Maintenance of hepatic nuclear factor 6 in postnatal islets impairs terminal differentiation and function of beta-cells.

The Onecut homeodomain transcription factor hepatic nuclear factor 6 (Hnf6) is necessary for proper development of islet beta-cells. Hnf6 is initially expressed throughout the pancreatic epithelium but is downregulated in endocrine cells at late gestation and is not expressed in postnatal islets. Transgenic mice in which Hnf6 expression is maintained in postnatal islets (pdx1(PB)Hnf6) show overt diabetes and impaired glucose-stimulated insulin secretion (GSIS) at weaning. We now define the mechanism whereby maintenance of Hnf6 expression postnatally leads to beta-cell dysfunction. We provide evidence that continued expression of Hnf6 impairs GSIS by altering insulin granule biosynthesis, resulting in a reduced response to secretagogues. Sustained expression of Hnf6 also results in downregulation of the beta-cell-specific transcription factor MafA and a decrease in total pancreatic insulin. These results suggest that downregulation of Hnf6 expression in beta-cells during development is essential to achieve a mature, glucose-responsive beta-cell.

Spermatogenesis of the Spider Crab Maja brachydactyla (Decapoda: Brachyura)

This study describes spermatogenesis in a majid crab (Maja brachydactyla) using electron microscopy and reports the origin of the different organelles present in the spermatozoa. Spermatogenesis in M. brachydactyla follows the general pattern observed in other brachyuran species but with several peculiarities. Annulate lamellae have been reported in brachyuran spermatogenesis during the diplotene stage of first spermatocytes, the early and mid-spermatids. Unlike previous observations, a Golgi complex has been found in midspermatids and is involved in the development of the acrosome. The Golgi complex produces two types of vesicles: light vesicles and electron-dense vesicles. The light vesicles merge into the cytoplasm, giving rise to the proacrosomal vesicle. The electron-dense vesicles are implicated in the formation of an electron-dense granule, which later merges with the proacrosomal vesicle. In the late spermatid, the endoplasmic reticulum and the Golgi complex degenerate and form the structures–organelles complex found in the spermatozoa. At the end of spermatogenesis, the materials in the proacrosomal vesicle aggregate in a two-step process, forming the characteristic concentric three-layered structure of the spermatozoon acrosome. The newly formed spermatozoa from testis show the typical brachyuran morphology.

Eimeria curvata n. sp.(Apicomplexa: Eimeriidae) in Columbina talpacoti and Scardafella squammata (Aves: Columbidae) from Brazil

Eimeria curvata is a new coccidian described in the doves Columbina talpacoti and Scardafella squammata from western of the State of São Paulo, Brazil. The oocysts are ovoid to ellipsoid, 18.3 (17-19) µm x 15.5 (15-17) µm, with a shape index of 1.2 (1.1-1.3). The wall is colorless, smooth and double-layered. A polar granule is present, but there is no micropyle or oocyst residuum. The sporocysts are elongate, 12.3 (11.5-13) µm x 5.8 (5.5-6) µm with a curved anterior portion and a smooth, thin, single-layered wall. The Stieda body is protuberant and nipple-like; there is no substieda body. The sporozoites lie head-to-tail in the sporocyst and contain a large refractile body at the extremities. The sporocyst residuum contains small granules uniformly distributed in the middle of the sporocyst. The prevalence of E. curvata n. sp. was 17.4% and 12.8% in C. talpacoti and S. squammata, respectively.

Morphological investigation of Toxoplasma gondii in vivo by a multiple beam interference microscope

A recently developed technique, namely multiple beam interference microscopy, has been applied to investigate the morphology of the parasite Toxoplasma gondii for the first time. The interference pattern obtained from the multiple internal reflection of a T. gondii, sandwiched between a glass plate and a cover plate, was focused on the objective of a conventional microscope. Because of the enhance contrast, several details of sub cellular structure and separating compartments are clearly visible. Details reveal the presence of a nucleus, lipid body, dense granule, rhoptry and amylopectin. The wall thickness of the membrane of the lipid body and the amylopectin is of the order of 0.02 µm and can be clearly distinguished with the help of the present technique. The same parasite has also been examined with the help of atomic force microscopy, and because of its thick membrane, the inner structural details were not observed at all. Sub cellular details of T. gondii observed with the present technique have been reported earlier only by low amplification transmission electron microscopy and not by any optical microscopic technique.

Distinct profiles of cytotoxic granules in memory CD8 T cells correlate with function, differentiation stage, and antigen exposure.

Cytotoxic CD8 T cells exert their antiviral and antitumor activity primarily through the secretion of cytotoxic granules. Degranulation activity and cytotoxic granules (perforin plus granzymes) generally define CD8 T cells with cytotoxic function. In this study, we have investigated the expression of granzyme K (GrmK) in comparison to that of GrmA, GrmB, and perforin. The expression of the cytotoxic granules was assessed in virus-specific CD8 T cells specific to influenza virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), or human immunodeficiency virus type 1 (HIV-1). We observed a dichotomy between GrmK and perforin expression in virus-specific CD8 T cells. The profile in influenza virus-specific CD8 T cells was perforin(-) GrmB(-) GrmA(+/-) GrmK(+); in CMV-specific cells, it was perforin(+) GrmB(+) GrmA(+) GrmK(-/+); and in EBV- and HIV-1-specific cells, it was perforin(-/+) GrmB(+) GrmA(+) GrmK(+). On the basis of the delineation of memory and effector CD8 T cells with CD45RA and CD127, the GrmK(+) profile was associated with early-stage memory CD8 T-cell differentiation, the perforin(+) GrmB(+) GrmA(+) profile with advanced-stage differentiation, and the GrmB(+) GrmA(+) Grmk(+) profile with intermediate-stage differentiation. Furthermore, perforin and GrmB but not GrmA and GrmK correlated with cytotoxic activity. Finally, changes in antigen exposure in vitro and in vivo during primary HIV-1 infection and vaccination modulated cytotoxic granule profiles. These results advance our understanding of the relationship between distinct profiles of cytotoxic granules in memory CD8 T cells and function, differentiation stage, and antigen exposure.

Isospora araponga sp. n. (Apicomplexa: Eimeriidae), a new species of Isospora Schneider from a bare-throated bellbird, Procnias nudicollis (Vieillot, 1817) (Passeriformes: Cotingidae) from Brazil

Faecal samples from a couple of bare-throated bellbirds Procnias nudicollis imported from Brazil to Barcelona Zoo contained oocysts of Isospora araponga n. sp. Sporulated oocysts were subspherical to broadly ellipsoidal, 19.5 (17-22) × 15.5 (14-16.5) µm, shape index (length:width ratio) 1.26 (1.13-1.38) with smooth and colourless bilayered wall, about 1 µm thick, and with varying number (1-3) of polar granule, but without a micropyle or residuum. The sporocysts were ellipsoidal, slightly asymmetric, 12.5 (12-13) × 8.5 (7.5-9) µm with barely visible Stieda body and indistinguishable substieda body. Sporozoites were elongated, possessing smooth surface and two distinct refractile bodies.

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

A new Eimeria (Apicomplexa: Eimeriidae), possessing mitra-shaped oocysts, from the Neotropical chelid turtle Batrachemys heliostemma (Testudines: Chelidae), and its comparison with Eimeria mitraria (Laveran & Mesnil 1902)

Eimeria jirkamoraveci sp. n. is described from faeces of two specimens of the toad-headed, side-necked turtle Batrachemys heliostemma collected at Iquitos in Peru. Oocysts are ovoid to almost spherical, 10.6 (8-12) × 8.9 (7-10) mum, without micropyle, polar granule and oocyst residuum. One conically stretched end and three blunt conical tubercles at the opposite end of oocyst give it mitra-like appearance. Sporocysts are elongated, ellipsoidal, 7.2 (6-8) × 4.1 (4-4.5) mum, with a small, knob-like Stieda body and sporocyst residuum composed of fine granules. To avoid possible conspecificity, the described new species is thoroughly compared with the most similar coccidium, E. mitraria, collected from its type host, Chinemys reevesii.

Three new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from the lesser seed-finch, Oryzoborus angolensis (Passeriformes: Emberizidae) from Brazil

Three new coccidian (Apicomplexa: Eimeriidae) species are reported from the lesser seed-finch, Oryzoborus angolensis from Brazil. Sporulated oocysts of Isospora curio n. sp. are spherical to subspherical; 24.6 × 23.6 (22-26 × 22-25) mum, shape-index (SI, length/width) of 1.04 (1.00-1.15). Oocyst wall is bilayerd, ~ 1.5 mum thick, smooth and colourless. Micropyle and oocyst residuum are absent. The sporocysts are ovoid, 13.2 × 10.9 (15-17 × 10-13) mum, SI = 1.56 (1.42-1.71), with a small Stieda body and residuum composed of numerous granules scattered among the sporozoites. Sporozoites are elongated and posses a smooth surface and two distinct refractile bodies. Oocysts of Isospora braziliensis n. sp. are spherical to subspherical, 17.8 × 16.9 (16-19 × 16-18) mum, with a shape-index of 1.06 (1.00-1.12) and a smooth, single-layered wall ~ 1 mum thick. A micropyle, oocyst residuum and polar granules are absent. Sporocysts are ellipsoid and slightly asymmetric, 13.2 × 10.8 (12-14 × 9-12) mum, SI = 1.48 (1.34-1.61). Each sporocyst contains a barely visible Stieda body and a residuum composed numerous of granules. Sporozoites are elongated and each of them contains two distinct refractile bodies. Oocysts of Isospora paranaensis n. sp. are subspherical to broadly ellipsoid 24.3 × 19.8 (22-26 × 18-22) mum, SI = 1.22 (1.15-1.38) with smooth single-layered wall ~ 1.5 mum thick. A micropyle and oocyst residuum are absent, but one distinct ellipsoid polar granule (2.5-3.5 × 1.5-2.5 mum) is present. Sporocyst are ovoid, 15.7 × 10.1 (14-18 × 8-12) mum, SI = 1.46 (1.31-1.72), with distinct Stieda and sub-Stieda bodies. Each sporocyst contains a spherical sporocyst residuum, 4 mum in diameter. All described isosporan species represent a possible cause of acute coccidiosis for O. angolensis in captivity.

Eimeria pavoaegyptica sp. nov. (Apicomplexa: Eimeriidae) in faeces of Indian peacocks, Pavo cristatus Linnaeus, 1758 (Galliformes: Phasianidae) from Egypt

Coprological examination of 15 Indian peacocks, Pavo cristatus, revealed the presence of a coccidium species of the genus Eimeria, which apparently represents a previously undescribed species. Sporulation is exogenous and fully developed oocysts of Eimeria pavoaegyptica sp. nov. are ellipsoidal, with a dimension of 15 (13-16) × 12 (10-12.9) μm and with a shape index of 1.25 (1-1.3). The sporulated oocysts have no micropyle but enclose one large rectangular-shaped polar granule and an oocyst residuum. The oocysts have a distinct two-layered wall, which is ~1.7 μm thick. The outer layer has a smooth texture; it fills ~¾ of the total thickness and appears bicolored. The sporocysts are boat-shaped, of about 10 (9-11) × 4 (4-4.7) μm; their average shape-index is 2.5 μm with a small pointed Stieda body and a smooth, thin single-layered wall. No substieda body is detected. The sporocysts contain numerous, nearly uniform granular residua. The sporozoites are banana-shaped, 6 × 3 μm and each has two different-sized refractile bodies.

Quantitative analysis of postnatal neurogenesis and neuron number in the macaque monkey dentate gyrus

The dentate gyrus is one of only two regions of the mammalian brain where substantial neurogenesis occurs postnatally. However, detailed quantitative information about the postnatal structural maturation of the primate dentate gyrus is meager. We performed design-based, stereological studies of neuron number and size, and volume of the dentate gyrus layers in rhesus macaque monkeys (Macaca mulatta) of different postnatal ages. We found that about 40% of the total number of granule cells observed in mature 5-10-year-old macaque monkeys are added to the granule cell layer postnatally; 25% of these neurons are added within the first three postnatal months. Accordingly, cell proliferation and neurogenesis within the dentate gyrus peak within the first 3 months after birth and remain at an intermediate level between 3 months and at least 1 year of age. Although granule cell bodies undergo their largest increase in size during the first year of life, cell size and the volume of the three layers of the dentate gyrus (i.e. the molecular, granule cell and polymorphic layers) continue to increase beyond 1 year of age. Moreover, the different layers of the dentate gyrus exhibit distinct volumetric changes during postnatal development. Finally, we observe significant levels of cell proliferation, neurogenesis and cell death in the context of an overall stable number of granule cells in mature 5-10-year-old monkeys. These data identify an extended developmental period during which neurogenesis might be modulated to significantly impact the structure and function of the dentate gyrus in adulthood.

A role for glutamate transporters in the regulation of insulin secretion.

In the brain, glutamate is an extracellular transmitter that mediates cell-to-cell communication. Prior to synaptic release it is pumped into vesicles by vesicular glutamate transporters (VGLUTs). To inactivate glutamate receptor responses after release, glutamate is taken up into glial cells or neurons by excitatory amino acid transporters (EAATs). In the pancreatic islets of Langerhans, glutamate is proposed to act as an intracellular messenger, regulating insulin secretion from β-cells, but the mechanisms involved are unknown. By immunogold cytochemistry we show that insulin containing secretory granules express VGLUT3. Despite the fact that they have a VGLUT, the levels of glutamate in these granules are low, indicating the presence of a protein that can transport glutamate out of the granules. Surprisingly, in β-cells the glutamate transporter EAAT2 is located, not in the plasma membrane as it is in brain cells, but exclusively in insulin-containing secretory granules, together with VGLUT3. In EAAT2 knock out mice, the content of glutamate in secretory granules is higher than in wild type mice. These data imply a glutamate cycle in which glutamate is carried into the granules by VGLUT3 and carried out by EAAT2. Perturbing this cycle by knocking down EAAT2 expression with a small interfering RNA, or by over-expressing EAAT2 or a VGLUT in insulin granules, significantly reduced the rate of granule exocytosis. Simulations of granule energetics suggest that VGLUT3 and EAAT2 may regulate the pH and membrane potential of the granules and thereby regulate insulin secretion. These data suggest that insulin secretion from β-cells is modulated by the flux of glutamate through the secretory granules.

Adult hippocampal neurogenesis inversely correlates with microglia in conditions of voluntary running and aging.

Adult hippocampal neurogenesis results in the formation of new neurons and is a process of brain plasticity involved in learning and memory. The proliferation of adult neural stem or progenitor cells is regulated by several extrinsic factors such as experience, disease or aging and intrinsic factors originating from the neurogenic niche. Microglia is very abundant in the dentate gyrus (DG) and increasing evidence indicates that these cells mediate the inflammation-induced reduction in neurogenesis. However, the role of microglia in neurogenesis in physiological conditions remains poorly understood. In this study, we monitored microglia and the proliferation of adult hippocampal stem/progenitor cells in physiological conditions known to increase or decrease adult neurogenesis, voluntary running and aging respectively. We found that the number of microglia in the DG was strongly inversely correlated with the number of stem/progenitor cells and cell proliferation in the granule cell layer. Accordingly, co-cultures of decreasing neural progenitor/glia ratio showed that microglia but not astroglia reduced the number of progenitor cells. Together, these results suggest that microglia inhibits the proliferation of neural stem/progenitor cells despite the absence of inflammatory stimulus.

Differential distribution of MAP1a and aldolase c in adult mouse cerebellum.

MAP1a is a microtubule-associated protein with an apparent molecular weight of 360 kDa that is found in the axonal and dendritic processes of neurons. Two monoclonal anti-MAP1a antibodies anti-A and anti-BW6, revealed different epitope distributions in the adult mouse cerebellum. Anti-A stained Purkinje and granule cells uniformly throughout the cerebellum. In contrast, anti-BW6 selectively stained the dendriites of a subset of Purkinje cells, revealing parasagittal bands of immunoreactivity in the molecular layer. The compartmentation of the BW6 epitope was compared to the Purkine cells as revealed by immunostaining with anti-zebrin II, a well known antigen expressed selectively by bands of Purkinje cells. The anti-BW6 staining pattern was complementary to the zebrin II bands, the zebrin II- Purkinjke cells having BW6+ dendrites. These results demonstrate that MAP1a is present in two forms in the mouse cerebellum, one of which is segregated into parasagittal bands. This may indicate a unique MAP1a isoform or may reflect differences in the metabolic states of Purkinje cell classes, and regional differences in their functions.

On the origin of the MR image phase contrast: an in vivo MR microscopy study of the rat brain at 14.1 T.

Recent studies at high magnetic fields using the phase of gradient-echo MR images have shown the ability to unveil cortical substructure in the human brain. To investigate the contrast mechanisms in phase imaging, this study extends, for the first time, phase imaging to the rodent brain. Using a 14.1 T horizontal bore animal MRI scanner for in vivo micro-imaging, images with an in-plane resolution of 33 microm were acquired. Phase images revealed, often more clearly than the corresponding magnitude images, hippocampal fields, cortical layers (e.g. layer 4), cerebellar layers (molecular and granule cell layers) and small white matter structures present in the striatum and septal nucleus. The contrast of the phase images depended in part on the orientation of anatomical structures relative to the magnetic field, consistent with bulk susceptibility variations between tissues. This was found not only for vessels, but also for white matter structures, such as the anterior commissure, and cortical layers in the cerebellum. Such susceptibility changes could result from variable blood volume. However, when the deoxyhemoglobin content was reduced by increasing cerebral blood flow (CBF) with a carbogen breathing challenge, contrast between white and gray matter and cortical layers was not affected, suggesting that tissue cerebral blood volume (and therefore deoxyhemoglobin) is not a major source of the tissue phase contrast. We conclude that phase variations in gradient-echo images are likely due to susceptibility shifts of non-vascular origin.