A novel intronic mutation and a missense mutation of MEN1 identified in two Chinese families with multiple endocrine neoplasia type 1.

BACKGROUND: Multiple endocrine neoplasia type 1 (MEN1) caused by MEN1 mutation is widely recognized. To date, 14 novel mutations were reported in Chinese and intronic mutations are getting more attention. AIM: To explore clinical features and MEN1 mutations in two Chinese families suffering from MEN1. METHODS: Nineteen individuals (10 males and 9 females) from two unrelated families with MEN1 were studied. Mutations of MEN1 were analyzed by direct sequencing of PCR products. In vitro splicing analysis was also performed with minigenes containing both wildtype and novel mutant fragments. Through the RNAstructure program, we analyzed the secondary structure of the wild type MEN1 pre-mRNA and then introduced T>G mutation at +2 donor splice site of intron 7. RESULTS: Clinical features of 3 patients in two families were described, and 5 individuals were proven to be carriers of MEN1 mutation without apparent symptoms. A novel splicing site mutation of the intron 7 (IVS7+2 T→G) was identified in the first family. In vitro analysis also verified this mutation caused the aberrant splicing of MEN1 mRNA. With the RNAstructure program, we could figure out that the global secondary structure as well as the number of stems and loops of pre-mRNA greatly changed after this mutation. The mutation c. 1227 C>A (C409X) was identified in another family, which also caused the truncation of menin. CONCLUSION: We reported a novel intronic mutation and a missense mutations in two Chinese families suffering from MEN1.

Identification of a locus (DSP2) for disseminated superficial porokeratosis at chromosome 12q21.2-24.21.

Porokeratosis is a rare disorder of epidermal keratinization that is characterized by the presence of a border called the cornoid lamella. Disseminated superficial porokeratosis (DSP) is a subtype of porokeratosis, which is inherited as an autosomal trait. The first locus for DSP was localized to chromosome 18p11.3, but no causative gene has yet been identified. In this study, we recruited and analysed a large six-generation Chinese family with autosomal dominant DSP. The genome-wide screening identified a maximum two-point LOD score of 3.06 at θ = 0.00 with the microsatellite marker D12S78. Fine mapping and haplotype analysis defined a critical region of 38 Mb between D12S326 and D12S79 on chromosome 12q21.2-24.21, which is a probable second locus identified for DSP (DSP2). We sequenced 50 candidate genes in this region, but no causative mutation was found. This study provides a map location for isolation of a gene causing DSP.

Aldosterone perturbs adiponectin and PAI-1 expression and secretion in 3T3-L1 adipocytes.

Aldosterone is considered as a new cardiovascular risk factor that plays an important role in metabolic syndrome; however, the underlying mechanism of these effects is not clear. Hypoadiponectinemia and elevated circulating concentration of plasminogen activator inhibitor-1 (PAI-1) are causally associated with obesity-related insulin resistance and cardiovascular disease. The aim of the present study is to investigate the effect of aldosterone on the production of adiponectin and PAI-1 in 3T3-L1 adipocytes. Northern and Western blot analyses revealed that aldosterone treatment inhibited adiponectin mRNA expression and secretion and simultaneously enhanced PAI-1 mRNA expression and secretion in a time- and dose-dependent manner. Rosiglitazone did not prevent aldosterone's effect on adiponectin or PAI-1 expression. In contrast, tumor necrosis factor (TNF)-α produced dramatic synergistic effects on adiponectin and PAI-1 expression when added together with aldosterone. Furthermore, the effects of aldosterone on adiponectin and PAI-1 expression appear to be mediated through glucocorticoid receptor (GR) but not mineralocorticoid receptor (MR). These results suggest that the effects of aldosterone on adiponectin and PAI-1 production are one of the underlying mechanisms linking it to insulin resistance, metabolic syndrome and cardiovascular disease.

Statistical optimization of process parameters on biohydrogen production from glucose by Clostridium sp. Fanp2.

Statistically based experimental designs were applied to optimizing process parameters for hydrogen production from glucose by Clostridium sp. Fanp2 which was isolated from effluent sludge of anaerobic hydrogen-producing bioreactor. The important factors influencing hydrogen production, which identified by initial screening method of Plackett-Burman, were glucose, phosphate buffer and vitamin solution. The path of steepest ascent was undertaken to approach the optimal region of the three significant factors. Box-Behnken design and response surface analysis were adopted to further investigate the mutual interaction between the variables and identify optimal values that bring maximum hydrogen production. Experimental results showed that glucose, vitamin solution and phosphate buffer concentration all had an individual significant influence on the specific hydrogen production potential (Ps). Simultaneously, glucose and vitamin solution, glucose and phosphate buffer were interdependent. The optimal conditions for the maximal Ps were: glucose 23.75 g/l, phosphate buffer 0.159 M and vitamin solution 13.3 ml/l. Using this statistical optimization method, the hydrogen production from glucose was increased from 2248.5 to 4165.9 ml H2/l.

Paramedian reticular nucleus--sympathetic inhibition in spontaneously hypertensive rats.

The cardiovascular reactivity of various areas in the medulla related to sympathetic or parasympathetic activation, or to sympathetic inhibition, was compared in spontaneously hypertensive rats (SHR) and in normotensive rats Wistar-Kyoto (WKY) or Sprague-Dawley (SD). In SHR, which has an elevated resting systemic arterial blood pressure (SAP), the sympathetic pressor responses elicited from electrical stimulation of the dorsomedial medulla (DMM), parvocellular lateral nucleus (PVC) or ventrolateral medulla (VLM) were more profound than those in WKY and SD. The depressor and bradycardia responses elicited from electrical stimulation of the paramedian reticular nucleus (PRN) (which exerts both sympathetic and parasympathetic inhibitions) or from the area of the solitary nucleus/dorsomotor nucleus of vagus (NTS/DMV) (where stimulation leads to both parasympathetic activation and sympathetic inhibition) were also more intensive in SHR than in WKY and SD. The elicited pressor and depressor responses, however, were not significantly different between WKY and SD. Our results are consistent with previous findings (15) that in SHR an increased sympathetic activity of the pressor areas of medulla contributes to the pathogenesis of hypertension. Sympathetic inhibition (PRN and NTS/DMV areas) and parasympathetic activation (NTS/DMV area) from these areas, however, may not be critically involved.

Existence of a powerful inhibitory mechanism in the medial region of caudal medulla--with special reference to the paramedian reticular nucleus.

Inhibitory actions of the medial trigon of the caudal medulla (ITM) with special reference to the paramedian reticular nucleus (PRN) were explored in cats under chloralose (40 mg/kg) and urethane (400 mg/kg) anesthesia. Stimulation with square wave pulses (80 Hz, 1 msec, 100-200 microA) produced a reduction of mean systemic arterial blood pressure (MSAP) of 15-90 mmHg, and change in heart rate (HR) that varied from mild increase of 15 to reduction of 85 beats/min. These responses were not affected by mid-collicular decerebration nor by bilateral vagotomy. Destruction of PRN did not change the resting MSAP, HR or baroreceptor reflex responses. Stimulation of PRN suppressed the sympathetic pressor and cardioacceleratory and the vagal bradycardia responses resulting from activating cardiovascular (CV) regulatory mechanisms in the hypothalamus, midbrain and medulla, or from activating the somatic or the baroreceptive afferents. Activation of the PRN suppressed the MSAP-increase produced by direct stimulation of the stellate or celiac ganglion. PRN stimulation could eliminate the pronounced CV reactions consequent either to asphyxial anoxia during occlusion of the trachea or to cerebral ischemia following occlusion of vertebral and carotid arteries. Furthermore, PRN activation could stop the general convulsion of the animal induced by picrotoxin, 4 mg/kg, IV. Our findings suggest that in the trigon area especially in the PRN, there resides an independent mechanism which exerts very powerful and broad inhibitory actions on the autonomic as well as somatic nervous system.

A cardioinhibitory area in the midbrain central tegmental field of cats.

A cardioinhibitory area in the central tegmental field of the midbrain (CIM) was studied in cats under chloralose-urethane anesthesia. Electrical and chemical (glutamate and DL-homocysteic acid) stimulations in this area produced marked bradycardia accompanied by mostly hypotension or minimal change in arterial pressure and by occasional hypertension particularly in the dorsal portion. CIM excitation potentiated the reflex bradycardia induced by IV phenylephrine, while bilateral electrolytic lesion of CIM neither changed the resting cardiovascular parameters nor the reflex bradycardia. The CIM bradycardia was not affected by supracollicular decerebration, but substantially reduced by unilateral vagotomy and completely eliminated by bilateral vagotomy. Destruction of the ambiguus nucleus (NA) and solitary and dorsal motor nuclei (NTS/DMV) abolished the bradycardia. Midline bisection at the midbrain-pontine level only slightly reduced the bradycardia while at the medullary level it was moderately attenuated. Electrolytic lesion of the cardioinhibitory area in gigantocellular reticular nucleus (GRN) abolished the bradycardia. These findings suggest that CIM is an independent mechanism which may send axons to GRN from which the axons may in turn synapse with the NTS/DMV complex and NA. Its final output may utilize both vagus nerves to modulate baroreceptor reflex in promoting bradycardia.

Afferent and efferent connections of the mesencephalic cardioinhibitory area (CIM) in the cat.

The afferent and efferent connections between the cardioinhibitory area in the midbrain tegmental field (CIM) and brain stem structures related to cardiovascular integration in cats were investigated by horseradish peroxidase (HRP) for both cell origins and axonal terminations and by chemical (kainic acid) lesion for topographic pathways. Retrogradely labeled neurons were observed in the gigantocellular reticular nucleus (GRN), the pontine reticular nucleus of the pons (PON), the ambiguus nucleus (AN) and the paramedian reticular nucleus (PRN). A few neurons were also labeled in the following structures i.e., ventrolateral medulla (VLM), dorsomedial medulla (DMM), dorsomotor nucleus of the vagus nerve (DMV) and nucleus of solitary tract (NTS). Anterograde HRP labeled terminals were found surrounding the cell bodies of the aforementioned structures. They were most abundant in VLM, DMM, DMV, NTS, moderate in GRN, PRN and AN, but only scanty in hypothalamus. The fiber pathway of CIM neurons was also traced by degenerating fibers consequent to kainic acid lesion and by means of silver stain. Degenerating fiber bundle was found primarily projecting through the medial portion of the mesencephalic pontine structures. As the bundle reached the medulla oblongata, it bifurcated into a dorsal and a ventral tracts on its course. The dorsal tract was primarily coursing through the dorsomedial area, including DMM, NTS and DMV, and the ventral tract was mainly passing VLM, AN and inferior olivary nucleus (ION) areas. The present findings suggest that neurons in the CIM may receive inputs from various cardiovascular-related structures and make output bilaterally to some of pontine and medullary structures to modulate cardiovascular functions. Based on the anatomical findings, the profound bradycardia produced by CIM stimulation may be mediated through the following mechanisms: A. Direct activation of vagal preganglionic neurons in DMV, AN and ION. B. Indirect activation of neurons in GRN for vagal activation and of neurons in PRN for sympathetic inhibition.

Afferent and efferent connections of the paramedian reticular nucleus in the brain stem of cats.

Anatomical connections of the paramedian reticular nucleus (PRN) of the caudal medulla were investigated using a bi-directional tracer, horseradish peroxidase (HRP). The followings were observed when the tracer was microinjected to PRN: A. Both labelled neurons and terminals were found in the areas of the mesencephalic cardioinhibitory mechanism (CIM), the gigantocellular reticular nucleus (GRN), the ambiguus nucleus (AN) and the contralateral PRN. B. Only labelled terminals were demonstrated in the area of the nucleus of solitary tract (NTS) and the intermedial lateral cell column (IML) of the spinal cord. C. Only retrogradely labelled neurons were observed in the areas of the dorsal and dorsomedial medulla (DM) and ventrolateral medulla (VLM). A few labelled neurons were observed in the periaqueductal gray, the cuneiform nucleus and superior colliculus of the mesencephalon as well as the alamina spinal trigeminal nucleus. When HRP was applied to the CIM, GRN or AN structures, respectively, both labelled cells and terminals were found in the PRN area. HRP injection in the VLM showed only labelled terminals in the PRN. However, injection of HRP to DM showed neither labelling neurons nor terminals in PRN. Results suggest that PRN projects to the pressor area of DM/NTS and IML through which PRN could exert its inhibitory functions on the sympathetic pressor actions. In addition, PRN may suppress the vagal bradycardiac action through its reciprocal connections with CIM, GRN and AN. No lateralization in the PRN pathway was evident.

Descending pressor pathways from the dorsomedial and ventrolateral medulla of cats.

Descending pathways of the pressor areas in dorsomedial (DM) and ventrolateral (VLM) medulla were studied physiologically and anatomically. The electrically induced pressor response from either DM or VLM that previously responded to glutamate (Glu) was slightly but not significantly decreased after splitting the medulla at midline. After electrolytic lesion in the ipsilateral lateral tegmental field (LTF) of caudal medulla, 4 mm caudal to the obex, electrically induced pressor responses from DM and VLM previously responded to Glu decreased significantly from 71% to 20% and from 57% to 7%, respectively. The dorsomedial LTF was found to be responsible for the DM responses, while the ventrolateral LTF for the VLM responses. Following electrolytic lesion in the ipsilateral dorsolateral funiculus (DLF) between C4 and C5, the magnitude of DM-induced pressor responses decreased significantly from 74% to 37% and that of the VLM from 75% to 14%. After electrolytic lesion at the ipsilateral intermediolateral cell column (IML) of T2 spinal cord, the pressor responses on DM and VLM stimulations also decreased significantly. Lesioning the remaining IML almost completely abolished the induced pressor responses. Seven to ten days following kainic acid injections into DM or VLM to destroy neurons therein, degenerative fibers (silver impregnation) were found to descend from the medulla to thoracic spinal cord, principally ipsilaterally, but with some fibers crossing to the controlateral medulla. Data from both physiological and anatomical studies suggest that fibers from DM and VLM descend ipsilaterally through different pathways and converge at LTF 4 min caudal to the obex, and then further descend to DLF before reaching the thoracic IML.

Population pharmacokinetic analysis of amikacin and validation on neonates using Monte Carlo method.

AIM: To make programs for population pharmacokinetic analysis and to assess the ability of this method in pharmacokinetic parameter estimation and in the prediction of serum concentrations. METHODS: Data of amikacin as a model drug were collected from 42 neonates with 142 serum samples. A one-compartment open model was used to describe the kinetics of amikacin after the intravenous infusion. Following Sheiner's idea of population pharmacokinetics, we made the programs to evaluate population parameter and individual parameter. The target function minimality was obtained from Monte Carlo algorithm. The validation of the population analysis was performed using classic pharmacokinetic program 3p87 for antithesis. The predictability of the developed method was evaluated by computing precision and accuracy of serum concentration predicted using the parameter estimates. RESULTS: The stability of our self-made program was good. The population parameters obtained from this approach were in conformity with those from 3p87, and the interindividual variability was relatively small. For the learning sample and the validation sample, predicted and observed concentrations were all close with correlation coefficient 0.995 and 0.990, respectively. Most of predicted errors were found < +/- 1 mg/L, and RMSD and BIAS were 0.58 and -0.07 for the validation sample, respectively. The choice of blood sampling time was an important factor for the predictive performance. An early sampling time after the infusion was observed to be the best sampling time. CONCLUSION: The estimation program of population parameter and individual parameter made by us ran stably, and allowed us to use sparse data to estimate population pharmacokinetic parameters. It provided accurate estimates of these parameters and satisfactory ability of serum concentration prediction. Therefore, it can be used for the population pharmacokinetic analysis and individualization of dosage regimen.

Predominance of vagal bradycardia mechanism in the brain stem of turtles.

Cardiovascular parameters of spontaneously breathing pond turtles (Cyclemys flavomarginata) anaesthetized with chloralose (4 mg 100 g-1) and urethane (40 mg 100 g-1), were examined during exploratory electrical stimulation of the brain stem. Turtles exhibited a low mean systemic arterial blood pressure (MSAP, average 25 mmHg) and slow heart rate (average 24 beats min-1). Upon stimulation, pressor (sympathetic), depressor (sympathetic inhibition), bradycardia and hypotensive (vagal) responses were elicited from regions of the brain stem extending from the hypothalamus to the medulla, principally in the medial region. The pressor response appeared after a longer latency than did the bradycardia and hypotensive responses. It developed rather slowly, and rarely attained a magnitude double its resting value. In contrast, stimulation of many points in the brain stem produced marked slowing or even cessation of the heart beat, and thus resulted in an immediate fall of the blood pressure even to zero. This cardio-inhibitory response depended on the integrity of the vagus nerves and was particularly marked upon stimulation in the caudal medulla, the areas of the ambiguus, solitary and dorsomotor nuclei of the vagus and the midline structures. When such an area was stimulated continuously the heart stopped beating throughout the stimulation. The longest period of cardiac arrest before the appearance of escape was 35 min. With continuous stimulation of the peripheral end of the cut vagus, the earliest escape beat occurred even later (65 min). Epinephrine given intravenously produced an increase of MSAP and force of cardiac contraction, although the slope of pressor rise was shallow. Reflex bradycardia, however, was not observed. These experiments show that a very prominent vagal bradycardia can be evoked from the turtle brain stem, which may contribute to its well-known capacity for tolerating anoxia.

[Staffing levels and patient needs in the intensive care unit].

The current study sought to utilize a patient classification system to investigate staffing and patient needs along with nursing care distribution in our intensive care units. The study employed a factor type analysis to design a patient need checklist (for our six ICUs) in order to determine staff load and nursing requirements. Snapshot observations were also taken to survey the distribution of nursing care time. The results of the two methods provide an estimate of current staffing needs, they also show that there is no significant difference between our surgical and internal medicine wards. On average, direct care accounts for 40.1% of the time schedule; indirect care, 37.3%; related care, 6.0%; and individual time, 16.6%. The average patient grade lies between I and III; and workload index, between 4.4 and 11.5. The average nursing time per patient per shift is 2.88 hours, and the average workload per person is estimated at 5.7-5.8 hours. By comparing the number of personnel currently employed and the estimated number needed, we discovered that two units are understaffed, and three are overstaffed. The understaffed units are all surgical units; the overstaffed ones, internal medicine units. To conclude the study, we examined the nature and complexity of nursing duties in the hopes of returning non-nursing responsibilities to the proper medical organizations. Our ultimate goal is to realize the full potential and improve the quality of our nursing personnel.

Pressor responses from electrical or glutamate stimulations of the dorsal or ventrolateral medulla.

In rats, rabbits, and cats anesthetized with alpha-chloralose and urethan, responses of the pressor areas of the dorsal portion (DM) and ventrolateral portion (VLM) of medulla and pons were compared. Electrical stimulation (monopolar square-wave pulses) on monosodium glutamate solution (Glu, 100-200 nl, 1 M) was delivered through an electrode-needle tubing connected to a Hamilton syringe for semimicroinjection. In all three of these species, pressor responses were elicited from both DM and VLM by either Glu or electrical stimulation. The most active parts of DM were found in the dorsomedial reticular formation of the rostral medulla to mid-medulla. In the pons and caudal medulla, the Glu-induced response was mild, although the electrically induced response was marked. Application of kainic acid (KA) to either DM or VLM produced an initial pressor response but was followed by a reduction of the pressure rise on subsequent electrical stimulation. Glu, unlike electrical stimulation, excites neural perikarya, not fibers of passage. KA initially excites the neural perikarya before causing damage that spares axons. These results thus suggest that both DM and VLM contain neural perikarya that mediate pressor effects.