Regulation of phospholamban and troponin-I phosphorylation in the intact rat cardiomyocytes by adrenergic and cholinergic stimuli: roles of cyclic nucleotides,calcium, protein kinases and phosphatases and depolarization

Protein phosphorylation was investigated in [³²P]-labeled cardiomyocytes isolated from adult rat heart ventricles. The -adrenergic stimulation (by isoproterenol, ISO) increased the phosphorylation of inhibitory subunit of troponin (TN-I), C-protein and phospholamban (PLN). Such stimulation was largely mediated by increased adenylyl cyclase (AC) activity, increased myoplasmic cyclic AMP and increased cyclic AMP dependent protein kinase (A-kinase)-catalyzed phosphorylation of these proteins in view of the following observations: (a) dibutyryl-and bromo-derivatives of cyclic AMP mimicked the stimulatory effect of ISO on protein phosphorylation while (b) Rp-cyclic AMP was found to attenuate ISO-dependent stimulation. Unexpectedly, 8-bromo cyclic GMP was found to markedly increase TN-I and PLN phosphorylation. Both ₁- and ₂-adrenoceptors were present and ISO binding to either receptor was found to stimulate myocyte AC. However, the stimulation of the ₂-AR only marginally increased while the stimulation of ₁-AR markedly increased PLN phosphorylation. Other stimuli that increase tissue cyclic AMP levels also increased PLN and TN-I phosphorylation and these included isobutylmethylxanthine (non-specific phosphodiesterase inhibitor), milrinone (inhibits cardiotonic inhibitable phosphodiesterase, sometimes called type III or IV) and forskolin (which directly stimulates adenylyl cyclase). Cholinergic agonists acting on cardiomyocyte M₂-muscarinic receptors that are coupled to AC via pertussis toxin(PT)-sensitive G proteins inhibited AC and attenuated ISO-dependent increases in PLN and TN-I phosphorylation. Thein vivo PT treatment, which ADP-ribosylated G_i-like protein(s) in the myocytes, markedly attenuated muscarinic inhibitory effect on PLN and TN-I phosphorylation on one hand and, increased the -adrenergic stimulation, on the other. Controlled exposure of isolated myocytes to N-ethyl maleimide, also led to the findings similar to those seen following the PT treatment. Exposure of myocytes to phorbol, 12-myristate, 13-acetate (PMA) increased the protein phosphorylation, augmenting the stimulation by ISO, and such augmentation was antagonized by propranolol suggesting modulation of the -adrenoceptor coupled AC pathway by PMA. Okadaic acid (OA) exposure of myocytes also increased protein phosphorylation with the results supporting the roles for type 1 and 2A protein phosphatases in the dephosphorylation of PLN and TN-I. Interestingly OA treatment attenuated the muscarinic inhibitory effect which was restored by subsequent brief exposure of myocytes to PMA. While the stimulation of alpha adrenoceptors exerted little effect on the phosphorylation of PLN and TN-I, inactivation of alpha adrenoceptors by chloroethylclonidine (CEC), augmented -adrenergically stimulated phosphorylation. KCl-dependent depolarization of myocytes was observed to potentiate ISO-dependent increase in phosphorylation (incubation period 15 sec to 1 min) as well as to accelerate the time-dependent decline in this phosphorylation seen upon longer incubation. Verapamil decreased ISO-stimulated protein phosphorylation in the depolarized myocytes. Depolarization was found to have little effect on the muscarinic inhibitory action on phosphorylation. Prior treatment of myocytes with PMA, was found to augment ISO-stimulated protein phosphorylation in the depolarized myocytes. Such augmented increases were completely blocked by propranolol. Forskolin also stimulated PLN and TN-I phosphorylation. Prior exposure of myocytes to forskolin followed by incubation in the depolarized and polarized media showed that PLN was dephosphorylated more rapidly in the depolarized myocytes. The results support the view that both cyclic AMP and calcium signals cooperatively increase the rates of phosphorylation of TN-I and PLN in the depolarized cardiomyocytes during -adrenergic stimulation. The results raise the additional possibility that the calcium signal may regulate the dephosphorylation of PLN in the depolarized cell. While muscarinic attenuation of -adrenergic action on protein phosphorylation was mediated, in part, by decreased AC activity, and muscarinic inhibition of AC and protein phosphorylation was not detectably influenced by the depolarization, the evidence was seen that muscarinic stimulation of dephosphorylation mechanisms are intimately involved. The postulate that the simultaneous stimulation of ₁-adrenoceptors inhibits -adrenergic stimulation of PLN and TN-I phosphorylation is supported.     

Resonance assignments, secondary structure and topology of leukaemia inhibitory factor in solution

The chemical shift assignments and secondary structure of a murine–human chimera,MH35, of leukaemia inhibitory factor (LIF), a 180-residue protein of molecular mass 20 kDa,have been determined from multidimensional heteronuclear NMR spectra acquired on auniformly 13C,15N-labelled sample. Secondary structure elements were defined on the basisof chemical shifts, NH-CH coupling constants, medium-range NOEs and the location ofslowly exchanging amide protons. The protein contains four -helices, the relativeorientations of which were determined on the basis of long-range, interhelical NOEs. The fourhelices are arranged in an up-up-down-down orientation, as found in other four-helical bundlecytokines. The overall topology of MH35-LIF is similar to that of the X-ray crystallographicstructure for murine LIF [Robinson et al. (1994) Cell, 77, 1101–1116]. Differencesbetween the X-ray structure and the solution structure are evident in the N-terminal tail, wherethe solution structure has a trans-Pro17 compared with the cis-Pro17 found in the crystalstructure and the small antiparallel -sheet encompassing residues in the N-terminus andCD loop in the crystal structure is less stable.     

Stimulative and inhibitory effects of bacteria on the growth of microalgae

Several examples of stimulative and inhibitoryeffects of bacteria on microalgal growth areintroduced, and the importance of bacteria in algalmass culture is investigated. Diatoms are often usedas live food for planktonic larvae of sea urchin andbivalves. Monodispersed Chaetoceros ceratosporum hasbeen cultivated by using clean, high nutrient content,deep seawater (DSW). However, the growth rate and cellyield of diatoms fluctuated, to relatively largeextent, with the season that DSW was collected. Whensome bacterial strains isolated from DSW were added tothe culture, diatom growth was often stimulated and arelatively constant cell yield was obtained. Anotherdiatom species, C. gracilis, was also stimulated byadding some bacterial strains to cultures. Thepositive effect of bacteria on diatoms was observednot only for planktonic species, but also on attachedspecies. A benthic diatom, Nitzschia sp., wasstimulated by a bacterial film of Alcaligenes on thesurface of the substratum. On the other hand, a strainof Flavobacterium sp. isolated from natural seawaterduring the decline period of an algal bloom had a strongalgicidal effect on the red tide plankton,Gymnodinium mikimotoi. Recent reports demonstratethat many bacterial strains have significantalgicidal effects on many species of red tideplankton. These results indicate that bacterialeffects should be taken into account to obtain stablemass culture of food microalgae.     

Roche Susceptibility Test (RST) medium,a defined formulation for susceptibility testing: I. Nutrient interaction and optimization studies

Roche Susceptibility Test (RST) Medium represents the most completely optimized and convenient fully defined medium described. It requires no post-autoclaving supplementation with vitamins, supports good growth of all common aerobic and anaerobic pathogens and may be used as a broth or agar gel on which the swarming of Proteus spp. is virtually eliminated. The broth as a superior buffering capacity to most complex media and an osmolality and pH close to those of human serum. RST is highly satisfactory for the susceptibility testing of commonly used antibiotics and meets the requirements of the National Committe for Clinical Laboratory Standards of the U.S.A. in almost every respect.     

Molecular cloning and physical mapping of the hyd gene of Escherichia coli K-12

Abstract Passive transfer between rates of protection against cholera toxin (CT) was studied. Extracts of various organs, obtained from CT-immunized rats, were injected intravenously into non-immunized recipient rats. The ability of the extracts to inhibit CT-induced secretion in ligated jejunal loop were tested. A significant inhibition of the response to CT was achieved by extracts from hypophysis, brain and jejunal mucosa. Extracts from pancreas, spleen or adrenal glands were without effect, as were all extracts obtained from control rats. The antisecretory effects of the hypophysis extracts became intensified with increasing numbers of immunizations, and the antisecretory effect was most pronounced when the extract was injected immediately before the CT challenge. The active component of the hypophysis extract was heat-labile and negatively charged, suggesting an acidic protein as the mediator of the protective effect against CT.     

The response of plasma gastric-inhibitory polypeptide (GIP) to slow and fast glucose ingestion in Billroth II resected patients and normal controls

Eight Billroth II resected patients and 8 normal controls were given two oral glucose loads, one ingested within 2 min, and the other ingested slowly over 80 min. In the Billroth II resected group, the integrated plasma GIP release was significantly higher after the fast than after the slow glucose ingestion. In this group the integrated plasma GIP release was also significantly higher than in the control group, but only after the fast glucose ingestion. These findings indicate that the rate of glucose delivery into the intestine may be of importance in the plasma GIP response to oral glucose.     

刺激家兔中脑导水管周围灰质抑制束旁核伤害性放电的脊髓上机制

本工作比较了家兔脊髓背侧1/2进行横切前后刺激中脑中央灰质对束旁核伤害性放电的抑制率的改变。实验表明中央灰质既可通过公认的脊髓下行纤维抑制脊髓水平的痛传递,减弱束旁核单位的痛敏放电,也可通过某种脊髓上机制实现对束旁核的抑制。我们对这两种机制的相对重要性作了分析。用直11个束旁核痛敏单位所作的计算表明,在脊髓切割前,刺激中央灰质可使束旁核痛敏放电抑制67.5%,脊髓背侧1/2横切后,同样的中脑刺激只能使痛敏放电抑制42.9%。如以切割前的抑制率作为直100%,则切割可使抑制减弱36.5%,这一部分抑制应当是由被切断的脊髓下行纤维实现的,其余63.5%则可能主要通过脊髓上机制来实现。考虑到脊髓背半部横切不可能全部切断下行纤维,故实际上脊髓上机制的重要性不会有上述数值表示的那么大。     

Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III

We have cloned in Escherichia coli and sequenced a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The resistance gene was located by analysis of the initiation and termination codons in an open reading frame (ORF) of 792 bp. The deduced gene product, a 3'5'-aminoglycoside phosphotransferase of type III, has an Mr of 29,200. Comparison of its amino acid sequence with those of type I (Oka et al., 1981) and type II (Beck et al., 1982) 3' phosphotransferase, from transposable elements Tn903 and Tn5, respectively, indicated a statistically significant structural relationship between these enzymes from phylogenetically remote bacterial genera. The degree of homology observed indicate that phosphotransferase type III and type I genes have diverged from a common ancestor and that the phosphotransferase type II gene has emerged more recently from the type I evolutionary pathway.     

Stimulatory and Inhibitory Effects of N-Methyl-D-Aspartate on 3H-Inositol Polyphosphate Accumulation in Rat Cortical Slices

The actions of the excitatory amino acid N-methyl-D-aspartate (NMDA) on the accumulation of 3H-inositol polyphosphate isomers in rat cerebral cortex slices have been examined over short (less than 5 min) incubation periods. NMDA caused the dose-dependent accumulation of only [3H]inositol monophosphate and [3H]inositol bisphosphate (maximal effect between 0.3 and 1 mM), with no increase in [3H]inositol trisphosphate ([3H]InsP3) and [3H]inositol tetrakisphosphate ([3H]InsP4). HPLC analysis confirmed this, showing no increases in the breakdown products of [3H]Ins(1,3,4,5)P4. When present with the muscarinic agonist carbachol (1 mM), high concentrations of NMDA (1 mM) could almost totally inhibit carbachol-induced accumulation of 3H-inositol polyphosphates. In contrast, at lower concentrations of NMDA (10 microM), the inhibitory effect was replaced with a synergistic accumulation of inositol polyphosphates, especially [3H]InsP4 and [3H]InsP3. The inhibitory effects of NMDA were only apparent when extracellular Ca2+ was present, although incubation in media with no added Ca2+ resulted in somewhat reduced stimulatory responses to NMDA alone, but suppressed totally the inhibitory effects of 1 mM NMDA and reduced the synergistic effects of 10 microM NMDA on carbachol responses. These studies, therefore, reveal Ca(2+)-dependent effects of NMDA indicative of indirect mechanisms of action and show that care must be made in interpreting the effects of NMDA on phosphoinositide metabolism unless the inositol polyphosphate composition has been fully characterised.