Conversion of β-alanine in vivo and in isolated liver tissue

Summary -alanine was incubated with chopped liver tissue of rats in oxygen for 160 minutes at 37°C. It was established that a certain part of added -alanine (about 12%) disappears, causing an excessive production of urea and of a small quantity of -alanine.In parenteral administration of -alanine an increased urinary excretion of this amino acid is observed in B₆-deficient rats in which the process of -alanine transformation is disturbed.     

Effects of kinase inhibitors on TGF-beta induced upregulation of Kv1.3 K+ channels in brain macrophages

Deactivation of brain macrophages (microglia) by transforming growth factor- (TGF-) is characterized by enhanced Kv1.3 K⁺ channel expression. The intracellular mechanisms by which TGF- causes K⁺ channel upregulation in microglia have remained unclear. We show here that the protein kinase inhibitor H7 abolishes TGF--induced increases in delayed rectifier K⁺ current density. However, this effect cannot be related to inhibition of protein kinase C (PKC) or protein kinase A (PKA) activity, because specific PKC and PKA inhibitors did not exhibit effects identical to H7. TGF--induced Kv1.3 channel expression was also unaffected by inhibitors of tyrosine kinase, Ca²⁺/calmodulin kinase II and mitogen-activated protein (MAP) kinase ERK. In contrast, delayed rectifier K⁺ current density was larger in TGF--stimulated cells pretreated with the p38 MAP kinase inhibitor SB203580 or the phosphatidylinositol 3-OH (PI3) kinase inhibitor wortmannin, suggesting that both p38 MAP kinase and PI3 kinase regulate negatively the upregulation of Kv1.3 K⁺ channels in TGF--treated microglial cells.     

Phenotypic characteristics of lewis lung carcinoma cells

We studied adhesive properties and physiological activity in vivo of cells from Lewis lung carcinoma and its metastases. These cells differed in tumorogenic activity and metastatic potential in the syngeneic system. In vivo non-metastasizing cells are characterized by a lower content of surface lectins to tetrasaccharides SiaLe^x [Neu5Ac2-3Gal1-4(Fuc1-3) GlcNAc] and SiaLe^a [Neu5Ac2-3Gal1-3(Fuc1-4)GlcNAc] and trisaccharide HSO₃Le^x [HSO₃2-3Gal1-4(Fuc1-3)GlcNAc] compared to cells forming metastases in the syngeneic system. Metastatic cells with low tumorogenic activity weakly expressed lectins to disaccharide ligands 6-SiaLac [Neu5Ac2-6Gal1-4Glc], 6-HSO₃LacNAc, and A-di [GalNAc 1-3Gal] and trisaccharides H-type 1 [Fuc1-2Gal1-3GlcNAc and Le^x [Fuc1-3(Gal 1-4)GlcNAc] compared to cells that initiated tumor formation in the syngeneic system (similarly to transplanted tumors). We hypothesized that cell receptors to these carbohydrate determinates are involved in the development and growth of primary tumors, while lectins to SiaLe^x, SiaLe^a, and HSO₃Le^x play a role in the progress of tumor process and metastasizing.     

Expression of growth factor receptors in injured nervous tissue. I. Axotomy leads to a shift in the cellular distribution of specific beta-nerve growth factor binding in the injured and regenerating PNS

Summary We have studied -nerve growth factor (-NGF) receptor expression in the injured and regenerating chick PNS using [¹²⁵I]-iodinated -NGF as a radioactive probe to map and quantitate autoradiographically thein situ distribution of specific [¹²⁵I] -NGF binding.Two different mechanisms are involved in the reappearance of specific [¹²⁵I] -NGF binding on the normally unlabelled adult peripheral nerves. The anterograde and retrograde axonal transport of -NGF binding sites leads to a rapid but transient accumulation of [¹²⁵I] -NGF binding on both sides of crushed or transected sciatic and brachial nerves. There is a dramatic decrease in the axonal transport of -NGF binding sites, starting 1 day after, nerve injury (1 DPO) and reaching basal levels of 10–20% of the control values at 3 to 10 DPO. Gradual but complete recovery of this axonal transport was noted in the sciatic neurites allowed to regain contact with their peripheral targets. A very different regulation pattern was observed for the local reappearance of specific [¹²⁵I] -NGF binding on the endoneurial Schwann cells throughout the distal part of the axotomized nerve. It was first observed at 4 DPO, becoming maximal at 6 DPO. Reinnervation of the nerve after crush led to a rapid decrease of this specific [¹²⁵I] -NGF binding, which followed a proximo-distal temporal gradient.These results show that axotomy leads to a drastic decrease in the axonal expression of [¹²⁵I] -NGF binding, while causing its appearance on the Schwann cells of the denervated endoneurium. They suggest that these endoneurial cells may become the primary target for -NGF following axotomy and during regeneration.     

Emergence of resistance to beta-lactam agents inPseudomonas aeruginosa with group I beta-lactamases in Spain

The contribution of induction and stable derepression of chromosomal class I -lactamases to -lactam antibiotic resistance was studied in clinical isolates ofPseudomonas aeruginosa collected from patients treated with -lactam antibiotics. Multiple isolates from the same patient were characterized by O-serotyping as a primary screen, combined with pyocin typing. Sonicated extracts of cells were assayed for chromosomal and plasmid-mediated -lactamases by isoelectric focusing and cloxacillin inhibition studies. The specific -lactamase activity, basal and induced, with cefoxitin was determined to differentiate strains with inducible or derepressed production of the enzyme. Beta-lactamase induction was performed in each strain against the -lactam agents used in the therapy of each patient. The observations showed that induction against older penicillins such as penicillin, amoxicillin, and amoxicillin/clavulanate resulted in a moderate to strong increase in -lactamase activity, whereas the results obtained with first-generation cephalosporins varied with the -lactam agent tested. Third-generation cephalosporins were weak inducers of -lactamases, and their use as therapy preceded the appearance of strains that produce chromosomal group I -lactamases constitutively. These strains showed a remarkable reduction in sensitivity to ureidopenicillins, carboxipenicillins, third-generation cephalosporins, and monobactams, but not to carbapenems.     

The effects of β-2-microglobulin on the infectivity of murine cytomegalovirus

Summary The role of -2-microglobulin (2m) in murine cytomegalovirus (MCMV) infection of susceptible (H-2^d) and resistant (H-2^k) murine embryo fibroblasts (MEF) and peritoneal macrophages was evaluated using serum-free virus (SF-MCMV). The infectivity of SF-MCMV was significantly lower than virus propagated in serum, although the concentrations of virions were similar. Infection of cells with SF-MCMV was assessed by measuring the proportion of cells expressing viral antigens, the sizes of plaques formed in fibroblast monolayers and TCID₅₀ titers. Infection of susceptible fibroblasts was significantly increased 1.6–4.7 fold by the addition of whole FCS, a<20 kDa FCS fraction, or purified human 2m. These supplements also significantly enhanced infection of susceptible macrophages and increased TCID₅₀ titers by 3.5–10 fold in susceptible MEF. In relatively resistant H-2^k cells, the TCID₅₀ titer and the proportion of cells expressing viral antigens after infection with SF-MCMV were not affected by 2m or FCS, but plaque sizes were increased 2.5–3 fold in resistant BALB.K MEF.When human or murine 2m was added to infected cultures, immunogold electron microscopy revealed these proteins to be always associated extracellularly with the tegument material of disrupted multicapsid virions, but rarely with the envelope of intact virions. However, no murine 2m was found in association with the envelope or tegument of SF-MCMV. These relatively modest effects of 2m which were restricted to genetically susceptible cells, may be due to tegument-bound 2m facilitating infection by capsids, or the stabilisation of the conformation of Class 1 molecules by exogenous 2m, promoting MCMV binding to the target cell.     

Activation of atrial muscarinic K+ channels by low concentrations ofβγ subunits of rat brain G protein

Effects of G protein subunits from rat brain on cardiac K⁺ channel was examined in single atrial cells of guinea-pig, using patch clamp techniques. We found that 10 pM concentration of rat brain subunits preparation could activate the atrial muscarine receptor-gated K⁺ channel (I_K.ACh). Neither the detergent, CHAPS, used to suspend nor the boiled preparation activated I_K.ACh. Furthermore, preincubation of subunits preparation in Mg²⁺-free solution, which easily inactivated -GTP-S, did not affect -activation of I_K.ACh. We concluded, therefore, that subunits themselves can activate I_K.ACh.Supported by the grants from the Ministry of Education, Culture and Science of Japan and from the Calcium Signal Workshop on Cardiovascular Systems     

Characterization of cell-surface β-adrenergic ([3H]CGP-12177) binding in adult rat ventricular myocytes: lack of regulation by β-agonists at physiological concentrations

The major focus of this paper is the characterization and quantification of rat cardiomyocyte, cell-surface -adrenergic receptors labelled with the hydrophilic radioligand [³H]CGP-12177. The ventricular cardiomyocytes used in these experiments have previously been extensively studied in our laboratory and confirmed to be functionally compatible with similar cells in vivo. Specific binding of [³H]CGP was stereospecific, saturable and of high affinity. Binding of [³H]CGP was also readily reversible, demonstrated appropriate drug specificity and positively correlated with increasing cell concentrations. The potency of the ₁-antagonist atenolol was almost 100 times higher than that of the ₂-antagonist ICI-118.551 in binding to the [³H]CGP binding site. This preparation appears ideal for the investigation of -adrenergic receptor regulation in heart cells. Indeed, our initial experiments show clearly that pharmacological concentrations of isoproterenol, and norepinephrine, can reduce (down-regulate) the number of specific [³H]CGP binding sites. This result is in agreement with many other reports on similar experiments in a variety of cell types. However, physiologically relevant concentrations of these two agonists (1–100 nM) do not induce down-regulation of the -adrenergic receptors in short-term (2 h) incubations. Nevertheless, the high-affinity receptors that we have described mediate a contractile response to isoproterenol in the nanomolar concentration range (EC₅₀ =3.6±0.3 nM). This is approximately 300 times lower than the concentration needed to produce down-regulation. Thus, our data indicate that short-term down-regulation of cardiomyocyte -adrenergic receptors can only be observed with high, pharmacological concentrations of isoproterenol. In summary, this preparation of cardiomyocytes is wellsuited for the further investigation of (patho)physiological regulation of -adrenergic receptors.     

Apparent superiority of H2-receptor stimulation and simultaneousβ-blockade over conventional treatment withβ-sympathomimetic drugs in post-acute myocardial infarction: Cardiac effects of impromidine — a new specific H2-receptor agonist — in the surviving catecholamine-insensitive myocardium

Left ventricular infarction (AMI) was produced in experimental animals and the contractile response to -adrenergic and H₂-histaminergic stimulation by isoproterenol and impromidine tested in the isolated perfused heart preparation. Adenylate cyclase activity as well as binding characteristics of [³H]-dihydroalprenolol ([³H]-DHA), [³H]-methyl-tiotidine ([³H]-TIOT) and [³H]-quinuclidinyl benzilate ([³H]-QNB) to cardiac ₁-, H₂- and cholinergic muscarinic receptors were determined in sarcolemmal membrane preparations of the right ventricle of the same hearts. In addition, an attempt was made to elucidate the therapeutic value of post-AMI treatment with impromidine in the presence and absence of-sympathomimetic, in contrast to administration of prenalterol and the conventional therapy with -sympathomimetic drugs, e.g. dobutamine. Three days post-AMI the dose-response curve for isoproterenol of right ventriculardP/dt _max was significantly depressed, while the inotropic effect of impromidine was not impaired. Stimulation of adenylate cyclase activity by isoproterenol was reduced by 80% whereas impromidine and NaF stimulation rates were unaltered. Receptor-binding studies indicated a 90% loss and 10-times lowered affinity (K _D) of the remaining -receptors while specific [³H]-TIOT- and [³H]-QNB-binding was unchanged.Administration of dobutamine increased mortality rates and extension of infarct size, led to a further decrease in contractile response to isoproterenol, induced complete insensitivity of adenylate cyclase to isoproterenol stimulation and caused pronounced additional reduction of number and affinity of [³H]-DHA-binding sites. In contrast, all above alterations were prevented by treatment with either prenalterol or combined administration of impromidine plus metoprolol. It is concluded, that these alterations in the non-ischemic, uninvolved myocardium post-AMI are the result of catecholamine-induced specific damage of sarcolemmal -receptors. Furthermore, treatment with H₂-agonists in combination with -blocking agents may have beneficial effects, whereas conventional therapy with -sympathomimetic drugs tends to worsen the already depressed function of the -adrenergic stimulation mechanism.Supported by grants Ba 666/1 and Ba 666/2-2 from the Deutsche Forschungsgemeinschaft (DFG).Data presented in this paper are part of a doctoral thesis by Dr S.B. Felix.     

Modulation of Nav1.5 by β1- and β3-subunit co-expression in mammalian cells

Cardiac sodium channels (Na_v1.5) comprise a pore-forming -subunit and auxiliary -subunits that modulate channel function. In the heart, 1 is expressed throughout the atria and ventricles, whilst 3 is present only in the ventricles and Purkinje fibers. In view of this expression pattern, we determined the effects of 3 and 1 co-expression alone, and in combination, on Na_v1.5 stably expressed in Chinese hamster ovary cells. The current/voltage relationship was shifted –5 mV with either 1 or 3 co-expression alone and –10 mV with co-expression of both 1 and 3. In addition, 3 and 1/3 co-expression accelerated macroscopic current decay. There were significant hyperpolarizing shifts in equilibrium gating relationships with co-expression of 1 and 3 alone and in combination. Co-expression of 1/3 together resulted in a greater hyperpolarizing shift in channel availability, and an increase in the slopes of equilibrium gating relationships. Co-expression of 3 and 1/3, but not 1, slowed recovery from inactivation at –90 mV. Development of inactivation at –70 and –50 mV was accelerated by -subunit co-expression alone and in combination. -Subunit co-expression also reduced the late Na current measured at 200 ms. In conclusion, -subunits modulate Na_v1.5 gating with important differences between co-expression of 1 and 3 alone and 1/3 together.