RASSF2 is a novel K-Ras-specific effector and potential tumor suppressor

Ras proteins regulate a wide range of biological processes by interacting with a broad assortment of effector proteins. Although activated forms of Ras are frequently associated with oncogenesis, they may also provoke growth-antagonistic effects. These include senescence, cell cycle arrest, differentiation, and apoptosis. The mechanisms that underlie these growth-inhibitory activities are relatively poorly understood. Recently, two related novel Ras effectors, NORE1 and RASSF1, have been identified as mediators of apoptosis and cell cycle arrest. Both of these proteins exhibit many of the properties normally associated with tumor suppressors. We now identify a novel third member of this family, designated RASSF2. RASSF2 binds directly to K-Ras in a GTP-dependent manner via the Ras effector domain. However, RASSF2 only weakly interacts with H-Ras. Moreover, RASSF2 promotes apoptosis and cell cycle arrest and is frequently down-regulated in lung tumor cell lines. Thus, we identify RASSF2 as a new member of the RASSF1 family of Ras effectors/tumor suppressors that exhibits a specificity for interacting with K-Ras.     

The effects of starvation and subsequent feeding on juvenile hormone synthesis and oöcyte growth in Schistocerca americana gregaria

The effect of starvation on the synthesis of C₁₆ juvenile hormone (JH) and the growth of terminal oöcytes was assessed in Schistocerca americana gregaria at two times during adult life: before activation of the corpora allata and during the first gonotrophic cycle. In both groups, starvation resulted in a decline in JH synthesis within 2–3 days and rates of synthesis remained low throughout the experimental period. The growth rate of oöcytes which were not vitellogenic at the time of starvation was depressed whereas the percentage of resorption of vitellogenic oöcytes increased dramatically with starvation. Although the percentage of resorption increased in animals with vitellogenic oöcytes, some mature oöcytes were produced, particularly in animals in which the oöcytes were greater than 5 mm in length at the time of starvation. This suggests that oöcyte maturation can be divided into two distinct phases—an early phase of vitellogenesis associated with high rates of JH synthesis and a late phase, in oöcytes greater than 5 mm, associated with much lower rates of JH synthesis.Stimulation of JH synthesis by farnesenic acid in 5-day starved animals resulted in high rates of JH synthesis, indicating that starvation did not appreciably alter the enzymic activities of the final two stages in JH synthesis. Thus rate limitation did not occur at these stages.Feeding of 5-day starved animals resulted in a transient increase in the rate of JH synthesis. However, rates of JH synthesis and oöcyte growth remained subnormal throughout the observation period, suggesting that the effects of starvation cannot be entirely reversed by feeding. Thus starvation may decrease the reproductive potential of the females.     

Quantitative trait loci for polyamine content in an RFLP-mapped potato population and their relationship to tuberization

DNA-based genetic markers are now widely used by geneticists to locate genes for quantitative traits, and may also serve as a valuable tool for dissecting complex physiological phenomena. Van den Berg et al. (1996a QTL analysis of potato tuberization. Theor Appl Gen 93: 307–316), using restriction fragment length polymorphism (RFLP)-mapped populations of potato, detected eleven quantitative trait loci (QTLs) for tuberization. Taylor et al. (1992 Expression and sequence analysis of cDNAs induced during the early stages of tuberisation in different organs of the potato plant [ Solanum tuberosum L.]. Plant Mol Biol 20: 641–651) have identified one of the genes associated with tuberization as that for the enzyme S-adenosylmethionine decarboxylase (SAMdc), an enzyme of the polyamine biosynthetic pathway. Chromosomal loci for SAMdc and arginine decarboxylase were established on the potato and tomato chromosomal maps, respectively, by hybridizing cDNA probes for these genes to RFLP digests. The polyamine content of leaves from an RFLP-mapped potato population was analyzed by fluorescence detection following HPLC, with quantitation using an internal standard. The data were analyzed by the 'qGene' statistical program, and QTLs for polyamines were detected on seven chromosomes. At least six QTLs were found for spermine, two for spermidine, and two for putrescine. A spermidine QTL was on chromosome 5 linked to marker TG441 , very close to the place where SAMdc mapped. There was some congruence between QTLs for spermine and those previously detected for tuberization and dormancy, but relationships were not consistent.     

Determination of XR510, a balanced angiotensin II receptor antagonist, in dog and rat plasma by combined liquid-liquid/solid-phase extraction and high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method for the determination of XR510 (I), a new non-peptide angiotensin II (AII) receptor antagonist with balanced affinity for AT₁ and AT₂ receptor subtypes is described. I and the internal standard, XR513, were extracted from acidified plasma by combined liquid-liquid/solid-phase extraction and chromatographed on a phenyl column with ultraviolet absorbance detection at a wavelength of 272 nm. The mobile phase consisted of a mixture of acetonitrile and sodium phosphate buffer. For both rat and dog plasma, the limit of quantitation was 5 ng/ml. This method has been validated and successfully utilized to investigate the disposition of I.     

Harassment and killing of porpoises (“phocoenacide”) by fish-eating Southern Resident killer whales (Orcinus orca)

Endangered Southern Resident killer whales (Orcinus orca) are fish-eaters that preferentially prey on adult Chinook salmon (Oncorhynchus tshawytscha). Despite being salmon specialists, individuals from all three killer whale pods (J, K, L) have been observed harassing and killing porpoises (family Phocoenidae) without consuming them. Retrospectively, we identified and analyzed 78 episodes of Southern Resident killer whales harassing porpoises between 1962 and 2020, of which 28 resulted in the porpoise's death (“phocoenacide”). Fifty-six episodes involved harbor porpoise (Phocoena phocoena), 13 involved Dall's porpoise (Phocoenoides dalli), and the porpoise species was unreported for nine episodes. Southern Resident killer whales often targeted young porpoises that were similar in size to adult Chinook salmon. Both sexes participated in porpoise harassment. Juveniles engaged in the behavior the most; however, their rates of engagement were not found to differ significantly from most other age classes. The behavior was passed through generations and social groupings, as it was first observed in L pod and spread to the other two pods. Killer whales are highly complex animals known to exhibit social learning and cultural transmission of learned behaviors, but the reason(s) for this behavior is unknown. Hypotheses include the social and developmental benefits of play, hunting practice, or displaced epimeletic behavior.     

NPC1 regulates the distribution of phosphatidylinositol 4‐kinases at Golgi and lysosomal membranes

Cholesterol and phosphoinositides (PI) are two critically important lipids that are found in cellular membranes and dysregulated in many disorders. Therefore, uncovering molecular pathways connecting these essential lipids may offer new therapeutic insights. We report that loss of function of lysosomal Niemann‐Pick Type C1 (NPC1) cholesterol transporter, which leads to neurodegenerative NPC disease, initiates a signaling cascade that alters the cholesterol/phosphatidylinositol 4‐phosphate (PtdIns4P) countertransport cycle between Golgi‐endoplasmic reticulum (ER), as well as lysosome‐ER membrane contact sites (MCS). Central to these disruptions is increased recruitment of phosphatidylinositol 4‐kinases—PI4KIIα and PI4KIIIβ—which boosts PtdIns4P metabolism at Golgi and lysosomal membranes. Aberrantly increased PtdIns4P levels elevate constitutive anterograde secretion from the Golgi complex, and mTORC1 recruitment to lysosomes. NPC1 disease mutations phenocopy the transporter loss of function and can be rescued by inhibition or knockdown of either key phosphoinositide enzymes or their recruiting partners. In summary, we show that the lysosomal NPC1 cholesterol transporter tunes the molecular content of Golgi and lysosome MCS to regulate intracellular trafficking and growth signaling in health and disease.     

LOCALIZATION OF PHYTOCHROME DURING PEANUT (ARACHIS HYPOGAEA) GYNOPHORE AND OVULE DEVELOPMENT

Previous studies indirectly indicated that phytochrome plays a role in peanut (Arachis hypogaea L. cv. Virginia) gynophore elongation and in ovule and embryo development. Recent advances in the use of monoclonal antibody procedures used in this study have allowed precise localization of phytochrome in the developing peanut gynophore and ovular tissues. Peanut phytochrome from etiolated tissues was found to have a molecular weight of 124 kD as determined by immunoblotting procedures using a monoclonal antibody to pea (Pisum sativum L. cv. Alaska) phytochrome. Immunoblotting procedures revealed that no detectable phytochrome was present in the gynophore tissues or immature ovules during the elongation of the peanut gynophores. After the gynophores penetrated the soil for 8–12 d, phytochrome was detected in increasing amounts in the ovular tissues but not the gynophore tissues. Immunohistological analysis revealed that phytochrome was localized in the developing embryo and adjacent integument tissues. These findings contradict earlier reports that suggested phytochrome was initially present in the gynophore tissues after fertilization where it was believed to inhibit ovular development and stimulate gynophore elongation.     

Structural Genomics of Minimal Organisms and Protein Fold Space

The initial aim of the Berkeley Structural Genomics Center is to obtain a near-complete structural complement of two minimal organisms, closely related pathogens Mycoplasma genitalium and M. pneumoniae. The former has fewer than 500 genes and the latter fewer than 700 genes. To achieve this goal, the current protein targets have been selected starting with those predicted to be most tractable and likely to yield new structural and functional information. During the past 3 years, the semi-automated structural genomics pipeline has been set up from cloning, expression, purification, and ultimately to structural determination. The results from the pipeline substantially increased the coverage of the protein fold space of M. pneumoniae and M. genitalium. Furthermore, about 1/2 of the structures of ‘unique’ protein sequences revealed new and novel folds, and over 2/3 of the structures of previously annotated ‘hypothetical proteins’ inferred their molecular functions.     

Reversion of CTL escape-variant immunodeficiency viruses in vivo

Engendering cytotoxic T-lymphocyte (CTL) responses is likely to be an important goal of HIV vaccines. However, CTLs select for viral variants that escape immune detection. Maintenance of such escape variants in human populations could pose an obstacle to HIV vaccine development. We first observed that escape mutations in a heterogeneous simian immunodeficiency virus (SIV) isolate were lost upon passage to new animals. We therefore infected macaques with a cloned SIV bearing escape mutations in three immunodominant CTL epitopes, and followed viral evolution after infection. Here we show that each mutant epitope sequence continued to evolve in vivo, often re-establishing the original, CTL-susceptible sequence. We conclude that escape from CTL responses may exact a cost to viral fitness. In the absence of selective pressure upon transmission to new hosts, these original escape mutations can be lost. This suggests that some HIV CTL epitopes will be maintained in human populations.