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There is a widespread belief that neurogenesis exists in adult human brain, especially in the dentate gyrus, and it is to be maintained and, if possible, augmented with different stimuli including exercise and certain drugs. Here, we examine the evidence for adult human neurogenesis and note important limitations of the methodologies used to study it. A balanced review of the literature and evaluation of the data indicate that adult neurogenesis in human brain is improbable. In fact, in several high-quality recent studies in adult human brain, unlike in adult brains of other species, neurogenesis was not detectable. These findings suggest that the human brain requires a permanent set of neurons to maintain acquired knowledge for decades, which is essential for complex high cognitive functions unique to humans. Thus, stimulation and/or injection of neural stem cells into human brains may not only disrupt brain homeostatic systems, but also disturb normal neuronal circuits. We propose that the focus of research should be the preservation of brain neurons by prevention of damage, not replacement.

At present, the guideline approach to the medical treatment and prevention of atherosclerotic cardiovascular disease (ASCVD) is to classify patients by risk and treat the known risk factors (contributory causes), e.g., hypertension, diabetes, obesity, smoking, and poor diet, as appropriate. All high-risk patients should receive statins. This approach has had substantial success but ASCVD still remains the number one cause of death in the United States. Until recently, the underlying cause of ASCVD remained unknown, although a potential dietary cause was suggested by the fact that vegetarians, especially vegans, have a much lower incidence of ASCVD than animal flesh eaters. Recently, consistent with the vegetarian data, substantial evidence for a cause of ASCVD in animals and humans has been discovered. Trimethylamine (TMA)-containing dietary compounds in meat, milk, and other animal foods (e.g., lecithin, choline, and carnitine) are converted by closely related gut bacterial TMA lyases to TMA, which is absorbed and converted predominantly by flavin mono-oxygenase 3 to the toxic trimethylamine N-oxide (TMAO). TMAO causes atherosclerosis in animals and is elevated in patients with coronary heart disease. Inhibition of bacterial lyases in mice prevents TMA and secondarily TMAO formation and atherosclerosis, strong evidence for the TMAO hypothesis. At present, the challenge for the pharmaceutical industry is to discover and develop a potent "broad spectrum" bacterial lyase inhibitor that, along with diet and exercise, could, if the TMAO hypothesis is correct, revolutionize the preventive treatment of ASCVD.

In animal models of acute ischemic stroke, intravenous dehydroascorbic acid (DHAA), unlike ascorbic acid (AA), readily enters brain and is converted in both normal and ischemic brain into protective ascorbic acid. When given parenterally DHAA minimizes infarct volume and facilitates functional recovery. I hypothesize the same effect will occur in humans with acute ischemic stroke. Efficacy in reducing infarct volume is demonstrable in mice and rats even when DHAA is infused three hours after the experimental infarct. Moreover, there is fivefold mechanistic rational for DHA beside excellent pharmacokinetics and rapid penetration of brain and conversion to protective AA: (1) in ischemic brain, there is a precipitous decline in AA which can be reversed by intravenous DHAA; (2) after reduction of DHAA to AA in both normal and ischemic brain, AA can reduce oxidized vitamin E and glutathione, other protectors of brain against damaging reactive oxygen species which build up in ischemic brain; (3) AA itself can protect brain against damaging reactive oxygen species; (4) AA is an essential cofactor for several enzymes in brain including ten-eleven translocase-2 which upregulates production of protective molecules like brain-derived neurotrophic factor; and (5) DHAA after conversion to AA prevents both lipid oxidation and presumably oxidation of other labile substances (e.g., dopamine) in ischemic brain. In terms of safety, based on all available animal information, DHAA is safe in the proposed dosing regimen. For human clinical trials, the methodology for conducting the proposed animal safety, clinical pharmacology and phase II efficacy studies is straightforward. Finally, if DHAA preserved brain substance and function in humans, it could be employed in pre-hospital stroke patients.

In this review, a companion piece to our recent examination of choroid plexus (CP), the organ that secretes the cerebrospinal fluid (CSF), we focus on recent information in the context of reliable older data concerning the composition and functions of adult human CSF. To accomplish this, we define CSF, examine the methodology employed in studying the CSF focusing on ideal or near ideal experiments and discuss the pros and cons of several widely used analogical descriptions of the CSF including: the CSF as the “third circulation,” the CSF as a “nourishing liquor,” the similarities of the CSF/choroid plexus to the glomerular filtrate/kidney and finally the CSF circulation as part of the “glymphatic system.” We also consider the close interrelationship between the CSF and extracellular space of brain through gap junctions and the paucity of data suggesting that the cerebral capillaries secrete a CSF-like fluid. Recently human CSF has been shown to be in dynamic flux with heart-beat, posture and especially respiration. Functionally, the CSF provides buoyancy, nourishment (e.g., vitamins) and endogenous waste product removal for the brain by bulk flow into the venous (arachnoid villi and nerve roots) and lymphatic (nasal) systems, and by carrier-mediated reabsorptive transport systems in CP. The CSF also presents many exogenous compounds to CP for metabolism or removal, indirectly cleansing the extracellular space of brain (e.g., of xenobiotics like penicillin). The CSF also carries hormones (e.g., leptin) from blood via CP or synthesized in CP (e.g., IGF-2) to the brain. In summary the CP/CSF, the third circulation, performs many functions comparable to the kidney including nourishing the brain and contributing to a stable internal milieu for the brain. These tasks are essential to normal adult brain functioning.

In the last decade, there has been substantial progress in understanding vectorial ligand transport through rodent and human choroid plexus (CP), the locus of the blood-CSF interface. In this Review, we enumerate the experimental data required to establish vectorial transport through CP and describe transporters involved in vectorial transport across CP. We also note how these transporters differ from those at the blood-brain barrier. The ligand (substrate) examples presented are methyltetrahydrofolate, methotrexate, leukotriene C(4), nucleosides, thiamine monophosphate, prostaglandins, and digoxin. Our focus is on more definitive experiments, including animal and human transporter "knock-outs." Finally, we discuss the neurochemical implications of vectorial transport through CP and the clinical implications of transporter polymorphisms and knockouts. Examples include descriptions of how vectorial transport through the CP for several micronutrients (e.g., methyltetrahydrofolate) nourishes the brain and how knowledge of CP vectorial transport can lead to important treatments.

In the last 40 years, especially with the application of new neurochemical and molecular biological techniques, there has been explosive progress in understanding how certain ligands and drugs are transported across the blood-brain barrier and choroid plexus out of brain and CSF. In the CNS, there are several separate efflux transporters with very broad specificity that are responsible for much of the efflux transport. This review focuses on three such transporters: organic acid transporter-3, peptide transporter-2 and P-glycoprotein for which there is substantial new information including 'knockout' models in mice and, in one case, dogs. Moreover, the structural biology and transport mechanism of P-glycoprotein at 3.8 angstroms is described. The overall objective is to show how this new knowledge provides a more thorough understanding (e.g., of molecular mechanisms) of efflux transport and in several cases leads to clinically relevant information that allows better treatment of certain CNS disorders (e.g., meningitis and brain cancer).

SUMMARY A large papilloma, presenting in a newborn child and showing historical features of malignancy, was studied by incubating fragments in a gaseous nutrient solution to which one of several substrates had been added. Folic acid, myoinositol, scyllitol and leucine were weakly accumulated, so that the concentration in the tissue was greater than in the bath. Penicillin entered the tissue by a saturable mechanism, but the concentration in the tissue was not greater than in the surrounding solution. Iodide was not accumulated. RESUME Accumulation de matŕriels par un papillome du plexus choroide in vitro Un large papillome prŕsent chez un nouveau-nŕ et montrant des caractŕristiques anamnestiques de malignitŕ a ŕtŕ ŕtudiŕ par 'incubation de fragments dans une solution nutritive gazeuse a laquelle ŕtait ŕtait ajoutŕ 'un de plusieurs substrats difftrents. 'acide folique, le myo-inositol, le scyllitol et la leucine ont ŕtŕ faiblement accumulŕs, la concentration dans le tissu ŕtant plus grande que dans la solution. La pŕnicilline a pŕnŕtrŕ le tissu par un mŕcanisme de saturation mais la concentration dans le tissu n'ŕtait pas plus forte que dans la solution environnante. Les corps iodŕs ne furent pas accumulŕs. ZUSAMMENFASSUNG Ein grol ses Papillorn, das bei einern Neugeborenen diagnostiziert wurde und eindeutige Zeichen der Malignitat bot, wurde weiteruntersucht, indem Teile davon in einem gashaltigen Medium, dem eine Losung mit mehreren Substraten zugesetzt war, inkubiert wurden. Folsaure, Myoinositol, Scyllitol und Leucin waren schwach erhoht, so dals die Konzentration Penicillin trat bis zur Sattigung ins Gwebe ein, die Konzentration im Gewebe war jedoch nicht groser als im umgebenden Medium. Jodid war nicht erhoht. RESUMEN Acumulacion de muteriales por un pupilomu de coroides de plexos coroideos in vitro Un gran papiloma aparecido en un reciŕn nacido y con caracteristicas en la historia de ma1 iniciaciŕn, fue estudiado incubando fragmentos en una solucion nutritiva gaseosa a la cual se habian anadido uno de una serie de substratos. Semanalmente se le fueron acumulando acido folico, mio-inositol, escillitol y leucina de forma tal que la concentracion en La penicilina entro en el tejido por mecanismo de saturacion, pero la concentracion en el tejido no era mayor que en la solucion de alrededor. El iodide no se acumulo.

With the application of genetic and molecular biology techniques, there has been substantial progress in understanding how vitamins are transferred across the mammalian blood-brain barrier and choroid plexus into brain and CSF and how vitamin homeostasis in brain is achieved. In most cases (with the exception of the sodium-dependent multivitamin transporter for biotin, pantothenic acid, and lipoic acid), the vitamins are transported by separate carriers through the blood-brain barrier or choroid plexus. Then the vitamins are accumulated by brain cells by separate, specialized systems. This review focuses on six vitamins (B(1), B(3), B(6), pantothenic acid, biotin, and E) and the newer genetic information including relevant 'knockdown' or 'knockout' models in mice and humans. The overall objective is to integrate this newer information with previous physiological and biochemical observations to achieve a better understanding of vitamin transport and homeostasis in brain. This is especially important in view of the newly described non-cofactor vitamin roles in brain (e.g. of B(1), B(3), B(6), and E) and the potential roles of vitamins in the therapy of brain disorders.

With application of molecular biology techniques, there has been rapid progress in understanding how many drugs and micronutrients (e.g., vitamins) are transferred across the choroid plexus (CP), the main transport locus of the blood-cerebrospinal fluid (CSF) barrier, and the renal tubular epithelial cells. In many cases, these molecules are transported by separate, specific carriers or receptors on the apical and/or basal side of the CP or renal epithelial cells. This commentary focuses on four micronutrient transport systems in CP (ascorbic acid, folate, inositol, and riboflavin), all of which have been recently cloned, expressed and for which knockout mice models were developed and transporter localization studies performed. Also reviewed is the recently cloned uric acid transport system in human kidney in which there exists a human "knockout" model. The implications of these transport systems for drug therapy of central nervous system and renal disorders are discussed, especially with regard to methods to circumvent the blood-brain and blood-CSF barriers to deliver drugs to the brain.

The unidirectional influx of D-pantothenic acid (PA) across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique using [3H]D-PA (1.1 Ci/mmol). PA was transported across the blood-brain barrier by a saturable system that could be described by a Michaelis-Menten transport model with a half-saturation concentration and maximal influx rate of 19 microM and 0.21 nmol/g of brain/min, respectively. PA (0.3 microM) transport through the blood-brain barrier was significantly inhibited by probenecid, nonanoic acid, and biotin (all less than or equal to 0.25 mM), but not by penicillin G, pyruvate, beta-hydroxybutyrate, L-leucine (all 1 mM), or poly-L-lysine HBr (1 mg/ml). Probenecid (0.25 mM), nonanoic acid (0.5 mM), and PA (1.0 mM) did not inhibit [3H]L-leucine transport through the blood-brain barrier, whereas 30 microM-L-leucine inhibited [3H]leucine transport to 23% of control values. Thus, PA is transported through the blood-brain barrier by a low-capacity, saturable transport system with a half-saturation concentration approximately 10 times the plasma PA concentration. Although involved in the transfer of PA from blood into brain, this system does not play an important regulatory role in the synthesis of CoA from PA in brain.

In vitro, the transport of [3H]pantothenic acid into and from rabbit brain slices was studied. In newborn rabbits and throughout development, forebrain and cerebellar slices were able to accumulate and phosphorylate PHJpantothenic acid comparably to slices from adults. The accumulation and phosphorylation of [3H]pantothenic acid by adult forebrain slices were not decreased by substitution of LiCl for NaCl in the artificial CSF or by addition of short-chain fuels (e.g., 5 mM pyruvate or acetoacetate) to the medium. However, probenecid and ouabain (both 1 mM) and medium-chain fatty acids (e.g., 0.1 mM octanoate, nonanoate, and decanoate) profoundly inhibited [3H]pantothenic acid accumulation by forebrain slices but not intracellular phosphorylation and conversion to [3H]CoA. These in vitro results suggest that brain slices accumulate pantothenic acid by a saturable system (probably facilitated diffusion) that is sensitive to inhibition by probenecid and medium-chain fatty acids.

Critical to the discovery, development and rational use of drugs and vaccines are the foundational principles and proper application of statistics. However, in too many cases, there has been misuse of statistics and/or overemphasis on statistical significance (p < 0.05), as though this criterion possessed truth-guaranteeing properties. To clarify confusion about the proper use of statistics in pharmacology, we summarize briefly the foundational principles of probability; the role of statistics in assessment of causality; the three basic uses of statistical methods, especially those employed in hypothesis testing; and current statistical issues in pharmacological research. We then review and provide examples of the meaning of statistical significance, the consequences of lack of randomization in epidemiology/observation studies, the criteria for measurement instrument validation, the problems with subgroup analyses, the need for multiple comparison statistical methods, and how to handle dropouts and missing data. Finally, based on sound experimental and statistical principles, we make a series of recommendations to both experimentalists and journal editors to improve published pharmacological experiments. These include widespread use of blinding and randomization and/or random selection of subjects in both basic and clinical pharmacology, mandatory use of rigorous evidentiary criteria in epidemiology/observation studies claiming causal associations, proper interpretation of statistical versus clinical/ pharmacological significance, appropriate interpretation of meta-analyses, meaningful validation of methods, and a more rational statistical approach to subgroup analyses and genetic association studies.

A vast and continuously growing amount of material on drugs exists in the literature to read and evaluate. Frequently, the papers and their recommendations are conflicting and contradictory. Readers are faced with the dilemma of deciding what to believe. The need for evidence-based medicine as a foundation for optimal clinical research and patient care requires application of the best scientific methods. Various methods are discussed. Generally, the most powerful method to test a clinical hypothesis is the randomized, controlled clinical trial. By contrast, epidemiology/observation studies have certain inherent weaknesses that can lead to erroneous conclusions. The examples of estrogen therapy in postmenopausal women and of vitamin E therapy to reduce cardiovascular risk are discussed extensively to provide historical perspective and to demonstrate erroneous conclusions reached using epidemiology/observation studies. The sociology of journal publication is briefly described, and an attempt is made to assess who benefits and who is harmed when leading medical journals publish erroneous results. Types of bias and confounding issues leading to errors are discussed, and the need is emphasized for publication of rigorous studies after careful evaluation by editors to avoid repetition of past mistakes and to ensure publication of correct medical information.

Background: In the last decade, childhood neurological disorders involving inadequate transport of thiamine, riboflavin or folates into brain have been defined. These involve two transporters of folate (the folate receptor α or the proton-coupled folate transporter), one of thiamine and two of the six riboflavin transporters. Depending on the mutations involved, without a specific diagnosis and treatment, the phenotypes vary from mild to severe and fatal.

The search for ideal drugs to improve patient care requires applications of modern scientific methods to both discovery and development. Using these modern methods, the pharmaceutical industry with strong academic and government collaboration has introduced in the last 25 years many new drugs that approach the ideal. After reviewing Björnsson’s classification of drug action and the notion of contributory causality, this commentary defines an ideal drug from the perspectives of pharmacodynamics, pharmacokinetics, and therapeutics. Examples of new drugs for hypertension, heart disease, stroke, osteoporosis, asthma, ulcer, and migraine headaches are described. Finally, the profound implications of progress in developing ideal drugs not only for the patient but also for the academic, educational, and regulatory establishments are briefly reviewed.

The need for evidence-based medicine as a foundation for optimal patient care requires application of the best scientific methods. Various methods used in the search for clinical truth are discussed. The most powerful method to test a clinical hypothesis is the randomized, controlled clinical trial. By contrast, epidemiology/observation studies (EOS) have inherent weaknesses that can lead to erroneous conclusions. Five such examples, two older and three very recent, are discussed to provide historical perspective and demonstrate potential problems with EOS. Each incident has produced widespread consternation or confusion among physicians. Types of bias leading to errors in EOS are discussed, and the need is emphasized for more rigorous evaluation of EOS by investigators, as well as editors, to avoid repetition of past mistakes and to ensure publication of correct medical information.

The rate of entry into and distribution of drugs and endogenous substances within the mammalian central nervous system (CNS) depend on the peculiar anatomy of the blood-brain and blood-cerebrospinal fluid barriers, the physicochemical nature of the substance or drug, and in many cases, multiple, complex specialized carriers. This review summarizes a systemic methodological and integrative approach to understanding the pharmacokinetic behavior of many drugs in the CNS in the context of the CNS barrier systems, focusing on recent data, several broad principles and selected examples. Standards of proof for valid conclusions are emphasized.

In 15 non-diabetic Type II hypercholesterolemic patients, the effect of 80 mg lovastatin daily on oral glucose tolerance was investigated. Using a randomized, double-blind, two-panel, parallel design, patients on a low cholesterol diet received lovastatin (n = 7) or placebo (n = 8) for 6 weeks. After 6 weeks of treatment, patients receiving lovastatin had a significant reduction in total cholesterol (30%), LDL-cholesterol (36%), and triglycerides (26%). Time courses of plasma glucose and serum insulin changes from baseline after the oral glucose tolerance test were evaluated by AUC. No statistically significant differences were observed in the AUC of changes from baseline between treatment groups or within either treatment group at prestudy, 6 weeks, and poststudy. No patient had a clinically important laboratory or clinical drug-related adverse effect during the study. This study demonstrated that short-term administration of 80 mg lovastatin daily effectively lowers cholesterol without having adverse effects on oral glucose tolerance.

Since the publication of the 4S trial in 1994, there has emerged a consensus that statins save lives and decrease myocardial infarctions and strokes in coronary artery disease (CAD) patients irrespective of baseline serum cholesterol. However, there is controversy over the correct dose and the utility of the treatment-to-goal (cholesterol, low-density lipoprotein) approach. To answer remaining questions about the optimal statin dose in CAD patients, we have performed simple and meta-analyses of 3 large long-term (approx. 5 years) dose-clinical response studies (TNT, IDEAL, and SEARCH) and compared the results with older data including long-term safety data. The results show that raising the dose of simvastatin or atorvastatin to 80 mg confers no mortality advantage, an increase in adverse reactions and only a slight decrease in myocardial infarctions and stroke versus a lower dose. These results suggest a cost-effective approach of a single safe dose (40 mg of inexpensive generic simvastatin or atorvastatin) for almost all CAD patients and makes treatment-to-goal and cholesterol monitoring (except to check for medication compliance) unnecessary; moreover, it is likely to improve the weakness in statin use – medication compliance.

In the last 40 years, especially with the application of new neurochemical and molecular biologic techniques, there has been an explosive progress in understanding how nutrients are transported across the blood-brain barrier and choroid plexus into brain and CSF, and how nutrient homeostasis in brain is achieved. In most cases, there are separate transporters, or in a few cases, systems that transport related substances (e.g., biotin, lipoic, and pantothenic acids). This review focuses on three crucial nutrients (glucose, ascorbic acid, and folates) for which there is substantial new information including 'knock down' and 'knockout' models in mice and/or humans. The overall objective is to show that this new knowledge leads not just to a more thorough understanding (e.g., of 'why' questions like: why do neurons require up to 10 mM ascorbic acid intracellulary?); but in some cases leads to clinically important predictions that allow treatment of heretofore devastating neurologic disorders.

The rate of entry into and distribution of many drugs in the mammalian brain cannot be explained by the physicochemical characteristics of these drugs taking into account the anatomy of the blood-brain barrier. Rather, specialized mechanisms (carriers) in the central nervous system have been sought after and characterized. These carriers explain the observed pharmacokinetic behavior of many drugs in brain. This review summarizes these data in the context of the blood-brain barrier and focuses on several broad principles and selected examples.

With the application of genetic and molecular biology techniques, there has been substantial progress in understanding how vitamins are transferred across the mammalian blood-brain barrier and choroid plexus into brain and CSF and how vitamin homeostasis in brain is achieved. In most cases (with the exception of the sodium-dependent multivitamin transporter for biotin, pantothenic acid, and lipoic acid), the vitamins are transported by separate carriers through the blood-brain barrier or choroid plexus. Then the vitamins are accumulated by brain cells by separate, specialized systems. This review focuses on six vitamins (B(1), B(3), B(6), pantothenic acid, biotin, and E) and the newer genetic information including relevant 'knockdown' or 'knockout' models in mice and humans. The overall objective is to integrate this newer information with previous physiological and biochemical observations to achieve a better understanding of vitamin transport and homeostasis in brain. This is especially important in view of the newly described non-cofactor vitamin roles in brain (e.g. of B(1), B(3), B(6), and E) and the potential roles of vitamins in the therapy of brain disorders.

Detection of DNA synthesis in brain employing ((3)H)thymidine (((3)H)dT) or bromo deoxyuridine (BrdU) is widely used as a measure of the "birth" of cells in brain development, adult neurogenesis and neuronal stem cell replacement strategies. However, recent studies have raised serious questions about whether this methodology adequately measures the "birth" of cells in brain either quantitatively or in an interpretable way in comparative studies, or in stem cell investigations. To place these questions in perspective, we review deoxynucleoside synthesis and pharmacokinetics focusing on the barriers interfacing the blood-brain (cerebral capillaries) and blood-cerebrospinal fluid (choroid plexus), and the mechanisms, molecular biology and location of the deoxynucleoside transport systems in the central nervous system. Brain interstitial fluid and CSF nucleoside homeostasis depend upon the activity of concentrative nucleoside transporters (CNT) on the 'central side' of the barrier cells and equilibrative nucleoside transporters (ENT) on their 'plasma side.' With this information about nucleoside transporters, blood/CSF concentrations and metabolic pathways, we discuss the assumptions and weaknesses of using ((3)H)dT or BrdU methodologies alone for studying DNA synthesis in brain in the context of neurogenesis and potential stem cell therapy. We conclude that the use of ((3)H)dT and/or BrdU methodologies can be useful if their limitations are recognized and they are used in conjunction with independent methods.

Critical to the discovery and development of drugs and vaccines is the rational selection of biochemical, immunologic or molecular targets. To understand the rationale for target selection, we review strengths and weaknesses of the four main approaches: whole animal disease models; molecular targeting; epidemiology/observation studies, and genomics. After classifying diseases into those with a relatively stable pathophysiology (e.g., hypertension and gout) versus those with an unstable pathophysiology (e.g., AIDS and influenza) to aid in understanding target selection, we provide examples of successful and unsuccessful selection of drug and vaccine targets, focusing on the molecular and epidemiological/observational approaches. We discuss the reasons that molecular targeting has led to successful control of many diseases, whereas the epidemiological/observational approach has had a checkered history. We also assess the potential power of the genomic approach, specifically the curative versus controlling/preventive strategies. With combined genetic and molecular approaches and judicious use of whole animal models and properly performed epidemiology/observation studies to select the appropriate targets, the future for controlling, preventing and even curing many diseases is very bright indeed.

Confusion exists concerning optimal selection of drugs for clinical practice. Many reasons contribute to this confusion which derives from various sources including industry, the FDA and often less than ideal education of physicians in clinical pharmacology during medical school and thereafter. This presentation does not focus on allocating blame for the current unsatisfactory situation, but rather offers a solution to improve drug therapy. Our educational solution consists of a paradigm for rational drug therapy, specifically a checklist of eight criteria for physicians to review before prescribing drugs. Such a review would facilitate better estimation by physicians of risk/benefit ratios involving new, often expensive and, in some cases, questionably effective and safe drugs.

The purpose of this review is to discuss the implications of the 2009 discovery of the sixth deoxyribonucleoside (dN) [5-hydroxymethyldeoxycytidine (hmdC)] in DNA which is the most abundant in neurons. The concurrent discovery of the three ten-eleven translocation enzymes (TET) which not only synthesize but also oxidize hmdC in DNA, prior to glycosylase removal and base excision repair, helps explain many heretofore unexplained phenomena in brain including: 1) the high concentration of ascorbic acid (AA) in neurons since AA is a cofactor for the TET enzymes, 2) the requirement for reduced folates and the dN synthetic enzymes in brain, 3) continued DNA synthesis in non-dividing neurons to repair the dynamic formation/removal of hmdC, and 4) the heretofore unexplained mechanism to remove 5-methyldeoxycytidine, the fifth nucleoside, from DNA. In these processes, we also describe the important role of choroid plexus and CSF in supporting vitamin homeostasis in brain: especially for AA and folates, for hmdC synthesis and removal, and methylated deoxycytidine (mdC) removal from DNA in brain. The nexus linking AA and folates to methylation, hydroxymethylation, and demethylation of DNA is pivotal to understanding not only brain development but also the subsequent function.

We and other investigators have postulated deterioration of essential choroid plexus (CP) functions in some elderly and especially Alzheimer’s disease patients based on apparent anatomical, histological and pathological changes in CP. We have termed this putative phenomenon CP failure. By focusing on four essential energy-requiring CP functions, specifically ascorbic acid (AA) and folate transport from blood into CSF, transthyretin synthesis and secretion into CSF, and electrolyte/acid–base balance in CSF, we were able to evaluate the hypothesis of CP failure by reviewing definitive human data. In both healthy elderly and Alzheimer’s disease patients, the CP functions normally to transport AA and folates actively from blood into CSF, synthesize and secrete transthyretin into CSF, and maintain CSF acid–base balance and ion concentrations. These human CSF compositional data provide no support for the notion of CP failure in elderly humans and Alzheimer’s disease patients.

Abstract We investigated the effects of angiotensin II (Ang II) type 1 receptor blockade with losartan on the renin-angiotensin-aldosterone system in hypertensive patients (supine diastolic blood pressure, 95 to 110 mm Hg). Qualifying patients (n=51) were allocated to placebo, 25 or 100 mg losartan, or 20 mg enalapril. Blood pressure, plasma drug concentrations, and renin-angiotensin-aldosterone system mediators were measured on 4 inpatient days: end of placebo run-in, after first dose, and 2 and 6 weeks of treatment. Plasma drug concentrations were similar after the first and last doses of losartan. At 6 weeks, 100 mg losartan and 20 mg enalapril showed comparable antihypertensive activity. Four hours after dosing, compared with the run-in day, 100 mg losartan increased plasma renin activity 1.7-fold and Ang II 2.5-fold, whereas enalapril increased plasma renin activity 2.8-fold and decreased Ang II 77%. Both drugs decreased plasma aldosterone concentration. For losartan, plasma renin activity and Ang II increases were greater at 2 than at 6 weeks. Effects of losartan were dose related. After the last dose of losartan, plasma renin activity and Ang II changes were similar to placebo changes by 36 hours. These results indicate that long-term blockade of the feedback Ang II receptor in hypertensive patients produces modest increases of plasma renin activity and Ang II that do not appear to affect the antihypertensive response to the antagonist.

Recently, substantial progress has been made in the therapy of bacterial meningitis related in part to better understanding of and predictions about the pharmacokinetics of antibiotics in the central nervous system. This review summarizes new data on pertinent anatomy and physiology of penicillin and cephalosporin transport in the central nervous system, focusing on the choroid plexus, which transports many antibiotics out of the central nervous system. These new pharmacological data provide the scientific basis for understanding recent advances in therapy of meningitis.

— The transport of two cyclohexitol stereoisomers, myo-inositol (inositol) and scyllo-inositol (scyllitol), from blood into the CNS in vivo and into the choroid plexus in vitro was studied. In vitro, the uptake of [3H]scyllitol or [3H]inositol by choroid plexuses, isolated from rabbits and incubated in artificial CSF, was measured. Both scyllitol and inositol inhibited [3H]scyllitol or [3H]inositol accumulation by the choroid plexus. Inositol competitively inhibited [3H]scyllitol accumulation and both isomers had a comparable affinity (Kt= 0.1 mm) for the single cyclohexitol accumulation system. The other 6 stereoisomers tested had an order of magnitude less affinity for the cyclohexitol accumulation system of choroid plexus. Thiol reagents that penetrate cells inhibited inositol accumulation by choroid plexus more effectively than nonpenetrating thiol reagents. In vivo, in unanesthetized rabbits. the transport of unmetabolized [3H]inositol from blood into CSF, choroid plexus and brain was readily saturated by increasing the plasma levels of myo-inositol but not by the stereoisomer d-chiroinositol. Similarly, the transport of unmetabolized [3H]scyllitol into CSF, brain and choroid plexus was readily saturated by increasing the plasma levels of myo-inositol. Beside documenting the stereospecificity and thiol reagent sensitivity of the inositol transport mechanism of the choroid plexus, these results provide further evidence that the choroid plexus is a locus for cyclohexitol transport between blood and CSF. Moreover, they show that scyllitol, which, like inositol, is present at a higher concentration in brain than plasma, can be transported from blood into CSF and brain by the same system that transports inositol.

The effects of changes in the concentrations of pyridoxal phosphate and blogenic amines in brain on: (I) pyridoxal kinase (EC 2.7.1.35) activity in brain and choroid plexus; and (2) vitamin B6 accumulation by brain slices and isolated, intact choroid plexuses were studied. New Zealand white rabbits were treated parenterally with 200 mg/kg pyridoxine-HCl for 3 days or 120 mg/kg 4-deoxypyridoxine HCI or 5 mg/kg reserpine I day before death. After these treatments the mean concentration of pyridoxal phosphate in brain was elevated by 39% by pyridoxine and decreased by 57% by 4-deoxypyridoxine. Reserpine had no effect. However, the ability of brain slices and isolated, intact choroid plexuses from the treated rabbits to accumulate [3H] vitamin B6 (with [3H]pyridoxine in the medium) was not different from untreated controls. Also, the specific activity of pyridoxal kinase in brain and choroid plexus of treated rabbits was not different from controls. These results show that vitamin B6 accumulation and pyridoxal kinase activity in brain and choroid plexus are independent of both pyridoxal phosphate and reserpine-sensitive biogenic amine concentrations in brain. In vitro studies with pyridoxal kinase showed that. in both choroid plexus and brain. pyridoxal kinase was a single enzyme with a molecular weight of 43.000 and a Km, for pyridoxine of 2.0 μM Crude and partially-purified pyridoxal kinase from brain was not inhibited by biogenic amines (1 mM) or pyridoxal phosphate (5 μM). These in vitro data are consistent with the lack of effect of changes in pyridoxal phosphate and biogenic amine concentrations (in brain) on pyridoxal kinase activity in brain in vivo.

The mechanisms by which deoxycytidine enters and leaves brain, choroid plexus, and CSF were investigated by injecting [3H]deoxycytidine intraarterially, intravenously, and intraventricularly. After intracarotid injection of deoxycytidine (1.0 microM) into rats, deoxycytidine did not pass through the blood-brain barrier at a faster rate than sucrose. [3H]Deoxycytidine, either alone or together with unlabeled deoxycytidine, was infused at a constant rate into conscious adult rabbits. At 130 min, [3H]deoxycytidine readily entered CSF, choroid plexus, and brain. In brain, approx. 60% of the nonvolatile radioactivity was attributable to [3H]deoxycytidine phosphates. The addition of 0.22 mmol/kg unlabeled deoxycytidine to the infusion syringe decreased the phosphorylation of [3H]deoxycytidine in brain by approx. 50%; the addition of 2.2 mmol/kg of unlabeled deoxycytidine to the infusion syringe decreased the relative entry of [3H]deoxycytidine into CSF and brain by approx. 50 and 75%, respectively. Two hours after the intraventricular injection of [3H]deoxycytidine, [3H]deoxycytidine was rapidly cleared from CSF, in part, to brain, where approx. 65% of the [3H]deoxycytidine was converted to [3H]deoxycytidine phosphates. The intraventricular injection of unlabeled deoxycytidine with the [3H]deoxycytidine decreased the phosphorylation of [3H]deoxycytidine in the brain significantly and also decreased the clearance of [3H]deoxycytidine from the CSF. These results were interpreted as showing that the entry of deoxycytidine from blood into CSF occurs by a saturable transport system within the choroid plexus. Once within the CSF, the deoxycytidine can enter brain, undergo phosphorylation to deoxycytidine phosphates, and subsequently be incorporated into DNA.

Recycling of methionine via homocysteine occurs in vivo in rat and rabbit brain. Methyltetrahydrofolate - homocysteiine methyltransferase (methionine synthetase, EC 2.1.1.13) is the only enzyme in the brain capable of remethylating homocysteine to methionine. The enzyme is uniformly distributed in various regions of the rabbit brain (except for the choroid plexus). The specific activity of the enzyme in glial cells is twice that of neurons. The specific activity and concentration of the enzyme in rabbit brain is highest at one day of age and then declines until it stabilizes in the adult at 50 percent of the original activity. Moderate folate deficiency did not affect the activity of the enzyme but brief treatment with nitrous oxide resulted in a 50 percent reduction in the enzyme level. We conclude that methionine recycling in mammalian brain occurs throughout the brain during the entire life span. Methionine and its active form S-adenosylmethionine (SAM) is one of Nature's most important molecules, serving as a methyl group donor and a precursor in the biosynthesis of polyamines (1,2). In the past, several authors (2,3,4) have suggested that, in brain, the biosynthesis of methionine occurs in both rabbit and rat brains in vivo via methionine synthetase. We will discuss the function, development and distribution of this enzyme in brain.

As in humans, theophylline intoxication in rabbits causes seizures and death. We studied whether the administration of phenobarbital or phenytoin following a toxic dose of theophylline would improve survival in rabbits. New Zealand white rabbits were infused intravenously with theophylline, 115 mg/kg over 50 minutes. Upon completion of the infusion, rabbits were randomized to receive either saline (control) (N = 60) or saline containing phenobarbital 20 mg/kg (N = 60), or phenytoin 12 mg/kg (N = 30), infused over 30 minutes. The number (and percentage) of rabbits surviving 24 hours in each group was: control 12 (20%), phenobarbital 30 (50%), and phenytoin 7 (23%) [X2; p less than 0.005; two-tailed]. In all fatal cases, death was preceded by a seizure; rabbits that survived did not seize. These results show that phenobarbital administered intravenously to theophylline-intoxicated rabbits prevented seizures and improved survival whereas phenytoin administration had no significant effect.

Kinetics and sedative and psychomotor effects of diphenhydramine were investigated in elderly Caucasian women (greater than 64 yr. old). In a double-blind trial, each of 12 healthy subjects received on one of three occasions 50 mg/70 kg IV or oral diphenhydramine HCl or oral placebo. Plasma levels of diphenhydramine were measured in six subjects and tests of sedation and psychomotor performance were performed hourly for 8 hr in all subjects. Kinetic analysis showed that the volume of distribution (295 +/- 50 [SEM] l/70 kg), clearance (42 +/- 5 l/70 kg/hr), and plasma t1/2 (4.9 +/- 0.7 hr) were of the same order as in young adults. As in young adults, there was minimal psychomotor impairment after oral and after intravenous diphenhydramine. In contrast to young adults, however, elderly women did not report significant sedation after diphenhydramine. These results suggest that diphenhydramine may not be an effective sedative/hypnotic in elderly women.

A folate-binding protein (binder) from human choroid plexus was solubilized with Triton X-100 and partially purified in three steps: (1) affinity chromatography, (2) Sephadex G-200 column chromatography, and (3) polyacrylamide gel electrophoresis. When the partially purified binder was subjected to sodium dodecyl sulfate--polyacrylamide gel electrophoresis, the binding activity was located in the region of the gel with a molecular weight between 45,000 and 60,000. The specific activity of the binder after the three purification steps was 1.2 mu g folic acid/mg protein, a 316-fold purification. Binding activity of the partially purified binder decreased below pH 6.0 and above pH 8.0, was unaffected by treatment with ribonuclease or deoxyribonuclease, but was abolished with trypsin, chymotrypsin, or protease (Streptomyces griesus). The binding of folic acid to the human binder was inhibited by folate Greater Than H4-folate Greater Than methyl-H4-folate approximately dihydrofolate approximately pteroic acid Greater Than methotrexate approximately aminopterin.

The unidirectional influx of niacinamide across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique employing [14C]niacinamide. Niacinamide was transported rapidly across the blood-brain barrier by a system that was not saturable with 10 mM niacinamide in the perfusate. However, with periods of perfusion longer than 30 seconds, there was substantial backflow of [14C]niacinamide into the perfusate. Niacinamide (1.7 microM) transport through the blood-brain barrier was not significantly inhibited by 3-acetylpyridine. Thus, niacinamide is transported rapidly and bidirectionally through the blood-brain barrier by a high capacity transport system. Although involved in the transfer of niacinamide between blood and brain, this transport system does not play an important regulatory role in the synthesis of NMN, NAD, and NADP from niacinamide in brain.

The ability of caramel color (ammonia process) (AC) to inhibit rabbit brain pyridoxal kinase (EC 2.7.1.35) and pyridoxine uptake and release by rabbit brain slices was investigated. AC contains a heatstable, competitive inhibitor of pyridoxal kinase that can be removed by dialysis or activated charcoal. A concentration of 0.6% AC in the pyridoxal kinase assay reduced activity by 50%. By Sephadex column chromatography, the apparent molecular weight of the inhibitor was ⋍360. AC at concentrations of 2 μl/ml medium also inhibited the accumulation of pyridoxine by brain slices and accelerated the release of vitamin B 6 from brain slices. AC is not a specific inhibitor of ATP-dependent kinases since AC had no effect on thymidine kinase activity. The inhibition of pyridoxine uptake and acceleration of vitamin B 6 release from brain slices by AC are probably related to the inhibition of pyridoxal kinase since accumulation and retention of vitamin B 6 in brain slices depend on pyridoxal kinase activity.

In adult New Zealand white rabbits, the effects of food deprivation and of massive elevations of plasma uridine or thymidine concentrations on CSF and plasma nucleoside and oxypurine concentrations were studied. Nucleoside and oxypurine levels were determined by high performance liquid chromatography using unequivocal methods of compound identification. After 48 and 96 h of food deprivation, the concentrations of uridine, cytidine, inosine, thymidine, deoxycytidine, deoxyuridine, hypoxanthine, xanthine, and uric acid in CSF and plasma were not different than in controls, except at 96 h, when the plasma uridine concentration was 35% lower (p less than 0.05). After elevation of the plasma and CSF thymidine concentrations to approximately 200 and 100 microM, respectively, with intravenous thymidine for 5 h, there was a large increase in CSF and plasma thymidine to approximately 100 microM and a smaller increase in plasma and CSF deoxyuridine concentrations. After elevation of the plasma and CSF uridine concentrations to 0.6 and 0.2 mM, respectively, there was a large increase in CSF and plasma uracil and a smaller increase in plasma and CSF deoxyuridine concentrations. Elevated plasma concentration of thymidine and uridine significantly decreased the CSF to plasma ratios of deoxyuridine and thymidine; however, only elevated plasma uridine concentrations decreased the CSF to plasma ratio of uridine. These results document the powerful homeostatic mechanisms that regulate the concentrations of the principal nucleosides and oxypurine bases in CSF.

A decrease in dietary protein intake lowers the clearance of a number of substances excreted principally by the kidney including uric acid and oxypurinol, the major metabolite of allopurinol. We studied the kinetics of uric acid and oxypurinol in seven healthy volunteers on a normal protein diet (2600 calories; 100 g protein) followed by a 400 calorie, protein-free diet. A 600 mg dose of allopurinol was given orally after 6 days of the normal protein diet and again after 2 days of the 400 calorie, protein-free diet. Two major findings emerged: first, the renal clearance of oxypurinol was reduced from 21.2 +/- 1.9 ml/min during the normal protein diet to 12.3 +/- 1.2 ml/min (P less than .05) during the 400 calorie, protein-free diet, and second, there was a striking diurnal difference in oxypurinol renal clearance with a 41% decrease in the oxypurinol clearance at night (8 PM to 8 AM) versus day (8 AM to 8 PM) on the 400 calorie, protein-free diet.

The kinetics of gentamicin accumulation by a sensitive strain of Escherichia coli were investigated at gentamicin concentrations from 0.02 to 200 microgram/ml. Accumulation with time shows two energy-dependent phases and is saturable. Sodium chloride delays the onset of the second more rapid energy-dependent phase and decreases the magnitude of gentamicin accumulation for incubations up to 60 minutes at all gentamicin concentrations tested. Simultaneous determinations of accumulation, cell viability, and growth inhibition indicate that antimicrobial activity is correlated with the magnitude of gentamicin accumulation. These observations suggest that altered bacterial accumulation of gentamicin explains the effect of sodium chloride on the antimicrobial activity of gentamicin.

A B S T R A C T The effect of intracisternal inoculation of bacteria on the choroid plexus system, which transports penicillin from cerebrospinal fluid (CSF) to blood, was studied in vitro and in vivo. Meningeal and choroid plexus inflammations as well as CSF pleocytosis were induced in rabbits with intracisternal inoculations of Hemophilus influenzae or Staphylococcus aureus. At various times after bacterial inoculation, the choroid plexuses of the inoculated rabbits were removed and incubated in artificial CSF containing ["C] penicillin. The ability of the choroid plexuses to accumulate pencillin in vitro was measured and was found to be depressed as compared with controls. This depression of choroid plexus uptake reversed with resolution of the inflammatory process. In vivo on the day after intracisternal inoculation of Hemophilus influenzae, a decrease in the disappearance of penicillin relative to inulin in the inoculated rabbits (as compared to the controls) was observed when ["C]penicillin and ['H]inulin were injected intraventricularly and cisternal CSF was sampled 2 h later. This decrease could not be explained by penicillin binding to the CSF exudate. However, the choroid plexus transport system for penicillin was only partially depressed in those inoculated rabbits with bacterially induced inflammation, since in vitro the choroid plexuses could still accumulate penicillin and in vivo CSF penicillin levels could be further increased with probenecid pretreatment. These results suggest that CSF penicillin levels are increased in this model due to three factors: a depression of active efflux of penicillin from the CSF, an increase in permeability to penicillin of inflamed meninges, and, less significantly, by CSF binding of penicillin.

Adult rabbit brain contains the enzymatic machinery to convert deoxyuridine to deoxyuridine triphosphate (dUTP). Although dUTP as dUMP can be readily incorporated into DNA in place of thymidine monophosphate, we detected no (3H)dUMP in newly synthesized (3H)DNA in adult rabbit brain after the intraventricular injection of (3H)deoxyuridine. Only (3H)thymidine was detected. The probable explanation for the lack of incorporation of uracil into adult rabbit brain DNA is the presence of a specific, high affinity dUTPase which converts dUTP to dUMP and PP. After homogenization and ammonium sulfate fractionation of adult rabbit brain (35 to 75% saturation), a high affinity, specific dUTPase was detected in the dialyzed enzyme preparation. The Km and Vmax of the dUTPase were 0.2 microM and 36 pmol/mg protein/min, respectively. No high affinity dUTPase activity was detectable in liver. In brain, another enzyme hydrolyzed dUTP and dTTP (NTPase( to their respective diphosphates. NTPase, unlike dUTPase, was not sensitive to heating at 65 degrees C for five minutes. Thus, brain, like other tissues, contains a high affinity, specific dUTPase presumably to "sanitize" the cells of dUTP and, thus, protect the integrity of newly synthesized DNA.

The development and regional distribution of deoxyuridine 5'-triphosphatase (dUTPase) in rabbit brain was studied. After partial purification of the dUTPase activity, newborn brain and liver activities fell from Vmax values of 1.0 and 6.9 nmol/mg protein/min, respectively, to adult levels of 0.1 (brain) and less than 0.01 (liver). The dUTPase activity was evenly distributed throughout the entire adult rabbit brain, and there was no difference in the Km of dUTPase in newborn and adult brain (0.1 microM). The low level of dUTPase in adult brain and the lack of activity in adult liver were not due to inhibitors or phenylmethylsulfonyl fluoride-sensitive proteases. dUTPase in brain, as in other tissues, probably inhibits the misincorporation of uracil into DNA.

A randomized crossover trial of the effect of oral activated charcoal on the kinetics of intravenous theophylline was carried out in six normal male subjects. After intravenous aminophylline (6 mg/kg), subjects received water or water with activated charcoal (140 gm) in divided doses over 12 hr. Serum theophylline concentrations were measured from 0 to 24 hr after the aminophylline infusion. Treatment with activated charcoal decreased the serum t 1/2 from 6.4 +/- 1.2 to 3.3 +/- 0.4 (SEM) hr and the serum AUC from 78 +/- 14 to 42 +/- 4 mg . hr/l. Percent decrease in AUC after treatment with charcoal correlated positively with the endogenous theophylline serum t 1/2 (r = 0.94). These results suggest that oral activated charcoal (1) enhanced the total body clearance of theophylline and (2) may be efficacious in the treatment of theophylline poisoning, especially in patients with prolonged serum t 1/2s of theophylline.