Lens epithelial cell elongation in the absence of microtubules: evidence for a new effect of colchicine.

Embryonic chick lens epithelial cells cultured in serum-supplemented medium elongated in the absence of microtubules after treatment with the antimicrotubule drug nocodazole. Colchicine, at concentrations lower than those that dissociate microtubules, blocks cell elongation and the associated increase in cell volume. These results indicate that an increase in cell volume, not microtubules, is responsible for lens cell elongation and suggest a previously undescribed effect of colchicine on cell volume regulation.

The ultrastructural morphology and distribution of trigemino-hypoglossal connections labeled with horseradish peroxidase.

Axon terminals projecting to the hypoglossal nucleus have been identified and characterized by electron microscopy following injections of horseradish peroxidase (HRP) into pars interpolaris of the spinal trigeminal nucleus (SPVN) in adult rats. Over 70% of the anterogradely labeled terminals contained spherical vesicles (S-terminals) and their synaptic densities were chiefly asymmetrical (Gray Type I). The rest (28%) of the labeled terminals had flattened vesicles (F-terminals) and predominantly established symmetrical (Gray Type II) synaptic contacts. The diameters of labeled terminals were 0.5-2.5 micron. Two-thirds of the S-terminals had diameters less than 1.25 micron, whereas, F-terminals were distributed equally in the higher (greater than 1.25) and lower (less than 1.25) diameter ranges. Most axon terminals ended on dendrites of hypoglossal neurons; some, chiefly F-terminals, formed axosomatic endings. Dendrites had diameters of 0.5-5 micron. The majority of S- and F-terminals ended on dendrites with diameters of less than 2.5 micron. However, more F-terminals (17%) than S-terminals (11%) were presynaptic to dendrites greater than 2.5 micron in diameter. Experiments in which anterograde HRP labeling of trigemino-hypoglossal projections was combined with retrograde WGA-HRP labeling of motoneurons projecting to the tongue, demonstrated that SPVN axons end on dendrites of these motoneurons. Whether some of the trigeminal fibers also terminate on intrinsic hypoglossal interneurons remains to be determined.

Glycogen, its transient occurrence in neurons of the rat CNS during normal postnatal development.

Progressive changes in the postnatal incidence, distribution and duration of glycogen in neurons of the pons, medulla and spinal cord were studied by light and electron microscopy using cytochemical and quantitative methods. Albino rats of 11 ages ranging from newborn to adult were used for this investigation. Methacrylate sections, stained with periodic acid-Schiff-dimedone (PAS) were surveyed to identify nerve cell groups containing the polysaccharide, glycogen. The PAS reaction was positive in neuronal cell groups of the hypoglossal nucleus, the mesencephalic nucleus of V, nucleus ambiguus, the abducens nucleus, the facial motor nucleus and anterior horn cells of the spinal cord. The intensity and duration of the PAS reaction appeared greatest in the hypoglossal nucleus. Neurons of the mesencephalic nucleus of V demonstrated a reaction of moderate intensity and duration. The remaining nerve cell groups exhibited a weak, diffuse reaction of brief duration. Postnatal differences in the incidence and patterns of disposition of glycogen were quantified using ultrathin sections of the hypoglossal nucleus, the site richest in glycogen. The presence of glycogen was verified by the periodic acid-thiosemicarbizide-silver proteinate (PA-TSC-SP) ultracytochemical stain. The incidence of glycogen in neuronal perikarya of hypoglossal nuclei was related to age. All neurons contained some glycogen during the first postnatal week. By 24 days postnatal (dpn), the majority of hypoglossal neurons lacked glycogen and all neurons of adult rats were glycogen-free.(ABSTRACT TRUNCATED AT 250 WORDS)

The ultrastructural identification of reticulo-hypoglossal axon terminals anterogradely labeled with horseradish peroxidase.

Injections of horseradish peroxidase (HRP) or wheat-germ agglutinin-horseradish peroxidase (WGA-HRP) into the nucleus reticularis parvocellularis (RPc) produced anterograde labeling of axon terminals within the hypoglossal nucleus. Based on morphological parameters of vesicle population, membrane specializations, and postsynaptic articulations, two types of axon terminals derived from neurons in RPc end on hypoglossal neurons. More than half of the terminals contained spherical vesicles (S-type), established asymmetrical membrane specializations and contacted proximal and medium-sized dendrites. The remaining labeled terminals had flattened vesicles (F-type), symmetrical membrane densities and apposed medium and small dendrites. The morphological differences expressed in the two types of terminals may reflect physiological and/or pharmacological differences in the action of RPc neurons on motoneurons in the hypoglossal nucleus.

Brain stem afferents of hypoglossal neurons in the rat.

The origin of afferent connections of the hypoglossal nucleus in rats was investigated using horseradish peroxidase (HRP) as a retrograde tracer. Pressure injections (0.15-0.17 mu1) of 15% HRP were introduced into the rostral, middle and caudal portions of the nucleus. Projections to the hypoglossal nucleus originated from 3 regions of the brainstem: the reticular formation, the spinal V complex and the nucleus of the solitary tract. Bilateral projections with ipsilateral predominance came from the lateral reticular formation: the dorsal aspect of the nucleus reticularis parvocellularis and its caudal continuation, the nucleus reticularis dorsalis. Fewer projections emerged from two nuclei of the medial reticular formation. The dorsal part of the nucleus reticularis ventralis at the spinomedullary junction contributed bilateral with mainly contralateral input to hypoglossal neurons. A few labeled neurons were situated bilaterally in the nucleus reticularis gigantocellularis of the rostral medulla. The input from the spinal V complex originated from the dorsal aspect along most of its length but particularly from the pars interpolaris and oralis subdivisions. Labeled neurons were located primarily in the posterior portion of the nucleus of the solitary tract. Projections from the spinal V complex and the solitary nucleus exhibited ipsilateral predominance. These results suggest that somatic and visceral centers of the rat brainstem play an important role in the control of the activity of hypoglossal motoneurons.

Factors affecting the ultrastructural pattern of anterograde labeling in axon terminals with HRP.

Comparisons of anterograde labeling of axon terminals originating from short and long projection neurons were made in the hypoglossal nucleus. Injections of dilute and concentrated horseradish peroxidase (HRP) or wheat germ-agglutinin-horseradish peroxidase (WGA-HRP) were made via a glass micropipette into the nucleus reticularis parvocellularis (RPc = short projection neurons) and the Spinal V trigeminal complex (Sp. V = long projection neurons). Axon terminals in the hypoglossal nucleus, a common projection site of the two efferent systems, were evaluated ultrastructurally using diaminobenzidine (DAB) as the chromogen for the cobalt-glucose oxidase (CO-GOD) method of HRP labeling. Labeled axon terminals from these two sources demonstrated different distribution patterns of the reaction product. For the short pathway, high concentrations of the tracers resulted in diffuse, agranular labeling in the majority of axon terminals. Dilute concentrations of the tracers were associated with membrane-bound, granular type of labeling. All anterograde labeling of terminals of long projection neurons (Sp. V) was membrane-bound and granular irrespective of the tracer concentration. The length of the pathway and the concentration of the enzyme tracers are factors that affect the pattern of anterograde label in axon terminals of hypoglossal afferents.

Reticulo- and trigemino-hypoglossal connections: a quantitative comparison of ultrastructural substrates.

Axon terminals were identified and characterized by electron microscopy after injections of horseradish peroxidase (HRP) into the spinal V nucleus (SPVN) or the medullary reticular formation adjacent to the XIIth nucleus. The synaptic organization and topology of these two different populations of hypoglossal afferents (T-XII and R-XII respectively) were determined by quantitative comparisons. Significant differences were obtained in the ratios of morphological types of terminals, sizes of axonal endings and their location on postsynaptic structures. Axon terminals containing spherical vesicles (S-terminals) and those with flattened/pleomorphic vesicles (F-terminals) were anterogradely labeled with HRP from both injection sites. However, the S/F ratio for R-XII terminals was 1.2:1 compared to 2.6:1 for T-XII afferents. Asymmetrical membrane densities (Gray Type I) were the predominant form of junctional specialization for S-terminal synapses. Asymmetrical densities with subjunctional dense bodies/bars (S-Taxi) were associated with a higher proportion of T-XII synapses than R-XII synapses. Almost all of the F-terminals from both sources had symmetrical densities (Gray Type II). The average diameter of R-XII terminals was greater than that of T-XII terminals. R-XII-F terminals were the largest terminals. The majority of axon terminals from both sources formed axodendritic synapses. However, R-XII terminals had a higher incidence (10% vs. 3%) of axosomatic contacts. The proportion of R-XII-F-terminals decreased from the central toward the distal dendrites, whereas the opposite was found for T-XII-F and T-XII-S-terminals. In contrast to these findings, R-XII-S-terminals were more uniformly distributed on dendrites of all sizes.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptome analyses of benign and malignant prostate epithelial cells in formalin-fixed paraffin-embedded whole-mounted radical prostatectomy specimens.

Formalin-fixed paraffin-embedded (FFPE) prostate specimens are rich sources of molecular pathological information. However, FFPE-based microarray analysis of tissue samples may be hampered by the degradation and chemical alteration of RNA molecules due to the preservation procedure. In this report, we employed a probe analyses of Affymetrix oligonucleotide arrays at individual probe level to compensate for the potential loss of gene identifications associated with compromised mRNA quality in FFPE preparations. Furthermore, to increase the sample quality, we utilized laser capture microdissection of prostate tumor and benign epithelial cells. Remarkably, combination of these approaches recapitulated the common prostate cancer-associated gene expression alteration. Identification of prostate cancer associated-gene expression alterations such as AMACR, Kallikrein gene family and genes associated with androgen signaling such as PDEF and STEAP were consistent with previous findings reported in prostate cancer. These data suggest that combination of laser capture dissection with computational enhancement of microarray data may be useful for the assessment of gene expression changes in FFPE prostate cancer specimens.

Particulate material in antihemophilic factor (AHF) concentrates.

Antihemophilic factor (AHF) concentrates contain noncellular particulate material which is retained by a screen filter of 40 mu pore size. This material is composed in part of IgG, fibrinoprotein, and cold insoluble globulin. The clinical significance of this material is uncertain, but a possible association with pulmonary and cerebral microemboli is suggested.

The progression of deafferentation as a retrograde reaction to hypoglossal nerve injury.

This study examined the fate of axon terminals of one of the major sources of hypoglossal afferents, the spinal V nucleus, after XIIth nerve resection in adult Sprague-Dawley rats. In order to anterogradely label trigemino-hypoglossal projections, small quantities of horse radish peroxidase were pressure-injected into the ipsilateral dorsal (mandibular) portion of the spinal V nucleus two days before the animals were killed. Survival periods ranged from 5 to 33 days after nerve injury (dpo). Axonal injury produced relative changes in the association of labelled axon terminals to structures in the hypoglossal nucleus on the injured side. The proportion of horse radish peroxidase-labelled spinal V nucleus terminals with spherical vesicles (S-terminals) that were unapposed to hypoglossal somata or dendrites increased rapidly and reached maximal levels by 11 dpo. By contrast, the isolation of labelled terminals with pleomorphic/flattened vesicles (P/F-terminals) from postsynaptic structures began later, advanced at a slower rate and did not attain maximal levels until 20 dpo. S-terminals not apposed to neuronal cell parts increased at a rate of 2.2 times greater than unapposed P/F-terminals. In addition, at peak levels, the proportion of labelled S-terminals that were detached from somata and dendrites was significantly greater than unapposed, labelled P/F-terminals. Axotomy did not alter the caliber of the labelled axon terminals. However, by 29 days after axotomy, the average diameter of dendrites remaining in contact with SPVN terminals was 1/3 the diameter of dendrites of uninjured neurons apposed to labelled axon terminals. These findings provide the morphological correlate for physiological and pharmacological evidence that the effectiveness of excitatory and inhibitory synapses are down-regulated in a coordinated manner after hypoglossal nerve injury.

Technical assessment of the affymetrix yeast expression GeneChip YE6100 platform in a heterologous model of genes that confer resistance to antimalarial drugs in yeast.

The advent of high-density gene array technology has revolutionized approaches to drug design, development, and characterization. At the laboratory level, the efficient, consistent, and dependable exploitation of this complex technology requires the stringent standardization of protocols and data analysis platforms. The Affymetrix YE6100 expression GeneChip platform was evaluated for its performance in the analysis of both global (6,000 yeast genes) and targeted (three pleiotropic multidrug resistance genes of the ATP binding cassette transporter family) gene expression in a heterologous yeast model system in the presence and absence of the antimalarial drug chloroquine. Critical to the generation of consistent data from this platform are issues involving the preparation of the specimen, use of appropriate controls, accurate assessment of experiment variance, strict adherence to optimized enzymatic and hybridization protocols, and use of sophisticated bioinformatics tools for data analysis.

Comparison of HIV-1 envelope-specific CD4+ T cell lines simultaneously established from peripheral blood mononuclear cells and lymph node biopsy in HIV-1-infected individuals.

HIV-1 envelope-specific CD4+ T cell lines were established simultaneously from PBMC and lymph node mononuclear cells of two HIV-1-infected patients. Three recombinant envelope proteins were used to establish the CD4+ T cell lines: gp160NL4-3, gp120IIIB, and gp120MN. Six T cell lines were established from the first patient, one for each Ag from each compartment, and four T cell lines, two per compartment, were established from the second patient. Each line was challenged with a panel of overlapping peptides spanning the entire gp120 sequence to define its T cell epitope specificity. The pattern of recognition for all the lines from any given patient was similar between compartments. Each patient had a different pattern of peptide recognition. TCR analysis showed a heterogeneous usage of Vbeta between lines with same peptide specificity and established from different compartments. These data suggest that the cellular immune response does not phenotypically vary between the peripheral blood and lymph node compartments, but demonstrates genotypic heterogeneity, showing possible redundancy of the immune response to HIV-1 gp160.

Performance of the Affymetrix GeneChip HIV PRT 440 platform for antiretroviral drug resistance genotyping of human immunodeficiency virus type 1 clades and viral isolates with length polymorphisms.

The performance of a silica chip-based resequencing method, the Affymetrix HIV PRT 440 assay (hereafter referred to as the Affymetrix assay), was evaluated on a panel of well-characterized nonclade B viral isolates and on isolates exhibiting length polymorphisms. Sequencing of human immunodeficiency virus type 1 (HIV-1) pol cDNAs from clades A, C, D, E, and F resulted in clade-specific regions of base-calling ambiguities in regions not known to be associated with resistance polymorphisms, as well as a small number of spurious resistance polymorphisms. The Affymetrix assay failed to detect the presence of additional serine codons distal to reverse transcriptase (RT) codon 68 that are associated with multinucleoside RT inhibitor resistance. The increasing prevalence of non-clade B HIV-1 strains in the United States and Europe and the identification of clinically relevant pol gene length polymorphisms will impact the generalizability of the Affymetrix assay, emphasizing the need to accommodate this expanding pool of pol genotypes in future assay versions.