Genetic and molecular analysis of the Enhancer (En) transposable element system of Zea mays

A newly isolated, unstable mutation wx-844::En-1 of Zea mays was proven to be caused by the insertion of the autonomous transposable element En into the Waxy (Wx) gene. Molecular analysis revealed that En-1 is 8.4 kb long, has a 13-bp long perfect inverted repeat at its termini and generates a 3-bp target site duplication. En-1 is integrated into an intron located approximately in the middle of the transcribed region of the Wx gene. Structural evidence is presented indicating that a receptor component (Inhibitor) can arise by internal deletion of an autonomous En element.     

Modes of spindle pole body inheritance and segregation of the Bfa1p-Bub2p checkpoint protein complex.

Yeast spindle pole bodies (SPBs) duplicate once per cell cycle by a conservative mechanism resulting in a pre-existing 'old' and a newly formed SPB. The two SPBs of yeast cells are functionally distinct. It is only the SPB that migrates into the daughter cell, the bud, which carries the Bfa1p-Bub2p GTPase-activating protein (GAP) complex, a component of the spindle positioning checkpoint. We investigated whether the functional difference of the two SPBs correlates with the time of their assembly. We describe that in unperturbed cells the 'old' SPB always migrates into the bud. However, Bfa1p localization is not determined by SPB inheritance. It is the differential interaction of cytoplasmic microtubules with the mother and bud cortex that directs the Bfa1p-Bub2p GAP to the bud-ward-localized SPB. In response to defects of cytoplasmic microtubules to interact with the cell cortex, the Bfa1p-Bub2p complex binds to both SPBs. This may provide a mechanism to delay cell cycle progression when cytoplasmic microtubules fail to orient the spindle. Thus, SPBs are able to sense cytoplasmic microtubule properties and regulate the Bfa1p-Bub2p GAP accordingly.     

Leishmania (Viannia) braziliensis: human mast cell line activation induced by logarithmic and stationary promastigote derived-lysates

Herein we investigate the ability of live promastigotes and total lysate of Leishmania (Viannia) braziliensis, derived from parasites in the logarithmic (L-Lb) or stationary phase (S-Lb), to induce human mast cell line (HMC-1) activation. In comparison with medium-treated cells, a significant histamine release was observed in HMC-1 cultures stimulated with S-Lb. Lipophosphoglycan also induced histamine release by HMC-1 cells. In immunocytochemical assays, we found a marked staining for tryptase in medium-treated HMC-1 cells, however, stimulation with L-Lb or S-Lb caused a marked decrease in the color reaction as well as in the number of tryptase-positive cells. L-Lb and S-Lb induced an evident decrease in the intracellular expression of IL-4 but not IL-12. Live stationary promastigotes were able to induce high levels of IL-4 release in HMC-1 cultures. Furthermore, these cells released significant amounts of IL-12 when incubated with both types of live promastigotes. These results indicate that L. (V.) braziliensis promastigotes differ in their ability to induce direct human mast cells activation, according to the growth phase of the parasite. Furthermore, the release of pro-inflammatory mediators and cytokines could represent an important phenomenon that might favor the initial establishment of the infection.     

An Acidic Cluster in the Cytosolic Domain of Human Cytomegalovirus Glycoprotein B Is a Signal for Endocytosis from the Plasma Membrane

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is transported to apical membranes in CMV-infected polarized retinal pigment epithelial (ARPE-19) cells and in Madin-Darby canine kidney (MDCK) epithelial cells constitutively expressing gB. The cytosolic domain of gB contains a cluster of acidic amino acids, a motif that plays a pivotal role in vectorial trafficking in polarized epithelial cells and may also function as a signal for entry into the endocytic pathway. Here we compared gB internalization and recycling to the plasma membrane in CMV-infected human fibroblasts (HF) and ARPE-19 cells by using antibody-internalization experiments. Immunofluorescence and quantitative assays showed that gB was internalized from the cell surface into clathrin-coated transport vesicles and then recycled to the plasma membrane. gB colocalized with clathrin-coated vesicles containing the transferrin receptor in the early endocytic/recycling pathway, indicating that gB traffics in this pathway. The specific role of the acidic cluster in regulating the sorting of gB-containing vesicles in the early endocytic/recycling pathway was examined in MDCK cells expressing mutated gB derivatives. Immunofluorescence assays showed that derivatives lacking the acidic cluster were impaired in internalization and failed to recycle. These findings, together with our earlier observation that the acidic cluster is a key determinant for targeting gB molecules to apical membranes in epithelial cells, establish that this signal is recognized by cellular proteins that participate in polarized sorting and transport in the early endocytic/recycling pathway.     

Seasonal shedding of multiple Cryptosporidium genotypes in California ground squirrels (Spermophilus beecheyi)

Twelve percent of 853 California ground squirrels (Spermophilus beecheyi) from six different geographic locations in Kern County, Calif., were found to be shedding on average 44,482 oocysts g of feces(-1). The mean annual environmental loading rate of Cryptosporidium oocysts was 57,882 oocysts squirrel(-1) day(-1), with seasonal patterns of fecal shedding ranging from <10,000 oocysts squirrel(-1) day(-1) in fall, winter, and spring to levels of 2 x 10(5) oocysts squirrel(-1) day(-1) in summer. Juveniles were about twice as likely as adult squirrels to be infected and shed higher concentrations of oocysts than adults did, with particularly high levels of infection and shedding being found among juvenile male squirrels. Based on DNA sequencing of a portion of the 18S small-subunit rRNA gene, there existed three genotypes of Cryptosporidium species in these populations of squirrels (Sbey03a, Sbey03b, and Sbey03c; accession numbers AY462231 to AY462233, respectively). These unique DNA sequences were most closely related (96 to 97% homology) to porcine C. parvum (AF115377) and C. wrairi (AF115378). Inoculating BALB/c neonatal mice with up to 10,000 Sbey03b or Sbey03c fresh oocysts from different infected hosts did not produce detectable levels of infection, suggesting that this common genotype shed by California ground squirrels is not infectious for mice and may constitute a new species of Cryptosporidium.     

Cytoprotective Agent in <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis> Extracts

Adenosine 5′-diphosphoribose (ADP-ribose) has been identified as a significant contributor to the anti-cytotoxic activity of Lactobacillus bulgaricus extracts. Although the biological activities associated with the administration of probiotic bacteria and components thereof are sometimes attributed to the peptidoglycans that comprise a substantial portion of the Gram-positive bacterial cell wall, we found that the beta-nicotine adenine dinucleotide (NAD) hydrolysis product ADP-ribose was a significant contributor to the observed anti-cytotoxicity in our L. bulgaricus extracts. The ADP-ribose was isolated, identified, and quantitated by high performance liquid chromatography (HPLC) and by nuclear magnetic resonance (NMR) spectroscopy. ADP-ribose levels as low as 5 mg/L exhibited a measurable inhibition of tumor necrosis factor alpha (TNF-α) mediated cytotoxicity in an in vitro cell assay, whereas the ADP-ribose content of the L. bulgaricus extracts often exceeded 5 mg/g dry weight.     

The yeast centrosome translates the positional information of the anaphase spindle into a cell cycle signal

The spindle orientation checkpoint (SPOC) of budding yeast delays mitotic exit when cytoplasmic microtubules (MTs) are defective, causing the spindle to become misaligned. Delay is achieved by maintaining the activity of the Bfa1-Bub2 guanosine triphosphatase-activating protein complex, an inhibitor of mitotic exit. In this study, we show that the spindle pole body (SPB) component Spc72, a transforming acidic coiled coil-like molecule that interacts with the gamma-tubulin complex, recruits Kin4 kinase to both SPBs when cytoplasmic MTs are defective. This allows Kin4 to phosphorylate the SPB-associated Bfa1, rendering it resistant to inactivation by Cdc5 polo kinase. Consistently, forced targeting of Kin4 to both SPBs delays mitotic exit even when the anaphase spindle is correctly aligned. Moreover, we present evidence that Spc72 has an additional function in SPOC regulation that is independent of the recruitment of Kin4. Thus, Spc72 provides a missing link between cytoplasmic MT function and components of the SPOC.     

Rotavirus and adenovirus in Rondônia

Acute gastroenteritis is one of the most common diseases in humans worldwide. Viral gastroenteritis is a global problem in infants and young children. In this study the incidence of diarrhea was assessed in 877 hospitalized children under five years old, over a period of 24 months and distributed in 470 cases of diarrhea and 407 age-matched group with other pathologies, as control group. Two antigen detection techniques based on enzyme immunoassay (EIA) and latex particles were used for detection of rotavirus and adenovirus. Rotavirus A was a major cause of gastroenteritis with 23.6% of cases, being 90% of these cases in young children. Adenovirus infections was detected by EIA with frequency of 6.4%. Rotavirus and adenovirus were detected in 10.1 and 1.7% of stools from control group, respectively. Interestingly, the frequency of the youngest children in the control group excreting Rotavirus A was comparable to that detected in stools from diarrheic children. We cannot rule out the existence of other enteric viruses because the etiology of 171 cases of diarrhea was not determined and active search for astrovirus and calicivirus was not done. This is the first study that shows the presence of enteric viruses in the infantile population from Western Brazilian Amazonia and it was important to help physicians in the treatment of viral gastroenteritis.     

Long-term effects of overfeeding during lactation on insulin secretion--the role of GLUT-2

Overnutrition during critical developmental periods is believed to be a risk factor for the emergence of metabolic disorders in adulthood. The present study investigated the effects of pups overfeeding during lactation on offspring's insulin secretion. To study the consequences of overnutrition early in life in rats, litter size reduction has been shown to be an appropriate experimental model. To induce early postnatal overnutrition, litter size was reduced to three pups per litter at the third day following birth [overfed group (OG)]. In the control group (CG), the litter size was adjusted to 10 pups per litter. Metabolic parameters and glucose-stimulated insulin secretion were assessed. OG pups ingested more milk at 10 and 21 days and had an augmented food intake at 1 year compared to the CG. Consistently, body weight, body fat, and fasting plasma levels of insulin were higher in 1-year-old OG rats. In addition, OG rats exhibited enhanced insulin secretion, accompanied by elevated content of GLUT-2 in pancreatic islets compared to CG. These findings indicate that early postnatal overnutrition during a critical developmental period in life may program permanent alterations in glucose-stimulated insulin secretion.