Biosynthetic pathways of two polypeptide subunits of the light-harvesting chlorophyll a/b protein complex.

We have used an in vitro reconstitution system, consisting of cell-free translation products and intact chloroplasts, to investigate the pathway from synthesis to assembly of two polypeptide subunits of the light-harvesting chlorophyll-protein complex. These polypeptides, designated 15 and 16, are integral components of the thylakoid membranes, but they are products of cytoplasmic protein synthesis. Double immunodiffusion experiments reveal that the two polypeptides share common antigenic determinants and therefore are structurally related. Nevertheless, they are synthesized in vitro from distinct mRNAs to yield separate precursors, p15 and p16, each of which is 4,000 to 5,000 daltons larger than its mature form. In contrast to the hydrophobic mature polypeptides, the precursors are soluble in aqueous solutions. Along with other cytoplasmically synthesized precursors, p15 and p16 are imported into purified intact chloroplasts by a post-translational mechanism. The imported precursors are processed to the mature membrane polypeptides which are recovered exclusively in the thylakoids. The newly imported polypeptides are assembled correctly in the thylakoid lipid bilayer and they bind chlorophylls. Thus, these soluble membrane polypeptide precursors must move from the cytoplasm through the two chloroplast envelope membranes, the stroma, and finally insert into the thylakoid membranes, where they assemble with chlorophyll to form the light-harvesting chlorophyll protein complex.

Cryptochromes: blue light receptors for plants and animals.

Cryptochromes are blue, ultraviolet-A photoreceptors. They were first characterized for Arabidopsis and are also found in ferns and algae; they appear to be ubiquitous in the plant kingdom. They are flavoproteins similar in sequence to photolyases, their presumptive evolutionary ancestors. Cryptochromes mediate a variety of light responses, including entrainment of circadian rhythms in Arabidopsis, Drosophila, and mammals. Sequence comparison indicates that the plant and animal cryptochrome families have distinct evolutionary histories, with the plant cryptochromes being of ancient evolutionary origin and the animal cryptochromes having evolved relatively recently. This process of repeated evolution may have coincided with the origin in animals of a modified circadian clock based on the PERIOD, TIMELESS, CLOCK, and CYCLE proteins.

Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1.

The arabidopsis thaliana HY4 gene encodes CRY1, a 75-kilodalton flavoprotein mediating blue light-dependent regulation of seedling development. CRY1 is demonstrated here to noncovalently bind stoichiometric amounts of flavin adenine dinucleotide (FAD). The redox properties of FAD bound by CRY1 include an unexpected stability of the neutral radical flavosemiquinone (FADH.). The absorption properties of this flavosemiquinone provide a likely explanation for the additional sensitivity exhibited by CRY1-mediated responses in the green region of the visible spectrum. Despite the sequence homology to microbial DNA photolyases, CRY1 was found to have no detectable photolyase activity.

The G-box: a ubiquitous regulatory DNA element in plants bound by the GBF family of bZIP proteins.

The G-box (CACGTG) is a ubiquitous, cis-acting DNA regulatory element found in plant genomes. Proteins known as G-box factors (GBFs) bind to G-boxes in a context-specific manner, mediating a wide variety of gene expression patterns. We suggest that, as for many biological systems, different combinations of these common elements can lead to diversity and specificity in the regulation of plant gene expression.

Photomorphogenesis. Seeing the light in plant development.

COP1, a protein thought to repress plant photomorphogenesis in the dark, is nuclear in the dark and cytoplasmic in the light. It may lie on the light signal transduction pathway and may be inactivated intracellularly by light.

(6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid effects on nucleotide metabolism in CCRF-CEM human T-lymphoblast leukemia cells.

(6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid [(6R)DDATHF] is a folate antimetabolite with activity specifically directed against de novo purine synthesis, primarily through inhibition of glycinamide ribonucleotide transformylase. This inhibition resulted in major changes in the size of the nucleotide pools in CCRF-CEM cells. After a 4-h incubation with 1 microM (6R)DDATHF, dramatic reductions in the ATP and GTP pools were observed, with almost no effect on CTP, UTP, and deoxyribonucleotide pools. When the incubation was continued in drug-free medium, recovery of ATP and GTP pools was protracted. ATP did not return to normal until 24-36 h, and GTP pools were only partially repleted by 48 h. The ATP and GTP pools were not affected when the initial 4-h incubation with (6R)DDATHF was conducted in the presence of 100 microM hypoxanthine. Addition of hypoxanthine to the medium after a 4-h incubation with (6R)DDATHF caused rapid recovery of the ATP and GTP pools. Similar effects were seen when the purine precursor aminoimidazole carboxamide was used in place of hypoxanthine. The effect of (6R)DDATHF on nucleotide pools and the capability of hypoxanthine or aminoimidazole carboxamide to prevent or reverse this phenomenon correlated directly with the inhibition of cell growth. Presumably as a consequence of the decrease in purine nucleotide triphosphate levels, the conversion of exogenously added uridine, thymidine, and deoxyuridine to nucleotides was markedly decreased. These effects were protracted for almost 48 h and were also reversed by hypoxanthine. Differential repletion of ATP and GTP pools after (6R)DDATHF pre-treatment demonstrated that diminished precursor phosphorylation is primarily a consequence of GTP rather than ATP starvation.

Inhibition of DNA synthesis in normal and malignant human cells by triazinate (Baker's antifol) and methotrexate.

Triazinate (TZT), a triazine folate antagonist, is a potent inhibitor of dihydrofolate reductase from mammalian cells. Because antitumor activity of triazinate in experimental tumors correlated closely with the in vitro inhibition of DNA synthesis in tumor cells derived from these tumors, we studied cells from patients with leukemia, solid tumor effusions, and cells from normal marrow to determine their in vitro sensitivity to TZT. DNA synthesis in cells from patients with acute leukemia was less sensitive to TZT than it was to methotrexate (MTX) at 2 X 10(-6) M concentration of the inhibitor, whereas the sensitivity was similar at 10(-5) M. This could be accounted for by the known greater sensitivity of dihydrofolate reductase to MTX than to TZT, and the observation that, whereas intracellular drug levels were similar at low (2 X 10(-6) M) extracellular concentrations of TZT or MTX, at the higher (10(-5) M) extracellular drug concentration intracellular TZT was greater than 3 times intracellular MTX. In vitro inhibition of DNA synthesis in cells obtained after patients were treated with TZT was correlated with drug serum concentration and with leukemia cell kill. The sensitivity of cells from solid tumor effusions to TZT was similar to the sensitivity to MTX. Since patients can tolerate doses of TZT five times higher than MTX with less toxicity, there may be advantage to the clinical use of TZT in some tumor cell types.

Clinical and pharmacological evaluation of triazinate in humans.

Twenty-four patients with advanced solid tumors and seven with acute leukemia were treated with a triazine folate antagonist, triazinate, to determine the toxicity spectrum, the maximum tolerated dose, and the pharmacological disposition of the drug. Negligible toxicity was seen with single doses of 20 to 225 mg/sq m given as a 0.5-hr infusion. Single doses of 300 to 600 mg/sq m infused over 0.5 to 3 hr caused moderate to severe central neurological impairment with light headedness, somnolence, visual disturbances, weakness, and in one patient, severe respiratory distress and cyanosis. Skin, mucous membrane, and bone marrow toxicity were mild to moderate with single doses. When triazinate was given by a multiple-dose schedule every 12 to 24 hr, there was no neurological toxicity, but mucositis, skin toxicity, and myelotoxicity were increased. Five patients developed an erythematous to desquamative rash at the site of previous or concurrent radiotherapy. Serum disappearance of triazinate was at least bionsiderable variation from patient to patient. Single i.v. doses of 300 mg/sq m resulted in serum levels of 10(-5) M or higher for 8 hr and, with repeated doses, this level could be maintained. Administration p.o. resulted in serum concentrations less than 10% of that achieved after i.v. administration. Cerebrospinal fluid concentrations were 2% or less of the serum levels in five or six patients, 1 to 4 hr after i.v. treatment. Urinary excretion varied from 12 to 71% (median, 43%) of the total dose injected during the first 24 hr. Measurable objective solid tumor responses were not seen in this Phase 1 study, although two patients had stabilization of previously advancing disease. Decreases in peripheral blasts occurred in both types of acute leukemia, but improvement in the bone marrow was not observed.

Pharmacology of a new triazine antifolate in mice, rats, dogs, and monkeys.

Triazinate (TZT), a potent inhibitor of dihydrofolate reductase, was selected for detailed investigation to determine its mechanism of selective action as well as its metabolic fate in mice, rats, dogs, and monkeys. The serum disappearance of TZT in normal and tumor-bearing mice was similar, with a rapid tissue equilibration phase and a slower elimination phase. Serum disappearance in normal and tumor-bearing rats was 1.5 to 2.2 hr. Serum disappearance in dogs and monkeys was similar, with half-lives of 3 to 4 and 2 to 4 hr, respectively. Urinary excretion of TZT at 24 hr was only 5 to 6% of the injected dose in mice and rats; in contrast, the dogs excreted 60% of the injected dose in 8 hr. TZT accumulated to comparable degrees in the organs of rats and mice, with progressively lesser concentrations in liver, kidney, spleen, and brain. Dihydrofolate reductase activity became almost undectectable in all tissues studied within 15 min after drug adminsitration. An important difference in drug accumulation was in the ascites cells of tumor-bearing animals: in mice, the drug level was consistently lower in the L1210 cells than in the ascites fluid; in contrast, by 30 min after treatment with TZT the drug level in Walker 256 cells was 10-fold higher than the level in the ascites fluid. No evidence for drug metabolism was found in extracts of urine, feces, or organ tissues from either mice or rats. TZT and two related triazines were studied for their ability to accumulate in the cerbrospinal fluid of dogs after i.v. administration. TZT achieved a cerebrospinal fluid level of approximately 15% of the serum concentration at 1 hr; in contrast, the other two triazines reached maximum cerebrospinal fluid values of 1% at 1 hr.

Multifactorial resistance to 5,10-dideazatetrahydrofolic acid in cell lines derived from human lymphoblastic leukemia CCRF-CEM.

5,10-dideaza-5,6,7,8-terrahydrofolic acid (DDATHF) is a potent antiproliferative agent in cell culture systems and in vivo in a number of murine and human xenograft tumors. In contrast to classical antifolates, which are dihydrofolate reductase inhibitors, DDATHF primarily inhibits GAR transformylase, the first folate-dependent enzyme along the pathway of de novo purine biosynthesis. The (6R) diastereomer of DDATHF (Lometrexol), currently undergoing clinical investigation, was used to develop CCRF-CEM human leukemia sublines resistant to increasing concentrations of the drug. Three cell lines were selected for ability to grow in medium containing 0.1 microM, 1.0 microM, and 10 microM of (6R)DDATHF, respectively. Impaired polyglutamylation was identified as a common mechanism of resistance in all three cell lines. A progressive decrease in the level of polyglutamylation was associated with diminished folylpolyglutamate synthetase activity and paralleled increasing levels of resistance to the drug. However, the expression of folylpolyglutamate synthetase RNA was not altered in the resistant cell lines compared to the parent cells. The most resistant cell subline also displayed an increased activity of gamma-glutamyl hydrolase. The sublines were scrutinized for other possible mechanisms of resistance. No alterations in drug transport or in purine economy were found. Modest increases were found in the activity of methylene tetrahydrofolate dehydrogenase but no alterations of other folate-dependent enzymes were observed. Increases in accumulation and conversion of folic acid to reduced forms, particularly 10-formyltetrahydrofolate, was also seen. The resistant cell lines were sensitive to dihydrofolate reductase inhibitors, methotrexate and trimetrexate, for a 72-h exposure period but showed cross-resistance to methotrexate for 4 and 24 h exposures. Cross-resistance was also shown toward other deazafolate analogues for both short- and long-term exposures.

Phase I studies with trimetrexate: clinical pharmacology, analytical methodology, and pharmacokinetics.

Twenty-two patients with advanced solid tumors were treated with a quinazoline folate antagonist, trimetrexate, to determine the toxicity spectrum, the maximal tolerated dose, and the pharmacokinetics of the drug. Negligible toxicity was seen with single doses of 10-70 mg/m2 given as a 1-h infusion. Single doses of 120 mg/m2 infused over 1 h caused moderate to grade 4 toxicity in five of nine patients treated. Two patients who had no toxicity at this level were escalated to a dose of 213 mg/m2 with mild to moderate toxicity. The primary dose-limiting toxicity was myelosuppression. Moderate transaminase elevations, rash, anorexia, nausea and vomiting, and mucositis were occasionally seen. Although there was variation in dose tolerance to this drug, with selected patients able to tolerate higher doses, we consider 120 mg/m2 every 2 weeks to be the maximal tolerated dose, and the recommended Phase II starting dose. Trimetrexate plasma concentration-time curves were best described as biphasic (N = 9) or triphasic (N = 5) in form. The half-life of the terminal elimination-phase was 16.4 h. The mean residence time was 17.8 h. The volume of distribution of the plasma compartment and the volume of distribution at steady-state were 0.17 and 0.62 liter/kg, respectively. Plasma clearance was 53 ml/min. Plasma concentrations as determined by dihydrofolate reductase enzyme inhibition assay and high-performance liquid chromatography were initially identical, but diverged at later times. Divergences were seen also in urinary recovery as determined by the two methods. Both results suggest the appearance of metabolite(s) of trimetrexate which can inhibit dihydrofolate reductase. Measurable objective solid tumor responses were not seen in this Phase I study, although three patients with colon cancer had stable disease lasting 18, 26, and 26 weeks, respectively.

Impaired polyglutamylation of methotrexate as a cause of resistance in CCRF-CEM cells after short-term, high-dose treatment with this drug.

Two methotrexate-resistant sublines, CCRF-CEM R3/7 and CCRF-CEM R30/6, were selected from the human leukemia T-lymphoblast cell line, CCRF-CEM, after repeated exposures (7 and 6 times, respectively) for 24 h to constant concentrations (3 and 30 microM) of the drug. Analysis of the mechanism of resistance revealed no differences in levels of dihydrofolate reductase activity, its binding affinity for methotrexate, or in methotrexate transport between the CCRF-CEM parent and methotrexate-resistant cell lines. The development of resistance to methotrexate was associated with a marked decrease in the intracellular level of methotrexate polyglutamates. Although the resistant sublines were able to form substantial amounts of folate polyglutamates when measured with [3H]folic acid, the level of polyglutamates formed was decreased to about 50% of that formed by the parent cell line. No qualitative differences in folate polyglutamates formed were noted between the parental and resistant sublines. This is the first example of a cell line which displays resistance which is solely attributable to defective methotrexate polyglutamate synthesis.

Pharmacology and toxicity of a potent "nonclassical" 2,4-diamino quinazoline folate antagonist, trimetrexate, in normal dogs.

The pharmacology of trimetrexate (JB-11, NSC 249008, 2,4-diamino-5-methyl-5-[(3,4,5-trimethoxyanilino)methyl]quinazoline), an antitumor agent effective against several mouse tumors, was studied in normal dogs. A high-performance liquid chromatographic technique with electrochemical detection, dihydrofolate reductase inhibition assay, and 14C-labeled drug were used to measure plasma disappearance, tissue distribution, excretion, and metabolism of the drug at doses from 0.5 to 6 mg/kg. Doses of 2 mg/kg were well tolerated without toxicity. Higher doses (3 to 6 mg/kg) produced mainly intestinal toxicity without significant hematological or liver abnormalities. The 6-mg/kg dose caused severe bloody diarrhea. After administration of 3 mg/kg, plasma concentrations of trimetrexate were 1 microM and were equal to or greater than 0.1 microM at 1 and 24 hr, respectively. The predominant pharmacokinetics of trimetrexate plasma disappearance was an elimination phase with a t1/2 of 3.5 hr. Concentrations in the cerebrospinal fluid were 2 to 5% of that in plasma and were maximum within 1 to 2 hr after i.v. administration. Highest tissue concentrations of drug were measured in liver and kidney; lowest were found in brain and lung. A dose equivalent to 3 mg/kg in humans (on a sq m basis) should produce adequate plasma concentrations (greater than 0.1 microM) for therapeutic effects.

Microheterogeneous Cytosolic High-Mobility Group Proteins from Broccoli Co-Purify with and Are Phosphorylated by Casein Kinase II.

A group of low molecular weight protein substrates was found to co-purify with casein kinase II from broccoli (Brassica oleracea var italica). These substrates showed very high affinity toward casein kinase II and were efficiently phosphorylated even in the presence of an excess of exogenous substrates. The broccoli substrates were purified from cytosolic extracts as a double band of related proteins migrating at 18.7 and 20 kD. Further microheterogeneity was revealed by anion-exchange high-performance liquid chromatography and mass spectroscopy. The actual molecular masses of the three major components identified by mass spectroscopy were determined to be 12,691, 13,256, and 14,128 D. The substrates showed characteristic amino acid composition with a high content of polar amino acids, including about 20% each of acidic and basic amino acids. They were soluble in 2% trichloroacetic acid. The substrates cross-reacted with an antibody against wheat high-mobility group protein d (HMGd) but not HMGa. The isolated broccoli HMGs showed general DNA-binding activity without preference for AT-rich DNA. The presence of these HMG proteins in the cytosolic fraction is similar to the distribution characteristics of the animal HMG-1 subgroup. On the basis of amino acid composition and DNA-binding specificity, the isolated broccoli HMGs resemble other plant HMGs homologous to the HMG-1 subgroup.

Molecular characterization and genetic mapping of two clusters of genes encoding chlorophyll a/b-binding proteins in Lycopersicon esculentum (tomato).

We have constructed a tomato genomic library in the gamma Charon 4 phage vector. The library was screened with a pea cDNA probe encoding a chlorophyll a/b-binding protein (CAB), and several recombinant phages containing tomato CAB genes were isolated and characterized by restriction mapping, heteroduplex analysis and nucleotide sequencing. Two phages with overlapping segments of the tomato genome contain a total of four CAB genes, all arranged in tandem. A third phase contains three CAB genes, two arranged in tandem and one in opposite orientation, and an additional, truncated CAB gene. Genetic mapping experiments showed that the four CAb genes on the first two phages belong to a locus, previously designated Cab-1, on chromosome 2. The CAB genes from the third phage belong to the Cab-3 locus on chromosome 3. Complete sequence determination of two CAB genes, one from each locus, and additional sequence determination of about 50% of each of the other five CAB genes showed that each gene within a CAB locus is more similar to other CAB genes in the same locus than it is to the CAB genes from the second locus. Furthermore, the polypeptides encoded by Cab-1 genes diverge significantly from those encoded by Cab-3 genes in the domains of transit peptide and the N terminus of the mature polypeptide but are essentially identical in the rest of the sequence.

Synthesis and evaluation of 2,4-diaminoquinazoline antifolates with activity against methotrexate-resistant human tumor cells.

In an attempt to find potent antifolates with selectivity against tumor cells with intrinsic or acquired resistance to methotrexate, eleven nonclassical 2,4-diaminoquinazoline antifolates were synthesized and tested as inhibitors of dihydrofolate reductase from L5178Y cells. Several compounds appeared to be good enzyme inhibitors, with I50 values around 1 nM. Two of the compounds were also good inhibitors of cell growth in vitro. One of these (PKC-32, 9-(2,4-diamino-5-methylquinazoline-6-methylene)aminophenanthren e) appeared to be 100-fold more potent than methotrexate as an inhibitor of growth of a methotrexate-resistant cell line with impaired transport for methotrexate. PKC-32 and PKC-155 were also tested against mouse tumors in vivo. PKC-32 was modestly active in vivo as compared with methotrexate. This drug may be a useful agent in the treatment of methotrexate-resistant tumors.

The role of methionine in methotrexate-sensitive and methotrexate-resistant mouse leukemia L1210 cells.

A mouse L1210 leukemia cell line was made 25-fold resistant to methotrexate (MTX) and had altered methionine transport and metabolism. L1210 cells resistant to methotrexate also had a 50-fold decrease in the exogenous methionine requirement for optimal cell growth compared to the parent cells. This change in methionine requirement was associated with differences in methionine metabolism between MTX-sensitive and MTX-resistant cell lines. Analysis of amino acid transport systems revealed different Kt and Vmax properties of methionine and nonmetbolizable amino acid analogues. There was a greater than twofold decrease in the initial sodium-dependent uptake of methionine in the resistant cells. Amino acid competition experiments revealed altered substrate specificities in the resistant cells. The cellular alterations occurring upon resistance may result from methotrexate-membrane interactions, and have been previously observed in cisplatinum-resistant cells. Thus modulation of methionine metabolism may provide the biochemical basis for MTX and cisplatinum collateral resistance.

Inhibition of methionine uptake by methotrexate in mouse leukemia L1210 cells.

Methionine-auxotrophic L1210 cells were used to study the effect of methotrexate (MTX) on methionine uptake and metabolism. MTX was shown to inhibit amino acid transport systems and cause a decrease of methionine uptake into L1210 cells. Conversely, a nonmetabolizable amino acid analogue reduced MTX uptake into L1210 cells. MTX also blocked the transfer of the beta carbon from serine into methionine. Therefore, methionine deprivation may be an additional mechanism of action for MTX in methionine-auxotrophic tumor cells.

In vivo pharmacokinetics of triazinate in L-1210 and W-256 cells.

A pharmacokinetic model for triazinate uptake in L-1210 cells in mice and W-256 cells in rats was developed to describe the observed concentration profiles with time in these cells following a 36 mg/m2 ip injection. The L-1210 cell permeability to triazinate was found to the approximately or 15 times smaller compared with W-256 cells. Similarly, the partition coefficient for L-1210 cells was calculated to be approximately or 175 times smaller than for W-256 cells. Cell membrane permeability appears to be the key parameter determining the drug transport at a short time after injection.