Production of Solvents by Clostridium acetobutylicum Cultures Maintained at Neutral pH

The formation of acetone and n-butanol by Clostridium acetobutylicum NCIB 8052 (ATCC 824) was monitored in batch culture at 35°C in a glucose (2% [wt/vol]) minimal medium maintained throughout at either pH 5.0 or 7.0. At pH 5, good solvent production was obtained in the unsupplemented medium, although addition of acetate plus butyrate (10 mM each) caused solvent production to be initiated at a lower biomass concentration. At pH 7, although a purely acidogenic fermentation was maintained in the unsupplemented medium, low concentrations of acetone and n-butanol were produced when the glucose content of the medium was increased (to 4% [wt/vol]). Substantial solvent concentrations were, however, obtained at pH 7 in the 2% glucose medium supplemented with high concentrations of acetate plus butyrate (100 mM each, supplied as their potassium salts). Thus, C. acetobutylicum NCIB 8052, like C. beijerinckii VPI 13436, is able to produce solvents at neutral pH, although good yields are obtained only when adequately high concentrations of acetate and butyrate are supplied. Supplementation of the glucose minimal medium with propionate (20 mM) at pH 5 led to the production of some n-propanol as well as acetone and n-butanol; the final culture medium was virtually acid free. At pH 7, supplementation with propionate (150 mM) again led to the formation of n-propanol but also provoked production of some acetone and n-butanol, although in considerably smaller amounts than were obtained when the same basal medium had been fortified with acetate and butyrate at pH 7.     

Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.     

Enumeration and identification of nitrogen-fixing bacteria from forage grass roots

Root-soil cores were collected from forage grasses growing in a subtropical region of Texas and tested for acetylene reduction activity. The population density of nitrogen-fixing bacteria was measured, using various media and incubation conditions. Bacteria were confirmed as nitrogen fixing, using the acetylene reduction assay, and were classified according to standard biochemical and cultural methods. The majority of the nitrogen-fixing bacteria isolated from roots were Enterobacter cloacae or Klebsiella pneumoniae. Root-associated, nitrogen-fixing bacteria were isolated from 21 of 24 root-soil cores. The population densities of nitrogen-fixing bacteria ranged from approximately 10 to 3 x 10 per g of root. Population density on roots was significantly correlated with the rate of acetylene reduction but the relationship was not linear.     

Failure of X inactivation in the autosomal segment of an X/A translocation.

A newborn with an X/A translocation (46,X,der X,t(X;17)(17pter leads to 17p13::Xp22 leads to Xqter) demonstrated multiple anomalies. X-replication studies in leukocytes of the patient with RBG (R Bands by BrdU using Giemsa stain) showed the abnormal X,t(X;17), to be late replicating except for the translocated segment. Clinical findings and replication studies suggest failure of inactivation of the translocated segment.     

Note on the Interpretation of Lateral Diffusion Coefficients

It is suggested that interpretation of lateral diffusion coefficients measured in membranes should include the effect of forces.     

Adenosine triphosphate induces a low [Ca2+]i sensitivity of phosphorylation and an unusual form of receptor desensitization in smooth muscle.

The contractile sensitivity of smooth muscle to changes in myoplasmic [Ca2+] is dependent on the form of stimulation. Both myosin phosphorylation and force are less sensitive to increases in [Ca2+]i derived from Ca2+ entry through L-type Ca2+ channels than to increases in [Ca2+] induced by agents which release internal Ca2+ stores. We hypothesized that activation of receptor-operated channels should produce a [Ca2+]i sensitivity similar to that induced by opening L channels. Aequorin-estimated myoplasmic [Ca2+] and myosin light chain phosphorylation were measured in swine carotid media tissues stimulated with ATP, an activator of the only known receptor-operated cation channel in smooth muscle. ATP, via activation of a P2x purinergic receptor, induced large, transient increases in [Ca2+]i, yet only small transient elevations in phosphorylation or force. Rapid desensitization to ATP was partially, but not completely, caused by hydrolysis of ATP into adenosine since 1) alpha-beta-methylene ATP (a poorly hydrolyzable analog of ATP) produced larger, yet still transient increases in [Ca2+]i, phosphorylation, and force; 2) BW A1433U, a P1 (adenosine) receptor antagonist, enhanced ATP-induced contractions; and 3) ATP, but not alpha-beta-methylene ATP increased bath [adenosine]. The [Ca2+]i sensitivity of phosphorylation during P2x receptor activation was similar to that observed with KCl-depolarization-induced opening of L channels, supporting the hypothesis that transplasmalemmal Ca2+ influx produces less phosphorylation and force than mobilization of intracellular Ca2+ stores. Cumulative additions of higher alpha-beta-methylene ATP concentrations induced repeated transient contractions, indicative of an unusual form of receptor desensitization which could be explained if the affinity of the P2x receptor for ATP, but not the receptor number were rapidly reduced.     

Partial trisomies in two spontaneously arising long-lived human keratinocyte lines

Summary During experiments concerning the introduction of oncogenes into normal human keratinocytes, we observed long-lived colonies arising spontaneously at the same low frequency in control cultures as in those transfected with Ha-rasEJ or activated c-myc or both. Two of these were karyotyped early in their life span and showed additional chromosomal material on the short arm of chromosome 9 in one case and of chromosome 18 in the other, whereas the parental cells had a normal karyotype. This indicates the presence of a partial trisomy in each line, although the origin of the extra chromosomal material is not known. A similarly long-lived human keratinocyte line containing an isochromosome of the long arm of chromosome 8 has been described elsewhere. Together these results suggest that the spontaneous occurrence of long-lived lines is more common in human keratinocytes than in fibroblasts and that a triple dose of one or more genes may be the initial event in this process. This work was supported by grant CA16754 from the National Cancer Institute, Bethesda, MD.     

Relation between calculated amide frequencies and solution structure in Ala-X peptides

Computational techniques have been used to aid interpretation of observed systematic shifts in the amide III frequencies of Ala-X peptides. Optimized structures and frequencies have been calculated for Ala-X peptides using GAUSSIAN86/88 with the 4-31G basis, MOPAC, and normal mode methods based on empirical force fields. We observe the following: (1) Frequencies calculated using scaled GAUSSIAN86 force constants correlate well with the experimental results. (2) Structures of the Ala-X peptides optimized by GAUSSIAN show a clear trend toward lower values of the dihedral angle phi as the X side chain becomes larger, while structures optimized here using semiempirical and empirical force fields do not show trends. (3) Computational changes in peptide conformations from beta-sheet to alpha-helix produce large changes in both amide I and amide III frequencies that are inconsistent with the experimental results. (4) Computational changes in the dihedral angle phi of Ala-Ala produce a change in the amide III frequency consistent with the experimental results. (5) The experimental frequency shifts cannot be attributed directly to the effects of changing mass.     

Numerical classification of some Rhodococci, Corynebacteria and related organisms

Nineteen strains of Corynebacterium sensu stricto, 23 received as Corynebacterium equi or Rhodococcus equi, marker cultures of Arthrobacter, Brevibacterium, Bacterionema matruchotii, Cellulomonas flavigena, Kurthia zopfii, Listeria denitrificans, Microbacterium lacticum, Rhodococcus rubropertinctus and 88 representatives of Mycobacterium, Nocardia, Rhodococcus and the 'aurantiaca' taxon were the subject of numerical phenetic analyses using 92 characters. The data were examined using the simple matching (SSM) and Jaccard (SJ) coefficients and clustering was achieved using the average linkage algorithm. With a single exception, strains containing meso-diaminopimelic acid, arabinose, galactose and mycolic acids were recovered in five aggregate clusters corresponding to Corynebacterium sensu stricto, Mycobacterium, Nocardia, Rhodococcus and the 'aurantiaca' taxon. Most of the Corynebacterium (Rhodococcus) equi strains formed a good taxospecies which included the type strain of Corynebacterium hoagii. The numerical data, and the results of earlier chemical and genetical studies, also provide sufficient evidence for the transfer of Bacterionema matruchotii to Corynebacterium sensu stricto as Corynebacterium matruchotii comb.nov. and for the recognition of Rhodococcus globerulus sp.nov. for some strains previously classified as Rhodococcus rubropertinctus (Hefferan) Goodfellow & Alderson. The classification of the remaining marker strains correlates well with other major developments in coryneform taxonomy.     

Capping of Viral RNA in Cultured Spodoptera frugiperda Cells Infected with Autographa californica Nuclear Polyhedrosis Virus

Viral RNA from fall armyworm (Spodoptera frugiperda) cells infected with Autographa californica nuclear polyhedrosis virus contains cap structures. Most of the cap labeled in vivo with [³H]methionine or ³²P_i cochromatographed on DEAE-cellulose with the −5 charge marker; a minor component appeared at −4 net charge. The former is probably a cap 1 structure (m⁷GpppX^m_pYp), and the latter is probably a cap 0 (m⁷GpppXp). On the basis of relative labeling of the two caps with [³H]adenosine and [³H]guanosine, we concluded that each cap contained at least one adenosine residue in addition to guanosine and, therefore, that cap 0 contained m⁷GpppAp. Cleavage of [³H]methionine-labeled viral RNA with tobacco acid pyrophosphatase released a labeled component that cochromatographed on polyethyleneimine-cellulose with m⁷Gp, confirming the m⁷GpppX linkage in the cap. These results were also seen with host polyadenylated RNA. The caps appeared in nearly equal abundance in viral polyadenylated and non-polyadenylated RNAs. The ratio of ³²P_i incorporated into the cap to that incorporated into mononucleotides suggested average lengths for the polyadenylated and non-polyadenylated RNAs of 1,800 and 1,200 nucleotides, respectively.