Chromium propionate in broilers: effect on insulin sensitivity.

The objective of this study was to evaluate the effects of dietary chromium (Cr), as chromium propionate, on measures of insulin sensitivity. Liver and muscle glycogen, and plasma glucose and non-esterified fatty acid (NEFA) concentrations were used as indicators of insulin sensitivity. In total, 288 newly hatched male Ross broilers were divided into 4 dietary treatments consisting of 0 (control diet analyzed 0.43 to 0.45 mg Cr/kg), 0.2, 0.4, or 0.6 mg supplemental Cr/kg diet, resulting in 4 treatments with 9 replicate pens per treatment containing eight birds per pen. At d 21, 2 birds per cage were removed based on the greatest deviation from pen mean BW, resulting in each pen containing 6 birds for the final analyses. Final BW were taken on d 40, and on d 42 two birds from each pen were sampled for plasma NEFA, glucose, and muscle and liver glycogen determination at the initiation and termination of a 22 h fast. The remaining 2 fasted birds were sampled after a 30 min refeeding period. No differences were observed in feed intake, BW gain, or feed efficiency on d 21 or d 40. Liver glycogen tended (P=0.10) to be greater in Cr-supplemented chicks in the fed state, and muscle glycogen concentrations tended (P=0.07) to be greater in Cr-supplemented chicks compared with controls following fasting and refeeding. Plasma glucose concentrations were not affected by dietary Cr in the fed, fasted, or refed state. Plasma NEFA levels were not affected by treatment in fed or fasted birds. However, plasma NEFA concentrations were lower (P<0.01) in chicks supplemented with Cr than in controls following fasting and refeeding, suggesting that Cr increased insulin sensitivity. No differences were detected among birds supplemented with 0.2 or 0.4 mg Cr/kg, and among those receiving 0.4 or 0.6 mg Cr/kg. Results of this study indicate that Cr propionate supplementation of a control diet containing 0.43 to 0.45 mg Cr/kg enhanced insulin sensitivity.

Incidental Findings from 16,400 Brain MRI Examinations of Research Volunteers.

BACKGROUND AND PURPOSE: Incidental findings are discovered in neuroimaging research, ranging from trivial to life-threatening. We describe the prevalence and characteristics of incidental findings from 16,400 research brain MRIs, comparing spontaneous detection by nonradiology scanning staff versus formal neuroradiologist interpretation. MATERIALS AND METHODS: We prospectively collected 16,400 brain MRIs (7782 males, 8618 females; younger than 1 to 94 years of age; median age, 38 years) under an institutional review board directive intended to identify clinically relevant incidental findings. The study population included 13,150 presumed healthy volunteers and 3250 individuals with known neurologic diagnoses. Scanning staff were asked to flag concerning imaging findings seen during the scan session, and neuroradiologists produced structured reports after reviewing every scan. RESULTS: Neuroradiologists reported 13,593/16,400 (83%) scans as having normal findings, 2193/16,400 (13.3%) with abnormal findings without follow-up recommended, and 614/16,400 (3.7%) with "abnormal findings with follow-up recommended." The most common abnormalities prompting follow-up were vascular (263/614, 43%), neoplastic (130/614, 21%), and congenital (92/614, 15%). Volunteers older than 65 years of age were significantly more likely to have scans with abnormal findings (P < .001); however, among all volunteers with incidental findings, those younger than 65 years of age were more likely to be recommended for follow-up. Nonradiologists flagged <1% of MRIs containing at least 1 abnormality reported by the neuroradiologists to be concerning enough to warrant further evaluation. CONCLUSIONS: Four percent of individuals who undergo research brain MRIs have an incidental, potentially clinically significant finding. Routine neuroradiologist review of all scans yields a much higher rate of significant lesion detection than selective referral from nonradiologists who perform the examinations. Workflow and scan review processes need to be carefully considered when designing research protocols.

Strategies for the structural analysis of multi-protein complexes: lessons from the 3D-Repertoire project.

Structural studies of multi-protein complexes, whether by X-ray diffraction, scattering, NMR spectroscopy or electron microscopy, require stringent quality control of the component samples. The inability to produce 'keystone' subunits in a soluble and correctly folded form is a serious impediment to the reconstitution of the complexes. Co-expression of the components offers a valuable alternative to the expression of single proteins as a route to obtain sufficient amounts of the sample of interest. Even in cases where milligram-scale quantities of purified complex of interest become available, there is still no guarantee that good quality crystals can be obtained. At this step, protein engineering of one or more components of the complex is frequently required to improve solubility, yield or the ability to crystallize the sample. Subsequent characterization of these constructs may be performed by solution techniques such as Small Angle X-ray Scattering and Nuclear Magnetic Resonance to identify 'well behaved' complexes. Herein, we recount our experiences gained at protein production and complex assembly during the European 3D Repertoire project (3DR). The goal of this consortium was to obtain structural information on multi-protein complexes from yeast by combining crystallography, electron microscopy, NMR and in silico modeling methods. We present here representative set case studies of complexes that were produced and analyzed within the 3DR project. Our experience provides useful insight into strategies that are more generally applicable for structural analysis of protein complexes.

Apollo 11: exposure of lower animals to lunar material.

Lunar material returned from the first manned landing on the moon was assayed for the presence of replicating agents possibly harmful to life on earth. Ten species of lower animals were exposed to lunar material for 28 days. No pathological effects attributable to contact with lunar material were detected.

Relationship between residual feed intake and lymphocyte mitochondrial complex protein concentration and ratio in crossbred steers.

Rate of oxygen uptake by muscle mitochondria and respiratory chain protein concentrations differed between high- and low-residual feed intake (RFI) animals. The hypothesis of this research was that complex I (CI), II (CII), and III (CIII) mitochondria protein concentrations in lymphocyte (blood) mitochondria were related to the RFI phenotype of beef steers. Daily feed intake (ADFI) was individually recorded for 92 Hereford-crossbreed steers over 63 d using GrowSafe individual feed intake system. Predicted ADFI was calculated as the regression of ADFI on ADG and midtest BW. Difference between ADFI and predicted ADFI was RFI. Lymphocytes were isolated from low-RFI (-1.32 ± 0.11 kg/d; = 10) and high-RFI (1.34 ± 0.18 kg/d; = 8) steers. Immunocapture of CI, CII, and CIII proteins from the lymphocyte was done using MitoProfile CI, CII, and CIII immunocapture kits (MitoSciences Inc., Eugene, OR). Protein concentrations of CI, CII, and CIII and total protein were quantified using bicinchoninic acid colorimetric procedures. Low-RFI steers consumed 30% less ( = 0.0004) feed and had a 40% improvement ( < 0.0001) in feed efficiency compared with high-RFI steers with similar growth ( = 0.78) and weight measurements ( > 0.65). High- and low-RFI steers did not differ in CI ( = 0.22), CII ( = 0.69), and CIII ( = 0.59) protein concentrations. The protein concentration ratios for CI to CII ( = 0.03) were 20% higher and the ratios of CI to CIII ( = 0.01) were 30% higher, but the ratios of CII to CIII ( = 0.89) did not differ when comparing low-RFI steers with high-RFI steers. The similar magnitude difference in feed intake, feed efficiency measurements, and CI-to-CIII ratio between RFI phenotypes provides a plausible explanation for differences between the phenotypes. We also concluded that mitochondria isolated from lymphocytes could be used to study respiratory chain differences among differing RFI phenotypes. Further research is needed to determine if lymphocyte mitochondrial complex proteins can be used for identification of RFI phenotype.

Determinants of potassium channel assembly localised within the cytoplasmic C-terminal domain of Kv2.1.

The C-terminal domain of the voltage-gated potassium channel Kv2.1 is shown to have a role in channel assembly using dominant negative experiments in Xenopus oocytes. Kv2.1 channel polypeptides were co-expressed with a number of polypeptide fragments of the cytosolic C-terminus and the assembly of functional channel homotetramers quantified electrophysiologically using the two electrode voltage clamp technique. Co-expression of C-terminal polypeptides corresponding to the final 440, 318, 220 and 150 amino acid residues of Kv2.1 all resulted in a significant reduction in the functional expression of the full-length channel. A truncated version of Kv2.1 lacking the final 318 amino acids of the C-terminal domain (Kv2. 11-535) exhibited similar electrophysiological properties to the full-length channel. Co-expression with either the 440 or 318 residue polypeptides resulted in a reduction in the activity of the truncated channel. In contrast, the 220 and 150 residue C-terminal fragments had no effect on Kv2.11-535 activity. These data demonstrate that C-terminal interactions are important for driving Kv2.1 channel assembly and that distinct regions of the C-terminal domain may have differential effects on the formation of functional tetramers.

N-desmethyldiazepam: a new metabolite of chlordiazepoxide in man.

Following administration of chlordiazepoxide HCl to man, N-desmethyldiazepam, a known metabolite of diazepam (Valium), was identified in plasma. The metabolite was identified on the basis of its thin-layer chromatographic (TLC) mobility, electron-capture gas-chromatographic (EC-GC) retention time, and mass spectrum relative to authentic N-desmethyldiazepam. Plasma levels of N-desmethyldiazepam in subjects receiving both single and chronic doses of chlordiazepoxide were determined by an EC-GC method with a limit of sensitivity of 10 ng/ml using 2-ml samples and by a radioimmunoassay procedure which had a limit of sensitivity of 20 ng/ml using a 0.1-ml sample. Both assay methods gave good agreement for the levels of N-desmethyldiazepam. In subjects receiving a single 30-mg oral or intravenous dose of chlordiazepoxide, measurable levels of N-desmethyldiazepam in plasma (10 to 60 ng/ml) were obtained 24 to 72 hr after administration. In 5 subjects receiving 10 mg of chlordiazepoxide three times a day, steady-state levels of N-desmethyldiazepam in plasma were reached after about 1 wk of administration. The mean maximum and minimum steady-state levels of N-desmethyldiazepam were 260 and 220 ng/ml of plasma, respectively. Similar steady-state levels were observed on treatment with 30 mg of chlordiazepoxide over 24 hr.

Species specificity of immunity to malignant transformed cultured insect cells.

The specificity of malignantly transformed cultured cells of the German cockroach was assayed by injecting the cells into eight additional species of cockroaches in three families. All species but the most advanced one were susceptible. The range of lethal times for the different species reflected their relationship to the species from which the cells originated. The abilities of the injected hosts to encapsulate implants of embryo fragments of the German cockroach and microcarrier beads paralleled their resistance against the malignant cells. The order of susceptibility of the cockroach species to a pathogenic amoeba paralleled their susceptibility to the malignant cells, but death from the amoeba occurred in a much shorter time. Hematocrit values were higher in the more resistant species.

Symbiosis and attenuation.

Parasitic protozoa and bacteria transmitted by vector insects and ticks resist destruction in the intermediate host, possibly through the possession of antigens similar to those of the host. There is no evidence that symbiotic microorganisms have evolved similar eclipsed antigens. Symbiotes are protected from destruction by the natural lytic agents in the host, because they remain for the most part inside special cells that have lost the ability to recognize the symbiotes as "nonself." Metallic ions may play an important role in attenuation through their effect on cell membranes and on the release of lytic agents. The symbiotes of insects are useful and interesting subjects for studying attenuation.

Anabolic effect of biosynthetic growth hormone in cystic fibrosis patients.

The purpose of this study was to determine whether GH treatment of cystic fibrosis (CF) patients can result in an anabolic effect, i.e., increased weight gain, improved growth rate, nitrogen retention, and improved pulmonary function. Nine prepubertal endocrinologically normal CF patients (3 girls, 6 boys; chronological age (CA) 5.5-9.8 years, and bone age (BA) 4.5-9.0 years), received recombinant human growth hormone (rhGH) 0.3 mg/kg/week subcutaneously for a period of 12 months (N = 8) or 9 months (N = 1). Normal glucose tolerance was determined before treatment. Pulmonary function studies and anthropometric measurements were done every 3 months. Thyroid status, somatomedin C (SmC), BA, and routine chemistries were evaluated every 6 months. The pretreatment growth velocity averaged 5.7 +/- 0.3 (SE) cm/year and significantly increased to 7.8 +/- 0.4 (SE) cm/year during therapy, (P < 0.01). Standard deviation scores (SDS) for height significantly increased during rhGH therapy as compared with pretreatment, (P < 0.05). Weight of the patients during rhGH therapy did not significantly change during or after rhGH therapy. After therapy, all patients showed a significant increase in arm muscle area (AMA) and a significant decrement in arm fat area (AFA) (P < 0.01). Net nitrogen anabolism was negative in all subjects before therapy but became more positive in five patients during rhGH therapy. Three patients achieved positive nitrogen retention. SmC values significantly increased from a mean value of 0.62 +/- 0.1 (SE) U/ml to 1.6 +/- 0.6 (SE) U/ml after therapy. BA advanced 1.0 +/- 0.1 SE per year after treatment. Of the seven patients able to perform adequate pulmonary function testing, improvement occurred in FVC, FEV1.0, and PEFR in 5, 5, and 4 patients, respectively, but these changes did not reach statistical significance. We conclude that biosynthetic rhGH therapy had a significant anabolic effect in CF patients as shown by increased growth velocity, SmC values, increased protein and decreased fet stores, and a positive or less negative net nitrogen retention in five of the patients.

Technical progress in parentage analysis.

At the turn of the 20th century, Mendel's laws were found to be applicable to human blood groups. Within two decades, blood group genetics were applied to problems of parentage. Expansion of immunohematology into leukocyte antigen identification produced the single most informative, expressed polymorphism. About the same time, analysis of a great number of soluble protein polymorphisms followed advances in electric separation methods, enzymology, and immunochemistry. As new, independent loci were discovered, the power to exclude the falsely accused increased, and it became possible to apply Bayesian principles to determined probabilities of biologic relationships. The revolution in nucleic acid technology has dramatically improved analysis and statistical inferences. By the turn of the 21st century, laboratories should be able to determine biologic parentage with virtual certainty.

The VP4 and VP7 of bovine rotavirus VMRI are antigenically and genetically closely related to P-type 5, G-type 6 strains.

We have previously reported the isolation of a bovine rotavirus, designated VMRI, with a super-short electropherotype. We have characterized this strain further as it has shown antigenic differences with the prototype G6 strain NCDV-Lincoln. In this communication, we report the antigenic and molecular characterization and the nucleotide sequence of the VP4 and VP7 genes of this strain. Virus neutralization tests indicated 2- to 13-fold differences in the titers between NCDV-Lincoln, B641 and VMRI strains. Northern blot hybridization results indicated a degree of heterogeneity in the VP4 gene of these strains which can be detected under conditions of high stringency. The VP4 and VP7 genes of the VMRI strain were cloned and sequenced and compared with the published sequences of other bovine rotavirus strains. The VP4 gene of VMRI had a high degree of homology with that of UK and B641 strains but differed significantly from that of both NCDV-Lincoln and B223 strains. Sequence analysis of the VP7 gene of VMRI and other strains indicated a high degree of conservation and the amino acid identity between the different strains was 96%. Sequence information regarding these strains and field isolates will assist in the generation of effective vaccination strategies for control of neonatal calf diarrhea.

Noninvasive polarographic measurement of drug dissolution.

Polarographic analysis was applied successfully to dissolution studies and content uniformity assessment of both capsules and tablets, using a dropping mercury electrode with the modified Levy beaker method. The described noninvasive technique places the polarographic sensor probe directly into the dissolution flask and thus simplifies dissolution measurement by eliminating transfer lines and pumps typically required with the invasive (sampling) mode of analysis. A continuous sampling flowcell with polarographic detection was also evaluated for invasive measurements. Continuous dissolution profiles and content uniformity were determined for chlordiazepoxide, trimethoprim, ornidazole, and isoniazid, using the invasive and noninvasive sampling modes. Results obtained for these drugs showed excellent precision with both sampling techniques. In addition, excellent correlation to UV spectrophotometric data was obtained.

High-pressure liquid chromatographic determination of amoxicillin in urine.

A rapid and specific high-pressure liquid chromatographic (HPLC) assay was developed for the simultaneous determination of amoxicillin and its pencilloic acid metabolite in urine. The two compounds, assayed directly in urine or after dilution with water-methanol (85:15), are separated by reversed-phase chromatography and quantitated spectrofluorometrically following postcolumn derivatization with fluorescamine. Linear calibration curves were measured in the ranges of 25-250 and 50-400 ng injected for amoxicillin and the penicilloic acid metabolite, respectively. The sensitivity limit of the assay is 2.5-5.0 microgram/ml of urine for amoxicillin and the penicilloic acid metabolite. Urine samples (0-8 hr) taken from six subjects following single 250-mg po doses and assayed by HPLC showed ranges of cumulative percent of the dose excreted as amoxicillin and the penicilloic acid metabolite (reported as amoxicillin equivalents) of 50.2-68.0, and 21.6-30.0%, respectively. An excellent correlation (r = 0.985) was demonstrated for the measurement of amoxicillin concentrations by the HPLC and microbiological assays.

Determination of nitroimidazoles in biological fluids by differential pulse polarography.

A sensitive differential pulse polarographic assay was developed for the determination of metronidazole, ornidazole, and 2-nitro-1H-imidazole-1-(3-methoxy-2-propanol) in plasma. The compounds are selectively extracted into ethyl acetate from a protein-free filtrate of plasma, buffered to pH 7.0 +/- 0.2. The residue of the ethyl acetate extract is dissolved in 0.1 N NaOH and analyzed by differential pulse polarography for the reduction of the nitro group at approximately -0.600 v versus the saturated calomel electrode. The overall recovery from plasma was about 55 +/- 3.0% (SD) for the three compounds investigated. A TLC step after the ethyl acetate extraction may also be included to ensure specificity. This step reduced the overall recovery to approximately 45%. The sensitivity limit of detection from plasma using a 2-ml sample is 0.1 mug/ml. The assay may also be employed for the analysis of urine. The urine is adjusted to pH 7.0 +/- 0.2 and extracted with ethyl acetate, and the residue is analyzed as described for plasma. The assay was applied to the determination of ornidazole in blood and urine in the dog following 10 mg/kg po.

Spectrofluorometric determination of the antibiotic lasalocid in blood.

A spectrofluorometric assay was developed for the determination of the antibiotic lasalocid in dog blood, based on the intrinsic fluorescence of the compound in ethyl acetate. The assay can measure "total" levels of drug and any metabolites present. The specificity of the assay was verified by TLC separation of the dog blood extract, which indicated the presence of only intact drug. The overall recovery (+/- SD) of lasalocid was 62.0 +/- 3.6% in the concentration range of 1.0-10 mug of compound/ml of dog blood. The sensitivity of the assay is 0.5 mug/ml. The assay was applied to the determination of blood levels of lasalocid in the dog following the intravenous administration of a 5-mg/kg dose.

Use of supercritical fluid extraction for sample preparation of sustained-release felodipine tablets.

Supercritical fluid extraction (SFE) was shown to be an accurate and precise alternative to liquid extraction for sample preparation of sustained-release felodipine tablets (5 mg potency) while realizing an 80% reduction in solvent consumption. Extractions of felodipine spiked on an inert support were used to evaluate the solubility of felodipine in CO2 as well as analyte trapping after SFE. Even though the pure drug was found to be soluble in pure CO2, extractions of felodipine from the tablet matrix required moderate modifier concentrations [8.7% (v/v) methanol in CO2] in order to overcome strong matrix-drug interactions. Sequential static/dynamic extraction steps were also required to quantitatively recover the drug from the tablet matrix, indicating that the drug extraction was diffusion-limited. Average recoveries (n = 5) for the optimized SFE method were determined to be 4.93 mg felodipine tablet (98.6% claim) with an RSD of 1.2% versus those for the liquid extraction procedure (n = 5, 4.98 mg/tablet, 99.6% claim, 2.4% RSD). Similar levels of drug degradation (0.12% expressed as felodipine) were also obtained with both the traditional liquid extraction and with the SFE method.

Determination of 1,4-benzodiazepines in biological fluids by differential pulse polarography.

The determination of various 1,4-benzodiazepines and their metabolites by differential pulse polarography is reviewed and compared with that by other methods, and the general applicability of the polarographic methods, in terms of simplicity and flexibility, is demonstrated.