Strain differences and effects of different stocking densities during rearing on the musculoskeletal development of pullets.

There are few published studies on the effect of stocking density (SD) of pullets, particularly between different genetic lines. The objectives of this study were to determine if strain or SD affects musculoskeletal development of pullets and determine any impact on the productivity and keel bone health of adult hens. Lohmann Selected Leghorn Lite (LSL), Dekalb White (DW), and Lohmann Brown (LB) pullets were reared at 4 different SD (247 cm2/bird, 270 cm2/bird, 299 cm2/bird, and 335 cm2/bird) in large cages furnished with elevated perches and a platform. At 16 wk of age, the keel bone, the muscles of the breast, wings, and legs, and the long bones of the wings and legs were collected to compare keel bone development, muscle growth, and bone breaking strength (BBS) between strain (adjusted for bodyweight) and SD treatments. Stocking density did not have an effect on the metasternum length, height, or area of the keel bone, the weights of the bicep brachii, pectoralis major or pectoralis minor, or the BBS of any of the selected bones. However, strain differences were found for all keel bone characteristics, all muscle weights, and the majority of BBS measures. The keel metasternum, height, and overall area of the keel bone were found to be smaller in LB pullets compared with LSL and DW pullets (P < 0.0001); however, cartilage length and overall percentage of the cartilage present on the keel bone was greatest in LB pullets (P < 0.0001). Leg muscles were heaviest in LB pullets (P < 0.05); however, breast muscles were heavier in LSL and DW pullets (P < 0.0001). Lohmann Brown pullets had lower BBS of the tibia (P < 0.0001) and femur (P < 0.0001) compared with LSL and DW pullets, whereas DW pullets had greater BBS of the humerus (P = 0.033). Additionally, there was a higher prevalence of keel bone fractures at 50 wk of age in LB hens compared with DW (P = 0.0144). Overall, SD during rearing used in this study had little impact on the musculoskeletal growth of pullets; however, significant differences were found between strains which may reflect strain-specific behavior. Additionally, differences in keel bone development between strains may lead to differences in keel bone damage in adult hens.

Membrane differentiations in freeze-fractured mammalian sperm.

A correlated thin-sectioning and freeze-fracturing study has been made of guinea pig and rat spermatozoa. In sections, the cell membrane over the acrosome has a concanavalin A and ruthenium red reactive glycocalyx which exhibits an ordered pattern related to the lattice of crystalline domains within the plane of the membrane revealed by freeze-fracturing. The cleaved acrosomal membrane also shows a finer linear periodicity in some areas. The membrane over the equatorial segment of the guinea pig acrosome is marked by a palisade of oblique ridges not observed in the rat. The plasmalemma of the postacrosomal region is rich in membrane intercalated particles, many randomly dispersed, others clustered in rectilinear arrays. A particle-poor zone is found just anterior to the posterior ring. The fold of redundant nuclear envelope posterior to the ring has many nuclear pores in close hexagonal array. The nuclear envelope lining the implantation fossa is devoid of pores. When cleaved it has a particle-free central area surrounded by a broad zone of large, closely packed, hollow particles. The membrane of the mid-piece in the guinea pig (but not the rat) contains linear strands of 6-8-nm particles oriented circumferentially. The membrane investing the principal piece exhibits the usual randomly distributed particles but in addition, a double row of larger (9 nm) particles runs longitudinally within the membrane over outer dense fiber 1. In the corresponding position in thin sections a local thickening of the membrane is discernible. These observations form a basis for further studies on the functional correlates of these regional specializations of the sperm membrane.

The ultrastructure of the cat myocardium. II. Atrial muscle.

The ultrastructure of the cells specialized for contraction in the atrium and ventricle of young adult cats are compared. The cells specialized for conduction are not included. In addition to possessing distinctive atrial granules, the cells of the atrium are smaller in diameter (5-6 micro) than ventricular cells (10-12 micro) and have strikingly fewer T tubules. These latter differences are discussed in terms of their possible significance for the rate of conduction of the action potential. It is suggested that the very small number of T tubules in atrial cells may compensate for the small cell diameter, and thus permit rapid conduction of the action potential across the surface of the atrium. Coated dense vesicles found in association with the sarcoplasmic reticulum at the level of the Z line in ventricular muscle are more evident in atrial cells. In the virtual absence of T tubules in atrial cells, the sub-sarcolemmal cisternae of the sarcoplasmic reticulum are almost exclusively at the cell periphery. The ends of the cells and their processes in ventricular muscle are rectilinear with the interdigitated portions of the intercalated discs oriented transversely, whereas those of the atrium are often oblique to the myofilament axis. This difference may be related to the lower mechanical tension on atrial cells.

The ultrastructure of the cat myocardium. I. Ventricular papillary muscle.

The ultrastructure of cat papillary muscle was studied with respect to the organization of the contractile material, the structure of the organelles, and the cell junctions. The morphological changes during prolonged work in vitro and some effects of fixation were assessed. The myofilaments are associated in a single coherent bundle extending throughout the fiber cross-section. The absence of discrete "myofibrils" in well preserved cardiac muscle is emphasized. The abundant mitochondria confined in clefts among the myofilaments often have slender prolongations, possibly related to changes in their number or their distribution as energy sources within the contractile mass. The large T tubules that penetrate ventricular cardiac muscle fibers at successive I bands are arranged in rows and are lined with a layer of protein-polysaccharide. Longitudinal connections between T tubules are common. The simple plexiform sarcoplasmic reticulum is continuous across the Z lines, and no circumferential "Z tubules" were identified. Specialized contacts between the reticulum and the sarcolemma are established on the T tubules and the cell periphery via subsarcolemmal saccules or cisterns. At cell junctions, a 20 A gap can be demonstrated between the apposed membranes in those areas commonly interpreted as sites of membrane fusion. In papillary muscles worked in vitro without added substrate, there is a marked depletion of both glycogen and lipid. No morphological evidence for preferential use of glycogen was found.

Structure and function of the undulating membrane in spermatozoan propulsion in the toad Bufo marinus.

Accessory fibers in most sperm surround the axoneme so that their function in propulsion is difficult to assess. In the sperm of the toad Bufo marinus, an accessory fiber is displaced from the axoneme, being connected to it by the thin undulating membrane in such a way that the movement of axoneme and accessory fiber can be viewed independently. The axoneme is highly convoluted in whole mounts, and the axial fiber is straight. Cinemicrographic analysis shows that it is the longer, flexuous fiber, the presumed axoneme, that move actively. The accessory fiber follows it passively with a lower amplitude of movement. The accessory fiber does not move independent of the axoneme, even after demembranation and reactivation of the sperm. On the basis of anatomical relations in the neck region, it appears that the accessory fibers of amphibians are analogous to the dense fibers of mammalian sperm. SDS polyacrylamide gel electrophoresis of demembranated toad sperm tails reveals two principal proteins in addition to the tubulins, the former probably arising from the accessory fibers and the matrix of the undulating membrane. The function of displacing an accessory fiber into an undulating membrane may be to provide stiffness for the tail without incurring an energy deficit large enough to require a long middle piece. A long middle piece is not present in toad sperm, in contrast to those sperm that have accessory fibers around the axoneme. However, the toad sperm suffers a reduction in speed of about one-third, compared with the speed expected for a sperm without an undulating membrane.

Changes in brain-behavior relationships following a 3-month pilot cognitive intervention program for adults with traumatic brain injury.

Facilitating functional recovery following brain injury is a key goal of neurorehabilitation. Direct, objective measures of changes in the brain are critical to understanding how and when meaningful changes occur, however, assessing neuroplasticity using brain based results remains a significant challenge. Little is known about the underlying changes in functional brain networks that correlate with cognitive outcomes in traumatic brain injury (TBI). The purpose of this pilot study was to assess the feasibility of an intensive three month cognitive intervention program in individuals with chronic TBI and to evaluate the effects of this intervention on brain-behavioral relationships. We used tools from graph theory to evaluate changes in global and local brain network features prior to and following cognitive intervention. Network metrics were calculated from resting state electroencephalographic (EEG) recordings from 10 adult participants with mild to severe brain injury and 11 age and gender matched healthy controls. Local graph metrics showed hyper-connectivity in the right inferior frontal gyrus and hypo-connectivity in the left inferior frontal gyrus in the TBI group at baseline in comparison with the control group. Following the intervention, there was a statistically significant increase in the composite cognitive score in the TBI participants and a statistically significant decrease in functional connectivity in the right inferior frontal gyrus. In addition, there was evidence of changes in the brain-behavior relationships following intervention. The results from this pilot study provide preliminary evidence for functional network reorganization that parallels cognitive improvements after cognitive rehabilitation in individuals with chronic TBI.

Orphan nuclear receptor ROR alpha-deficient mice display the cerebellar defects of staggerer.

It has recently been shown that the neurological mutant mouse staggerer (sg) harbors a deletion within the Rora gene that encodes the orphan nuclear receptor ROR alpha. This deletion removes an exon encoding part of the ligand binding domain of the putative receptor, generating an ROR alpha truncated protein (ROR alpha(sg)). It is unknown whether sg acts as a null or highly hypomorphic allele. To address this question, we have generated a null mutation of Rora by targeted disruption of its DNA binding domain in ES cells. The Rora-/- mice are viable but display tremor, body imbalance, small size and die between 3-4 weeks, similar to the sg mouse. Histological examination of the cerebellum of Rora-/- and sg mice showed similar defects, including small size and fewer ectopically localized Purkinje cells. Northern blot analysis of cerebellar RNA showed that ROR alpha transcripts are still expressed in the Rora-/- and sg mutants, although with altered mobilities. However, the cerebellum of the Rora-/- mutant does not express the ROR alpha protein. Attempts to complement the defect of the Rora-/- with sg failed, demonstrating conclusively that the sg defects are caused by the absence of functional ROR alpha.

The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro. Immunoelectron microscopic observations.

In an electron microscopic investigation of the entry of sporozoites of Theileria parva into bovine lymphocytes, the fate of the surface coat of the parasite was traced by immunocytochemical methods. A monoclonal antibody (MAbD1) raised in mice and directed against a surface antigen of sporozoites, was applied to ultrathin frozen sections of bovine lymphocytes infected in vitro. Sites of binding of MAbD1 were localized using a protein A-colloidal gold conjugate as an electron-dense label. The surface of all free sporozoites was labelled. Sporozoites in the process of entering were labelled only on that portion of the membrane not yet tightly bound to the lymphocyte membrane. No label was detected on sporozoites that had completed entry. After fixation with formaldehyde, but not with glutaraldehyde, local areas of labelling were found on lymphocytes in contact with sporozoites and on cells already invaded. The sporozoite organelles, called micronemes, occasionally appeared to contain labelled antigen. No label was found on sporozoites or lymphocytes in control preparations previously exposed to non-specific antibody or treated with protein A-colloidal gold alone. The findings support the conclusion that the sporozoite surface coat, containing the antigen recognized by MAbD1, is shed as the sporozoite enters the host cell.

The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro. Electron microscopic observations.

The entry of sporozoites of Theileria parva into bovine lymphoid cells in vitro was studied with the electron microscope. Endocytosis is completed in less than 10 min. No local mobilization of actin or other cytoskeletal elements is detected in the cytoplasm of the cell being invaded and no engulfing pseudopods are formed. At the site of initial contact, the membranes of parasite and host cell come into very close apposition. As the zippering up of the membranes spreads laterally, the sporozoite sinks into a progressively deepening recess in the surface of the host cell until the rim of the invagination closes and fuses over the parasite. The observation that sporozoites are interiorized at 1-2 degrees C as well as at 37 degrees C suggests that endocytosis depends mainly upon a ligand-receptor interaction of the parasite and host cell membranes and requires little energy. Sporozoites may enter in any orientation, unlike other sporozoan parasites in which the membrane overlying an apical complex is invariably the site of attachment. 24 h after entry, the sporozoite is located in the Golgi region and the investing host cell membrane acquired during endocytosis has disappeared. The Golgi complex has been activated to form small lysosomes which gather around the parasite but are ineffective for lack of a membrane which they can fuse. It is suggested that removal of the investing host-cell membrane permits the parasite to evade destruction by the phagolysosomal system of the host cell. Persistence of micronemes after entry of the sporozoite and their subsequent disappearances invites the speculation that these parasite organelles may play a role in dispersal of the invaginated host cell membrane.

Immunocytochemical localization of androgen-binding protein in the male rat reproductive tract.

The localization of androgen-binding protein (ABP) in the reproductive tract of young adult male rats was studied with the peroxidase-antiperoxidase technique using frozen sections and light microscopy. Within the seminiferous tubules, a positive reaction was noted in the apical portion of the epithelium, apparently in spermatids and/or Sertoli cells. ABP was localized in granules in the apical cytoplasm of the principal epithelial cells of the proximal part of the caput epididymis and in the epithelial cells of the ductuli efferentes. The cells in the distal part of the caput as well as the corpus and cauda of the epididymis did not contain ABP. Numerous coated vesicles and multivesicular bodies were present in the supranuclear cytoplasm of the epididymal epithelium where ABP was taken up. The results indicate that ABP is taken up from the lumen by epithelial cells of the ductuli efferentes and proximal part of the caput epididymis.

Short-term changes in energy intake and serum insulin, neutral amino acids, and urinary catecholamine excretion in women.

The effect of short-term undereating (4.2 MJ [1000 kcal] for 4 d) followed by overeating (12.6 MJ [3000 kcal] for 2 d) on fasting and 2-h postprandial serum glucose, insulin, and neutral amino acids and on urinary free and total norepinephrine and dopamine excretion was studied in 12 normal women. Protein and sodium intake was constant throughout the study. Serum glucose concentration was not affected by diet but the serum total neutral amino acids (ie, sum of valine, leucine, isoleucine, and phenylalanine) tended to increase during undereating and decrease during overeating. Serum tryptophan concentration, relative to the remaining neutral amino acids, was consequently lower during undereating than overeating. The postprandial increase in serum insulin level was greater during overeating than undereating. Urinary free norepinephrine and total dopamine levels were also increased during overeating, suggesting both sympathetic and dopaminergic activation during overeating after undereating.

Molecular cloning, sequence analysis and expression of putative chicken insulin-like growth factor-I cDNAs.

A cDNA library was constructed from mRNA isolated from the liver of a 5-week-old female broiler chicken; at this age the level of insulin-like growth factor-I (IGF-I) mRNA was expected to be high. Three clones, of sizes 2.3, 0.86 and 0.2 kb, were isolated by using a homologous human (IGF-I) probe. The DNA sequence of these clones has been determined and the potential amino acid sequence deduced. The sequence of the mature chicken IGF-I peptide shows a high degree of homology with IGF-I from other species, providing evidence for the identity of these clones. Alternative splicing of the chicken IGF-I mRNA has been found in the region potentially encoding the leader peptide. This may give rise to two forms of prepeptide, differing in the length and nature of their leader peptide. The 0.86 kb cDNA has been used as a probe to Northern blots of chicken mRNA. A major band of approximately 0.65-0.85 kb was seen, plus several minor bands of larger molecular weights. Analysis of genomic Southern blots shows that there is one copy of the chicken IGF-I gene.

Pharmacokinetic approach to the estimation of hepatic removal of insulin.

We simultaneously measured hepatic insulin removal invasively and estimated hepatic clearance and extraction of insulin pharmacokinetically from cardiac output and peripheral plasma concentrations (relatively) noninvasively. The invasive methods involved continuous electromagnetic measurements of portal venous and hepatic arterial blood flow and simultaneous intermittent sampling of blood from the portal and hepatic veins and femoral artery for assay of insulin concentrations. The noninvasive method assumed that hepatic plasma flow is proportional to cardiac output and that hepatic clearance is a constant fraction of total body clearance of insulin. In anesthetized dogs (n = 6), endogenous insulin was suppressed with somatostatin (800 ng/kg/min) while biosynthetic human insulin (0.25, 0.50, and 1.00 mU/kg/min) was infused to steady state during three consecutive 90-min periods. Insulin concentrations were directly proportional to the infusion rate (p less than 0.01). Hepatic blood flow accounted for 20 +/- 2% of cardiac output. Measured hepatic clearance accounted for 51 +/- 5% of total body clearance of insulin and correlated with the pharmacokinetic estimates (p less than 0.01); the estimates of hepatic clearance ranged from 91 to 114% of the measured values. We conclude that this pharmacokinetic approach, which requires only samples of peripheral blood and estimates of hepatic blood flow, may be used to study the hepatic removal of insulin relatively noninvasively.

Accuracy of C-peptide:insulin molar ratio as a measure of hepatic removal of insulin.

We measured transhepatic C-peptide and insulin concentrations in plasma, and hepatic removal of insulin, to examine whether the practice of reporting the C-peptide:insulin molar ratio as a measure of the hepatic removal of insulin is valid. In anesthetized dogs (n = 6), during electromagnetic hepatic blood flow monitoring, endogenous insulin was suppressed with somatostatin, while equimolar proportions of porcine insulin and simian C-peptide (2.4 and 6.0 pmol/kg.min) were infused during two consecutive 45-min periods. Insulin reached steady state within 20 min (t1/2 = 4.5 min); however, C-peptide concentrations continued to rise (t1/2 V 12.5 min). The ratio decreased when the peptide infusion was changed to the higher rate and increased when it was stopped, reflecting the more rapid removal of insulin than of C-peptide. Hepatic removal of insulin remained constant during the two infusion periods (average 60% extraction) and never correlated with the changing molar ratios. Hepatic net flux of insulin correlated with the ratio (P less than 0.05) only while plasma insulin concentrations were rising during constant-rate infusion. We therefore conclude that the molar ratio is not a reliable measure of the hepatic removal of insulin during non-steady states of insulin or C-peptide.

Interaction of sporozoites of Theileria parva with bovine lymphocytes in vitro. I. Early events after invasion.

Sporozoites of Theileria parva rapidly enter bovine lymphocytes by a mechanism of passive endocytosis that depends upon progressive circumferential binding of ligands on the parasite to receptors on the host-cell membrane. Within 10 min of entry, the micronemes of the sporozoite discharge their content and the enveloping host-cell membrane is lysed. The host cell responds within 30 min of invasion by polymerization of microtubules arrayed tangential to the sporozoite and converging upon the cytocentrum. Multivesicular bodies and lysosomes are generated and gather around the parasite but are ineffective in the absence of an endocytotic membrane with which they can fuse. Thus Theileria parva can be added to the category of obligate intracellular parasites that ensure their survival by lysis of the parasitophorous vacuole.

Changes in distribution of nuclear pores during differentiation of the male germ cells.

Changes in number of nuclear pores in different states of physiologica activity have been reported, but little is known about changing patterns of distribution in the course of cell differentiation. Pore distribution in male germ cells was studied in freeze fracture preparations of immature and mature rodent testis. As in other somatic cells, pores were uniformly and apparently randomly distributed in Sertoli cell nuclei. The nucleus of gonocytes and spermatogonia showed varying degrees of pore clustering. Spermatocytes invariably exhibited very striking pore aggregation with close hexagonal packing in pore-rich areas, and large pore-free areas. In early spermatids, pores appeared to be randomly distributed. As the acrosome formed and spread over the apical pole of the nucleus, pores disappeared ahead of its advancing margin and became more concentrated in the post-acrosomal region. The relationship of pore complexes to the chromosomes and the role of the fibrous lamina are discussed. The question as to whether the changing patterns observed involve movement of pores within fluid nuclear membranes, or a dissolution and reformation of new pores remains unanswered.

Salivary gland of the tick vector of East Coast fever. III. The ultrastructure of sporogony in Theileria parva.

Sporogony of the sporozoan Theileria parva in the salivary gland of the tick vector of East Coast fever was studied in electron micrographs. The findings differ in several respects from previous interpretations based upon light microscopy. Cytokinesis of the primary sporoblast to form secondary and tertiary sporoblasts is not substantiated. Instead it is suggested that the parasite develops as a ramifying, multinucleate syncytium rapidly increasing in size and complexity until it gives rise to myriad sporozoites in a terminal episode of cytoplasmic fission. The proliferating nuclei initially occupy peripheral lobules that are continuous with a central labyrinth of branching and anastomosing processes which present a very large surface area for interchange of metabolites with the host cell cytoplasm. The membrane of the labyrinth is rich in cytostomes, but no evidence if found to bulk uptake of host cytoplasmic matrix or organelles into food vacuoles. Rhoptries are the first of the polar organelles of the parasite to develop and are associated with dense plaques irregularly distributed on the inner aspect of the parasite membrane. Micronemes form independently of the rhoptries at a later stage. After 3-4 days of tick feeding, sporogeny is complete and the infected salivary gland cell contains up to 50, 000 spherical or ovoid sporozoites about 1 micrometer in diameter. These are limited by a simple plasma membrane. The inner layer of the 'pellicle', the polar ring, and the conoid described for zoites of other Apicomplexa are lacking. Maturational changes are noted in sporozoites after sporogony is completed. Micronemes appear to increase in size, and possibly in number, from days 3-5 and the majority take up positions immediately subjacent to the plasmalemma.

Salivary gland of the tick vector of East Coast fever. IV. Cell type selectivity and host cell responses to Theileria parva.

Responses of cells in the tick salivary gland to parasitism by Theileria parva were studied by electron microscopy. The gland is composed of three distinct types of acini (I, II, III) which together include ten or more different cell types. Of some 30 infected cells observed in the present study, all were E-cells of acinus III. The parasite thus exhibits a high degree of selectivity for acinus and cell type. The glandular cell invaded undergoes massive hypertrophy and accumulates glycogen deposits in its cytoplasm which may serve as an energy source for the growing intracellular parasite. As synthesis of its secretory material declines the product is packaged in progressively smaller secretory granules. The extensive arrays of endoplasmic reticulum are dismantled and eliminated in autophagic vacuoles. Excess secretory granules are also broken down by crinophagy. After 4 days, sporogony is completed and the host cell contains 30,000-50,000 sporozoites in an electron-lucent cytoplasm largely devoid of cytomembranes and secretory granules. Mitochondria are still present and normal in appearance. The loss of basophilia and secretory granules observed heretofore by light microscopy have been attributed to ingestion and destruction of host organelles by the parasite. The pallid appearance of the cytoplasm has been interpreted as a sign of impending degeneration of the host cell. In electron micrographs no ingestion of organelles by the parasite or degenerative changes were found. The host cell clearly remains viable and metabolically active throughout sporogony. The striking changes in its ultrastructure result from active elimination of organelles and inclusions by the host cell itself in response to parasitism.

Salivary gland of the tick vector (R. appendiculatus) of East Coast fever. I. Ultrastructure of the type III acinus.

The brown ear tick Rhipicephalus appendiculatus is the vector for East Coast fever, a disease that seriously limits livestock production in East Africa. The sporozoites of the infectious agent Theileria parva develop in the tick salivary gland. This paper describes the organization of the type III acinus of the gland and establishes unambiguous ultrastructural criteria for identification of the three secretory cell types: the d-cell, e-cell and f-cell. These observations are basic to exploration of possible cell-type specificity of the invading theileria and other aspects of host-parasite relations.