Alterations at the intercalated disk associated with the absence of muscle LIM protein.

In this study, we investigated cardiomyocyte cytoarchitecture in a mouse model for dilated cardiomyopathy (DCM), the muscle LIM protein (MLP) knockout mouse and substantiated several observations in a second DCM model, the tropomodulin-overexpressing transgenic (TOT) mouse. Freshly isolated cardiomyocytes from both strains are characterized by a more irregular shape compared with wild-type cells. Alterations are observed at the intercalated disks, the specialized areas of mechanical coupling between cardiomyocytes, whereas the subcellular organization of contractile proteins in the sarcomeres of MLP knockout mice appears unchanged. Distinct parts of the intercalated disks are affected differently. Components from the adherens junctions are upregulated, desmosomal proteins are unchanged, and gap junction proteins are downregulated. In addition, the expression of N-RAP, a LIM domain- containing protein located at the intercalated disks, is upregulated in MLP knockout as well as in TOT mice. Detailed analysis of intercalated disk composition during postnatal development reveals that an upregulation of N-RAP expression might serve as an early marker for the development of DCM. Altered expression levels of cytoskeletal proteins (either the lack of MLP or an increased expression of tropomodulin) apparently lead to impaired function of the myofibrillar apparatus and to physiological stress that ultimately results in DCM and is accompanied by an altered appearance and composition of the intercalated disks.

In vitro and in vivo properties of the bovine antimicrobial peptide, Bactenecin 5.

Antimicrobial peptides (AMP), part of the innate immune system, are well studied for their ability to kill pathogenic microorganisms. However, many also possess important immunomodulatory effects, and this area has potential for the development of novel therapies to supplement traditional methods such as the use of antibiotics. Here, we characterise the microbicidal and immunomodulatory potential of the proline-rich bovine AMP, Bactenecin 5 (Bac5). We demonstrate broad antimicrobial activity, including against some mycobacterial species, which are important pathogens of fish, cattle and humans. Bac5 is able to activate macrophage-like THP-1 cells and can synergistically trigger the upregulation of tnf-α when co-stimulated with M. marinum. Furthermore, Bac5 sensitises A549 epithelial cells to stimulation with TNF-α. For the first time, we characterise the activity of Bac5 in vivo, and show it to be a potent chemokine for macrophages in the zebrafish (Danio rerio) embryo model of infection. Bac5 also supports the early recruitment of neutrophils in the presence of M. marinum. In the absence of host adaptive immunity, exogenous injected Bac5 is able to slow, although not prevent, infection of zebrafish with M. marinum.

Altered focal adhesion regulation correlates with cardiomyopathy in mice expressing constitutively active rac1.

The ras family of small GTP-binding proteins exerts powerful effects upon cell structure and function. One member of this family, rac, induces actin cytoskeletal reorganization in nonmuscle cells and hypertrophic changes in cultured cardiomyocytes. To examine the effect of rac1 activation upon cardiac structure and function, transgenic mice were created that express constitutively activated rac1 specifically in the myocardium. Transgenic rac1 protein was expressed at levels comparable to endogenous rac levels, with activation of the rac1 signaling pathway resulting in two distinct cardiomyopathic phenotypes: a lethal dilated phenotype associated with neonatal activation of the transgene and a transient cardiac hypertrophy seen among juvenile mice that resolved with age. Neither phenotype showed myofibril disarray and hypertrophic hearts were hypercontractilein working heart analyses. The rac1 target p21-activated kinase translocated from a cytosolic to a cytoskeletal distribution, suggesting that rac1 activation was inducing focal adhesion reorganization. Corroborating results showed altered localizations of src in dilated cardiomyopathy and paxillin in both cardiomyopathic phenotypes. This study, the first examination of rac1-mediated cardiac effects in vivo, demonstrates that dilation and hypertrophy can share a common molecular origin and presents evidence that both timing and concurrent signaling from multiple pathways can influence cardiac remodeling.

Left ventricular hypertrophy in ascending aortic stenosis mice: anoikis and the progression to early failure.

BACKGROUND: To determine potential mechanisms of the transition from hypertrophy to very early failure, we examined apoptosis in a model of ascending aortic stenosis (AS) in male FVB/n mice. METHODS AND RESULTS: Compared with age-matched controls, 4-week and 7-week AS animals (n=12 to 16 per group) had increased ratios of left ventricular weight to body weight (4.7+/-0.7 versus 3.1+/-0.2 and 5. 7+/-0.4 versus 2.7+/-0.1 mg/g, respectively, P<0.05) with similar body weights. Myocyte width was also increased in 4-week and 7-week AS mice compared with controls (19.0+/-0.8 and 25.2+/-1.8 versus 14. 1+/-0.5 microm, respectively, P<0.01). By 7 weeks, AS myocytes displayed branching with distinct differences in intercalated disk size and staining for beta(1)-integrin on both cell surface and adjacent extracellular matrix. In vivo left ventricular systolic developed pressure per gram as well as endocardial fractional shortening were similar in 4-week AS and controls but depressed in 7-week AS mice. Myocyte apoptosis estimated by in situ nick end-labeling (TUNEL) was extremely rare in 4-week AS and control mice; however, a low prevalence of TUNEL-positive myocytes and DNA laddering were detected in 7-week AS mice. The specificity of TUNEL labeling was confirmed by in situ ligation of hairpin oligonucleotides. CONCLUSIONS: Our findings indicate that myocyte apoptosis develops during the transition from hypertrophy to early failure in mice with chronic biomechanical stress and support the hypothesis that the disruption of normal myocyte anchorage to adjacent extracellular matrix and cells, a process called anoikis, may signal apoptosis.

Ultrastructural localization of laminin on in vivo embryonic, neonatal, and adult rat cardiac myocytes and in early rat embryos raised in whole-embryo culture.

The temporal and spatial distribution of the basement membrane component laminin was examined in vivo in developing rat hearts at 11.5 and 15 days of embryonic development (ED), and in neonates and adults, by pre-embedding ultrastructural immunocytochemistry. In addition, the patterns observed at 11.5 days ED were compared to the distribution of laminin in embryos maintained in whole-embryo culture. At 11.5 days ED laminin was localized in punctate patches on the surface of the plasma membrane, with large gaps between areas of staining. The development of myocytes and localization of laminin in the whole embryo-cultured embryos was similar to that found in the in vivo embryos. At 15 days ED, laminin localization was limited to distinct patches of developing extracellular matrix material associated with the sarcolemma. Gaps between areas of localization were shorter than in the 11.5-day hearts. In neonates, distribution of laminin localization was more extensive with fewer gaps and was associated with the developing basement membrane. In adult hearts, laminin was localized along the entire length of the basement membrane and was heaviest in areas of morphological specialization, such as Z-bands, where collagen bundles contacted the sarcolemma.

Distribution of beta-1 integrin in the developing rat heart.

Cell-cell and cell-matrix interactions play critical roles in various developmental processes including differentiation, proliferation, and migration. Members of the integrin family of cell surface components are important mediators of these cell-extracellular matrix (ECM) contacts or interactions. The ECM provides signals to individual cells essential for development and differentiation and plays essential roles in establishing and maintaining the complex structure of the vertebrate heart. Integrins provide a fundamental link for transduction of developmental signals to cells. Integrin expression by cardiac myocytes is altered during neonatal heart development and disease; however, little is known regarding the spatial and temporal patterns of integrin expression during embryonic and fetal heart development. Essential to understanding the role of integrins in the organization of the heart, the present studies have localized beta-1 integrin protein and mRNA in fetal and neonatal rat hearts. Beta-1 integrin is predominantly found in regions of remodeling (trabeculae) in the early heart (10-13 days of gestation). Later in development (15 days of gestation onward), beta-1 integrin is abundant in regions containing an elaborate ECM, such as the valves. These studies further support the hypothesis that the expressions of integrins and ECM are coordinately regulated in the developing heart.

Increased co-localization of connexin43 and ZO-1 in dissociated adult myocytes.

In the heart, the intercellular geometry of myocyte coupling by Connexin43-gap junctions (Cx43-gjs) is a determinant of normal and abnormal patterns of propagation of electrical excitation. ZO-1 has been suggested to play a role in determining the pattern of intercellular coupling between myocytes. We therefore investigated the co-distribution of Cx43 with ZO-1 in ventricular myocytes of the adult rat using quantitative immunoconfocal microscopy. Our data indicates that low-moderate levels of co-immunolocalization occur between Cx43 and ZO-1 in normal ventricular myocardium. However, rapid and significant increases in relative co-localization occur between Cx43 and ZO-1 following dissociation of myocytes from ventricular myocardium--a treatment inducing internalization of Cx43-gjs. This increased relative co-localization may represent an increase in Cx43-ZO-1 interaction, suggesting a role for ZO-1 in the remodeling of myocardial Cx43-gjs. A more comprehensive study, including immunoprecipitation and immunoelectron microscopy analyses has been carried out (Barker et al. Circ. Res., in press, 2002 and as presented to the 2001 International GJ Conference). This study further assesses the biological relevance of the increased association between ZO-1 and Cx43 accompanying internalization of Cx43-gjs.

Mechanical regulation of cardiac myofibrillar structure.

The excitation-contraction coupling cycle (ECC) consists of a complex cascade of electrochemical and mechanical events; however, the relative contributions of these different processes in the regulation of cardiac myofibrillar structure are not well understood. There is extensive evidence to suggest that the mechanical aspects of the ECC play a crucial role in controlling the availability of contractile proteins for myofibrillar assembly. To examine if these physical forces might also serve to stabilize the structure of preexisting myofibrils, beating and nonbeating cultures of neonatal cardiac myocytes (NCM) were subjected to a 5% static stretch. Contractile arrest was achieved by treating NCM with 12 microM nifedipine, which resulted in immediate and sustained contractile arrest and initiated the evolution of marked myofibrillar abnormalities within 24 hours. As judged by scanning confocal and transmission electron microscopic examination, an external load appears to partially stabilize myofibrillar structure in nonbeating NCM. These results suggest that the maintenance of myofibrillar structure may be highly dependent upon the mechanical aspects of ECC.

Microtubules have special physical associations with smooth endoplasmic reticula and mitochondria in axons.

Ultrastructural morphometry was used to document the non-random spatial distributions of organelles within the compact myelinated region of avian oculomotor axons. These regions contain large numbers of loosely packed neurofilaments (NFs) (241/microns 2) and only a relatively small number of microtubules (MTs) (4/microns 2), mitochondria (0.6/microns 2), and smooth endoplasmic reticulum (SER) (1.6/microns 2). Random co-occurrences between the relatively sparsely distributed MTs, mitochondria, and SER are probably infrequent in these axons. The actual co-occurrences of MTs, mitochondria, and SER with MTs were counted and compared to the co-occurrences expected in a random Poisson distribution. At long distances (200 nm), the co-occurrences were random. At shorter distances (40 nm and less), MTs were still randomly associated with other MTs. However, at these shorter distances, the spatial associations of mitochondria with MTs and of SER with MTs were not random; such preferential stable associations may be produced by specific MT associated cross-bridging proteins. In axons, MTs tend to be clustered together, giving the appearance of MT bundles. We propose that the MT-MT bundling is an indirect result of MT concentration along the continuous intra-axonal SER network, to which the MTs are apparently tied directly by dynamic molecular cross-bridges.

Neurofilaments assume a less random architecture at nodes and in other regions of axonal compression.

Neurofilament distributions were mathematically characterized in four chicken somatic motor axons at each of four histologically distinct regions: compact myelinated regions, compact myelinated regions associated with Schwann cell nuclei, Schmidt-Lanterman clefts, and nodes of Ranvier. Compact myelinated regions had the largest cross-sectional areas, the lowest neurofilament densities, and the most random neurofilament organizations--nodes of Ranvier had the smallest cross-sectional areas, the highest neurofilament densities, and the most ordered architectures. In these myelinated axons, the closest natural neurofilament spacing was 25 nm. Mathematical analyses of serial sections suggested that neurofilament interactions are sufficiently weak and transient to permit a full range of variation from random to ordered cytoskeletal architectures as the neurofilaments move longitudinally through the few micron span of the paranodal-nodal region of a single axon.

Internal axonal cytoarchitecture is shaped locally by external compressive forces.

The cross-sectional architecture of the axon and the area of its surrounding Schwann cell were quantified at selected histological regions along the length of avian myelinated axons. The number of neurofilaments (NFs), the density of NFs, axoplasmic area, and Schwann cell cross-sectional area were measured. These parameters were examined at Schmidt-Lanterman (S-L) clefts, at paranodal-nodal regions, and at regions of compact myelin Schwann cell nuclei. The results were then compared with the same parameters in adjacent compact myelinated regions of the same axons. At S-L clefts, paranodal-nodal regions, and Schwann cell nuclei, the axonal areas were smaller and the NF densities were higher than at compact myelinated regions. From other studies, it has been suggested that NF organization is responsive to local compressive forces--NF packing density tends to increase with increasing compression of the axon. We found that the NF packing densities were relatively small and the axon diameters were relatively large in the compact myelinated regions; this result suggests that in these axonal regions external constraints on axonal architecture are minimal. The higher NF packing densities and smaller axon diameters in the other histological regions suggest that external compressive effects on the axon increase in the following order: simple compact myelin less than Schwann cell nucleus less than S-L cleft less than paranodal-nodal region. Ultrastructural comparisons of these 4 histological regions show that the Schwann cell cross-sectional areas differ reproducibly, and this is consistent with the idea that variations in the organization of extra-axonal elements that envelop the axon produce different amounts of physical constraint on the axon and that this can affect the amount of external pressure on the internal architecture of the axon.

Contact lenses in the management of myopic anisometropic amblyopia.

We treated 16 patients aged 1 1/2 to 11 years with myopic anisometropic amblyopia with contact-lens correction of refractive error and occlusion. The degree of visual improvement compared favourably with that reported with the use of spectacles. There were no complications from contact lenses, and only one patient required anesthesia for fitting. Contact lenses appear to be more satisfactory than spectacle lenses in the management of myopic anisometropic amblyopia in regard to cosmesis, comfort, and treatment compliance. Patients with myelinated retinal nerve fibers and unilateral severe myopia had a poor visual prognosis. Treatment should not be abandoned in these children, but prolonged occlusion is not indicated. The presence of strabismus at the start of treatment appeared to have little effect on the final visual results. Most patients with strabismus responded well to occlusion, and all such children should be considered candidates for treatment of their amblyopia.

Spontaneously regressing retinoblastomas, retinoma, or retinoblastoma group 0.

A 5-year-old boy had a gray, translucent retinal mass containing calcified nodules and surrounded by retinal pigment clumping and atrophy. The eye was enucleated and the patient has remained well for seven years. Microscopic examination disclosed an intraretinal tumor composed of benign-appearing cells in a bed of well-vascularized ground substance with calcific foci. There was surrounding retinal pigment epithelial hyperplasia but no peripheral necrosis or signs of tumor regression. There were no mitoses, cellular pleomorphism, nuclear atypia, rosettes, or other characteristics of malignancy. Tumors with this typical fundus appearance have been termed spontaneously regressing retinoblastoma or retinoma. Although the tumor in this patient was histopathologically benign, it carries the same genetic risk as a retinoblastoma. A better term for this lesion, therefore, is retinoblastoma group 0.

Interocular transfer of the motion aftereffect in strabismus.

The interocular transfer of the motion aftereffect (MAE) was measured in three groups of strabismic subjects (six with monofixation, nine with alternation, four with anomalous retinal correspondence) and compared with a group of four normal subjects. The duration of the MAE was measured using sinusoidal gratings of 0.5 c/deg subtending a visual angle of 8. Contrary to previous findings, a substantial amount of MAE transfer was found in some stereoblind individuals with strabismus. The mean amount of transfer for each group was found to correlate with binocularity; the order of decreasing transfer being: normal, monofixation, alternation, and anomalous retinal correspondence (ARC). This reduction in transfer which accompanied the loss of binocularity was asymmetric, that is, right-left transfer did not equal left-right transfer. The amount of asymmetry was found to correlate inversely with the amount of transfer. The direction in which the greatest transfer occurred did not correlate with visual acuity. These data substantiate the overall poor binocularity in subjects with ARC, and the relatively good binocularity of subjects with monofixation. Furthermore, the substantial amount of transfer found in subjects with alternating strabismus may be a measure of potentially achievable binocularity.

Temporal and spatial patterns of phosphotyrosine immunolocalization during cardiac myofibrillogenesis of the chicken embryo.

To investigate the possible role of phosphorylation of protein tyrosine during myofibrillogenesis (6- to 13-somite stages) of the chicken embryonic heart tube, immunolocalization of phosphotyrosine (P-Tyr) and the relationship between P-Tyr and developing myofibrils were studied by means of confocal scanning laser microscopy and immuno-electron microscopy. The staining pattern of P-Tyr varied in different sites of myocytes at different stages of embryonic development: At the cell-cell boundaries, P-Tyr was localized at the adhesion belt of outer myocardial layer cells (6- to 13-somite stages), non-junctional cell-cell contacts (6- to 13-somite stages) and early intercalated disks of both the outer and inner myocardial layer cells (8- to 13-somite stages). At the cell-extracellular matrix boundaries of inner layer cells, the first stages of myofibril formation appeared as serially aligned areas of P-Tyr localization closely associated with circumferentially aligned thick actin bundles (8- to 9-somite stages). This P-Tyr immunostaining decreased when the thick actin bundles developed into mature striated myofibrils at the 10- to 13-somite stages. These findings suggest that the phosphorylation of protein tyrosine residues is primarily concentrated at the modulating cell-cell and cell-matrix adhesion sites of developing myocytes and myofibrils.

Effect of ammoniation of aflatoxin B1-contaminated cottonseed feedstock on the aflatoxin M1 content of cows' milk and hepatocarcinogenicity in the trout bioassay.

The effectiveness of ammonia in inactivating aflatoxins in contaminated cottonseed was investigated. Two aflatoxin-contaminated cottonseed lots were treated separately using an atmospheric pressure, ambient temperature ammoniation procedure (APAT) or a high pressure, high temperature ammoniation procedure (HPHT), and incorporated into dairy cow rations. Isocalorific diets containing 25% defatted, dried milk from cows fed aflatoxin-contaminated cottonseed without or with APAT or HPHT treatment, or an aflatoxin-free human grade commercial milk powder, were then fed for 12 months to rainbow trout (Oncorhynchus mykiss). Aflatoxin M1 (AFM1) concentrations in milk powders without and with seed treatment were: APAT, 85 and < 0.05 microgram/kg; HPHT, 32 and < 0.05 microgram/kg. In the APAT experiment, trout consuming the diet containing milk from cows fed the aflatoxin-contaminated cottonseed had a 42% incidence of hepatic tumours; APAT cottonseed treatment reduced this to 2.5%. Positive controls were included to demonstrate trout responsiveness. AFB1 fed continuously for 12 months at 4 micrograms/kg resulted in a 34% tumour incidence, whereas positive controls fed 20 micrograms AFB1/kg, 80 micrograms AFM1/kg, or 800 micrograms AFM1/kg for 2 wk and killed 9 months later had a 37, 5.7 and 50% incidence of tumours, respectively. These data demonstrate that APAT ammonia treatment of aflatoxin-contaminated dairy cattle cottonseed feedstock abolished the detectable transfer of AFM1 or AFB1 into milk powder, and greatly reduced the carcinogenic risk posed by any carry-over of aflatoxins or their derivatives into milk. In addition, the results confirm AFM1 to be a lower level hepatocarcinogen in comparison with AFB1 in the trout carcinogenicity assay. In the separate HPHT experiment, no tumours were observed in the livers of trout fed diets containing milk from either the ammonia-treated or untreated source, or the control diet containing 8 micrograms AFM1/kg. Positive controls fed 64 micrograms AFB1/kg for 2 wk exhibited a 29% tumour incidence 12 months later. Thus in this experiment, neither AFM1 at 8 micrograms/kg nor any HPHT-derived aflatoxin derivatives that might have been carried over into milk, represented a detectably carcinogenic hazard to trout.

Reduction of risks associated with fumonisin contamination in corn.

Fumonisins, produced by Fusarium moniliforme, have been recognized as an important group of chemicals which cause health risks in domestic animals and humans. Decontamination procedures for fumonisin B1 (FB1) were evaluated to determine chemical modification and reduction in toxic/carcinogenic potentials. Ammoniation, a procedure used for decontamination of aflatoxins, yielded a 79% reduction in FB1 levels in naturally contaminated corn. Authentic FB1 and FB1-contaminated corn were exposed to alternative treatments containing various combinations of Ca(OH)2, NaHCO3, and H2O2 simulating a modified nixtamalization procedure. Treatments also included NH4Cl alone or in combination with H2O2 or horseradish peroxidase. The brine shrimp assay (Artemia spp.) was used to monitor toxicity of reaction products and the Salmonella/microsomal mutagenicity assay, using tester strains TA-100 and TA-102, was used to evaluate mutagenicity. Treatments of FB1-contaminated corn simulating modified nixtamalization (Ca(OH)2 alone or with Na-HCO3 + H2O2) gave 100% reduction of FB1 and reduced brine shrimp toxicity by ca. 40%. The positive mutagenic potential (without S-9) for extracts of corn naturally contaminated with FB1 was eliminated following exposure to modified nixtamalization. Reaction products formed when pure FB1 was treated with Ca(OH)2 and H2O2/NaHCO3 were inhibitory to Bacillus cereus, B. subtilis, and B. megaterium. No inhibitory potential was evident for contaminated corn extracts following the chemical treatments.

Myxobolus microcystus sp. N. (Protozoa: Myxosporida) from the gills of Micropterus salmoides (Lacépède 1802) in southern Illinois.

Myxobolus microcystus sp. n. is described from the gills of the largemouth bass, Micropterus salmoides. Mean spore dimensions (in micrometers) are as follows: spore length 12.5; spore width 7.5; spore thickness 5.5; polar capsule length 6.5; polar capsule width 2.5; extended length of polar filaments 21 to 37; polar filaments with 6 to 7 coils. In histological sections, hyperplasia of infected filaments, atrophy of lamellae, partial occlusion of the efferent artery, and reduction in the number of peripheral goblet cells were observed.

Enhanced functions of osteoblasts on nanostructured surfaces of carbon and alumina.

It is of the utmost importance to increase the activity of bone cells on the surface of materials used in the design of orthopaedic implants. Increased activity of such cells can promote either integration of these materials into surrounding bone or complete replacement with naturally produced bone if biodegradable materials are used. Osteoblasts are bone-producing cells and, for that reason, are the cells of interest in initial studies of new orthopaedic implants. If these cells are functioning normally, they lay down bone matrix onto both existing bone and prosthetic materials implanted into the body. It is generally accepted that a successful material should enhance osteoblast function, leading to more bone deposition and, consequently, increased strength of the interface between the material and juxtaposed bone. The present study provided the first evidence of greater osteoblast function on carbon and alumina formulations that mimic the nano-dimensional crystal geometry of hydroxyapatite found in bone.

Comparison of digestibility, nutritive value, and storage characteristics of different forms of cottonseed in diets fed to lactating dairy cows.

Thirty-two Holstein cows averaging 70 d postpartum were used to compare digestibility of whole short staple cottonseed (SS) to whole Pima (WP), coarsely cracked Pima (CrP), and ground Pima (GP) cottonseeds and their effects on lactational performance. Cottonseed was fed at 15% of diet DM. Milk and solids-corrected milk (SCM) yields were higher for cows fed GP than for cows fed CrP or WP but were not different from yields of cows fed SS. Feed efficiency (SCM/DMI) was higher for cows fed GP and SS than for those fed WP. Milk of cows fed GP was lower in stearic acid but higher in linoleic and linolenic acids than milk of cows fed SS. Whole seeds passing into the feces (percentage of consumed) were higher for WP (12.3%) than for SS (6.2%). Total tract digestibility of ether extract was lower for WP than for other diets. Digestibilities of other nutrients were not different. To test storage characteristics, samples of cottonseed were incubated at 32 degrees C and 30% relative humidity for 0, 10, 20, and 30 d, or stored at ambient temperatures in covered buckets for 9 mo. No differences in amount of free fatty acids between incubated samples were noted, and only CrP stored in buckets for 9 mo was significantly higher in free fatty acids than initial or frozen seed. No differences in aflatoxin were detected, and levels were very low. Milk yield of cows fed GP diets was similar to that of cows fed SS diets and slightly higher than those of cows fed CrP or WP diets. Processing the Pima seed increased feed efficiency.