Paroxysmal neuronal depolarizations in the rat motor cortex in vivo: intracellular injection of the calcium agonist BAY K 8644

Summary Epileptic activity was elicited in the rat's motor cortex by local application of penicillin. At the neuronal level it consisted of typical paroxysmal depolarization shifts. The calcium agonist BAY K 8644 was injected into neurons showing such a discharge pattern. The application of this drug increased amplitude and afterdepolarization of paroxysmal neuronal depolarizations.     

Purification of bovine conglutinin using pepsin digestion

This paper describes a new method for the purification of bovine conglutinin based on the relative resistance of this protein to pepsin digestion. First, conglutinin is purified by absorption on yeast, then the preparation is treated with 2% pepsin (w/w) at 4°C for 18 hr, and finally gel filtrated on agarose A5m. The yield is 60–75% and conglutinin thus prepared appears physically, immunochemically and functionally intact. This procedure allows for a rapid production of sufficient amounts of conglutinin for immune complex detection or purification methods.     

Circadian variation of blood clotting time and circulating vitamin K in the athletic horse

In equine sport medicine, blood clotting and fibrinolysis variations are well investigated, given the practical implications of several pathophysiological conditions affecting the athlete horse such as exercise-induced pulmonary haemorrhage (EIPH) and other bleeding disorders whose etiology and pathogenesis mechanisms are not yet clearly understood. The purpose of the present investigation was to gain evidence of a daily rhythm of several blood clotting indices such as prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), plasma fibrinogen concentration and serum vitamin K concentration in the athletic horses. Blood samples from five thoroughbred mares were collected at 4-h intervals for 48 h (starting at 08:00 h on day 1 and finishing at 4:00 on day 2 via an intravenous catheter inserted into the jugular vein. Prothrombin time, activated partial thromboplastin time, thrombin time and plasma fibrinogen concentration were assessed by means of a Seac Clot 2 coagulometer (SEAC, Italy), while serum vitamin K concentration was measured by HPLC. Data analysis was conducted by one-way repeated analysis of variance (ANOVA) and by the single cosinor method. ANOVA showed a significant influence of time on all parameters investigated, in all horses, on either day. Cosinor analysis defined the periodic parameters and their acrophases (expressed in hours) during the 2 days of monitoring. PT showed a nocturnal acrophase, whereas serum vitamin K concentration acrophase occurred during the evening. The results of this study reflect the physiological peculiarities of the horse that is subjected to a number of exogenous (environmental, nutritional, physical) and endogenous stimuli capable of entraining the circadian rhythm specifically and thus producing time-dependent variations not always comparable with those observed in humans or laboratory animals.     

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.     

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.     

Different specificities of an HLA-DRw6 haplotype detected by alloreactive T lymphocytes

DRw6 has been difficult to define serologically. In the present experiments we have developed T cell lines in order to characterize the components of a DRw6 haplotype. This was accomplished by priming T cells with allogeneic mononuclear cells mismatched for DRw6, Dw6, and MT2. Subsequently, three sublines with distinct reactivity patterns were derived by limiting dilution. The specificities detected by these sublines included: (a) a specificity found on a subset of cells positive for DRw6 which was inhibited by monoclonal antibodies against DS(DC), the human homologue of the murine IA-encoded molecules, (b) another DRw6-associated specificity blocked by an MT2-like antibody, and (c) an MT2-like specificity blocked by monoclonal antibodies reactive with a different MT2-associated determinant. These results show that more than one IE-like, as well as the DS/DC (IA-like) molecules, carry distinctive antigenic epitopes that can be recognized by allogeneic T cells. Primed T cell lines may be useful for a better definition of certain haplotypes which are at present difficult to characterize with serological reagents alone.     

In vivo and in vitro actions of mammalian tachykinins

Summary Recently, two kassinin-like tachykinins have been isolated from mammalian nervous tissue. The potencies of these peptides, substance K (neurokinin ) and neuromedin K (neurokinin ) were compared with those of substance P, eledoisin, and kassinin in various pharmacological systems in vivo and in vitro.In contracting the isolated guinea-pig ileum and rabbit jejunum the potencies of eledoisin, kassinin, substance K, and neuromedin K were 13–80% that of substance P. In the rat vas deferens substance K and neuromedin K potentiated the electrically induced contractions with potencies similar to those of eledoisin and kassinin; they were 46–236 times as potent as substance P.In stimulating salivation in the rat after intravenous injection, eledoisin, kassinin, and substance K were respectively 2.3, 1.3 and 0.33 times as potent as substance P. In contrast, neuromedin K exhibited negligible activity. Each peptide tested led to a short fall in blood pressure after intravenous injection in the rabbit, substance P being 12–250 times as potent as the other peptides. Substance P was 20 times as potent as substance K or neuromedin K in inducing vasodilatation in the rat hind paw in vivo.Of the peptides tested, only substance P (10 nmol/min) significantly increased the release of histamine from the rat isolated hindquarter preparation.The results are discussed with respect to several theories of tachykinin receptor heterogeneity.The work was supported by the Austrian Scientific Research Funds, grant No. P5616, by the Austrian National Bank, grant No. 2216, and the Pain Research Commission of the Austrian Academy of Sciences