Over-expression of StZFP2 in Solanum tuberosum L. var. Kennebec (potato) inhibits growth of Tobacco Hornworm larvae (THW, Manduca sexta L.).

Tobacco hornworm (Manduca sexta, THW) is a voracious pest of tomato and potato. StZFP2 is a Q-type C2H2 zinc finger transcription factor (TF) that is induced upon wounding and infestation. Previous work has shown that Q-type C2H2 TFs are involved in stress responses and when over expressed, can enhance protection against drought, salinity or pathogen infection. Twelve transgenic lines (S1-S12) were tested that over-express StZFP2. Feeding S6 or S8 to THW significantly lowered larval weight (21-37%) as well as increased expression of StPIN2 in comparison to untransformed Kennebec. The increase in StPIN2, a classic marker for insect defense in potato, is consistent with the decreases in larval weight gain.

Systemic carboplatin for retinoblastoma: change in tumour size over time.

BACKGROUND/AIM: Chemotherapy for intraocular retinoblastoma is used to shrink individual retinal tumours to a size amenable to focal treatments. Quantitative data regarding retinal tumour response following treatment with primary systemic carboplatin are reported. METHODS: Changes in area and largest basal diameter of tumours that were exposed to carboplatin, had no concomitant focal treatment, and had digital funduscopic photography performed before and after treatment, were measured. Response was evaluated. RESULTS: 36 tumours were measured following one treatment: 34/36 (94.4%) responded, with a 37.1% mean decrease in area (median = 37.0%; range 4.0%-76.7%). Mean reduction in basal diameter was 21.3% (med = 21.0%; -7.9%-52.5%). 20 tumours were treated with a second cycle: 15/20 (75.0%) responded. Mean decrease in area was 17.8% (med = 15.3%; -7.0%-49.7%). The mean cumulative decrease in area after two treatments was 55.1% (med = 56.2%; 33.0%-74.5%). Mean cumulative reduction in basal diameter was 33.6% (med = 33.6%; 10.9%-53.2%). 12 tumours were treated with a third cycle: 3/12 (25.0%) responded, 8/12 were stable, and one progressed. Mean decrease in area was 5.4% (med = 7.2%; -17.7%-20.6%). Cumulative decrease in area after three treatments was 58.1% (med = 57.3%; 34.8%-77.2%). Mean cumulative reduction in basal diameter was 38.8% (med = 38.2%; 19.1%-54.1%). CONCLUSIONS: Carboplatin caused measurable shrinkage of retinoblastoma tumours. Response was greatest following the initial treatment and decreased with subsequent treatments.

Transit peptide mutations that impair in vitro and in vivo chloroplast protein import do not affect accumulation of the gamma-subunit of chloroplast ATPase.

We have begun to take a genetic approach to study chloroplast protein import in Chlamydomonas reinhardtii by creating deletions in the transit peptide of the gamma-subunit of chloroplast ATPase-coupling factor 1 (CF1-gamma, encoded by AtpC) and testing their effects in vivo by transforming the altered genes into an atpC mutant, and in vitro by importing mutant precursors into isolated C. reinhardtii chloroplasts. Deletions that removed 20 or 23 amino acid residues from the center of the transit peptide reduced in vitro import to an undetectable level but did not affect CF1-gamma accumulation in vivo. The CF1-gamma transit peptide does have an in vivo stroma-targeting function, since chimeric genes in which the stroma-targeting domain of the plastocyanin transit peptide was replaced by the AtpC transit peptide-coding region allowed plastocyanin to accumulate in vivo. To determine whether the transit peptide deletions were impaired in in vivo stroma targeting, mutant and wild-type AtpC transit peptide-coding regions were fused to the bacterial ble gene, which confers bleomycin resistance. Although 25% of the wild-type fusion protein was associated with chloroplasts, proteins with transit peptide deletions remained almost entirely cytosolic. These results suggest that even severely impaired in vivo chloroplast protein import probably does not limit the accumulation of CF1-gamma.

A vegetative storage protein homolog is expressed in the growing shoot apex of hybrid poplar.

The ability of poplars (Populus deltoides Bartr. ex Marsh., and Populus trichocarpa Torr. and Gray) to sequester nitrogen in stems in preparation for winter has been associated with the massive accumulation of protein bodies in the bark and xylem ray parenchyma. These protein bodies contain a bark storage protein (BSP) that can account for up to 30% of the total soluble bark protein during the winter months. Perhaps the plant's ability to efficiently cycle nitrogen through BSP is an important aspect of its growth potential. Sequence analysis of BSP led to the identification of a leaf-associated homolog, win4, which was initially isolated because its transcript increased in abundance upon mechanical wounding. The goal of this work was to characterize this putative leaf-associated vegetative storage protein, and determine whether it might perform a storage role in vivo. Antibodies, produced against protein synthesized upon over-expression of the win4 coding region in Escherichia coli, were used to examine the relative abundance of WIN4 protein in response to supplemental nitrogen, and during development. The transcript and protein were most abundant in the youngest leaves and also increased with nitrogen fertilization. Immunolocalization of the protein was performed and showed that WIN4 was associated with cells surrounding the vasculature, and cells of the lower epidermis and stipules of immature leaves. Under moderate nitrogen fertilization regimes, WIN4 accounted for only about 2% of total soluble leaf protein; however, given the cellular specificity and enhancement with nitrogen, the protein is regulated in a manner similar to other vegetative storage proteins. Since poplar is amenable to DNA transformation and regeneration, it is now possible to ask direct questions about the role these proteins play in nitrogen storage in rapidly expanding or in dormant tissue. This type of analysis could determine whether these proteins mainly ameliorate the toxic effects of excess nitrogen, if they are instrumental in controlling nitrogen allocation or if they simply represent an efficient method for sequestering this valuable nutrient.

Alterations in the Chlamydomonas plastocyanin transit peptide have distinct effects on in vitro import and in vivo protein accumulation.

Nucleus-encoded chloroplast proteins that reside in the thylakoid lumen are synthesized as precursors with bipartite transit peptides that contain information for uptake and intra-chloroplast localization. We have begun to apply the superb molecular and genetic attributes of Chlamydomonas to study chloroplast protein import by creating a series of deletions in the transit peptide of plastocyanin and determining their effects on translocation into isolated Chlamydomonas chloroplasts. Most N-terminal mutations dramatically inhibited in vitro import, whereas replacement with a transit peptide from the gamma-subunit of chloroplast ATPase restored uptake. Thus, the N-terminal region has stroma-targeting function. Deletions within the C-terminal portion of the transit peptide resulted in the appearance of import intermediates, suggesting that this region is required for lumen translocation and processing. Thus, despite its short length and predicted structural differences, the Chlamydomonas plastocyanin transit peptide has functional domains similar to those of vascular plants. Similar mutations have been analyzed in vivo by transforming altered genes into a mutant defective at the plastocyanin locus (K. L. Kindle, manuscript in preparation). Most mutations affected in vitro import more severely than plastocyanin accumulation in vivo. One exception was a deletion that removed residues 2-8, which nearly eliminated in vivo accumulation but had a modest effect in vitro. We suggest that this mutant precursor may not compete successfully with other proteins for the translocation pathway in vivo. Apparently, in vivo and in vitro analyses reveal different aspects of chloroplast protein biogenesis.

Chromoplast development in ripening tomato fruit: identification of cDNAs for chromoplast-targeted proteins and characterization of a cDNA encoding a plastid-localized low-molecular-weight heat shock protein.

During tomato fruit ripening, photosynthetically competent thylakoid membranes are broken down and replaced by membranous deposits of carotenoids. Few of the proteins involved in this transition have been identified. We have used chloroplast protein import assays as a means to identify two cDNAs that encode proteins destined for the developing chromoplast. One of the cDNAs had unexpected properties and its biological function has not been determined. However, the other cDNA encodes a plastid-localized low-MW heat shock protein (hsp). The steady-state level of RNA corresponding to this cDNA increased several-fold during tomato ripening, and the amount of RNA induced by heat stress increased dramatically during this process. These observations suggest a new role for this stress protein in protecting the plastid during the dismantling of the thylakoid membranes or during the buildup of carotenoids.

Two cDNA clones encoding 14-3-3 homologs from tomato fruit.

Two tomato cDNA clones with homology to the 14-3-3 family of proteins were identified through immunoscreening a ripening tomato fruit library (Clontech). These two clones share approx. 71% identity at the nucleotide level and 84% identity at the deduced amino acid level, with radical amino acid substitutions clustering at the acidic carboxy-terminus. Southern hybridization data indicate that each clone represents a unique gene. Distinct transcript accumulation patterns during tomato fruit ripening together with the homology to brain regulatory proteins suggest potential involvement in fruit development.

Psychological and social effects of orthodontic treatment.

Adolescents with commonly occurring forms of malocclusion often are presumed to be at risk for negative self-esteem and social maladjustment. A randomized control group design was used to assess the psychosocial effects of orthodontic treatment for esthetic impairment. Ninety-three participants, 11 to 14 years old, with mild to moderate malocclusions, were randomly assigned to receive orthodontic treatment immediately or after serving as delayed controls. A battery of psychological and social measures was administered before treatment, during treatment, and three times after completion of treatment, the last occurring one year after termination. Repeated measures analyses of variance assessed group differences at the five time points. Parent-, peer-, and self-evaluations of dental-facial attractiveness significantly improved after treatment, but treatment did not affect parent- and self-reported social competency or social goals, nor subjects' self-esteem. In summary, dental-specific evaluations appear to be influenced by treatment, while more general psychosocial responses are not.

Chromoplast-Targeted Proteins in Tomato (Lycopersicon esculentum Mill.) Fruit.

The chloroplast to chromoplast transition during tomato (Lycopersicon esculentum Mill.) fruit ripening is characterized by a dramatic change in plastid structure and function. We have asked whether this process is mediated by an increase in the steady-state level of RNA for plastid targeted proteins. Assays for import of radiolabeled translation products into isolated pea (Pisum sativum L.) chloroplasts were used to monitor levels of chromoplast-targeted proteins at four stages of tomato fruit development. We have found striking increases during development in levels of translatable RNA for two such proteins. Additionally, the import of in vitro translation products was examined for seven individual cDNA clones known to encode RNA that increase during fruit ripening. Three of these clones produced in vitro translation products that were imported into pea chloroplasts. This implies that there is synthesis and import of new proteins during the transition from chloroplast to chromoplast and that the plastid conversion is an active developmental program rather than a simple decline in synthesis of the photosynthetic apparatus. Furthermore, our results demonstrate the utility of this method for identification of structural genes involved in plastid morphogenesis.

Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants.

A method for Agrobacterium-mediated transformation of Citrus and organogenic regeneration of transgenic plants is reported. Internodal stem segments were co-cultured with Agrobacterium harboring binary vectors that contained the genes for the scorable marker ß-glucuronidase (GUS) and the selectable marker NPT-II. A low but significant percentage (≤ 5%) of the shoots regenerated in the presence of 100 µg/ml kanamycin were GUS(+). Polymerase chain reaction (PCR) analysis confirmed that GUS(+) shoots contained T-DNA. Two plants established in soil were shown to be transgenic by Southern analysis.

Molecular cloning and nucleotide sequencing of the coat protein gene of citrus tristeza virus.

Citrus tristeza virus (CTV) contains approximately 20,000 bases of positive-sense ssRNA, encapsidated by a coat protein of approximately 25,000 Mr that has previously been reported to consist of at least two size variants, cp1 and cp2. In the present study, a cDNA library of the T36 isolate of CTV was prepared in a protein expression vector and screened with a polyclonal antibody against the coat protein. Five immunopositive clones produced proteins in Escherichia coli that reacted with monoclonal as well as polyclonal antibodies to the CTV coat protein. Nucleotide sequence analysis of a region common to the five clones revealed the presence of a 669 nucleotide open reading frame flanked by numerous in-frame termination codons. The encoded protein has a predicted Mr of 24,909 and an amino acid composition consistent with that previously reported for the CTV coat protein. Comparison of the predicted amino acid sequence of the coat protein with the amino-terminal sequences of cp1 and cp2 indicated that these coat protein species arise from the same primary translation product, as a result of post-translational proteolysis at sites approximately 12 to 15 and 26 amino acids from the amino terminus respectively. These results are the first reported cloning and sequencing of a CTV gene and provide evidence that CTV may be translated using subgenomic RNA.

Cooperation of adolescents in orthodontic treatment.

Cooperation of 39 adolescents with orthodontic treatment was examined 8-10 months into treatment and again at completion. Early in treatment, parental attitudes served as the best predictors of cooperation. By the end of active treatment, however, the adolescent patients' own cognitions were the most salient predictors of cooperation. Prior to beginning orthodontic treatment, subjects and their parents completed a battery of psychosocial and orthodontic-specific measures. Results of stepwise multiple regression analyses showed that only the Parent Positive Attitude Toward Braces measure significantly predicted orthodontic cooperation early in treatment, while External-Powerful Others (Professionals) attributions of control, External-Chance attributions of control, and the initial assessment of cooperation significantly predicted cooperation over longer periods of time.

Chemical quantification of unscheduled DNA synthesis in cultured hepatocytes as an assay for the rapid screening of potential chemical carcinogens.

Technical modifications of the quantitative determination of unscheduled DNA synthesis in cultured hepatocytes are described which allow for the rapid identification of potentially carcinogenic chemicals on a large-scale screening basis. The test is based on the biochemical quantification of [methyl-3H]thymidine incorporation into DNA in the presence of hydroxyurea following isolation of nuclei from hepatocytes treated with the agent under study. This procedure ("nuclei procedure") eliminates most of the background radioactivity which otherwise obscures the stimulation of DNA repair synthesis by agents that induce a relatively weak response. By combining the nuclei procedure with a double-labeling technique, test results can be obtained within a few hr after exposure of hepatocytes to the test agents. A test series involving 41 agents confirmed the reliability of the nuclei procedure for the assay of DNA repair synthesis. In addition, chemicals which had yielded conflicting results previously in the autoradiographic hepatocyte DNA repair test, such as 4-acetylaminofluorene, or which had passed unrecognized in previous in vitro tests, such as the potent liver carcinogen methapyrilene hydrochloride, scored clearly positive in our test protocol.

ADP-ribosyltransferase activity in cultured hepatocytes. Interactions with DNA repair.

A rapid increase in ADP-ribosyltransferase activity was observed when freshly isolated hepatocytes derived from adult rats were established in primary monolayer culture. (ADP-ribose)n-degrading activity remained constant over a period of 48 h of culture. Inhibition of ADP-ribosyltransferase activity with pyridine derivatives, 3-aminobenzamide, theophylline, or thymidine, was accompanied by an enhanced DNA repair synthesis in response to the direct-acting carcinogen, methyl methanesulfonate, or UV irradiation. Three aminobenzamides differing only in the position of the amino group exhibited the same structure-activity relationship in regard to their action on DNA repair synthesis and ADP-ribosyltransferase. Spermine treatment of hepatocytes apparently had an inverse effect on both these cellular functions. The removal of DNA strand breaks following methyl methanesulfonate treatment was accelerated by inhibitors of ADP-ribosyltransferase. The results suggest that ADP-ribosylation interacts with late stages in the process of DNA repair. This interaction apparently is dependent on the nature of damage imposed on chromatin since repair synthesis in response to a number of carcinogens is unaffected by inhibitors of ADP-ribosyltransferase.

DNA damage induced by the antihistaminic drug methapyrilene hydrochloride.

Treatment of primary cultures of rat hepatocytes with the antihistaminic drug, methapyrilene hydrochloride, stimulated DNA-repair synthesis up to 7-fold and caused the formation of alkaline-labile lesions in hepatocellular DNA. These data clearly demonstrate that methapyrilene hydrochloride is a DNA damaging agent. In view of a recent report and our own findings we suggest that this antihistamine has the properties of a complete carcinogen.