Effects of dietary supplementation of an enzyme blend on the ileal and fecal digestibility of nutrients in growing pigs.

The objective of this experiment was to determine the effect of a beta-glucanase-protease enzyme blend product (EBP) on fecal digestibility (FD), apparent ileal digestibility (AID), standardized ileal digestibility, and digestibility in the hindgut of growing pigs. Twelve ileal-cannulated, growing barrows (38.2 +/- 0.5 kg) were housed in individual metabolism crates, blocked by previous feed intake into 3 groups with 4 pigs each, and randomly assigned to 1 of 4 treatments within a square (group) of 3 replications of 4 x 4 Latin square design. Treatments were basal diet (Basal), Basal + 0.05% of EBP (0.05% EBP), Basal + 0.10% of EBP (0.10% EBP), and hydrolyzed casein for measurement of endogenous amino acids. The Basal diet consisted of corn and soybean meal and was calculated to have 3.36 Mcal of ME/kg and 1.1% of total lysine, as-fed basis. Feed intake of each replicate of the Latin square during the first period was 85% of the minimum feed intake of the 4 pigs during the preliminary period and was equalized within each square. The feeding level was increased by 100 g/d in each subsequent period. Each of the experimental periods was 14 d, including 4 d of dietary adaptation, 5 d of fecal collection, 3 d of transition period, and 2 d of ileal collection. Ileal effluents were collected continuously for the same 12-h interval each day. Pigs fed the EBP demonstrated increased (P < 0.05) FD of DM, OM, energy, CP, nonfiber carbohydrate, total dietary fiber, insoluble dietary fiber, acid-hydrolyzed fat, ash, Ca, and P compared with pigs fed Basal. The AID of NDF and hemicellulose was increased (P < 0.05) by supplying the EBP either at 0.05 or 0.10% in the diets, but AID of DM and energy was not increased. The AID of acid-hydrolyzed fat tended to be greater (P = 0.051) for the pigs fed the EBP than for those fed Basal. Ileal digestibility of most amino acids was not affected by treatment, but the EBP reduced the apparent and standardized digestibility of methionine, alanine, and serine (P < 0.05). The difference between FD and AID of hemicellulose was lower (P < 0.05) for the pigs fed the EBP than for those fed Basal. These results demonstrated that the EBP fed to growing pigs improved the FD of DM, OM, energy, CP, nonfiber carbohydrate, total dietary fiber, acid-hydrolyzed fat, Ca, and P, and the AID of NDF and hemi-cellulose, but the standardized ileal digestibility of amino acids was not improved by supplying the EBP in corn-soybean meal-based diets of growing pigs.

Protein cross-linking in food.

The aims of this paper are (1) to probe the relationship between molecular structure and protein cross-linking ability for a range of small molecules; (2) to establish whether this relationship holds within a food matrix; and (3) to test the impact of Maillard cross-linking on food functionality, particularly texture, in wheat- and soy-based food systems. A variety of molecules were obtained, either commercially or via organic synthesis. Cross-linking ability was tested using our standard model system, employing ribonuclease A and analyzing the results by SDS-PAGE. Molecules of varying reactivity were tested in wheat- and soy-based products, and the changes in functionality were correlated with changes in protein cross-linking. No simple relationship was found between molecular structure and ability to cross-link ribonuclease. Only the most reactive reagents were able to cross-link within the food matrix. Nevertheless, a low degree of cross-linking was shown to have significant consequences on the properties of wheat- and soy-based foods, suggesting that the Maillard reaction may represent a means to control food texture.

MlrA, a novel regulator of curli (AgF) and extracellular matrix synthesis by Escherichia coli and Salmonella enterica serovar Typhimurium.

Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium) is associated with extracellular matrix production and is optimal at low temperature during stationary phase. Curli and extracellular matrix synthesis involves a complex regulatory network that is dependent on the CsgD (AgfD) regulator. We have identified a novel regulator, termed MlrA, that is required for curli production and extracellular matrix formation. Two cosmids from a genomic library of avian pathogenic E. coli chi7122 conferred mannose-resistant haemagglutination (HA) and curli production to E. coli HB101, which is unable to produce curli owing to a defective regulatory pathway. The rpoS gene, encoding a known positive regulator of curli synthesis, and the E. coli open reading frame (ORF) of unknown function, yehV, identified on each of these cosmids, respectively, conferred curli production and HA to E. coli HB101. We have designated yehV as the mlrA gene for MerR-like regulator A because its product shares similarities with regulatory proteins of the MerR family. HA and curli production by strain chi7122 were abolished by disruption of rpoS, mlrA or csgA, the curli subunit gene. Both csgD and csgBA transcription, required for expression of curli, were inactive in an mlrA mutant grown under conditions that promote curli production. An mlrA homologue was identified in S. typhimurium. Analysis of mlrA-lac operon fusions demonstrated that mlrA was positively regulated by rpoS. mlrA mutants of wild-type S. typhimurium SL1344 or SR-11 no longer produced curli or rugose colony morphology, and exhibited enhanced aggregation and extracellular matrix formation when complemented with the mlrA gene from either S. typhimurium or E. coli present on a low-copy-number plasmid. However, inactivation of mlrA did not affect curli production and aggregative morphology in an upregulated curli producing S. typhimurium derivative containing a temperature- and RpoS-independent agfD promoter region. These results indicate that MlrA is a newly defined transcriptional regulator of csgD/agfD that acts as a positive regulator of RpoS-dependent curli and extracellular matrix production by E. coli and S. typhimurium.

The propeptide of the transforming growth factor-beta superfamily member, macrophage inhibitory cytokine-1 (MIC-1), is a multifunctional domain that can facilitate protein folding and secretion.

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-beta (TGF-beta) superfamily. While it is synthesized in a pre-pro form, it is unique among superfamily members because it does not require its propeptide for correct folding or secretion of the mature peptide. To investigate factors that enable these propeptide independent events to occur, we constructed MIC-1/TGF-beta1 chimeras, both with and without a propeptide. All chimeras without a propeptide secreted less efficiently compared with the corresponding constructs with propeptide. Folding and secretion were most affected after replacement of the predicted major alpha-helix in the mature protein, residues 56-68. Exchanging the human propeptide in this chimera with either the murine MIC-1 or TGF-beta1 propeptide resulted in secretion of the unprocessed, monomeric chimera, suggesting a specific interaction between the human MIC-1 propeptide and mature peptide. Propeptide deletion mutants enabled identification of a region between residues 56 and 78, which is important for the interaction between the propeptide and the mature peptide. Cotransfection experiments demonstrated that the propeptide must be in cis with the mature peptide for this phenomenon to occur. These results suggest a model for TGF-beta superfamily protein folding.

Epitope mapping of the transforming growth factor-beta superfamily protein, macrophage inhibitory cytokine-1 (MIC-1): identification of at least five distinct epitope specificities.

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-beta (TGF-beta) superfamily whose increased expression is associated with macrophage activation and which is expressed highly in placenta as compared to other tissues. There are two known allelic forms of human MIC-1 due an amino acid substitution at position 6 of the mature protein. We have raised four monoclonal antibodies (MAbs) and one polyclonal antiserum to the mature protein region of human MIC-1 and have used an extensive panel of MIC-1 relatives, mutants, and chimeras to map their epitopes. None of the MAbs were able to cross-react with either the murine homologue of MIC-1 or with hTGF-beta1, and all of the MAb epitopes were conformation-dependent. A distinct cross-reactivity pattern with the various antigens was observed for each of the monoclonal and polyclonal antibodies suggesting the presence of at least five immunogenic regions on the MIC-1 surface. One of the MAbs is directed against the amino terminus of the protein and can distinguish between the two allelic forms of MIC-1. The epitopes for the other three MAbs were located near the tips of the so-called "fingers" of the protein and appeared to be partially overlapping as each involved amino acids in the region 24-37. In one case, it was possible to mutate murine MIC-1 so that it could be recognized by one of the MAbs. Finally, the use of another mutant in which Cys 77 was replaced by serine enabled confirmation of the location of the MIC-1 interchain disulfide bond.

Expression of a TGF-beta superfamily protein, macrophage inhibitory cytokine-1, in the yeast Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, has been used to express both human and murine macrophage inhibitory cytokine-1 (MIC-1), a transforming growth factor beta (TGF-beta) superfamily cytokine. This is the first report of the expression of a correctly folded TGF-beta superfamily protein in a microbial organism. The protein is secreted in its correctly folded dimeric form at milligram per litre quantities, which are significantly higher than we have been able to achieve using mammalian expression systems. Purification schemes are described, and the purified protein is immunologically identical to protein produced in a mammalian expression system. Protein expression was influenced by a number of factors, most significantly by the concentration of methanol used during the induction phase. However, with very high levels of MIC-1 induction, substantial amounts of MIC-1 monomer were also secreted.

The transforming growth factor-ss superfamily cytokine macrophage inhibitory cytokine-1 is present in high concentrations in the serum of pregnant women.

Macrophage inhibitory cytokine-1 (MIC-1) is a recently described divergent member of the transforming growth factor-ss superfamily. MIC-1 transcription up-regulation is associated with macrophage activation, and this observation led to its cloning. Northern blots indicate that MIC-1 is also present in human placenta. A sensitive sandwich enzyme-linked immunosorbent assay for the quantification of MIC-1 was developed and used to examine the role of this cytokine in pregnancy. High levels of MIC-1 are present in the sera of pregnant women. The level rises substantially with progress of gestation. MIC-1 can also be detected, in large amounts, in amniotic fluid and placental extracts. In addition, the BeWo placental trophoblastic cell line was found to constitutively express the MIC-1 transcript and secrete large amounts of MIC-1. These findings suggest that the placental trophoblast is a major source of the MIC-1 present in maternal serum and amniotic fluid. We suggest that MIC-1 may promote fetal survival by suppressing the production of maternally derived proinflammatory cytokines within the uterus.

Hypersegmented megakaryocytes and megakaryocytes with multiple separate nuclei in dogs treated with PNU-100592, an oxazolidinone antibiotic.

Megakaryocytes in normal dogs have a variety of nuclear forms. The most common variations are a single large multilobed nucleus or a segmented nucleus consisting of irregular nuclear lobes joined by strands of chromatin. Exaggerated segmentation (hypersegmentation) of the nucleus occurs in a small number of megakaryocytes. Megakaryocytes with multiple separate nuclei are infrequently observed. In a 1-mo toxicology study in young adult beagle dogs with PNU-100592, a new oxazolidinone antibacterial agent, a large increase in the number of megakaryocytes with hypersegmented nuclei and multiple separate nuclei was observed. The group mean platelet count was slightly decreased for most PNU-100592-treated groups. Siderocytes were observed on peripheral blood smears, and ring sideroblasts were present on bone marrow smears. Minimal to mild toxicologic lesions were observed in the large intestine, rectum, kidneys, liver, and testes, primarily in the high-dose group. PNU-100592 may be useful in the study of the regulation of endomitosis during megakaryocytopoiesis in the dog.

Unique chromosomal regions associated with virulence of an avian pathogenic Escherichia coli strain.

The avian pathogenic Escherichia coli strain (chi)7122 (serotype O78:K80:H9) causes airsacculitis and colisepticemia in chickens. To identify genes associated with avian disease, a genomic subtraction technique was performed between strain (chi)7122 and the E. coli K-12 strain (chi)289. The DNA isolated using this method was found only in strain (chi)7122 and was used to identify cosmid clones carrying unique DNA from a library of (chi)7122 that were then used to map the position of unique DNA on the E. coli chromosome. A total of 12 unique regions were found, 5 of which correspond to previously identified positions for unique DNA sequence in E. coli strains. To assess the role each unique region plays in virulence, mutants of (chi)7122 were constructed in which a segment of unique DNA was replaced with E. coli K-12 DNA by cotransduction of linked transposon insertions in DNA flanking the unique sequence. The resulting replacement mutants were assessed for inability to colonize the air sac and cause septicemia in 2-week-old white Leghorn chickens. Two mutants were found to be avirulent when injected into the right caudal air sac of 2-week-old chickens. One avirulent mutant, designated (chi)7145, carries a replacement of the rfb locus at 44 min, generating a rough phenotype. The second mutant is designated (chi)7146, and carries a replacement at position 0.0 min on the genetic map. Both mutants could be complemented to partial virulence by cosmids carrying sequences unique to (chi)7122.

Use of Salmonella phage P22 for transduction in Escherichia coli.

A cosmid (pPR1347) carrying both the rfb gene cluster and the rfc gene of a Salmonella group B serovar has been constructed; Escherichia coli K-12 strains carrying this cosmid produce long-chain O antigen, are sensitive to phage P22, and can be transduced by P22. Some of the benefits of P22 transduction are now available for studying E. coli and potentially other genera.

Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length.

We report the identification and sequence from Escherichia coli and Salmonella enterica strains of the cld gene, encoding the chain-length determinant (CLD) which confers a modal distribution of chain length on the O-antigen component of lipopolysaccharide (LPS). The distribution of chain lengths in the absence of this gene fits a model in which as the chain is extended there is a constant probability of 0.165 of transfer of growing chain to LPS core, with termination of chain extension. The data for E. coli O111 fit a model in which the CLD reduces this probability for short chains and increases it to 0.4 for longer chains, leading to a reduced number of short chain molecules but an increase in numbers of longer molecules and transfer of essentially all molecules by chain length 21. We put forward a model for O-antigen polymerase which resembles the ribosome and fatty acid synthetase in having two sites, with the growing chain being transferred from a D site onto the new unit at the R site to extend the chain and then back to the D site to repeat the process. It is proposed that the CLD protein and polymerase form a complex which has two states: 'E' facilitating extension and 'T' facilitating transfer to core. The complex is postulated to enter the E state as O-antigen polymerization starts, and to shift to the T state after a predetermined time, the CLD acting as a molecular clock. The CLD is not O-antigen or species-specific but the modal value does depend on the source of the cld gene.

Molecular analysis of the rfb gene cluster of Salmonella serovar muenchen (strain M67): the genetic basis of the polymorphism between groups C2 and B.

The rfb (O antigen) gene cluster of group C2 Salmonella differs from that of group B in a central region of 12.4 kb: we report the sequencing of this region of strain M67 (group C2) and a subsequent comparison with the central region of strain LT2 (group B). We find a block of seven open reading frames unique to group C2 which encode the O antigen polymerase (rfc) and the transferases responsible for assembly of the group C2 O antigen. The remaining rfb genes are common to strains M67 and LT2, but rfbJ (CDP-abequose synthase) and rfbM and rfbK (GDP-mannose synthesis), which are immediately adjacent to the central region, are highly divergent. All these genes have a low G+C content and appear to have been recent additions to Salmonella enterica. We discuss the evolutionary significance of the arrangement and divergence of the genes in the polymorphism of the rfb cluster.

Molecular cloning and genetic characterization of the rfb region from Yersinia pseudotuberculosis serogroup IIA, which determines the formation of the 3,6-dideoxyhexose abequose.

The rfb region of Yersinia pseudotuberculosis serogroup IIA has been cloned and expression of O antigen in Escherichia coli K12 was demonstrated. Transposon mutagenesis analysis confined the DNA region required for O antigen expression to a 19.3 kb fragment, and the O antigen expressed was visualized by SDS-PAGE and silver staining. Southern hybridization analysis demonstrated significant levels of similarity between the Yersinia rfb region and the 3,6-dideoxyhexose pathway genes rfbF and rfbG, previously isolated from Salmonella enterica LT2, but no similarity to the abequose synthase gene rfbJ of the same strain or the paratose synthase gene rfbS isolated from S. enterica Ty2. The evolutionary relationship between the abequose biosynthetic genes of the two species of Salmonella and Yersinia is discussed.

Cloning of the rfb gene cluster of a group C2 Salmonella strain: comparison with the rfb regions of groups B and D.

We report the cloning and mapping of the entire rfb gene cluster of a group C2 Salmonella strain. Comparison with the rfb region of group B strain LT2 and group D strain Ty2 reveals an 11.8 kb central region of limited similarity flanked by regions of high similarity. The genes from the central region confer a group C2 O-antigen structure on a Salmonella LT2 partial delete strain. The significance of this region in relation to function and evolutionary origin is discussed. We also report evidence for the existence of an O-antigen chain-length determinant in Escherichia coli K12 and propose a model for a possible mechanism by which a preferred chain length is determined.

Clinical measurement of glare effect in cataract patients.

Glare complaints are common for patients with good visual acuity. Spot glare sensitivity is a sensitive measure of intraocular scattering caused by cataracts. The TVA functional vision tester was used to measure contrast sensitivity with and without spot glare. The case studies presented compare relative spot glare disability for cataractous and normal patients with the same visual acuity. The glare measurements show distinct attenuation of contrast sensitivity for cataract patients over patients with no ocular pathology.

Use of a rapid and highly sensitive fluorescamine-based procedure for the assay of plasma lipoproteins.

A rapid and sensitive method for determining protein concentrations using fluorescamine has been characterized for use in the analysis of intact lipoproteins. It was shown that there is no interference with the assay due to the presence of lipid-associated turbidity or primary amine content. The assay was shown to be sensitive to as little as 0.3 microgram of lipoprotein and to yield similar results when compared to the Lowry method.

Diet induced atherogenic hyperlipoproteinaemia and liver injury in cynomolgus macaques.

This study was conducted to determine the efficacy of an experimental anti-atherosclerosis drug in the adult male cynomolgus monkey. A semipurified diet containing 0.5% cholesterol and 25.5% butter was fed to groups of 20, each, drug and placebo-treated animals for 18 months. Similar liver and arterial changes were present in both groups. However, we report here tissue changes seen in animals given placebo only, with plasma lipid and lipoprotein values of placebo-treated animals compared to those in animals fed nonatherogenic commercial ration. Animals fed atherogenic diet had enlarged livers (mean 3.9% b.w.), and all had evidence of hepatocellular lipid accumulation which was often marked and diffuse. Cholangitis was common including mononuclear cell infiltration, bile ductule proliferation and portal tract fibrosis. Five animals had severe portal fibrosis with bands of connective tissue extending into and around lobules (bridging fibrosis). All animals fed atherogenic diet developed hypercholesterolemia (greater than 600 mg/dl) which was the result of a three-fold increase in five cholesterol and cholesterol ester. Oleic acid was increased and linoleic acid was reduced in plasma phospholipids and cholesterol esters. Plasma lipoprotein distribution was altered with a marked increase in low density lipoproteins, increased very low density lipoproteins and decreased high density lipoproteins. These changes were undoubtedly caused by diet, i.e., high in cholesterol and saturated fat and limiting in linoleic acid. It is probable that diet-induced liver injury would affect the pathogenesis of atherosclerosis in this model since the liver is central in the synthesis and metabolism of lipoproteins.

Properties of the visual pigments of the moth Manduca sexta and the effects of two detergents, digitonin and chaps.

The three known visual pigments (P520, P450, P357) of the moth, Manduca sexta (Lepidoptera, Sphingidae), were extracted in two different detergents (2% digitonin, 6 or 12 mM CHAPS). As is the case in unextracted membranes, the metarhodopsins are quite stable in CHAPS extracts, while in digitonin the metarhodopsins of P520 and P450 decay rapidly at 15 degrees C to opsin and free retinal. The relative absorbance ratios are: 1.0:1.6 (P520:M485), 1.0:1.1 (P450:M485), and 1.0:0.8 (P357:M470). The relative amounts of the visual pigments found in digitonin extracts is 100:25:8 (P520:P450:P357); about 60 picomoles of P520 can be extracted from one Manduca retina.

The macular pigment. I. Absorbance spectra, localization, and discrimination from other yellow pigments in primate retinas.

The nonbleaching yellow pigments of the primate fovea were studied by microspectrophotometry (MSP). Retinas fixed with glutaraldehyde/paraformaldehyde mixtures retained yellow pigments with absorbance spectra very similar to those recorded by MSP in fresh retinas. This allowed the authors to prepare retinal sections for localization of the pigments. The spectrum of the macular pigment in fixed tissue is shifted slightly (about 6 nm) toward longer wavelengths, with maximum absorbance at 460 nm. Two short-wavelength yellow pigments also have been identified, with absorbance maxima at 410 nm ( P410 ) and 435 nm ( P435 ), respectively. All three yellow pigments are present in the fovea. The short-wavelength pigments are detected more easily outside the central foveal region because the macular pigment does not obscure them there. They are especially apparent when the MSP beam is confined to the outer nuclear layer or the inner segment layer of retinal sections. The macular pigment is most dense in the fiber layers (receptor axon layer and inner plexiform layer); its density declines markedly with retinal eccentricity. The maximal absorbance of P410 and P435 is usually lower than that of the macular pigment in the central fovea, but their densities and relative proportions change more gradually with eccentricity. Consequently, their maximal absorbance is higher than that of the macular pigment outside the foveal center. The P410 and P435 pigments may be two different oxidation states of one or more respiratory hemoproteins. Commonly used procedures for estimating the absorbance spectrum of the macular pigment by comparing the foveal center with a parafoveal region may be influenced by the amounts and the oxidation states of the short-wavelength pigments in the living eye.

Ultrastructure of the eye of a euphausiid crustacean.

The compound eye of the Antarctic euphausiid Euphausia superba is a spherical clear zone eye. The dioptric system consists of a hexagonally-faceted cornea, two corneagenous cells, two crystalline cone cells which form the bipartite crystalline cone, and two accessory cone cells. The dioptric system of each ommatidium is separated from that of adjacent ommatidia by six distal pigment cells and a basement membrane. The proximal tip of the crystalline cone is cupped by the distal ends of the seven retinula cells whose nuclei are arranged in a staggered array slightly distal to the middle of the clear zone. In the distal half of the clear zone, each narrow retinula cell column is surrounded by large proximal extensions of the six distal pigment cells. The pigment cells narrow more proximally and terminate at the proximal basement membrane. A specialized axial channel complex extends from the crystalline cone through the clear zone, and is continuous with a conical refractive element which caps the distal end of the rhabdom. The rhabdom is fused, and made up of alternating highly birefringent layers of orthogonally-oriented microvilli. It is surrounded by a narrow extra-cellular space which is continuous with the distal refractive element and a second conical refractive element at the proximal end of the rhabdom.