Salt-wasting congenital adrenal hyperplasia: detection of mutations in CYP21B gene in a Chilean population.

The steroid 21-hydroxylase deficiency (21OHD) is the most frequent cause of congenital adrenal hyperplasia. We have characterized the disease-causing mutations in the 21-hydroxylase genes of 63 patients with salt-wasting congenital adrenal hyperplasia from a Chilean population of Hispanic origin, a group that has been scarcely evaluated. Using allele-specific PCR, lesions were identified in 97 chromosomes out of 126 tested (77%). The most frequent findings were the gene deletion or large gene conversion (LGC) = 22.9%, I2 splice = 19%, R357W = 12.7%, and Q319X = 10.5%. We did not find alleles with the mutation F308insT and we found three alleles with the cluster E6. The frequency of the point mutation R357W was at least two times more frequent than the one found in Caucasians populations, but similar to that communicated in Asian populations; this finding may be explained by the Asian ancestry of our South-Amerindian population. The frequency of Q319X was also high, similar only to those patients studied in Italy and in a neighboring Argentinian population. In summary, this is a genetic characterization of 21OHD made in an almost pure Hispanic population in Latin America. The high frequency of deletion of CYP21B gene, I2 splice, R357W, and Q319X mutations probably reflects the European-Caucasian-Spanish influence of the conquerors, mixed with Amerindians of Asian ancestry and modulated by other European immigrations.

Primary hyperaldosteronism in essential hypertensives: prevalence, biochemical profile, and molecular biology.

There is evidence that primary aldosteronism (PA) may be common in patients with essential hypertension (EH) when determinations of serum aldosterone (SA), plasma renin activity (PRA), and the SA/PRA ratio are used as screening. An inherited form of primary hyperaldosteronism is the glucocorticoid-remediable aldosteronism (GRA) caused by an unequal crossing over between the CYP11B1 and CYP11B2 genes that results in a chimeric gene, which has aldosterone synthase activity regulated by ACTH. The aim of this study was to evaluate the prevalence of PA and the GRA in 305 EH patients and 205 normotensive controls. We measured SA (1-16 ng/dL) and PRA (1-2.5 ng/mL x h) and calculated the SA/PRA ratio in all patients. A SA/PRA ratio level greater than 25 was defined as being elevated. PA was diagnosed in the presence of high SA levels (>16 ng/dL), low PRA levels (<0.5 ng/mL x h), and very high SA/PRA ratio (>50). Probable PA was diagnosed when the SA/PRA ratio was more than 25 but the other criteria were not present. A Fludrocortisone test was done to confirm the diagnosis. GRA was differentiated from other forms of PA by: the aldosterone suppression test with dexamethasone, the high levels of 18-hydroxycortisol, and the genetic detection of the chimeric gene. In EH patients, 29 of 305 (9.5%) had PA, 13 of 29 met all the criteria for PA, and 16 of 29 were initially diagnosed as having a probable PA and confirmed by the fludrocortisone test. Plasma potassium was normal in all patients. The dexamethasone suppression test was positive for GRA in 10 of 29 and 18-hydroxycortisol levels were high in 2 of 29 patients who had also a chimeric gene. In normotensive subjects, 3 of 205 (1.46%) had PA, and 1 of 205 had a GRA. In summary, we found a high frequency of normokalemic PA in EH patients. A high proportion of PA suppressed SA with dexamethasone, but only a few had a chimeric gene or high levels of 18-hydroxycortisol. These results emphasize the need to further investigate EH patients.

A(-6)G variant of angiotensinogen gene and aldosterone levels in hypertensives.

Recently, a novel mutation in the promoter region of the angiotensinogen gene that involves the presence of an adenine instead of a guanine 6 bp upstream from the transcription initiation site (A(-6)G) has been shown to induce an increase in gene transcription. The aim of this study was to determine the prevalence of the A(-6)G mutation in essential hypertensive patients and to correlate it with aldosterone and renin activity levels. We studied 191 hypertensives. We measured levels of aldosterone (plasma and urinary) and plasma renin activity. We determined the variants A and G using a mutagenically separated polymerase chain reaction technique. In 191 hypertensives, the A variant was detected in 266 of 382 (69.6%) and the G variant in 116 of 382 alleles (30.4%). Plasma aldosterone was significantly higher in patients homozygous for AA than in those homozygous for GG (369+/-208 versus 246+/-142 pmol/L). Urinary aldosterone was significantly higher in homozygous AA than in AG or GG patients (62.4+/-39.4 versus 50.8+/-25.2 and 37.4+/-22.3 nmol/d, respectively). When the patients were grouped according to the presence or absence of the A allele, the aldosterone levels and the plasma aldosterone/plasma renin activity ratio were significantly higher in patients with the A allele. The presence of the A variant was associated with higher levels of aldosterone. These results suggest that the presence of the A variant could determine the appearance of arterial hypertension through higher transcription activity of the angiotensinogen gene and concomitant aldosterone production.

Metabotropic glutamate receptors group I are involved in cochlear neurotransmission.

All three types of ionotropic glutamate receptors, AMPA, NMDA and kainate, contribute to the neurotransmission between inner hair cells (IHC) and afferent neurons in the mammalian cochlea. We used microiontophoretic techniques to investigate whether metabotropic glutamate receptors group I (mGluR I) are also involved in the transmission of IHC afferents of the guinea pig. The mGluR I agonist DHPG produced an increase in afferent firing, which lasted significantly longer than that of the ionotropic agonists AMPA and NMDA. The activation was reversibly blocked by the mGluR I antagonist AIDA in a dose-dependent manner. AIDA also diminished spontaneous activity, but only slightly affected the AMPA- or NMDA-induced firing rate. Our results suggest that mGluR I are involved in peripheral auditory processing.

Degeneration of nigrostriatal dopaminergic neurons increases iron within the substantia nigra: a histochemical and neurochemical study.

Parkinson's-diseased (PD) brains have increased levels of iron in the zona compacta of the substantia nigra (SNc). To determine whether these elevated nigral iron levels may be caused secondarily by degeneration of nigrostriatal dopaminergic (NS-DA) neurons, the NS-DA pathway was unilaterally lesioned in rats through 6-hydroxydopamine (6-OHDA) infusion and nigral iron levels evaluated three weeks later. A significant increase was observed in both iron concentration (+35%) and iron content (+33) within the substantia nigra (SN) ipsilateral to comprehensive 6-OHDA lesions. Moreover, ferric iron staining was dramatically increased within the SNc following 6-OHDA lesions, primarily due to the appearance of iron-positive SNc neurons and infiltrating reactive glial cells. Iron staining in the SN zona reticularis was modestly increased after 6-OHDA lesions, but staining in the neostriatum and globus pallidus was unaffected. These results indicate that loss of NS-DA neurons is associated with increased iron levels in the SN. This suggests that increased nigral iron levels in PD may be secondary to some neurodegenerative process. Nonetheless, even a secondary increase in nigral iron levels may be of pathogenic importance in PD because of iron's ability to catalyze neurotoxic free radical formation and perpetuate neurodegeneration.

Neuroprotection by dopamine agonists.

Research on Parkinson's disease has led to new hypotheses concerning the mechanisms of neurodegeneration and to the development of neuroprotective agents. Recent findings of impaired mitochondrial function, altered iron metabolism and increased lipid peroxidation in the substantia nigra of parkinsonian patients emphasize the significance of oxidative stress and free radical formation in the pathogenesis of Parkinson's disease. Present research is therefore focussing on improvements in neuroprotective therapy to prevent or slow the rate of progression of the disease. Possible neuroprotective strategies include free radical scavengers, monoamine oxidase-B inhibitors, iron chelators and glutamate antagonists. Recent studies point to the possibility of achieving neuroprotection in ageing and parkinsonism by the administration of dopamine agonists. In the rat, the dopamine agonist pergolide appears to preserve the integrity of nigrostriatal neurones with ageing. The prevention of age-related degeneration may be achieved as a result of a decreased dopamine turnover and reduced conversion of dopamine to toxic compounds. In our own study, bromocriptine treatment prevented the striatal dopamine reduction following MPTP administration in the mouse. These results suggest that the neurotoxic effects of MPTP can be prevented by bromocriptine. Monotherapy with the dopamine agonist lisuride in the early stages of Parkinson's disease delays the need for the initiation of levodopa treatment to a similar extent as has been reported for L-deprenyl. It remains to be shown whether this is due to neuroprotective efficacy of the dopamine agonist or to a direct symptomatic effect.

Dopamine regulates the glutamatergic inner hair cell activity in guinea pigs.

Recent immunocytochemical and biochemical studies support a possible neurotransmitter function of dopamine (DA) in the efferent olivocochlear innervation of the guinea pig cochlea. However, the physiological role of DA in cochlear neurotransmission remains unknown. In the present study microiontophoretic techniques were used for testing the action of DA as well as D1- and D2-agonists and -antagonists on spontaneous and N-methyl-D-aspartic acid (NMDA)-, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-, kainic acid- or glutamate-induced firing of afferent fibres in the dendritic region of inner hair cells. Subsynaptic spike activities of anaesthetised guinea pigs were recorded after exposing the third or fourth turn of the cochlea for electrode penetration. Application of DA alone had very little effect on the spontaneous afferent firing rate. In contrast, firing induced by NMDA or AMPA could be depressed by additional administration of DA in a dose-dependent manner. A similar reduction of the induced spike activity was seen after co-administration of D1- or D2-agonists. The action of DA on glutamate agonist-induced firing could be blocked by D1- as well as D2-antagonists. These results show that DA can depress the activated firing rate of the afferent fibres and that this action is mediated by both D1- and D2-receptor subtypes.

GABA(A) receptor modulates the activity of inner hair cell afferents in guinea pig cochlea.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is mediated by two main categories of receptors: the GABA(A) and GABA(B) receptor. Recent immunocytochemical and electron microscopical studies revealed the existence of GABA at the efferent olivocochlear innervation of the guinea pig cochlea. In this microiontophoretic study we examined the effect of GABA on spontaneous and glutamate or acetylcholine induced activity of afferent fibres in the dendritic region of inner hair cells. Furthermore, the receptor subtypes being responsible for this GABA action were analysed using specific agonists and antagonists on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) induced activity. The spike activities of the subsynaptic area were recorded in the third or fourth turn of the cochlea of anaesthetised guinea pigs. Application of GABA had little effect on spontaneous activity whereas the glutamate or acetylcholine induced firing rate could be depressed by GABA. AMPA and NMDA induced activity was reduced by the GABA(A) agonist muscimol but not by the GABA(B) agonist baclofen. The GABA(A) antagonist blocked the inhibition of both GABA and the GABA(A) agonist. In contrast, the GABA(B) antagonist saclofen was without effect. These results reveal that GABA reduces the activated firing rate of inner hair cell afferents mediated by the GABA(A) receptor subtype.

New approaches for inner ear therapy with glutamate antagonists.

In the mammalian cochlea neurotransmission between inner hair cells and afferent auditory neurons is probably mediated by glutamate or another related excitatory amino acid. Neurotoxicity induced by excessive glutamate release seems to play a crucial role in some pathological conditions of the cochlea, such as ischaemia or noise trauma. Thus, glutamate antagonists may be a new therapeutic strategy for different inner ear diseases. Because of their potential severe side-effects only a few glutamate antagonists have so far been adopted for clinical use. We used microiontophoretic techniques to compare the effects of memantine and caroverine on the glutamatergic transmission of inner hair cells of the guinea pig and tested the possibility of a local administration of memantine to the cochlea with a micropump. Memantine selectively inhibited the NMDA (N-methyl-D-aspartate) stimulated activity while caroverine blocked NMDA as well as AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) induced activity of inner hair cell afferents. With a flow rate of 1 microl/h the local administration of memantine via a cochleotomy was succeeded in a reversible blockade of the spontaneous and the NMDA induced firing of inner hair cell afferents. These results suggest that local application to the cochlea could be a feasible way to administer glutamate antagonists in sufficient amounts while avoiding systemic side-effects.

Alpha and beta subunits of acetylcholine receptors in the human inner ear.

The localization and distribution of nicotinic acetylcholine receptors (n-ACh-r) was characterized by studying alpha and beta subunits in the adult human inner ear by FITC fluorescence technique. In the cochlea, distinct fluorescence staining occurred for beta subunits in outer hair cells (OHCs), but no alpha subunits were identified. Beta subunits differ quantitatively between the three rows of OHCs, decreasing along a base-to-apex gradient in the cochlea. Both alpha and beta subunits were identified on spiral ganglion cells, adjacent nerve fibres and in vestibular hair cells (HCs). It would appear that they form an active complex in n-ACh-r at these locations.

A microcomputer program for fitting enzyme inhibition rate equations.

This paper presents a computer program written in BASIC for fitting different enzyme inhibition kinetic models. The program, based on a nonlinear least-squares regression, can be run on any microcomputer with the CP/M operating system. Weighting of observed initial velocities is decided by the user by assessing constant variance, proportional variance or by incorporation of the variances calculated by a subroutine. The program also uses robust regression by bisquare weighting. All questions concerning data input, type of rate function, type of weight and the use of bisquare regression appear on the video display unit.

A microcomputer program for fitting two-substrate enzyme rate equations.

This paper describes a microcomputer program written in BASIC for fitting different two-substrate enzyme kinetic mechanisms. The present program is a complement of one which was recently published and was developed to fit different models of enzyme inhibition. The complete program resulting from this complementation allows the fitting of the most usual rate equations found in steady-state studies of enzyme kinetics. The program based on a non-linear least-squares regression, can be run on any microcomputer having the CP/M operating system. The initial rate equation for a steady-state random bisubstrate mechanism was chosen to evaluate the performance of the program, with contrived data of known error structure.

Pocket computer program for fitting the Hill equation.

This paper presents a program written in the BASIC language for the Radio Shack TRS-80 PC-1 or the Sharp PC 1211 pocket computers for fitting the Hill equation to experimental data. The program is based on a procedure involving a combination of an optimization technique and non-linear regression. The program determines precisely all three parameters of the Hill equation and in addition, it performs a simulation of the kinetic experiment using the parameter values previously estimated, calculating the sum of squares of residuals.

Pocket computer program for fitting the Michaelis-Menten equation.

This paper presents a program written for the Radio Shack TRS-80 or the Sharp PC 1211 pocket computers for fitting the Michaelis-Menten equation to experimental data by a non-linear regression analysis. The program determines the kinetic parameters Km and Vm, their standard errors and the residual standard error. In addition it performs a simulation of a kinetic experiment using the parameter values previously determined.

Squamous cell carcinoma and Zenker diverticulum.

Squamous cell carcinoma in a Zenker diverticulum is a very rare condition. We report a case of a patient with a Zenker carcinoma, who was primarily functionally inoperable and therefore received neoadjuvant radiochemotherapy before cardiac bypass surgery. After a complicated course with cardiogenic shock and myocardial infarction, a re-evaluation of functional risk analysis and the tumor situation revealed operability. Subsequently, partial hypopharyngectomy and partial cervical esophageal resection with lymphadenectomy was performed. Reconstruction of the gastrointestinal continuity was made by interposition of a free small bowel graft and microvascular anastomosis. The postoperative course showed a small anastomotic leakage of the hypopharyngeal-small bowel anastomosis, which was successfully treated conservatively.

Estradiol increases proteinuria and angiotensin II type 1 receptor in kidneys of rats receiving L-NAME and angiotensin II.

Prospective, placebo-controlled clinical trials suggest that estrogen may have adverse effects on the vascular system in women. The goal of this study was to determine if 17beta-estradiol (E2) would have adverse effects on the renovasculature in a rat model of renal injury characterized by low nitric oxide (NO) and high angiotensin II (AngII). We studied female Wistar rats that were sham-operated (sham), ovariectomized (OVX), or ovariectomized and replaced with E2 (OVX/E2). All rats were maintained on a high salt diet and renovascular injury was caused by treating rats with an inhibitor of NO synthase, N(omega)-nitro-L-arginine-methyl-ester (L-NAME), for 14 days, plus AngII on days 11 through 14. L-NAME/AngII treatment, as compared to placebo, caused proteinuria, glomerular injury, and fibrinoid necrosis of renal arterioles in sham-operated rats. Ovariectomy reduced L-NAME/AngII-induced renal damage, whereas E2 treatment increased L-NAME/AngII-induced damage in OVX rats. In rats treated with L-NAME/AngII, levels of AngII type 1 receptor (AT(1)R) protein were higher in the renal cortex of sham and OVX/E2 rats than in OVX rats. AT(1)R protein correlated with renal injury. E2 treatment also increased expression of AT(1)R mRNA. Thus, under conditions of low NO and high AngII, E2 exacerbated renal injury. E2-mediated increases in renal cortical AT(1)R expression may represent a novel mechanism for the adverse renovascular effects of estrogen.

Structural brain changes in tinnitus.

Tinnitus is a common but poorly understood disorder characterized by ringing or buzzing in the ear. Central mechanisms must play a crucial role in generating this auditory phantom sensation as it persists in most cases after severing the auditory nerve. One hypothesis states that tinnitus is caused by a reorganization of tonotopic maps in the auditory cortex, which leads to an overrepresentation of tinnitus frequencies. Moreover, the participation of the limbic system in generating tinnitus has been postulated. Here we aimed at identifying brain areas that display structural change in tinnitus. We compared tinnitus sufferers with healthy controls by using high-resolution magnetic resonance imaging and voxel-based morphometry. Within the auditory pathways, we found gray-matter increases only at the thalamic level. Outside the auditory system, gray-matter decrease was found in the subcallosal region including the nucleus accumbens. Our results suggest that reciprocal involvement of both sensory and emotional areas are essential in the generation of tinnitus.

[Impalement injury of the neck]. / Pfählungsverletzung des Halses.

In comparison to the United States or South Africa, penetrating injuries of the neck are rare in Europe. Most of these traumas are due to sharp perforation mechanisms. We report on a 43-year-old man who was admitted to the emergency room because of an impressive transcervical penetrating neck trauma inflicted by a chisel. He survived the trauma since the chisel missed all important structures of the neck. The diagnostic strategy to evaluate the dimension of the trauma was primarily based on endoscopic and surgical exploration.

Effects of temperature on the mitochondrial malate dehydrogenase of adult muscle of Toxocara canis.

Purified mitochondrial malate dehydrogenase isoenzyme (m-MDH) of Toxocara canis muscle presented maximum activity at 48 degrees C. A clear change in slope of the Arrhenius plot was observed. The energy of activation calculated for the catalytic process showed values of 3.2 kcal/mol and 10.5 kcal/mol. Thermal inactivation of m-MDH showed that it is more thermolabile than the s-isoenzyme. The inactivation of the enzyme by heat could be reduced at least in part by the addition of 0.1 mM NADH. The heat denaturation showed to be a first-order process. The rate constant (k) was calculated as being of the order of 5.28 X 10(-4) s-1 at 40 degrees C. The activation energy for the heat inactivation process was 16.45 kcal/mol between 30 degrees C and 40 degrees C and 13.79 kcal/mol between 40 degrees C and 48 degrees C.

Inhibition of horse liver alcohol dehydrogenase and rabbit muscle lactate dehydrogenase by phenylhydrazine.

The activity of horse liver alcohol dehydrogenase was inhibited by phenylhydrazine. Kinetic experiments showed that this compound produced linear competitive inhibition with respect to NAD+ and linear noncompetitive inhibition with respect to ethanol. These results suggested that the inhibitor competes with NAD+ for the coenzyme binding site of alcohol dehydrogenase, forming a dead-end complex with the free form of the enzyme. A Ki value of 393 +/- 51 microM was estimated for the enzyme-inhibitor complex. Further evidence for this mechanism of inhibition arose from the fact that the same kind of inhibition was found for rabbit muscle lactate dehydrogenase. The Ki value for the lactate dehydrogenase-phenylhydrazine complex was 43.41 +/- 2.10 mM. The significant difference between these Ki values is explained in terms of known differences in hydrophobicity of the nicotinamide binding region in the two enzymes.