Control of mitotic exit in budding yeast. In vitro regulation of Tem1 GTPase by Bub2 and Bfa1

The elimination of mitotic kinase activity at the end of mitosis is essential for progression to the next stage of the eukaryotic cell cycle. In budding yeast, this process is controlled by a regulatory cascade called the mitotic exit network. Extensive genetic data indicate that mitotic exit network activity is determined by a GTP-binding protein, Tem1, and its putative regulators, Bub2, Bfa1, and Lte1. Here we describe the purification and in vitro activities of Tem1, Bub2, and Bfa1. We describe the nucleotide binding properties of Tem1 and characterize its intrinsic GTPase activity. The combination of Bfa1 and Bub2 acts as a two-component GTPase-activating protein for Tem1. In the absence of Bub2, Bfa1 inhibits the GTPase and GTP exchange activities of Tem1. This inhibition is elicited by either the N- or C-terminal regions of Bfa1, which also retain some ability to co-activate GTPase activity in the presence of Bub2. Although the C-terminal region of Bfa1 binds to Bub2, no interaction of the N-terminal half of Bfa1 with Bub2 was detected despite their combined GAP activity. Therefore, we propose that Bfa1 acts both as an adaptor to connect Bub2 and Tem1 and as an allosteric effector that facilitates this interaction.     

Spontaneous pancreatic islet amyloidosis in 40 baboons

Spontaneous amyloidosis occurs in many nonhuman primate species but remains difficult to diagnose and treat. Nonhuman primates continue to offer promise as animal models in which to study amyloidosis in humans. Amyloidosis was not diagnosed clinically but was found histologically in four male and 36 female baboons. The baboons averaged 18 years of age at death (range, 7-28 years). Clinical signs, if present, were hyperglycemia and cachexia. Blood glucose values were elevated in 12 of 30 baboons with available clinical pathology data. Four baboons had been clinically diagnosed as diabetic and three were treated with insulin. Amyloid was found in the islets of Langerhans of the pancreas in 40 baboons; 35 baboons had amyloid only in the islets of Langerhans. Amyloid was found in nonislet tissue of baboons as follows: five, nonislet pancreas; four, intestine and adrenal; three, kidney; two, prostate and spleen; and one each, lymph node, liver, gall bladder, stomach, tongue, urinary bladder, and salivary gland. Sections of paraffin-embedded tissues were evaluated for amyloid with hematoxylin and eosin (HE) and congo red (CR) staining, and using immunohistochemistry for human islet amyloid polypeptide (IAPP), calcitonin gene-related peptide (CGRP), glucagon, pancreatic polypeptide (PP), somatostatin (SS), and porcine insulin. Islet amyloid was positive with HE in 40 baboons, with CR in 39 baboons, and with IAPP and CGRP in 35 baboons. IAPP and CGRP only stained islet amyloid. PP, SS, glucagon, and porcine insulin did not stain amyloid. Islet amyloidosis in the baboon appears to be difficult to diagnose clinically, age-related, and similar to islet amyloidosis in other species. The baboon may be a good model for the study of islet amyloidosis in humans.     

Contrasting effects of UV-A and UV-B on photosynthesis and photoprotection of beta-carotene in two Dunaliella spp

Photosynthetic and antioxidant responses following exposure to either ultraviolet-A or ultraviolet-B were contrasted in two species of the unicellular green alga, DUNALIELLA: Species selection was based on the ability of Dunaliella bardawil (UTEX 2538) to accumulate inter-thylakoid beta-carotene when subjected to environmental stress while Dunaliella salina (UTEX 200) lacks this ability. Cells were cultured in high and low levels of visible light (150 and 35 micro mol photons m(-2 )s(-1), respectively) and then either ultraviolet-A (320-400 nm) or ultraviolet-B (290-320 nm) was added to visible light for 24-h exposure. A potassium chromate solution was found to be an ideal screen for removal of ultraviolet-A and ultraviolet-C from ultraviolet-B radiation. There were no significant changes in photosynthetic or antioxidant parameters following exposure to ultraviolet-B. Ultraviolet-A exposure significantly decreased photosynthetic parameters (>70% decrease in Fv/Fm and the ratio of light-limited to light-saturated photosynthesis in low beta-carotene cells) and resulted in 50% increases in ascorbate peroxidase activity and ascorbate concentrations. The results suggest exposure to ultraviolet-A (but not ultraviolet-B) directly affects photosynthesis, observed as a loss of photosystem II electron transport efficiency and increased radical formation. This research indicates that the accumulated beta-carotene in D. bardawil prevents UV-related photosynthetic damage through blue-light/ultraviolet-A absorption (supported by trends observed for antioxidant enzyme responses).     

Syntenic relationships between Medicago truncatula and Arabidopsis reveal extensive divergence of genome organization

Arabidopsis and Medicago truncatula represent sister clades within the dicot subclass Rosidae. We used genetic map-based and bacterial artificial chromosome sequence-based approaches to estimate the level of synteny between the genomes of these model plant species. Mapping of 82 tentative orthologous gene pairs reveals a lack of extended macrosynteny between the two genomes, although marker collinearity is frequently observed over small genetic intervals. Divergence estimates based on non-synonymous nucleotide substitutions suggest that a majority of the genes under analysis have experienced duplication in Arabidopsis subsequent to divergence of the two genomes, potentially confounding synteny analysis. Moreover, in cases of localized synteny, genetically linked loci in M. truncatula often share multiple points of synteny with Arabidopsis; this latter observation is consistent with the large number of segmental duplications that compose the Arabidopsis genome. More detailed analysis, based on complete sequencing and annotation of three M. truncatula bacterial artificial chromosome contigs suggests that the two genomes are related by networks of microsynteny that are often highly degenerate. In some cases, the erosion of microsynteny could be ascribed to the selective gene loss from duplicated loci, whereas in other cases, it is due to the absence of close homologs of M. truncatula genes in Arabidopsis.     

Hematology and blood biochemistry in infant baboons (Papio hamadryas)

Although published normative reference standards for hematologic and clinical chemistry measures are available for adult baboons, their applicability to infants has not been addressed. We analyzed these measures in 110 infant baboons (55 females and 55 males) from a large breeding colony at the Southwest Regional Primate Research Center in San Antonio, Texas. The sample consists of olive baboons and olive/yellow baboon hybrids, 1 week to 12 months of age. We produced cross-sectional reference values and examined the effects of age, sex, and subspecies on these variables. Hematology reference ranges for infant baboons are similar to, but wider than, those for adults. Reference ranges for blood biochemistry measures are generally more dissimilar to adults, indicating that for many variables, reference ranges for adult baboons are not adequate for infants. Although sex and subspecies differences are rare, age accounts for more than 10% of the variance in many of the variables.     

Targeting of cancer cells with monoclonal antibodies specific for C3b(i)

Purpose: The goal of this research is to determine the feasibility of an immunotherapeutic approach based on the use of monoclonal antibodies (mAb) to target complement activation fragments on opsonized cancer cells. Methods: We investigated whether treatment of LNCaP and C4-2 human prostate cancer cell lines with normal human serum would allow for deposition of sufficient amounts of the complement-activation protein C3b and its fragments [collectively referred to as C3b(i)] such that these proteins could serve as cancer-cell-associated antigens for targeting by mAb. Radioimmunoassays, flow cytometry, and magnetic purging with specific immunomagnetic beads were used for the analyses. Results: In vitro opsonization of human prostate cancer cells with normal human serum resulted in deposition of C3b(i) in sufficient quantity (approx. 100,000 molecules/cell) for the cells to be targeted in a variety of protocols. We found that ⁵¹Cr-labeled and C3b(i)-opsonized cancer cells could be specifically purged at high efficiency (95%–99%) using anti-C3b(i) mAb covalently coupled to magnetic beads. Flow-cytometry experiments indicated that most normal white cells were not removed under similar conditions. Opsonization of cancer cells with sera from men with prostate cancer led to lower levels of cell-associated IgM and, subsequently, lower amounts of C3b(i) deposited than in normal subjects. Prototype experiments suggested that this deficiency could be corrected by addition of IgM from normal donor plasma. Conclusion: mAb directed against complement-activation products may provide new opportunities to deliver diagnostic and therapeutic agents selectively to cancer cells and tumor deposits. These opportunities may include ex vivo purging of C3b(i)-opsonized cancer cells prior to autologous bone marrow or stem cell transplantation. Received: 17 February 2000 / Accepted: 1 August 2000     

The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.     

Metabolism and function of phenazines in bacteria: impacts on the behavior of bacteria in the environment and biotechnological processes

Phenazines constitute a large group of nitrogen-containing heterocyclic compounds produced by a diverse range of bacteria. Both natural and synthetic phenazine derivatives are studied due their impacts on bacterial interactions and biotechnological processes. Phenazines serve as electron shuttles to alternate terminal acceptors, modify cellular redox states, act as cell signals that regulate patterns of gene expression, contribute to biofilm formation and architecture, and enhance bacterial survival. Phenazines have diverse effects on eukaryotic hosts and host tissues, including the modification of multiple host cellular responses. In plants, phenazines also may influence growth and elicit induced systemic resistance. Here, we discuss emerging evidence that phenazines play multiple roles for the producing organism and contribute to their behavior and ecological fitness.