Purification and some properties of the squalene-tetrahymanol cyclase from Tetrahymena thermophila

The membrane-bound enzyme from Tetrahymena thermophila responsible for the conversion of squalene into the quasi-hopanoid tetrahymanol was purified 297-fold to near homogeneity. Purification involved solubilization by octylthioglucoside, chromatography on DEAE-trisacryl, hydroxyapatite and FPLC ion-exchange on Mono Q. The apparent KM was found to be 18 microM. 2,3-Iminosqualene and N,N-dimethyldodecylamine-N-oxide are effective inhibitors of the cyclase with I50 values of 50 and 30 nM, respectively. The cyclase has a molecular mass of 72 kDa as judged by electrophoresis in polyacrylamide gels under denaturating conditions. The optimal enzymatic activity was obtained at pH 7.0 and 30 degrees C. The solubilized enzyme needs the presence of detergent for maintaining activity. The influence of different detergents on cyclase activity was studied. Triton X-100 proved to be a strong inactivator of the enzyme. Solubilization of the cyclase in Tween 80 and digitonin inactivates the enzyme. However, its activity can be recovered by complementation of the assay buffer with octylthioglucoside above its critical micellar concentration. We suggest that this approach might be applicable to other membrane-bound proteins.     

Biosynthesis of triterpenoid sapogenols in soybean and alfalfa seedlings

By incubation of germinating soybeans with mevalonate-[2-¹⁴C] (MVA), radioactivity was incorporated into four sapogenols which were identified by TLC. Unequivocal evidence for the identity of three of the four sapogenols was provided by co-crystallization to constant specific radioactivity. The partition of incorporated radioactivity into lipid- and water-soluble fractions and the pattern of radioactivity of individual sapogenols varied with the mode of administering labeled substrates to soybean seedlings, such as incubation of germinating soybeans with MVA-[2-¹⁴C], immersion of roots into MVA-[2-¹⁴C] or foliar application of squalene-[¹⁴C]. When alfalfa seedlings were incubated with MVA-[2-¹⁴C], about two-thirds of the radioactivity incorporated into the sapogenols was associated with medicagenic acid.     

Long-Lasting Maintenance of Learning-Induced Enhanced Neuronal Excitability: Mechanisms and Functional Significance

Pyramidal neurons in the piriform cortex of olfactory discrimination trained rats show enhanced intrinsic neuronal excitability that lasts for several days after learning. Such enhanced intrinsic excitability is mediated by long-term reduction in the postburst after hyperpolarization which is generated by repetitive spike firing. The molecular machinery underlying such long-lasting modulation of intrinsic excitability, as well as its exceptional durability, is yet to be fully described. In this review, we present recent advancements that reveal the identity of the current that is modulated after learning and the second messenger system by which enhanced excitability is maintained. We also discuss the significance of such long-lasting modulation to the local network’s sensitivity to noradrenaline, a major learning-relevant neuromodulator.     

The ADNP Derived Peptide,NAP Modulates the Tubulin Pool: Implication for Neurotrophic and Neuroprotective Activities

Microtubules (MTs), key cytoskeletal elements in living cells, are critical for axonal transport, synaptic transmission, and maintenance of neuronal morphology. NAP (NAPVSIPQ) is a neuroprotective peptide derived from the essential activity-dependent neuroprotective protein (ADNP). In Alzheimer’s disease models, NAP protects against tauopathy and cognitive decline. Here, we show that NAP treatment significantly affected the alpha tubulin tyrosination cycle in the neuronal differentiation model, rat pheochromocytoma (PC12) and in rat cortical astrocytes. The effect on tubulin tyrosination/detyrosination was coupled to increased MT network area (measured in PC12 cells), which is directly related to neurite outgrowth. Tubulin beta3, a marker for neurite outgrowth/neuronal differentiation significantly increased after NAP treatment. In rat cortical neurons, NAP doubled the area of dynamic MT invasion (Tyr-tubulin) into the neuronal growth cone periphery. NAP was previously shown to protect against zinc-induced MT/neurite destruction and neuronal death, here, in PC12 cells, NAP treatment reversed zinc-decreased tau-tubulin-MT interaction and protected against death. NAP effects on the MT pool, coupled with increased tau engagement on compromised MTs imply an important role in neuronal plasticity, protecting against free tau accumulation leading to tauopathy. With tauopathy representing a major pathological hallmark in Alzheimer''s disease and related disorders, the current findings provide a mechanistic basis for further development. NAP (davunetide) is in phase 2/3 clinical trial in progressive supranuclear palsy, a disease presenting MT deficiency and tau pathology.     

Dahlia spectabilis (Asteraceae, Coreopsideae), a new species from San Luis Potosí, Mexico

Dahlia spectabilis (Asteraceae, Coreopsideae), a new species from San Luis Potosí, Mexico, is described and illustrated. The plant is distinguished by its large habit, big flowering heads, and overall lack of trichomes. It occurs within the natural range for the genus and is known from only one location where it is under heavy grazing pressure.

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Resumen   Dahlia spectabilis (Asteraceae, Coreopsideae), una especie nueva del estado de San Luis Potosí, México, es descrita e ilustrada. Se distingue por su hábito más grande, su grandes inflorescencias y la falta de tricomas. Ocuriendo dento del rango natural del género, conocida solo por un poblacón, la cual está sufriendo por presión de ganado.

     

DNA-free recombinant SV40 capsids protect mice from acute renal failure by inducing stress response, survival pathway and apoptotic arrest

Viruses induce signaling and host defense during infection. Employing these natural trigger mechanisms to combat organ or tissue failure is hampered by harmful effects of most viruses. Here we demonstrate that SV40 empty capsids (Virus Like Particles-VLPs), with no DNA, induce host Hsp/c70 and Akt-1 survival pathways, key players in cellular survival mechanisms. We postulated that this signaling might protect against organ damage in vivo. Acute kidney injury (AKI) was chosen as target. AKI is critical, prevalent disorder in humans, caused by nephrotoxic agents, sepsis or ischemia, via apoptosis/necrosis of renal tubular cells, with high morbidity and mortality. Systemic administration of VLPs activated Akt-1 and upregulated Hsp/c70 in vivo. Experiments in mercury-induced AKI mouse model demonstrated that apoptosis, oxidative stress and toxic renal failure were significantly attenuated by pretreatment with capsids prior to the mercury insult. Survival rate increased from 12% to >60%, with wide dose response. This study demonstrates that SV40 VLPs, devoid of DNA, may potentially be used as prophylactic agent for AKI. We anticipate that these finding may be projected to a wide range of organ failure, using empty capsids of SV40 as well as other viruses.     

Gonoid thelytoky in soft scale insects (Coccidae: Homoptera)

Cytological analysis of the thelytokous soft-scale insects Coccus hesperidum L. (2n=14) and Saissetia coffeae (Walker) (2n=16) revealed that while in both species the chromosomes did not pair during prophase I, meiosis consisted of two divisions, the chromosome number was reduced, and diploidy was restored by the fusion of the female pronucleus with the polar nucleus II. The difficulty of trying to classify this type of thelytoky as either automictic or apomictic led to the proposal that a new criterion and new terms be used to classify thelytoky (and parthenogenesis). The new criterion is whether the number of chromosome elements present in the first (or only) metaphase of oogenesis is the same as that present in the oogonia (gonoid thelytoky) or different from it (agonoid thelytoky). The new criterion is superior to the existing criteria because it is unambiguous, and because it groups together forms with a similar tendency towards heterozygosity (or homozygosity). The possible evolution of the forms analyzed as well as the two other thelytokous forms of each species described by Thomsen (1927) are discussed. Another soft-scale insect, Physokermes hemicryphus Dalam, consisted of a diploid (2n=18) and a triploid (3n=27) form, in both of which the chromosomes also did not pair. Each of the three species contained a strain in which only a single nucleolus was present per cell. In C. hesperidum some strains with two nucleoli differed in the size of the nucleoli.     

CD8+ T cell epitope-flanking mutations disrupt proteasomal processing of HIV-1 Nef

CTL play a critical role in the control of HIV and SIV. However, intrinsic genetic instability enables these immunodeficiency viruses to evade detection by CTL through mutation of targeted antigenic sites. These mutations can impair binding of viral epitopes to the presenting MHC class I molecule or disrupt TCR-mediated recognition. In certain regions of the virus, functional constraints are likely to limit the capacity for variation within epitopes. Mutations elsewhere in the protein, however, might still enable immune escape through effects on Ag processing. In this study, we describe the coincident emergence of three mutations in a highly conserved region of Nef during primary HIV-1 infection. These mutations (R69K, A81G, and H87R) flank the HLA B*35-restricted VY8 epitope and persisted to fixation as the early CTL response to this Ag waned. The variant form of Nef showed a reduced capacity to activate VY8-specific CTL, although protein stability and expression levels were unchanged. This effect was associated with altered processing by the proteasome that caused partial destruction of the VY8 epitope. Our data demonstrate that a variant HIV genotype can significantly impair proteasomal epitope processing and substantiate the concept of immune evasion through diminished Ag generation. These observations also indicate that the scale of viral escape may be significantly underestimated if only intraepitope variation is evaluated.     

Conservation of an open-reading frame as an element affecting 5' splice site selection

Splice site selection is a key element of pre-mRNA splicing and involves specific recognition of consensus sequences at the 5(') and 3(') splice sites. Evidently, the compliance of a given sequence with the consensus 5(') splice site sequence is not sufficient to define it as a functional 5(') splice site, because not all sequences that conform with the consensus are used for splicing. We have previously hypothesized that the necessity to avoid the inclusion of premature termination codons within mature mRNAs may serve as a criterion that differentiates normal 5(') splice sites from unused (latent) ones. We further provided experimental support to this idea, by analyzing the splicing of pre-mRNAs in which in-frame stop codons upstream of a latent 5(') splice site were mutated, and showing that splicing using the latent site is indeed activated by such mutations. Here we evaluate this hypothesis by a computerized survey for latent 5(') splice sites in 446 protein-coding human genes. This data set contains 2311 introns, in which we found 10490 latent 5(') splice sites. The utilization of 10045 (95.8%) of these sites for splicing would have led to the inclusion of an in-frame stop codon within the resultant mRNA. The validity of this finding is confirmed here by statistical analyses. This finding, together with our previous experimental results, invokes a nuclear scanning mechanism, as part of the splicing machine, which identifies in-frame stop codons within the pre-mRNA and prevents splicing that could lead to the formation of a prematurely terminated protein.