In vitro processing activity of Bacillus subtilis polynucleotide phosphorylase

A phosphate-dependent exonuclease activity was identified in purified protein fractions from Bacillus subtilis that were selected for binding to poly(I)-poly(C) agarose. Based on the characteristics of the degradation products and the absence of this activity in a pnpA strain, which contains a transposon insertion in the B. subtilis PNPase gene (Luttinger et al ., 1996 — accompanying paper), this exonuclease activity was shown to be due to polynucleotide phosphorylase (PNPase). Processive 3'-to-5' exonucleolytic degradation of an SP82 phage RNA substrate was stalled at a particular site. Structure probing of the RNA showed that the stall site was downstream of a particular stem-loop structure. A similar stall site was observed for an RNA that comprised the intergenic region between the B. subtilis rpsO and pnpA genes. The ability to initiate degradation of a substrate that had a stem structure at its 3' end differed for the B. subtilis and Escherichia coli PNPase enzymes.     

Codon-acticodon recognition in the valine codon family.

An in vitro protein-synthesizing system completely dependent on added valine tRNA (valyl-tRNAval) and programmed with RNA from the phage MS2 has been used to investigate the incorporation into MS2 coat protein of valine from isoaccepting valyl-tRNAsval with the anticodons U AC (U represents 5-oxyacetic acid uridine monophosphate), GAC, and IAC in response to the four valine codons GUU, GUC, GUA, and GUG. By examining the incorporation of valine into NH2-terminal and internal positions of three tryptic peptides from the MS2 coat protein it has been established that these anticodons each recognize all four valine codons. We therefore conclude that under our conditions of in vitro protein synthesis the genetic code, as far as the valine codons are concerned, is operationally a two letter code, i.e. the third codon nucleotide has no absolute discriminating function.     

The role of DNA polymerase I-associated 5'-exonuclease in replication of coliphage M13 replicative-form DNA.

The conversion of both parental- and progeny-nascent open circular M13 RF DNA into covalently closed RF I is drastically reduced in an $\text{E. coli}$ mutant deficient in the 5′ → 3′ exonuclease associated with DNA polymerase I. The nascent progeny RF DNA also contains a significant proportion of fragments of smaller than unit length.     

An isoflavone glycoside from the heartwood of Pterocarpus marsupium

A new isoflavone, 5,4′-dimethoxy-8-methylisoflavone, has been identified from the heartwood of Pterocarpus marsupium.     

Respiratory responses in reversible diaphragm paralysis

The effects of diaphragm paralysis on respiratory activity were assessed in 13 anesthetized, spontaneously breathing dogs studied in the supine position. Transient diaphragmatic paralysis was induced by bilateral phrenic nerve cooling. Respiratory activity was assessed from measurements of ventilation and from the moving time averages of electrical activity recorded from the intercostal muscles and the central end of the fifth cervical root of the phrenic nerve. The degree of diaphragm paralysis was evaluated from changes in transdiaphragmatic pressure and reflected in rib cage and abdominal displacements. Animals were studied both before and after vagotomy breathing O2, 3.5% CO2 in O2, or 7% CO2 in O2. In dogs with intact vagi, both peak and rate of rise of phrenic and inspiratory intercostal electrical activity increased progressively as transdiaphragmatic pressure fell. Tidal volume decreased and breathing frequency increased as a result of a shortening in expiratory time. Inspiratory time and ventilation were unchanged by diaphragm paralysis. These findings were the same whether O2 or CO2 in O2 was breathed. After vagotomy, no significant change in phrenic or inspiratory intercostal activity occurred with diaphragm paralysis in spite of increased arterial CO2 partial pressure. Ventilation and tidal volume decreased significantly, and respiratory timing was unchanged. These results suggest that mechanisms mediated by the vagus nerves account for the compensatory increase in respiratory electrical activity during transient diaphragm paralysis. That inspiratory time is unchanged by diaphragm paralysis whereas the rate or rise of phrenic nerve activity increases suggest that reflexes other than the Hering-Breuer reflex contribute to the increased respiratory response.     

Analysis of a case of somatic instability in a strain of maize from India

A case of somatic instability affecting aleurone colour in a strain of maize from India with flint background was analysed. The somatic instability is localized to theC ¹ (Inhibitor) allele ofC locus on the short arm of chromosome 9. Molecular tests indicated thatAc is not present in the Indian stock and the evidence is consistent with the involvement of theEn (Spm) transposable element in the instability. The presence of theEn (Spm)-like element in the stock would suggest that these elements have been present in the maize genome for a long time. A new allele ofshrunken (sh1) gene with a somewhat unorthodox breeding behaviour is also described.     

Effects of muscarinic receptor agonists and antagonists on response of non-extensor rats to maximal electroshock

Rats which do not respond consistently to maximal electroshock by exhibiting the classical hindlimb extensor response, are designated as 'flexors', and can serve as a useful experimental model for investigating seizure mechanisms. 20-25% Charles Foster rats exhibit the flexor status and were used in this study. The flexor rats were converted to extensors by acetylcholine (icv), physostigmine (ip) and the selective muscarinic M1 receptor agonists, arecholine (ip) and McN-A-343 (icv). This conversion of flexors to extensors was significantly attenuated by M1 receptor antagonists scopolamine (ip) and pirenzepine (icv). The M2 receptor agonist, carbachol (icv), had no effect in lower doses but induced conversion of flexor rats to the extensor status only in very high doses which may be due to loss of receptor specificity on dose increment. The M2 receptor antagonists, gallamine (icv) and AF-DX 116 (ip), also induced significant conversion of flexors to extensors, which was dependent upon the availability of neuronal acetylcholine since the effects were attenuated following pretreatment with hemicholinium, an inhibitor of acetylcholine synthesis. The results suggest that the central cholinergic system has a facilitatory pro-convulsant effect, mediated through the muscarinic M1 receptors, an action modulated by the M2 receptors.     

Photoproduction of hydrogen from sewage by immobilized cells of Chromatium species IA.

Immobilized cells of two Chromatium species produced hydrogen continuously for more than 160 hr in 60% and 80% sewage. One strain showing high optimum range of sulfide tolerance (up to 9 mM) produced more hydrogen in 80% sewage while the less sulfide tolerating strain (up to 6 mM) showed hydrogen photoproduction in 60% sewage. Cells were immobilized in alginate and stable hydrogen photoproduction was observed for more than one week. Appropriate strategy necessary for the treatment of sewage and similar industrial effluents for energy reclamation is discussed.     

Wild-type and mutant bacterioopsins D85N, D96N, and R82Q: high-level expression in Escherichia coli

The integral membrane protein bacterioopsin, found in the extremely halophilic archaebacterium Halobacterium halobium, was expressed in Escherichia coli as a fusion protein containing 13 heterologous amino acids at the amino terminus. The expressed protein was localized primarily to the E. coli cytoplasmic membrane (greater than 80%) and had an in vivo half-life of 26 min. The amount of bacterioopsin in E. coli crude lysates was quantitated immunologically from Western blots and was expressed at 10-20-fold higher levels than seen previously (i.e., 17 mg/L; 5.6% of the total protein). Three distinct forms of the protein were detected immunologically: two of the forms were generated by the removal of either one or four amino acid residues at the amino terminus; the third form remained unaltered.     

Horseradish peroxidase catalyzed oxidation of thiocyanate by hydrogen peroxide: comparison with lactoperoxidase-catalysed oxidation and role of distal histidine

Horseradish peroxidase-catalysed oxidation of thiocyanate by hydrogen peroxide has been studied by 15N-NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pH values. The extent of the oxidation and the identity of the oxidized product of the thiocyanate has been investigated in the SCN-/H2O2/HRP system and compared with the corresponding data on the SCN-/H2O2/LPO system. The NMR studies show that (SCN)2 is the oxidation product of thiocyanate in the SCN-/H2O2/HRP system, and its formation is maximum at pH less than or equal to 4 and that the oxidation does not take place at pH greater than or equal to 6. Since thiocyanate does not bind to HRP at pH greater than or equal to 6 (Modi et al. (1989) J. Biol. Chem. 264, 19677-19684), the binding of thiocyanate to HRP is considered to be a prerequisite for the oxidation of thiocyanate. It is further observed that at [H2O2]/[SCN-] = 4, (SCN)2 decomposes very slowly back to thiocyanate. The oxidation product of thiocyanate in the SCN-/H2O2/LPO system has been shown to be HOSCN/OSCN- which shows maximum inhibition of uptake by Streptococcus cremoris 972 bacteria when hydrogen peroxide and thiocyanate are present in equimolar amounts (Modi et al. (1991) Biochemistry 30, 118-124). However, in case of HRP no inhibition of oxygen uptake by this bacteria was observed. Since thiocyanate binds to LPO at the distal histidine while to HRP near 1- and 8-CH3 heme groups, the role of distal histidine in the activity of SCN-/H2O2/(LPO, HRP) systems is indicated.