Modified inoculum for radiometric susceptibility testing ofMycobacterium tuberculosis

Two-hundred and fifteen isolates ofMycobacterium tuberculosis were evaluated with the BACTEC 460 radiometric method for susceptibility to isoniazid, rifampin, ethambutol, and streptomycin (SM); a revised protocol for inoculum preparation was used. Fresh clinical isolates were subcultured into 7H9 broth and then photometrically adjusted to the equivalent of a 0.5 McFarland standard, one-half the recommended inoculum density. This method produced an overall 98.3% correlation with a conventional agar method. The sensitivity of this procedure was good for all drugs tested except for the lowest concentration of SM (2 g/ml). Specificity was excellent for all drugs tested. After repeat testing, only four discrepancies were found, yielding a 99.8% correlation between the two systems. The time required for susceptibility tests averaged 4.6 days. This method for inoculum preparation effectively minimized the number of susceptibility tests exceeding the threshold value before the fourth day of incubation. This allowed for definite trends of the growth index values to become established before interpretation of results.     

DNA sequence analysis of spontaneous histidine mutations in a polA1 strain of Escherichia coli K12 suggests a specific role of the GTGG sequence

Summary Spontaneously arising histidine mutations in an Escherichia coli K12 strain deficient for DNA polymerase I were analysed at the DNA sequence level. We screened approximately 150000 colonies and isolated 106 histidine auxotrophs. Of these, 98 were unstable hisC mutations; 12 representative mutants analysed were shown to have arisen by the excision of a single quadruplet repeat in the sequence 5-GCTGGCTGGCTGGCTG-3. Of the eight mutations at other sites, three hisA deletions and one hisD deletion occurred as a consequence of misalignment of tandemly repeated pentamers (hisD) or decamers (hisA). A single hisA point mutation was found to be a missense mutation. Two extended deletions, covering the his operon were not analysed. We could not identify the hisC deletion by sequencing. We conclude that polA1 is a strong imitator that induces mutations mostly of the minus frameshift and deletion type by a Streisinger-type of mispairing in repetitive DNA sequences. Finally, the possible role of a 5-GTGG-3 sequence and its inverted or direct complements, which are found in the vicinity of all the deletions and frameshifts, is discussed.     

The nucleotide sequence of Euglena cytoplasmic phenylalanine transfer RNA. Evidence for possible classifications of Euglena among the animal rather than the plant kingdom

The nucleotide sequence of cytoplasmic phenylalanine tRNA from Euglena gracilis has been elucidated using procedures described previously for the corresponding chloroplastic tRNA [Cell, 9, 717 (1976)]. The sequence is: pG-C-C-G-A-C-U-U-A-m(2)G-C-U-Cm-A-G-D-D-G-G-G-A-G-A-G-C-m(2)2G-psi-psi-A-G-A-Cm -U-Gm-A-A-Y-A-psi-C-U-A-A-A-G-m(7)G-U-C-*C-C-U-G-G-T-psi-C-G-m(1)A-U-C-C-C-G-G- G-A-G-psi-C-G-G-C-A-C-C-A. Like other tRNA Phes thus far sequenced, this tRNA has a chain length of 76 nucleotides. The sequence of E. gracilis cytoplasmic tRNA Phe is quite different (27 nucleotides out of 76 different) from that of the corresponding chloroplastic tRNA but is surprisingly similar (72 out of 76 nucleotides identical) to that of tRNA Phe from mammalian cytoplasm. This extent of sequence homology even exceeds that found between E. gracilis and wheat germ cytoplasmic tRNA Phe. These findings raise interesting questions on the evolution of tRNAs and the taxonomy of Euglena.     

NEUROTOXICITY OF A NON-METABOLIZABLE AMINO ACID, 1-AMINOCYCLOPENTANE-1-CARBOXYLIC ACID: ANTAGONISM BY AMINO ACIDS IN CULTURES OF CEREBELLUM

Abstract— The non-metabolizable amino acid 1-aminocyclopentane-1-carboxylic acid (ACPC) induced degeneration of myelinated axons but spared nerve cell bodies in well myelinated organotypic cultures of cerebellum. The ACPC concentrations used were comparable to those which induce axonal degeneration in vivo. Developing unmyelinated cultures were more sensitive to ACPC than mature cultures and newly myelinating axons appeared to be particularly affected. Supplementing the medium with amino acids, but not with vitamins, prevented toxicity at the lower concentrations of ACPC and afforded considerable protection against the highest concentrations. The protective effect of amino acids could not be accounted for by inhibition of intracellular ACPC transport. These results are considered in terms of other evidence indicating defective protein metabolism in ACPC-treated mice.     

Rhizobacteria associated with Miscanthus x giganteus improve metal accumulation and plant growth in the flotation tailings

Plant and Soil - Flotation tailings represent an extremely unfriendly substrate for plant colonization due to toxic metal concentrations and marked macronutrient deficiencies. The perennial grass...     

Antimicrobial and antiviral activity-guided fractionation from Scutia buxifolia Reissek extracts

Antimicrobial and antiviral activities of the fractions from Scutia buxifolia stem bark and leaves were evaluated. Best antimicrobial results occurred with the ethyl acetate (EA) and n-butanolic (NB) fractions from the leaves against Micrococcus sp. (minimal inhibitory concentration—MIC = 62.5 μg/ml), and NB fraction from stem bark and leaves against Klebsiella pneumoniae and Enterococcus faecalis (MIC = 62.5 μg/ml). The most active fractions were selected and fractioned into silica column to perform an in vitro antibiofilm assay, which evidenced subfractions EA2 and EA3 as the more active against Candida albicans (biofilm inhibitory concentration—BIC = 582 ± 0.01 μg/ml) and Staphylococcus aureus (BIC = 360 ± 0.007 μg/ml), respectively. The NB (selectivity index—SI = 25.78) and the EA (SI = 15.97) fractions from the stem bark, and the EA (SI = 14.13) fraction from the leaves exhibited a potential antiviral activity towards Herpes Simplex Virus type 1 whereas EA2 and EA3 subfractions from leaves (SI = 12.59 and 10.06, respectively), and NB2 subfraction from stem bark (SI = 12.34) maintained this good activity. Phenolic acids and flavonoids (gallic acid, chlorogenic acid, caffeic acid, rutin, isoquercitrin, quercitrin and quercetin) were identified by HPLC and may be partially responsible for the antimicrobial and antiherpes activities observed. The results obtained in this study showed that Scutia buxifolia has antibiofilm and anti-herpetic activities and that these properties are reported for the first time for this species.     

The extent of whole‐genome copy number alterations predicts aggressive features in primary melanomas

Recent evidence indicates that melanoma comprises distinct types of tumors and suggests that specific morphological features may help predict its clinical behavior. Using a SNP‐array approach, we quantified chromosomal copy number alterations (CNA) across the whole genome in 41 primary melanomas and found a high degree of heterogeneity in their genomic asset. Association analysis correlating the number and relative length of CNA with clinical, morphological, and dermoscopic attributes of melanoma revealed that features of aggressiveness were strongly linked to the overall amount of genomic damage. Furthermore, we observed that melanoma progression and survival were mainly affected by a low number of large chromosome losses and a high number of small gains. We identified the alterations most frequently associated with aggressive melanoma, and by integrating our data with publicly available gene expression profiles, we identified five genes which expression was found to be necessary for melanoma cells proliferation. In conclusion, this work provides new evidence that the phenotypic heterogeneity of melanoma reflects a parallel genetic diversity and lays the basis to define novel strategies for a more precise prognostic stratification of patients.