Crystalline tubes of myosin subfragment-2 showing the coiled-coil and molecular interaction geometry

We have produced crystalline tubes of chicken breast myosin long subfragment-2 that show order to resolutions better than 2 nm. The tubes were formed from a thin sheet in which the myosin long subfragment-2 molecules were arranged on an approximately rectangular crystalline lattice with a = 14.1 +/- 0.2 nm and b = 3.9 +/- 0.1 nm in projection. Shadowing indicated that the tube wall was approximately 7 nm thick and that the sheets from which it was formed followed a right-handed helix. Superposition of the lattices from the top and bottom of the tube produced a moire pattern in negatively stained material, but images of single sheets were easily obtained by computer image processing. Although several molecules were superimposed perpendicular to the plane of the sheet, the modulation in density due to the coiled-coil envelope was clear, indicating that the coiled-coils in these molecules were in register (or staggered by an even number of quarter pitches). In projection the coiled-coil had an apparent pitch of 14.1 nm (the axial repeat of the unit cell), but the small number of molecules (probably four) superimposed perpendicular to the plane of the sheet meant that pitches within approximately 1 nm of this value could have shown a modulation. Therefore, a more precise determination of the coiled-coil pitch must await determination of the sheet's three-dimensional structure. The coiled-coils of adjacent molecules within the plane of the sheet were staggered by an odd number of quarter pitches. This arrangement was similar to that between paramyosin molecules in molluscan thick filaments and may have features in common with other coiled-coil protein assemblies, such as intermediate filaments. Each molecule in the crystal had two types of neighbor: one staggered by an odd number of quarter pitches and the other by an even number of quarter pitches, as has been proposed for the general packing of coiled-coils (Longley, W., 1975, J. Mol. Biol., 93:111-115). We propose a model for the detailed packing within the sheet whereby molecules are inclined slightly to the plane of the sheet so that its thickness is determined by the molecular length.     

The cloning of chromosomal DNA associated with methicillin and other resistances in Staphylococcus aureus

Competitive hybridization was used to detect the deletion of chromosomal DNA accompanying the loss of resistance to methicillin (and concomitantly, to cadmium, mercury and tetracycline) from a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA). The method was also used to screen a partial plasmid library of chromosomal HindIII fragments from the MRSA strain. Eight recombinant plasmid clones were identified as containing DNA included in the deletion. These clones were used as probes to screen a phage library of the total DNA of the same MRSA strain, resulting in the isolation of overlapping recombinant phage clones carrying 24 kb of the deleted DNA. Two of the cloned HindIII fragments were associated closely with methicillin resistance, as shown by probing DNA from an independent methicillin-sensitive/resistant transduced strain pair and from two MRSA strains following growth in the presence of high concentrations of methicillin. The endonuclease map of the cloned DNA indicates the presence of four copies of a direct repeat less than 1 kb in size. The map is also consistent with the presence in the chromosome of sequences for mercury resistance (mer A mer B) and for tetracycline-resistance plasmid pT181.     

Serum lipids and lipoproteins in the atherosclerosis prone LA/N corpulent rat

The LA/N rat is one of two congenic strains bred from the original obese, hyperphagic and hypertensive rats of Koletsky. With the exception of hypertension the LA/N strain, when homozygous for the corpulent gene, is phenotypically similar to the parent Koletsky strain and prone to the development of vascular and myocardial lesions. Here we report a detailed analysis of the serum lipids, lipoproteins and apolipoproteins B, E and A-I levels in young adult homozygous corpulent (cp/cp) rats of both sexes and in lean males of the same age which were demonstrable non-carriers (+/+) of the cp gene. Both male and female cp/cp rats were hypertriglyceridemic (282-512 mg/100 ml) and moderately hypercholesterolemic (74-84 mg/100 ml). Elevations in these lipids reflected the presence of large (622 A), triacylglycerol-rich and apoprotein-poor VLDL containing both apolipoproteins Bh and B1 and increased phospholipid-rich HDL. Similar, but less pronounced, elevations in serum apolipoproteins B and E in the cp/cp rats when compared to the +/+ animals were also noted. Apolipoproteins A-I levels were 2.7-3-fold higher in cp/cp rats. The levels of VLDL were significantly higher in female cp/cp rats; however, the levels of IDL (intermediate-density lipoproteins), LDL and HDL were significantly lower than in the more atherosclerosis prone male cp/cp rats. Similarly, apolipoprotein A-I was higher and apolipoprotein B lower in the male cp/cp than in the female cp/cp rats. The LDL (d = 1.030-1.063 g/ml) in cp/cp rats, like that in normal animals, was heterogeneous and contained apolipoproteins Bh, E, A-I and C. This fraction was significantly elevated in male cp/cp rats when compared to females but still represented less than 13% of the total serum cholesterol and less than 6% of the total serum lipids in 3-month-old cp/cp animals. The ratio of cholesterol to phospholipids was significantly lower for all lipoproteins in cp/cp rats when compared to +/+ males and these ratios for female cp/cp rats were in all cases lower than those of male cp/cp animals.     

Variation in Male Fertilities and Pairwise Mating Probabilities in Picea glauca

Frequencies of multilocus male gametes in seeds collected from clones in several blocks of a white spruce seed orchard were analyzed as part of a 2-yr study of mating system variation in this species. Observed frequencies of male gamete types departed significantly from those expected assuming equal male fertilities among clones. Male gamete frequencies in seed crops were significantly heterogeneous among clones within blocks, and among blocks within clones. Clonal male fertilities were estimated from male gamete frequency data. These estimates were highly skewed, with a small proportion of the clones contributing male gametes to the majority of the seed. The estimates were significantly heterogeneous among clones within blocks, and among blocks within clones. Between-year variation in clonal male fertilities was also pronounced, with male fertilities of some clones changing by as much as three orders of magnitude. Clonal male fertility was significantly correlated with clonal male cone production in both years. These results are important with regard to assumptions made for the estimation of general combining ability, average genetic correlation among progeny from single parents, and expected response to selection in open-pollinated plant populations.     

The role of enzymatic processing in the biological actions of substance P

There is considerable evidence that substance P (SP) is a neurotransmitter in the CNS. Current findings suggest that the effects of synaptically released SP are terminated by enzymatic breakdown, primarily by endopeptidase 3.4.24.11 (endo 24.11). The products of cleavage by endo 24.11 include the amino-terminal fragment SP(1-7). Evidence suggests that SP is involved in mediating baroreceptor reflex activity in the nucleus of the solitary tract (NTS). Microinjection of SP into the NTS lowered blood pressure and heart rate. Microinjection of SP(1-7) into the NTS reproduced the effects of SP on both heart rate and blood pressure. Intra-NTS injection of phosphoramidon, an inhibitor of endo 24.11 activity, completely blocked the effects of a subsequent injection of SP. This blocking effect of phosphoramidon was unaltered by pretreatment with the opiate inhibitor naloxone. In contrast, phosphoramidon failed to block the depressor and bradycardic effects of SP(1-7). The implications of these findings regarding the role of endo 24.11 in the metabolism of SP are discussed.     

A myosin isoform repressed in hypertrophied ALD muscle of the chicken reappears during regeneration following cold injury

A library of monoclonal antibodies specific for myosin heavy chain (HC) was used to study myosin expression in regenerating fibers. The response to cold injury of slow skeletal ALD muscle previously induced to eliminate SM1 myosin by weight overload was compared to that of its contralateral control. Native gel electrophoresis combined with immunoblotting demonstrated that slow SM1 myosin HC eliminated from hypertrophic muscle reappeared both at the site of active regeneration and unexpectedly, also distal to the site of injury. The regeneration response of hypertrophied muscles was similar to that of the controls. In addition to SM1 myosin HC, ventricular-like and embryonic/fast isoforms were also expressed in both muscles during the early stages of regeneration and disappeared as the muscle fibers matured. These observations demonstrate that regenerating slow muscle fibers reexpress myosins' characteristic of developing muscle irrespective of the myosin phenotype prior to injury. The reappearance of repressed myosin HC in the hypertrophied ALD muscle is consistent with the presence of newly differentiated myonuclei.     

Common expression of a tumor necrosis factor resistance mechanism among gynecological malignancies

Summary The efficacy of tumor necrosis factor (TNF) as an anticancer agent is limited. This limitation might be related to the expression of a protein-synthesis-dependent resistance mechanism that prevents the lysis of tumor cells by TNF. To test this possibility eight randomly selected human cell lines, three derived from ovarian carcinomas and five derived from cervical carcinomas, were tested for their in vitro sensitivity to TNF-mediated lysis. The results of this analysis showed that all eight cell lines are normally resistant to lysis by TNF. However, in the presence of inhibitors of protein synthesis, seven of them showed a significant increase in TNF-mediated lysis. Measurement of protein synthesis showed that there is a linear correlation between the level of inhibition of protein synthesis and the level of TNF-mediated lysis. The fact that seven of eight randomly selected cell lines are resistant to TNF because they express a protein-synthesis-dependent resistance mechanism suggests that this mechanism of resistance may be common among gynecological cancers. The results also suggest that a therapy involving TNF and inhibitors of protein synthesis might be useful for the treatment of gynecological malignancies.