Subunit interactions in myosin subfragment 1. Subunit scrambling is independent of catalytic center involvement

Evidence is presented that, under conditions of 4.7 M NH4Cl and 10 mM Mg-ATP where no subunit dissociation can be detected by transport methods, a dynamic equilibrium exists in subfragment 1 between the associated and dissociated subunits. This is readily discerned by the formation of hybrid subfragment 1 species when a subfragment 1 isozyme is incubated with excess free light chains of the alternate isozyme. A similar process occurs with p-N,N'-phenylenedimaleimide (pPDM)-modified subfragment 1 containing [14C]Mg-ADP, but in this case, although extensive amounts of hybrid are formed, no loss of the trapped nucleotide is observed. Subunit scrambling without loss of the trapped nucleotide is apparent from incubating pPDM-SF1(A2)-[14C]Mg-ADP with unmodified SF1(A1) under similar conditions since the mixture subsequently contains SF1(A1), SF1(A2)h, pPDM-SF1(A1)h-[14C]Mg-ADP and pPDM-SF1(A2)-[14C]Mg-ADP. These data show that the nucleotide trapped in the presumptive active site does not escape during the dissociation-reassociation cycle, and suggest that the ATPase site resides solely on the heavy chain.     

Drosophila X virus RNA polymerase: tentative model for in vitro replication of the double-stranded virion RNA.

An RNA polymerase activity was found to be associated with the infectious drosophila X virus particles extracted from infected flies. The rate of synthesis was at first linear as a function of time, and then a plateau was reached. During the linear phase of the synthesis, the template and product were associated as replicative intermediates which were larger than the double-stranded RNA of the drosophila X virus genome, but the final product of the reaction was indistinguishable from the RNA genome with respect to its density, sedimentation coefficient, electrophoretic mobility, and RNase resistance. The results indicated that both strands of the genomic RNA were copied. The implications of these findings with regard to other virion polymerases are discussed.     

Plaquing procedure for infectious hematopoietic necrosis virus.

A single overlay plaque assay was designed and evaluated for infectious hematopoietic necrosis virus. Epithelioma papillosum carpio cells were grown in normal atmosphere with tris(hydroxymethyl)aminomethane- or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered media. Plaques were larger and formed more quickly on 1- to 3-day-old cell monolayers than on older monolayers. Cell culture medium with a 10% addition of fetal calf serum (MEM 10) or without serum (MEM 0) were the most efficient virus diluents. Dilution with phosphate-buffered saline, saline, normal broth, or deionized water reduced plaque numbers. Variations in the pH (7.0 to 8.0) of a MEM 0 diluent did not affect plaque numbers. Increasing the volume of viral inoculum above 0.15 ml (15- by 60-mm plate) decreased plaquing efficiency. Significantly more plaques occurred under gum tragacanth and methylcellulose than under agar or agarose overlays. Varying the pH (6.8 to 7.4) of methylcellulose overlays did not significantly change plaque numbers. More plaques formed under the thicker overlays of both methylcellulose and gum tragacanth. Tris(hydroxymethyl)aminomethane and HEPES performed equally well, buffering either medium or overlay. Plaque numbers were reduced when cells were rinsed after virus adsorption or less than 1 h was allowed for adsorption. Variation in adsorption time between 60 and 180 min did not change plaque numbers. The mean plaque formation time was 7 days at 16 degrees C. The viral dose response was linear when the standardized assay was used.     

Spectral sensitivities of retinular cells measured in intact,living flies by an optical method

Summary The spectral sensitivity of the peripheral retinular cells R1–6 in nine species of intact flies was determined using non-invasive, optical measurements of the increase in reflectance that accompanies the pupillary response. Our technique is to chronically illuminate a localized region of the eye with a long wavelength beam, adjusted to bring pupillary scattering above threshold, then, after stabilization, to stimulate with monochromatic flashes. A criterion increase in scattering is achieved at each wavelength by adjusting flash intensity. Univariance of the pupillary response is demonstrated by Fig. 3.Action spectra measured with this optical method are essentially the same as the published spectral sensitivity functions measured with intracellular electrophysiological methods (Fig. 4 forCalliphora, Fig. 5 forDrosophila, Fig. 7 forEristalis, and Fig. 8 forMusca). This holds for both the long wavelength peak and the high sensitivity in the UV as was consistently found in all investigated fly species.Spectral sensitivity functions for R1–6 of hover flies (family Syrphidae) are quite different in different regions of the same eye. There can also be substantial differences between the two sexes of the same species. The ventral pole of the eye of femaleAllograpta (Fig. 10) contains receptors with a major peak at 450 nm, similar to those ofEristalis. However, the dorsal pole of the same eye contains receptors with a major peak at 495 nm, similar to those ofCalliphom. Both dorsal and ventral regions of the maleToxomerus eye, and the ventral region of the female eye, contain only the 450 nm type of R1-6 (see Fig. 12). However, the dorsal region of the female eye also contains another spectral type of receptor that is maximally sensitive at long wavelength. Eyes of both sexes ofAllograpta (Figs. 10 and 11) contain a mixture of spectral types of receptors R1-6.We thank Dr. Chris Maier of the Connecticut Agricultural Experiment Station, for determination of the Syrphidae. This work was supported by grants EY01140 and EY00785 from the National Eye Institute, U.S.P.H.S., (to GDB), by the Connecticut Lions Eye Research Foundation (to GDB), and by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.), (to DGS).     

Septic superficial thrombophlebitis: a major threat from a minor lesion

Sites of insertion of peripheral intravenous catheters remain an important but often overlooked source of hospital-acquired septicemia and the most important source of staphylococcal septicemia. Septic superficial thrombophlebitis is characterized by severe local pain and is readily evident clinically. Care of the intravenous line can prevent this complication. To be diagnosed, it must be suspected. Treatment consists of removal of the intravenous line and administration of heparin and antibiotics effective against penicillinase-producing organisms. Occasionally the vein must be ligated or, preferably, removed.     

Causes and consequences of Robertsonian exchange

At least two types of Robertsonian exchange are now known in the acrocentric chromosomes of man. Both types involve breakage in the arms adjacent to the centromere. Evidence is presented for a third type of exchange, one involving breakage within the centromere itself, in the grasshopper Percassa rugifrons. In this species, which is regularly homozygous for a single fusion metacentric, the eighteen rod autosomes have small but pronounced granules at the centric end of the chromosome. When C-banded these granules show differential Giemsa staining and appear to represent centromeric chromomeres; these chromomeres are lacking in the metacentric fusion product. Equivalent fusions may have occurred in some mammal species too and possible examples of this are discussed in sheep and mice. The Percassa fusion has led to a modification in both the frequency and the distribution of chiasmata as judged by a comparison of these properties in the metacentric relative to the two next smallest rod equivalents. Comparable modifications are known to occur in other naturally occurring fusions but these changes are certainly not automatic consequences of fusion since they are not shown in at least some newly produced fusion mutants.     

The metabolism of steviol to 13-hydroxylated ent-Gibberellanes and ent-kauranes

Steviol(ent-13-hydroxykaur-16-en-19-oic acid) is rapidly metabolised by the mutant B1-41a of Gibberellafujikuroi. The initial product is the ent- 7-α-hydroxy derivative which is then further metabolised to gibberellins A₁, A₁₈, A₁₉, A₂₀, 13-hydroxy GA₁₂, the ent-6α, 7α, 13- and ent-6β, 7α, 13 (19,6-lactone)-trihydroxykaurenoic acids, and a seco-ring B diacid. This apparently low substrate specificity of the enzymes operative beyond the block in the mutant B1-41a provides a useful model for the biosynthetic pathways to 13-hydroxylated gibberellins of higher plants and a preparative route to these plant gibberellins.