P53 mutation and response to hepatic arterial floxuridine in patients with colorectal liver metastases.

PURPOSE: To establish the relationship between the number and site of p53 genomic mutations in metastatic colorectal cancer, and the response to hepatic arterial floxuridine. METHODS: Liver metastasis biopsies were collected, at the time of laparotomy for hepatic arterial cannulation. in 28 patients with metachronous colorectal liver metastases. p53 Gene mutations were assessed using reverse transcription, nested polymerase chain reaction, single strand conformational polymorphism and gene sequencing. Chemotherapy response was determined from computerised liver tomograms after 4 months of treatment. RESULTS: Liver metastasis p53 mutation was identified in 21 (75%), and p53 "hot spot" mutation in 11 (39%) patients. There was a significantly lower prevalence (Fisher's, P=0.001) of patients with p53 "hot spot"-mutated liver metastases in stable disease and partial response (5/22) than in progressive (6/6) disease groups. Significantly fewer (Mann-Whitney U, P=0.002) p53 "hot spot" mutations/biopsy were observed in liver metastases from stable disease and partial response (median 0, iqr. 0-0) than in progressive (median 1, iqr 1-2) disease patients. p53 "Hot spot"-mutated liver metastases were associated with significantly shorter survival times (logrank P=0.05) after hepatic arterial floxuridine. Significant response or survival-time differences by total or L2/L3 zinc-binding site p53 mutations were not detected. CONCLUSIONS: The results support a role for p53 "hot spot" mutations in colorectal liver metastasis resistance to fluorinated pyrimidines, and suggest that the presence of such mutations may be a contra-indication to treatment of colorectal liver metastases with hepatic arterial floxuridine.

Newly described weathering pattern in pili annulati hair shafts: a scanning electron microscopic study.

Certain scalp hair shafts from 2 of 10 cases of pili annulati examined by scanning electron microscopy exhibited an unusual weathering pattern. The majority of affected hair shafts showed minor surface abnormalities at regular intervals (nodes) associated with the underlying spaces. However, in a few examples, there was marked damage to the cuticle at the nodes exposing the underlying cortex; in severe cases there was cracking of both cuticle and cortex. These damaged nodes were also associated with trichorrhexis nodosa-like breaks of the hair shaft. This study shows that the nodes in pili annulati may have some inherent weakness that could result in breaks in the hair shaft exposed to physical trauma.

Overexpression of DWARF4 in the brassinosteroid biosynthetic pathway results in increased vegetative growth and seed yield in Arabidopsis.

Plants unable to synthesize or perceive brassinosteroids (BRs) are dwarfs. Arabidopsis dwf4 was shown to be defective in a steroid 22alpha hydroxylase (CYP90B1) step that is the putative rate-limiting step in the BR biosynthetic pathway. To better understand the role of DWF4 in BR biosynthesis, transgenic Arabidopsis plants ectopically overexpressing DWF4 (AOD4) were generated, using the cauliflower mosaic virus 35S promoter, and their phenotypes were characterized. The hypocotyl length of both light- and dark-grown AOD4 seedlings was increased dramatically as compared to wild type. At maturity, inflorescence height increased >35% in AOD4 lines and >14% in tobacco DWF4 overexpressing lines (TOD4), relative to controls. The total number of branches and siliques increased more than twofold in AOD4 plants, leading to a 59% increase in the number of seeds produced. Analysis of endogenous BR levels in dwf4, Ws-2 and AOD4 revealed that dwf4 accumulated the precursors of the 22alpha-hydroxylation steps, whereas overexpression of DWF4 resulted in increased levels of downstream compounds relative to Ws-2, indicative of facilitated metabolic flow through the step. Both the levels of DWF4 transcripts and BR phenotypic effects were progressively increased in dwf4, wild-type and AOD4 plants, respectively. This suggests that it will be possible to control plant growth by engineering DWF4 transcription in plants.

Expression of an immediate early gene, IEX-1, in human tissues.

IEX-1 is an immediate early gene that is induced by ionizing radiation, ultraviolet radiation, and a variety of growth factors. It plays an important role in the regulation of cellular growth. Earlier, we performed studies on the distribution of IEX-1 messenger RNA in different tissues and on the subcellular localization of IEX-1 protein. No reports, however, have appeared concerning the distribution of IEX-1 protein in a variety of human tissues. We raised a polyclonal antibody against a synthetic IEX-1 peptide (amino acids 51-75) and used the antibody to study the distribution of the protein in human tissues. We demonstrate that IEX-1 is strongly expressed in epithelia of the skin, trachea, gastrointestinal, and genitourinary systems, as well as in the pancreas and breast. Endothelial cells within the vasculature of most tissue/organs also strongly express IEX-1. Liver, lung, lymph nodes, and placenta stain weakly. No IEX-1 epitopes were detected in the thymus, testes, ovary, myocardium, skeletal muscle, or spleen. We conclude that IEX-1 is widely expressed in epithelial and endocrine tissues, as well as in vascular endothelium.

Cuticular waxes on eceriferum mutants of Arabidopsis thaliana.

We present cuticular wax chemical profiles for the leaves and stems of Arabidopsis wildtype Landsberg erecta and eleven isogenic eceriferum mutants: cer5, cer10 to cer15, and cer17 to cer20. These cer mutants have wax profiles that are different from those of wildtype in chemical chain length distribution, amount per chemical class, and/or total wax load. Analyses of detailed leaf and stem wax profiles for these cer mutants have allowed us to place some of these mutants at specific steps in wax production. The cer13 gene is predicted to affect release of the 30 carbon fatty acid from the elongation complex or the reduction of C30 fatty acid to C30 aldehyde. The CER19 gene product is predicted to be involved in C28 to C30 fatty acyl-CoA elongation. The CER20 gene is predicted to affect the oxidation of C29 alkane to C29 secondary alcohol. Several predicted gene products affect only stem specific steps in the wax pathway.

Cytochrome P450s as genes for crop improvement.

In the past year, several cytochrome P450 genes have been identified that will be important for generating crop protectants and natural medicinal products. Among these are the 2-hydroxyisoflavone synthase (CYP93C) and the indole-3-acetaldoxime N-hydroxylase (CYP83B1) genes, which catalyze the formation of isoflavones and glucosinolates, respectively.

CYP83B1, a cytochrome P450 at the metabolic branch point in auxin and indole glucosinolate biosynthesis in Arabidopsis.

Auxins are growth regulators involved in virtually all aspects of plant development. However, little is known about how plants synthesize these essential compounds. We propose that the level of indole-3-acetic acid is regulated by the flux of indole-3-acetaldoxime through a cytochrome P450, CYP83B1, to the glucosinolate pathway. A T-DNA insertion in the CYP83B1 gene leads to plants with a phenotype that suggests severe auxin overproduction, whereas CYP83B1 overexpression leads to loss of apical dominance typical of auxin deficit. CYP83B1 N-hydroxylates indole-3-acetaldoxime to the corresponding aci-nitro compound, 1-aci-nitro-2-indolyl-ethane, with a K(m) of 3 microM and a turnover number of 53 min(-1). The aci-nitro compound formed reacts non-enzymatically with thiol compounds to produce an N-alkyl-thiohydroximate adduct, the committed precursor of glucosinolates. Thus, indole-3-acetaldoxime is the metabolic branch point between the primary auxin indole-3-acetic acid and indole glucosinolate biosynthesis in Arabidopsis.

TRH1 encodes a potassium transporter required for tip growth in Arabidopsis root hairs.

Root hair initiation involves the formation of a bulge at the basal end of the trichoblast by localized diffuse growth. Tip growth occurs subsequently at this initiation site and is accompanied by the establishment of a polarized cytoplasmic organization. Arabidopsis plants homozygous for a complete loss-of-function tiny root hair 1 (trh1) mutation were generated by means of the T-DNA-tagging method. Trichoblasts of trh1 plants form initiation sites but fail to undergo tip growth. A predicted primary structure of TRH1 indicates that it belongs to the AtKT/AtKUP/HAK K(+) transporter family. The proposed function of TRH1 as a K(+) transporter was confirmed in (86)Rb uptake experiments, which demonstrated that trh1 plants are partially impaired in K(+) transport. In line with these results, TRH1 was able to complement the trk1 potassium transporter mutant of Saccharomyces, which is defective in high-affinity K(+) uptake. Surprisingly, the trh1 phenotype was not restored when mutant seedlings were grown at high external potassium concentrations. These data demonstrate that TRH1 mediates K(+) transport in Arabidopsis roots and is responsible for specific K(+) translocation, which is essential for root hair elongation.

Biosynthetic pathways of brassinolide in Arabidopsis.

Our previous studies on the endogenous brassinosteroids (BRs) in Arabidopsis have provided suggestive evidence for the operation of the early C6-oxidation and the late C6-oxidation pathways, leading to brassinolide (BL) in Arabidopsis. However, to date the in vivo operation of these pathways has not been fully confirmed in this species. This paper describes metabolic studies using deuterium-labeled BRs in wild-type and BR-insensitive mutant (bri1) seedlings to establish the intermediates of the biosynthetic pathway of BL in Arabidopsis. The first evidence for the conversion of campestanol to 6-deoxocathasterone and the conversion of 6-deoxocathasterone to 6-deoxoteasterone is provided. The later biosynthetic steps (6-deoxoteasterone --> 3-dehydro-6-deoxoteasterone --> 6-deoxotyphasterol --> 6-deoxocastasterone --> 6alpha-hydroxycastasterone --> castasterone --> BL) were demonstrated by stepwise metabolic experiments. Therefore, these studies complete the documentation of the late C6-oxidation pathway. The biosynthetic sequence involved in the early C6-oxidation pathway (teasterone --> 3-dehydroteasterone --> typhasterol --> castasterone --> BL) was also demonstrated. These results show that both the early and late C6-oxidation pathways are functional in Arabidopsis. In addition we report two new observations: the presence of a new branch in the pathway, C6 oxidation of 6-deoxotyphasterol to typhasterol, and increased metabolic flow in BR-insensitive mutants.

Lesions in the sterol delta reductase gene of Arabidopsis cause dwarfism due to a block in brassinosteroid biosynthesis.

The brassinosteroid (BR) biosynthetic pathway, and the sterol pathway which is prerequisite to the BR pathway, are rapidly being characterized because of the availability of a large number of characteristic dwarf mutants in Arabidopsis. Here we show that the Arabidopsis dwarf5 mutants are disrupted in a sterol Delta7 reduction step. dwf5 plants display the characteristic dwarf phenotype typical of other BR mutants. This phenotype includes small, round, dark-green leaves, and short stems, pedicels, and petioles. Metabolite tracing with 13C-labeled precursors in dwf5 verified a deficiency in a sterol Delta7 reductase activity. All six independent alleles contain loss-of-function mutations in the sterol Delta7 reductase gene. These include a putative mRNA instability mutation in dwf5-1, 3' and 5' splice-site mutations in dwf5-2 and dwf5-6, respectively, premature stop codons in dwf5-3 (R400Z) and dwf5-5 (R409Z), and a mis-sense mutation in dwf5-4 (D257N). The dwf5 plant could be restored to wild type by ectopic overexpression of the wild-type copy of the gene. Both the Arabidopsis dwf5 phenotype and the human Smith-Lemli-Opitz syndrome are caused by loss-of-function mutations in a sterol Delta7 reductase gene, indicating that it is required for the proper growth and development of these two organisms.

Brassinosteroid-insensitive dwarf mutants of Arabidopsis accumulate brassinosteroids.

Seven dwarf mutants resembling brassinosteroid (BR)-biosynthetic dwarfs were isolated that did not respond significantly to the application of exogenous BRs. Genetic and molecular analyses revealed that these were novel alleles of BRI1 (Brassinosteroid-Insensitive 1), which encodes a receptor kinase that may act as a receptor for BRs or be involved in downstream signaling. The results of morphological and molecular analyses indicated that these represent a range of alleles from weak to null. The endogenous BRs were examined from 5-week-old plants of a null allele (bri1-4) and two weak alleles (bri1-5 and bri1-6). Previous analysis of endogenous BRs in several BR-biosynthetic dwarf mutants revealed that active BRs are deficient in these mutants. However, bri1-4 plants accumulated very high levels of brassinolide, castasterone, and typhasterol (57-, 128-, and 33-fold higher, respectively, than those of wild-type plants). Weaker alleles (bri1-5 and bri1-6) also accumulated considerable levels of brassinolide, castasterone, and typhasterol, but less than the null allele (bri1-4). The levels of 6-deoxoBRs in bri1 mutants were comparable to that of wild type. The accumulation of biologically active BRs may result from the inability to utilize these active BRs, the inability to regulate BR biosynthesis in bri1 mutants, or both. Therefore, BRI1 is required for the homeostasis of endogenous BR levels.

The Arabidopsis dwarf1 mutant is defective in the conversion of 24-methylenecholesterol to campesterol in brassinosteroid biosynthesis.

Since the isolation and characterization of dwarf1-1 (dwf1-1) from a T-DNA insertion mutant population, phenotypically similar mutants, including deetiolated2 (det2), constitutive photomorphogenesis and dwarfism (cpd), brassinosteroid insensitive1 (bri1), and dwf4, have been reported to be defective in either the biosynthesis or the perception of brassinosteroids. We present further characterization of dwf1-1 and additional dwf1 alleles. Feeding tests with brassinosteroid-biosynthetic intermediates revealed that dwf1 can be rescued by 22alpha-hydroxycampesterol and downstream intermediates in the brassinosteroid pathway. Analysis of the endogenous levels of brassinosteroid intermediates showed that 24-methylenecholesterol in dwf1 accumulates to 12 times the level of the wild type, whereas the level of campesterol is greatly diminished, indicating that the defective step is in C-24 reduction. Furthermore, the deduced amino acid sequence of DWF1 shows significant similarity to a flavin adenine dinucleotide-binding domain conserved in various oxidoreductases, suggesting an enzymatic role for DWF1. In support of this, 7 of 10 dwf1 mutations directly affected the flavin adenine dinucleotide-binding domain. Our molecular characterization of dwf1 alleles, together with our biochemical data, suggest that the biosynthetic defect in dwf1 results in reduced synthesis of bioactive brassinosteroids, causing dwarfism.

The Arabidopsis dwf7/ste1 mutant is defective in the delta7 sterol C-5 desaturation step leading to brassinosteroid biosynthesis.

Lesions in brassinosteroid (BR) biosynthetic genes result in characteristic dwarf phenotypes in plants. Understanding the regulation of BR biosynthesis demands continued isolation and characterization of mutants corresponding to the genes involved in BR biosynthesis. Here, we present analysis of a novel BR biosynthetic locus, dwarf7 (dwf7). Feeding studies with BR biosynthetic intermediates and analysis of endogenous levels of BR and sterol biosynthetic intermediates indicate that the defective step in dwf7-1 resides before the production of 24-methylenecholesterol in the sterol biosynthetic pathway. Furthermore, results from feeding studies with 13C-labeled mevalonic acid and compactin show that the defective step is specifically the Delta7 sterol C-5 desaturation, suggesting that dwf7 is an allele of the previously cloned STEROL1 (STE1) gene. Sequencing of the STE1 locus in two dwf7 mutants revealed premature stop codons in the first (dwf7-2) and the third (dwf7-1) exons. Thus, the reduction of BRs in dwf7 is due to a shortage of substrate sterols and is the direct cause of the dwarf phenotype in dwf7.

Systematic reverse genetics of transfer-DNA-tagged lines of Arabidopsis. Isolation of mutations in the cytochrome p450 gene superfamily.

We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics. Despite their importance, progress in assigning enzymatic function to individual P450 gene products has been slow. Here we report the isolation of the first 12 such lines, including one (CYP83B1-1) that displays a runt phenotype (small plants with hooked leaves), and three insertions in abundantly expressed genes. The DNAs used in this study are publicly available and can be used to systematically isolate mutants in Arabidopsis.

Chloroplast development at low temperatures requires a homolog of DIM1, a yeast gene encoding the 18S rRNA dimethylase.

Poikilothermic organisms require mechanisms that allow survival at chilling temperatures (2 to 15 degreesC). We have isolated chilling-sensitive mutants of Arabidopsis, a plant that is very chilling resistant, and are characterizing them to understand the genes involved in chilling resistance. The T-DNA-tagged mutant paleface1 (pfc1) grows normally at 22 degrees C but at 5 degrees C exhibits a pattern of chilling-induced chlorosis consistent with a disruption of chloroplast development. Genomic DNA flanking the T-DNA was cloned and used to isolate wild-type genomic and cDNA clones. The PFC1 transcript is present at a low level in wild-type plants and was not detected in pfc1 plants. Wild-type Arabidopsis expressing antisense constructs of PFC1 grew normally at 22 degrees C but showed chilling-induced chlorosis, confirming that the gene is essential for low-temperature development of chloroplasts. The deduced amino acid sequence of PFC1 has identity with rRNA methylases found in bacteria and yeast that modify specific adenosines of pre-rRNA transcripts. The pfc1 mutant does not have these modifications in the small subunit rRNA of the plastid.