Effect of sulfur availability on the integrity of amino acid biosynthesis in plants.

Amino acid levels in plants are regulated by a complex interplay of regulatory circuits at the level of enzyme activities and gene expression. Despite the diversity of precursors involved in amino acid biosynthesis as providing the carbon backbones, the amino groups and, for the amino acids methionine and cysteine, the sulfhydryl group and despite the involvement of amino acids as substrates in various downstream metabolic processes, the plant usually manages to provide relatively constant levels of all amino acids. Here we collate data on how amino acid homeostasis is shifted upon depletion of one of the major biosynthetic constituents, i.e., sulfur. Arabidopsis thaliana seedlings exposed to sulfate starvation respond with a set of adaptation processes to achieve a new balance of amino acid metabolism. First, metabolites containing reduced sulfur (cysteine, glutathione, S-adenosylmethionine) are reduced leading to a number of downstream effects. Second, the relative excess accumulation of N over S triggers processes to dump nitrogen in asparagine, glutamine and further N-rich compounds like ureides. Third, the depletion of glutathione affects the redox and stress response system of the glutathione-ascorbate cycle. Thus, biosynthesis of aromatic compounds is triggered to compensate for this loss, leading to an increased flux and accumulation of aromatic amino acids, especially tryptophan. Despite sulfate starvation, the homeostasis is kept, though shifted to a new state. This adaptation process keeps the plant viable even under an adverse nutritional status.

Modifications of cellulose synthase confer resistance to isoxaben and thiazolidinone herbicides in Arabidopsis Ixr1 mutants.

In many higher plants, cellulose synthesis is inhibited by isoxaben and thiazolidinone herbicides such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidinone. Semidominant mutations at the IXR1 and IXR2 loci of Arabidopsis confer isoxaben and thiazolidinone resistance. Isolation of the IXR1 gene by map-based cloning revealed that it encodes the AtCESA3 isoform of cellulose synthase. The two known mutant alleles contain point mutations that replace glycine 998 with aspartic acid, and threonine 942 with isoleucine, respectively. The mutations occur in a highly conserved region of the enzyme near the carboxyl terminus that is well separated from the proposed active site. Although the IXR1 gene is expressed in the same cells as the structurally related RSW1 (AtCESA1) cellulose synthase gene, these two CESA genes are not functionally redundant.

Positional cloning in Arabidopsis. Why it feels good to have a genome initiative working for you.

Positional (or map-based) cloning techniques are widely used to identify the protein products of genes defined by mutation. In Arabidopsis the information generated by the Genome Initiative is giving this approach a decisive boost. A wealth of sequence polymorphisms and molecular markers is now available and can be exploited for fine mapping with technically simple and robust polymerase chain reaction-based methods. As a result it has become possible to complete positional cloning projects in a short time and with relatively little effort.

The irregular xylem3 locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis.

The irregular xylem3 (irx3) mutant of Arabidopsis has a severe deficiency in secondary cell wall cellulose deposition that leads to collapsed xylem cells. The irx3 mutation has been mapped to the top arm of chromosome V near the marker nga106. Expressed sequence tag clone 75G11, which exhibits sequence similarity to cellulose synthase, was found to be tightly linked to irx3, and genomic clones containing the gene corresponding to clone 75G11 complemented the irx3 mutation. Thus, the IRX3 gene encodes a cellulose synthase component that is specifically required for the synthesis of cellulose in the secondary cell wall. The irx3 mutant allele contains a stop codon that truncates the gene product by 168 amino acids, suggesting that this allele is null. Furthermore, in contrast to radial swelling1 (rsw1) plants, irx3 plants show no increase in the accumulation of beta-1,4-linked glucose in the noncrystalline cell wall fraction. IRX3 and RSW1 fall into a distinct subgroup (Csa) of Arabidopsis genes showing homology to bacterial cellulose synthases.

Nitrate Acts as a Signal to Induce Organic Acid Metabolism and Repress Starch Metabolism in Tobacco.

Nia30(145) transformants with very low nitrate reductase activity provide an in vivo screen to identify processes that are regulated by nitrate. Nia30(145) resembles nitrate-limited wild-type plants with respect to growth rate and protein and amino acid content but accumulates large amounts of nitrate when it is grown on high nitrate. The transcripts for nitrate reductase (NR), nitrite reductase, cytosolic glutamine synthetase, and glutamate synthase increased; NR and nitrite reductase activity increased in leaves and roots; and glutamine synthetase activity increased in roots. The transcripts for phosphoenolpyruvate carboxylase, cytosolic pyruvate kinase, citrate synthase, and NADP-isocitrate dehydrogenase increased; phosphoenolpyruvate carboxylase activity increased; and malate, citrate, isocitrate, and [alpha]-oxoglutarate accumulated in leaves and roots. There was a decrease of the ADP-glucose pyrophosphorylase transcript and activity, and starch decreased in the leaves and roots. After adding 12 mM nitrate to nitrate-limited Nia30(145), the transcripts for NR and phosphoenolpyruvate carboxylase increased, and the transcripts for ADP-glucose pyrophosphorylase decreased within 2 and 4 hr, respectively. Starch was remobilized at almost the same rate as in wild-type plants, even though growth was not stimulated in Nia30(145). It is proposed that nitrate acts as a signal to initiate coordinated changes in carbon and nitrogen metabolism.

Identification and purification of translation initiation factor 2 (IF2) from Thermus thermophilus.

Translation initiation factor 2 (IF2) is one of three protein factors required for initiation of protein synthesis in eubacteria. The protein is responsible for binding the initiator RNA to the ribosomal P site. IF2 is a member of the GTP GDP-binding protein superfamily. In the extreme thermophilic bacterium Thermus thermophilus, IF2 was identified as a 66-kDa protein by affinity labeling and immunoblotting. The protein was purified to homogeneity. The specific activity indicates a stoichiometric IF2-mediated binding of formylmethionine-tRNA to 70S ribosomes. The N-terminal amino acid sequences of the intact protein and of two proteolytic fragments of 25 kDa and 40 kDa were determined. Comparison with other bacterial IF2 sequences indicates a similar domain architecture in all bacterial IF2 proteins.

Tobacco mutants with a decreased number of functional nia genes compensate by modifying the diurnal regulation of transcription, post-translational modification and turnover of nitrate reductase.

Although nitrate reductase (NR. EC 1.6.6.1) is thought to control the rate of nitrate assimilation, mutants with 40-45% of wildtype (WT) NR activity (NRA) grow as fast as the WT. We have investigated how tobacco (Nicotiana tabacum L. cv. Gatersleben) mutants with one or two instead of four functional nia genes compensate. (i) The nia transcript was higher in the leaves of the mutants. However, the diurnal rhythm was retained in the mutants, with a maximum at the end of the night and a strong decline during the photoperiod. (ii) Nitrate reductase protein and NRA rose to a maximum after 3-4 h light in WT leaves, and then decreased by 50-60% during the second part of the photoperiod and the first part of the night. Leaves of mutants contained 40-60% less NR protein and NRA after 3-4 h illumination, but NR did not decrease during the photoperiod. At the end of the photoperiod the WT and the mutants contained similar levels of NR protein and NRA. (iii) Darkening led to a rapid inactivation of NR in the WT and the mutants. However, in the mutants, this inactivation was reversed after 1-3 h darkness. Calyculin A prevented this reversal. When magnesium was included in the assay to distinguish between the active and inactive forms of NR, mutants contained 50% more activity than the WT during the night. Conversion of [15N]-nitrate to organic compounds in leaves in the first 6 h of the night was 60% faster in the mutants than in the WT. (iv) Growth of WT plants in enhanced carbon dioxide prevented the decline of NRA during the second part of the photoperiod, and led to reactivation of NR in the dark. (v) Increased stability of NR in the light and reversal of dark-inactivation correlated with decreased levels of glutamine in the leaves. When glutamine was supplied to detached leaves it accelerated the breakdown of NR, and led to inactivation of NR, even in the light. (vi) Diurnal changes were also investigated in roots. In the WT, the amount of nia transcript rose to a maximum after 4 h illumination and then gradually decreased. The amplitude of the changes in transcript amount was smaller in roots than in leaves, and there were no diurnal changes in NRA. In mutants, nia transcript levels were high through the photoperiod and the first part of the night. The NRA was 50% lower during the day but rose during the night to an activity almost as high as in the WT. The rate of [15N]-nitrate assimilation in the roots of the mutants resembled that in the WT during the first 6 h of the night. (vii) Diurnal changes were also compared in Nia30(145) transformants with very low NRA, and in nitrate-deficient WT plants. Both sets of plants had similar low growth rates. Nitrate reductase did not show a diurnal rhythm in leaves or roots of Nia30(145), the leaves contained very low glutamine, and NR did not inactivate in the dark. Nitrate-deficient WT plants were watered each day with 0.2 mM nitrate. After watering, there was a small peak of nia transcript NR protein and NRA and, slightly later, a transient increase of glutamine and other amino acids in the leaves. During the night glutamine was low, and NR did not inactivate. In the roots, there was a very marked increase of nitrate, nia transcript and NRA 2-3 h after the daily watering with 0.2 mM nitrate. (viii) It is concluded that WT plants have excess capacity for nitrate assimilation. They only utilise this potential capacity for a short time each day, and then down-regulate nitrate assimilation in response, depending on the conditions, to accumulation of the products of nitrate assimilation or exhaustion of external nitrate. Genotypes with a lower capacity for nitrate assimilation compensate by increasing expression of NR and weakening the feedback regulation, to allow assimilation to continue for a longer period each day.

Ultrasonography of the popliteal fossa and lower extremities.

In this article, the numerous proven applications of diagnostic ultrasound to the assessment of disease processes affecting the popliteal fossa and lower extremities have been discussed. Popliteal cysts and aneurysms, hematomas, cellulitis, abscesses, soft tissue neoplasms, and other space-occupying fluid collections are readily identified using this technique. In addition, post-traumatic and inflammatory disorders affecting tendons, synovial thickening and effusion, derangement of articular cartilage, meniscal tears, and alterations in soft tissue thickness as well as foreign bodies are amenable to detection by sonography. With future technologic improvements in transducer design and computerized image reconstruction, diagnostic capabilities of this method in the lower extremities should continue to expand.

The subplacental complex: further sonographic observations.

The subplacental complex, which represents vascular channels at the placental-myometrial junction, is a structure that is routinely seen sonographically in patients with posteriorly placed placentas, but not in those with anteriorly placed placentas. This study shows that the subplacental complex is a constant anatomic feature of the placenta regardless of whether it develops on the anterior or posterior uterine wall, and can be visualized in most patients if the subplacental complex is in the focal plane of the transducer. This has important implications in patients with anterior placentas that are being evaluated for abruption or in patients in whom fetal parts lie just beneath the anterior myometrial wall.

Large umbilical cord: a normal finding in some fetuses.

We report two cases in which the umbilical cords of fetuses were large on prenatal ultrasound scans. The pregnancies terminated uneventfully, however, and the babies were healthy at birth and on follow-up visits. We discuss the various etiologies of a large umbilical cord and conclude that, although a large cord may suggest the presence of an underlying abnormality, in some instances it may represent a normal finding.

Ultrasonography of the salivary glands.

Sonography and computed tomography play complementary roles in imaging mass lesions in the salivary glands and their surroundings. Ninety per cent of parotid tumors originate from the superficial lobe, and we consider sonography to be the method of choice for imaging these lesions. The sensitivity of high-resolution sonography in detecting intraparotid tumors approaches 100 per cent, and ultrasound is therefore an excellent method to evaluate patients with parotid swelling. It may provide clinically useful information by precisely outlining the tumor borders or by detecting multiple or bilateral lesions. We restrict our use of CT to tumors that appear to extend beyond the borders of the parotid gland with possible invasion of surrounding soft tissues or bone. CT is also the method of choice for differentiating lesions of the deep lobe from parapharyngeal tumors and for staging carcinomas. Although ultrasound supplemented with computed tomography should replace conventional sialography in the work-up of parotid neoplasms, sialography remains the method of choice for evaluating patients with chronic sialadenitis, autoimmune diseases, and sialolithiasis. The role of sonography in these conditions is limited to ruling out a parotid neoplasm, assessing the extent of abscess formation, or assisting in localizing calculi in selected patients.

Neuroblastoma metastatic to the testis: ultrasonographic screening as an aid to clinical staging.

Secondary testicular tumors are rare, and to our knowledge only a few cases of metastatic neuroblastoma to the testicle have been presented in the literature. We report two cases in which high resolution ultrasonography was helpful in establishing the diagnosis of metastatic neuroblastoma. Because accurate staging is important in the choice of therapy, an aggressive search for the presence of metastatic disease is essential. The small size of the testes in young children makes physical examination difficult, and sonography can be of value.

Intrauterine/newborn testicular torsion.

Although spermatic cord torsion in the neonate is uncommon, early recognition with prompt surgical exploration and detorsion of the cord can result in preservation of some testicular tissue. Palpation of the infantile testes can be difficult, and distinguishing testicular from extratesticular abnormalities may not be possible. High-resolution real-time ultrasonography is an effective, noninvasive method of differentiating between the two. If sonography demonstrates and abnormal testicle, prompt surgical exploration is indicated.

Comparison of high resolution real time ultrasonography and high resolution computed tomography in an infant with spinal dysraphism.

An infant with a lumbosacral spinal subcutaneous and intraspinal lipoma involving the conus medullaris was studied with computed tomography and high resolution real time ultrasonography to determine the extent and location of the disease before operation. The high resolution ultrasonogram demonstrated the abnormality with good detail and the added advantage of real time display of the lack of pulsatile mobility of the neural elements, confirming spinal cord tethering. Spinal high resolution ultrasonography is a noninvasive diagnostic tool in infants with spinal abnormalities and should be an important part of a neurosurgeon's diagnostic support system.

Occult spinal dysraphism in infants: screening with high-resolution real-time ultrasound.

Spinal dysraphism refers to incomplete embryologic development of the dorsal midline. Frequently it is not clinically obvious, and lack of treatment can lead to progressive neurological damage. Early diagnosis of occult defects can allow appropriate surgical intervention and prevent neurological deficits. The authors have used high-resolution real-time ultrasound to screen infants with various cutaneous lumbosacral markers, all of whom were at risk of occult spinal dysraphism. Preliminary results suggest that ultrasound is a reliable screening procedure and could obviate the need for more costly and invasive tests.

Sonography of jugular vein thrombosis.

The sonographic findings in three patients with jugular vein thrombosis are described. In each case, intraluminal clot appeared as a solid mass composed of midamplitude echoes. In one patient, near complete resolution of the thrombus was documented by serial sonograms over a 3 month period. High-resolution sonography of the neck is a reliable method to establish the diagnosis of jugular vein thrombosis.

High-resolution real-time ultrasonography of neonatal kidneys.

A high-resolution real-time ultrasound system was used to evaluate 43 neonates who had problems referable to the kidney. In addition to a review of normal anatomic features portrayed by this instrument, a variety of pathologic states are illustrated. The system offers the dual advantages of superior spatial resolution and rapid survey capability when compared with traditional articulated-arm contact scanners. As with all sonographic investigations, functional immaturity or depressed organ function is no hindrance to obtaining diagnostic examinations.