Use of green fluorescent protein (GFP) as a molecular reporter is restricted by several environmental factors, such as its requirement for oxygen in the development of the fluorophore, and its poor fluorescence at low pH. There are conflicting data on these limitations, however, and systematic studies to assess the importance of these factors for growing bacterial cultures are lacking. In the present study, homogeneous expression of the gfpmut3* gene directed by a synthetic constitutive lactococcal promoter was demonstrated in batch cultures and in biofilms of Streptococcus gordonii DL1. A lower limit of oxygen concentration for maturation of the GFP fluorophore was determined: fluorescence was emitted at 0.1 p.p.m. dissolved oxygen (in conventionally prepared anaerobic media lacking reducing agents), whereas no fluorescence was detected in the presence of 0.025 p.p.m. dissolved oxygen (obtained by addition of L-cysteine as reducing agent). When an anaerobically grown (non-fluorescent) >50 microm thick biofilm was shifted to aerobic conditions, fluorescence could be detected within 4 min, reaching a maximum over the next 16 min. It was not possible to detect any fluorescence gradients (lateral or vertical) within the >50 microm thick biofilm, and fluorescence development after the shift to aerobic conditions occurred throughout the biofilm (even at the substratum). This suggests that oxygen gradients, which might result in reduced GFP fluorescence, did not exist in the >50 microm thick biofilm of this organism. Production of lactic acid and the subsequent acidification in batch cultures of S. gordonii DL1 led to a decrease in fluorescence intensity. However, severe pH reduction was prevented when the bacterium was grown as a biofilm in a flowcell, and a homogeneous distribution of a strong fluorescence signal was observed. These findings show that GFP can be applied to studies of oxygen-tolerant anaerobic bacteria, that densely packed, flowcell-grown biofilms of S. gordonii do not develop oxygen gradients inhibitory to GFP fluorescence development, and that the often transient nature of GFP fluorescence in acid-producing bacteria can be overcome in flowcells, probably by the elimination of metabolic by-product accumulation.

The phagocytosis and intracellular destruction of bacteria by rabbit polymorphonuclear leucocytes has been studied in vitro under defined conditions. The efficient and continuing ingestion of bacteria was dependent upon (a) opsonic factors present in fresh rabbit serum as well as upon, (b) the availability of an adequate supply of glucose in the medium. The effects of selected enzymatic inhibitors on the metabolic and functional activities of the leucocytes was investigated. Cyanide which inhibited oxygen consumption had no effect on the ingestion or inactivation of bacteria, Iodoacetate and arsenite which blocked glycolysis produced a marked inhibition in particle ingestion. 2,4-Dinitrophenol which stimulated both oxygen consumption and glycolysis, depressed phagocytosis after a 1 hour latent period. It was concluded that phagocytosis was an energy-requiring process in which glycolysis served as the most important source of energy. Leucocytes which were ingesting heat-killed bacteria exhibited increases in oxygen consumption, glucose utilization, and lactic acid synthesis. The effect of particle ingestion on glycogen metabolism was characterized by an initial period of glycogenolysis followed by an enhanced rate of glycogen synthesis. Leucocytes which had previously ingested heat-killed bacteria also demonstrated increased rates of phagocytosis.

Concentrations of bacteria in cerebrospinal fluid ranged from 4.5 X 10(3) to 3 X 10(8) colony-forming units/ml in 27 patients with bacterial meningitis before antibiotic therapy and from 4 X 10(1) to 1.4 X 10(6) CFU/ml in four patients after one to two days of antibiotic therapy. All patients with persistent positive cultures had pretreatment concentrations of 10(7) CFU/ml or greater. A significant association was observed between cerebrospinal fluid lactic acid dehydrogenase activity and concentrations of bacteria (p less than 0.01). Large inocula of Hemophilus influenzae type b (10(7)) increased the minimal inhibitory concentration for penicillin and ampicillin but not for chloramphenicol. The minimal inhibitory concentration of each of the three antibiotics increased when group B streptococci were assayed. These data indicate that persistence of a positive culture may be related to large initial concentrations of bacteria. The relative "resistance" in vitro of large inocula possibly contributes to this persistence. These observations are also consistent with the hypothesis that lactic acid dehydrogenase activity in cerebrospinal fluid is derived from bacteria.

The adhering human Lactobacillus acidophilus strain LA1 inhibits the cell association and cell invasion of enteropathogens in cultured human intestinal Caco-2 cells (M. F. Bernet, D. Brassard, J. R. Neeser, and A. L. Servin, Gut 35:483-489, 1994). Here, we demonstrate that strain LA1 developed its antibacterial activity in conventional or germ-free mouse models orally infected by Salmonella typhimurium. We present evidence that the spent culture supernatant of strain LA1 (LA1-SCS) contained antibacterial components active against S. typhimurium infecting the cultured human intestinal Caco-2 cells. The LA1-SCS antibacterial activity was observed in vitro against a wide range of gram-negative and gram-positive pathogens, such as Staphylococcus aureus, Listeria monocytogenes, S. typhimurium, Shigella flexneri, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter cloacae. By contrast, no activity was observed against species of the normal gut flora, such as lactobacilli and bifidobacteria. The LA1-SCS antibacterial activity was insensitive to proteases and independent of lactic acid production.

This study was undertaken to elucidate whether eating a fermented milk containing Lactobacillus acidophilus La1 and bifidobacteria could induce changes in intestinal flora and modulate the immune response in man. Volunteers consumed a fermented milk containing L. acidophilus La1 and bifidobacteria over a period of three weeks during which an attenuated Salmonella typhi Ty21a was administered to mimic an enteropathogenic infection. A control group ate no fermented foods but received the S. typhi Ty21a. Faecal flora analyses showed an increase in L. acidophilus and bifidobacterial counts during fermented milk intake. The specific serum IgA titre rise to S. typhi Ty21a in the test group was>> 4-fold and significantly higher (P = 0.04) than in the control group. An increase in total serum IgA was also observed. These results indicate that lactic acid bacteria which can persist in the gastrointestinal tract can act as adjuvants to the humoral immune response.

Spoilage of fresh and lightly preserved fish products is caused by microbial action. This paper reviews the current knowledge in terms of the microbiology of fish and fish products with particular emphasis on identification of specific spoilage bacteria and the qualitative and quantitative biochemical indicators of spoilage. Shewanella putrefaciens and Pseudomonas spp. are the specific spoilage bacteria of iced fresh fish regardless of the origin of the fish. Modified atmosphere stored marine fish from temperate waters are spoiled by the CO2 resistant Photobacterium phosphoreum whereas Gram-positive bacteria are likely spoilers of CO2 packed fish from fresh or tropical waters. Fish products with high salt contents may spoil due to growth of halophilic bacteria (salted fish) or growth of anaerobic bacteria and yeasts (barrel salted fish). Whilst the spoilage of fresh and highly salted fish is well understood, much less is known about spoilage of lightly preserved fish products. It is concluded that the spoilage is probably caused by lactic acid bacteria, certain psychotrophic Enterobacteriaceae and/or Photobacterium phosphoreum. However, more work is needed in this area.

In the past decade it has become clear that the lactic acid bacterium Lactobacillus plantarum occupies a diverse range of environmental niches and has an enormous diversity in phenotypic properties, metabolic capacity and industrial applications. In this review, we describe how genome sequencing, comparative genome hybridization and comparative genomics has provided insight into the underlying genomic diversity and versatility of L. plantarum. One of the main features appears to be genomic life-style islands consisting of numerous functional gene cassettes, in particular for carbohydrates utilization, which can be acquired, shuffled, substituted or deleted in response to niche requirements. In this sense, L. plantarum can be considered a "natural metabolic engineer".

Polylactide polymers have gained enormous attention as a replacement for conventional synthetic packaging materials in the last decade. By being truly biodegradable, derived from renewable resources and by providing consumers with extra end-use benefits such as avoiding paying the "green tax" in Germany or meeting environmental regulations in Japan, polylactides (PLAs) are a growing alternative as a packaging material for demanding markets. The aim of this paper is to review the production techniques for PLAs, summarize the main properties of PLA and to delineate the main advantages and disadvantages of PLA as a polymeric packaging material. PLA films have better ultraviolet light barrier properties than low density polyethylene (LDPE), but they are slightly worse than those of cellophane, polystyrene (PS) and poly(ethylene terephthalate) (PET). PLA films have mechanical properties comparable to those of PET and better than those of PS. PLA also has lower melting and glass transition temperatures than PET and PS. The glass transition temperature of PLA changes with time. Humidity between 10 and 95% and storage temperatures of 5 to 40 degrees C do not have an effect on the transition temperature of PLA, which can be explained by its low water sorption values (i.e. <100 ppm at Aw = 1). PLA seals well at temperatures below the melting temperature but an appreciable shrinking of the films has been noted when the material is sealed near its melting temperature. Solubility parameter predictions indicate that PLA will interact with nitrogen compounds, anhydrides and some alcohols and that it will not interact with aromatic hydrocarbons, ketones, esters, sulfur compounds or water. The CO2, O2 and water permeability coefficients of PLA are lower than those of PS and higher than those of PET. Its barrier to ethyl acetate and D-limonene is comparable to PET. The amount of lactic acid and its derivatives that migrate to food simulant solutions from PLA is much lower than any of the current average dietary lactic acid intake values allowed by several governmental agencies. Thus, PLA is safe for use in fabricating articles for contact with food.

BACKGROUND

Genome-scale flux models are useful tools to represent and analyze microbial metabolism. In this work we reconstructed the metabolic network of the lactic acid bacteria Lactococcus lactis and developed a genome-scale flux model able to simulate and analyze network capabilities and whole-cell function under aerobic and anaerobic continuous cultures. Flux balance analysis (FBA) and minimization of metabolic adjustment (MOMA) were used as modeling frameworks.

RESULTS

The metabolic network was reconstructed using the annotated genome sequence from L. lactis ssp. lactis IL1403 together with physiological and biochemical information. The established network comprised a total of 621 reactions and 509 metabolites, representing the overall metabolism of L. lactis. Experimental data reported in the literature was used to fit the model to phenotypic observations. Regulatory constraints had to be included to simulate certain metabolic features, such as the shift from homo to heterolactic fermentation. A minimal medium for in silico growth was identified, indicating the requirement of four amino acids in addition to a sugar. Remarkably, de novo biosynthesis of four other amino acids was observed even when all amino acids were supplied, which is in good agreement with experimental observations. Additionally, enhanced metabolic engineering strategies for improved diacetyl producing strains were designed.

CONCLUSIONS

The L. lactis metabolic network can now be used for a better understanding of lactococcal metabolic capabilities and potential, for the design of enhanced metabolic engineering strategies and for integration with other types of 'omic' data, to assist in finding new information on cellular organization and function.

OBJECTIVE

We conducted this study to determine the result of prolonged cardiopulmonary resuscitation (CPR) with extracorporeal membrane oxygenation (ECMO) and the predictive factors for hospital discharge and ECMO weaning.

BACKGROUND

Prolonged CPR carries considerable associated mortality and morbidity. As yet, ECMO for prolonged CPR has no definite results. Only small groups of patients and no detailed analysis have been reported.

METHODS

Candidates for ECMO resuscitation were patients in cardiac arrest receiving CPR >10 min without return of spontaneous circulation and no absolute contraindication. Venoarterial ECMO was set up during CPR. We reviewed the data of 57 prolonged CPR patients who received ECMO during CPR over a six-year period.

RESULTS

The mean duration of CPR was 47.6 +/- 13.4 min and that of ECMO was 96.1 +/- 87.9 h. The rate of weaning was 66.7%, and the survival rate was 31.6%. Multiple-organ failure was the major reason for mortality, despite successful weaning. Among survivors, long-term follow-up revealed 88.9% survival, and only 5.6% had a severe neurologic deficit. The results indicate that a shorter CPR duration, postcardiotomy arrest, myocardial indicators, a hepatic indicator, and lactic acid are significantly correlated with both weaning and survival, whereas late damage (level on the third or seventh day of reperfusion) rather than initial damage (level on the first day) was more predictive of the results.

CONCLUSIONS

Prolonged CPR rescue by ECMO provides an acceptable survival rate and outcome in survivors. Our results of the selected cases encourage further investigations of the wider application of ECMO in CPR.

In the 1920s, Otto Warburg observed that tumor cells consumed a large amount of glucose, much more than normal cells, and converted most of it to lactic acid. This phenomenon, now known as the "Warburg effect," is the foundation of one of the earliest general concepts of cancer: that a fundamental disturbance of cellular metabolic activity is at the root of tumor formation and growth. In the ensuing decades, as it became apparent that abnormalities in chromosomes and eventually individual genes caused cancer, the "metabolic" model of cancer lost a good deal of its appeal, even as emerging technologies were exploiting the Warburg effect clinically to detect tumors in vivo. We now know that tumor suppressors and proto-oncogenes influence metabolism, and that mutations in these genes can promote a metabolic phenotype supporting cell growth and proliferation. Thus, these advances have unified aspects of the metabolic and genetic models of cancer, and have stimulated a renewed interest in the role of cellular metabolism in tumorigenesis. This review reappraises the notion that dysregulated cellular metabolism is a key feature of cancer, and discusses some metabolic issues that have escaped scrutiny over the years and now deserve closer attention.

Controlled-release drug delivery systems have many applications, including treatments for hormone deficiencies and chronic pain. A biodegradable device that could provide multi-dose drug delivery would be advantageous for long-term treatment of conditions requiring pulsatile drug release. In this work, biodegradable polymeric microchips were fabricated that released four pulses of radiolabelled dextran, human growth hormone or heparin in vitro. Heparin that was released over 142 days retained on average 96 +/- 12% of its bioactivity. The microchips were 1.2 cm in diameter, 480-560 microm thick and had 36 reservoirs that could each be filled with a different chemical. The devices were fabricated from poly(L-lactic acid) and had poly(D,L-lactic-co-glycolic acid) membranes of different molecular masses covering the reservoirs. A drug delivery system can be designed with the potential to release pulses of different drugs at intervals after implantation in a patient by using different molecular masses or materials for the membrane.

The in vitro degradation behaviour of a wide range of poly(D,L-lactic-co-glycolic acid) (PLGA) has been examined in terms of degree of degradation and morphological change during an incubation period of up to 53 d. Gel permeation chromatography and differential scanning calorimetry were employed to characterize their degradation profiles. It was found that amorphous PLGA exhibited a transient multiple crystallization behaviour of D- or L-lactic acid oligomers during degradation. This indicated that the hydrolytic scission of ester bonds tends to primarily target the linkage between glycolic acid and D- or L-lactic acid or glycolic acid. In addition, two distinctive glass transition temperatures appeared when these crystallization phenomena occurred, suggesting the transient presence of fast and slowly eroding polymer domains within microspheres during the degradation. This study supports the heterogeneous bulk degradation for PLGA microspheres which has been proposed recently for a large specimen.

It is increasingly recognized that material surface topography is able to evoke specific cellular responses, endowing materials with instructive properties that were formerly reserved for growth factors. This opens the window to improve upon, in a cost-effective manner, biological performance of any surface used in the human body. Unfortunately, the interplay between surface topographies and cell behavior is complex and still incompletely understood. Rational approaches to search for bioactive surfaces will therefore omit previously unperceived interactions. Hence, in the present study, we use mathematical algorithms to design nonbiased, random surface features and produce chips of poly(lactic acid) with 2,176 different topographies. With human mesenchymal stromal cells (hMSCs) grown on the chips and using high-content imaging, we reveal unique, formerly unknown, surface topographies that are able to induce MSC proliferation or osteogenic differentiation. Moreover, we correlate parameters of the mathematical algorithms to cellular responses, which yield novel design criteria for these particular parameters. In conclusion, we demonstrate that randomized libraries of surface topographies can be broadly applied to unravel the interplay between cells and surface topography and to find improved material surfaces.

Metabolic acidosis is characterized by a primary reduction in serum bicarbonate (HCO(3)(-)) concentration, a secondary decrease in the arterial partial pressure of carbon dioxide (PaCO(2)) of approximately 1 mmHg for every 1 mmol/l fall in serum HCO(3)(-) concentration, and a reduction in blood pH. Acute forms (lasting minutes to several days) and chronic forms (lasting weeks to years) of the disorder can occur, for which the underlying cause/s and resulting adverse effects may differ. Acute forms of metabolic acidosis most frequently result from the overproduction of organic acids such as ketoacids or lactic acid; by contrast, chronic metabolic acidosis often reflects bicarbonate wasting and/or impaired renal acidification. The calculation of the serum anion gap, calculated as [Na(+)] - ([HCO(3)(-)] + [Cl(-)]), aids diagnosis by classifying the disorders into categories of normal (hyperchloremic) anion gap or elevated anion gap. These categories can overlap, however. Adverse effects of acute metabolic acidosis primarily include decreased cardiac output, arterial dilatation with hypotension, altered oxygen delivery, decreased ATP production, predisposition to arrhythmias, and impairment of the immune response. The main adverse effects of chronic metabolic acidosis are increased muscle degradation and abnormal bone metabolism. Using base to treat acute metabolic acidosis is controversial because of a lack of definitive benefit and because of potential complications. By contrast, the administration of base for the treatment of chronic metabolic acidosis is associated with improved cellular function and few complications.

The anaerobic threshold (AnT) is defined as the highest sustained intensity of exercise for which measurement of oxygen uptake can account for the entire energy requirement. At the AnT, the rate at which lactate appears in the blood will be equal to the rate of its disappearance. Although inadequate oxygen delivery may facilitate lactic acid production, there is no evidence that lactic acid production above the AnT results from inadequate oxygen delivery. There are many reasons for trying to quantify this intensity of exercise, including assessment of cardiovascular or pulmonary health, evaluation of training programs, and categorization of the intensity of exercise as mild, moderate, or intense. Several tests have been developed to determine the intensity of exercise associated with AnT: maximal lactate steady state, lactate minimum test, lactate threshold, OBLA, individual anaerobic threshold, and ventilatory threshold. Each approach permits an estimate of the intensity of exercise associated with AnT, but also has consistent and predictable error depending on protocol and the criteria used to identify the appropriate intensity of exercise. These tests are valuable, but when used to predict AnT, the term that describes the approach taken should be used to refer to the intensity that has been identified, rather than to refer to this intensity as the AnT.

The newly cloned proton-linked monocarboxylate transporter MCT3 was shown by Western blotting and immunofluorescence confocal microscopy to be expressed in all muscle fibers. In contrast, MCT1 is expressed most abundantly in oxidative fibers but is almost totally absent in fast-twitch glycolytic fibers. Thus MCT3 appears to be the major MCT isoform responsible for efflux of glycolytically derived lactic acid from white skeletal muscle. MCT3 is also expressed in several other tissues requiring rapid lactic acid efflux. The expression of both MCT3 and MCT1 was decreased by 40-60% 3 weeks after denervation of rat hind limb muscles, whereas chronic stimulation of the muscles for 7 days increased expression of MCT1 2-3-fold but had no effect on MCT3 expression. The kinetics and substrate and inhibitor specificities of monocarboxylate transport into cell lines expressing only MCT3 or MCT1 have been determined. Differences in the properties of MCT1 and MCT3 are relatively modest, suggesting that the significance of the two isoforms may be related to their regulation rather than their intrinsic properties.

The purpose of this review article on the lactic acid bacteria grew from an early curiosity and a desire to convey and impart the broad scope of literary information on their functions as starter cultures, in the manufacture of fermentation products such as dairy products and alcoholic beverages, as well as their contribution to better health. This review article is an attempt to empower the reader and to circumvent the difficult task in acquiring and elucidating a large body of information. The intent is to familiarize the reader with the various lactic species, their habitat or source, associated food, physiological characteristics, colonial morphology, biochemical characteristics, culture media (enrichment, nonselective, and selective), classic description, and taxonomy. This review provides information on Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Carnobacterium, and Enterococcus. Trends are presented, such as the use of nisin to extend food shelf-life and the current research premise that Probiotic strains may alter the intestinal flora and thus prevent intestinal wall penetration by pathogens.

The anti-human immunodeficiency virus (-HIV) nucleoside analogs azidothymidine (AZT), dideoxycytidine (ddC), dideoxyinosine (ddl), dideoxydidehydrothymidine (D4T), and dideoxydidehydrocytidine (D4C) and the anticancer drug cytosine arabinoside (AraC) were compared for their effects on the mitochondrial DNA (mtDNA) content in a human lymphoblastoid cell line, CEM. The potency of these compounds in reducing mtDNA content was in the order of ddC greater than D4C greater than D4T greater than AZT greater than ddl. AraC did not have a significant effect on mtDNA content. All of the compounds tested, except AraC, stimulated lactic acid production at concentrations that inhibited mtDNA synthesis. The action of ddC and ddl occurred at concentrations that did not affect cell growth significantly in 4 days but retarded cell growth by day 6. D4T and D4C decreased mtDNA content by 50% at doses lower than those that inhibited cell growth by 50% in 4 days (ID50). However, AZT required a dose higher than the ID50 to exert similar effects on mtDNA content. The decrease of mtDNA content caused by ddC also occurred in nerve growth factor-treated PC12 cells, which differentiate to neuron-like cells upon treatment with nerve growth factor. The preferential inhibition of mtDNA, compared with cell growth, by some of these anti-HIV nucleoside analogs correlates well with their ability to cause drug-limiting delayed toxicity, such as peripheral neuropathy, in patients. These data suggest that the selective mitochondrial toxicity could be responsible for the delayed toxicity caused by these anti-HIV analogs.

Lactic acid bacteria (LAB) have a very long history of use in the manufacturing processes of fermented foods and a great deal of effort was made to investigate and manipulate the role of LAB in these processes. Today, the diverse group of LAB includes species that are among the best-studied microorganisms and proteolysis is one of the particular physiological traits of LAB of which detailed knowledge was obtained. The proteolytic system involved in casein utilization provides cells with essential amino acids during growth in milk and is also of industrial importance due to its contribution to the development of the organoleptic properties of fermented milk products. For the most extensively studied LAB, Lactococcus lactis, a model for casein proteolysis, transport, peptidolysis, and regulation thereof is now established. In addition to nutrient processing, cellular proteolysis plays a critical role in polypeptide quality control and in many regulatory circuits by keeping basal levels of regulatory proteins low and removing them when they are no longer needed. As part of the industrial processes, LAB are challenged by various stress conditions that are likely to affect metabolic activities, including proteolysis. While environmental stress responses of LAB have received increasing interest in recent years, our current knowledge on stress-related proteolysis in LAB is almost exclusively based on studies on L. lactis. This review provides the current status in the research of proteolytic systems of LAB with industrial relevance.

Phage-host interactions remain poorly understood in lactic acid bacteria and essentially in all Gram-positive bacteria. The aim of this study was to identify the phage genetic determinant (anti-receptor) involved in the recognition of Streptococcus thermophilus hosts. The complete genomic sequence of the lytic S. thermophilus phage DT1 was determined previously, and bioinformatic analysis indicated that orf18 might be the anti-receptor gene. The orf18 of six additional S. thermophilus phages was determined (DT2, DT4, MD1, MD2, MD4 and Q5) and compared with the orf18 of DT1. The deduced ORF18 was divided into three domains. The first domain, which contains the N-terminal part of the protein, was conserved in all seven phages. The second domain was detected in only two phages and flanked by a motif called collagen-like repeats. The second domain also contained a variable region (VR1). All seven phages had a third domain that consisted of the C-terminal section of the protein as well as another variable region (VR2). Chimeric DT1 phages were constructed by recombination; a portion of its orf18 was replaced by the corresponding section in orf18 of the phage MD4. All DT1 chimeric phages acquired the host range of phage MD4. Analysis of the orf18 in the chimeric phages revealed that host specificity in phages DT1 and MD4 resulted from VR2. This is the first report on the identification and characterization of a phage gene involved in the host recognition process of Gram-positive bacteria.

The common preference of cancers for lactic acid-generating metabolic energy pathways has led to proposals that their reprogrammed metabolism confers growth advantages such as decreased susceptibility to hypoxic stress. Recent observations, however, suggest that it generates a novel way for cancer survival. There is increasing evidence that cancers can escape immune destruction by suppressing the anti-cancer immune response through maintaining a relatively low pH in their micro-environment. Tumours achieve this by regulating lactic acid secretion via modification of glucose/glutamine metabolisms. We propose that the maintenance by cancers of a relatively low pH in their micro-environment, via regulation of their lactic acid secretion through selective modification of their energy metabolism, is another major mechanism by which cancers can suppress the anti-cancer immune response. Cancer-generated lactic acid could thus be viewed as a critical, immunosuppressive metabolite in the tumour micro-environment rather than a 'waste product'. This paradigm shift can have major impact on therapeutic strategy development.

Neuroblastomas produce high amounts of lactic acid and upregulate the H(+)-linked monocarboxylate transporter isoform 1 (MCT1/SLC16A1). We found elevated MCT1 mRNA levels in fresh neuroblastoma biopsy samples that correlated positively with risk of fatal disease and amplification of the "proto-oncogenic" transcription factor MYCN. We further investigated MCT as a potential therapeutic target in vitro. The neuroblastoma cell lines evaluated were Sk-N-SH, CHP134, IMR32, and NGP. All lines exhibited decreased intracellular pH at low tumor-like extracellular pH. Lonidamine or exogenous lactate further lowered intracellular pH. Immediate early lowering of intracellular pH with lonidamine or lactate at extracellular pH 6.5 correlated positively with diminished cell viability within 48 h. These findings indicate that MCT1 is a potential therapeutic target and that neuroblastoma therapy may be enhanced by therapeutic strategies to inhibit or overwhelm MCT. Additional experiments indicated that the mechanism of cell death by lonidamine or exogenous lactate is similar to that obtained using alpha-cyano-4-OH-cinnamate, a well established MCT inhibitor. Because lactate production is also high in melanoma and many other tumor types, MCT inhibitors may have broad application in cancer treatment. Such treatment would have selectivity by virtue of the acidic milieu surrounding tumors, because MCT is increasingly active as extracellular pH decreases below 7.0 and lactic acid production increases.

The kinetics and activation energy of entry of pyruvate and lactate into the erythrocyte were studied at concentrations below 4 and 15mM respectively. The Km and Vmax. values for both substrates are reported, and it is shown that pyruvate inhibits competitively with respect to lactate and vice versa. In both cases the Km for the carboxylate as a substrate was the same as its Ki as an inhibitor. Alpha-Cyano-4-hydroxycinnamate and its analogues inhibited the uptake of both lactate and pyruvate competitively. Inhibition was also produced by treatment of cells with fluorodinitrobenzene but not with the thiol reagents or Pronase. At high concentrations of pyruvate or lactate (20mM), uptake of the carboxylate was accompanied by an efflux of Cl-ions. This efflux of Cl- was inhibited by alpha-cyano-4-hydroxycinnamate and picrate and could be totally abolished by very low (less than 10 muM) concentrations of the inhibitor of Cl- transport, 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid. This inhibitor titrated out the chlordie efflux induced by pyruvate, bicarbonate, formate and fluoride, in each case total inhibition becoming apparent when approximately 1.2x10(6) molecules of inhibitor were present per erythrocyte, that is, about one inhibitor molecule per molecule of the Cl- carrier. Evan when Cl- efflux was totally blocked pyruvate and lactate uptake occurred. Kinetic evidence is presented which suggests that the Cl- carrier can transport pyruvate and lactate with a high Km and high Vmax., but that an additional carrier with a low Km and a low Vmax. also exists. This carrier catalyses the exchange of small carboxylate anions with intracellular lactate, is competitively inhibited by alpha-cyano-4-hydroxycinnamate and non-competitively inhibited by picrate. The Cl- carrier shows a reverse pattern of inhibition. It is concluded that net efflux of lactic acid from the cell must occur on the Cl- carrier and involve exchange with HCO3 - followed by loss of CO2. The low Km carrier might be used in pyruvate/lactate or acetoacetate/beta-hydroxybutyrate exchanges involved in transferring reducing power across the cell membrane. The possibility that the Cl- carrier exists in cells other than the erythrocyte is discussed. It is concluded that its presence in other cell membranes together with a low intracellular Cl- concentration would explain why the pH in the cytoplasm is lower than that of the blood, and why permeable carboxylate anions do not accumulate within the cell when added from outside.