Salt bridges do not stabilize polyproline II helices

Interactions between side chains, and in particular salt bridges, have been shown to be important in the stabilization of secondary structure. Here we investigate the contribution of a salt bridge formed between a lysine and a glutamate to the polyproline II (P(II)) helical content of proline-rich peptides. Since this structure has precisely three residues per turn, charged residues spaced three residues apart are on the same side of the helix and are best situated to interact. By contrast, computer simulations show that charged residues spaced four residues apart are both too far apart to interact strongly and are oriented such that interactions are unlikely. We have measured the P(II) content of peptides containing a lysine and glutamate pair spaced three or four residues apart using circular dichroism spectroscopy. Somewhat surprisingly we find that the P(II) content is insensitive to both the spacing and the pH. These findings indicate that i --> i + 3 salt bridges do not stabilize the P(II) helical conformation. The implications of these observations for both P(II) helix formation and denatured protein conformations are discussed.     

The Na,K-ATPase alpha 2 isoform is expressed in neurons,and its absence disrupts neuronal activity in newborn mice

Na,K-ATPase is an ion transporter that impacts neural and glial physiology by direct electrogenic activity and the modulation of ion gradients. Its three isoforms in brain have cell-type and development-specific expression patterns. Interestingly, our studies demonstrate that in late gestation, the alpha2 isoform is widely expressed in neurons, unlike in the adult brain, in which alpha2 has been shown to be expressed primarily in astrocytes. This unexpected distribution of alpha2 isoform expression in neurons is interesting in light of our examination of mice lacking the alpha2 isoform which fail to survive after birth. These animals showed no movement; however, defects in gross brain development, muscle contractility, neuromuscular transmission, and lung development were ruled out. Akinesia suggests a primary neuronal defect and electrophysiological recordings in the pre-B?tzinger complex, the brainstem breathing center, showed reduction of respiratory rhythm activity, with less regular and smaller population bursts. These data demonstrate that the Na,K-ATPase alpha2 isoform could be important in the modulation of neuronal activity in the neonate.     

Improving male mating competitiveness and survival in the field for medfly,Ceratitis capitata (Diptera: Tephritidae) SIT programs

The success of the sterile insect technique (SIT) depends critically upon mating between released sterilized males and wild females. In Hawaii, improvements in the efficiency of sterile males were attempted on two separate fronts – mating enhancement and survival improvement. In the former, two methods have been investigated – selective breeding and aromatherapy. In the latter, flies which survived in field cages for several days were selected and bred to produce progeny with enhanced survival ability compared to control flies. Regarding mating selection, standard laboratory-reared males that successfully mated with wild females in field cages were allowed to breed. F1 offspring were inbred, then the selection procedure was repeated for four additional cycles. In the aromatherapy procedure, laboratory-reared males were exposed to ginger root oil for several hours 1 day prior to testing in field cages. Compared to controls, the selected flies improved the mating competitiveness of male flies ca. 3-fold, irradiation reduced this increase to ca. 2.5-fold. Exposing the selected, hybrid strain raised the fitness of the lab males to ca. 9-fold that of wild males. In the ongoing survival selection study, we have obtained lines in which the selected males survived ca. 2-fold better than laboratory control males over several days in an outdoor field cage, with food and water provided. The goal is to combine the traits of higher survival and mating ability into a single strain for SIT release.     

Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration

To test the hypothesis that inhibition of axonal transport is sufficient to cause motor neuron degeneration such as that observed in amyotrophic lateral sclerosis (ALS), we engineered a targeted disruption of the dynein-dynactin complex in postnatal motor neurons of transgenic mice. Dynamitin overexpression was found to disassemble dynactin, a required activator of cytoplasmic dynein, resulting in an inhibition of retrograde axonal transport. Mice overexpressing dynamitin demonstrate a late-onset progressive motor neuron degenerative disease characterized by decreased strength and endurance, motor neuron degeneration and loss, and denervation of muscle. Previous transgenic mouse models of ALS have shown abnormalities in microtubule-based axonal transport. In this report, we describe a mouse model that confirms the critical role of disrupted axonal transport in the pathogenesis of motor neuron degenerative disease.     

Amylase hyperproduction by deregulated mutants of the thermophilic fungus Thermomyces lanuginosus

Thermomyces lanuginosus was subjected to three cycles of mutagenesis (UV/NTG) and a selection procedure to develop amylase-hyperproducing, catabolite-repression-resistant and partially constitutive strains. One of the selected derepressed mutant strain III₅₁, produced ∼7- and 3-fold higher specific activity of α-amylase (190 U/mg protein) and glucoamylase (105 U/mg protein), respectively, compared to a wild-type parental strain. Further, the effect of production parameters on mutant strain III₅₁ was studied using a Box–Behnken design. The regression models computed showed significantly high R ² values of 96 and 97% for α-amylase and glucoamylase activities, respectively, indicating that they are appropriate for predicting relationships between corn flour, soybean meal and pH with α-amylase and glucoamylase production. Journal of Industrial Microbiology & Biotechnology (2002) 29, 70–74 doi:10.1038/sj.jim.7000270 Received 05 July 2001/ Accepted in revised form 16 April 2002     

Use of expression constructs to dissect the functional domains of the CHS/beige protein: identification of multiple phenotypes

The Chediak-Higashi Syndrome (CHS) and the orthologous murine disorder beige are characterized at the cellular level by the presence of giant lysosomes. The CHS1/Beige protein is a 3787 amino acid protein of unknown function. To determine functional domains of the CHS1/Beige protein, we generated truncated constructs of the gene/protein. These truncated proteins were transiently expressed in Cos-7 or HeLa cells and their effect on membrane trafficking was examined. Beige is apparently a cytosolic protein, as are most transiently expressed truncated Beige constructs. Expression of the Beige construct FM (amino acids 1-2037) in wild-type cells led to enlarged lysosomes. Similarly, expression of a 5.5-kb region (amino acids 2035-3787) of the carboxyl terminal of Beige (22B) also resulted in enlarged lysosomes. Expression of FM solely affected lysosome size, whereas expression of 22B led to alterations in lysosome size, changes in the Golgi and eventually cell death. The two constructs could be used to further dissect phenotypes resulting from loss of the Beige protein. CHS or beigej fibroblasts show an absence of nuclear staining using a monoclonal antibody directed against phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5) P2]. Transformation of beige j fibroblasts with a YAC containing the full-length Beige gene resulted in the normalization of lysosome size and nuclear PtdIns(4,5)P2 staining. Expression of the carboxyl dominant negative construct 22B led to loss of nuclear PtdIns(4,5)P2 staining. Expression of the FM dominant negative clone did not alter nuclear PtdIns(4,5) P2 localization. These results suggest that the Beige protein interacts with at least two different partners and that the Beige protein affects cellular events, such as nuclear PtdIns(4,5)P2 localization, in addition to lysosome size.     

Inhibition of microsurgical thrombosis by the platelet glycoprotein IIb/IIIa antagonist SR121566A

Despite major improvements in tools and significant refinements of techniques, microsurgical anastomosis still carries a significant risk of failure due to microvascular thrombosis. The key to improving the success of microvascular surgery may lie in the pharmacologic control of thrombus formation. Central to pathologic arterial thrombosis are platelets. Glycoprotein IIb/IIIa is a highly abundant platelet surface receptor that plays a major role in platelet aggregation by binding platelets to each other through the coagulation factor fibrinogen. To explore the ability of antithrombotic agents to prevent microvascular thrombosis, a rabbit ear artery model was used in which a standardized arterial injury results in predictable thrombus formation. This model was used to examine whether SR121566A, a specific and potent glycoprotein IIb/IIIa inhibitor, can successfully prevent microsurgical thrombosis.Using a coded, double-blind experimental design, 20 rabbits (40 arteries) were assigned to four treatment groups: (1) saline injection (n = 10), (2) acetylsalicylic acid 10 mg/kg (n = 10), (3) heparin 0.5 mg/kg bolus with subsequent intermittent boluses of 0.25 mg/kg every 30 minutes (n = 10), and (4) SR121566A 2 mg/kg bolus (n = 10). After vessel damage and clamp release, arteries were assessed for patency at 5, 30, and 120 minutes by the Acland refill test. Coagulation assays, in vivo bleeding times, and ex vivo platelet aggregation studies were also conducted. Scanning electron microscopy was used to examine mural thrombus composition.A significant, fourfold increase in vessel patency following administration of SR121566A over saline control (80 percent versus 20 percent patency, respectively, at 35 minutes after reperfusion, p < 0.01) was noted. This was correlated with marked inhibition of ex vivo platelet aggregation. This antiplatelet treatment did not prolong coagulation assays (mean international normalized ratio: saline, 0.66 +/- 0.04; SR121566A, 0.64 +/- 0.03; mean thromboplastin time: saline, 19.63 +/- 0.67; SR121566A, 17.87 +/- 3.27) and bleeding times (mean bleeding time: saline, 42 +/- 4; SR121566A, 48 +/- 6). Scanning electron microscopy demonstrated extensive platelet and fibrin deposition in control vessel thrombi. In contrast, thrombi from SR121566A-treated vessels demonstrated predominance of fibrin with few platelets when examined under scanning electron microscopy.Administration of SR121566A was associated with a significant increase in vessel patency, without deleterious effects on coagulation assays or bleeding times. The increase in vessel patency was correlated with inhibition of platelet aggregation and decreased platelet deposition, as demonstrated by scanning electron microscopy. Glycoprotein IIb/IIIa antagonists represent a new class of anti-platelet agents that may be suited for inhibiting microsurgical thrombosis. This study supports further investigation into the use of these agents in microsurgery.     

Microbial populations stimulated for hexavalent uranium reduction in uranium mine sediment

Uranium-contaminated sediment and water collected from an inactive uranium mine were incubated anaerobically with organic substrates. Stimulated microbial populations removed U almost entirely from solution within 1 month. X-ray absorption near-edge structure analysis showed that U(VI) was reduced to U(IV) during the incubation. Observations by transmission electron microscopy, selected area diffraction pattern analysis, and energy-dispersive X-ray spectroscopic analysis showed two distinct types of prokaryotic cells that precipitated only a U(IV) mineral uraninite (UO(2)) or both uraninite and metal sulfides. Prokaryotic cells associated with uraninite and metal sulfides were inferred to be sulfate-reducing bacteria. Phylogenetic analysis of 16S ribosomal DNA obtained from the original and incubated sediments revealed that microbial populations were changed from microaerophilic Proteobacteria to anaerobic low-G+C gram-positive sporeforming bacteria by the incubation. Forty-two out of 94 clones from the incubated sediment were related to sulfate-reducing Desulfosporosinus spp., and 23 were related to fermentative Clostridium spp. The results suggest that, if in situ bioremediation were attempted in the uranium mine ponds, Desulfosporosinus spp. would be a major contributor to U(VI) and sulfate reduction and Clostridium spp. to U(VI) reduction.     

The microtubule plus-end proteins EB1 and dynactin have differential effects on microtubule polymerization

Several microtubule-binding proteins including EB1, dynactin, APC, and CLIP-170 localize to the plus-ends of growing microtubules. Although these proteins can bind to microtubules independently, evidence for interactions among them has led to the hypothesis of a plus-end complex. Here we clarify the interaction between EB1 and dynactin and show that EB1 binds directly to the N-terminus of the p150(Glued) subunit. One function of a plus-end complex may be to regulate microtubule dynamics. Overexpression of either EB1 or p150(Glued) in cultured cells bundles microtubules, suggesting that each may enhance microtubule stability. The morphology of these bundles, however, differs dramatically, indicating that EB1 and dynactin may act in different ways. Disruption of the dynactin complex augments the bundling effect of EB1, suggesting that dynactin may regulate the effect of EB1 on microtubules. In vitro assays were performed to elucidate the effects of EB1 and p150(Glued) on microtubule polymerization, and they show that p150(Glued) has a potent microtubule nucleation effect, whereas EB1 has a potent elongation effect. Overall microtubule dynamics may result from a balance between the individual effects of plus-end proteins. Differences in the expression and regulation of plus-end proteins in different cell types may underlie previously noted differences in microtubule dynamics.     

Alternative mating strategies in a desert grasshopper: a transitional analysis

We describe between- and within-individual variation in signalling behaviour for a population of male Ligurotettix coquilletti in a North American desert. For each observation date individual males were categorized as actively-signalling or inactive by the amount and regularity of their stridulation and their signalling location. Few males were active or inactive on all dates they were observed. By comparing the behaviour of individuals on consecutive observation dates, we identified the primary ways that individuals changed between active signalling and inactive behaviour. Behavioural transitions were frequently associated with movement between bushes. Inactive males became active upon moving to a vacant bush, and lone active males often became inactive after moving to a bush containing other males. Male-male aggression (primarily chases involving no contact between the participants, or only very brief contact) had a pronounced, short-term effect upon the signalling behaviour of males that received an attack. Approximately 33% of these recipients that had been stridulating became silent immediately following the chase. However, chases did not markedly alter the behaviour of recipients on the following observation date. Recipients were no more likely to change from inactive to active behaviour than vice versa between the observation dates immediately preceding and following the day of the chase. Although the transition analysis failed to detect marked behavioural changes among recipients, analyses based on longer periods of time revealed that males observed to be recipients were more likely to display inactive behaviour and less likely to mate than males observed to be initiators of aggression.