Phosphorus nuclear magnetic resonance studies of phosphorus metabolites in frog muscle.

31P-nuclear magnetic resonance was applied to living muscles of bullfrogs, and the time courses of metabolic changes of ATP, creatine phosphate, inorganic phosphate, and sugar phosphates were studied under anaerobic and aerobic conditions. A decrease in creatine phosphate was observed in the resting muscle under anaerobic conditions with a concomitant decrease in the intracellular pH, while the ATP level remained constant. With the use of 2,4-dinitro-1-fluorobenzene and iodoacetic acid, ATP disappeared quickly. When the resting muscle was perfused with oxygen-saturated glucose-Ringer's solution, the amount of creatine phosphate increased gradually. These findings indicate that anaerobic glycolysis is insufficient for even the resting energy consumption whereas oxidative phosphorylation is sufficient. The effects of tetanic stimulation on living muscles were also studied. When glycolysis and oxidative phosphorylation were suppressed, the intracellular energy store was depleted by the tetanic contraction. Anaerobic glycolysis produced rapid recovery of the energy store level, although it was insufficient to reach the initial level. Aerobic oxidative phosphorylation produced sufficient energy to reach the initial level, and this level was never exceeded. This finding suggests the existence of a regulatory mechanism for the energy store level.     

Non-invasive measurement of adrenocortical activity in male and female rats

Rats are widely used in biomedical research as animal models for human diseases. However, due to their small body size, blood sampling is complicated and invasive and thereby can seriously interfere with endocrine functions and possibly compromise the animals' welfare. Therefore, a non-invasive technique to monitor stress hormones in these animals is highly desired. Our study aimed to gain general information about corticosterone metabolism and excretion and to validate a 5alpha-pregnane-3beta,11beta,21-triol-20-one enzyme immunoassay (EIA) to reliably measure faecal corticosterone metabolites (CMs) in laboratory rats. In total, 18 rats were administered 2.3 MBq of (3)H-corticosterone intravenously and per os, respectively (intravenous: 6 males and 6 females; per os: 3 males and 3 females). Subsequently, all voided excreta were frequently collected for five days. About 75+/-9% of the recovered CMs were found in the faeces. Peak concentrations of radiolabelled steroids appeared in the urine after 1.7+/-0.6 h in males and after 6.0+/-3.5 h in females. In faeces, maxima were observed after 14.7+/-2.4 h in both sexes. In principle, the time course and delay for both routes of administration (intravenous or per os) were the same, except for a delay of peak concentrations in urine (4.5+/-2.1 h) in per os administered males. Using high-performance liquid chromatography (HPLC), faecal (3)H-CMs were characterized and differences were found between the sexes. In both sexes, corticosterone was extensively metabolized, but while males showed only minor variations in their CM patterns, those of females differed largely between individuals. To validate the mentioned EIA, we investigated the diurnal variation (DV) of glucocorticoids as well as effects of the injection procedure itself and conducted an adrenocorticotropic hormone challenge test and a dexamethasone suppression test, using six male and six female rats each. Our results demonstrated that pharmacological stimulation, suppression and DV of adrenocortical activity were accurately reflected by means of CM measurement in faeces. By successful physiological validation, we proved for the first time the suitability of an immunoassay to non-invasively monitor adrenocortical activity in rats of both sexes. This method opens up new perspectives for biomedical and pharmacological investigations as well as for animal welfare related issues.     

In vitro tracheal mechanics by nuclear magnetic resonance imaging

Images of rabbit tracheal cross sections were obtained at a series of transmural pressures ranging from 22 to -95 cmH2O by use of a nuclear magnetic resonance imaging microscope. The excised, washed tracheas were immersed in a solution of phosphate-buffered saline made up in deuterium oxide (D2O, pH 7.3). The images are maps of proton density in the image slice (2.5 mm thick). All but one series of images showed a collapse process in which the trachealis muscle invaginated asymmetrically, i.e., the muscle appeared to favor one side of the cartilage ring system more than the other. The connecting tissue between the cartilage rings appeared to be more compliant than the rings themselves, thus suggesting that the tracheal lumen became corrugated at negative pressures. In the plane of a cartilage ring, the lumen appeared to remain patent at pressures as low as -95 cmH2O. However, between rings, where the tracheal wall was more compliant, the lumen appeared to be totally occluded at -53 cmH2O. Lumen areas in both the plane of the cartilage rings and in a plane between rings were measured from each series of printed images for six tracheas. These measurements, when normalized, averaged, and plotted against transmural pressure gave asymptotic logarithmic compliances (n1 in the model of Lambert et al., J. Appl. Physiol. 52: 44-56, 1982) of 1.2 +/- 0.4 and 20 +/- 7 for the interring and ring regions, respectively. These values are greater than the critical value of 0.5 (J. Appl. Physiol. 62: 2426-2435, 1987) and are thus consistent with wave speed flow limitation being possible anywhere in the trachea during forced expiration.     

Identification of experimentally induced colitis by in vitro nuclear magnetic resonance

The present study determined whether in vitro nuclear magnetic resonance could be used to assess experimentally induced colitis in rats. Acute colitis was induced in 6 Sprague-Dawley rats by acetic acid enema, while 6 control animals received saline enemas. All animals were sacrificed 24 hours post-enema, and NMR relaxation times, T1 and T2, of colonic samples were determined on a 10 MHz spin analyzer (RADX, Houston, TX). Colonic water content was determined on the same samples by desiccation. Colitis animals showed significantly higher T1 and T2 relaxation times and tissue water content than controls. T1 and T2 times correlated significantly with tissue water content. Twelve additional animals were studied histologically, six of which received acetic acid enemas and showed extensive transmural colitis. Our results suggest that in vivo proton NMR might be a useful means of non-invasively assessing the degree of colonic inflammation.     

Conformational analysis by nuclear magnetic resonance: insulin

High-resolution 270-MHz proton nuclear magnetic resonance (NMR) spectra of the native two-zinc insulin hexamer at pH 9 have been obtained, and assignments of key resonances have been made. Spectra of zinc-free insulin titrated with Zn2+ are unchanged after the addition of 1 equiv of zinc per insulin hexamer, indicating that the conformation of the hexamer is fixed at this point and that the second zinc ion does not significantly change the conformation. Titration of the two-zinc insulin hexamer with anions high on the Hofmeister series such as SCN- causes marked changes in the NMR spectra which are interpreted as the result of major conformational changes to a new hexameric form of insulin having a twofold axis perpendicular to the threefold axis. Analysis of difference spectra indicates that this new hexamer (which should be capable of binding six zinc ions) binds 2 equiv of SCN- at two sites which are assumed to be identical and independent (K1 = 10(3), K2 = 2.5 X 10(2) M-1).     

Phosphorus-31 nuclear magnetic resonance analysis of internal pH during photosynthesis in the cyanobacterium Synechococcus

Phosphorus-31 nuclear magnetic resonance (31P NMR) spectra were obtained from actively photosynthesizing and darkened suspensions of the unicellular cyanobacterium Synechococcus. These spectra show intracellular resonances belonging to inorganic phosphate (Pi), a sugar phosphate (sugar-P), nucleotide di- and triphosphates, and poly-phosphates. The pH-dependent chemical shifts of Pi and sugar-P allowed the estimation of intracellular pH. When irradiated with high-intensity tungsten-halogen light (100 x 10(4) ergs . cm-2 . s-1, measured in the visible range), concentrated cell suspensions in the NMR spectrometer incorporated NaH14CO3 at approximately two-thirds the rate shown by a dilute suspension of cells at saturating light intensity. On the basis of NaH14CO3 incorporation, the effective light intensity obtained under NMR conditions would support growth at approximately one-fourth the maximum rate in dilute suspensions of cells. Irradiated cells maintained a cytoplasmic pH of 7.1--7.3 when exposed to an external pH from 6.4 to 8.3. At an external pH of 6.7, a darkness to light shift caused a 0.4 pH unit alkalinization of the cytoplasm. Treatment of cell suspensions with the uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), in light or darkness, collapsed the internal pH to the level of the external pH. The results suggest a strong light- or energy-dependent buffering of the cytoplasm over a range of external pH. The study demonstrates that 31P NMR can be used to investigate intracellular events in an actively photosynthesizing microorganism.     

Phosphate metabolites in single barnacle muscle fibers investigated by phosphorus-31 nuclear magnetic resonance

• 1.1. A study has been made of phosphate-containing metabolites in single barnacle muscle fibers using phosphorus-31 nuclear magnetic resonance.
• 2.2. Spectra from single fibers (∼50 mg in wet weight) show major resonances from sugar phosphates, inorganic phosphate, arginine phosphate and the α, β and γ phosphorus atoms of ATP.
• 3.3. The approximate “free” concentration of each metabolite was determined by integration of the spectrum, using a sample of 1 M-methylene diphosphonic acid as a reference. A notable feature of the results obtained is that the concentrations of SP&P_i in freshly dissected fibers are low.
• 4.4. Time-dependent changes in ³¹P-NMR spectra indicate that ArP declines fairly slowly, while SP and P_i rises. The half-life of ArP at 26°C turns out to be about 8 hr. ATP remains relatively constant for the first 8 hr but disappears following the disappearance of ArP. As the intensity of the P_i resonance increases with time, it broadens and moves upfield, suggesting internal acidosis.
• 5.5. These results demonstrate that ³¹P-NMR can provide useful information about metabolism and its regulation in single barnacle muscle fibers.

     

Use of two replacements of serum during bovine embryo culture in vitro

This study evaluated the effect of two commercial serum replacements (Ultroser G and CPSR-3 on in vitro bovine embryo culture. In Experiment 1, zygotes were cultured in SOF+Ultroser G (2, 4 and 6%), SOF+CPSR-3 (2, 4 and 6%), and SOF+5% FCS (control). Blastocyst rates obtained after culturing with Ultroser G were lower than those with FCS. However, blastocyst rates for CPSR-3 were similar to those for serum. In addition, embryos produced in SOF+CPSR-3 had the same proportion inner cell mass number and total cell number as embryos cultured with FCS. In Experiment 2, a combination of serum replacements during different periods showed that treatment before the five-to eight-cell stages had no effect on further embryo development. However, treatments up to the morula stage affected blastocyst formation. The concentration of supplement and the timing of its inclusion in culture markedly affected embryo development. The serum replacement CPSR-3 can supplement embryo culture with blastocyst rates and quality similar to those for serum.     

Identification of metabolites of importance in the pathogenesis of pulmonary cryptococcoma using nuclear magnetic resonance spectroscopy

Primary lung infection with Cryptococcus neoformans is characterised by circumscribed lesions (cryptococcomas). To identify cryptococcal and/or host products of importance in pathogenesis, we applied proton nuclear magnetic resonance (NMR) spectroscopy, which identifies mobile compounds present in complex mixtures, to experimental pulmonary cryptococcomas from rats. Magnetic resonance experiments were performed on cryptococcomas (n = 10) and healthy lungs (n = 8). Signal assignment to key metabolites was confirmed by homo-nuclear and hetero-nuclear NMR correlation spectroscopy. Cryptococcal metabolites, dominating spectra from cryptococcomas included the stress protectants, trehalose and mannitol, acetate, and in some animals, ethanol. Glycerophosphorylcholine was also abundant in cryptococcomas, consistent with hydrolysis of phospholipids in vivo by the cryptococcal enzyme, phospholipase B (PLB). PLB has been identified by molecular studies as a cryptococcal virulence determinant. We propose that PLB secreted by cryptococci promotes tissue invasion by hydrolysing host phospholipids, such as dipalmitoyl phosphatidyl choline, which is abundant in pulmonary surfactant, and lung cell membrane phospholipids. Our results confirm the utility of NMR spectroscopy in studies of microbial pathogenesis.     

Non-invasive histochemistry of plant materials by magnetic resonance microscopy

Summary We have combined nuclear magnetic resonance (NMR) imaging on the microscopic scale with chemical shift selection to demonstrate the application of magnetic resonance imaging (MRI) to plant histochemistry. As an example of the method we have obtained separate images of the distribution of reserve oil and anethole in dried fennel mericarps. The technique can be employed to separately image the distribution of aromatics, carbohydrates, oils, water and possibly fatty acids in suitable plant materials.Abbreviations NMR nuclear magnetic resonance - MRI magnetic resonance imaging - COSY correlation spectroscopy - TMS tetramethylsilane     

A sample preparation protocol for H nuclear magnetic resonance studies of water-soluble metabolites in blood and urine

We describe a general protocol for preparing protein-containing biofluids for ¹H nuclear magnetic resonance (NMR) metabolomic studies. In this protocol, untreated samples are diluted in deuterated solvents to precipitate proteins and recover metabolites quantitated relative to standard reference compounds such as 3-trimethylsilylpropionic acid (TSP) and 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS). The efficacy of this protocol was tested using a bovine serum albumin/metabolite mix and human serum samples. This sample preparation method can be readily applied to any protein-containing biofluid for ¹H NMR studies.     

Identification and quantitation of phosphorus metabolites in yeast neutral pH extracts by nuclear magnetic resonance spectroscopy.

(31)P NMR spectroscopy offers a possibility to obtain a survey of all low-molecular-weight phosphorylated compounds in yeast. The yeast cells have been extracted using chloroform into a neutral aqueous phase. The use of high fields and the neutral pH extracts, which are suitable for NMR analysis, results in well-resolved (31)P NMR spectra. Two-dimensional NMR experiments, such as proton-detected heteronuclear single quantum ((1)H-(31)P HSQC) and (31)P correlation spectroscopy ((31)P COSY), have been used to assign the resonances. In the phosphomonoester region many of the signals could be assigned to known metabolites in the glycolytic and pentose phosphate pathways, although some signals remain unidentified. Accumulation of ribulose 5-phosphate, xylulose 5-phosphate, and ribose 5-phosphate was observed in a strain lacking transketolase activity when grown in synthetic complete medium. No such accumulation occurred when the cells were grown in yeast-peptone-dextrose medium. Trimetaphosphate (intracellular concentration about 0.2 mM) was detected in both cold methanol-chloroform and perchloric acid extracts.     

Review: magnetic resonance imaging of male/female differences in human adolescent brain anatomy

ABSTRACT: Improvements in neuroimaging technologies, and greater access to their use, have generated a plethora of data regarding male/female differences in the developing brain. Examination of these differences may shed light on the pathophysiology of the many illnesses that differ between the sexes and ultimately lead to more effective interventions. In this review, we attempt to synthesize the anatomic magnetic resonance imaging (MRI) literature of male/female brain differences with emphasis on studies encompassing adolescence -- a time of divergence in physical and behavioral characteristics. Across all ages total brain size is consistently reported to be about 10 % larger in males. Structures commonly reported to be different between sexes include the caudate nucleus, amygdala, hippocampus, and cerebellum -- all noted to have a relatively high density of sex steroid receptors. The direction and magnitude of reported brain differences depends on the methodology of data acquisition and analysis, whether and how the subcomponents are adjusted for the total brain volume difference, and the age of the participants in the studies. Longitudinal studies indicate regional cortical gray matter volumes follow inverted U shaped developmental trajectories with peak size occurring one to three years earlier in females. Cortical gray matter differences are modulated by androgen receptor genotyope and by circulating levels of hormones. White matter volumes increase throughout childhood and adolescence in both sexes but more rapidly in adolescent males resulting in an expanding magnitude of sex differences from childhood to adulthood.