

Effect of milking frequency and diet on milk production, energy balance, and reproduction in dairy cows.

The objective of this study was to determine the effects of reduced milking frequency and increased dietary energy density in early lactation on milk production, energy balance, and subsequent fertility. Sixty-six spring-calving, multiparous Holstein-Friesian cows were assigned to 1 of 3 treatment groups: once-daily milking on a standard diet (1xST); 3-times daily milking on a standard diet (3xST); and 3-times daily milking on a high-energy diet. Treatments were imposed for the first 28 d of lactation, after which all groups were milked twice daily and fed the standard diet. During the treatment period, the 1xST cows had 19.6% lower milk yield and higher milk fat and milk protein concentrations (15.7 and 10.2%, respectively) compared with 3xST. Dry matter (DM) intake was similar between 1xST and 3xST during the treatment period (12.64 vs. 13.25 kg/ d; SED = 0.82). Daily energy balance was less negative for 1xST compared with 3xST during wk 1 to 3 of lactation [-3.92 vs. -5.30 unité fourragère lait (UFL)/d; SED = 0.65; 1 UFL is equal to the net energy for lactation of 1 kg of standard air-dry barley]. During the treatment period, the cows on the high-energy diet had 17% higher milk yield, higher DM intake (15.5 vs. 13.9 kg/d; SED = 0.71), and similar energy balance (-4.45 vs. -4.35 UFL/d; SED = 0.65) compared to 3xST. Diet had no significant effect on any of the fertility variables measured. The interval to first ovulation was shorter for 1xST than 3xST (18.3d vs. 28.6d; SED = 1.76). In conclusion, once-daily milking in early lactation may promote earlier resumption of ovarian cyclicity, mediated through improved nutritional status.

An immediate early human gene encodes an Id-like helix-loop-helix protein and is regulated by protein kinase C activation in diverse cell types.

Transcription factors characterized by the presence of a helix-loop-helix (HLH) domain play a central role in the regulation of cell growth/differentiation and tumorigenesis. We report here the cDNA sequence of a human early-response gene, designated HLH 1R21, encoding a 15-kDa HLH protein that lacks a basic, DNA-binding domain and which by a number of criteria appears to be the human homologue of mouse HLH 462. Like its murine counterpart, HLH 1R21 protein functions as an Id (inhibitor of DNA binding) transcription factor by inhibiting the binding of E2A-containing protein complexes to muscle creatine kinase E-box enhancer oligonucleotide in vitro. However HLH 1R21 does not inhibit the binding of HLH Max protein to a Max-binding oligonucleotide in vitro, indicating that it has limited promiscuity in its ability to antagonize the function of other HLH transcription factors. In addition, HLH 1R21 mRNA transcripts are regulated by phorbol ester treatment of a diverse range of human cell lines and, when overexpressed in mouse NIH3T3 cells, HLH 1R21 induces a morphologically transformed phenotype.

Cell-type-specific early response gene expression during plasmacytoid differentiation of human B lymphocytic leukemia cells.

Monoclonal B lymphocytes from B cell chronic lymphocytic leukemia (B-CLL) can be induced to undergo plasmacytoid differentiation in vitro by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). By differential screening of a cDNA library derived from cells treated with phorbol ester we have isolated and characterised a set of early response genes (ERGs) displaying rapid transient up-regulation of expression in response to PMA. Cross-hybridisation studies showed that PMA probably induces the expression of over one hundred distinct genes, implying an ERG complexity comparable to that activated by mitogenic stimulation of fibroblasts and normal T lymphocytes. Of 13 genes analysed in detail, most were induced by PMA without a requirement for de novo protein synthesis, whilst nuclear run-on analysis showed that at least some of the more abundant classes of ERG were up-regulated through transcriptional mechanisms. In a proliferating variant B-CLL population, few differences in ERG expression were seen, suggesting that these genes are part of a gene regulatory pathway coupled to the differentiative rather than the proliferative response of B-CLL cells. However, studies in a range of cell types revealed a surprisingly diverse pattern of PMA-induced expression where most ERGs were relatively B-CLL-specific. This implies an extreme diversity of gene regulatory pathways activated in the primary response by phorbol ester generally and suggests that the onset of PMA-induced plasmacytoid differentiation of B-CLL cells is preceded by activation of a complex gene regulatory program that is largely unique to this maturation-arrested B cell.

Evidence for protein kinase C--independent pathways mediating phorbol ester induced plasmacytoid differentiation of B chronic lymphocytic leukemia cells.

The effects of phorbol esters on many cell types are known to be mediated through activation of the protein kinase C (PKC) signal transduction pathway. By using the specific inhibitor of this enzyme 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine dihydrochloride (H7) we have assessed the role of PKC activation in phorbol ester (phorbol 12-myristate 13-acetate, PMA)-induced plasmacytoid differentiation of B chronic lymphocytic leukemia cells (B-CLL) as a model of terminal differentiation of human B lymphocytes. H7 affected a dose-dependent inhibition of PMA-induced thymidine and uridine uptake with ID50 values of 41 microM and 32 microM, respectively. A comparable ID50 value (34 microM) was obtained for H7 inhibition of B-CLL PKC activity in a cell-free system. PMA-induced changes in cell morphology, expression of CD20, CD37 and FMC7 surface antigens together with increased secretion of immunoglobulin were variably abrogated by H7 suggesting that PKC activation is more important in B cell activation/DNA synthesis than in the differentiative response. Consistent with this, expression of a sizable proportion of PMA-inducible genes was not significantly affected by H7. These data are consistent with the existence of a PMA-activated, PKC-independent signal transduction pathway which may be important, though by itself apparently insufficient, for eliciting full terminal differentiation in B lymphocytes.

A B cell specific immediate early human gene is located on chromosome band 1q31 and encodes an alpha helical basic phosphoprotein.

We have determined the cDNA sequence of a human B cell specific, immediate early gene, designated 1R20, which is inducible in response to several B cell activation signals. The cDNA sequence predicts a 196 amino acid open reading frame comprising numerous highly basic residues and the predicted structure contains several potential alpha helical domains together with eight consensus protein phosphorylation sites. The 1R20 gene has been localised by fluorescence in situ hybridisation to chromosome band 1q31, a region known to be implicated in the pathogenesis of haemopoietic malignancies.

Torsade de pointes induced by N-acetylprocainamide.

N-Acetylprocainamide (NAPA), a class III antiarrhythmic drug, caused torsade de pointes in a 72 year old woman who had this arrhythmia on two previous occasions while being treated with quinidine and disopyramide. Initial evaluation with an intravenous infusion of NAPA indicated a favorable antiarrhythmic response. The QTC interval was prolonged, but the 2.4 ms/microgram per ml incremental QTC interval lengthening caused by NAPA was not greater than usual. During subsequent oral therapy with NAPA, torsade de pointes developed at plasma levels of this drug that appeared to be well tolerated during the initial evaluation.

Effects of diets fed to dairy cows before and after calving on their plasma progesterone profiles after calving.

Four weeks before their predicted mean second lactation calving date, 60 spring-calving Holstein-Friesian cows were blocked into groups of six on the basis of their predicted calving date and body condition score, and allocated at random to one of six dietary treatments in a factorial design: ad libitum grass silage, ad libitum grass silage plus barley straw or ad libitum grass silage plus 3 kg of concentrates, was offered for four weeks before the expected calving date, and after calving they were offered either 4 kg or 8 kg of concentrates plus ad libitum grass silage for eight weeks. On average, the first luteal activity occurred in all the groups at 29 days after calving. Seventeen of the cows had an atypical first plasma progesterone profile; 12 were anovulatory, three had prolonged luteal phases and two became anovulatory after having been cyclic. The cows offered grass silage only before calving had a significantly shorter mean (se) interval between calving and second luteal activity (44.9 [2.1] days), than the cows offered grass silage and straw (53.5 [1.9] days) or grass silage and concentrates (51.5 [3.2] days) (P<0.05). After calving none of the 28 cows offered grass silage and 4 kg of concentrates started cycling before day 21, whereas five of the 30 cows offered grass silage and 8 kg of concentrates cycled before day 21 (P<0.05).

Coupling of early response gene expression to distinct regulatory pathways during alpha-interferon and phorbol ester-induced plasmacytoid differentiation of B chronic lymphocytic leukaemia cells.

Phorbol esters (phorbol 12-myristate 13-acetate; PMA) and alpha-interferon (alpha-IFN) act through divergent signal transduction pathways to induce B chronic lymphocytic leukaemia cells (B-CLL) to undergo plasmacytoid differentiation in vitro. By using a panel of PMA-inducible early response gene probes we show that these two different effectors are coupled to second messenger pathways that do not converge on a common gene regulatory programme in differentiation of B-CLL cells. Moreover, using the calcium ionophore, A23187, four distinct regulatory classes of early response gene could be defined implying that multiple regulatory pathways may mediate the process of terminal differentiation in B lymphocytes.