High resolution experimental and theoretical thermal denaturation studies on small overlapping restriction fragments containing the Escherichia coli lactose genetic control region

The distribution of thermal stability in the Escherichia coli lac control region is evaluated from the melting behavior of 5 short (80-219 base pairs (bp)) sequenced DNA restriction fragments containing various parts of this sequence. The thermal denaturation of these fragments was measured at 3 salt concentrations. The previous notion that the melting curves for small fragments are sharp and asymmetric in 0.01 M Na+ and broadened and less asymmetric at 0.105 and 0.505 M Na+ is confirmed and the possible explanations are discussed. The existence of two thermodynamic boundaries in this region is also confirmed. The exact location of the boundary upstream of the cyclic AMP receptor protein (CAP) binding site is accurately determined from melting experiments at 260 and 282 nm. The secondary boundary located between the promoter and operator sequence is apparent at the two higher salt concentrations and begins to disappear at the lower salt concentration. The physical interpretation of the melting experiments is compared to the results of theoretical predictions derived from the known sequence of the fragments.     

Fine Structure of Extracellular Polysaccharide of Erwinia amylovora

Virulent E₉ and avirulent E₈ strains of Erwinia amylovora were shown by means of light, transmission, and scanning microscopy to be, respectively, encapsulated and unencapsulated. Difficulty was encountered in stabilizing the fibrillar-appearing capsular extracellular polysaccharide. We suggest that the ephemeral nature of extracellular polysaccharide is due to the collapse of its extended structure upon dehydration. This occurs when bacteria are prepared for either transmission or scanning electron microscopy. The electron micrographs support our previous biochemical and immunological studies contending that the capsule is composed of tightly bound and loosely held components. The preparation of bacteria in freeze-dried colonies has permitted us to observe and explain the fluidity of the encapsulated strain. We suggest that this fluidity is a reflection of the loosely held extracellular polysaccharide or slime.     

The effect of ecdysterone on nonhistone proteins in salivary gland chromosomes of Sciara coprophila.

Synthesis and transport of proteins to the cell nucleus during puff induction was studied in S. coprophila. Changes in grain distribution along chromosomes (L-methionine [35S] incorporation into protein) were correlated with puffs induced by ecdysterone in vitro; A pattern of specific labelling at the sites of incipient puffs was noted within 2 h after the addition of the hormone, i.e. grains on the chromosomes were in clusters, characteristic for this time point and not seen in the controls (where only non-specific labeling was noted 0-4 h). Characteristic chromosomal puffs appeared between 3-4 h after the addition of ecdysterone. It was concluded that during ecdysterone-induced puff formation in salivary gland chromosomes, proteins which had been previously synthesized were selectively transported from the cytoplasm to specific sites on the chromosomes.     

Studies on the transport of vitamin D and of 25-hydroxyvitamin D in human plasma.

A study was conducted to investigate whether human plasma contains one or more than one protein for the transport of vitamin D and of 25-hydroxyvitamin D (25-OH-D).Serum was labeled in vivo with a mixture of radioactive vitamin D3 (derived from orally administered tracer vitamin D3) and of endogenously synthesized labeled 25-OH-D3. Samples of such serum were subjected to several different protein fractionation procedures. Only a single peak of protein-bound radioactivity was observed after each of these procedures. The fraction comprising the ascending and the descending limbs of the single peak of protein-bound radioactivity (after each procedure) were separately pooled. In each instance the ratio of radioactive 25-OH-D3 to radioactive vitamin D3 was found to be almost identical in both the ascending and the descending limbs. Taken together, these findings provide strong evidence that human serum contains only a single binding protein responsible for the normal transport of both vitamin D and 25-OH-D. Plasma labeled in vitro with added 3H-labeled 25-OH-D3 was subjected to gel filtration on Sephadex G-200 and to chromatography on columns of DEAE-cellulose and of SP-Sephadex. After each of these procedures a single peak of protein-bound radioactivity was observed, with elution profiles of protein and of radioactivity that were identical with those observed with in vivo labeled serum. These data indicate that tracer 25-OH-D3 added to plasma in vitro binds to the same plasma protein normally responsible for the transport of vitamin D and of 25-OH-D.     

Effect of particle size on the anthelmintic efficacy of mebendazole against Nippostrongylus brasiliensis in the rat.

The effect of reduction in particle size on the anthelmintic efficacy of mebendazole (methyl 5 (6)- benzoyle 1–2 benzimidazole carbamate) was studied in rats undergoing a primary infection with N. brasiliensis. A single oral treatment with fine ground mebendazole (particle size spectrum—54·95 per cent of particles less than 10·62 μ dia.; 86·06 per cent less than 21·27 μ) removed more than 98 per cent of adult worms from the intestine at a dose rate of 12·5 mg/kg body wt. On the other hand the best result achieved with coarse ground mebendazole (18·47 per cent of particles less than 10·62 μ dia; 42·26 per cent less than 21·27 μ dia) was 58 per cent of adult worms removed at a dose rate of 100 mg/kg body wt. It was also shown that fine ground mebendazole adversely affected migrating third stage larvae of N. brasiliensis.     

Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures.

Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules.Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10⁻² μm) is smaller than the random coil segment value b (7·17 × 10⁻² μm) was observed, while circular structures of the dimer of the same molecule (12 × 10⁻² μm) were detected.     

Cultivation of Mesophilic Soil Crenarchaeotes in Enrichment Cultures from Plant Roots

Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division Crenarchaeota over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two dominant phylotypes of Crenarchaeota that colonizes the roots of tomato plants grown in soil from a Wisconsin field is selectively enriched in mixed cultures amended with root extract. Clones recovered from enrichment cultures were found to group phylogenetically with sequences from clade C1b.A1. This work corroborates and extends our recent findings, indicating that the diversity of the crenarchaeal soil assemblage is influenced by the rhizosphere and that mesophilic soil crenarchaeotes are found associated with plant roots, and provides the first evidence for growth of nonthermophilic crenarchaeotes in culture.     

Differential loss of enzyme activity by vitC and iron containing proteins

Our earlier studies showed that rabbit muscle phosphoglucomutase was irreversibly inactivated by exposure to a mixture of vitamin C, FeCl3 and O2. The enzyme lost about 70% of its phosphate (V.V. Desphande and J.G. Joshi, J. Biol. Chem. 260, 754-764, 1985). The present report shows that several other iron proteins can substitute for FeCl3 to a varying degree. The rate of inactivation by FeCl3 greater than ferritin greater than hemoglobin = hemerythrin greater than transferrin = ferridoxin = vitamin C. These iron compounds also produced dephosphoenzyme but did not dephosphorylate ATP, ADP, AMP or phospholipids.     

Invertebrate Iridescent Virus 6, a DNA Virus,Stimulates a Mammalian Innate Immune Response through RIG-I-Like Receptors

Insects are not only major vectors of mammalian viruses, but are also host to insect-restricted viruses that can potentially be transmitted to mammals. While mammalian innate immune responses to arboviruses are well studied, less is known about how mammalian cells respond to viruses that are restricted to infect only invertebrates. Here we demonstrate that IIV-6, a DNA virus of the family Iridoviridae, is able to induce a type I interferon-dependent antiviral immune response in mammalian cells. Although IIV-6 is a DNA virus, we demonstrate that the immune response activated during IIV-6 infection is mediated by the RIG-I-like receptor (RLR) pathway, and not the canonical DNA sensing pathway via cGAS/STING. We further show that RNA polymerase III is required for maximal IFN-β secretion, suggesting that viral DNA is transcribed by this enzyme into an RNA species capable of activating the RLR pathway. Finally, we demonstrate that the RLR-driven mammalian innate immune response to IIV-6 is functionally capable of protecting cells from subsequent infection with the arboviruses Vesicular Stomatitis virus and Kunjin virus. These results represent a novel example of an invertebrate DNA virus activating a canonically RNA sensing pathway in the mammalian innate immune response, which reduces viral load of ensuing arboviral infection.