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The specific antigenicity of pentaglycine bridges in cell walls ofS. aureus was investigated. The antistaphylococcal sera (against11 Oeding strains ofS. aureus) were found to contain antibodies of two different specificities against polyglycyl peptides; one type reacted with polyglycyl peptides with a free amino terminus and the other with polyglycyl peptides with a free carboxyl terminus. These antibodies were assayed by passive hemagglutination with tanned sheep red cells sensitized with N-polyglycyl-BSA (containing polyglycyl peptides with a free amino terminus) or with C-polyglycyl-BSA (containing polyglycyl peptides with a free carboxyl terminus). Only the antibodies against N-polyglycyl peptides were absorbed byS. aureus strains and byS. epidermidis. Heterologous bacterial species failed to absorb antibodies of either specificity. Antibodies against polyglycyl peptides were used to confirm the mechanism of action of penicillin onS. aureus cell wall.

A finite element model of a complete vertebra was formulated from a previously developed bilaterally symmetric model. This model was verified by comparing its results to those of the previous model and by ensuring that the output was bilaterally symmetric. The applied nodal loads were checked against experimentally measured pressures on the vertebral endplate. Simulations of complete and partial laminectomies on one side of a vertebra were performed. High tensile stresses occurred in the pedicle and in the region of the pars interarticularis as a result of surgery, particularly when the superior half of the lamina was removed.

In heme-deficient reticulocyte lysates a translational inhibitor which regulates protein synthesis is formed or activated. To define the mechanism of action of the translational inhibitor (RI), RI was partially purified. We have utilized the isolated RI to examine its relationship to the translational inhibitor formed in situ in heme-deficiency, some quantitative aspects of inhibition of protein synthesis, and the relationship of RI concentration to the initiation factor (IF-MP) which forms a ternary complex with Met-tRNAf and GTP (IF-MP-Met-tRNAf-GTP). The results demonstrate that the activity of isolated RI is related to the in situ heme-deficiency inhibitor by several criteria: (a) the biphasic kinetics of inhibition manifested by RI in lysates containing optimal levels of hemin are very similar to those observed in heme-deficiency, i.e., an initial period in which several rounds of protein synthesis proceed at the control rate followed by an abrupt decline in the rate of protein synthesis. (b) Both inhibitions are accompanied by the disaggreagation of polyribosomes with a concomitant increase in 80S ribosomes. (c) Both inhibitions are reversed by IF-MP. The isolated RI blocked protein synthesis in lysates at temperatures ranging from 15 degrees to 30 degrees. Although the rate of protein synthesis was a function of the temperature of incubation, the number of rounds of protein synthesis prior to shut-off was essentially the same at various temperatures. When RI was added to lysates, at increasing intervals after the start of incubation, the period of synthesis before shut-off (lag) progressively decreased. The inhibition of protein synthesis by RI was immediately reversed by the addition of IF-MP. The extent of reversal increased with increasing concentrations of IF-MP; at low levels of RI almost complete reversal of inhibition by IF-MP was obtained. However, at high levels of RI which did not appreciably increase the degree of inhibition of protein synthesis, equivalent amounts of IF-MP were less effective in reversing inhibition. These results suggest that the inhibition of protein synthesis by the isolated inhibitor involves the initiation factor IF-MP.

The heme-regulated translational inhibitor (HRI) has been purified 4800-fold. On electrophoresis in sodium dodecyl sulfate/polyacrylamide gel, the purified HRI showed one major polypeptide band. The purified HRI inhibits protein synthesis in lysates containing optimal levels of hemin with inhibition kinetics which parallel those observed in heme-deficiency. Data are presented which are consistent with an enzymatic function of HRI in the inhibition of protein synthesis. The HRI is an adenosine 3':5'-cyclic monophosphate independent protein kinase which phosphorylates the small subunit (38,000) but not the large subunits (52,000 and 50,000) of the initiation factor which forms a ternary complex with Met-tRNAf and GTP. This evidence supports the hypothesis that inhibition of protein synthesis by HRI involves the phosphorylation of the initiation factor. These findings are discussed in relation to various models for the regulation of protein kinase activity by heme. (see article).

Summary 80S ribosomes from skeletal muscle of diabetic rats were more susceptible than normal to dissociation by the initiation factor EIF-3; the increased sensitivity to dissociation was a property of the 60S subunit. Increased dissociation may be a reflection of the instability of diabetic 80S couples resulting from a change in the structure of the large subunit.

In the absence of added hemin, protein synthesis in rabbit reticulocyte lysates proceeds at maximal linear rates for several minutes and then ceases abruptly. Inhibition involves the action of a translational inhibitor whose formation is regulated by hemin. Addition of the isolated inhibitor to hemin-supplemented lysates produces an inhibition of protein chain initiation similar to that observed in heme-deficiency. The inhibitor has been purified over 300-fold and contains a protein kinase activity that copurifies with the inhibitory function. With calf thymus histone II as the phosphate receptor, the inhibitor-associated protein kinase requires ATP as the phosphorylating agent. Cycle AMP stimulates kinase activity 5- to 8-fold; the concentration of cycle AMP required for halfmaximal activity is 4 X 10-8 M. Preincubation of the inhibitor in the presence of cyclic AMP significantly reduces cyclic AMP-dependent phosphorylation and inhibitory activity. The corresponding protein kinase activity from hemin-supplemented lysates displays reduced cyclic AMP-dependency and little or no inhibitory activity. These findings suggest that the protein kinase activity associated with the purified translational inhibitor is involved in the mechanism of inhibition of initiation observed in hemedeficient reticulocyte lysates.

Physical, dielectric, piezoelectric and electromechanical parameters have been reported for bone and its two major constituents i.e. collagen and apatite, for their characterization as ultrasonic transducer materials. Collagen and apatite have been extracted from full bone using well known methods. These materials have been used to prepare simple disc-shaped test pieces (dimensions: diameter 10 mm and thickness 1.0 ± 0.01 mm). The variation of various electrical parameters with frequency in the region (1–108 MHz) is examined for these materials. These include impedance, phase angle, relative voltage output, quality factor ‘Q’, dielectric constant and resistivity. The data so obtained are compared with those for ceramic and quartz transducers. The observations on impedance, phase and relative voltage output for bone materials indicate that the first resonance peak falls around 56 MHz followed by second and third harmonics around 112 MHz and 168 MHz respectively. A low ‘Q’ value suggests a fairly wide band transducer, while other parameters compare favourably with ceramic and quartz materials. Bone material has also been used to obtain a transducer in the standard configuration and velocity dispersion in the frequency range 1–108 MHz is examined.

A finite element model of a complete vertebra has been formulated. It is used to simulate the laminectomy and Harrington rod procedures. The results indicate that high tensile stresses occurred on the affected side in the region of the pars interarticularis after laminectomy. High tensile stresses near the pars interarticularis and at the posterior junction of lamina and pedicles are predicted due to Harrington rod loading.

Vanadate inhibits protein chain initiation in rabbit reticulocyte lysates. The evidence that supports this conclusion is as follows: (i) the biphasic kinetics of inhibition in which protein synthesis is maintained at the control rate for 1-2 min is followed by an abrupt decline in the rate of synthesis; (ii) inhibition is associated with a marked disaggregation of polyribosomes and a concomitant increase in 80S ribosomes; and (iii) vanadate concentrations that inhibit protein chain initiation do not inhibit polypeptide chain elongation or the aminoacylation of tRNA. In partial reactions of protein chain initiation, vanadate concentrations that inhibit protein synthesis have no detectable effect on the formation of eukaryotic initiation factor eIF-2-promoted ternary complex with Met-tRNAf and GTP and on the assembly of 40S ribosomal subunit-Met-tRNAf complexes. On the addition of mRNA, the 40S ribosomal subunit-Met-tRNAf complexes also are transformed into 80S ribosome-mRNA-Met-tRNAf complexes, termed 80S initiation complexes. In vanadate-treated samples, however, these 80S initiation complexes are defective and unable to proceed beyond this step.

The effect of various sulfhydryl reagents on the ribosomes of Bacillus subtilis was studied. The 70 S ribosomes were completely dissociated into 30 S and 50 S subunits by appropriate concentrations of p -chloromercuribenzoic acid (PCMB) and 5,5′-dithio- bis -(2-nitro-benzoic acid). The N -ethylmaleimide and iodoacetamide failed to dissociate the ribosomes even at relatively high concentrations. The rate of dissociation of ribosomes by PCMB varied with the concentration of ribosomes. A progressive decrease in the rate of dissociation was observed as the concentration of ribosomes in the reaction mixture was increased. The PCMB-induced ribosomal subunits were unable to reassociate into 70 S monomers unless they were dialyzed against buffer containing β-mercaptoethanol. On the average, four molecules of PCMB per 70 S ribosome and two molecules of PCMB per each 30 S and 50 S subunit were bound. The number of PCMB molecules bound per ribosome did not change with increasing concentrations of PCMB, even though higher concentrations of PCMB resulted in dissociation of ribosomes into subunits.

A methodology is described for measuring the centre of pressure for successive steps during stance phase. Three persons were assessed, one normal, one below knee amputee and one having suffered from poliomyelitis. The clinical significance of this parameter of gait as measured by the miniature shoe-borne load cells is highlighted.

Discrimination between eukaryotic and prokaryotic, and formylated and non-formylated, initiator tRNAs by eukaryotic initiation factor EIF-3

Man has recognized carbon as an implant material well tolerated by the body for many years. Indeed, carbon in an impure form (Indian ink) was first used in the making of tatoos. Most recently carbon has been used in a filamentous form of high strength. These carbon fibers are produced in long tows of 40,000 fibers, each with a cross sectional area of about 7μ (Figure 1), by heating acrylic fibers to high temperature. This causes the noncarbon components to be removed while maintaining the molecular structure (scaffold) of the acrylic. Fibers can be chopped into short lengths and used to reinforce polyethylenes (8, 11, 12). These reinforced plastics have-twice the strength and ten times the stiffness of their non-reinforced counterparts, but suffer only one tenth as much creep when subjected to a load (11). Long tows of carbon fibers are used in tendon and ligament replacement (1, 2, 4, 5, 6, 7, 9, 10). This application warrants further investigation because of the apparent ability of the carbon fibers to induce the formation of ligaments and tendons (3, 4, 5, 6, 7).

Protein chain initiation in reticulocyte lysates is inhibited by (a) heme-deficiency, (b) low levels of double-stranded RNA, and (c) a purified translational inhibitor isolated from heme-deficient lysates. Previous studies have shown that the inhibitions produced by heme-deficiency and double-stranded RNA are prevented by 3': 5'-cyclic AMP, and that GTP, but not ATP, prevents the inhibition of heme-deficiency. In view of the recent finding that the inhibitor purified from heme-deficient lysates is associated with a protein kinase which appears to be involved in the mechanism of inhibition, the effects of cyclic AMP, GTP, and ATP on the three modes of inhibition were examines. In all three types of inhibition, cyclic AMP or GTP (a) prevents the onset of inhibition when added at zero time, and (b) restores protein synthesis in inhibited lysates. In contrast to these effects, ATP potentiates each inhibition, and blocks reversal of inhibition by cyclic AMP or GTP. On the basis of these and earlier findings, we propose that (a) these inhibitions involve the phosphorylation by protein kinases of the Met-tRNAf binding factor and/or a related site(s) on the 40S ribosomal subunit; and (b) cyclic AMP, GTP, and ATP exert their effects by their actions on this phosphorylation mechanism.

Fracture of the fibrous networks of normal and irradiated rat dermis have been studied using the SEM. The implications of these observations are discussed with regard to the mechanical behaviour of the tissue in the living animal. It is shown that after radiation the normal fibrous structure of collagen is replaced by a solid mat which is ill adapted to support stress, so that rupture at low stress levels is inevitable.

The increasing use of metals as implant materials in orthopaedic surgery has stimulated a considerable amount of interest among researchers and orthopaedic surgeons enabling the study of corrosion, wear, hypersensitivity, toxcity and carcinogenicity (Furst and Haro, 1960; Mears, 1979; Woodman, 1980). Partly, the corrosion Phenomena is due to the metal implant ionization in living tissue (Ferguson et al, 1960; Lautenschlager et al, 1974). Corrosion is a complex electrochemical deterioration of metals (Clark and Hickman, 1953). Futhermore, corrosion is referred to as a combination of oxidation and reduction of reactions at the implant-tissue interface. The basic reactions in corrosion are the removal of positive metal ions from their positions in the metal crystal lattice. This is called an anodic reaction. Similarly, the freed electrons, as a result of removed cations, also react to oxygen and hydrogren molecules in tissues to form hydroxyle. This is defined as cathodic reactions at the metal-tissue interface (Mears, 1979).

To define the mechanism of regulation of the protein kinase that is activated in heme deficiency and that inhibits initiation of protein synthesis, we have isolated and purified the heme-reversible form of the protein kinase from rabbit reticulocytes. The inhibitory activity is found in a single band after polyacrylamide gel electrophoresis under nondenaturing conditions. It migrates as a 95,000-dalton polypeptide in 15% sodium dodecyl sulfate/polyacrylamide gels. This purified inhibitor becomes self-phosphorylated in the presence of ATP; the phosphorylated protein and the inhibitory activity copurify. The inhibitor produces characteristic biphasic kinetics of inhibition in reticulocyte lysates and phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2); the inhibition is reversed by added eIF-2. In contrast to the heme-irreversible inhibitor, this heme-reversible inhibitor is no longer inhibitory after incubation with 20 micron hemin. Incubation with hemin also inhibits self-phosphorylation. Preincubation of the heme-reversible inhibitor in the presence of ATP potentiates the inhibition of protein synthesis in the subsequent incubation, as does treatment with N-ethylmaleimide. Phosphorylation of the heme-reversible inhibitor and inhibition of protein synthesis in the lysate due to phosphorylation of eIF-2 appear to be related. These findings suggest that hemin acts directly on the heme-reversible inhibitor.

Heme deficiency in rabbit reticulocytes and their lysates leads to the activation of a heme-regulated translational inhibitor (HRI) which causes the cessation of polypeptide initiation. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 binds Met-tRNA f and GTP in ternary complex. As a continuation of the studies on the molecular basis of the inhibition of the formation of 40S ribosomal subunit-Met-tRNA f complexes by HRI [Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., Roy, R. & Gupta, N. K. (1978) Proc. Natl. Acad. Sci. USA 75, 745-749], we describe here the isolation and some characteristics of a factor that is required for the HRI-catalyzed inhibition of eIF-2-promoted ternary complex formation. In the presence of 1 mM Mg 2+ , ternary complex formation by eIF-2 is dependent on the presence of this stabilization factor (SF). Under these conditions, SF increases the rate and the extent of ternary complex formation. This finding suggests that the interaction of SF with eIF-2 causes a conformational change that stabilizes eIF-2 and promotes efficient ternary complex formation by increasing the affinity of eIF-2 for GTP and Met-tRNA f . In the absence of Mg 2+ , however, eIF-2 efficiently forms the ternary complex and SF has little effect on its ternary complex formation capacity—hence, the name eIF-2 stabilization factor (SF). In the presence of SF, HRI markedly inhibits (70-80%) the ternary complex formation capacity of eIF-2. The inhibitory effect requires both HRI and ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of eIF-2. Both the rate and the extent of the SF-dependent ternary complex formation are inhibited. These findings are consistent with the idea that phosphorylation causes a conformational change in eIF-2 such that its interactions with other initiation factors in the formation and the binding of ternary complex to 40S ribosomal subunits are inhibited.

Publisher Summary This chapter describes the regulation of eukaryotic protein chain initiation by protein kinases and heme. Reticulocyte lysates provide a model system for studies on the regulation of protein synthesis owing to several unique properties. They include an efficient rate of initiation, the extensive synthesis of one major protein species, and the complete dependence of protein synthesis on exogenous hemin. In the presence of optimal concentrations of added hemin, protein synthesis proceeds at linear rates for prolonged periods. During the period of linear synthesis, the polyribosomes are maintained, and initiation factors are utilized catalytically. In efficient lysates, the rate of globin synthesis is comparable to the in vivo rate. In the absence of added hemin, protein synthesis proceeds at the control rate for several minutes, and then declines abruptly yielding characteristic biphasic kinetics of inhibition. The studies have established that the inhibition is mediated through a translational inhibitor which is rapidly activated in the absence of heme.

In vitro thermal properties of human cortical bone have been measured using a specially developed technique. The technique enables accurate measurement of the coefficient of thermal expansion and contraction and the average values have been measured to be 27.5 ± 3.9 × 10−6 mm/°C for expansion and 27.2 ± 5.2 × 10−6 mm/°C for contraction.

A previous study demonstrated that the translational inhibitor from lysates of heme-deficient rabbit reticulocytes is associated with a protein kinase activity. Chromatography of this inhibitor preparation on phosphocellulose yields two distinct protein kinase activities, PC1 and PC2. PC1, which consitutes about 90% of the activity in the unresolved preparation, does not inhibit protein synthesis in lysates, but actively phosporylates calf thymus histone II in a 3':5'-cyclic AMP-denpendent reaction. PC2 contains the translational inhibitor, phosphorylates histone poorly, and is not cyclic AMP-dependent. While [gamma-32P]ATP as the phosphate donor, the two kinase fractions were analyzed with the putative substrates, salt-washed 40S ribosomal subunits, and the initiation factor that mediates the binding of Met-tRNAf to the 40S subunit. PC1 is inactive with the initiation factor, but phosphorylates 40S subunits at a single major site that migrates as a 31,000-dalton band in sodium dodecyl sulfate-acrylamide gels; phosphorylation requires cyclic AMP. Similar phosphorylation of the reticulocyte 40S site (31,000 daltons) can be demonstrated with other cyclic AMP-dependent kinases from reticulocytes, rat liver, and bovine heart muscle. PC2 phosphorylates the small subunit (38,000 daltons) but not the large subunit(s) of the initiation factor; the reaction does not require cyclic AMP. PC2 does not phosphorylate 40S subunits. In the presence of 40S subunits, the initiation factor appears to be rapidly bound in a manner that effectively blocks phosphorylation of the initiation factor by PC2; under the same conditions phosphorylation of the 40S subunit by PC1 is not affected. The initiation factor has been shown to reverse the inhibitions of protein chain initiation induced in lysates by heme deficiency, double-stranded RNA, oxidized glutathione, or the purified translational inhibitor. The observation that the Met-tRNAf binding factor is phosphorylated by PC2 supports the hypothesis that this initiation factor is a target for the action of the translational inhibitor activated in heme deficiency.

An engineering approach to functional assessment has been utilized to assist the moderate-to-severely physically handicapped. These computer-aided assessments are directed at work, keyboard operation and driving. In order to improve the quality of life for this population, independent living systems, recreational and mobility aids, and automated retrieval systems for information on assistive devices have been developed. This paper discusses their implementation and applications.

Pressures in the nucleus pulposus and under the annulus fibrosus have been measured, using the specially developed miniature pressure transducers, when the vertebral segment was subjected to physiological loading. Strain data and stress-relaxation were also measured. Results show that pressures are linear with loading. Pressure variations within the nucleus are due to disc degeneration.

Protein synthesis in rabbit reticulocytes and their lysates is regulated by heme. In heme-deficient reticulocyte lysates, protein synthesis proceeds at the initial rate for several minutes and then declines abruptly. Inhibition of protein synthesis is due to the activation of a heme-regulated translational inhibitor (HRI) which blocks the initiation of protein synthesis. Addition of the isolated HRI to hemin-supplemented lysates causes inhibition of initiation similar to that observed in heme-deficiency. HRI has been shown to be a protein kinase that specifically phosphorylates the Met-tRNA f binding factor (eIF-2). We have isolated an inhibitor (LI) of protein chain initiation from rat liver which displays properties similar to those of HRI: ( i ) the chromatographic behavior of LI on DEAE-Sephadex, DEAE-cellulose, and phosphocellulose is similar to that of HRI; ( ii ) both LI and HRI inhibit protein chain initiation in rabbit reticulocyte lysates with the same kinetics of inhibition—i.e., an initial period of synthesis for several minutes at the control rate followed by an abrupt decline in the rate of initiation; ( iii ) both inhibitions are prevented or reversed by eIF-2; ( iv ) GTP (2 mM) prevents, and ATP (2 mM) potentiates, the inhibition of protein synthesis induced by either inhibitor; ( v ) LI is associated with a protein kinase that also phosphorylates the 38,000-dalton subunit of elF-2. These findings indicate that a mechanism for the regulation of protein synthesis similar to that found in rabbit reticulocytes may be present in rat liver.

Skin is a viscoelastic material, whose properties modify with age, some diseases and certain therapeutic treatments.

Purified eukaryotic initiation factor, EIF-3, prepared from ascites cells dissociated rat liver 80S ribosomes into subunits. Ribosomes bearing endogenous mRNA and nascent peptide were not dissociated by EIF-3. When 80S ribosomes reconstituted from subunits were used as substrate the reaction had the following characteristics: Dissociation was rapid--the reaction being completed within 2 min at 30°. The extent of dissociation was directly proportional to the amount of EIF-3; with 21 μg of EIF-3 about 70% (or 10.5 μg) of the 80S monomers were dissociated. The dissociation of 80S monomers by EIF-3 decreased with increasing concentrations of magnesium. The reaction was not catalytic: 28 moles of EIF-3 were required to dissociate 1 mole of 80S ribosomes. The characteristic of the dissociation reaction promoted by EIF-3 and by E. coli initiation factor IF-3 are remarkably similar. The dissociation reaction provides a practical assay for EIF-3 independent of complimentation of other initiating factors.

The biological activities of Bacillus subtilis ribosomes pretreated with 0.5, 1, or 2 m m p -chloromercuribenzoate (PCMB) were examined. The ribosomes treated with 1 or 2 m m PCMB lost their capacity to synthesize polyphenylalanine as well as the capacity to bind phenylalanyl-transfer ribonucleic acid. On the other hand, approximately 70% of the polyphenylalanine synthesis capacity was lost with ribosomes pretreated with 0.5 m m PCMB. This inhibition was found to be due to a specific inhibition of the translocation step. The effect of 0.5 m m PCMB was reversed by 20 m m Mg 2+ without the loss of PCMB bound to ribosomes, while β-mercaptoethanol partially reversed the inhibitory effect with concomitant loss of bound PCMB.

Tanned sheep erythrocytes have been considered incapable of sensitization with the precipitinogen of protein A of Staphylococcus aureus , so that the activity of this antigen in serological reactions has so far been studied by means of the agar diffusion test (ADT) only. The precipitinogen of the protein A in this study was found to become attached to the tanned erythrocytes and to sensitize them for the passive hemagglutination test (PHT). It was determined that, in contrast to nonspecific reactivity between normal human serum and the precipitinogen in the ADT, the reaction in the PHT was of specific nature. Of seven species studied, all normal human, dog, and hog sera tested were positive in the PHT. However, the hemagglutinin titers of the sera of the two animal species by far exceeded those of the human sera. The data emphasized the usefulness of the highly sensitive PHT for assaying antibodies to protein A.