Hopping and swimming in the leopard frog Rana pipiens: II. A comparison of muscle activities

Electromyography (EMG) was used to examine muscle activity of the major hip, knee, and ankle extensors during both hopping and swimming in leopard frogs. Chronic EMG electrodes were implanted for periods of 7–10 days. This permitted us to record EMG activities during both hopping and swimming from the same electrode, allowing a direct comparison of the timing and amplitudes of muscle activity between the two behaviors. We could then relate these activities to the kinematics of locomotion. In both behaviors, all three extensors were synchronously activated 30–50 ms before limb extension began. However, the hip extensor turned on relatively earlier in hopping than in swimming when on time was expressed as percent of stride. The hip and knee extensors were activated relatively longer in hopping and the ankle extensor relatively longer in swimming. The amplitudes of the rectified, integrated EMG signals were roughly twice as large in hopping as in swimming for all three muscles, supporting the notion that propulsion in hopping requires more force than in swimming. The EMG burst durations differed little between the muscles or, in relative duration, between the behaviors. As has been found in other quadrupeds, the EMG bursts began before visible movement and ceased at or before hindlimb extension was completed. In our animals, however, we found a consistent, low level (10–30% of maximum amplitude) of EMG activity that continued 60–200 ms past the end of the burst and into the suspension periods in both hopping and swimming. We hypothesize that this unusual activity may be present in frogs so that the hind limb remains aero(hydro)dynamically stable as the frog arches through its leap or glides in swimming following completed limb extension. Thus, the timing and pattern of the EMG bursts are consistent with those present in other tetrapods and support conservatism of neural control. However, the prolonged low-level activity suggests flexibility in the control pattern and variation according to specific behaviors. © 1996 Wiley-Liss, Inc.     

Hopping and swimming in the leopard frog,Rana pipiens: I. Step cycles and kinematics

This study presents a model for the step cycle patterns used during both hopping and swimming by the leopard frog, Rana pipiens. The two behaviors are essentially similar in movement pattern and in the ways they are modified from quadrupedal gaits. In hopping, there is marked hind limb extension throughout stance. The swing begins with a suspension equivalent to the leap that occurs in a galloping or bounding quadruped. Following suspension, as the frog descends from the apex of its leap, the hind limbs remain posterior and in line with the spine while they flex. Near the end of flexion, there is a rapid downward rotation of the hindquarters to bring the hind feet underneath the body. This movement utilizes the planted forelimb as a pivot. A similar pattern of movement occurs in swimming; the stance (propulsion) phase involves extension at all hind limb joints. The swing (recovery) phase begins with the hind feet fully extended and includes a protracted gliding phase, equivalent to the suspension in the hop. The hind limb then recovers to its initial position during a flexion phase. Since there is no landing and the hind limbs remain lateral rather than ventral to the pelvis, less flexion occurs in the spine or the limb joints. In both behaviors, the extensor muscles of hip (M. semimembranosus), knee (M. cruralis), and ankle (M. plantaris longus) achieve their longest lengths, when they likely can produce near maximal force, at the beginning of extension. All three muscles shorten during extension, but, because they are multiple-joint muscles, the amount of shortening is relatively small (≈ 15%). Hopping and swimming in frogs are comparable asymmetrical gaits with the same relative contact intervals (25% of stride). The step cycles in both gaits are modified from quadrupedal locomotion in the same ways: by 1) loss of knee and ankle extension toward the ground prior to landing (or end of flexion in swimming), 2) loss of a yield phase on landing (or end of flexion in swimming), and 3) inclusion of extended suspensions in both gaits. © 1996 Wiley-Liss, Inc.     

Quinazolinone-based rhodanine-3-acetic acids as potent aldose reductase inhibitors: Synthesis,functional evaluation and molecular modeling study

A series of quinazolinone-based rhodanine-3-acetic acids was synthesized and tested for in vitro aldose reductase inhibitory activity. All the target compounds displayed nanomolar activity against the target enzyme. Compounds 3a, 3b, and 3e exhibited almost 3-fold higher activity as compared to the only marketed reference drug epalrestat. Structure-activity relationship studies indicated that bulky substituents at the 3-phenyl ring of the quinazolinone moiety are generally not tolerated in the active site of the enzyme. Insertion of a methoxy group on the central benzylidene ring was found to have a variable effect on ALR-2 activity depending on the nature of peripheral quinazolinone ring substituents. Removal of the acetic acid moiety led to inactive or weakly active target compounds. Docking and molecular dynamic simulations of the most active rhodanine-3-acetic acid derivatives were also carried out, to provide the basis for further structure-guided design of novel inhibitors.     

Potato bacterial wilt suppression and plant health improvement after application of different antioxidants

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease that often threatens potato production and exportation. The potential of four antioxidants (seaweed extract (SWE), yeast, chitosan and ascorbic acid (ASA)) in controlling the disease was evaluated in vitro, under glasshouse and field conditions. The field experiment was conducted in two naturally infested locations: Wardan, Giza (sandy soil), and Talia, Minufiya (silty clay soil). Only chitosan showed antibacterial properties against the pathogen in vitro. SWE, yeast and chitosan showed disease suppression under both glasshouse and field conditions. The disease suppression was accompanied by an increase in the ratio of soil copiotrophic to oligotrophic bacteria. The three antioxidants increased plant nitrogen content, decreased soil OM content and decreased C/N ratio. Disease suppression after chitosan application was clearly observed only in Wardan area, which was characterized by a higher soil alkalinity. A high percentage of antagonistic fluorescent strains similar to Pseudomonas putida group were detected for chitosan‐treated plants in Wardan area (sandy soil). ASA drastically decreased the count of the pathogen in soil, but was conducive to the pathogen in plant tissues. A remarkable increase in microbial (bacterial and fungal) soil and rhizosphere diversity as indicated by PCR‐DGGE analysis for bacterial 16S rRNA and fungal 18S rRNA was recorded. In Talia area (silty clay soil), the soil microbial community was more stable and was in general resistant to the disease where the soils were characterized by high electrical conductivity. SWE, yeast and ASA significantly increased crop production in Talia area only.     

Does habitat fragmentation reduce genetic diversity and subpopulation connectivity?

The estimation of levels of genetic variation has received considerable attention because it is generally thought to be indicative of overall species vitality and the potential for evolutionary responses to environmental changes. Here, we use allozymes markers and two distinct collections of Cakile maritima, an annual species from sandy coastal habitats (2000 generation and 2005 generation collected from 9 populations in their natural habitats), to assess the magnitude of expected genetic change. We compared genetic diversity between generations (all populations combined), and then between populations at each generation. Based on 13 loci scored from the eight enzymes examined, a high genetic diversity was detected at both the population and generation level as compared to other herbaceous species. However, allelic richness reduction in the 2005 generation suggested restricted gene flow and a high risk of future genetic bottlenecks, if larger tracts of coastal areas disappear. Most loci showed deviation from Hardy‐Weinberg equilibrium due to excess of heterozygotes in all populations suggesting that this species has an allogamic mode of reproduction. It appears most likely that this species has experienced a recent decrease in population size, and that genetic drift in small populations has resulted in a loss of alleles occurring at low frequency. Despite the deterioration process, maintenance of high genetic diversity suggests that there are some ecological factors determining population structure.     

A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

Mutations in the apically located Na⁺-K⁺-2Cl⁻ co-transporter, NKCC2, lead to type I Bartter syndrome, a life-threatening kidney disorder, yet the mechanisms underlying the regulation of mutated NKCC2 proteins in renal cells have not been investigated. Here, we identified a trihydrophobic motif in the distal COOH terminus of NKCC2 that was required for endoplasmic reticulum (ER) exit and surface expression of the co-transporter. Indeed, microscopic confocal imaging showed that a naturally occurring mutation depriving NKCC2 of its distal COOH-terminal region results in the absence of cell surface expression. Biotinylation assays revealed that lack of cell surface expression was associated with abolition of mature complex-glycosylated NKCC2. Pulse-chase analysis demonstrated that the absence of mature protein was not caused by reduced synthesis or increased rates of degradation of mutant co-transporters. Co-immunolocalization experiments revealed that these mutants co-localized with the ER marker protein-disulfide isomerase, demonstrating that they are retained in the ER. Cell treatment with proteasome or lysosome inhibitors failed to restore the loss of complex-glycosylated NKCC2, further eliminating the possibility that mutant co-transporters were processed by the Golgi apparatus. Serial truncation of the NKCC2 COOH terminus, followed by site-directed mutagenesis, identified hydrophobic residues ¹⁰⁸¹LLV¹⁰⁸³ as an ER exit signal necessary for maturation of NKCC2. Mutation of ¹⁰⁸¹LLV¹⁰⁸³ to AAA within the context of the full-length protein prevented NKCC2 ER exit independently of the expression system. This trihydrophobic motif is highly conserved in the COOH-terminal tails of all members of the cation-chloride co-transporter family, and thus may function as a common motif mediating their transport from the ER to the cell surface. Taken together, these data are consistent with a model whereby naturally occurring premature terminations that interfere with the LLV motif compromise co-transporter surface delivery through defective trafficking.The Na-K-2Cl co-transporter, NKCC2, provides the major route for sodium/chloride transport across the apical plasma membrane of the thick ascending limb (TAL)³ of the kidney (1). This co-transporter is critical for salt reabsorption, acid-base regulation, and divalent mineral cation metabolism (2). The prominent importance of NKCC2 in renal functions is evidenced by the effect of loop diuretics, which as pharmacologic inhibitors of NKCC2, are extensively used in the treatment of edematous states (2). Even more impressive, inactivating mutations of the NKCC2 gene in humans causes Bartter syndrome type 1 (BS1), a life-threatening renal tubular disorder for which the diagnosis is usually made in the antenatal-neonatal period, due to the presence of polyhydramnios, premature delivery, salt loss, hypokalemia, metabolic alkalosis, hypercalciuria, and nephrocalcinosis (3). Without appropriate treatment, patients with BS1 will not survive the early neonatal period (4). In congruence with the severity of the symptoms and the uniformity of the clinical picture, functional analysis of diverse NKCC2 mutants consistently revealed a loss of function effect of the tested mutations (5, 6). However, regulatory characterizations of mutants NKCC2 were limited to Xenopus laevis oocytes. Indeed, studies aimed at understanding the post-translational regulation of NKCC2 have been hampered by the difficulty of expressing the co-transporter protein in mammalian cells (7, 8). As a consequence, our knowledge of the molecular mechanisms underlying membrane trafficking of mutated NKCC2 proteins in mammalian cells is nil. Increasing our understanding of the molecular determinants underlying NKCC2 expression in renal cells is essential for elucidating the pathophysiology of BS1 and for improving the available treatments (9, 10). Undeniably, only analysis of the expression such NKCC2 of mutants in renal cells would definitively establish their cellular fate.NKCC2 belongs to the superfamily of electroneutral cation-coupled chloride (CCC) co-transporters (SLC12A) (1). The cation-chloride co-transporters (CCCs) family comprises two principal branches of homologous membrane proteins. One branch includes the Na⁺-dependent chloride co-transporters composed of the Na⁺-K⁺-2Cl⁻ co-transporters (NKCC1 and NKCC2) and the Na⁺-Cl⁻ co-transporter (NCC). The second branch includes the Na⁺-independent K⁺-Cl⁻ co-transporters composed of at least four different isoforms: KCC1 KCC2, KCC3, and KCC4 (11). Within the families, the CCCs share 25–75% amino acid identity. All of these co-transporters exhibit similar hydropathy profiles with 12 transmembrane-spanning domains, an amino terminus of variable length, and a long cytoplasmic carboxyl terminus. Because the COOH-terminal domain of NKCC2 is the predominant cytoplasmic region, it is likely to be a major factor in the trafficking of the NKCC2 protein. Moreover, there have been several reports demonstrating that COOH-terminal residues are important for correct protein targeting (12–14). Occasionally, COOH-terminal mutations are known to cause genetic disorders (15–17). Although studies of other ion transporters support the importance of the COOH-terminal signals in protein stability, maturation, surface delivery, and ER export (18–22), little is known about the role of COOH-terminal signals in the biogenesis of NKCC2.We were recently able to express NKCC2 protein in mammalian cells (23), providing therefore a powerful tool to study and understand the molecular mechanisms underlying the co-transporter expression and regulation in renal cells. This allowed us, in this study, to take the advantage of the existence of natural mutants altering the COOH-terminal tail of the co-transporter to investigate the role of the COOH terminus in the biogenesis of NKCC2 and to explore possible mechanisms implicated in BS1. The results demonstrate the importance of the COOH terminus in normal maturation of the NKCC2 protein. Indeed, we identified a motif of three hydrophobic residues, ¹⁰⁸¹LLV¹⁰⁸³, highly conserved in the COOH-terminal tails of all members of the CCC family, that controls the rate of ER export and thus of surface expression of NKCC2. Loss of the motif disrupts glycosylation and plasma membrane localization of NKCC2. Therefore, we propose abnormal trafficking as a common BS1 mechanism associated with mutations depriving NKCC2 of its COOH terminus.     

A multi-target antisense approach against PDE4 and PDE7 reduces smoke-induced lung inflammation in mice

Background

Recent development in the field of COPD has focused on strategies aimed at reducing the underlying inflammation through selective inhibition of the phosphodiesterase type IV (PDE4) isoform. Although the anti-inflammatory and bronchodilator activity of selective PDE4 inhibitors has been well documented, their low therapeutic ratio and dose-dependent systemic side effects have limited their clinical utility. This study examined the effect of 2''-deoxy-2''-Fluoro-β-D-Arabinonucleic Acid (FANA)-containing antisense oligonucleotides (AON) targeting the mRNA for the PDE4B/4D and 7A subtypes on lung inflammatory markers, both in vitro and in vivo.

Methods

Normal human bronchial epithelial (NHBE) cells were transfected with FANA AON against PDE4B/4D and 7A alone or in combination. mRNA levels for target PDE subtypes, as well as secretion of pro-inflammatory chemokines were then measured following cell stimulation. Mice were treated with combined PDE4B/4D and 7A AON via endo-tracheal delivery, or with roflumilast via oral delivery, and exposed to cigarette smoke for one week. Target mRNA inhibition, as well as influx of inflammatory cells and mediators were measured in lung lavages. A two-week smoke exposure protocol was also used to test the longer term potency of PDE4B/4D and 7A AONs.

Results

In NHBE cells, PDE4B/4D and 7A AONs dose-dependently and specifically inhibited expression of their respective target mRNA. When used in combination, PDE4B/4D and 7A AONs significantly abrogated the cytokine-induced secretion of IL-8 and MCP-1 to near baseline levels. In mice treated with combined PDE4B/4D and 7A AONs and exposed to cigarette smoke, significant protection against the smoke-induced recruitment of neutrophils and production of KC and pro-MMP-9 was obtained, which was correlated with inhibition of target mRNA in cells from lung lavages. In this model, PDE AONs exerted more potent and broader anti-inflammatory effects against smoke-induced lung inflammation than roflumilast. Moreover, the protective effect of PDE4B/4D and 7A AON was maintained when a once-weekly treatment schedule was used.

Conclusion

These results indicate that inhaled AON against PDE4B/4D and 7A have unique effects on biomarkers that are believed to be important in the pathophysiology of COPD, which supports further development as a potential therapy in this disease.