The direct effect of carbon monoxide on KCa channels in vascular smooth muscle cells

 The vasorelaxation induced by carbon monoxide (CO) has been demonstrated previously. Both a guanosine cyclic monophosphate (cGMP) signalling pathway and cGMP-independent mechanisms have been proposed to be responsible for the vascular action of CO. A direct effect of CO on the activity of calcium-activated K (K_Ca) channels in vascular smooth muscle cells (SMCs) and the underlying mechanisms were investigated in the present study. It was found that CO hyperpolarized single SMCs isolated from rat tail arteries. The whole-cell outward K⁺ channel currents in vascular SMCs, but not in neuroblastoma cells, were enhanced by CO. Extracellularly or intracellularly applied CO increased the open probability of single high-conductance K_Ca channels concentration-dependently without affecting the single channel conductance. Although it did not increase the resting level of intracellular free calcium concentration, CO significantly enhanced the calcium sensitivity of single K_Ca channels in SMCs. Furthermore, the effect of CO on K_Ca channels was not mediated by cGMP or guanine nucleotide-binding proteins (G proteins, G_i/G_o or G_s) in excised membrane patches. Our results suggest that the direct modulation of high-conductance K_Ca channels in vascular SMCs by CO may constitute a novel mechanism for the vascular effect of CO. Received: 9 January 1997 / Received after revision: 21 February 1997 / Accepted: 10 March 1997     

Temporal relationship between noradrenaline release in the central amygdala and plasma noradrenaline secretion in rats and tree shrews

In pentobarbital anesthetized Wistar rats and tree shrews (Tupaia belangeri) the hypothalamus, the hippocampus and the amygdala were simultaneously superfused through push-pull cannulae with artificial cerebrospinal fluid. Blood samples were withdrawn in order to make an attempt to correlate release rates of noradrenaline (NA), adrenaline and dopamine in the above-mentioned areas with plasma catecholamine levels. A strong, positive correlation was found between NA release in central amygdala and NA concentrations in peripheral blood suggesting a functional relationship between noradrenergic systems in discrete brain areas and the activity of the peripheral sympathetic nervous system.     

Electrical behaviour in a line of anterior pituitary cells (GH cells) and the influence of the hypothalamic peptide, thyrotrophin releasing factor

Anterior pituitary cells of the GH line, which secrete prolactin spontaneously, showed spontaneous action potential activity. Thyrotrophin releasing factor, which increases secretion in these cells, caused a prompt increase of action potential frequency. Potassium, another secretagogue, depolarized the cells and sometimes initiated a burst of action potentials at the onset of this effect. The action potentials persisted in tetrodotoxin-containing and Na-free media, but were suppressed by the Ca-channel blocker, methoxyverapamil. Moreover, elevating the extracellular Ca²⁺ concentration increased the amplitude of the action potentials. These action potentials therefore have a prominent Ca component. This endows them with a particular interest since secretory activity of these cells is known to be dependent on extracellular Ca²⁺. Ba²⁺, which can substitute for Ca²⁺ in maintaining secretion, also substituted for Ca²⁺ in the maintenance of the action potentials. In addition, Ba²⁺ prolonged action potentials remarkably: tetraethylammonium was less effective in this regard.The several parallels between known secretory behaviour and electrical phenomena encourage the view that analysis of electrical activity in anterior pituitary cells may provide useful clues to events involved in stimulus-secretion coupling and in the secretory control exerted by the brain.     

Effect of hindlimb unloading on resting intracellular calcium in intrafusal fibers and ramp-and-hold stretches evoked responsiveness of soleus muscle spindles in conscious rats

The present study was to investigate and evaluate the dynamic changes of calcium homeostasis of soleus muscle spindle for the exploration of the potential mechanisms of muscle spindle degeneration induced by hindlimb unloading. We systematically observed the changes in immunoreactivity of calbindin D28K (CaBP-D28K), intracellular resting calcium in intrafusal fibers of soleus muscle spindle, and the responsiveness of muscle spindles to ramp-and-hold stretches after short- and long-term (3, 7, 14 d) hindlimb unloading. The immunoreactivity of CaBP-D28K started to decrease after 7-d hindlimb unloading, while its decrease was obviously different compared with the control group 14 d following the hindlimb unloading. The resting calcium concentration was increased significantly at 3 d, and reached the peak level 14 d after the hindlimb unloading. The responsiveness of muscle spindles, assessed by investigating Index3-L₂, Index3-L₃, and Index5, to ramp-and-hold stretches started to decrease during the period of 7-d hindlimb unloading. All Indexs, in particular Index3-L₃ and Index5, were significantly decreased at 14 d after the hindlimb unloading. The data suggest the disturbance of calcium homeostasis in intrafusal fibers during the exposure to hindlimb unloading might gradually influence the structure and function of muscle spindles.     

A simple and reliable method to screen isolates of Escherichia coli and Klebsiella pneumoniae for the production of TEM- and SHV-derived extended-spectrum β-lactamases

Objective: To evaluate which of 24 β-lactams used in susceptibility tests best discriminated between strains of Klebsiella pneumoniae and Escherichia coli that produce extended spectrum β-lactamases (ESBLs) from strains that produce older, more familiar, plasmid-mediated β-lactamases such as TEM-1 and SHV-1.
Methods: Susceptibility to the 24 β-lactam agents was determined by agar dilution and disk diffusion methodologies, using 27 strains of K. pneumoniae and E. coli that produced 22 different older plasmid-mediated β-lactamases and 28 strains that produced 17 different ESBLs.
Results: In general, strains that produced ESBLs were intermediate or resistant to cefpodoxime, whereas those that produced other β-lactamases were susceptible to this agent. The agar dilution test exhibited 96% sensitivity and 100% specificity in discriminating these two groups of organisms. The disk diffusion test exhibited 100% sensitivity and 96% specificity. All other β-lactam agents tested were inferior discriminators between the two groups of organisms.
Conclusions: Agar dilution and disk diffusion tests with cefpodoxime can be used to discriminate strains of K. pneumoniae and E. coli that produce ESBLs from those that produce older, plasmid-mediated β-lactamases.     

Tranexamic acid: Preoperative prophylactic therapy for patients with hereditary angioneurotic edema

Short-term therapy of 96-hr duration with tranexamic acid was prophylactically effective as defined by the absence of attacks of angioedema in 14 patients with hereditary angioedema undergoing 10 dental and 4 general surgical procedures. Eight of the 14 patients had previously undergone dental extractions without prophylactic therapy with antifibrinolytic agents and each had experienced one or more attacks of angioedema. Seven of these 8 patients had a cumulative experience of 13 episodes of laryngeal edema after dental extractions and the eighth had a bout of cutaneous angioedema. Although the number of dental extractions conducted without prophylactic antifibrinolytic therapy cannot be accurately defined in retrospect, the prominence of laryngeal edema in this circumstance is striking when compared with the absence of attacks in the presence of prophylaxis with tranexamic acid. Methyltestosterone and impeded androgens are now known to be effective prophylaxis for spontaneous and, presumably, postoperative attacks when employed chronically because their administration is associated with correction of the biochemical defect of hereditary angioneurotic edema, but their chronic administration to children and women of childbearing age requires further definition because of their potential pituitary suppressive action. Tranexamic acid prophylaxis makes it possible to offer to untreated patients with hereditary angioneurotic edema dental work and other operative procedures that in the past were withheld or conducted with considerable risk.     

The use of detergents and immunoperoxidase reagents for the ultrastructural demonstration of internal immunoglobulin in lymph cells.

Lymph-borne immunoblasts were fixed in dilute glutaraldehyde and then treated with saponin. This treatment made most parts of the cells permeable to ferritin, so that anti-immunoglobulin (Ig) antibodies which had been conjugated to horse radish peroxidase (HRP) had no difficulty in gaining access to Ig which thus could be demonstrated at an ultrastructural level. Best results were obtained by fixing the cells in 0.1% glutaraldehyde for 7 min and then treating them with a 1% solution of saponin for 100 min at 55 degrees C before exposing them to the Ig-HRP conjugate. The method yielded reproducible results and although it causes a small amount of ultrastructural damage, it may be of value in detecting a variety of intracellular antigens.     

TUBB3 E410K syndrome: Case report and review of the clinical spectrum of TUBB3 mutations

The tubulinopathies refer to a wide range of brain malformations caused by mutations in one of the seven genes encoding different tubulin's isotypes. The β‐tubulin isotype III (TUBB3) gene has a primary function in nervous system development and axon generation and maintenance, due to its neuron‐specific expression pattern. A recurrent heterozygous mutation, c.1228G > A; p.E410K, in TUBB3 gene is responsible of a rare disorder clinically characterized by congenital fibrosis of the extraocular muscle type 3 (CFEOM3), intellectual disability and a wide range of neurological and endocrine abnormalities. Other mutations have been described spanning the entire gene and genotype–phenotype correlations have been proposed. We report on a 3‐year‐old boy in whom clinical exome sequencing allowed to identify a de novo TUBB3 E410K mutation as the molecular cause underlying a complex phenotype characterized by a severe bilateral palpebral ptosis refractory to eye surgery, psychomotor delay, absent speech, hypogonadism, celiac disease, and cyclic vomiting. Brain MRI revealed thinning of the corpus callosum with no evidence of malformation cortical dysplasia. We reviewed available records of patients with TUBB3 E410K mutation and compared their phenotype with the clinical outcome of patients with other mutations in TUBB3 gene. The present study confirms that TUBB3 E410K results in a clinically recognizable phenotype, unassociated to the distinct cortical dysplasia caused by other mutations in the same gene. Early molecular characterization of TUBB3 E410K syndrome is critical for targeted genetic counseling and prompt prospective care in term of neurological, ophthalmological, endocrine, and gastrointestinal follow‐up.     

Sequence of centromere separation: Occurrence,possible significance,and control

This review describes the existence of a phenomenon, sequential separation of centromeres, in mitotic cells of various species including both animals and plants. Critical observations at metaanaphase show that the centromeres of chromosomes in a given genome do not separate into two sister units randomly, but that there is a genetically controlled, nonrandom, species-specific sequence which is independent of the length of the chromosome or the position of the centromere. A stricter control appears to exist for late-separating than for early-separating chromosomes. At early stages of metaanaphase several chromosomes initiate onset of separation simultaneously or in rapid succession, but late-separating chromosomes are better defined in their sequential position. The effect of Colcemid on the sequence of separation is minimal. It is proposed that aneuploidy in humans and other organisms may result from out-of-phase separation of a given chromosome. With the exception of chromosome No. 16, it appears that very early- or very late-separating centromeres are involved in human trisomies more often than those in between.Perhaps one function of centromeric heterochromatin is the control of centromere separation. The amount of such chromatin shows a positive correlation with the timing of separation of the centromeres. Superimposed upon this quantitative influence is the qualitative aspect, as discussed for various genomes. This suggestion explains a lack of extremely large quantities of heterochromatin near the centromere. Its existence in the form of homogeneously staining regions distal to the centromere, as in some cancer cells or in sex chromosomes, seemingly has no influence on the separation of centromeres.A brief discussion of centromere separation errors in human disease is provided, and suggestions for further studies are made.     

Neuropeptide Y regulates rat renal tubular Na,K-ATPase through several signalling pathways

Neuropeptide Y (NPY) has at least three receptors (Y₁, Y₂ and Y₃) through which it influences different mechanisms in many cell types. Previous data suggest that the Y₂ receptor may be divided into prejunctional and postjunctional subgroups. We have examined the intracellular signalling pathways of the postjunctional Y₂ receptor in rat renal proximal tubules. The results indicate that NPY regulates Na⁺,K⁺-ATPase through several signalling pathways: (1) In proximal tubule (PT) cells NPY increased intracellular calcium. The response was blocked by removing extracellular calcium and was also blocked by using nifedipine. This suggests that calcium was increased by influx from the extracellular space through L -type calcium channels. (2) NPY increased Na⁺,K⁺-ATPase activity in PT segments and this effect was also blocked by nifedipine. CaMKII-Ala²⁸⁶[281–302] a blocker of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibited the NPY-stimulated Na⁺,K⁺-ATPase activity. This implies that increased intracellular calcium activates CaMKII which subsequently increases Na⁺,K⁺-ATPase activity. CaMKII thus appear to act similar to what has been proposed for protein phosphatase 2B. (3) Calphostin C, an inhibitor of protein kinase C (PKC), did not inhibit NPY-stimulated Na⁺,K⁺-ATPase activity. PKC is, therefore, unlikely to be involved. (4) Y₂ receptors are negatively coupled to the cAMP pathway. NPY attenuated forskolin-stimulated cAMP production in renal tubules and exogenous cAMP counteracted the NPY-stimulated Na⁺,K⁺-ATPase activity. This illustrated the importance of NPY for the regulation of renal sodium handling. We also propose that the renal tubule cell is a good model for studying the function and mechanisms of postjunctional Y₂ receptors.