Gas Chromatography-Mass Spectrometry of N- Heptafluorobutyryl Isobutyl Esters of Amino Acids in the Analysis of the Kinetics of [N]H(4) Assimilation in Lemna minor L

Rapid, sensitive, and selective methods for the determination of the ¹⁵N abundance of amino acids in isotopic tracer experiments with plant tissues are described and discussed. Methodology has been directly tested in an analysis of the kinetics of [¹⁵N]H₄⁺ assimilation in Lemna minor L. The techniques utilize gas chromatography-mass spectrometry selected ion monitoring of major fragments containing the N moiety of N-heptafluorobutyryl isobutyl esters of amino acids. The ratio of selected ion pairs at the characteristic retention time of each amino acid derivative can be used to calculate ¹⁵N abundance with an accuracy of ±1 atom% excess ¹⁵N using samples containing as little as 30 picomoles of individual amino acids. Up to 11 individual amino acid derivatives can be selectively monitored in a single chromatogram of 30 minutes. It is suggested that these techniques will be useful in situations where the small quantities of N available for analysis have hitherto hindered the use of ¹⁵N-labeled precursors.     

Aberrant metabolism of matrix components in neonatal fibular cartilage of brachypod (bpH) mice.

Collagen and the acid mucopolysaccharides (AMPS) were studied in the fibular cartilage of brachypod ($\text{bp}{}^{\mathrm{<mtext>H</mtext>}}\text{bp}{}^{\mathrm{<mtext>H</mtext>}}$) and normal (+/+) newborn mice. At this age the mutant fibulae are still cartilaginous and are comprised of closely packed chondrocytes, homogenous in size and shape.Brachypod fibular chondrocytes synthesized a normal cartilage-type collagen molecule but at half the normal rate. Incorporation of tryptophan indicated this was related to a depression of general protein synthesis rather than being specific for collagen. Pulse-chase experiments showed that collagen degradation over a 3-day culture period was 15% slower than normal thus accounting for the higher collagen content in mutant fibulae.AMPS synthesis in normals and brachypods was nearly equal; however, in pulse-chase experiments radioactivity could not be chased out of the mutant tissue. The failure of AMPS degradation also accounted for greater than normal quantity of AMPS in the mutant cartilage. Characterization of the AMPS led to the discovery of a small population of unsulfated chondroitin molecules in normal, but not brachypod cartilage. The importance of a coordinated metabolism of matrix products during limb development is discussed.     

Impaired ribosome biogenesis disrupts the integration between morphogenesis and nuclear duplication during the germination of Aspergillus fumigatus

Aspergillus fumigatus is an important opportunistic fungal pathogen that is responsible for high mortality rates in the immunosuppressed population. CgrA, the A. fumigatus ortholog of a Saccharomyces cerevisiae nucleolar protein involved in ribosome biogenesis, contributes to the virulence of this fungus by supporting rapid growth at 37 degrees C. To determine how CgrA affects ribosome biogenesis in A. fumigatus, polysome profile and ribosomal subunit analyses were performed on both wild-type A. fumigatus and a DeltacgrA mutant. The loss of CgrA was associated with a reduction in the level of 80S monosomes as well as an imbalance in the 60S:40S subunit ratio and the appearance of half-mer ribosomes. The gene expression profile in the DeltacgrA mutant revealed increased abundance of a subset of translational machinery mRNAs relative to the wild type, suggesting a potential compensatory response to CgrA deficiency. Although DeltacgrA conidia germinated normally at 22 degrees C, they swelled excessively when incubated at 37 degrees C and accumulated abnormally high numbers of nuclei. This hypernucleated phenotype could be replicated pharmacologically by germinating wild-type conidia under conditions of reductive stress. These findings indicate that the germination process is particularly vulnerable to global disruption of protein synthesis and suggest that CgrA is involved in both ribosome biogenesis and polarized cell growth in A. fumigatus.     

Substantial Metabolic Activity of Human Brown Adipose Tissue during Warm Conditions and Cold-Induced Lipolysis of Local Triglycerides

1. Download high-res image (141KB)
2. Download full-size image

     

Effect of variable suckling intensity and estrogen administration upon serum luteinizing hormone in Brahman cows

Ten mature Brahman cows were randomly allotted within calving intervals to either a suckled (S) or nonsuckled (NS) treatment group. All cows received a 20 mg intramuscular injection of estradiol-17beta (E2), suspended in 2 ml of corn oil, to determine the effect of suckling on the estrogen induced LH surge. Starting on day 21 postpartum the S cows were suckled at six hour intervals for 24 hours, at which time they were challenged with a 20 mg E2 injection. The suckling regimen was continued for 48 hours postinjection. The NS cows were separated from their calves on day 21 postpartum and received no suckling stimulus for 72 hours. At 24 hours after calf separation, the NS cows were challenged with a 20 mg E2 injection. Blood samples were removed at two hour intervals beginning 10 hours post E2 injection until 36 hours postinjection, at which time blood samples were removed at four hour intervals until 48 hours postinjection. Blood samples were processed to yield serum and assayed for luteinizing hormone (LH) via radioimmunoassay. The injection of a 20 mg dose of E2 induced an LH surge in all cows. The NS cows were found to exhibit a longer (P<.05) duration of the estrogen induced LH surge than the S cows, 15.6 +/- .98 and 12.4 +/- .75 hours, respectively. The timing parameters (time to start of LH surge, time to peak LH value and time to end of surge) and LH concentration parameters (LH concentration at start of LH surge, peak value of LH surge and LH concentration at end of LH surge) were not different between suckling regimens. No blockage of the LH response to estrogen challenge was found on day 22 postpartum. Suckling did depress the duration of the LH surge indicating some blockage due to suckling stimuli.     

Amino Acid Metabolism of Lemna minor L. : II. Responses to Chlorsulfuron

Chlorsulfuron, an inhibitor of acetolactate synthase (EC 4.1.3.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemna minor at concentrations of 10⁻⁸ molar and above, but had no inhibitory effects on growth at 10⁻⁹ molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10⁻⁸ molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as α-amino-n-butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of α-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10⁻⁴ molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 10⁴ times greater than that required to inhibit growth. Tracer studies with ¹⁵N demonstrate that chlorsulfuron inhibits the incorporation of ¹⁵N into valine, leucine, and isoleucine. The α-amino-n-butyrate accumulated in the presence of chlorsulfuron and [¹⁵N]H₄⁺ was heavily labeled with ¹⁵N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isoleucine branch of the pathway, and transamination of 2-oxobutyrate to α-amino-n-butyrate by a constitutive transaminase utilizing either glutamate or alanine as α-amino-N donors.     

Metabolic Changes in Excised Fruit Tissue. IV. Changes Occurring in Discs of Apple Peel During the Development of the Respiration Climacteric

It is shown that a sequential development of a series of enzyme systems occurs in the peel of the apple as the respiration climacteric develops in the whole fruit. The sequence of development of these systems, i.e. acetate incorporation into lipid, production of ethylene, incorporation of amino acid into protein and, finally, the decarboxylation of added malate (malate effect) is the same as that shown earlier for the short term (24 hr) aging of peel discs from pre-climacteric apples. As these systems appear in the initial discs from fruit passing through the climacteric they gradually cease to increase during the 24 hour aging period. Uptake studies show that none of the changes in these systems can be due solely to changes in the permeability of the tissue over the climacteric period. On the basis of these results it is tentatively suggested that the aging of discs from pre-climacteric tissue might provide a model system for a detailed study of the physiological and biochemical changes occurring during the climacteric of apple fruits.