bluuebunny/crossref_metadata_2025 huggingface dataset:
A simple dot-immunobinding assay for quantification of synaptophysin-like immunoreactivity in human brain.
Authors: Alford M F , Masliah E , Hansen L A , Terry R D
Published: April 2011

Neocortical decreases in synaptic density correlate significantly with the cognitive impairment seen in Alzheimer disease. Recently available monoclonal antibodies (MAb) have made possible the highly specific and sensitive detection of synapse-associated proteins in immunocytochemical and immunochemical techniques. We describe a simple yet highly sensitive dot-immunobinding assay for relative quantification of the synapse marker protein synaptophysin in human brain homogenate fractions with the mouse MAb SY38. Fractions prepared from control and Alzheimer specimens were blotted to nitrocellulose membranes and reacted with SY38, rabbit secondary antibody, and iodinated protein A. A relative standard curve was constructed to normalize results from multiple assay runs. We correlated the results with the more complex immunocytochemical synaptic density measurement technique of immunolabeling coupled with laser confocal imaging, showing good correlation at r = 0.821. Results from Alzheimer cases showed a 40% decrease in synaptophysin immunoreactivity in midfrontal cortex compared with normal controls.
Nitric oxide synthase in motor neurons after axotomy.
Authors: Yu W H
Published: April 2011

Nitric oxide synthase (NOS), an enzyme involved in synthesis of nitric oxide (NO), has been localized in many diverse cell types. In the CNS and PNS, discrete neuron cell groups express NOS constitutively. Recent evidence indicates that NOS is inducible in neurons normally not expressing NOS. After transection of peripheral nerves, NOS expression was significantly up-regulated in the axotomized sensory ganglion cells, whereas in the corresponding motor neurons NOS was not induced unless axon regeneration was prevented and ensuing neuron death became massive. Studies on axotomy-induced NOS have been limited largely to spinal nerves, with only one reported in the vagus nerve. The aim of this study was to determine whether NOS induction in motor neurons of the brainstem after axotomy is regulated in a manner similar to that of the spinal cord. By NADPH-diaphorase histochemistry and NOS immunocytochemistry, the status of NOS in neurons of the hypoglossal nucleus, dorsal motor nucleus of the vagus, and motor nucleus of the facial nerve was examined 2 weeks after unilateral transection of the respective cranial nerves, and the results were compared with those of spinal motor neurons after transection of the sciatic nerve. NOS, undetectable in neurons of the three cranial motor nuclei of sham-operated animals, was observed in about 30-50% of neurons in the cranial motor nuclei ipsilateral to axotomy, but it was not detected in spinal motor neurons after axotomy. NOS localized in axotomized cranial motor neurons was unrelated to NOS of macrophages or endothelial cells. There was no appreciable cell loss from axotomy at this period except in the dorsal motor nucleus of the vagus, where some loss was observed. The results indicate that there is a fundamental difference in the regulation of NOS expression between motor neurons of the cranial and spinal nerves. The possible role of NOS/NO acting as cytoprotective or cytotoxic agent on injured motor neurons is discussed. Motor neurons of cranial and spinal nerves may serve as a useful model to further define the roles of NOS/NO in neurons, especially after traumatic injury.
Localization and quantification of the insulin-like growth factor-1 receptor in mouse articular cartilage by confocal laser scanning microscopy.
Authors: Verschure P J , van Marle J , Joosten L A , Van den Berg W B
Published: April 2011

Insulin-like growth factor-1 (IGF-1) is an essential anabolic growth factor in the regulation of cartilage metabolism and exerts its effects by binding to the IGF-1 Type 1 receptor on the chondrocyte membrane. We have localized and quantified in situ IGF-1 receptor expression in intact articular cartilage of normal mice. The IGF-1 receptor was detected immunohistochemically with antibodies to the IGF-1 receptor and visualized with conventional light microscopy and confocal laser scanning microscopy (CLSM). CLSM analysis enabled us to distinguish IGF-1 receptor immunoreactivity on the chondrocyte cell membrane from intracellular staining. We have established two approaches to quantify in confocal images low levels of fluorescence intensity of the immunolocalized IGF-1 receptor at the chondrocyte membrane, i.e., mean pixel measurement and area measurement. The majority of IGF-1 receptor fluorescence intensity was localized on chondrocytes in the middle and deeper zones of cartilage, whereas chondrocytes in the surface zone exhibited negligible fluorescence. The variable distribution of IGF-1 receptor in chondrocytes of articular cartilage suggests that effects of IGF-1 on chondrocytes may be distinctly heterogeneous in the different mouse articular cartilage zones.
Synthesis and storage in rat thyroid C-cells.
Authors: Thomas G A , Neonakis E , Davies H G , Wheeler M H , Williams E D
Published: April 2011

Thyroid C-cells have the capacity to produce a variety of peptides, as do C-cell tumors. The cellular content of one such peptide, somatostatin, is restricted to a minority of C-cells in rat and human. We set out to clarify whether the synthesis of somatostatin is equally restricted and to study changes that occur in somatostatin synthesis with age. We used immunocytochemistry to localize somatostatin and calcitonin in conjunction with in situ hybridization, using digoxigenin-labeled oligoprobes to localize somatostatin and calcitonin mRNAs in serial sections of formalin-fixed, paraffin-embedded rat thyroid, and correlated peptide and mRNA content in individual cells. All C-cells synthesize and store calcitonin, and somatostatin synthesis, as shown by mRNA content, is limited to the subset of cells containing immunoreactive somatostatin. The numbers of C-cells in general and of the subset synthesizing somatostatin increase between juvenile and adult animals, but the somatostatin cells remain confined to a small area of the gland. These findings support the proposal that somatostatin production is not facultative, but that C-cells differentiate into two distinct subsets of cells, only one of which synthesizes and stores somatostatin.
Composition of the transfer medium is crucial for high-resolution immunocytochemistry of cryosectioned human neutrophils.
Authors: Takizawa T , Robinson J M
Published: April 2011

One of the more routine steps associated with cryosectioning of ultra-thin and semi-thin sections for immunocytochemistry is the transfer of sections to EM grids or coverslips. In a study of the granules of human neutrophils, we have found that the composition of the transfer medium was crucial to the success of these experiments.
An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry.
Authors: Phend K D , Rustioni A , Weinberg R J
Published: April 2011

Immunocytochemistry for amino acids with post-embedding gold is compatible with glutaraldehyde fixation, osmication, and embedding in epoxy-based plastics, but immunogold detection of larger molecules in the central nervous system commonly requires special procedures, e.g. minimizing exposure to glutaraldehyde, eliminating osmium, cryosectioning, and/or embedding in acrylic plastics. These make samples more difficult to prepare and view and may compromise structural preservation. We report a new technique, fixing with high levels of glutaraldehyde, replacing osmium with tannic acid followed by other heavy metals and p-phenylenediamine, and embedding in Epon. This method optimizes antigenicity while retaining the structural preservation and convenient handling of standard embedding techniques. Compared to standard Epon embedment, labeling for neuropeptides in brain and spinal cord is improved. Moreover, the present method yields excellent labeling of glutamate receptors (difficult to identify with traditional post-embedding techniques) and enables simultaneous visualization of associated neurotransmitters.
Ultrastructural localization of protein kinase C in human sperm.
Authors: Kalina M , Socher R , Rotem R , Naor Z
Published: April 2011

We localized protein kinase C (PKC) in human sperm cells at the ultrastructural level by the immunogold technique. The sperm head PKC was localized in the acrosome, equatorial segment, and post-acrosomal region. In the flagellum, PKC was associated with the segmented column of the neck and was distributed along the mid, principal, and end pieces. Immunoreactive sites were observed in patches along the axoneme and outer dense fibers and were evenly distributed between these regions. Pre-absorption of the antibody used with rat brain PKC (alpha and beta) eliminated gold labeling of the sperm head but only reduced labeling of the sperm tail. The co-localization of PKC with various cytoskeletal and other structural elements suggests that the proteins involved are potential substrates for sperm PKC subspecies. The localization of PKC in distinct structures of the human sperm (head, neck, and tail) strongly suggests a role for this enzyme in various aspects of sperm physiology.
Chondrocyte heterogeneity: immunohistologically defined variation of integrin expression at different sites in human fetal knees.
Authors: Salter D M , Godolphin J L , Gourlay M S
Published: April 2011

During development and at maturity different forms of cartilage vary in morphology and macromolecular content. This reflects heterogeneity of chondrocyte activity, in part involving differential interactions with the adjacent extracellular matrix via specialized cell surface receptors such as integrins. We undertook an immunohistological study on a series of human fetal knee joints to assess variation in the expression of integrins by chondrocytes and potential matrix ligands in articular, epiphyseal, growth plate, and meniscal cartilage. The results show that articular chondrocytes (beta 1+, beta 5 alpha V+, alpha 1+, alpha 2+/-, alpha 5+, weakly alpha 6+, alpha V+) differed from epiphyseal (beta 1+, beta 5 alpha V+, alpha 1+/-, alpha 2+/-, alpha 5+, alpha 6+, alpha V+) growth plate (beta 1+, beta 5 alpha V+, alpha 1-, alpha 2-, alpha 5+, alpha 6+, alpha V+), and meniscal cells (beta 1+, beta 5 alpha V+, alpha 1+, strongly alpha 2+, alpha 5+, alpha 6+, alpha V+ in expression of integrin subunits. There was no expression of beta 3, beta 4, beta 6, or alpha 3 by chondrocytes. These results differ from previous reports on the expression of integrins by adult articular cartilage, where alpha 2 and alpha 6 are not seen. Variation in distribution of matrix ligands was also seen. Fibronectin, laminin and Type VI collagen were expressed in all cartilages but there was restricted expression of tenascin, ED-A and ED-B fibronectin isoforms (articular cartilage and meniscus), and vitronectin (absent from growth plate cartilage). Regulated expression of integrins by chondrocytes, associated with changes in the pericellular matrix composition, is of potential importance in control of cartilage differentiation and function in health and disease.
Improved resolution of fibronectin mRNA expression in the inner ear using laser scanning confocal microscopy.
Authors: Woolf N K , Jaquish D V , Koehrn F J , Woods V L , Peterson D A
Published: April 2011

We describe a modified in situ hybridization protocol for localizing and quantifying fibronectin gene expression at the cellular level in paraffin sections of rat temporal bone. When combined with a novel analytical approach using laser scanning confocal microscopy (LSCM), this protocol significantly improved the resolution, sensitivity, and specificity of existing procedures for evaluating fibronectin synthesis in developing inner ear. For simultaneous viewing of cochlear anatomy and the autoradiographic signal, transmitted light images of the cochlea were collected separately from LSCM reflected light images of the autoradiographic silver grains and then the two images were electronically merged. Within the first 2 microns below the surface of the emulsion, silver grains were clustered specifically over hybridized cells. In contrast, nonspecific silver grain development (i.e., background noise) was confined primarily to the lower 5 microns of the emulsion adjacent to the tissue section. Limiting the volume of the emulsion examined in the LSCM analysis, i.e., restricting the range of optical sectioning to the first 2 micron below the surface of the emulsion, effectively minimized nonspecific background noise and maximized the specificity of the hybridization signal. The improvements offered by the described methodological approaches are equally appropriate for non-calcified tissues.
A reexamination of ganglioside GD3-immunopositive glial cells in the forebrains of rats and mice less than 1 week of age.
Authors: Cammer W , Zhang H
Published: April 2011

Before examining remyelination in the brains of rats and mice in vivo, we evaluated the criteria for immature cells in both species that belong to the oligodendrocyte lineage. In the forebrains and cerebella of neonatal rats, ganglioside GD3 has been used as an early immunocytochemical marker for such cells. Using tissue sections from brains of perinatal rats and neonatal mice, we performed double immunofluorescence staining of GD3 with markers for brain macrophages and microglia (both termed "microglia" below) and for the early oligodendrocyte marker O4, respectively. In the rat brains, intense GD3 immunofluorescence was observed in cells also bearing the microglial marker ED1, but not in O4-bearing immature oligodendrocytes. In the rat corpus callosum there also were faintly GD3-positive longitudinal processes that did not co-stain with ED1 or BSI-B4. Some additional GD3-positive structures were observed, mostly in gray matter. In the brains of neonatal and 3-day-old mice, the most intensely GD3-positive structures were elongated longitudinal and radial processes, and the BSI-B4-positive microglia were GD3-negative. In both rat and mouse brains there were at least two additional types of GD3-positive structures, which were ovoid or had a diaphanous appearance, respectively. The present results suggest that there are GD3-positive microglia in immature rat brains and, in both rat and mouse brains, GD3-positive structures that have not yet been identified precisely but which may be radial glial or microglial processes. Accordingly, it is inappropriate to use GD3 as a specifically oligodendrocytic marker in vivo.
Tissue-specific expression of two Artemia franciscana sarco/endoplasmic reticulum Ca-ATPase isoforms.
Authors: Escalante R , Sastre L
Published: April 2011

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) gene from Artemia franciscana is transcribed into two mRNAs that code for two different enzyme isoforms. We investigated the tissue-specific expression of each mRNA by in situ hybridization of larval tissue sections. One of the isoforms is expressed in the muscle fibers of the appendages. The other isoform is generally expressed throughout all tissues of the larvae. The tissue distribution of these two isoforms is very similar to the one described for the two homologous isoforms generated from the vertebrate SERCA 2 gene, and shows the evolutionarily conserved nature of their tissue-specific expression.
A quantitative study of the segmental distribution of somitic cells in the developing chick limb bud using laser-scanning confocal microscopy.
Authors: Ewan K B , Everett A W
Published: April 2011

Our aim was to map the segmental distribution of somitic cells in the limb before and after the fusion of these cells into myotubes. Somitic cells of the brachial somites were labeled by injection of DiI and DiO into the somitoceles of embryonic Day 2 (E2) embryos. The quantitative distribution of dye-labeled cells from injected somites was examined in whole mounts of E4 wing buds and in sections of E5 wing buds using confocal microscopy. Dye derived from cells of anterior brachial somites 16 and 17 was highly concentrated in the anterior half of E4 wings, whereas dye from posterior brachial somites 20 and 21 was concentrated in the posterior half of the wing. Not more than 14% (on average) of the dye in the wing was found outside the preferred half. Dye was equally dispersed in the anterior and posterior halves of the wing when the middle two somites contributing to the wing musculature, numbers 18 and 19, were injected. The total amount of dye in the whole limb at E4 was not significantly different after injection of similar amounts of dye into either pair of brachial somites 16/17, 18/19, or 20/21. In E5 embryos the distribution of labeled cells in the newly formed muscle masses, identified with antibody 2H2 to myosin light chains, was examined in cryostat cross-sections of the wing. Dye was more widely distributed in the wing than at E4. As much as 32% (on average) of the dye was found in the muscle mass outside the preferred half after administration of dye into the anterior or posterior pair of somites. We conclude that the branchial somites contribute similar numbers of cells to the wing musculature and that the segmental origin of these cells is rigidly maintained during their lateral migration into the limb. Cells from adjacent segments then "mix" when they fuse to form myotubes at E5. In addition, we found a significantly greater amount of dye per unit of muscle mass in the proximal compared with distal parts of the limb. We argue that this finding is due to division of myogenic cells during the course of their migration in the wing.
Detection of plasma proteins in CNS neurons: conspicuous influence of tissue-processing parameters and the utilization of serum for blocking nonspecific reactions.
Authors: Moos T , Høyer P E
Published: April 2011

Despite the presence of a blood-brain barrier (BBB), plasma proteins have been detected intraneuronally in regions with axonal projections confined to the CNS. This finding raises the question of whether plasma proteins are taken up from the brain interstitium or whether the results are due to experimental artifact. We examined the effect of various protocols for tissue processing on the intraneuronal distribution of plasma proteins using immunohistochemistry. The detection level of plasma proteins decreased after prolonged fixation, irrespective of the fixative and embedding method employed. In cryostat sections, attempts to block nonspecific staining by serum protein caused considerable nonspecific staining in itself. When nonspecific staining was blocked with a serum-free buffer, specifically labeled neuronal perikarya were found in cryostat sections of brains fixed by perfusion with paraformaldehyde without postfixation. Albumin and IgG occurred predominantly in neurons having projections beyond the BBB but also sparsely in neurons having projections confined to the CNS. Transferrin was evenly distributed within neuronal somata, irrespective of the orientation of projections. The immunoreaction product of the three plasma proteins exhibited a specific intraneuronal localization in the differently projecting neurons. In circumventricular organs, plasma proteins were observed extracellularly and in projecting fibers. In conclusion, plasma proteins are present in neurons with projections confined to the CNS and are probably taken up from the brain interstitium.
Regulation of Na+,K+-ATPase in submandibular glands of hypophysectomized male mice by steroid and thyroid hormones.
Authors: Kurihara K , Maruyama S , Hosoi K , Sato S , Ueha T , Gresik E W
Published: April 2011

The effects of thyroid hormone, androgen, glucocorticoid, and mineralocorticoid on Na+,K+-ATPase activity and on levels of its alpha-subunit protein (alpha 1 isoform) in mouse submandibular gland (SMG) were studied by enzyme assay for ouabain-sensitive ATP hydrolysis, by quantitative densitometric scanning of Western blots, and by immunohistochemistry. To define the specific regulatory effects of various pituitary-dependent hormones on expression of Na+,K+-ATPase in the SMG, we treated hypophysectomized (hypox) male mice with triiodo-L-thyronine (T3), 5 alpha-dihydrotestosterone (DHT), dexamethasone (Dex), and aldosterone (Ald), injected singly or in combination. Na+,K+-ATPase was confined to the duct system of the SMG. In intact mice there was a gender difference in SMG Na+,K+-ATPase, with levels of the enzyme's activity and of its alpha 1-subunit being less in the glands of males. In males, hypophysectomy caused a rise in levels of Na+,K+-ATPase activity and in levels of the alpha 1-subunit protein of this enzyme, and in intensity of immunocytochemical staining for this subunit but there were no such changes in the SMG of hypox females. Changes caused by hormonal replacement to hypox males in Na+,K-ATPase activity, levels of its alpha 1-subunit, or the intensity of immunocytochemical staining for this subunit were complex. Ald had no effect. T3 or dexamethasone, given alone, induced Na+,K+-ATPase activity above control values (hypox males) and increased levels of its alpha 1-subunit protein and immunohistochemical staining for this subunit. By contrast, DHT did not cause a decline in any of these parameters. However, when treatment with T3 was combined with administration of Dex or DHT, enzymatic activity of Na+,K+-ATPase decreased but levels of the alpha 1-subunit protein and immunohistochemical staining for this subunit increased. Therefore, inductions of the alpha 1-subunit of this enzyme are not always correlated with increases in levels of activity of Na+,K+-ATPase, and we propose that both enzymatic and immunochemical analyses are essential for evaluation of hormonal regulation of Na+,K+-ATPase in salivary gland and in other tissues.
In vivo modulation of proliferating cell nuclear antigen in growth plate chondrocytes from normal, hypophysectomized, growth hormone-treated hypophysectomized rats: a comparative immunohistochemical study with image analysis.
Authors: Tajima Y , Kato K , Maruyama S , Hosoi K
Published: April 2011

Growth hormone (GH) regulates the proliferation and maturation of chondrocytes in the epiphyseal growth plate, in which a temporal dimension is superimposed on the septal organization of the tissue. In this study we investigated the in vivo effects of hypophysectomy (Hypox) and injection of GH into Hypox animals (Hypox + GH) on the proliferative activity of the growth plate chondrocytes. We assessed the immunohistochemical expression of proliferating cell nuclear antigen (PCNA) in paraffin-embedded tissues, using monoclonal antibody PC 10 against PCNA combined with immunogold-silver staining. We subjected the immunostained sections to computer-based image analysis by ACAS 570 interactive laser cytometry employing a conventional microscopic light source. Hypox was carried out on 20 rats at 8 weeks of age, half of which received a hypodermic injection of human GH at a dose of 1 IU/kg twice a day for 1 week after the operation. Another group of five rats of the same age were used as normal controls. In normal rats, a distinct PCNA immunoreaction was observed in the proliferative zone, whereas a remarkable diminution of the number of immunoreactive cells in this zone was apparent in Hypox animals. On the other hand, marked hyperplasia of PCNA-positive cells was seen in the proliferative zone of the Hypox + GH rat growth plate. The immunoreactive cells of this zone in Hypox + GH animals exhibited increased nuclear size and staining intensity of PCNA compared with those in normal and Hypox rats. These findings were further confirmed by computer-based image analysis of the specimens in terms of cell integrated value, area, perimeter, and shape factor. These different patterns of PCNA expression may imply that GH specifically promotes the proliferation phase of the chondrocytes in the proliferative zone. The data also suggest that GH influences not only cell replication activity but also cell kinetics of chondrocytes in the growth plate during their lifespan.
Quantitation of L-selectin distribution on human leukocyte microvilli by immunogold labeling and electron microscopy.
Authors: Bruehl R E , Springer T A , Bainton D F
Published: April 2011

L-Selectin is a leukocyte cell adhesion receptor that contributes to neutrophil (PMN) rolling on activated endothelium at sites of inflammation and mediates lymphocyte attachment to high endothelial venules in peripheral lymph nodes. Localization of this receptor to the tips of PMN and lymphocyte microvilli has been demonstrated. However, its distribution on these cells has not been quantified, and its localization on other leukocytes and the morphometry of microvilli on different leukocyte subpopulations have not been previously examined. In this study, PMN and mononuclear leukocytes were isolated from anticoagulated blood by dextran sedimentation and density centrifugation, fixed in 2% paraformaldehyde and 0.05% glutaraldehyde, immunogold-labeled for L-selectin, and embedded in Epon resin. The distribution of L-selectin was determined by counting gold particles on the plasma membrane of sectioned cells, and the surface microstructure of these cells was surveyed on two-dimensional transmission electron micrographs. On average, 78% of PMN, 72% of monocyte, and 71% of lymphocyte L-selectin was observed on the microvilli, with more variance on lymphocytes than the other cell types. Typical PMN and monocyte sections had 26 microvilli, whereas typical lymphocyte sections had 23. Quantitation of the distribution of L-selectin and leukocyte surface topology offers a foundation from which to study the requirement of microvilli or microvillus-localized L-selectin for leukocyte tethering and rolling in model systems that mimic microvascular environments.
A COMPARISON BETWEEN THE HISTOCHEMICAL DEMONSTRATION OF NON-SPECIFIC ESTERASE ACTIVITY BY 5-BROMOINDOXYL ACETATE, α-NAPHTHYL ACETATE AND NAPHTHOL AS ACETATE
Authors: PEARSON BJARNE , DEFENDI VITTORIO
Published: April 2011

5-Bromoindoxyl acetate was used as a substrate for the histochemical demonstration of esterases.

The pH optimum for this salt as used in this study was approximately 5. This differed from the optimum when α-naphthyl and naphthol AS acetates were used, which showed an optimum as determined by color reaction of between 7.3 and 8.4. This latter may be an artifact due to non-enzymatic hydrolysis. Naphthol binding may also vitiate the results. If these hydrolytic artifacts could be eliminated, the true optimum may be of a lower order.

No hydrolysis or diffusion artifacts occur in any pH range with the 5-bromoindoxyl acetate. This may occur with the unsubstituted indoxyl acetate.

The esterase reactions in various tissues following the use of 5-bromoindoxyl acetate was similar to the α-naphthyl and naphthol AS acetates with the exception of the large intestine which was negative with the 5-bromoindoxyl acetate.

The reaction with 5-bromoindoxyl acetate was enhanced by taurocholate in the pancreas and inhibited in the kidney, liver and intestine. This seems to indicate that 5-bromoindoxyl acetate is hydrolyzed by both lipases and esterases from the data of this study.

Because of the fine and uniform granularity of the resulting 5,5' dibromoindigo crystals, this substrate should be valuable in the study of esterases.
HISTOCHEMICAL STUDY OF A FLUORIDE RESISTANT ACID PHOSPHATASE REACTION IN THE MOUSE DUODENUM
Authors: BURT ROBERT C. , MEREDITH BARBARA R. , GRAUER ROBERT C.
Published: April 2011

A fluoride resistant acid phosphatase reaction (pH 5.0) has been demonstrated histochemically in the cuticular border of the mouse duodenum. This reaction was shown to be inactivated by the metal chelating agent, ethylene diamine tetraacetic acid, and reactivated by the following metallic salts : calcium chloride, cobalt(ous) nitrate, cupric sulfate, ferric chloride, ferric ammonium sulfate, ferrous sulfate, lead nitrate, lithium sulfate, magnesium sulfate, mercuric chloride, manganous chloride and zinc acetate. The two ferric salts produced the greatest amount of reactivation and cupric sulfate and manganous chloride produced the least amount of reactivation.

These characteristics of inactivation by a metal chelating agent and metal reactivation plus the fluoride resistance of the acid phosphatase reaction present in the cuticular border of the mouse duodenum are features more typical of an alkaline type of phosphatase rather than the acid variety. This suggests that alkaline type phosphatase may be demonstrable histochemically in the cuticular border of the mouse duodenum in the acid pH range.
QUANTITATIVE HISTOCHEMISTRY OF THE DEVELOPING HAMSTER TOOTH: ALKALINE PHOSPHATASE AND LACTIC DEHYDROGENASE
Authors: NUKI KLAUS , BONTING SJOERD L.
Published: April 2011

1. It has been possible to adapt the quantitative histochemical technique of microdissection and microchemical assay for the study of the quantitative distribution of alkaline phosphatase and lactic dehydrogenase activity in the first maxillary molar of the hamster, between birth and 4 days of age.

2. The zones of the tooth buds dissected out and assayed were: stellate reticulum, stratum intermedium, ameloblasts, dentine plus enamel, odontoblasts, three areas of the dental papilla, and the cervical loop.

3. The quantitative results for the alkaline phosphatase distribution were found to agree qualitatively with the results obtained by the Gomori and azo dye staining techniques, except that the staining techniques gave falsely negative reactions at low activities.

4. Alkaline phosphatase activity was lowest in the zones furthest from dentine and enamel formation, while the two zones of cells most intimately concerned in the formation of dentine and enamel did not show the highest activity; in fact the ameloblast zone was one of the lowest in activity. The highest zones of activity, stratum intermedium and sub-odontoblastic papilla, were immediately next to the cells forming the matrices of dentine and enamel.

5. These facts are considered to be supporting evidence for the theory of Neuman and Neuman regarding the "detoxifying" properties of alkaline phosphatase in a "barrier" surrounding the sites of calcification.

6. The levels of lactic dehydrogenase activity were of the same order of magnitude in all the cellular zones. The zones furthest removed from enamel and dentine formation occupied the lower part of the range found.
STARCH GEL ELECTROPHORESIS OF BRAIN ESTERASES
Authors: BARRON KEVIN D. , BERNSOHN JOSEPH I. , HESS ADELINE
Published: April 2011

1. Starch gel electrophoresis of rat brain homogenates has demonstrated 9 esterases which hydrolyze α-naphthyl acetate and naphthol AS-acetate. One of these has been identified as acetylcholinesterase. The nature of the others is undefined. In addition an enzyme (presumably pseudocholinesterase) acting against butyrylthiocholine iodide has been located at the site of sample insertion.

2. Homogenates of human white matter have yielded 9 esterases active against the same naphthol substrates. None of these is a cholinesterase. However, with acetylthiocholine iodide as substrate, acetylcholinesterase is demonstrable in human material. Enzymic activity against butyrylthiocholine iodide is demonstrable at the origin in human material, also.

3. Best results are obtained by use of homogenates centrifuged at 20,000 x g for 60 minutes.
THE HISTOCHEMISTRY OF GLUCOSE-6-PHOSPHATASE IN THE EPIDIDYMIS OF THE MOUSE
Authors: ALLEN JOHN M.
Published: April 2011

The distribution and properties of an enzyme hydrolyzing ghucose-6-phosphate have been studied by cytochemical and quantitative methods in the epididymis of the mouse. The enzyme hydrolyzed all hexose-6-phosphate esters at comparable rates and showed lower activity against α-glycerophosphate and fructose-1-phosphate. It also hydrolyzed ribose-5-phosphate. Both cytochemical and quantitative determinations suggested optimal activity at pH 5.5. The enzyme could be distinguished from acid phosphatase on the basis of a different pH optimum, on the basis of its insensitivity to d-tartaric acid, and by its tissue and cellular distribution. The enzyme hydrolyzing ghucose-6-phosphate was distributed homogeneously within the cytoplasm of reactive cells as contrasted to a localization associated with the Golgi region for acid phosphatase. Acid phosphatase was totally inhibited by 0.002 M d-tartaric acid while this concentration had no effect upon activity against ghucose-6-phosphate. It was concluded that the enzyme studied was similar to the glucose-6-phosphatase present in other tissues.
THE PRESENCE OF GLYCOGEN IN THE RAT LIVER FOLLOWING IN VITRO PROCESSING IN DECALCIFYING AGENTS
Authors: TROTT J. R.
Published: April 2011

It has been shown that glycogen can be adequately retained in rat livers after fixation with acetic alcohol formalin for 6 hours, 24 hours, 48 hours or 7 days, followed by treatment with disodium ethylenediaminetetraacetate adjusted to pH 7.5. Formic acid-sodium citrate or 5% trichloracetic acid solutions remove the glycogen following fixation.
Characterization and treatment of chronic active Epstein-Barr virus disease: a 28-year experience in the United States
Authors: Cohen Jeffrey I. , Jaffe Elaine S. , Dale Janet K. , Pittaluga Stefania , Heslop Helen E. , Rooney Cliona M. , Gottschalk Stephen , Bollard Catherine M. , Rao V. Koneti , Marques Adriana , Burbelo Peter D. , Turk Siu-Ping , Fulton Rachael , Wayne Alan S. , Little Richard F. , Cairo Mitchell S. , El-Mallawany Nader K. , Fowler Daniel , Sportes Claude , Bishop Michael R. , Wilson Wyndham , Straus Stephen E.
Published: April 2011

Chronic active EBV disease (CAEBV) is a lymphoproliferative disorder characterized by markedly elevated levels of antibody to EBV or EBV DNA in the blood and EBV RNA or protein in lymphocytes in tissues. We present our experience with CAEBV during the last 28 years, including the first 8 cases treated with hematopoietic stem cell transplantation in the United States. Most cases of CAEBV have been reported from Japan. Unlike CAEBV in Japan, where EBV is nearly always found in T or natural killer (NK) cells in tissues, EBV was usually detected in B cells in tissues from our patients. Most patients presented with lymphadenopathy and splenomegaly; fever, hepatitis, and pancytopenia were common. Most patients died of infection or progressive lymphoproliferation. Unlike cases reported from Japan, our patients often showed a progressive loss of B cells and hypogammaglobulinemia. Although patients with CAEBV from Japan have normal or increased numbers of NK cells, many of our patients had reduced NK-cell numbers. Although immunosuppressive agents, rituximab, autologous cytotoxic T cells, or cytotoxic chemotherapy often resulted in short-term remissions, they were not curative. Hematopoietic stem cell transplantation was often curative for CAEBV, even in patients with active lymphoproliferative disease that was unresponsive to chemotherapy. These studies are registered at http://www.clinicaltrials.gov as NCT00032513 for CAEBV, NCT00062868 and NCT00058812 for EBV-specific T-cell studies, and NCT00578539 for the hematopoietic stem cell transplantation protocol.
Heterozygous ITGA2B R995W mutation inducing constitutive activation of the αIIbβ3 receptor affects proplatelet formation and causes congenital macrothrombocytopenia
Authors: Kunishima Shinji , Kashiwagi Hirokazu , Otsu Makoto , Takayama Naoya , Eto Koji , Onodera Masafumi , Miyajima Yuji , Takamatsu Yasushi , Suzumiya Junji , Matsubara Kousaku , Tomiyama Yoshiaki , Saito Hidehiko
Published: April 2011

Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. αIIbβ3 has not been implicated in these conditions. We identified a novel, conserved heterozygous ITGA2B R995W mutation in 4 unrelated families. The surface expression of platelet αIIbβ3 was decreased to 50% to 70% of control. There was spontaneous PAC-1 and fibrinogen binding to resting platelets without CD62p expression. The activation state of αIIbβ3 in 293T cells was higher for αIIb-W995 than for β3-H723 but was weaker than for β3-N562. FAK was spontaneously phosphorylated in αIIb-W995/β3-transfected 293T cells. These results indicate that αIIb-W995/β3 has a constitutive, activated conformation but does not induce platelet activation. αIIb-W995/β3-transfected CHO cells developed membrane ruffling and abnormal cytoplasmic protrusions. The increased size and decreased number of proplatelet tips in αIIb-W995/β3-transduced mouse fetal liver-derived megakaryocytes indicate defective proplatelet formation. We propose that activating mutations in ITGA2B and ITGB3 represent the etiology of a subset of congenital macrothrombocytopenias.
A novel vascular disrupting agent plinabulin triggers JNK-mediated apoptosis and inhibits angiogenesis in multiple myeloma cells
Authors: Singh Ajita V. , Bandi Madhavi , Raje Noopur , Richardson Paul , Palladino Michael A. , Chauhan Dharminder , Anderson Kenneth C.
Published: April 2011

Previous studies have established a role of vascular-disrupting agents as anti- cancer agents. Plinabulin is a novel vascular-disrupting agent that exhibits potent interruption of tumor blood flow because of the disruption of tumor vascular endothelial cells, resulting in tumor necrosis. In addition, plinabulin exerts a direct action on tumor cells, resulting in apoptosis. In the present study, we examined the anti–multiple myeloma (MM) activity of plinabulin. We show that low concentrations of plinabulin exhibit a potent antiangiogenic action on vascular endothelial cells. Importantly, plinabulin also induces apoptotic cell death in MM cell lines and tumor cells from patients with MM, associated with mitotic growth arrest. Plinabulin-induced apoptosis is mediated through activation of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase cleavage. Moreover, plinabulin triggered phosphorylation of stress response protein JNK, as a primary target, whereas blockade of JNK with a biochemical inhibitor or small interfering RNA strategy abrogated plinabulin-induced mitotic block or MM cell death. Finally, in vivo studies show that plinabulin was well tolerated and significantly inhibited tumor growth and prolonged survival in a human MM.1S plasmacytoma murine xenograft model. Our study therefore provides the rationale for clinical evaluation of plinabulin to improve patient outcome in MM.
β2-Glycoprotein I: a novel component of innate immunity
Authors: Ağar Çetin , de Groot Philip G. , Mörgelin Matthias , Monk Stephanie D. D. C. , van Os Gwendolyn , Levels Johannes H. M. , de Laat Bas , Urbanus Rolf T. , Herwald Heiko , van der Poll Tom , Meijers Joost C. M.
Published: April 2011

Sepsis is a systemic host response to invasive infection by bacteria. Despite treatment with antibiotics, current mortality rates are in the range of 20%-25%, which makes sepsis the most important cause of death in intensive care. Gram-negative bacteria are a prominent cause of sepsis. Lipopolysaccharide (LPS), one of the major constituents of the outer membrane of Gram-negative bacteria, plays a major role in activating the host's immune response by binding to monocytes and other cells. Several proteins are involved in neutralization and clearance of LPS from the bloodstream. Here, we provide evidence that β2-glycoprotein I (β2GPI) is a scavenger of LPS. In vitro, β2GPI inhibited LPS-induced expression of tissue factor and IL-6 from monocytes and endothelial cells. Binding of β2GPI to LPS caused a conformational change in β2GPI that led to binding of the β2GPI-LPS complex to monocytes and ultimately clearance of this complex. Furthermore, plasma levels of β2GPI were inversely correlated with temperature rise and the response of inflammatory markers after a bolus injection of LPS in healthy individuals. Together, these observations provide evidence that β2GPI is involved in the neutralization and clearance of LPS and identify β2GPI as a component of innate immunity.
Disease properties, geography, and mitigation strategies in a simulation spread of rinderpest across the United States
Authors: Manore Carrie , McMahon Benjamin , Fair Jeanne , Hyman James M , Brown Mac , LaBute Montiago
Published: April 2011

For the past decade, the Food and Agriculture Organization of the United Nations has been working toward eradicating rinderpest through vaccination and intense surveillance by 2012. Because of the potential severity of a rinderpest epidemic, it is prudent to prepare for an unexpected outbreak in animal populations. There is no immunity to the disease among the livestock or wildlife in the United States (US). If rinderpest were to emerge in the US, the loss in livestock could be devastating. We predict the potential spread of rinderpest using a two-stage model for the spread of a multi-host infectious disease among agricultural animals in the US. The model incorporates large-scale interactions among US counties and the small-scale dynamics of disease spread within a county. The model epidemic was seeded in 16 locations and there was a strong dependence of the overall epidemic size on the starting location. The epidemics were classified according to overall size into small epidemics of 100 to 300 animals (failed epidemics), epidemics infecting 3 000 to 30 000 animals (medium epidemics), and the large epidemics infecting around one million beef cattle. The size of the rinderpest epidemics were directly related to the origin of the disease and whether or not the disease moved into certain key counties in high-livestock-density areas of the US. The epidemic size also depended upon response time and effectiveness of movement controls.
Hospital-based, prospective, multicentre surveillance to determine the incidence of intussusception in children aged below 15 years in Germany
Authors: Bissantz Nicolai , Jenke Andreas C , Trampisch Matthias , Klaaßen-Mielke Renate , Bissantz Kathrin , Trampisch Hans-Joachim , Holland-Letz Tim
Published: April 2011

Background

A new vaccine against Rotavirus (RV) gastroenteritis was introduced in Germany in 2006. In 1997 the first RV vaccine was withdrawn due to an increased incidence in intussusception (IS). Thus, an accurate estimation of the incidence of IS is important for post-licensure surveillance.

Methods

IS-Data were obtained from the 'Erhebungseinheit für seltene pädiatrische Erkrankungen Deutschland' (ESPED, German surveillance unit for rare pediatric diseases) collaborations' central register where all cases of intussusception in Germany for the years 2006 and 2007 are collected (n = 1200). In order to obtain an unbiased estimate of the incidence, it is necessary to determine the population under risk out of which these cases originated, and the proportion of real cases not reported to the registry (underreporting). In order to assess underreporting, a random sample of 31 hospitals was re-assessed by an outside reviewer. The estimation of incidence was done using a single Maximum-Likelihood (ML) estimator based on data from both the registry and the sample.

Results

The uncorrected observed incidence was calculated to be 26.6/100,000 child-years for children below 1 year old, 23.8 for those below 2 years old, and 5.2 for those below 15 years old. The review revealed a mean reporting quota of about 41% and the ML approach yielded an incidence of 51.5/100,000 child-years (95%CI [41.7;61.1]) for children below 2 years of age.

Conclusions

While substantial under-reporting led to very conservative estimates of the IS incidence, the approach described here allows an accurate estimation of IS incidence including corresponding confidence bands. Therefore, ML estimation is a straightforward instrument to derive stable, unbiased estimates in epidemiological studies with incomplete data.
Interactions between genetic admixture, ethnic identity, APOE genotype and dementia prevalence in an admixed Cuban sample; a cross-sectional population survey and nested case-control study
Authors: Teruel Beatriz Marcheco , Rodríguez Juan J Llibre , McKeigue Paul , Mesa T Teresa Collazo , Fuentes Evelyn , Cepero A Adolfo Valhuerdi , Hernandez Milagros A Guerra , Copeland JRM John RM , Ferri Cleusa P , Prince Martin J
Published: April 2011

Background

The prevalence and incidence of dementia are low in Nigeria, but high among African-Americans. In these populations there is a high frequency of the risk-conferring APOE-e4 allele, but the risk ratio is less than in Europeans. In an admixed population of older Cubans we explored the effects of ethnic identity and genetic admixture on APOE genotype, its association with dementia, and dementia prevalence.

Methods

A cross-sectional catchment area survey of 2928 residents aged 65 and over, with a nested case-control study of individual admixture. Dementia diagnosis was established using 10/66 Dementia and DSM-IV criteria. APOE genotype was determined in 2520 participants, and genetic admixture in 235 dementia cases and 349 controls.

Results

Mean African admixture proportions were 5.8% for 'white', 28.6% for 'mixed' and 49.6% for 'black' ethnic identities. All three groups were substantially admixed with considerable overlap. African admixture was linearly related to number of APOE-e4 alleles. One or more APOE-e4 alleles was associated with dementia in 'white' and 'black' but not 'mixed' groups but neither this, nor the interaction between APOE-e4 and African admixture (PR 0.52, 95% CI 0.13-2.08) were statistically significant. Neither ethnic identity nor African admixture was associated with dementia prevalence when assessed separately. However, considering their joint effects African versus European admixture was independently associated with a higher prevalence, and 'mixed' or 'black' identity with a lower prevalence of dementia.

Conclusions

APOE genotype is strongly associated with ancestry. Larger studies are needed to confirm whether the concentration of the high-risk allele in those with African ancestry is offset by an attenuation of its effect. Counter to our hypothesis, African admixture may be associated with higher risk of dementia. Although strongly correlated, effects of admixture and ethnic identity should be distinguished when assessing genetic and environmental contributions to disease risk in mixed ancestry populations.