30 months follow-up of an early enzyme replacement therapy in a severe Morquio A patient: About one case.

Patients under 5 years were not evaluated in the phase-3 study for enzyme replacement therapy (ERT) in MPS IV A. Here we describe the evolution of a severe Morquio A pediatric patient who was diagnosed at 19 months old and treated by ERT at 21 months old for the next 30 months. Applying the standard ERT protocol on this very young patient appeared to reduce his urinary excretion of glycosaminoglycans (GAGs); the improvements in both the 6 minute-walk test (6MWT) and the stair climb test, however, were no different than those reported in the nature history study. Additionally, this young patient experienced many ERT-associated side effects, and as a result a specific corticosteroid protocol (1 mg/kg of betamethasone the day before and 1 h before the ERT infusion) was given to avoid adverse events. Under these treatments, the height of this patient increased during the first year of the ERT although no more height gain was observed thereafter for 18 months. However, despite of ERT, his bone deformities (including severe pectus carinatum) actually worsened and his medullar cervical spine compression showed no improvement (thus needed decompression surgery). CONCLUSION: early ERT treatment did not improve the bone outcome in this severe MPS IV A patient after the 30 months-long treatment. A longer term follow up is required to further assess the efficacy of ERT on both the motor and the respiratory function of the patient.

Phytobeneficial Properties of Bacteria Isolated from the Rhizosphere of Maize in Southwestern Nigerian Soils.

Biocontrol agents isolated outside Africa have performed inconsistently under field conditions in Africa. The development of indigenous phytobeneficial microbial strains that suit local environments may help enhance competitiveness with in situ microorganisms and effectiveness at suppressing local pathogen strains. We isolated bacteria from the rhizosphere of maize growing in southwestern Nigeria and assessed them for growth-promoting characteristics. The best isolates were characterized using 16S rRNA genes and were further evaluated in the greenhouse on maize seedlings. Four isolates (EBS8, IGBR11, EPR2, and ADS14) were outstanding in in vitro assays of antagonistic activity against a local strain of Fusarium verticillioides, phosphate solubilization efficiency, chitinase enzyme activity, and indole-3-acetic acid production. Inoculation of maize seeds with these isolates resulted in ≥95% maize seed germination and significantly enhanced radicle and plumule length. In the greenhouse, maize seedling height, stem girth, number of leaves, leaf area, shoot mass (dry matter), and nutrient contents were significantly enhanced. The bioprotectant and phytobeneficial effects were strongest and most consistent for isolate EBS8, which was identified as a Bacillus strain by 16S rRNA gene analysis. As a bacterial strain that exhibits multiple growth-promoting characteristics and is adapted to local conditions, EBS8 should be considered for the development of indigenous biological fertilizer treatments.

Undiagnosed phenylketonuria in parents of phenylketonuric patients, is it worthwhile to be checked?

In our phenylketonuria (PKU) cohort of 120 patients, we uncovered a couple of cases of undiagnosed mild phenylketonuria (mPKU)/hyperphenylalaninemia (mHPA) in maternal parents of the PKU cohort. This finding prompted us to evaluate the risk of either mild phenylketonuria or mild hyperphenylalaninemia in the parent population whose children were diagnosed with hyperphenylalaninemia (HPA). Taking into account the phenylalanine hydroxylase (PAH) mutation carrier frequency and the PAH mild mutation rate, we estimated that the prevalence of the parental mPKU/mHPA varied widely, from 1/74 in Turkey to 1/708 in Lithuania. The benefits of the parental detection procedure described here are the prevention of further maternal PKU syndrome, the follow-up of the newly detected patients and the accuracy of the genetic counseling provided to these families. This very simple procedure should be incorporated into neonatal PKU management of the hospitals in countries where a routine systematic neonatal screening is operational.

Decreased vitamin B12 availability induces ER stress through impaired SIRT1-deacetylation of HSF1.

Vitamin B12 (cobalamin) is a key determinant of S-adenosyl methionine (SAM)-dependent epigenomic cellular regulations related to methylation/acetylation and its deficiency produces neurodegenerative disorders by elusive mechanisms. Sirtuin 1 deacetylase (SIRT1) triggers cell response to nutritional stress through endoplasmic reticulum (ER) stress. Recently, we have established a N1E115 dopaminergic cell model by stable expression of a transcobalamin-oleosin chimera (TO), which impairs cellular availability of vitamin B12, decreases methionine synthase activity and SAM level, and reduces cell proliferation. In contrast, oleosin-transcobalamin chimera (OT) does not modify the phenotype of transfected cells. Presently, the impaired cellular availability of vitamin B12 in TO cells activated irreversible ER stress pathways, with increased P-eIF-2α, P-PERK, P-IRE1α, ATF6, ATF4, decreased chaperon proteins and increased pro-apoptotic markers, CHOP and cleaved caspase 3, through reduced SIRT1 expression and consequently greater acetylation of heat-shock factor protein 1 (HSF1). Adding either B12, SIRT1, or HSF1 activators as well as overexpressing SIRT1 or HSF1 dramatically reduced the activation of ER stress pathways in TO cells. Conversely, impairing SIRT1 and HSF1 by siRNA, expressing a dominant negative form of HSF1, or adding a SIRT1 inhibitor led to B12-dependent ER stress in OT cells. Addition of B12 abolished the activation of stress transducers and apoptosis, and increased the expression of protein chaperons in OT cells subjected to thapsigargin, a strong ER stress stimulator. AdoX, an inhibitor of methyltransferase activities, produced similar effects than decreased B12 availability on SIRT1 and ER stress by a mechanism related to increased expression of hypermethylated in cancer 1 (HIC1). Taken together, these data show that cellular vitamin B12 has a strong modulating influence on ER stress in N1E115 dopaminergic cells. The impaired cellular availability in vitamin B12 induces irreversible ER stress by greater acetylation of HSF1 through decreased SIRT1 expression, whereas adding vitamin B12 produces protective effects in cells subjected to ER stress stimulation.

Clinical and microbiological characteristics of community-acquired thoracic empyema or complicated parapneumonic effusion caused by Klebsiella pneumoniae in Taiwan.

Klebsiella pneumoniae is the major cause of community-acquired pyogenic infections in Taiwan and is becoming an increasing problem in acute thoracic empyema. This study evaluated the clinical and microbiological characteristics of community-acquired thoracic empyema or complicated parapneumonic effusion caused by K. pneumoniae in Taiwanese adults treated during the period 2001-2008 at a tertiary medical center. All clinical isolates were examined for capsular serotypes K1/K2, and pulsed-field gel electrophoresis (PFGE) was performed on strains of the same serotype. K. pneumoniae was the most frequent cause of community-acquired thoracic empyema or complicated parapneumonic effusion. It was associated with high mortality (32.4%) and was an independent risk factor for fatal outcome. Diabetes mellitus, liver cirrhosis, and bronchogenic carcinoma were independent risk factors for K. pneumoniae infection. Serotypes K1 (9/37, 24.3%) and K2 (13/37, 35.1%) were the prevalent strains but did not predispose patients to poor outcome compared with other non-K1/K2 serotypes. There was no major cluster of isolates found among serotype K1/K2 strains. In summary, physicians should be aware of the risk factors for thoracic empyema or complicated parapneumonic effusion caused by K. pneumoniae and the associated high mortality, and monitor these patients more closely.

Study on hydrogen production with hysteresis in UASB.

This paper uses a 10-l UASB (upflow anaerobic sludge blanket) bench-scale reactor to treat the esterification wastewater of a polyethylene terephthalate manufacturing plant. Two organic loading rates are used to evaluate the effect on H2 production of temperature gradually step-down and step-up in the range of 11-25 degrees C. Experimental results show that H2 production is positively related to temperature. H2 production increases with temperature at the higher organic loading rate (4.5 kg COD m(-3)d(-1)). However, the H2 produced does not go back to its original concentration but rather follows a hysteresis curve. This hysteresis also occurs in the corresponding concentrations of COD, acetate, propionate and butyrate. As in the H2 profiles, these parameter curves return clockwise during the temperature step-up. At the lower organic loading rate (2.2 kg COD m(-3)d(-1)), no obvious hysteresis is observed for H2 curve. The pattern of other parameters, except for the propionate, returns counterclockwise resulting in the hysteresis phenomena.

The effectiveness of Tsu-San-Li (St-36) and Tai-Chung (Li-3) acupoints for treatment of acute liver damage in rats.

This study investigated the effects of acupuncture on carbon tetrachloride (CCl4) induced acute liver injury in male rats (n=36). The experimental groups were injected with CCl4 before, during, or after acupuncture therapy. Acupoints similar to the human Tsu-San-Li (St-36) and Tai-Chung (Li-3) were needled bilaterally. Rats treated with CCl4 had higher levels of serum glutamate-oxalate-transaminase (sGOT) and serum glutamate-pyruvate-transaminase (sGPT). Comparing the experimental groups, biochemical and pathological parameters of liver injury were significantly reduced when rats were acupunctured after, not before, CCl4-induced hepatotoxicity. Acupuncture at the Tsu-San-Li and Tai-Chung acupoints cannot prevent acute liver injury but may be effective in treating liver injury induced by carbon tetrachloride in rats.

Induction of persistent sodium current by exogenous and endogenous nitric oxide.

Most voltage-gated Na(+) channels inactivate almost completely at depolarized membrane potentials, but in some cells a residual Na(+) current is seen that is resistant to inactivation. This persistent Na(+) current can have a profound impact on the electrical behavior of excitable cells, and the regulation of this property could have important biological consequences. However, the biological signaling mechanisms that regulate the persistence of Na(+) channels are not well understood. This study showed that in nerve terminals and ventricular myocytes nitric oxide (NO) reduced the inactivation of Na(+) current. This effect was independent of cGMP, was blocked by N-ethylmaleimide, and could be elicited in cell-free outside-out patches. Thus, a reactive nitrogen species acts directly on the channel or closely associated protein. Persistent Na(+) current could also be induced by endogenous NO generated enzymatically by NO synthase (NOS). Application of ionomycin to raise the intracellular Ca(2+) concentration in myocytes activated NOS. The NO produced in response to ionomycin was detected with an NO-sensitive fluorescent dye. Persistent Na(+) current was enhanced by the same treatment, and NOS inhibitors abolished both the elevation of NO and the induction of persistent Na(+) current. These experiments show that NO is a potential endogenous regulator of persistent Na(+) current under physiological and pathophysiological conditions.

Direct actions of nitric oxide on rat neurohypophysial K+ channels.

1. Nitric oxide (NO) has been shown to modulate neuropeptide secretion from the posterior pituitary. Here we show that NO activates large-conductance Ca2+-activated K+ (BK) channels in posterior pituitary nerve terminals. 2. NO, generated either by the photolysis of caged-NO or with chemical donors, irreversibly enhanced the component of whole-terminal K+ current due to BK channels and increased the activity of BK channels in excised patches. NO also inhibited the transient A-current. The time courses of these effects on K+ current were very different; activation of BK channels developed slowly over several minutes whereas inhibition of A-current immediately followed NO uncaging. 3. Activation of BK channels by NO occurred in the presence of guanylyl cyclase inhibitors and after removal of ATP or GTP from the pipette solution, suggesting a cGMP-independent signalling pathway. 4. The sulfhydryl alkylating agent N-ethyl maleimide (NEM) increased BK channel activity. Pretreatment with NEM occluded NO activation. 5. NO activation of BK channels occurred independently of voltage and cytoplasmic Ca2+ concentration. In addition, NO removed the strict Ca2+ requirement for channel activation, rendering channels highly active even at nanomolar Ca2+ levels. 6. These results suggest that NO, or a reactive nitrogen byproduct, chemically modifies nerve terminal BK channels or a closely associated protein and thereby produces an increase in channel activity. Such activation is likely to inhibit impulse activity in posterior pituitary nerve terminals and this may explain the inhibitory action of NO on secretion.

Ultra-violet light-induced changes in membrane properties in secretory cells.

Illumination with ultra-violet is used widely in physiological experiments for the photolysis of caged compounds. In the peptidergic cells of the pituitary gland, as well as cultured PC12 cells, ultra-violet light was found to produce changes in a number of membrane properties. Light of sufficient intensity to produce rapid photolysis of commonly used caged compounds induced changes in K+ and Ca2+ current, as well as changes in membrane capacitance. All responses to light showed a rapid timecourse, activating in a few ms and decaying within 10-50 ms after illumination ended. Experiments with radical scavengers and with inhibitors of cytochrome p450 and phospholipase A2 failed to block the light responses. These rapid responses to light emphasize that experiments employing ultra-violet light in the photorelease of physiological and pharmacological agents require special care for control of light artifacts.

Production and characterization of monoclonal antibodies against Tal1 oncoprotein.

TAL1 (or SCL) oncogene functions as a transcription regulatory factor that is necessary for the early development of all hematopoietic lineages. Aberrant expression of Tall protein in T cells is the cause of T-cell acute lymphoblastic leukemia (T-ALL) in children. We generated two hybridomas that secreted monoclonal antibodies (MAbs) that reacted strongly with GST-TAL(1-210) but not with GST fusion protein in ELISA assay. These two MAbs, TWN3 and TWN60, recognized TALI oncoprotein expressed in transfected COS-1 cells by Western blotting. In addition, both MAbs were also effective in detecting TAL1 oncoprotein in leukemic cells by immunoprecipitation and immunocytochemistry. Therefore, they should be useful in the studies of Tall protein functions in both hematopoietic development and oncogenesis.

Membrane excitability and secretion from peptidergic nerve terminals.

1. Thin slices of the posterior pituitary can be used as a preparation for the study of biophysical mechanisms underlying neuropeptide secretion. Patch-clamp techniques in this preparation have revealed the properties of ion channels that control the excitability of the nerve terminal membrane and have clarified the relation between Ca2+ and exocytosis. 2. Repetitive electrical activity at high frequencies broadens action potentials to allow more Ca2+ entry and thus enhance exocytosis. Action potential broadening results from the inactivation of a voltage-dependent K+ channel. 3. When repetitive electrical activity is sustained, secretion is depressed. This depression can be attributed in part to action potential failure caused by the opening of a Ca(2+)-activated K+ channel. This channel can be modulated by protein kinases, phosphatases, and G-proteins. 4. The inhibitory neurotransmitter GABA activates a GABAA receptor in the nerve terminal membrane. The gating of the associated Cl- channel depolarizes the membrane slightly to inactivate voltage-gated Na+ channels and block action potential propagation. 5. The response of the nerve terminal GABAA receptor is enhanced by neuroactive steroids and this can potentiate the inhibition of neurosecretion by GABA. The action of neurosteroids at this site could play a role in changes in neuropeptide secretion associated with reproductive transitions. 6. Ca2+ channels in the nerve terminal membrane are inactivated by sustained depolarization and by trains of brief pulses. Ca2+ entry promotes Ca2+ channel inactivation during trains by inhibiting the recovery of Ca2+ channels from inactivation. The inactivation of Ca2+ channels can play a role in defining the optimal frequency and train duration for evoking neuropeptide secretion. 7. Measurements of membrane capacitance in peptidergic nerve terminals have revealed rapid exocytosis and endocytosis evoked by Ca2+ entry through voltage-gated Ca2+ channels. Exocytosis is too rapid to account for the delays in neuropeptide secretion evoked by trains of action potentials. Endocytosis sets in rapidly after exocytosis with a time course comparable to that of the rapid endocytosis observed in nerve terminals at rapid synapses. Our results support the finding in rapid synaptic nerve terminals that endocytosis is inhibited by intracellular Ca2+. Multiple pools of vesicles were revealed, and these pools may reflect different stages in the mobilization and release of neuropeptide.

Dopamine D4 receptor mediated inhibition of potassium current in neurohypophysial nerve terminals.

Dopamine influences the release of neurohypophysial peptides in vivo. However, the extent to which this effect is caused by a direct dopaminergic action within the neurohypophysis remains unclear. With use of the patch-clamp technique on thin slices of rat posterior pituitary glands, we now provide evidence that dopaminergic agonists inhibit potassium current (IK) in neurohypophysial nerve terminals. Superfusion with the dopamine receptor agonist, (+/-)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin (PPHT), causes a reversible inhibition of whole-terminal IK under voltage clamp. This effect is concentration-dependent, with a maximal inhibition of 40 +/- 5% and an EC50 of 1.8 +/- 1.0 microM. It can be blocked with either a nonselective D2-like antagonist (100 microM eticlopride) or with the highly selective D4 antagonist, RBI-257 (10 microM). U101958 (a derivative of RBI-257) exhibits agonist activity similar to PPHT. Neither SKF 38393 (a D1/D5 agonist) nor quinpirole (a D2/D3 agonist) had any effect on whole-terminal IK in this preparation. Kinetic analysis demonstrated that the amplitude of both the rapidly and slowly inactivating phases of neurohypophysial IK are reduced by D4 receptor activation. These two separate current components have previously been shown to represent current through two distinct potassium channels, an A-current channel and a high-conductance Ca++-activated K+ channel. Thus, both channel types can be modulated by D4 receptors. This effect is likely to enhance the release of neurohypophysial peptides in vivo.

Aplasia cutis congenita: report of one case.

We present a female newborn with a skin defect on the vertex. Aplasia cutis congenita was diagnosed. No associated anomaly was found. There was no family or drug history noted. At one-year follow-up, the lesion tended to contract and became epithelialized. We report this case and also review the literature, and then we suggest a checklist for aplasia cutis congenita.

The differential transforming activities of Tal1 oncoproteins.

TAL1 (Or SCL) oncogene produces both full-length pp42 and truncated pp22 phosphoproteins in T-cell acute lymphoblastic leukemia cells. To investigate the transforming properties of these two oncoproteins, permanent transfected NIH3T3 cell lines were generated that constitutively express either pp42 or pp22. Both cell lines exhibited similar morphology as parental NIH3T3 cells. However, they showed differential anchorage independent growth in soft-agar. The clonogenicity of pp22 expressing cells was ten folds higher than that of pp42 expressing cells. This result might implicate the differential transformation potential of the two Tal1 oncoproteins.

Adaptation of Ca(2+)-triggered exocytosis in presynaptic terminals.

Rapid increases in Ca2+ concentration, produced by photolysis of caged Ca2+, triggered exocytosis in squid nerve terminals. This exocytosis was transient in nature, decaying with a time constant of approximately 30 ms. The decay could not be explained by a decline in presynaptic Ca2+ concentration, depletion of synaptic vesicles, or desensitization of postsynaptic receptors. Experiments in which Ca2+ was increased either in a series of steps or continuously at different rates suggested that the decay is caused by adaptation of the exocytotic Ca2+ receptor to higher levels of Ca2+. This adjustable sensitivity to Ca2+ represents a novel property of the triggering mechanism that can be used to evaluate molecular models of exocytosis. Adaptation can limit the amount of transmitter released by a nerve terminal and permit the speed of a presynaptic Ca2+ rise to serve as a critical determinant of synaptic efficacy.

Rapid exocytosis and endocytosis in nerve terminals of the rat posterior pituitary.

1. Ca(2+)-induced exocytosis and endocytosis were studied by measuring the membrane capacitance of voltage-clamped peptidergic nerve terminals in slices prepared from the rat posterior pituitary. 2. Depolarizing pulses produced rapid increases in capacitance. These increases varied in parallel with Ca2+ current as voltage was varied. Elimination of Ca2+ current blocked depolarization-induced capacitance changes. 3. Depolarization-induced capacitance changes increased with pulse duration. Capacitance changes also increased with integrated Ca2+ influx, but saturated at high levels of Ca2+ entry. This saturation allowed us to estimate a pool size of 190 vesicles, assuming each vesicle has a capacitance of 1 fF. Vesicles from this pool fused with a time constant of 0.43 s. The capacitance change increased with the first power of integrated Ca2+ influx. 4. Experiments with briefer pulses revealed a rapid component of exocytosis comprising a pool of forty vesicles that fuse with a time constant of 14 ms. This rapid process may reflect a final Ca(2+)-regulated triggering step, which is distinct from the slower kinetic step revealed by longer duration pulses. The slower step may reflect a priming of vesicles prior to exocytosis. 5. Depolarization-induced capacitance increases in most cases were followed by a rapid decay in capacitance, reflecting membrane reuptake tightly coupled to exocytosis. A variable amount of rapid endocytosis followed depolarization-induced capacitance increases. The time constant for rapid endocytosis to baseline was 0.44 s. Excess endocytosis was occasionally observed, with capacitance decaying below the pre-stimulus baseline with a time constant of 2.1 s. 6. Rapid endocytosis was slower after pulses that produced greater increases in intracellular Ca2+, consistent with the hypothesis that intracellular Ca2+ inhibits rapid endocytosis. 7. Exocytosis follows depolarization with no detectable delay, indicating that Ca2+ triggers neuropeptide secretion from nerve terminals with kinetics comparable to that observed in other rapidly secreting systems.

The effect of amphotericin B on the K-channel activity of MDCK cells.

By using the whole-cell patch technique, it is shown that the total outward current is increased, as a function of time, after the addition of amphotericin B to the bathing solution. The whole-cell current is shown to be primarily a K-channel current by the blockage of this current upon application of TEA to the bathing solution. Single K-channel studies, using the outside-out patch-clamp technique, reveal that the single K-channel opening probability increases by a factor of six after the addition of amphotericin B. In addition, single K-channel voltage dependent studies, using the inside-out patch-clamp technique, demonstrate that this increase in opening probability is due to an increase in the amplitude of Po(v). In contrast to the present belief that amphotericin B simply creates pores in a cell's membrane, these results suggest that amphotericin B can also influence the function of the cell's K-channel proteins.

Oesophageal trauma in patients with spinal cord injury.

Oesophageal perforations associated with cervical fractures occur from a variety of injuries. Fractures of the cervical spine, blunt trauma and penetrating injuries such as gunshot wounds, knives and missiles, perforate the cervical oesophagus. This retrospective study consists of 24 patients with an oesophageal perforation and cervical fracture. Motor vehicle accidents were responsible for 54% of the oesophageal perforations. The other oesophageal injuries were related to anterior spine surgery, gunshot wounds and sports-related activities. The clinical features related to these injuries included the obvious signs of an oesophageal perforation as well as fever of unknown origin, leukocytosis and unexplained persistent tachycardia. A variety of techniques was used to establish the diagnosis. All the patients had treatment for the cervical fracture and 20 patients required surgical repair of the oesophagus. The most common oesophageal complications were stricture of the oesophagus (54%) and oesophageal diverticulum (10%). The other complications were atelectasis, pneumonia, tracheobronchitis, pulmonary embolism, cervical osteomyelitis, cervical abscess, mediastinitis, septicemia and cervical fistulae. These patients have a serious life-threatening illness that may be difficult to diagnose and treat.

A quantitative electromyographic index that is independent of the force of contraction.

Quantitative EMG has been used to improve the accuracy of the diagnosis of myopathy. Equipment is available commercially that utilizes the Willison method of quantification, but the technique has not been widely used because of the necessity of measuring muscle contraction force. An analysis of the basic parameters of the Willison method of quantification was performed for forces of 10, 20, 40 and 60% of maximum for 16 normal subjects for the deltoid muscle. An index was derived from the basic parameters that is independent of the force of contraction. The index is a standardized value for turns/sec expressed as a logarithm. The index has a mean of 1.171 with a standard error of 0.059 for a sample size of 10 muscle sites. Quantitative EMG can be utilized in routine clinical examinations by applying mild to moderate resisting forces to patients, and the proposed index can be determined graphically. The force applied to patients can be maintained within the range of the forces investigated by estimating the force of contraction, evaluating patient effort and fatigue, and monitoring the parameter of total amplitude per second.