Diversity in meiotic spindle origin and determination of cytokinetic planes in sporogenesis of complex thalloid liverworts (Marchantiopsida)

As the earliest divergent land plants, bryophytes (mosses, hornworts, and liverworts) provide insight into the evolution of the unique plant process of sporogenesis by which meiosis results in heavy walled spores. New immunohistochemical data on microtubules and γ-tubulin in four genera of complex thalloid liverworts combined with previously published data on another four genera demonstrate grades in the evolution of spindle organization in meiosis. We have discovered that all recognized forms of microtubule organizing centers (MTOCs) in plant cells (plastid MTOCs, spheroid cytoplasmic MTOCs, polar organizers, and nuclear envelope MTOCs) occur in organization of the meiotic spindle of complex thalloid liverworts. In addition, all aspects of pre-meiotic preparation for quadripartitioning of the sporocyte into a tetrad of spores occur, with the exception of pre-meiotic wall precursors found in certain simple thalloids. The preparation includes morphogenetic plastid migration, cortical bands of microtubules that mark future cytokinetic planes in pre-meiosis, quadrilobing of the cytoplasm during meiotic prophase, and quadripolar microtubule systems that are transformed into functionally bipolar metaphase I spindles. Quadripolar spindle origin is typical of bryophyte sporogenesis even though the MTOCs involved may differ. However, in certain crown taxa of complex thalloids the spindle develops with no traces of quadripolarity and placement of intersporal walls is determined after meiosis, as is typical of higher plants.     

VEGF-dependent plasticity of fenestrated capillaries in the normal adult microvasculature

Unlike during development, blood vessels in the adult are generally thought not to require VEGF for normal function. However, VEGF is a survival factor for many tumor vessels, and there are clues that some normal blood vessels may also depend on VEGF. In this study, we sought to identify which, if any, vascular beds in adult mice depend on VEGF for survival. Mice were treated with a small-molecule VEGF receptor (VEGFR) tyrosine kinase inhibitor or soluble VEGFRs for 1-3 wk. Blood vessels were assessed using immunohistochemistry or scanning or transmission electron microscopy. In a study of 17 normal organs after VEGF inhibition, we found significant capillary regression in pancreatic islets, thyroid, adrenal cortex, pituitary, choroid plexus, small-intestinal villi, and epididymal adipose tissue. The amount of regression was dose dependent and varied from organ to organ, with a maximum of 68% in thyroid, but was less in normal organs than in tumors in RIP-Tag2-transgenic mice or in Lewis lung carcinoma. VEGF-dependent capillaries were fenestrated, expressed high levels of both VEGFR-2 and VEGFR-3, and had normal pericyte coverage. Surviving capillaries in affected organs had fewer fenestrations and less VEGFR expression. All mice appeared healthy, but distinct physiological changes, including more efficient blood glucose handling, accompanied some regimens of VEGF inhibition. Strikingly, most capillaries in the thyroid grew back within 2 wk after cessation of treatment for 1 wk. Our findings of VEGF dependency of normal fenestrated capillaries and rapid regrowth after regression demonstrate the plasticity of the adult microvasculature.     

Mycotoxin-producing Fusarium Species Occurring in Winter Wheat in Belgium (Flanders) During 2002–2005

Deoxynivalenol (DON) content and Fusarium spp. ( Fusarium graminearum , Fusarium culmorum , Fusarium avenaceum , Microdochium nivale and Fusarium poae ) of mycotoxin-producing Fusarium species in winter wheat in Belgium (Flanders) were determined. Field trials were set up in the varietal testing network of Flanders Agricultural Centre for Small Grains (Roeselare – Beitem, Belgium) and followed up during growing seasons 2001–2002, 2002–2003, 2003–2004 and 2004–2005. Fusarium infection and DON contamination were mainly influenced by location and environmental parameters. Mean DON levels ranged from 0 to 15 mg/kg. Over the period of four growing seasons cvs Deben, Ordeal and Napier had the highest DON contamination. Seasonal and local weather conditions before and during flowering together with local crop husbandry measures (crop rotation, soil preparation) seemed to be of great importance in explaining the variation in results obtained. At Bottelare a positive correlation between disease index and DON content was found for the growing seasons 2001–2002 and 2002–2003, but not the season 2003–2004. Fusarium graminearum and F. culmorum were in general the most frequently occurring Fusarium spp. in Flanders over the 4 years but the composition of the Fusarium population varied strongly from location to location and from year to year. Fusarium graminearum predominated in areas especially where maize was cultivated, whereas in areas with more small cereals in the crop rotation more F. culmorum was present. Also temperature played a role in the composition of Fusarium spp.     

Induction of Integral Membrane PAM Expression in AtT-20 Cells Alters the Storage and Trafficking of POMC and PC1

Peptidylglycine α-amidating monooxygenase (PAM) is an essential enzyme that catalyzes the COOH-terminal amidation of many neuroendocrine peptides. The bifunctional PAM protein contains an NH₂-terminal monooxygenase (PHM) domain followed by a lyase (PAL) domain and a transmembrane domain. The cytosolic tail of PAM interacts with proteins that can affect cytoskeletal organization. A reverse tetracycline-regulated inducible expression system was used to construct an AtT-20 corticotrope cell line capable of inducible PAM-1 expression. Upon induction, cells displayed a time- and dose-dependent increase in enzyme activity, PAM mRNA, and protein. Induction of increased PAM-1 expression produced graded changes in PAM-1 metabolism. Increased expression of PAM-1 also caused decreased immunofluorescent staining for ACTH, a product of proopiomelanocortin (POMC), and prohormone convertase 1 (PC1) in granules at the tips of processes. Expression of PAM-1 resulted in decreased ACTH and PHM secretion in response to secretagogue stimulation, and decreased cleavage of PC1, POMC, and PAM. Increased expression of a soluble form of PAM did not alter POMC and PC1 localization and metabolism. Using the inducible cell line model, we show that expression of integral membrane PAM alters the organization of the actin cytoskeleton. Altered cytoskeletal organization may then influence the trafficking and cleavage of lumenal proteins and eliminate the ability of AtT-20 cells to secrete ACTH in response to a secretagogue.     

Development of endosperm in Arabidopsis thaliana

 The process of endosperm development in Arabidopsis was studied using immunohistochemistry of tubulin/microtubules coupled with light and confocal laser scanning microscopy. Arabidopsis undergoes the nuclear type of development in which the primary endosperm nucleus resulting from double fertilization divides repeatedly without cytokinesis resulting in a syncytium lining the central cell. Development occurs as waves originating in the micropylar chamber and moving through the central chamber toward the chalazal tip. Prior to cellularization, the syncytium is organized into nuclear cytoplasmic domains (NCDs) defined by nuclear-based radial systems of microtubules. The NCDs become polarized in axes perpendicular to the central cell wall, and anticlinal walls deposited among adjacent NCDs compartmentalize the syncytium into open-ended alveoli overtopped by a crown of syncytial cytoplasm. Continued centripetal growth of the anticlinal walls is guided by adventitious phragmoplasts that form at interfaces of microtubules emanating from adjacent interphase nuclei. Polarity of the elongating alveoli is reflected in a subsequent wave of periclinal divisions that cuts off a peripheral layer of cells and displaces the alveoli centripetally into the central vacuole. This pattern of development via alveolation appears to be highly conserved; it is characteristic of nuclear endosperm development in angiosperms and is similar to ancient patterns of gametophyte development in gymnosperms. Received: 21 September 1998 / Revision accepted: 17 November 1998     

Treatment of recurrent glioma with intracavitary alloreactive cytotoxic T lymphocytes and interleukin-2

 For a single-dose toxicity assessment, five patients with recurrent malignant glioma (ages 29–46 years) were treated with intracavitary alloreactive cytotoxic T lymphocytes (CTL) and interleukin-2 (IL-2). The trial tested the hypothesis that alloreactive CTL, sensitized to the major histocompatibility complex (MHC) proteins of the patient, offer selective, targeted killing of glioma cells that express MHC. Patient lymphocytes, which also express MHC, were irradiated and placed into CellMax artificial capillary systems with lymphocytes from MHC-disparate donors and CTL developed over a 2- to 3-week period with a low concentration of IL-2. The CTL largely expressed CD3 and CD11a/CD8 markers and lysed targets displaying patient MHC. CTL were implanted into the tumor bed at surgery and a catheter was used for subsequent infusions. Patients received one to five treatment cycles every other month; one cycle generally consisted of two or three CTL infusates administered within a 1- to 2-week period. Different unrelated donors were used for each cycle. Treatment was well tolerated; transient toxicity at grades 1–3 was recorded by NCI Common Toxicity Scale criteria. Two glioblastoma patients have died; one from tumor recurrence locally and the other from recurrence at a site distant from the treatment. Two of the five patients completed five cycles; one anaplastic oligodendroglioma patient shows no evidence of tumor 30 months from the start of immune therapy and an anaplastic astrocytoma patient shows stable disease 28 months after initiation of therapy. One anaplastic oligodendroglioma patient, who dropped the protocol during her second treatment cycle, has no evidence of tumor 28 months after recurrence. Received: 21 May 1997 / Accepted: 17 July 1997     

Novel Asn-linked oligosaccharides terminating in GaINAc{beta}(1->4)[Fuca(1->3)]GlcNAcB(1->{bullet}) are present in recombinant human Protein C expressed in human kidney 293 cells

Recombinant human Protein C (rHPC), expressed in human kidney293 cells, has a higher anticoagulant activity than plasma HPC,while its in vivo circulatory half-life is essentially unalteredcompared to that of the natural protein. In seeking to elucidatethe molecular basis for the improved efficacy of the recombinantantithrombotic drug, we focused on the carbohydrate moiety ofrHPC. Protein C is a heavily post-translationally modified serineprotease with four N-glycosylation sites. Glycosyl compositionanalysis of rHPC revealed a 5-fold higher fucose content anda 2-fold lower sialic acid content compared to plasma HPC. Inaddition, we found that rHPC contains N-acetylgaiac-tosamine(2.6 mol GalNAc/mol rHPC) in its Asn-linked oligosaccharides,while plasma HPC is devoid of GalNAc. The Asn-linked oligosaccharidesof rHPC were released by N-glycanase and separated into 25 fractionsby high-pH anion-exchange chromatography. The most abundantoligosaccharides were structurally characterized by glycosylcomposition and linkage analysis, in conjunction with ¹H-NMRspectroscopy at 600 MHz. The structure of the major neutraloligosaccharide in rHPC was determined to be: Two representatives of the sialylated oligosaccharides in rHPCare: and Thus, many of the Asn-linked oligosaccharides in rHPC were foundto terminate in GaINAcß(1     

A study of calcium pump activity of lysosomes from rat renal cortex

Fractions rich in either primary or secondary lysosomes were prepared from rat renal cortex by differential centrifugation and evaluated for their capacity for net calcium uptake. No uptake was observed in the absence of ATP. A vigorous uptake did take place in the presence of ATP but it was largely prevented by azide and other inhibitors of mitochondrial calcium uptake, suggesting that it was attributable to contamination by mitochondria. Evidence was obtained for an inhibitory influence of the secondary lysosomal fraction on mitochondrial calcium uptake.