Abscisic acid is involved in the response of grape (Vitis vinifera L.) cv. Malbec leaf tissues to ultraviolet-B radiation by enhancing ultraviolet-absorbing compounds, antioxidant enzymes and membrane sterols.

We investigated the interactions of abscisic acid (ABA) in the responses of grape leaf tissues to contrasting ultraviolet (UV)-B treatments. One-year-old field-grown plants of Vitis vinifera L. were exposed to photosynthetically active radiation (PAR) where solar UV-B was eliminated by using polyester filters, or where PAR was supplemented with UV-B irradiation. Treatments combinations included weekly foliar sprays of ABA or a water control. The levels of UV-B absorbing flavonols, quercetin and kaempferol were significantly decreased by filtering out UV-B, while applied ABA increased their content. Concentration of two hydroxycinnamic acids, caffeic and ferulic acids, were also increased by ABA, but not affected by plus UV-B (+UV-B) treatments. Levels of carotenoids and activities of the antioxidant enzymes, catalase, ascorbate peroxidase and peroxidase were elevated by +ABA treatments, but only if +UV-B was given. Cell membrane beta-sitosterol was enhanced by ABA independently of +UV-B. Changes in photoprotective compounds, antioxidant enzymatic activities and sterols were correlated with lessened membrane harm by UV-B, as assessed by ion leakage. Oxidative damage expressed as malondialdehyde content was increased under +UV-B treatments. Our results suggest that the defence system of grape leaf tissues against UV-B is activated by UV-B irradiation with ABA acting downstream in the signalling pathway.

Association of a protease with polytene chromosomes of Drosophila melanogaster.

Incubation of Drosophila salivary glands with radioactive diisopropyl fluorophosphate results in the uniform labeling of polytene chromosomes. Extensive labeling is seen only when chromosome squashes are prepared by a formaldehyde fixation procedure and not by standard acetic acid techniques. The labeling is inhibited in the presence of tosylphenylalanine chloromethyl ketone and phenylmethane sulfonylfluoride but not by tosyllysine chloromethyl ketone, suggesting that a chymotrypsin-like serine protease is associated with the chromosomes. Protease inhibitors show no apparent effect on heat-shock specific puffing.

Reproductive biology of the native forage grass Trichloris crinita (Poaceae, Chloridoideae).

Trichloris crinita is a perennial forage grass species native to arid regions of the American continent. Due to its extensive area of distribution, good forage quality and resistance to drought and grazing, this species is widely utilised as forage and for revegetation purposes in environments with low water availability. Despite its importance, genetic improvement of T. crinita has been very limited, partly as consequence of the lack of knowledge on its mode of reproduction. In the present work, we studied the reproductive biology of T. crinita by means of embryological analyses, flow cytometric seed screen (FCSS), self-compatibility tests and progeny testing with morphological and molecular markers. Cytological analyses revealed embryo sacs with eight nuclei and of Polygonum type for all T. crinita accessions analysed. FCSS histograms exhibited two clear peaks corresponding to 2C and 3C DNA content, indicating embryo sacs of sexual origin. Controlled pollination experiments designed to evaluate seed set (%) demonstrated that T. crinita is self-compatible, whereas results from morphological and simple sequence repeat (SSR) marker analysis of progeny revealed lack of outcrossing. Together, these results indicate that T. crinita reproduces sexually. It is a self-compatible and autogamous species. It is expected that these data will have a positive impact in the genetics and breeding of this species, and therefore contribute to its proper utilisation in arid regions.

Progress and challenges in the preclinical assessment of cytokines.

This presentation focuses on the history and status of the preclinical toxicologic assessment of recombinant proteins. Cytokines and growth factors are used as examples. There has been an evolution of thought over the past dozen years on the testing of these substances that has ranged from their being considered nontoxic, human-specific proteins for which no predictive testing could be done in animals, to the present view that they can be toxic, relevant testing may be possible in animals, and assessment approaches should be science-based and case-by-case. The challenge of appropriate testing of recombinant proteins has caused toxicologists in both industry and in regulatory authorities to reconsider not only testing procedures but the purpose of preclinical assessment. The form of regulatory guidelines (guidance vs. inflexible testing protocols) has been questioned, and the need for interaction between scientists in industry and regulatory agencies has been strengthened. This process has advanced the science of toxicology.

Distribution of C3 and C4 grasses at different altitudes in a temperate arid region of Argentina.

The distribution of native C3 and C4 grasses in a temperate arid region of Mendoza, Argentina, was studied in six areas at different altitudes. C4 species predominate at low elevations in both relative species abundance and plant cover. At high elevations C3 species are dominant in cover and composition. At medium altitudes (1100-1600 m) grass species composition is balanced but plant cover of C3 species is greater. Of 31 genera in the whole area, 19 were C4. Only the genera Stipa (C3) and Aristida (C4) were present in all the six areas surveyed. The pattern of grass distribution shows high correlation with evapotranspiration and temperature parameters, but low correlation with rainfall. The relation between grass distribution and different climatic parameters is discussed.

Immunotoxicology and the new biotechnology.

The use of recombinant DNA techniques, cell fusion and novel bioprocessing in the pharmaceutical industry has assisted the development of many kinds of diagnostic and therapeutic products. Some directly affect the immune system (e.g. interleukins, interferons, tumour necrosis factor, and colony stimulating factors). Others (e.g. peptides, cytokines, growth factors, H2-receptor antagonists, non-steroidal anti-inflammatory drugs and neurohormonal agents) have immunological reactivity even though they are not designed to be immune modulators. The need to define the immunotoxicological potential of these products during preclinical safety evaluation was among the topics discussed at a recent meeting.

Molecular basis for the bidirectional modulation of the neuroendocrine and the immune systems.

Experimental evidence in many fields point towards the existence of a bidirectional communication between the neuroendocrine and immune systems. The immune response unifies the endocrine, nervous and immune systems. This integrated microenvironment includes lymphoid cell, nonlymphoid cells, cholinergic and adrenergic neurons and their neurohumoral products, biologically active substances including the cytokines and lymphokines produced by lymphoid and nonlymphoid cells, hormones and neuropeptides released by endocrine glands and regulatory cells of the brain, membrane and intracellular receptors which make possible the immune connections, and ions which are involved in the transmission of information and the higher nervous system activity which influences the immune microenvironment. Neuroendocrine circuits constitute only one type of efferent link between the brain and the immune compartment. The autonomic nervous system, via its innervation of many peripheral target tissues throughout the body, might also prove to be an important link to the immune system. While the precise mechanism(s) of neuroendocrine-immune relationships may not as yet completely defined, it is apparent that such interrelationships exist.

Interrelationships between water and cellular metabolism in Artemia cysts. IV. Adenosine 5'-triphosphate and cyst hydration.

The concentration of ATP in cysts of the brine shrimp, Artemia salina, has been studied as a function of cyst hydration. Cysts dried over CaSO4 contain 0.02 gH2O/g cysts and 0.54 +/- 0.05 (S.D.) mumoles of ATP/g dried cysts. Addition of water up to 0.05 g/g cysts produced no net change in the level of ATP during incubation. Hydration levels between 0.05 and 0.62 g/g cysts resulted in a net loss of ATP, whereas above 0.65 g/g cysts a net increase was observed with incubation time. No net change in the amount of ATP, compared with dried cysts, was detected between the latter two hydrations. These results, when integrated with those from previous work, indicate that conventional aerobic energy metabolism does not begin until cyst hydrations of about 0.65 g/g are achieved. The fate of ATP in cysts hydrated to levels lower than 0.65 g/g was discussed.

Bidirectional regulatory circuit between the immune and neuroendocrine systems.

The immune system and the neuroendocrine system affect each other via molecules and receptors shared by both systems. Activation of the neuroendocrine system results in a generalized alteration in host homeostasis and immune response. The interaction appears to be a complete circuit in that products of the immune system can also modulate nervous and neuroendocrine system responses. Since different hormones/soluble mediators are produced by neuroendocrine and immune cells, the particular alteration is a function of the induction stimulus.

Neuroendocrine-immune interactions: immunoregulatory signals mediated by neurohumoral agents.

While we generally think of the brain and nervous system as central to most basic life processes, the concept of immune regulation or modulation is a relatively new idea. The novelty of such an association may be rooted in classic concepts of neurologic function describing direct innervation of controlled tissues and stimulation across synapses. The involvement of the neuroendocrine system in the precise control of metabolic and a variety of cellular functions should preface its involvement in defense against and/or surveillance for aberrant cell replication. Moreover, a principal characteristic of any control mechanism is feedback from the affected system (be it an organ or single cell). In this framework, it is not unreasonable to expect bidirectional interactions between the nervous and immune systems. Direct innervation of lymphoid tissues was described a number of years ago. More recently, immunoregulatory function has been demonstrated in vitro with a variety of neuroendocrine molecules such as the biogenic amines, SP, CGRP, SOM, vasopressin, ACTH, the endorphins, enkephalins, neurotensin, NGF and VIP. Now it has been shown that many of these same or similar neuroregulatory molecules are produced by cells of the immune system. The possibility that neurotransmitters or peptides, or both, may play a role in vivo in the maintenance of immunocompetence is supported by the finding that specific receptors for the neurohumoral modulators are present on the surface of immunocompetent cells. Current hypotheses speculate that feedback control mechanisms are manifested through the production of lymphokines, PGs and leukotrienes. Though it has not been possible to clearly demonstrate the reciprocal interaction between the neuroendocrine and immune systems in vivo, the evidence to date points to its inevitability.

Norcocaine nitroxide. A potential hepatotoxic metabolite of cocaine.

Norcocaine nitroxide was found to be produced via the one-electron oxidation of N-hydroxynorcocaine by hepatic microsomal enzymes from induced and noninduced rats, hamsters, and mice in the presence of an NADPH-generating system. This reaction was demonstrated to be mediated by cytochrome P-450 as suggested by induction experiments using phenobarbital, which markedly enhanced the production of this nitroxide, and by the inhibition of this monooxygenase by metyrapone, which depressed the formation of this free radical. Unlike other nitroxides, norcocaine nitroxide was rapidly reduced by flavoproteins such as cytochrome P-450 reductase and FAD-monooxygenase, but not cytochrome P-450. We believe that since NADPH is consumed during the futile cycling of N-hydroxynorcocaine/norcocaine nitroxide and since NADPH is an essential cofactor of the glutathione reductase system, diminished reduced nucleotide may lead to depressed levels of cellular glutathione. In this manner, we theorize that cocaine initiates hepatotoxicity.

Design of biological equivalence programs for therapeutic biotechnology products in clinical development: a perspective.

The determination of biological equivalence requires that studies are conducted to establish that two molecules, two formulations, of two dosing regimens, for example, are indistinguishable with respect to safety and efficacy profiles that have been previously established. The criteria that are used to establish biological equivalence will depend on the nature of the change (e.g., molecular, process, formulation), the stage of the development program, the duration of treatment, and the intended clinical indications. Key components of an equivalence program include chemical characterization, in vitro and in vivo bioactivity against reference material, pharmacokinetics, and safety. Special considerations for patient populations, endogenous concentrations, environmental factors, immunogenicity, assay methodology, biochemical identity, pharmacodynamic equivalence, and statistical methodology are discussed. In addition, the role of preclinical in vivo assessments is addressed. Specific case studies provide insight into the varied nature of approaches that are currently employed.

Regulatory considerations for evaluating the pharmacology and toxicology of antisense drugs.

This article focuses on pharmacology and toxicology data that should be included in an Investigational New Drug Application (IND), a request to use an investigational drug in clinical trials. In general, pharmacology and toxicology testing programs for antisense compounds are held to the same regulatory standards applied to other new therapeutic classes. Biological properties of oligonucleotide therapeutics are mentioned where they may pertain to clinical safety issues. Nonclinical data submitted to the IND should characterize the pharmacology, disposition, and toxicology of a new drug; these data form the basis for clinical risk assessment. Concomitant evaluation of pharmacokinetics allows for better interpretation of in vivo studies and increased accuracy of dose extrapolation to humans. Recommendations for nonclinical drug development will be modified as new information regarding the biological properties of oligonucleotides becomes available.

Toxicologic evaluations of an immunotoxin, H65-RTA.

A series of acute and multiple dose toxicology studies were performed to support the clinical dose and to evaluate the systemic toxicity of an immunotoxin, H65-RTA. H65-RTA consists of a murine anti-CD5 monoclonal antibody and ricin A chain (RTA). The LD50 of H65-RTA was estimated to be between 60 and 62.5 mg/kg in the rat. H65-RTA was administered to the rat and the monkey as a bolus injection at doses of 0.1, 0.5, and 2 mg/kg and over 1-hr infusion at 0.2 and 2 mg/kg, respectively. Two to three weeks of postdosing recovery was included in the study design. Following repeated doses of H65-RTA, the following findings were demonstrated: peripheral edema, decreased body weight, decreased body temperature (monkey only) in addition to a general inflammatory reaction evidenced by changes in hematology, clinical chemistry, and urinalysis parameters. Histopathologically, chronic inflammation in the nonarticular soft tissue was found in the rat at doses of 0.1 mg/kg and higher and monkeys developed much more severe toxicity when compared to the rat at the same doses. Inflammation, hemorrhage, and/or edema were evident in a variety of tissues. Myeloid hyperplasia was also evident. Additional findings resulting from the drug-related stress involved adrenals, spleen, thymus, and lymph nodes. All toxicity was reversible. The antibody response was evident in rats at doses of 0.5 mg/kg and higher and in all monkeys at doses of 0.2 and 2 mg/kg. In conclusion, since H65 antibody does not cross-react with the T cells from either the rat or the cynomolgus monkey, the toxicity observed in the studies described above was not related to T lymphocytes and was probably due to a series of acute to subacute inflammatory reactions caused largely by the RTA moiety of H65-RTA.

Regulatory concerns in the development of topical recombinant ophthalmic and cutaneous wound healing biologics.

The use of recombinant DNA technology has enabled the development of an increasing number of endogenous growth regulatory peptides for potential use as therapeutic biologics. Numerous such recombinant peptides are now licensed, and many are in various stages of pharmaceutical development. Although currently there are a number of "Points to Consider" and related guidance documents available concerning various issues of biotechnology-derived products, the purpose of this article is to focus on the use of these biologics in topical ophthalmic and chronic cutaneous wound healing. Regulatory expectations with respect to product quality, safety, and efficacy that may be particularly germane to these products will be discussed. Providing regulatory guidance on these issues may not only facilitate the introduction of safe and effective new biologic therapies into clinical trials at the investigational level but also provide appropriate information to aid in their eventual approval for licensure and widespread clinical use.