Radiobiology of ultrasoft X rays. I. Cultured hamster cells (V79)

Ultrasoft X rays (approximately less than keV) provide a useful probe for the study of the physical parameters associated with the induction of biological lesions because the spatial scale of their energy depositions is of nanometer dimensions, comparable to that of critical structures within the cell. We report on cell-killing experiments using cultured hamster cells (V79) exposed to carbon K (0.28 keV), aluminum K (1.5 keV), copper K (8.0 keV), and 250 kVp X rays, under oxic and hypoxic conditions, and as a function of cell-cycle phase. Our principal results are: RBE increases with decreasing X-ray energy; OER decreases with decreasing X-ray energy; and cell-cycle response is similar for all X-ray energies. Our RBE results confirm earlier observations using ultrasoft X rays on mammalian cells. The shapes of fitted curves through the data for each energy are statistically indistinguishable from one another, implying that the enhanced effectiveness is purely dose modifying. The results reported herein generally support the view that single-track effects of radiation are predominantly due to very local energy depositions on the nanometer scale, which are principally responsible for observed radiobiological effects.     

Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.     

Molecular cloning of Clostridium difficile toxin A gene fragment in lambda gt11

Toxin A of Clostridium difficile has been purified and monospecific antiserum produced. A reliable procedure for isolation and restriction of C. difficile chromosomal DNA was developed which allowed for the construction of a genomic library in lambda gt11. Approx. 35,000 plaques were screened using anti-toxin A which resulted in the identification of one stable positive clone, lambda cd19. Verification of the immunological identity of the isolated toxin A gene fragment in lambda cd19 was determined by affinity purifying toxin A antibodies specific for lambda cd19 gene product, and using these selected antibodies to probe a Western blot of purified toxin A. The insert in lambda cd19 was demonstrated to be a 0.3 kb fragment by restriction digestion, and by hybridization of the clone to a chromosomal digest of C. difficile. The peptide coded for by the toxin A gene fragment in lambda cd19 was not cytotoxic for 3T3 mammalian tissue culture cells.     

Primary structure of an analog of crustacean pigment-dispersing hormone from the lubber grasshopper Romalea microptera

An octadecapeptide capable of inducing pigment dispersion in the chromatophores of the fiddler crab Uca pugilator has been isolated from lyophilized heads of the lubber grasshopper Romalea microptera. This pigment-dispersing factor (PDF) was purified by gel filtration, ion-exchange chromatography, partition chromatography, and reversed-phase high performance liquid chromatography. Automated gas-phase sequencing, followed by the identification of the carboxyl-terminal amide, established the primary structure of this PDF as Asn-Ser-Glu-Ile-Ile-Asn-Ser-Leu-Leu-Gly-Leu-Pro-Lys-Leu-Leu-Asn-Asp-Ala- NH2. This structure was confirmed by chemical synthesis and by demonstrating that the synthetic and native PDF displayed identical chromatographic behavior and biological activity. The Romalea PDF is structurally related to the crustacean pigment-dispersing hormones (PDHs), which are also octadecapeptides. The sequence of grasshopper PDF shows 78% homology with beta-PDH (from the crabs U. pugilator and Cancer magister) and 50% homology with alpha-PDH (from the prawn Pandalus borealis). This study provides the first direct chemical evidence for the structural relatedness of insect PDF to the crustacean PDHs, thus identifying them as an authentic family of arthropod peptides.     

Partial repair of deamidation-damaged calmodulin by protein carboxyl methyltransferase

Modification of calmodulin by protein carboxyl methyltransferase requires deamidation of one or more labile asparagine residues (Johnson, B.A., Freitag, N. E., and Aswad, D. W. (1985) J. Biol. Chem. 260, 10913-10916). We now show that deamidation results in the generation of two altered forms of calmodulin, designated A and B, which can be separated by electrophoresis under nondenaturing conditions. The A form is characterized by a larger apparent molecular radius, has only 10% the activity of native calmodulin when assayed for its ability to activate a Ca2+/calmodulin-dependent protein kinase from rat brain, and serves as an excellent substrate for the methyltransferase. The B form more closely resembles native calmodulin: it has an apparent molecular radius more like the native, exhibits about 40% the activity of native calmodulin, and is a relatively poor methyl acceptor. Evidence suggests that the A and B forms probably contain isoaspartate (A) and aspartate (B) in place of Asn-60 and/or Asn-97. Incubation of the A form with methyltransferase and S-adenosyl-L-methionine converts about half of the A form to an electrophoretic band indistinguishable from the B form. The activity of this partly converted calmodulin rises to 30-50% that of native calmodulin. These observations imply that the methyltransferase may have a biological role in restoring activity to proteins which contain abnormal isoaspartyl peptide bonds resulting from asparagine deamidation.     

Allosteric behavior in transducin activation mediated by rhodopsin. Initial rate analysis of guanine nucleotide exchange

Photolyzed rhodopsin acts in a catalytic manner to mediate the exchange of GTP for GDP bound to transducin. We have analyzed the steady-state kinetics of this activation process in order to determine the molecular mechanism of interactions between rhodopsin, transducin, and guanine nucleotides. Initial velocities (Vo) of the exchange reaction catalyzed by rhodopsin were measured for various transducin concentrations at several fixed levels of the GTP analog, [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). The initial rate data analysis rigorously demonstrates that rhodopsin mediates the activation of transducin by a double-displacement catalytic mechanism. The Michaelis-Menten curves determined as a function of [transducin] reveal remarkable allosteric behavior; analysis of this data yields a Hill coefficient of 2. Lineweaver-Burk plots of Vo-1 versus [transducin]-1 display curvilinearity indicative of positive cooperativity and a series of parallel lines are generated by plotting Vo-1 as a function of [transducin]-2. The plots of Vo-1 versus [GTP gamma S]-1 show no evidence of allosterism and are a parallel series. Furthermore, the allosteric behavior observed in the activation of transducin is also witnessed in the rhodopsin-catalyzed guanine nucleotide exchange of the G protein's purified alpha subunit in the absence of the beta X gamma subunit complex. The latter observation implies that the molecular basis for allosterism in the activation process resides in the interactions between the photoreceptor and transducin's alpha subunit.     

Class I-like MHC molecules expressed by baboon placental syncytiotrophoblast

Human placental villous trophoblast is known to be unreactive with W6/32 and other monoclonal antibodies recognizing monomorphic determinants of human class I MHC heavy chains, whereas extravillous cytotrophoblast in the placental bed is W6/32-reactive by immunohistology. We have now demonstrated, in contrast, that syncytiotrophoblast is the only cellular component of baboon early placental villous tissue which is reactive with any of these antibodies. Radioimmunoprecipitation of detergent-solubilized baboon placental membrane preparations, and subsequent SDS-PAGE, has shown the W6/32-reactive component to have an m.w. of 41,000 and to be associated with beta 2-microglobulin, whereas baboon peripheral lymphocytes express 45,000 m.w. W6/32-reactive antigens comparable with the HLA-A,B,C heavy chains of human lymphocytes.     

Trifluoperazine Activates and Releases Latent ATP-Generating Enzymes Associated with the Synaptic Plasma Membrane

Neurone-specific enolase (NSE) and the brain form of creatine phosphokinase (CPK-BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two-dimensional gel (2-D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be "cryptic" unless the SPM were treated with Triton X-100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for "unmasking" the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK-BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2-D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70-kilodalton heat-shock cognate protein. Tubulin, synapsin I, and a 35-kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase on the basis of 2-D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.     

Loading patterns and jaw movements during mastication in Macaca fascicularis: a bone-strain, electromyographic, and cineradiographic analysis

Rosette strain gage, electromyography (EMG), and cineradiographic techniques were used to analyze loading patterns and jaw movements during mastication in Macaca fascicularis. The cineradiographic data indicate that macaques generally swallow frequently throughout a chewing sequence, and these swallows are intercalated into a chewing cycle towards the end of a power stroke. The bone strain and jaw movement data indicate that during vigorous mastication the transition between fast close and the power stroke is correlated with a sharp increase in masticatory force, and they also show that in most instances the jaws of macaques are maximally loaded prior to maximum intercuspation, i.e. during phase I (buccal phase) occlusal movements. Moreover, these data indicate that loads during phase II (lingual phase) occlusal movements are ordinarily relatively small. The bone strain data also suggest that the duration of unloading of the jaw during the power stroke of mastication is largely a function of the relaxation time of the jaw adductors. This interpretation is based on the finding that the duration from 100% peak strain to 50% peak strain during unloading closely approximates the half-relaxation time of whole adductor jaw muscles of macaques. The EMG data of the masseter and medial pterygoid muscles have important implications for understanding both the biomechanics of the power stroke and the external forces responsible for the "wishboning" effect that takes place along the mandibular symphysis and corpus during the power stroke of mastication. Although both medial pterygoid muscles reach maximum EMG activity during the power stroke, the activity of the working-side medial pterygoid peaks after the balancing-side medial pterygoid. Associated with the simultaneous increase of force of the working-side medial pterygoid and the decrease of force of the balancing-side medial pterygoid is the persistently high level of EMG activity of the balancing-side deep masseter (posterior portion). This pattern is of considerable significance because the direction of force of both the working-side medial pterygoid and the balancing-side deep masseter are well aligned to aid in driving the working-side lower molars across the upper molars in the medial direction during unilateral mastication.(ABSTRACT TRUNCATED AT 400 WORDS)