Effects of Porcine Circovirus Type 2 (PCV2) Maternal Antibodies on Experimental Infection of Piglets with PCV2

To determine the effects of porcine circovirus type 2 (PCV2) maternal antibodies on and response to experimental PCV2 infection, 24 piglets were divided into four groups on the basis of the enzyme-linked immunosorbent assay titers of PCV2 maternal antibodies: group A (n = 6; sample/positive [S/P] ratio, <0.2), group B (n = 5; S/P ratio, >0.2 to <0.5), and groups C (n = 8) and D (n = 5) (S/P ratio, >0.5). Piglets in groups A, B, and C were inoculated with PCV2 at day 0 and challenged with PCV2 at day 42. Group D piglets were not exposed to PCV2 at day 0 but were challenged at day 42. Before challenge, seroconversion to PCV2 antibodies occurred in fiveofsix group A piglets, and the antibody level rose above the cutoff level in oneoffive group B piglets. Viremia was detected in fiveofsix, fouroffive, and twoofeight pigs in groups A, B, and C, respectively. After challenge, PCV2 DNA was detectable from 7 to 21 days postchallenge in the sera from sixofsix, fouroffive, threeofeight, and fiveoffive pigs in groups A, B, C, and D, respectively. The results indicated that protection against PCV2 infection conferred by maternal antibodies is titer dependent: higher titers are generally protective, but low titers are not.     

Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha

Telomere proteins protect the chromosomal terminus from nucleolytic degradation and end-to-end fusion, and they may contribute to telomere length control and the regulation of telomerase. The current studies investigate the effect of Oxytricha single-stranded telomere DNA-binding protein subunits α and β on telomerase elongation of telomeric DNA. A native agarose gel system was used to evaluate telomere DNA-binding protein complex composition, and the ability of telomerase to use these complexes as substrates was characterized. Efficient elongation occurred in the presence of the α subunit. Moreover, the α–DNA cross-linked complex was a substrate for telomerase. At higher α concentrations, two α subunits bound to the 16-nucleotide single-stranded DNA substrate and rendered it inaccessible to telomerase. The formation of this α·DNA·α complex may contribute to regulation of telomere length. The α·β·DNA ternary complex was not a substrate for telomerase. Even when telomerase was prebound to telomeric DNA, the addition of α and β inhibited elongation, suggesting that these telomere protein subunits have a greater affinity for the DNA and are able to displace telomerase. In addition, the ternary complex was not a substrate for terminal deoxynucleotidyltransferase. We conclude that the telomere protein inhibits telomerase by rendering the telomeric DNA inaccessible, thereby helping to maintain telomere length.     

CpG Islands of the Pig

We describe an analysis of the CpG islands (CGIs) of the pig. We have used both database survey and a porcine genomic library that is enriched for CGIs. Approximately half of 41 pig genomic database sequences had CGIs with an average G+C content of 65.3%, an average CpG observed/expected frequency of 0.85, and an average size of 978 bp. Of 27 CGI library clones, 16 were nonrepetitive, nonribosomal DNA and CGI-like. CGI library clones had similar average values for G+C and CpG frequency to CGIs of database genes, and an average size of 670 bp, as MseI cuts within some islands. Library clones were also shown to be low copy number and unmethylated in genomic DNA. The presence in the library of seven previously known CGI sequences was confirmed as was the absence of one nonisland sequence. The CGI library exhibits an R-band pattern for many chromosomes in FISH analysis. The pig chromosome arms that show the most dense CGI population are homologous to segments of human chromosomes that are known to be gene rich.     

Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence

The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10⁻⁶ must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or “PCR noise”. Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5×10⁻⁷, and five of its more frequent mutations (hot spots) consisted of three transversions (GC→TA, AT→TA, and AT→CG), one transition (AT→GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.     

Overcoming cellular senescence in human cancer pathogenesis

Elevation of p16, the CDKN2/p16 tumor suppressor gene (TSG) product, occurs at senescence in normal human uroepithelial cells (HUC). Immortal HUCs and bladder cancer cell lines show either alteration of p16 or pRb, the product of the retinoblastoma (RB) TSG. In addition, many human cancers show p16 or pRb alteration along with other genetic alterations that we associated with immortalization, including +20q and −3p. These observations led us to hypothesize that p16 elevation plays a critical role in senescence cell cycle arrest and that overcoming this block is an important step in tumorigenesis in vivo, as well as immortalization in vitro. Using a novel approach, we tested these hypotheses in the present study by examining p16 and pRb status in primary culture (P0) and after passage in vitro of transitional cell carcinoma (TCC) biopsies that represented both superficial bladder tumors and invasive bladder cancers. We demonstrated that all superficial TCCs showed elevated p16 after limited passage in vitro and then senesced, like normal HUCs. In contrast, all muscle invasive TCCs contained either a p16 or a pRb alteration at P0 and all spontaneously bypassed senescence (P=0.001). Comparative genomic hybridization (CGH) was used to identify regions of chromosome loss or gain in all TCC samples. The application of a statistical model to the CGH data showed a high probability of elevated alteration rates of +20q11−q12 (0.99) and +8p22−pter (0.94) in the immortal muscle invasive TCCs, and of −9q (0.99) in the superficial TCCs. Three myoinvasive TCCs lost 3p13−p14. In this study, four of six myoinvasive TCCs also showed TP53 mutation that associated well with genome instability (P=0.001), as previously hypothesized. Notably, TP53 mutation, which has been used as a marker of tumor progression in many human cancers, was less significant in associating with progression in this study (P=0.04) than was p16 or pRb alteration (P=0.001). Thus, these data support a new model in which overcoming senescence plays a critical role in human cancer pathogenesis and requires at least two genetic changes that occur in several combinations that can include either p16 or pRb loss and at least one additional alteration, such as +20q11−q12, −3p13−p14, or −8p21−pter.     

Association of TPH1 with suicidal behaviour and psychiatric disorders in the Chinese population

Tryptophan hydroxylase (TPH), the rate limiting enzyme in serotonin biosynthesis, is one of the most important regulating factors in the serotonergic system. Recently, polymorphisms of the TPH gene have been identified as being associated with suicide, but the evidence is inconsistent. To investigate the role in suicide of one of the isoforms, TPH1, we examined the association of five single nucleotide polymorphisms (SNPs) in the promoter region and in intron 7 of the TPH1 gene based on a sample from the Chinese population of 810 subjects, of whom 329 had made no suicide attempts (NSA), 297 had made suicide attempts (SA), and 184 were healthy subjects (HS). In this study, we observed statistically significant differences between NSA and HS subjects in allele distributions on one marker, −6526A (p=0.0329; odds ratio (OR) 1.36; 95% confidence interval (CI) 1.01 to 1.81). No significant difference in genotype distribution or allele frequencies of other polymorphisms was found between the suicide victims and the controls. The overall haplotype frequency was significantly different between cases and healthy controls (p=0.000024 NSA v HS; p<0.000001, SA v HS; p<0.000001, cases v HS). We found the haplotype TCAAA of −7180/−7065/−6526/218/779 to be strongly associated with suicidal behaviour and psychiatric disorders (p=0.00243; OR=1.62; 95% CI 1.17 to 2.24 and p=0.018; OR=1.41; 95% CI 1.05 to 1.91), which suggests an association of TPH1 with suicidal behaviour and indicates that TPH1 may play a significant role in the aetiology of psychiatric disorders in the Han Chinese population.     

Acute Plasmodium chabaudi chabaudi Malaria Infection Induces Antibodies Which Bind to the Surfaces of Parasitized Erythrocytes and Promote Their Phagocytosis by Macrophages In Vitro

CBA/Ca mice infected with 5 × 10⁴ Plasmodium chabaudi chabaudi AS-parasitized erythrocytes experience acute but self-limiting infections of relatively short duration. Parasitemia peaks (~40% infected erythrocytes) on day 10 or 11 and is then partially resolved over the ensuing 5 to 6 days, a period referred to as crisis. How humoral and cellular immune mechanisms contribute to parasite killing and/or clearance during crisis is controversial. Humoral immunity might be parasite variant, line, or species specific, while cellular immune responses would be relatively less specific. For P.c. chabaudi AS, parasite clearance is largely species and line specific during this time, which suggests a primary role for antibody activity. Accordingly, acute-phase plasma (APP; taken from P.c. chabaudi AS-infected mice at day 11 or 12 postinfection) was examined for the presence of parasite-specific antibody activity by enzyme-linked immunosorbent assay. Antibody binding to the surface of intact, live parasitized erythrocytes, particularly those containing mature (trophozoite and schizont) parasites, was demonstrated by immunofluorescence in APP and the immunoglobulin G (IgG)-containing fraction thereof. Unfractionated APP (from P.c. chabaudi AS-infected mice), as well as its IgG fraction, specifically mediated the opsonization and internalization of P.c. chabaudi AS-parasitized erythrocytes by macrophages in vitro. APP from another parasite line (P.c. chabaudi CB) did not mediate the same effect against P.c. chabaudi AS-parasitized erythrocytes. These results, which may represent one mechanism of parasite removal during crisis, are discussed in relation to the parasite variant, line, and species specificity of parasite clearance during this time.     

Sour Cherry (Prunus cerasus L) Anthocyanins as Ingredients for Functional Foods

In the recent years many studies on anthocyaninshave revealed their strong antioxidant activity and their possibleuse as chemotherapeutics. The finding that sour cherries(Prunus cerasus L) (also called tart cherries) containhigh levels of anthocyanins that possess strong antioxidant andanti-inflammatory properties has attracted muchattention to this species. Here we report the preliminary resultsof the induction of anthocyanin biosynthesis in sour cherrycallus cell cultures. The evaluation and characterization of thein vitro produced pigments are compared to those of theanthocyanins found in vivo in fruits of several sour cherrycultivars. Interestingly, the anthocyanin profiles found in wholefruit extracts were similar in all tested genotypes but weredifferent with respect to the callus extract. The evaluation ofantioxidant activity, performed by ORAC and TEAC assays, revealeda relatively high antioxidant capacity for the fruitextracts (from 1145 to 2592μmol TE/100g FW) and alower one for the callus extract (688μmol TE/100g FW).     

A Low Concentration of Ethanol Reduces the Chemiluminescence of Human Granulocytes and Monocytes but Not the Tumor Necrosis Factor Alpha Production by Monocytes after Endotoxin Stimulation

The ability of polymorphonuclear neutrophils (PMNs) and monocytes (M) to produce reactive oxygen species (ROS) has been related closely to their potential in the killing of microorganisms. Ethanol has been shown to impair the generation of ROS in these phagocytes after stimulation with some immunogens and to increase the susceptibility of alcohol abusers to infectious diseases. As endotoxemia is common in alcohol abusers, we investigated the effect of ethanol (21.7 mmol/liter) on the luminol-amplified chemiluminescence of PMNs and M after endotoxin stimulation and the release of tumor necrosis factor alpha (TNF-α) from M. Further, the efficiency of ethanol to inactivate chemically generated ROS was tested. Significant stimulation of ROS release occurred at endotoxin concentrations of 1 ng/ml or higher in both PMNs and M. Ethanol significantly suppressed the formation of ROS in both cell types, the decrease being more pronounced in M (−73.8%) than in PMNs (−45.7%). The correlations between endotoxin concentration and the amount of released ROS showed a dose-dependent, sigmoidal course. Concentrations of endotoxin necessary for half-maximum stimulation were nearly identical (6 to 8 ng/ml) in both PMNs and M, independent of the presence of ethanol. In contrast to ROS formation, ethanol had no effect on the amount of TNF-α produced by endotoxin-stimulated M. Ethanol was shown to be unable to decrease the levels of chemically generated ROS under physiological conditions. Therefore, ethanol cannot be assumed to be an “antioxidative” compound but rather seems to modify processes of endotoxin recognition, intracellular signal transduction, or metabolism.     

Serum Cytokine Responses during Acute Human Granulocytic Ehrlichiosis

Human granulocytic ehrlichiosis (HGE) is caused by obligate intracellular bacteria in the Ehrlichia phagocytophila group. The disease ranges from subclinical to fatal. We speculated that cell-mediated immunity would be important for recovery from and potentially in the clinical manifestations of HGE; thus, serum tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), gamma interferon (IFN-γ), IL-10, and IL-4 concentrations were studied. IFN-γ (1,035 ± 235 pg/ml [mean ± standard error of the mean]) and IL-10 (118 ± 46 pg/ml) concentrations were elevated in acute-phase sera versus convalescent sera and normal subjects (P≤0.013 and P≤0.018, respectively). TNF-α, IL-1β, and IL-4 levels were not elevated. Cytokine levels in severely and mildly affected patients were not different. HGE leads to induction of IFN-γ-dominated cell-mediated immunity associated with clinical manifestations, recovery from infection, or both.     

Evaluation of the In Vitro Pyrogen Test System Based on Proinflammatory Cytokine Release from Human Monocytes: Comparison with a Human Whole Blood Culture Test System and with the Rabbit Pyrogen Test

The reliability of an in vitro pyrogen test system based on proinflammatory cytokine release from human monocytic cells was assessed by comparison with a test system based on a human whole blood culture as well as with the conventional rabbit pyrogen test. The human cells used as the pyrogen indicator cells were newly selected by subcloning of a human monocytic cell line, Mono-Mac-6. The selected cells, named MM6-CA8, responded to various pyrogens, including endotoxin, peptidoglycan (PG), Staphylococcus aureus Cowan 1 (SAC), and poly(I·C), with a high sensitivity and produced proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha. Among these cytokines, IL-6 was produced most sensitively in response to traces of the pyrogens and detected in the largest quantities in the culture medium. The cytokine-producing responses of MM6-CA8 cells correlated significantly with the responses of cultured human whole blood, which represents an ex vivo culture test system reproducing pyrogen-induced cytokine production in the human body. In terms of cytokine inducibility, the pyrogens were ranked in the order endotoxin > PG > poly (I·C) > SAC in both culture systems, a ranking which almost agreed with the ranking of their pyrogenicity as assessed by the rabbit pyrogen test. These results suggest that the in vitro responsiveness of MM6-CA8 cells to various pyrogens is highly relevant for human pyrogenic reactions. Therefore, the in vitro test system is useful and reliable for detecting the presence of materials that are pyrogenic for humans.     

papG Alleles among Escherichia coli Strains Causing Urosepsis: Associations with Other Bacterial Characteristics and Host Compromise

Alleles I, II, and III of the P adhesin gene papG were sought by PCR among 75 Escherichia coli blood isolates from adults with urosepsis, and the papG genotype was compared with associated bacterial characteristics and host compromise status. Allele II predominated over allele III in the total population (71% of the strains versus 7% for allele III; P<0.01). Allele I was not encountered. In comparison with allele II, allele III was significantly associated with the presence of hly and the absence of iuc (which encode hemolysin and aerobactin biosynthesis), nonagglutination of digalactoside-coated beads, absence of aerobactin production, membership in serogroups O6 and O18, and host compromise, particularly cancer and upper urinary tract abnormalities.     

Effect of Grape Seed Extract and Quercetin on Cardiovascular and Endothelial Parameters in High-Risk Subjects

Grape seed extract (GSE) has in vitro antioxidant activity but whether or notit works in vivo is not clear. In a fully randomised, crossovertrial with 4-week treatment periods on 36 men and womenwith above-average vascular risk, we aimed to demonstrate that2g/day of GSE (1g of polyphenols) alone,or with 1g/day of added quercetin in yoghurt, favourably altersvascular function, endothelial function, and degree of oxidative damagein comparison to a control yoghurt. GSE alone improved flow-mediateddilatation determined ultrasonically by an absolute 1.1% comparedwith control. There was no effect of the combination of GSE withquercetin. No other blood or urine measure was altered. Thussufficient polyphenols from GSE appear to be absorbed to influenceendothelial nitric oxide production, and GSE has the potential tofavourably influence vascular function.     

Caffeoylquinic Acids Generated In Vitro in a High-Anthocyanin-Accumulating Sweet potato Cell Line

Accumulation of phenolic compounds has beenmonitored in a suspension culture of anthocyanin-accumulatingsweet potato cell line grown under the conditions ofmodified Murashige and Skoog high-anthocyanin productionmedium (APM) over a period of 24 days. Tissue samplesextracted with 15% acetic acid were analysed using HPLC at adetection wavelength of 326nm. Among others, the followingderivatives of caffeoylquinic acids were detected:4,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid,3,4-dicaffeoylquinic acid, and 3,4,5-tricaffeoylquinic acid. Theirtotal amount reached a maximum of 110mg/gFW between the 4thand the 15th day of culture growth on APM. The major compound ofthe phenolic mixture was 3,5-dicaffeoylquinic acid with maximumaccumulation level of 80mg/100gFW. The potentialeffects of targeted phenolic compounds on the nutraceuticalqualities of in vitro produced anthocyanin-rich extracts arediscussed.     

Effects of Dietary Copper Loading on Livers of Rats: I. Changes in Subcellular Acid Phosphatases and Detection of an Additional Acid p-Nitrophenylphosphatase in the Cellular Supernatant During Copper Loading

Significant changes occurred in lysosomal structure and function as copper was metabolized by rat livers. Hepatic total acid p-nitrophenylphosphatase (pNPase) activity was markedly increased in copper-loaded rats, and this increase was almost completely accounted for by heat- and formalin-stable (HFS) acid pNPase. Heat- and formalin-labile acid pNPase was essentially unchanged. On a subcellular level, the microsomal and supernatant fractions reflected the greatest relative increase in HFS acid pNPase. Increases in lysosomal, HFS acid pNPase in the large-granule fractions correlated with increase in solubilized large-granule enzymes, LG I, with mol wt > 200,000. LG II, representng solubilized large-granule enzymes with mol wt < 200,000, remained unchanged. Marked increases in supernatant acid pNPase were principally accounted for by a sevenfold increase in a supernatant lysosomal-like enzyme, DEAE Pk 1, separated by DEAE cellulose chromatography. An additional enzyme, DEAE Pk 2A′, that was hardly or not detectable in normal rats, was consistently demonstrated and increased in copper-loaded rats. Serum HFS acid pNPase increased in copper-loaded rats, suggesting that the increased hepatic supernatant acid pNPase in part escaped into the circulating fluid. Copper was principally associated with cytoplasmic organelles and was highest in mitochondrial and lysosomal fractions.     

The Change of Total Anthocyanins in Blueberries and Their Antioxidant Effect After Drying and Freezing

This study examined the effects of freezing, storage, and cabinetdrying on the anthocyanin content and antioxidant activity ofblueberries (Vaccinium corymbosum L). Fresh samples werestored for two weeks at 5^°C while frozen samples were keptfor up to three months at −20^°C. There were two dryingtreatments, one including osmotic pretreatment followed bycabinet drying and the other involving only cabinet drying.Total anthocyanins found in fresh blueberries were 7.2 ±0.5mg/g dry matter, expressed as cyanidin 3-rutinosideequivalents. In comparison with fresh samples, total anthocyaninsin untreated and pretreated dried blueberries were significantlyreduced to 4.3 ± 0.1mg/g solid content, 41% loss, and3.7 ± 0.2mg/g solid content, 49% loss, respectively.Osmotic treatment followed by a thermal treatment had a greatereffect on anthocyanin loss than the thermal treatment alone. Incontrast, the frozen samples did not show any significantdecrease in anthocyanin level during three months of storage.Measurement of the antioxidant activity of anthocyanin extractsfrom blueberries showed there was no significant differencebetween fresh, dried, and frozen blueberries.     

Effect of Synthetic Truncated Apolipoprotein C-I Peptide on Plasma Lipoprotein Cholesterol in Nonhuman Primates

The present studies were conducted to determinewhether a synthetic truncated apoC-I peptide thatinhibits CETP activity in baboons would raise plasmaHDL cholesterol levels in nonhuman primates with lowHDL levels. We used 2 cynomolgus monkeys and 3baboons fed a cholesterol- and fat-enriched diet. Incynomolgus monkeys, we injected synthetic truncatedapoC-I inhibitor peptide at a dose of 20mg/kgand, in baboons, at doses of 10, 15, and 20mg/kgat weekly intervals. Blood samples were collected 3times a week and VLDL + LDL and HDL cholesterolconcentrations were measured. In cynomolgus monkeys,administration of the inhibitor peptide caused arapid decrease in VLDL + LDL cholesterolconcentrations (30%–60%) and an increase in HDLcholesterol concentrations (10%–20%). VLDL + LDLcholesterol concentrations returned to baselinelevels in approximately 15days. In baboons,administration of the synthetic inhibitor peptidecaused a decrease in VLDL + LDL cholesterol (20%–60%)and an increase in HDL cholesterol (10%–20%). VLDL+ LDL cholesterol returned to baseline levels byday 21, whereas HDL cholesterol concentrationsremained elevated for up to 26days. ApoA-Iconcentrations increased, whereas apoE andtriglyceride concentrations decreased. Subcutaneousand intravenous administrations of the inhibitorpeptide had similar effects on LDL and HDLcholesterol concentrations. There was no change inbody weight, food consumption, or plasma IgGlevels of any baboon during the study. Thesestudies suggest that the truncated apoC-I peptide canbe used to raise HDL in humans.     

Mechanical work and efficiency in level walking and running

1. The mechanical power spent to accelerate the limbs relative to the trunk in level walking and running, _int, has been measured at various `constant' speeds (3-33 km/hr) with the cinematographic procedure used by Fenn (1930a) at high speeds of running.

2. _int increases approximately as the square of the speed of walking and running. For a given speed _int is greater in walking than in running.

3. In walking above 3 km/hr, _int is greater than the power spent to accelerate and lift the centre of mass of the body at each step, _ext (measured by Cavagna, Thys & Zamboni, 1976b). In running _int < _ext up to about 20 km/hr, whereas at higher speeds _int > _ext.

4. The total work done by the muscles was calculated as W_tot = |W_int| + |W_ext|. Except that at the highest speeds of walking, the total work done per unit distance W_tot/km is greater in running than in walking.

5. The efficiency of positive work was measured from the ratio W_tot/Net energy expenditure: this is greater than 0·25 indicating that both in walking and in running the muscles utilize, during shortening, some energy stored during a previous phase of negative work (stretching).

6. In walking the efficiency reaches a maximum (0·35-0·40) at intermediate speeds, as may be expected from the properties of the contractile component of muscle. In running the efficiency increases steadily with speed (from 0·45 to 0·70-0·80) suggesting that positive work derives mainly from the passive recoil of muscle elastic elements and to a lesser extent from the active shortening of the contractile machinery. These findings are consistent with the different mechanics of the two exercises.

     

Two Spectrophotometric Assays for Dopamine Derivatives in Pharmaceutical Products and in Biological Samples of Schizophrenic Patients Using Copper Tetramine Complex and Tri-iodide Reagent

Two simple, rapid, and sensitive spectrophotometric methods areproposed for the determination of levodopa (LD). The first methodis based on coupling of 4-aminoantipyrine (4-AAP) with one of thedopamine derivatives (LD, CD) to give a new ligand that reactswith copper tetramine complex to give intenselycolored chelates. The colored products are quantifiedspectrophotometrically at 525 and 520nm for LD and CD,respectively. The optimization of the experimental conditions isdescribed. The method has been used for the determination of19.7–69.0 and 18.1–54.3μg mL⁻¹ of LDand CD, respectively. The accuracy of the method is achieved bythe values of recovery (100 ± 0.2%) and the precision issupported by the low standard deviation (SD = 0.17–0.59) andrelative standard deviation (CV = 0.4%–1.54%) values. Thesecond method is based on the formation of ion-pair iodinatedinner sphere or outer sphere colored complexes between the LDand triiodide ions at pH 5 and room temperature (23 ± 3^°C). This method has been used for the determination of LDwithin the concentration range 39.44–78.88μgmL⁻¹ with SD = 0.22–0.24 and recovery percent = 100 ± 0.3%.The sensitivity of the two methods is indicated bySandell's sensitivity of 0.014–0.019g cm⁻². Theresults of the two methods are compared with those of theofficial method. The interference of common drug additives,degradation products, and excipients was also studied. Theproposed methods were applied successfully to the determinationof the LD-CD synthetic mixture and Levocare drug. Thedetermination of LD in urine of some schizophrenic patients wasapplied with good precision and accuracy. The reliability of themethods was established by parallel determinations against theofficial British pharmacopoeia method.     

Oxygen intake in track and treadmill running with observations on the effect of air resistance

1. The relation of _O2 and speed was measured on seven athletes running on a cinder track and an all-weather track. The results were compared with similar observations on four athletes running on a treadmill.

2. In treadmill running the relation was linear and the zero intercept coincided with resting _O2.

3. In track running the relation was curvilinear, but was adequately represented by a linear regression over a range of speeds extending from 8·0 km/hr (2·2 m/sec) to 21·5 km/hr (6·0 m/sec). The slope of this line was substantially steeper than the regression line slope for treadmill running.

4. The influence of air resistance in running was estimated from measurements of _O2 on a subject running on a treadmill at constant speed against wind of varying velocity.

5. The extra O₂ intake (Δ_O2) associated with wind increased as the square of wind velocity. If wind velocity and running velocity are equal, as in running on a track in calm air, Δ_O2 will increase as the cube of velocity.

6. It was estimated that the energy cost of overcoming air resistance in track running is about 8% of total energy cost at 21·5 km/hr (5000 m races) and 16% for sprinting 100 m in 10·0 sec.