Joseph Bonaventura

Barnacle in vitro assays for biologically active substances: Toxicity and Settlement inhibition assays using mass cultured Balanus amphitrite amphitrite darwin

The development of non‐toxic or non‐polluting antifouling additives that can be formulated in practical coatings requires assays involving target organisms. Assays that test both for the effective and toxic concentrations of active compounds are useful. It is also desirable if the assay can provide information regarding the performance that can be expected if the compounds are incorporated into different matrices. Described here are the simple laboratory assays that have been developed using the barnacle, Balanus amphitrite, a common fouling organism found throughout temperate and tropical seas. One of the assays depends on synchronous year‐round mass culture, the procedure for which is described, of cypris larvae. The laboratory assays provide quantitative estimates of toxicity and settlement inhibition of barnacles. The methods described provide an excellent system for the use of barnacles to study the interaction of the test compounds and surfaces.

Ascaris haemoglobin is a nitric oxide-activated ‘deoxygenase’

The parasitic nematode Ascaris lumbricoides infects one billion people worldwide. Its perienteric fluid contains an octameric haemoglobin that binds oxygen nearly 25,000 times more tightly than does human haemoglobin. Despite numerous investigations, the biological function of this molecule has remained elusive. The distal haem pocket contains a metal, oxygen and thiol, all of which are known to be reactive with nitric oxide. Here we show that Ascaris haemoglobin enzymatically consumes oxygen in a reaction driven by nitric oxide, thus keeping the perienteric fluid hypoxic. The mechanism of this reaction involves unprecedented chemistry of a haem group, a thiol and nitric oxide. We propose that Ascaris haemoglobin functions as a ‘deoxygenase’, using nitric oxide to detoxify oxygen. The structural and functional adaptations of Ascaris haemoglobin suggest that the molecular evolution of haemoglobin can be rationalized by its nitric oxide related functions.

NO is necessary and sufficient for egg activation at fertilization.

The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest—particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.

Human erythrocyte catalase: an improved method of isolation and a reevaluation of reported properties.

An improved method for the isolation of human erythrocyte catalase from large volumes of cells is described. Time of isolation has been reduced and the need for larger equipment has been minimized. The yield is 200–300 mg per liter of packed cells. Amino acid composition, molecular weight, spectral constants, heme content, activity, and N-terminal residues have been determined. Although the isolative procedure is a modification of that of Morikofer-Zwez et al. (1969) (Eur. J. Biochem.11:49), the lability of the product toward oxygen as reported by them has not been observed nor has it been possible to produce extensive changes in properties of the material by subjecting it to the adverse conditions that they describe. However, by chromatographic procedures, it has been shown that the catalase immediately after isolation has a heterogeneity different than they describe and that alteration with time does occur. The cause of the heterogeneity has not been determined.

The complete amino acid sequence of bovine liver catalase and the partial sequence of bovine erythrocyte catalase.

Abstract The data upon which the sequence of the 506 residues in the subunit of bovine liver catalase (BLC) is based are presented in detail. A partial sequence of bovine erythrocyte catalase (BEC) which accounts for 493 residues shows complete concordance with the BLC data. On the other hand, BEC has at least 517 residues, that is, an extension beyond the C terminus of the BLC data. Although normally BLC has only 506 residues, there is evidence that, at some point in its history, it also had the C-terminal extension. It is speculated that this extension is lost in BLC either through a different processing of the molecule in liver than in erythrocytes or by partial degradation in the first stages of catabolism.

Rapid identification of monospecific monoclonal antibodies using a human proteome microarray

To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His6 fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.

Barnacle cement: a polymerization model based on evolutionary concepts

SUMMARY Enzymes and biochemical mechanisms essential to survival are under extreme selective pressure and are highly conserved through evolutionary time. We applied this evolutionary concept to barnacle cement polymerization, a process critical to barnacle fitness that involves aggregation and cross-linking of proteins. The biochemical mechanisms of cement polymerization remain largely unknown. We hypothesized that this process is biochemically similar to blood clotting, a critical physiological response that is also based on aggregation and cross-linking of proteins. Like key elements of vertebrate and invertebrate blood clotting, barnacle cement polymerization was shown to involve proteolytic activation of enzymes and structural precursors, transglutaminase cross-linking and assembly of fibrous proteins. Proteolytic activation of structural proteins maximizes the potential for bonding interactions with other proteins and with the surface. Transglutaminase cross-linking reinforces cement integrity. Remarkably, epitopes and sequences homologous to bovine trypsin and human transglutaminase were identified in barnacle cement with tandem mass spectrometry and/or western blotting. Akin to blood clotting, the peptides generated during proteolytic activation functioned as signal molecules, linking a molecular level event (protein aggregation) to a behavioral response (barnacle larval settlement). Our results draw attention to a highly conserved protein polymerization mechanism and shed light on a long-standing biochemical puzzle. We suggest that barnacle cement polymerization is a specialized form of wound healing. The polymerization mechanism common between barnacle cement and blood may be a theme for many marine animal glues.

Structural basis for the root effect in haemoglobin.

The remarkable ability of Root effect haemoglobins to pump oxygen against high O2 gradients results from extreme, acid-induced reductions in O2 affinity and cooperativity. The long-sought mechanism for the Root effect, revealed by the 2 Å crystal structure of the ligand-bound haemoglobin from Leiostomus xanthurus at pH 7.5, unexpectedly involves modulation of the R-state. Key residues strategically assemble positive-charge clusters across the allosteric β1β2-interface in the R-state. At low βH, protonation of the βN terminus and His 147(HC3)β within these clusters is postulated to destabilize the R-state and promote the acid-triggered, allosteric R→T switch with concomitant O2 release. Surprisingly, a set of residues specific to Root effect haemoglobins recruit additional residues, conserved among most haemoglobins, to produce the Root effect.

Nitric Oxide is Required for Tactile Learning in Octopus vulgaris

Nitric oxide, produced by nitric oxide synthase in brain tissue, is essential for several different kinds of learning in vertebrates. We present the first evidence that it is also essential for learning in an invertebrate. Intramuscular injections of an inhibitor of the enzyme completely block touch learning in Octopus vulgaris. Eight control animals learned a touch paradigm, but none of eight synthase-inhibited ones learned it.

How are climate and marine biological outbreaks functionally linked

Since the mid-1970s, large-scale episodic events such as disease epidemics, mass mortalities, harmful algal blooms and other population explosions have been occurring in marine environments at an historically unprecedented rate. The variety of organisms involved (host, pathogens and other opportunists) and the absolute number of episodes have also increased during this period. Are these changes coincidental? Between 1972 and 1976, a global climate regime shift took place, and it is manifest most clearly by a change in strength of the North Pacific and North Atlantic pressure systems. Consequences of this regime shift are: (1) prolonged drought conditions in the Sahel region of Africa; (2) increased dust supply to the global atmosphere, by a factor of approximately four; (3) increased easterly trade winds across the Atlantic; (4) increased eolian transport of dust to the Atlantic and Caribbean basins; and (5) increased deposition of iron-rich eolian dust to typically iron-poor marine regions. On the basis of well-documented climate and dust observations and the widely accepted increase in marine outbreak rates, this paper proposes that the increased iron supply has altered the micronutrient factors limiting growth of opportunistic organisms and virulence of pathogenic microbes, particularly in macronutrient-rich coastal systems.