Pupillometric assessment of dysautonomia in pediatric bowel and bladder dysfunction: a pilot study.

Bowel and bladder dysfunction (BBD) refers to a heterogeneous group of voiding disorders, accounting for an estimated 40% of pediatric urology visits. Symptoms of BBD include enuresis, urgency, and urinary retention, often accompanied by constipation. The aim of this pilot study was to explore whether a pupillary response can be characterized for BBD, by examining the pupillary light reflex (PLR) before and after voiding among patients with BBD. A total of 28 patients aged from 7 to 21 years were recruited from the Wetting, Infections, and Stooling Help clinic at Children's National Medical Center. An infrared pupilometer was used to assess the PLR. Both baseline static and dynamic pupillometry assessments were obtained before and after voiding. Measurements were also taken after 5 min in the supine position, followed by 5 min standing to induce an orthostatic stressor. Visual inspection of the graphed data revealed a characteristic shape in 11 of 28 patients with voiding symptoms. In these 11 patients, the redilation arm of the PLR shows a 'notch,' or a brief reconstriction of the pupil before resting pupil size is reestablished (figure). This feature of the PLR has not been seen in previous and parallel studies using pupillometry to evaluate other populations. The results of this study suggest that a subset of patients with BBD may have a significant perturbation of autonomic regulation, identifiable through analysis of the PLR. To our knowledge, this 'notch' during redilation has not been previously described or seen in other patient populations and may represent a distinctive and readily identifiable physiologic marker of disease. These results are broadly aligned with results of other studies that have examined ANS activity in patients with BBD, although further study is needed to confirm the results of this pilot study and to assess relative contributions of sympathetic and parasympathetic function in producing pupillary abnormalities. This study has several limitations, including the small sample size, the absence of data on severity and duration of symptoms, and the absence of a control group of patients without any voiding symptoms. A simple tool for diagnosing BBD and for monitoring response to treatment could significantly improve the quality of treatment for one of the most common pediatric urologic complaints. Given the heterogeneity of symptoms under the BBD umbrella, pupillometric data could guide selection of treatment options, as well as assess adequacy of response to pharmacologic therapy.

Long term effects of lipopolysaccharide on satellite glial cells in mouse dorsal root ganglia.

Lipopolysaccharide (LPS) has been used extensively to study neuroinflammation, but usually its effects were examined acutely (24h<). We have shown previously that a single intraperitoneal LPS injection activated satellite glial cells (SGCs) in mouse dorsal root ganglia (DRG) and altered several functional parameters in these cells for at least one week. Here we asked whether the LPS effects would persist for 1 month. We injected mice with a single LPS dose and tested pain behavior, assessed SGCs activation in DRG using glial fibrillary acidic protein (GFAP) immunostaining, and injected a fluorescent dye intracellularly to study intercellular coupling. Electron microscopy was used to quantitate changes in gap junctions. We found that at 30 days post-LPS the threshold to mechanical stimulation was lower than in controls. GFAP expression, as well as the magnitude of dye coupling among SGCs were greater than in controls. Electron microscopy analysis supported these results, showing a greater number of gap junctions and an abnormal growth of SGC processes. These changes were significant, but less prominent than at 7 days post-LPS. We conclude that a single LPS injection exerts long-term behavioral and cellular changes. The results are consistent with the idea that SGC activation contributes to hyperalgesia.

Effects of dry period length on milk production, body condition, metabolites, and hepatic glucose metabolism in dairy cows.

Dry period (DP) length affects energy metabolism around calving in dairy cows as well as milk production in the subsequent lactation. The aim of the study was to investigate milk production, body condition, metabolic adaptation, and hepatic gene expression of gluconeogenic enzymes in Holstein cows (>10,000 kg milk/305 d) with 28- (n=18), 56- (n=18), and 90-d DP (n=22) length (treatment groups) in a commercial farm. Cows were fed total mixed rations ad libitum adjusted for far-off (not for 28-d DP) and close-up DP and lactation. Milk yield was recorded daily and body condition score (BCS), back fat thickness (BFT), and body weight (BW) were determined at dry off, 1 wk before expected and after calving, and on wk 2, 4, and 8 postpartum (pp). Blood samples were taken on d -56, -28, -7, 1, 7, 14, 28, and 56 relative to calving to measure plasma concentrations of metabolites and hormones. Liver biopsies (n=11 per treatment) were taken on d -10 and 10 relative to calving to determine glycogen and total liver fat concentration (LFC) and to quantify mRNA levels of pyruvate carboxylase (PC), cytosolic phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase. Time course of milk yield during first 8 wk in lactation differed among treatment. Milk protein content was higher in 28-d than in 90-d DP cows. Milk fat to protein ratio was highest and milk urea was lowest in 90-d DP cows. Differences in BW, BFT, and BCS were predominantly seen before calving with greatest BW, BFT, and BCS in 90-d DP cows. Plasma concentrations of NEFA and BHBA were elevated during the transition period in all cows, and the greatest increase pp was seen in 90-d DP cows. Plasma glucose concentration decreased around calving and was greater in 28-d than in 90-d DP cows. Dry period length also affected plasma concentrations of urea, cholesterol, aspartate transaminase, and glutamate dehydrogenase. Plasma insulin concentration decreased around calving in all cows, but insulin concentration pp was greater in 28-d than in 56-d DP cows. Hepatic glycogen concentration decreased and LFC increased after calving in all cows, and LFC was greater pp in 90-d DP than in 28-d DP cows. Hepatic PC mRNA abundance pp tended to increase most in 90-d DP cows. Changes on glucose metabolism were more balanced in cows with a reduced DP, whereas cows with extended DP and elevated body condition indicated greatest metabolic changes according to lipid and glucose metabolism during the transition period.

Systemic inflammation alters satellite glial cell function and structure. A possible contribution to pain.

Local peripheral injury activates satellite glial cells (SGCs) in sensory ganglia, which may contribute to chronic pain. We hypothesized that systemic inflammation affects sensory ganglia like local injury. We induced systemic inflammation in mice by injecting lipopolysaccharide (LPS) intraperitoneally, and characterized SGCs and neurons in dorsal root ganglia (DRG), using dye injection, calcium imaging, electron microscopy (EM), immunohistochemistry, and electrical recordings. Several days post-LPS, SGCs were activated, and dye coupling among SGCs increased 3-4.5-fold. EM showed abnormal growth of SGC processes and the formation of new gap junctions. Sensitivity of SGCs to ATP increased twofold, and neuronal excitability was augmented. Blocking gap junctions reduced pain behavior in LPS-treated mice. Thus, changes in DRG due to systemic inflammation are similar to those due to local injury, which may explain the pain in sickness behavior and in other systemic diseases.

Asymmetric adaptation to indolic and aliphatic glucosinolates in the B and Q sibling species of Bemisia tabaci (Hemiptera: Aleyrodidae).

The role glucosinolates play in defending plants against phloem feeders such as aphids and whiteflies is currently not clear as these herbivores may avoid bringing glucosinolates from the phloem sap into contact with myrosinase enzymes. Here, we investigated the effects of high levels of aliphatic and indolic glucosinolates on life history traits and detoxification gene expression in two sibling species, B and Q, of the whitefly Bemisia tabaci. High levels of aliphatic glucosinolates decreased the average oviposition rate of both species and reduced the survival and developmental rate of Q nymphs. High levels of indolic glucosinolates decreased the oviposition rate and survival of nymphal stages of the B species and the developmental rate of both species. Molecular analyses revealed two major asymmetries between the B and Q species. First, specific GST genes (BtGST1 and BtGST2) were significantly induced during exposure to indolic glucosinolates only in Q. This may reflect the genes putative involvement in indolic glucosinolates detoxification and explain the species' good performance on plants accumulating indolic glucosinolates. Second, the constitutive expression of eight of the 10 detoxification genes analysed was higher in the Q species than in the B species. Interestingly, four of these genes were induced in B in response to high levels of glucosinolates. It seems, therefore, that the B and Q species differ in their 'optimal defence strategy'. B utilizes inducible defences that are profitable if the probability of experiencing the stress is small and its severity is low, while Q invests significant resources in being always 'ready' for a challenge.

Augmentation in gap junction-mediated cell coupling in dorsal root ganglia following sciatic nerve neuritis in the mouse.

Recent findings highlight the participation of central glial cells in chronic pain, but less is known of a comparable role for satellite glial cells (SGCs), in dorsal root ganglia (DRG). Our previous work showed that sciatic nerve axotomy augmented SGC coupling by gap junctions. The aim of the present research was to find out whether similar changes occur in a mouse inflammation model. Sciatic nerve neuritis was induced by complete Freund's adjuvant (CFA), and isolated ganglia were examined 1 week later. Cell coupling was monitored by intracellular injection of the fluorescent dye Lucifer Yellow. Changes in gap junctions were assessed quantitatively by electron microscopy. Withdrawal threshold in the foot on the side of the inflamed nerve decreased from an average of 3.9 g in control to 0.94 g using Von Frey hairs (P<0.05). In CFA-treated animals dye coupling incidence between SGCs belonging to different glial envelopes increased from 6.9% in controls to 22.5% (P<0.05). Whereas in controls there was no coupling between neurons or between neurons and SGCs, after CFA application the incidence of neuron-neuron and neuron-SGC coupling was 8%. Electron microscopy showed formation of bridges between SGC sheaths surrounding different neurons, which were completely absent in controls. The mean number of gap junctions/100 microm(2) of surface of the section occupied by SGCs increased from 0.215 in controls to 0.709 (P<0.01) in CFA-treated mice. The size of individual gap junctions remained the same. This is the first evidence for ultrastructural changes in SGCs following inflammation. The results support the idea that SGCs are sensitive to a variety of peripheral nerve injuries. We propose that the observed changes may alter signal transmission in DRG and thus may contribute to chronic pain.

Noncanonical cell death programs in the nematode Caenorhabditis elegans.

Genetic studies of the nematode Caenorhabditis elegans have uncovered four genes, egl-1 (BH3 only), ced-9 (Bcl-2 related), ced-4 (apoptosis protease activating factor-1), and ced-3 (caspase), which function in a linear pathway to promote developmental cell death in this organism. While this core pathway functions in many cells, recent studies suggest that additional regulators, acting on or in lieu of these core genes, can promote or inhibit the onset of cell death. Here, we discuss the evidence for these noncanonical mechanisms of C. elegans cell death control. We consider novel modes for regulating the core apoptosis genes, and describe a newly identified cell death pathway independent of all known C. elegans cell death genes. The existence of these noncanonical cell death programs suggests that organisms have evolved multiple ways to ensure appropriate cellular demise during development.

A musculo-skeletal model of rat ankle motion and its experimental test on rat.

A biomechanical musculo-skeletal model of functional electrical stimulation (FES)-induced rat ankle motion was implemented and tested in rat experiments. The muscle model is a new Hill-based model which includes established physiological relations of force-velocity and force-length-frequency. However, the series-elastic component and the activation component of previous Hill-based models are replaced by a new component which accounts for dynamic time delays and recruitment that occur in real muscle force generation during limb movements. The skeletal model includes gravity and dynamic forces that occur in real rat ankle motions. In computer simulations, various FES patterns were applied to the tibialis anterior (TA) and soleus (SO) model muscles to produce walk-like ankle motions. In lab experiments, the same stimulation patterns were applied by epimysial electrodes implanted in the TA and SO muscles of live rats cordotomized at level T7. The resulting rat motions were recorded by video camera. Video data was converted to ankle angle-vs-time files for comparison with corresponding model angle-vs-time files. Over a physiologically significant range of ankle motions, model parameters were adjustable to yield model motions that agreed with rat motions to within 2 degrees (root mean square differences of rat and model ankle angles). This is shown in plots of model and rat motions presented here for representative cases of FES. The accuracy of our model in reproducing real ankle motions supports the hypothesis that our new muscle model generates correct muscle forces over a useful range of limb motions. It suggests that the model may be useful in the design of FES neural prostheses.

exl, an exchangeable genetic island in Neisseria meningitidis.

The genetic structure and evolution of a novel exchangeable meningococcal genomic island was defined for the important human pathogen Neisseria meningitidis. In 125 meningococcal strains tested, one of three unrelated nucleotide sequences, designated exl (exchangeable locus), was found between a gene required for heme utilization, hemO, and col, encoding a putative Escherichia coli collagenase homologue. The 5' boundary of each exl cassette was the stop codon of hemO, whereas the 3' boundary was delineated by a 33-bp repeat containing neisserial uptake sequences located downstream of col. One of the three alternative exl cassettes contained the meningococcal hemoglobin receptor gene, hmbR (exl3). In other meningococcal strains, hmbR was absent from the genome and was replaced by either a nucleotide sequence containing a novel open reading frame, exl2, or a cassette containing exl3. The proteins encoded by exl2 and exl3 had no significant amino acid homology to HmbR but contained six motifs that are also present in the lipoprotein components of the lactoferrin (LbpB), transferrin (TbpB), and hemoglobin-haptoglobin (HpuA) uptake systems. To determine the evolutionary relationships among meningococci carrying hmbR, exl2, or exl3, isolates representing 92 electrophoretic types were examined. hmbR was found throughout the population structure of N. meningitidis (genetic distance, >0.425), whereas exl2 and exl3 were found in clonal groups at genetic distances of <0.2. The commensal neisserial species were identified as reservoirs for all of the exl cassettes found in meningococci. The structure of these cassettes and their correlation with clonal groups emphasize the extensive gene pool and frequent horizontal DNA transfer events that contribute to the evolution and virulence of N. meningitidis.

Antimicrobial susceptibility of staphylococci isolated from otitis externa in dogs.

Samples were obtained from 65 unmedicated adult dogs, processed for isolation of Staphylococcus species and tested for susceptibility to penicillin G, gentamicin, oxacillin, tetracycline, trimethoprim-sulphamethoxazole, streptomycin, ampicillin and rifampin. Forty-four isolates were obtained, which represents 67.7% of samples. Coagulase-negative species were most commonly found, and the most frequently isolated staphylococcus species were Staph. epidermidis and Staph. aureus. Other species, such as Staph. simulans, Staph. haemolyticus, Staph. saprophyticus and Staph. intermedius were also isolated. Resistance to antibiotics was frequently observed, with 90.9% of the isolates showing resistance to at least one drug. The most active antimicrobial agents against staphylococci isolated from otitis externa of dogs were rifampin and oxacillin. Multidrug resistance was a common finding, and one strain of Staph. haemolyticus species, was resistant to all tested antimicrobial agents. Resistance to three or more different drugs was a common finding, observed in 16 strains (36.4%) of both coagulase-positive and coagulase-negative staphylococci. This study highlights the emergence of cases of otitis externa determined by coagulase-negative staphylococcus strains and once more emphasizes the need for bacterial culture with species identification and susceptibility testing of swab specimens from the ear canal in order to choose appropriate antimicrobial agents.

Polyadenylation promotes degradation of 3'-structured RNA by the Escherichia coli mRNA degradosome in vitro.

Polyadenylation contributes to the destabilization of bacterial mRNA. We have investigated the role of polyadenylation in the degradation of RNA by the purified Escherichia coli degradosome in vitro. RNA molecules with 3'-ends incorporated into a stable stem-loop structure could not readily be degraded by purified polynucleotide phosphorylase or by the degradosome, even though the degradosome contains active RhlB helicase which normally facilitates degradation of structured RNA. The exoribonucleolytic activity of the degradosome was due to polynucleotide phosphorylase, rather than the recently reported exonucleolytic activity exhibited by a purified fragment of RNase E (Huang, H., Liao, J., and Cohen, S. N. (1998) Nature 391, 99-102). Addition of a 3'-poly(A) tail stimulated degradation by the degradosome. As few as 5 adenosine residues were sufficient to achieve this stimulation, and generic sequences were equally effective. The data show that the degradosome requires a single-stranded "toehold" 3' to a secondary structure to recognize and degrade the RNA molecule efficiently; polyadenylation can provide this single-stranded 3'-end. Significantly, oligo(G) and oligo(U) tails were unable to stimulate degradation; for oligo(G), at least, this is probably due to the formation of a G quartet structure which makes the 3'-end inaccessible. The inaccessibility of 3'-oligo(U) sequences is likely to have a role in stabilization of RNA molecules generated by Rho-independent terminators.

Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome.

The Escherichia coli RNA degradosome is the prototype of a recently discovered family of multiprotein machines involved in the processing and degradation of RNA. The interactions between the various protein components of the RNA degradosome were investigated by Far Western blotting, the yeast two-hybrid assay, and coimmunopurification experiments. Our results demonstrate that the carboxy-terminal half (CTH) of ribonuclease E (RNase E) contains the binding sites for the three other major degradosomal components, the DEAD-box RNA helicase RhlB, enolase, and polynucleotide phosphorylase (PNPase). The CTH of RNase E acts as the scaffold of the complex upon which the other degradosomal components are assembled. Regions for oligomerization were detected in the amino-terminal and central regions of RNase E. Furthermore, polypeptides derived from the highly charged region of RNase E, containing the RhlB binding site, stimulate RhlB activity at least 15-fold, saturating at one polypeptide per RhlB molecule. A model for the regulation of the RhlB RNA helicase activity is presented. The description of RNase E now emerging is that of a remarkably complex multidomain protein containing an amino-terminal catalytic domain, a central RNA-binding domain, and carboxy-terminal binding sites for the other major components of the RNA degradosome.

Polyphosphate kinase is a component of the Escherichia coli RNA degradosome.

The Escherichia coli degradosome is a multienzyme complex with four major protein components: the endoribonuclease RNase E, the exoribonuclease PNPase, the RNA helicase RhlB and enolase. The first three of these proteins are known to have important functions in mRNA processing and degradation. In this work, we identify an additional component of the degradosome, polyphosphate kinase (PPK), which catalyses the reversible polymerization of the gamma-phosphate of ATP into polyphosphate (poly(P)). An E. coli strain deleted for the ppk gene showed increased stability of the ompA mRNA. Purified His-tagged PPK was shown to bind RNA, and RNA binding was prevented by hydrolysable ATP. Chemical modification of RNA by PPK, for example the addition or removal of 3' or 5' terminal phosphates, could not be detected. However, polyphosphate was found to inhibit RNA degradation by the degradosome in vitro. This inhibition was overcome by the addition of ADP, required for the degradation of polyphosphate and for the regeneration of ATP by PPK in the degradosome. Thus, PPK in the degradosome appears to maintain an appropriate microenvironment, removing inhibitory polyphosphate and NDPs and regenerating ATP.

Association of mitogen-activated protein kinases with microtubules in mouse macrophages.

Taxol, a microtubule-binding diterpene, mimics many effects of lipopolysaccharide (LPS) on mouse macrophages. The LPS-mimetic effects of taxol appear to be under the same genetic control as responses to LPS itself. Thus we have postulated a role for microtubule-associated proteins (MAP) in the response of macrophages to LPS. Stimulation of macrophages by LPS quickly induces the activation of mitogen-activated protein kinases (MAPK). MAPK are generally considered cytosolic enzymes. Herein we report that much of the LPS-activatable pool of MAPK in primary mouse peritoneal macrophages is microtubule associated. By immunofluorescence, MAPK were localized to colchicine- and nocodazole-disruptible filaments. From both mouse brain and RAW 264.7 macrophages, MAPK could be coisolated with polymerized tubulin. Fractionation of primary macrophages into cytosol-, microfilament-, microtubule-, and intermediated filament-rich extracts revealed that approximately 10% of MAPK but none of MAPK kinase (MEK1A and MEK2) was microtubule bound. Exposure of macrophages to LPS did not change the proportion of MAPK bound to microtubules, but preferentially activated the microtubule-associated pool. These findings confirm the prediction that LPS activates a kinase bound to microtubules. Together with LPS-mimetic actions of taxol and the shared genetic control of responses to LPS and taxol, these results support the hypothesis that a major LPS-signaling pathway in mouse macrophages may involve activation of one or more microtubule-associated kinases.

PCR-directed preparation and single-step purification of highly active histidine-tagged restriction endonuclease HgiBI (GGWCC).

The polymerase chain reaction was used to produce His6 fusion proteins via deletion of an intervening piece of DNA. The generally applicable method was performed using a standard primer with the advantage that the fusion does not produce additional amino acids. In a single-step purification highly purified, enzymatically active restriction endonuclease was obtained.

Organization and gene expression within restriction-modification systems of Herpetosiphon giganteus.

We have characterized a family of related restriction-modification (R-M) systems from the soil bacterium Herpetosiphon giganteus (Hgi). A comparison of their genetic organization reveals two types of regulatory proteins, called controlling ORF C. While one of these small reading frames derived from RM.HgiCI seems to be an enhancer of its own promoter, evidence is provided for a silencer function of the other ORF C derived from the closely related AvaII-type systems RM.HgiBI/CII/EI. The respective silencer function is detected during our various attempts to clone three isoschizomers with unusually high differences in their specific activity. Sequencing and site-directed mutagenesis revealed just two amino acids as being responsible for a massive increase in specific activity of these endonucleases.

PCR directed preparation and single step purification of highly active histidine-tagged restriction endonuclease HgiBI (GGWCC).

The polymerase-chain-reaction technique is used to produce fusion proteins via deletion of any intervening piece of DNA. Here a stretch of six histidine codons is fused to the 3'-terminus of any defined gene using a standard plasmid vector or a derivative thereof. The advantage over existing methods is that no other amino acids besides the six histidines are added to the protein terminus and only one oligonucleotide needs to be synthesized as special primer. Genes of interest must only be cloned in the correct orientation into a universal multilinker. Using just one specific primer derived from the 3'-terminus of the gene and one standard primer derived from the six histidine codons the fusion is performed by amplifying the entire vector system as described for inverse PCR. As an example, we report on the modification and purification of the restriction endonuclease HgiBI (GGWCC). Enzymatically active protein was obtained in a single step purification under nondenaturating conditions with a purity greater than 95% according to polyacrylamide gel electrophoresis.

The development of autonomy and superego precursors.

Superego precursors appear in the latter part of the first year, concomitant with the behavioural control necessitated and made possible by ego development, particularly of intentionality, communication, and mobility. During the second year of life wilful strivings intensify, complicated by the power struggles of the rapprochement crisis. A subtle process of negotiation accompanies the learning of parental rules and regulations, and the infant also learns that some rules and limits are non-negotiable. The development of the superego depends not only on 'identification with the aggressor' and self-directed aggression, but on positive identifications and internalizations of approval. While the real attitudes of the caregiver are communicated and are important, superego precursors, because of the infant's drives, defences, and ego immaturity, may be far removed from the reality of the caregiver's attitudes and intent. The parents' and infants' inter-identifications and empathy for each other contribute to the capacity for self-criticism, guilt, and remorse, attributes of the developing superego.