Effects of abomasal lipid infusion on liver triglyceride accumulation and adipose lipolysis during fatty liver induction in dairy cows.

The objective was to determine the effects of abomasal infusion of linseed oil on liver triglyceride (TG) accumulation and adipose tissue lipolysis during an experimental protocol for induction of fatty liver. Eight nonpregnant, nonlactating Holstein cows were randomly assigned to treatments in a replicated 4 x 4 Latin square design. Treatments were abomasal infusion of water (W), tallow (T), linseed oil (LO), or half linseed oil and half tallow (LOT) at a rate of 0.56 g/kg of body weight per day. Each experimental period consisted of a 4-d fast concurrent with administration of treatments into the abomasum in 6 equal doses per day (every 4 h). Cows were fed ad libitum for 24 d between periods of fasting and lipid infusion. Infusion of linseed oil (LO and LOT) increased alpha-linolenic acid (C18:3n-3) content in serum (12.2, 10.4, 4.2, and 4.6 g/100 g of fatty acids for LO, LOT, T, and W, respectively), but not in the nonesterified fatty acid (NEFA) fraction of plasma. Treatments had no effect on plasma NEFA concentrations. Abomasal infusion of lipid increased in vitro stimulated lipolysis in subcutaneous adipose tissue, compared with W (4,294, 3,809, 4,231, and 3,293 nmol of glycerol released x g(-1) tissue x 2 h(-1) for LO, LOT, T, and W, respectively), but there was no difference between fat sources. Hepatic TG accumulation over 4-d fast was 2.52, 2.60, 2.64, and 2.09 +/- 0.75 microg of TG/microg of DNA for W, LO, LOT, and T, respectively, which did not differ. Abomasal infusion of LO did not reduce liver TG accumulation, plasma NEFA concentration, or alter in vitro adipose tissue lipolysis when compared with T. These results contrast with a previous study involving i.v. infusion of lipid emulsion derived from LO. Discrepancies might be explained by the use of different administration routes and a relatively modest induction of liver TG accumulation in the current experiment.

Effect of dry period length on reproduction during the subsequent lactation.

Days dry may influence reproductive measures such as days to first postpartum ovulation, days open, and pregnancy per artificial insemination (AI). Holstein cows (n = 781) from an approximately 3,000-cow commercial dairy operation were randomly assigned to 1 of 2 treatments with different targeted dry period (DP) lengths. Treatments were 1) a traditional DP of 55 d (T) or 2) a shortened DP of 34 d (S). All dry cows on T were fed a low-energy diet until 35 d before expected calving, and then at 34 d before expected calving, cows on T and S were fed a moderate energy diet until parturition. After parturition, all cows consumed the same diets that included a postcalving diet followed by a lactation diet. Actual days dry for each treatment were close to expected values, 34 and 56 d for S and T, respectively. Median days until first postpartum ovulation occurred sooner for S compared with T (35 vs. 43 d). The percentage of cows that were classified anovular by 70 d in milk (DIM) was more than 2-fold greater for cows on T than S (18 vs. 8%). Cows received AI after standing estrus starting at d 45, and the percentage of cows pregnant at 70 DIM tended to be greater for S than T; younger cows were similar (20.2 vs. 18.8%), but there was a difference between S and T in older cows (20.3 vs. 10.6%). Similarly, median days open tended to be fewer for cows on S than T. At 300 DIM, 85% of cows in both treatments were pregnant. Combining data from first and second service, pregnancies per AI were greater in older cows on S than T (32 vs. 24%). Thus, shortening the DP appeared to increase reproductive efficiency in older cows by shortening time to first ovulation, reducing numbers of anovular cows, and improving fertility. Future studies at more locations with varying reproductive management strategies are needed to confirm and provide the mechanistic basis for these results.

Effects of dry period length on milk production and health of dairy cattle.

Holstein cows (n = 781) in a commercial dairy herd were used in a randomized design to evaluate 2 dry period (DP) management strategies on milk production, milk components, milk quality, colostrum quality, and incidence of metabolic disorders. Cows were randomly assigned to a traditional 55 d (T) or shortened 34 d (S) DP. Cows assigned to T were fed a low-energy diet until 34 d before expected calving at which time all cows were fed a moderate-energy transition diet until calving. Postpartum, cows assigned to T produced more milk and tended to produce more solids-corrected milk than cows on S. Treatment differences in milk and solids-corrected milk yield were accounted for by cows in their second lactation. Milk fat percentage did not differ between treatments, but milk protein percentage was greater for cows assigned to S. Colostrum quality measured as IgG concentration did not differ between management strategies. Somatic cell score and cases of mastitis were not affected by management strategy. There was a tendency for prepartum nonesterified fatty acid (NEFA) to be lower for cows assigned to T compared with S. However, postpartum, cows assigned to S had significantly lower NEFA concentrations than those assigned to T. The incidences of ketosis, retained placenta, displaced abomasum, and metritis did not differ between treatments. Postpartum energy balance, as indicated by plasma NEFA, may have been improved for cows assigned to S; there was no detectable effect on animal health.

Effects of abomasal infusion of linseed oil on responses to glucose and insulin in holstein cows.

The objective was to study the effects of abomasal infusion of linseed oil, a source rich in n-3 C18:3, on whole-body response to insulin (experiment 1) and on insulin antilipolytic effects during feed restriction (experiment 2). In experiment 1, eight nonlactating, non-gestating cows were assigned to a crossover design, fed to meet maintenance requirements, and infused abomasally with either linseed oil (LIN) or tallow (TAL) at a rate of 0.54 g/kg of body weight per d for 5.5 d. Infusions were performed every 8 h during the first 3 d of each period and every 4 h thereafter. Intravenous glucose tolerance tests (IVGTT) were performed on d 5 of each period, followed by i.v. insulin challenges (IC) 12 h later. In experiment 2, six nonlactating, nongestating cows were assigned to a replicated 3 x 3 Latin square design. The experimental protocol included a water (WTR) treatment and feeding was suspended on d 3, leading to 50 and 62 h of feed restriction before IVGTT and IC, respectively. Clearance of glucose during IVGTT and IC was not affected by treatments in either experiment. However, LIN had an insulin sensitizing effect in experiment 1, because a lower insulin concentration led to the same clearance of glucose as TAL. In experiment 1, plasma nonesterified fatty acid (NEFA) concentration was low, reflecting a postprandial state, but NEFA was greater for LIN than TAL during IVGTT (108 vs. 88 +/- 4 microEq/L) and IC (133 vs. 83 +/- 9 microEq/L). In experiment 2, insulin concentrations during IVGTT did not differ across treatments. Basal plasma NEFA concentration before IVGTT tended to be greater for LIN than for TAL (612 vs. 508 microEq/L). Plasma NEFA clearance rate during IVGTT was greater for LIN than for TAL (2.8 vs. 2.5%/min), leading to a shorter time to reach half NEFA concentration (25 vs. 29 min) and greater absolute value of NEFA response area under the curve [AUC; -64,150 vs. -46,402 (microEq/L) x 180 min]. Data suggest that LIN enhanced the antilipolytic effects of insulin. Yet, other factors could have been involved because plasma NEFA concentration before IVGTT was 104 muEq/L greater for LIN than TAL for unknown reasons.

Technical note: Effect of sampling protocol on plasma nonesterified fatty acid concentration in dairy cows.

The objective of these experiments was to determine effects of sampling protocol on plasma nonesterified fatty acid (NEFA) concentration. In experiment 1, 8 nonlactating, nongestating dairy cows were blood sampled from a jugular vein catheter (basal, 0 min), moved to an exercise lot for 15 min, returned to stanchions, and sampled immediately and at 5, 15, 30, 60, and 120 min following return to their stalls. Following 15 min of exercise, plasma NEFA concentration increased, peaking at 5 min (225 microEq/L) and returning to basal (84 microEq/L) by 30 min (110 microEq/L). Cows were then moved to box stalls overnight, and 24 h after the basal sample, they were locked up and sampled again. Housing cows in a box stall overnight and locking them in headlocks increased plasma NEFA concentration (184 microEq/L). In a second experiment at a large free-stall commercial dairy, 11 late-gestation nonlactating dairy cows were locked in headlocks at feeding, blood was sampled from the coccygeal artery or vein (0 min), and cows were then released and allowed to finish eating and return to their stalls. Cows were then herded to headlocks and sampled immediately at 120 min after initial sampling and at 135, 150, and 180 min. Plasma NEFA concentration was highest at initial lockup (0 min; 284 microEq/L), lowest at 180 min (178 microEq/L), and intermediate at time points in between. A second group of 10 late-gestation nonlactating dairy cows were locked in headlocks at feeding, and blood was sampled immediately and at 5, 15, 30, and 60 min. Plasma NEFA concentration was highest 15 min after being placed in headlocks and lowest 60 min after lockup (221 and 113 microEq/L, respectively). At each time point in experiments 1 and 2, a behavior score was given (1 to 10; 1 = calm; 10 = extremely excited). In both experiments, there was a significant correlation between the plasma NEFA concentration and behavior score. In conclusion, plasma NEFA concentration was affected by sampling protocol.

Feasibility of using genetic linkage analysis to identify the genes encoding T cell-defined minor histocompatibility antigens.

We have evaluated the utility of genetic linkage analysis to identify genes that encode minor histocompatibility antigens using vaccinia virus vectors as a simple and convenient method for transient expression of class I MHC molecules in lymphoblastoid cell lines. As a test case, we used a CTL clone that recognizes HA-8, a minor histocompatibility antigen encoded by the KIAA0020 gene and presented by HLA-A*0201. EBV-transformed B cell lines from individuals in three large pedigrees from the CEPH reference family collection were infected with a recombinant vaccinia virus vector encoding an HLA-A*0201 transgene, which led to high level expression of the MHC restricting allele HLA-A*0201 on the cell surface. HA-8 expression in the vaccinia-infected target cells was then determined using standard in vitro cytotoxicity assays. Pairwise linkage analysis of the segregation of HA-8 expression in these pedigrees demonstrated that the HA-8 gene was tightly linked with a cluster of marker loci located on the distal portion of chromosome 9p. Analysis of 9p marker haplotypes for individuals in the three families identified several individuals with recombinant haplotypes, and these recombination events were used to refine the precision of the HA-8 gene localization further. The data collectively indicate that the HA-8 gene is localized to a 10.3 cM (corresponding to 3.9 Mb) interval of distal 9p that is thought to encode at least 11 genes, including KIAA0020. These results demonstrate that linkage analysis can be used to map minor histocompatibility genes with high precision and accuracy. Over the next years, refinement and annotation of the human genome sequence will undoubtedly increase the utility of linkage analysis as a tool for identifying minor histocompatibility antigen genes.

TSG-6 is concentrated in the extracellular matrix of mouse cumulus oocyte complexes through hyaluronan and inter-alpha-inhibitor binding.

During development of ovarian follicles in mammals, cumulus cells and the oocyte form a mucoelastic mass that detaches itself from peripheral granulosa cell layers upon an ovulatory surge. The integrity of this cumulus-oocyte complex (COC) relies on the cohesiveness of a hyaluronan (HA)-enriched extracellular matrix (ECM). We previously identified a serum glycoprotein, inter-alpha-inhibitor (IalphaI), that is critical in organizing and stabilizing this matrix. Following an ovulatory stimulus, IalphaI diffuses into the follicular fluid and becomes integrated in the ECM through its association with HA. TSG-6 (the secreted product of the tumor necrosis factor-stimulated gene 6), another HA binding protein, forms a complex with IalphaI in synovial fluid. The purpose of this study was to investigate whether TSG-6 is involved in the ECM organization of COCs. Immunolocalization of TSG-6 and IalphaI in mouse COCs at different ovulatory stages was analyzed by immunofluorescence and laser confocal microscopy. IalphaI, TSG-6, and HA colocolized in the cumulus ECM. Western blot analyses were consistent with the presence of both TSG-6 and TSG-6/IalphaI complexes in ovulated COCs. These results suggest that TSG-6 has a structural role in COC matrix formation possibly mediating cross-linking of separate HA molecules through its binding to IalphaI.

The immunogenicity of a new human minor histocompatibility antigen results from differential antigen processing.

Minor histocompatibility antigens (mHAgs) present a significant impediment to organ and bone marrow transplantation between HLA-identical donor and recipient pairs. Here we report the identification of a new HLA-A*0201-restricted mHAg, HA-8. Designation of this mHAg as HA-8 is based on the nomenclature of Goulmy (Goulmy, E. 1996. Curr. Opin. Immunol. 8:75-81). This peptide, RTLDKVLEV, is derived from KIAA0020, a gene of unknown function located on chromosome 9. Polymorphic alleles of KIAA0020 encode the alternative sequences PTLDKVLEV and PTLDKVLEL. Genotypic analysis demonstrated that the HA-8-specific cytotoxic T lymphocyte (CTL) clone SKH-13 recognized only cells that expressed the allele encoding R at P1. However, when PTLDKVLEV was pulsed onto cells, or when a minigene encoding this sequence was used to artificially translocate this peptide into the endoplasmic reticulum, it was recognized by CTLs nearly as well as RTLDKVLEV. This indicates that the failure of CTLs to recognize cells expressing the PTLDKVLEV-encoding allele of KIAA0020 is due to a failure of this peptide to be appropriately proteolyzed or transported. Consistent with the latter possibility, PTLDKVLEV and its longer precursors were transported poorly compared with RTLDKVLEV by transporter associated with antigen processing (TAP). These studies identify a new human mHAg and provide the first evidence that minor histocompatibility differences can result from the altered processing of potential antigens rather than differences in interaction with the relevant major histocompatibility complex molecule or T cell receptor.

A duodenum-specific enhancer regulates expression along three axes in the small intestine.

Adenosine deaminase (ADA) is expressed at high levels in the epithelium of proximal small intestine. Transgenic mice were used to characterize the regulatory region governing this activation. A duodenum-specific enhancer is located in intron 2 of the human ADA gene at the central site among a cluster of seven DNase I-hypersensitive sites present in duodenal DNA. Flanking DNA, including the remaining hypersensitive sites, is required for consistent high-level enhancer function. The enhancer activates expression in a pattern identical to endogenous ADA along both the anterior-posterior axis of the small intestine and the crypt-villus differentiation axis of the intestinal epithelium. Timing of activation by the central enhancer mimics endogenous mouse ADA activation, occurring at 2-3 wk of age. However, two upstream DNA segments, one proximal and one distal, collaborate to change enhancer activation to a perinatal time point. Studies with duodenal nuclear extracts identified five distinct DNase I footprints within the enhancer. Protected regions encompass six putative binding sites for the transcription factor PDX-1, as well as proposed CDX, hepatocyte nuclear factor-4, and GATA-type sites.

Genomic sequence comparison of the human and mouse adenosine deaminase gene regions.

A challenge for mammalian genetics is the recognition of critical regulatory regions in primary gene sequence. One approach to this problem is to compare sequences from genes exhibiting highly conserved expression patterns in disparate organisms. Previous transgenic and transfection analyses defined conserved regulatory domains in the mouse and human adenosine deaminase (ADA) genes. We have thus attempted to identify regions with comparable similarity levels potentially indicative of critical ADA regulatory regions. On the basis of aligned regions of the mouse and human ADA gene, using a 24-bp window, we find that similarity overall (67.7%) and throughout the noncoding sequences (67.1%) is markedly lower than that of the coding regions (81%). This low overall similarity facilitated recognition of more highly conserved regions. In addition to the highly conserved exons, ten noncoding regions >100 bp in length displayed >70% sequence similarity. Most of these contained numerous 24-bp windows with much higher levels of similarity. A number of these regions, including the promoter and the thymic enhancer, were more similar than several exons. A third block, located near the thymic enhancer but just outside of a minimally defined locus control region, exhibited stronger similarity than the promoter or thymic enhancer. In contrast, only fragmentary similarity was exhibited in a region that harbors a strong duodenal enhancer in the human gene. These studies show that comparative sequence analysis can be a powerful tool for identifying conserved regulatory domains, but that some conserved sequences may not be detected by certain functional analyses as transgenic mice.

Regulation of duodenal specific expression of the human adenosine deaminase gene.

Formation of the mammalian gastrointestinal tract is an ordered process of development and differentiation. Yet, the adult small intestine also retains the plasticity to respond to cues both internal and environmental to modulate intestinal function. The components that regulate this development, differentiation, and modulation at the molecular level are only now being elucidated. We have used the human adenosine deaminase (ADA) gene as a model to identify potential cis-regulatory components involved in these processes within the small intestine. In mammals, high levels of ADA in the small intestine are limited specifically to the differentiated enterocytes within the duodenal region. These studies describe the identification of a region of the human ADA gene, completely distinct from the previously identified T-cell enhancer, which is capable of directing the human intestinal expression pattern in the intestine of transgenic mice. The reporter gene expression pattern observed in these transgenic mice is identical to the endogenous gene along both the cephalocaudal and crypt/villus axis of development. Timing of this transgene activation, however, varies from that of the endogenous mouse gene in that the transgene is activated approximately 2 weeks earlier in development. Even so, this precocious activation is also limited to the epithelium of the developing villi strictly within the duodenal region of the small intestine.

Identification of a murine homolog of the human adenosine deaminase thymic enhancer.

We have identified a 236-bp first intron segment of the mouse adenosine deaminase gene (ADA) that shares 71.1% identity with the human ADA thymic enhancer. This segment has the same natural orientation as the human enhancer and a relative location within the first intron very analogous to that of the human enhancer. Four highly conserved regions were defined within this segment, including a 72-bp region having 83.6% identity with a segment containing the critical human enhancer core. Several consensus binding sequences were also conserved within these regions. Transient transfection assays in human and murine T-cell lines revealed that a 1.8-kb murine genomic fragment harboring the 236-bp segment functions as a weak activator of both the human and mouse ADA promoters. In contrast, a 2.3-kb human enhancer fragment exhibited high-level activation in conjunction with either the human or mouse ADA promoter in both the MOLT 4 (human) and S49 (murine) T-cell lines. Interestingly, the murine ADA promoter is significantly stronger than the human promoter in driving cat expression in transient transfection assays in all the T-cell lines tested.

Clinical diagnoses and EEG interpretation.

A sample of 62 electroencephalographers in the United States evaluated 10-second samples of eight electroencephalograms. The evaluations were performed with and without knowledge of the clinical history. Evaluations consisted of multiple choice questions related to electroencephalographic observations, clinical diagnosis, and requests for additional tests such as computerized tomography and cerebrospinal fluid studies. The results indicate that clinical history influences interpretation, with considerable variation among readers in the number and type of additional tests requested.

Interobserver variability in EEG interpretation.

A random sample of 100 active electroencephalographers in the United States evaluated 10-second samples of 12 selected EEGs. The evaluations consisted of multiple-choice questions related to the age of the patient, EEG finding, artifact, and consciousness of the patient. The rate of reporting the "correct" response was examined in terms of various respondent characteristics such as EEG board certification, age, percent of time in clinical EEG work, and number of recordings interpreted annually. This study indicates that, even today, there is considerable variability in EEG interpretation, and that this variability is influenced by specific reader characteristics.

Functional domains of the alpha subunit of the eighth component of human complement: identification and characterization of a distinct binding site for the gamma chain.

The purified gamma subunit of the eighth component of human complement (C8) was used to characterize its site of interaction within C8 and to probe the ultrastructure of membrane-bound C5b-8 and C5b-9 complexes. Purification of gamma was accomplished by separating the disulfide-linked alpha-gamma subunit from the noncovalently associated beta chain and subjecting the former to limited reduction, alkylation, and ion-exchange chromatography. Upon mixing, purified alpha and gamma exhibited a high affinity for each other, as evidenced by their ability to form a noncovalent, equimolar complex at dilute concentrations and in the presence of excess serum albumin. Purified gamma also exhibited an affinity for C8', a previously described derivative that is functionally similar to C8 although it is composed of only alpha and beta. These results indicate that alpha possesses a specific site for interaction with gamma and that this site is preserved in the isolated subunit. Furthermore, this site remains accessible when alpha is associated with beta. In related experiments, gamma was found to specifically associate with membrane-bound C5b-8' and C5b-(8')9 complexes. These results indicate that the site for gamma interaction remains accessible on alpha in C5b-8' and is not shielded by C9 within C5b-(8')9. It is concluded that the gamma subunit of C8 is located on the surface of membrane-bound C5b-8 and C5b-9.

Measurement of the ratio of the eighth and ninth components of human complement on complement-lysed membranes.

The mole ratio of the eighth (C8) and ninth (C9) components of human complement on membranes carrying the cytolytic C5b-9 complex was measured by direct binding assays. Erythrocytes from two different species were used as the membrane system. Antibody-treated sheep erythrocytes carrying a relatively small number of precursive membrane-bound C5b-7 complexes were prepared by exposure to human C8-depleted serum. These complexes were subsequently converted to C5b-8 by addition of saturating amounts of C8. Parallel binding assays using 125I-C8 were used to determine the exact amount bound and thus the number of C5b-8 complexes per cell. These cells were subsequently incubated with excess 125I-C9 and the amount bound relative to C8 on the membrane was measured. Results indicated the C8:C9 ratio remained constant at approximately 1:4 as the number of complexes varied from 40 to 310 per cell. Similar results were obtained regardless of whether C8 and C9 were added sequentially or simultaneously to cells bearing C5b-7. For comparison, experiments were also performed using membranes that contained a high number of complexes. Here, rabbit erythrocytes which carried approximately 25 000 C5b-7 per cell were incubated with limited amounts of C8 to form C5b-8 complexes on the membrane surface, the exact number of which was measured by 125I-C8 binding assays. When erythrocytes prepared in this manner were incubated with excess 125I-C9, the ratio of C8:C9 on the membrane was found to be essentially constant at approximately 1:3 as the number of these complexes varied from 50 to 4000 per cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Function of subunits within the eighth component of human complement: selective removal of the gamma chain reveals it has no direct role in cytolysis.

The eighth component of human complement (C8) consists of a disulfide-linked alpha-gamma dimer that is noncovalently associated with beta. Previous results from this laboratory established that two of these subunits have distinct roles in the cytolytic function of C8. Binding of C8 to the precursive C5b-7 complex is mediated strictly through beta while interaction between C8 and the lipid bilayer of target membranes occurs primarily through alpha. In the present study, we examined the importance of the gamma subunit in cytolysis by characterizing functional properties of C8', a derivative of C8 that lacks gamma. Preparation of this derivative was accomplished by limited cleavage of disulfide bonds in purified alpha-gamma and separation of alpha from gamma. When mixed, purified alpha and beta combined to form C8'. When tested for functional similarity to normal C8, the following results were obtained. (1) Specific and saturable binding of C8' to the C8 binding site on C5b-7 could be achieved. Significantly, the resulting C5b-8' supported subsequent C9 binding and cell lysis, which was equivalent to that observed with C5b-8. (2) Complement activation of (C8 + C9)-depleted serum containing C8' resulted in formation of SC5b-8'. Inclusion of C9 in the serum resulted in further conversion to SC5b-(8')9. These observations indicate that C8' is functionally similar to C8. Furthermore, they indicate that unlike alpha and beta, the gamma subunit has no direct role in facilitating C8 interaction with the nascent cytolytic complex or in mediating C9 binding and membrane lysis.(ABSTRACT TRUNCATED AT 250 WORDS)