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.     

Role of actin microfilament in osmotic stretch-induced increase of voltage-operated calcium channel current in guinea-pig gastric myocytes

 Using the whole-cell patch clamp technique, the role of actin microfilament in hyposmotic increase of voltage-operated calcium channel current (I _Ba) was studied in guinea-pig gastric myocytes. Hyposmotic superfusate (212 mOsm) increased peak I _Ba amplitude by 32.7 ± 6.5%; when cytochalasin-D (Cyt-D, 20 μM), an actin cytoskeleton disruptor, was used, an increase of only 9.7 ± 3.1% was seen. I _Baresponse to osmotic stress was potentiated (45.1 ± 4.1% increase) by 20 μM phalloidin, an actin microfilament stabilizer. However, colchicine (100 μM), an microtubule cytoskeleton disruptor, had no effect on either I _Ba or its response to hyposmotic solution. Phalloidin also induced a rightward shift of the I/V relationship of I _Ba, while Cyt-D itself had no effect. These results suggest that actin cytoskeleton may mediate hyposmotic stretch-induced I _Ba increase in gastric smooth muscle. Received: 26 March 1997 / Received after revision: 28 May 1997 / Accepted: 3 June 1997     

Isolation and radiation hybrid mapping of a dinucleotide repeat polymorphism at the human calcium-sensing receptor (CASR) locus

Calcium-sensing receptor (CASR) in parathyroid gland regulates calcium homeostasis by sensing decreases in extracellular calcium levels and effecting an increase in secretion of parathyroid hormone. A polymorphic dinucleotide (CA) sequence was isolated from a genomic clone containing the human CASR gene and was mapped to 3q13.3–q21. This polymorphism will be useful in the genetic study of disorders affecting calcium metabolism, such as hypercalcemia, hypocalcemia, osteoporosis, hyperparathyroidism, and hypoparathyroidism. Received: June 2, 1998 / Accepted: June 24, 1998     

Functional AMPA/kainate receptors in human embryonic and foetal central nervous system

Here, functional AMPA/kainate receptors in human embryonic (5.5–7.5 gestational weeks) and foetal (8–10 gestational weeks) central nervous system tissue, shown by the cobalt labeling method, are reported. Specific agonist-induced cobalt incorporation was detected in brainstem and spinal cord cells, even in the youngest embryo studied. T-AMPA or kainate, but also vegetal toxins such as L-BOAA or acromelate, induced accumulation of cobalt. In contrast, no labeling was observed after exposure to KCl or NMDA. Cobalt labeled cells were particularly prominent in motor regions of brainstem and spinal cord. Co-application of the diuretic agent cyclothiazide, a desensitization blocker at AMPA receptors, dramatically increased the number of stained cells, which was particularly obvious in sensory regions, suggesting different receptor properties in motor versus sensory regions. This is the first study providing evidence for functional AMPA/kainate receptors, permeable to divalent cations, in brainstem and spinal cord at an early stage of human central nervous system development. Since many developmental processes are influenced by the modulation of cytosolic calcium, exposure at critical stages of embryogenesis to food or drug substances modifying the activity of AMPA/kainate receptors may alter brain development.     

Slow frequency-dependence of action potential afterhyperpolarization in bullfrog sympathetic ganglion neurones

The afterhyperpolarization (AHP) which follows the action potential (AP) in bullfrog sympathetic ganglion B-cells involves activation of Ca²⁺-sensitive K⁺ conductances following Ca²⁺ influx via Ca²⁺ channels. The duration of AHPs evoked at 2-s stimulus intervals were 70.05±3.76% of those evoked at 90-s stimulus intervals (n=35). Since there was no consistent effect of ryanodine (5 M), ruthenium red, (300 M) or dantrolene Na (35 M) on this frequency dependence, it is unlikely to result from release of Ca²⁺ from intracellular stores. Ca²⁺ currents (I _Ca, studied by means of the whole-cell patch-clamp technique, exhibited a slow frequency dependence as a result of a slow inactivation process which was independent of Ca²⁺-induced I _Ca inactivation and I _Ca run-down. There was excellent correlation (r=0.964) between the estimated changes in Ca²⁺ influx and the expected activation of the Ca²⁺-sensitive K⁺ current, I _AHP. This result is consistent with the hypothesis that the frequency dependence of the AHP is a consequence of the slow inactivation of I _Ca.     

Identification of parasite proteins in a membrane preparation enriched for the surface membrane of erythrocytes infected with Plasmodium knowlesi

A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.     

Suppression of charge movement by calcium antagonists is not related to calcium channel block

The calcium channel-inhibiting drugs nitrendipine and diltiazem represent two important classes of organic calcium antagonists. In the present study, the effect of these drugs on calcium currents and charge displacement currents in bullfrog semitendinosus muscle fibers was examined using a vaseline gap voltage clamp. Nitrendipine (10 M) reduced the quantity of charge that moved both during the ON phase (Q_ON) and the OFF phase (Q_OFF) of charge movement. This action appeared to be most selective for Q_ON. However, at this same concentration, nitrendipine had no blocking action on inward calcium currents. In contrast to these findings, diltiazem blocked calcium currents in a concentration-dependent manner, while slightly increasing the quantity of charge moved during Q_ON and Q_OFF. The enhancement of charge movement by diltiazem resulted from two actions. First, diltiazem shifted the voltage-dependence of charge movement to more negative potentials. Second, diltiazem increased the maximum amount of charge moved. (Supported by NIH NS 03178 and HL 07382.)     

Homo- and heteromeric assembly of TRP channel subunits

Mammalian homologues of the Drosophila melanogaster transient receptor potential (TRP) channels are the second largest cation channel family within the superfamily of hexahelical cation channels. Most mammalian TRP channels function as homooligomers and mediate mono- or divalent cation entry upon activation by a variety of stimuli. Because native TRP channels may be multimeric proteins of possibly complex composition, it is difficult to compare cation conductances in native tissues to those of clearly defined homomeric TRP channel complexes in living cells. Therefore, the possibility of heteromeric TRP channel assembly has been investigated in recent years by several groups. As a major conclusion of these studies, most heteromeric TRP channel complexes appear to consist of subunit combinations only within relatively narrow confines of phylogenetic subfamilies. Although the general capability of heteromer formation between closely related TRP channel subunits is now clearly established, we are only beginning to understand whether these heteromeric complexes are of physiological significance. This review summarizes the current knowledge on the promiscuity and specificity of the assembly of channel complexes composed of TRPC-, TRPV- and TRPM-subunits of mammalian TRP channels.     

Monoclonal antibodies to three distinct epitopes on human IgE: their use for determination of allergen-specific IgE

Three different monoclonal antibodies (MAb) against human immunoglobulin E have been obtained which specifically bind to human myeloma and polyclonal IgE. The antibodies showed high avidities for soluble IgE (0.7 X 10(9) to 3.3 X 10(9) M-1). These MAb defined three distinct epitopes on IgE. A mixture of these antibodies in combination with an 125I-labelled anti-mouse Kappa chain MAb has been used to measure allergen-specific IgE. This determination was performed by a solid-phase radioimmunoassay using allergen extracts coated to either chemically activated paper discs or to polyvinyl chloride wells. This method is 4-10 times more sensitive than other previously reported procedures. A similar technique has also been applied to detect individual allergens in immunoblots of allergen extracts.     

Associated changes in Ca2+ and Sr2+ activation properties and fiber proteins in cross-reinnervated rabbit soleus

Changes in the Ca²⁺ and Sr²⁺ activation properties of functionally skinned slow-twitch soleus fibers were measured and compared with those of normal fast-twitch extensor digitorum longus (EDL) following cross-reinnervation of soleus with the nerve to EDL. Most of the fibers showed either complete transformation of activation properties (66%) or remained unchanged (34%). The change in sensitivity to divalent cations was correlated with changes in the proteins present in fibers pooled on the basis of their activation properties. The banding patterns of the 35,000- and 37,500-dalton proteins (tropomyosin and troponin T) in cross-reinnervated soleus were correspondingly transformed to those of normal EDL. Slow and fast myosin light chains were present in the pooled cross-reinnervated fibers. Fiber distributions based on activation properties were confirmed by histochemical features. For the first time it has been demonstrated that cross-reinnervation produced changes in the activation properties of soleus fibers and associated changes in the regulatory proteins measured.