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l h e claim that succinate and malate can directly stimulate the activity of the alternative oxidase i n plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1 042) was reinvestigated using sweet potato (Ipomoea batatas 1.) mitochondria. I n whole mitochondria, succinate (in the presence of malonate) and both i-and D-malate stimulated respiration via alternative oxidase in a pH-(and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both i-and D-malate, although the latter at only a low rate and only at acid pH. I n submitochondrial particle preparations with negligible malic enzyme activity, neither i-nor o-malate stimulated alternative oxidase activity. However, even i n the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither i-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. I n contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 p~ in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion.We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intramitochondrial generation of pyruvate via malic enzyme.

A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly. Lactococcus lactis, Streptococcus thermophilus, and Leuconostoc spp. all produced folate, while most Lactobacillus spp., with the exception of Lactobacillus plantarum, were not able to produce folate. Folate production was further investigated in L. lactis as a model organism for metabolic engineering and in S. thermophilus for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production. S. thermophilus excreted more folate than L. lactis, presumably as a result of differences in the number of glutamyl residues of the folate produced. In S. thermophilus 5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in L. lactis 5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in S. thermophilus, but pH had no effect on folate excretion by L. lactis. Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of p-aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis.

Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches 'bottle-necks' are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected 'rate-limiting' steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http ://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75 % of measured product formation rates.

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Regulatory approaches for allergen immunotherapy (AIT) products and the availability of high-quality AIT products are inherently linked to each other. While allergen products are available in many countries across the globe, their regulation is very heterogeneous. First, we describe the regulatory systems applicable for AIT products in the European Union (EU) and in the United States (US). For Europe, a depiction of the different types of relevant procedures, as well as the committees involved, is provided and the fundamental role of national agencies of the EU member states in this complex and unique network is highlighted. Furthermore, the regulatory agencies from Australia, Canada, Japan, Russia, and Switzerland provided information on the system implemented in their countries for the regulation of allergen products.While AIT products are commonly classified as biological medicinal products, they are made available by varying types of procedures, most commonly either by obtaining a marketing authorization or by being distributed as named patient products. Exemptions from marketing authorizations in exceptional cases, as well as import of allergen products from other countries, are additional tools applied by countries to ensure availability of needed AIT products. Several challenges for AIT products are apparent from this analysis and will require further consideration. K E Y W O R D Sallergen immunotherapy, allergic diseases, allergy, regulation | INTRODUCTIONThe availability of medicinal products to provide a reliable diagnosis of clinical allergy and effective treatment(s) is of critical importance for patients with suspected or proven allergy. Products for allergen immunotherapy (AIT) have been approved by national competent authorities in different regions of the world. However, the regulatory landscape governing the approval of these products is enormously heterogeneous-both within the European Union (EU) and even more so when looking globally-thereby rendering it extremely complicated and challenging to develop a harmonized, international approach to regulating these products.Pharmaceutical companies are increasingly focused on global strategies to develop and market their products. It is therefore very important to understand the current regulatory situation for allergen products from an international perspective, as this will have a direct impact on the availability of these medicinal products to patients throughout the world. Certain regulatory patterns can be observed on a global scale. For example, whereas AIT was previously mainly used and placed on the market on the basis of expert opinions with limited regulatory oversight, the requirements for high-quality clinical data for granting market access have greatly increased during the last 20 years. In the EU, legislation applicable for new and existing products 1,2 has been in force since 1989 demanding that allergen products are registered as medicinal products with corresponding requirements for clinical data. The development of the guid...

Adequate quality is essential for any medicinal product to be eligible for marketing. Quality includes verification of the identity, content and purity of a medicinal product in combination with a specified production process and its control. Allergen products derived from natural sources require particular considerations to ensure adequate quality. Here, we describe key aspects of the documentation on manufacturing and quality aspects for allergen immunotherapy products in the European Union and the United States. In some key parts, requirements in these areas are harmonized while other fields are regulated separately between both regions. Essential differences are found in the use of Reference Preparations, or the requirement to apply standardized assays for potency determination. As the types of products available are different in specific regions, regulatory guidance for such products may also be available in one specific region only, such as for allergoids in the European Union. Region-specific issues and priorities are a result of this. As allergen products derived from natural sources are inherently variable in their qualitative and quantitative composition, these products present special challenges to balance the variability and ensuring batch-to-batch consistency. Advancements in scientific knowledge on specific allergens and their role in allergic disease will consequentially find representation in future regulatory guidelines.

Lactococcus lactis NZ9010 in which the las operon-encoded ldh gene was replaced with an erythromycin resistance gene cassette displayed a stable phenotype when grown under aerobic conditions, and its main end products of fermentation under these conditions were acetate and acetoin. However, under anaerobic conditions, the growth of these cells was strongly retarded while the main end products of fermentation were acetate and ethanol. Upon prolonged subculturing of this strain under anaerobic conditions, both the growth rate and the ability to produce lactate were recovered after a variable number of generations. This recovery was shown to be due to the transcriptional activation of a silent ldhB gene coding for an Ldh protein (LdhB) with kinetic parameters different from those of the native las operon-encoded Ldh protein. Nevertheless, cells producing LdhB produced mainly lactate as the end product of fermentation. The mechanism underlying the ldhB gene activation was primarily studied in a single-colony isolate of the recovered culture, designated L. lactis NZ9015. Integration of IS981 in the upstream region of ldhB was responsible for transcription activation of the ldhB gene by generating an IS981-derived ؊35 promoter region at the correct spacing with a natively present ؊10 region. Subsequently, analysis of 10 independently isolated lactate-producing derivatives of L. lactis NZ9010 confirmed that the ldhB gene is transcribed in all of them. Moreover, characterization of the upstream region of the ldhB gene in these derivatives indicated that site-specific and directional IS981 insertion represents the predominant mechanism of the observed recovery of the ability to produce lactate.Homolactic fermentation by lactic acid bacteria involves the classical Embden-Meyerhoff-Parnas pathway leading to pyruvate, which is converted to lactic acid by lactate dehydrogenase. This enzyme and the gene that encodes it have been studied in many lactic acid bacteria, including Lactococcus lactis (11, 34), Streptococcus thermophilus (19), and various lactobacilli (2,15,47,51). L. lactis is the best-studied representative of this group, and the complete and partial genomes of several strains have been determined (4, 29). The gene encoding L. lactis Ldh was identified and characterized by Llanos and coworkers in 1992 (33, 34). The ldh gene is the last gene of the so-called lactic acid synthesis or las operon, which also encodes the glycolytic enzymes phosphofructokinase and pyruvate kinase. Transcription of the las operon was shown to yield a polycistronic transcript encompassing all three genes. But under some conditions, transcripts representing only two genes (pfk and pyk or pyk and ldh) or even a single gene (ldh) of the operon were also detected, which probably resulted from RNA processing upstream of the pyk and ldh genes (38). It has been shown that the las operon is subject to CcpAmediated carbon catabolite transcriptional activation, and a CcpA target site (cre sequence) was found within the las promoter region (38). The...