Analyzing the joint effects of two antibodies and the design of molecularly engineered vaccines

A new analytical approach to determine how antibodies relate to each other in producing immunity is presented. One purpose of this analysis is to decide on the antigenic composition of vaccines. Regression analyses and single discrete analyses are shown to be inappropriate to this task. The proposed analyses makes multiple cut points on two pre-exposure antibody levels. At each resulting discrete classification, additive and multiplicative interaction parameters are calculated. Various models of joint antibody action are shown to produce two general patterns of interaction terms with this type of analysis. One pattern characterizes situations where each antibody, or something substituting for the action of each antibody, is always needed to eliminate all infections. This may be because the antigens to which the antibodies are directed are on separate microbes or because microbes can only be neutralized by the joint action of both antibodies. A second pattern characterizes situations where both antibodies act cooperatively but given high enough levels only one may be needed. This may be because the antibodies have the same molecular effect or because they act by separate means against the same organisms in an innoculum.     

A favourable assessment of the O.P.T.--histamine reaction in the method of von Redlich and Glick

During the course of an investigation of the properties of triglyceride lipase in nerve endings of the central nervous system (1) there arose a need for rapid determinations of a lipase in a large number of membrane preparations. This communication reports a procedure for the determination of glyceride lipase in which fatty acid, formed from a radioactive substrate by the lipase, is separated from glycerides on DEAE-cellulose paper.     

Mutagenesis by 4-nitrobenzofurazans and furoxans.

15 nitrobenzofurazans and 10 nitrobenzofuroxans synthesized primarily for testing as potential anti-rheumatic drugs were also tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100. The method used involved placing each compound in a "well" cut out of a plate of selective medium previously seeded with the appropriate tester strain, and then adding a rat liver microsome/cofactor mixture to one of the two wells on each plate. This method is considerably cheaper and more convenient than the conventional agar overlay technique, but in the present series of experiments failed to detect 4 compounds which could be detected by the overlay technique. Using a combination of the two techniques, 21 of the 25 compounds tested were found to be mutagenic. All 10 benzofuroxans and 9 of the 15 benzofurazans were detected as direct-acting mutagens with at least one of the two tester strains. Two of the benzofurazans gave positive results only in the presence of rat liver microsomes, and hence are pro-mutagens. One of the 4 benzofurazans which gave negative results for mutagenicity in these tests was found to be an efficient inhibitor of a neutral protease activity found in rheumatic synovial fluid, and may therefore have some potential as an anti-rheumatic drug.     

The ontogeny of social relationships with group companions among free-ranging infant rhesus monkeys II. Differentiation and attractiveness

Infants are frequently with their mothers when troop members initiate interaction with them. To what extent are the infants' companions attracted specifically to them rather than to the mother? A simple method is presented for approaching this question and for assessing the extent to which the relationships of 11 free-ranging rhesus (Macaca mulatta) infants on Cayo Santiago have acquired qualities independent of (differentiated from) those of the mother. Companions of all ages tend to be attracted specifically to infants between 1 and 14 weeks of age and not simply to their mothers. However, this tendency is less marked for sibling companions than for more distantly related companions. No differences are found between infants of high- and low-ranking mothers. Infantile attractiveness is not necessarily sustained when infants are 17 to 30 weeks of age. The degree to which relationships with infant companions and with older female companions have, differentiated by 17 to 30 weeks is negatively related to the companion's age. Infants show early and increasingly marked tendencies to develop differentiated relationships with one another; however, they show no signs of doing so with adult female companions by 30 weeks of age.     

The ontogeny of social relationships with group companions among free-ranging infant rhesus monkeys I. Social networks and differentiation

Social networks of infant rhesus monkeys (Macaca mulatta) in a free-ranging, lineage-based group on Cayo Santiago are described by assessing the extent to which four measures of positive interaction between infants and finely-divided categories of companions are associated with (1) degree of relatedness through maternal lines; (2) sex of the companion; (3) age of the companion; and (4) dominance rank of the infant's lineage. The results suggest that the infant's social network mirrors that of its mother both in the first weeks of life and as late as 30 weeks of age. Infants have more positive social interaction with close kin than with distant kin or with unrelated individuals, and thus function as members of their lineage from the beginning. They associate more with female companions than with male companions, and more with younger immatures than with older immatures. Finally, infants in the top-ranking lineage spend more time with their own relatives than do infants in other lineages. The fact that these patterns change little as the infant gains independence from the mother supports suggestions that early maternal influence serves to pass on aspects of the mother's social network. It is suggested that the ontogeny of early social relationships resembles a process of differentiation.     

A combined theoretical and experimental study of the interaction of metrizamide with proteins

A theory of isopycnic sedimentation of interacting systems provides a theoretical explanation for the isopycnic behavior of catalase and other proteins in metrizamide gradients of pH 7.0–7.5, particularly the bimodal banding patterns reported by some investigators. As for whether or not the model is realistic, a spectroscopic investigation provides an unambiguous demonstration of a reversible interaction between metrizamide and the catalase dimers formed by dissociation of catalase tetramer at pH 3. The metrizamide-catalase dimer complex(es) is of high bouyant density. In addition, metrizamide at pH 7.4 interacts with heat-denatured ovalbumin. These observations suggest that the denser band in the experimental bimodal isopycnic patterns might be a complex(es) of metrizamide with a comformationally altered state of the protein.     

Limiting nutrient patchiness and its rôle in phytoplankton ecology

Limiting nutrient patchiness is examined as a factor affecting the community structure and species succession of natural phytoplankton communities held in ammonia limited continuous culture at a dilution rate of 0.3 day^?1. It was found that under a homogeneous distribution of the limiting nutrient members of genus Chaetoceros dominated and when ammonia was added daily (patchy distribution), Skeletonema dominated. Intermediate patchiness gave rise to an assemblage dominated by both Chaetoceros and Skeletonema. The nutrient uptake ability of each assemblage was determined three weeks after experiment initiation. Each assemblage was best able to optimize uptake of ammonia under its particular patchy nutrient regime. Optimization of a patchy environment took place by an increased maximal uptake rate (V_max) while optimization of a homogeneous environment appeared to take place by increased substrate affinity (i.e., low K_s).This study demonstrates that limiting nutrient patchiness can alter the relative abundance of populations within a community based on each population's ability to exploit the limiting resource under a particular degree of patchiness. We also show that coexistence of two populations might be expected due to the patchiness of a single limiting nutrient. The importance of patchiness in relation to other factors which determine community structure is discussed.     

Neonatal heart rate variability and its relation to respiration

The heart rate and respiration signals from nine healthy full term neonates were studied using autoregressive spectral analysis and cross-correlation techniques. The heart rate spectra could be divided into three regions of activity: a very low frequency (VLF) region from 0-0.04 Hz; a low frequency (LF) band from 0.04-0.20 Hz; and a high frequency (HF) region above 0.20 Hz. The newborns exhibited very little respiratory sinus arrhythmia in their heart rate variability in contrast to the situation for adults and older infants. However, variations in heart rate correlated strongly with changes in the breath amplitude, leading to what may be termed a breath amplitude sinus arrhythmia. The neonatal heart rate behaviour under stable conditions of oscillation could be simulated with a nonlinear control model provided the delay time in the baroreceptor loop of the model was taken to be approximately 2 seconds longer than in adults. This is consistent with the immature neurological status of neonates.     

Conditions for the various types of co-operativity allowed by the adair,equation and their relation with the algebraic character of binding polynomials

A convenient way to obtain for any number, n, of sites, the functions of the constants of the Adair equation that decide the type of co-operativity of ligand binding to a non-dissociating protein is given and is illustrated by the examples n = 4 and n = 5. These functions are invariants of the binding polynomial and various of its derivatives.Although there are some simple sufficient conditions (inequalities relating successive Adair constants) for some co-operativity types, the full necessary and sufficient conditions even for uniform positive and negative co-operativity depend on very complicated functions of the constants for n > 4.However there are alternative ways of writing binding polynomials known as canonical forms. Up to at least n = 5, and probably beyond, the conditions that are complicated in terms of Adair constants are very simple in terms of the constants of canonical forms. For instance any fourth-degree polynomial can be written in the form p(x - α)⁴ + q(x - β)⁴ + 6μ (x - α)²(x - β)² although in three different ways. For one of these ways, the sign of μ distinguishes between mixed and uniform co-operativity. For any kind of mixed co-operativity μ > 0, while μ < 0 corresponds to uniform co-operativity. Advantages of the use of canonical forms are briefly commented on.     

Unusual origins of isotope effects in enzyme-catalysed reactions

High hydrostatic pressure is a neglected tool for probing the origins of isotope effects. In chemical reactions, normal primary deuterium isotope effects (DIEs) arising solely from differences in zero point energies are unaffected by pressure; but some anomalous isotope effects in which hydrogen tunnelling is suspected are partially suppressed. In some enzymatic reactions, high pressure completely suppresses the DIE. We have now measured the effects of high pressure on the parallel 13C heavy atom isotope effect of yeast alcohol dehydrogenase and found that it is also suppressed by high pressure and, similarly, suppressed in its entirety. Moreover, the volume changes associated with the suppression of both deuterium and heavy atom isotope effects are virtually identical. The equivalent decrease in activation volumes for hydride transfer, when one mass unit is added to the carbon end of a scissile C-H bond as when one mass unit is added to the hydrogen end, suggests a common origin. Given that carbon is highly unlikely to undergo tunnelling, it follows that hydrogen is not doing so either. The origin of these isotope effects must lie elsewhere. We offer protein domain motions as a possibility.     

Protein symmetry and the co-operative ligand-binding behaviour predicted by allosteric Koshland models

It is shown that the nearest-neighbour interaction two-conformation allosteric models of Koshland, Nemethy & Filmer (1966) predict binding curves with a centre of symmetry when the protein is also symmetrical and induced-fit is assumed. When nonexclusive binding to both conformations is assumed, the models predict that the family of homotropic binding curves obtained by varying the heterotropic ligand has a centre of symmetry. It is argued that the symmetry or asymmetry of binding curves is the main experimentally verifiable prediction of allosteric models insofar as they are models of interaction between protein subunits.Symmetry in a binding curve greatly simplifies the analysis of cooperative behaviour. The co-operative features possible with a symmetric binding curve for a four-site protein are analysed. The sign of co-operativity may either be uniform or change twice as saturation increases; the conditions for the various possibilities are given. For example, in terms of the intrinsic binding constants per site A^∥₁, A2, etc. the necessary and sufficient condition for positive macroscopic co-operativity over the whole symmetric binding curve is A^∥₁≤ A2, A ^∥₁ ≤ A3 which should be contrasted with the obvious A^∥₁ ≤ Al, AZ ≤A^∥₃ (positive microscopic co-operativity) which is only a sufficient but not a necessary condition. A symmetric curve may have one or three, but no more, extrema of the “Hill coefficient” h. For three extrema a change of sign of microscopic (but not necessarily macroscopic) co-operativity is necessary but not sufficient. In the case where there are off-centre maxima of h, then h < 2 everywhere on the curve.The Koshland models predict qualitative and quantitative restrictions on the forms of binding curves additional to that of symmetry. In tetrameric induced fit models, negative co-operativity in the mid-region of the curve and positive co-operativity in the outside regions is possible, but not the opposite, and three extrema of h are possible with uniform positive but not with uniform negative co-operativity.Thus by recognising the importance of symmetry it has been possible to describe and categorise all the co-operativity behaviour possible with the most plausible Koshland tetrameric models. Several experimental examples of probable non-exclusive binding to proteins and enzymes are discussed, and it is shown how the symmetry point of view illuminates their interpretation.     

Biosynthesis of 2-methylalkanes in the crickets Nemobius fasciatus and Gryllus pennsylvanicus.

The biosynthesis of 2-methylalkanes was studied in the crickets $\text{Nemobius}\text{fasciatus}$ and $\text{Gryllus}\text{pennsylvanicus}$. Labelled acetate, valine, and isobutyric acid were incorporated into the cuticular hydrocarbon of $\text{N.}\text{fasciatus}$ at levels of 6.0 ± 1, 6.5 ± 2, and 1.5 ± 0.7 percent respectively. The hydrocarbons of this insect are 20 percent 2-methylalkanes, primarily of even numbered carbon chain lengths, and 80% n-alkanes. Of the label incorporated into the hydrocarbon fraction, 28 ± 2 percent of sodium [1-¹⁴C] acetate, 98 ± 1 percent of L-[G-³H] valine, and 75 ± 10 percent of [1-¹⁴C] isobutyric acid were incorporated into the 2-methylalkanes. This suggests that valine is converted to isobutyric acid and is incorporated into the even numbered carbon chain length 2-methylalkanes during the initial stages of chain elongation. Similar data obtained in $\text{G.}\text{pennsylvanicus}$ suggests that leucine is converted to isovaleric acid which is then incorporated into the odd numbered carbon chain length 2-methylalkanes.     

Intramolecular determination of primary kinetic isotope effects in hydroxylations catalyzed by cytochrome P-450

Isotope effects for hydroxylation reactions catalyzed by cytochrome P-450 have usually been measured by comparing the overall reaction velocities of deuterated and nondeuterated substrates. Since the rate-limiting step is probably not the single reaction involving covalent bond cleavage, such an approach does not yield information about the primary isotope effect. We measured the primary kinetic isotope effect for benzylic hydroxylation by a method utilizing intramolecular competition, using the symmetrical substrate 1,3-diphenylpropane-1,1-d₂. These experiments yield a value of $\text{k}{}_{\mathrm{<mtext>H</mtext>}}\text{k}{}_{\mathrm{D}}\text{= 11}$, a larger effect than has previously been reported for benzylic hydroxylations.     

Interactions of bovine fibrinogen and thrombin. Early events in the development of clot structure.

Interactions which determine the rate of conversion of fibrinogen into monomer fibrin and the retention of monomer fibrin in a noncompactible form through interaction with residual fibrinogen (solution stabilization) were examined through the kinetics of formation of equilibrium compactible network at pH 7 and ionic strength 0.15. For studies of conversion, reactions with thrombin were at 29 or 2 °C, hirudin was added at successive times to inhibit thrombin, and compactible network was equilibrated at 2 °C, where solution stabilization is negligible. A substrate dependency of initial rate is interpreted on the basis of inactive complex formation between thrombin and both fibrinogen and monomer fibrin. At 29 or 2 °C specific rate constants are 32 or 2.9 × 10⁶ liter/mol, and association constants for inactive complex formation are 5.2 or 2.0 × 10⁵ liter/mol. The second peptide-A is removed from fibrinogen ~ 40-fold as rapidly as the first.With equilibration at 29 °C, compactible network does not appear until the solution stabilization ratio of residual fibrinogen/monomer fibrin is four. Thereafter, increasing amounts of compactible network appear. However, the stabilization ratio progressively decreases to approximately two, a situation which indicates the complexity of the stabilization mechanism.The thrombin-hirudin association constant is estimated to be 4.9 or 17 × 10¹¹ liter/mol at 29 or 2 °C.     

An observational study of scent-marking in coyotes, Canis latrans

Urination and defaecation patterns of free-ranging coyotes (Canis latrans) were studied in the Grand Teton National Park, Jackson, Wyoming, for two years. The vast majority of urinations by adult males and females were involved in ‘marking’, and differentiating between ‘marking’ and ‘elimination’ may not be necessary. Our results may be summarized as follows: (1) raised-leg urinations (RLU) performed by males were most frequently used in marking. (2) Females marked throughout the year using the squat (SQU) posture. (3) Snow tracking and reading snow sign resulted in a gross underestimate of the relative frequency of SQU's and a large overestimate in the relative frequency of defaecations (DEF) when compared to results obtained by direct observation. (4) There was sexual dimorphism for the contexts in which marking occurred. Overall, marking by males was associated with courtship and mating, with travelling, and with aggression. Marking by females was associated with the acquisition and possession of food and with the denning season. (5) Marking rates per coyote increased in groups larger than two animals. (6) RLU marking rates were greatest in areas of high intrusion when compared to denning areas and areas in which non-group members infrequently tresscent odours are important in orienting individuals in space but do not represent in and of themselves barriers to movement.     

Determination of uronic acid in uronides by application of a new microgram-scale isotope dilution method for carbon dioxide.

Conditions studied earlier by Tracey [(1948) Biochem. J.43, 185] are used for acid decarboxylation in sealed tubes of uronide samples supplemented with 6-¹⁴C-labeled uronic acid. The specific activity of the CO₂ evolved is measured as the ratio of radioactivity to area of the CO₂ peak obtained in a gas chromatogram. By appropriate standardization, samples containing some 60 nmol of uronic acid can be analyzed with reproducibility and apparent accuracy of about ±2% (mean deviation). The techniques developed for uronic acid analysis should apply with minor modification to any problem requiring accurate measurement of CO₂ in small amounts.     

Facilitation of adenylate cyclase stimulation in macrophages by lectins.

Preincubation of guinea pig peritoneal macrophages with concanavalin A (Con A) markedly enhanced the accumulation of 3′,5′-cyclic-adenosine monophosphate (cAMP) in response to the adenylate cyclase (AC) stimulators prostaglandin E₁ (PGE₁) and isoproterenol (IP). Basal cAMP levels were not altered. Maximal enhancement of cAMP accumulation was induced by preincubation with 50–100 μg/ml Con A for 10 min at 37 °C. Con A-induced facilitation of macrophage responsiveness was prevented by α-methyl-d-mannoside (αMM). No facilitation was induced by the divalent derivative, succinyl-Con A or by Con A immobilized on Sepharose beads. Con A-induced facilitation developed normally in macrophages treated with the microfilament blocking agent, cytochalasin B. The responsiveness of macrophages to PGE₁ and IP was also augmented by phytohemagglutinin (PHA) but wheat germ agglutinin (WGA), soy bean agglutinin (SBA), pokeweed mitogen (PWM), and Lotus tetragonolobus lectin (LL) showed no enhancing effect. The effect of Con A on cAMP levels was the result of augmented cAMP synthesis and not of reduced degradation or a block in cAMP egress from the cells. Lectin-induced facilitation of AC stimulation could be mediated via one of the following mechanisms: (i) induction of receptor clustering; (ii) causing a conformational change in the receptors; (iii) inhibition of negative cooperativity; (iv) causing an increase in membrane fluidity; (v) disruption of microtubules by acting as a Ca²⁺ ionophore; or (vi) inactivation of a sugar-containing inhibitor of AC.     

Horseradish peroxidase. XXV. An electron spin resonance study of the low temperature photochemical reaction of compound I.

The insoluble acrosome granule content of sea urchin sperm consists of a single 30,500 dalton protein named bindin. Bindin mediates species-specific recognition and adhesion of sperm to the egg surface. Bindin from $\text{Strongylocentrotus purpuratus}$ (Sp) and $\text{Strongylocentrotus franciscanus}$ (Sf) have tyrosine as their single N-terminal amino acid. The pI of Sp bindin is 6.62 and of Sf 6.59. Amino acid analysis reveals almost identical composition between the two species for 16 amino acids. Only two (or three) amino acids, Pro and Asx, show large species differences. Tryptic peptide maps of the two species of bindin show very similar patterns with 24 spots of identical correspondence.