Ribozymes targeted to stearoyl-ACP delta9 desaturase mRNA produce heritable increases of stearic acid in transgenic maize leaves.

Ribozymes are RNAs that can be designed to catalyze the specific cleavage or ligation of target RNAs. We have explored the possibility of using ribozymes in maize to downregulate the expression of the stearoyl-acyl carrier protein (Delta9) desaturase gene. Based on site accessibility and catalytic activity, several ribozyme constructs were designed and transformed into regenerable maize lines. One of these constructs, a multimer hammerhead ribozyme linked to a selectable marker gene, was shown to increase leaf stearate in two of 13 maize lines. There were concomitant decreases in Delta9 desaturase mRNA and protein. The plants with the altered stearate phenotype were shown to express ribozyme RNA. The ribozyme-mediated trait was heritable, as evidenced by stearate increases in the leaves of the R1 plants derived from a high-stearate line. The increase in stearate correlated with the presence of the ribozyme gene. A catalytically inactive version of this ribozyme did not produce any significant effect in transgenic maize. This is evidence that ribozymes can be used to modulate the expression of endogenous genes in maize.

Development of an end-stage renal disease managed care demonstration.

At present, end-stage renal disease (ESRD) beneficiaries cannot enroll in health maintenance organizations (HMOs) or social health maintenance organizations (SHMOs), but HMO members who develop ESRD may remain enrolled, and the Health Care Financing Administration (HCFA) pays the HMO a state-specific, but otherwise unadjusted, capitation rate that is 95% of fee-for-service (FFS) costs. Thus, more than 6,000 ESRD beneficiaries were enrolled in HMOs in 1993, when Congress mandated an ESRD SHMO demonstration in which not only Medicare-covered services, but extra benefits were to be provided to Medicare beneficiaries, with the SHMO receiving a capitation rate based on 100% of FFS costs. The demonstration will test (1) the feasibility of year-round open enrollment of ESRD beneficiaries in HMOs; (2) a capitation system based on treatment status--dialysis, transplant, or functioning graft--and adjusted for age and whether diabetes was the cause of renal failure; (3) the effect of the additional benefits; and (4) whether managed care can improve ESRD quality outcomes. HCFA made demonstration awards in September 1996 to Kaiser-Permanente in Southern California; Health Options in Southern Florida; and Phoenix Healthcare in Central Tennessee. The sites are expected to have 1 year of planning and development before beginning the congressionally mandated 3 years of service delivery. There will be an independent evaluation.

cDNA cloning and characterization of a putative 1,3-beta-D-glucanase transcript induced by fungal elicitor in bean cell suspension cultures.

Synthetic oligonucleotides based on similarity between tobacco 1,3-beta-D-glucanase and barley 1,3-1,4-beta-D-glucanase were used to prime the synthesis and amplification of a 162 bp bean (Phaseolus vulgaris L.) beta-glucanase cDNA by the polymerase chain reaction (PCR). The PCR product was used to isolate a near full-length beta-glucanase cDNA corresponding to an approximately 1400 bp full-length transcript, from a library containing cDNA sequences complementary to mRNA from fungal elicitor-treated bean cells. At the amino acid level, the bean beta-glucanase cDNA was 59% similar to tobacco 1,3-beta-D-glucanase, 46% similar to barley 1,3-beta-D-glucanase and 46% similar to barley 1,3-1,4-beta-D-glucanase. At the nucleotide level, the similarities were 65, 50 and 53% respectively. The beta-glucanase appeared to be encoded by a single gene with similar genomic organization in bean cultivars Canadian Wonder, Imuna and Saxa. On the basis of predicted Mr, isoelectric point, sequence similarity, and comparisons of rate of transcript appearance with induced enzyme activity, it was concluded that the cDNA encodes the basic bean endo-1,3-beta-D-glucanase. Glucanase transcripts were induced, from very low basal levels, with similar kinetics to chitinase transcripts in elicitor-treated bean cell suspension cultures.

Induction of a chicken small heat shock (stress) protein: evidence of multilevel posttranscriptional regulation.

A novel form of regulation of expression of a vertebrate heat shock gene is described. A cDNA clone encoding human Hsp27 was shown to specifically recognize chicken Hsp23 RNA by Northern (RNA) blot analysis and hybrid-select translation. This probe was then used to measure chicken hsp23 gene activity in control and heat-stressed cells. The hsp23 gene(s) was transcriptionally active in non-heat-stressed cells, and its rate of transcription did not increase significantly upon heat shock. Cytoplasmic Hsp23 mRNA, which was metabolically very stable in nonstressed cells, underwent a fourfold increase in amount after a 1-h heat shock, resulting in a twofold increase in Hsp23 mRNA in polysomes. Hsp23 mRNA was relatively abundant and translationally active even in non-heat-shocked cells. Taken together, these data implicated posttranscriptional nuclear events as an important control point for induction of Hsp23 RNA transcripts. The protein half-life of Hsp23 increased from approximately 2 h in control cultures to 13 h in heat-shocked cells, revealing a second major control point. Hsp23 which was synthesized prior to heat shock also increased in stability and contributed to the overall accumulation of Hsp23 in heat-shocked cells. Cycloheximide had no effect on this change in Hsp23 half-life, while dactinomycin blocked the stabilization of Hsp23, suggesting a need for newly synthesized RNA. These data indicated that stabilization of Hsp23 protein and posttranscriptional nuclear events resulting in increased production of Hsp23 mRNA were primarily responsible for a 13-fold increase in the accumulation of newly synthesized Hsp23 after 1 h of heat shock. The regulation of the hsp23 gene is discussed in comparison with several other posttranscriptionally regulated genes, including the proto-oncogene c-fos, the developmentally regulated chicken delta-crystallin gene, and regulation of cellular gene expression by the proto-oncogene c-myc.

Stress Responses in Alfalfa (Medicago sativa L.): I. Induction of Phenylpropanoid Biosynthesis and Hydrolytic Enzymes in Elicitor-Treated Cell Suspension Cultures.

Alfalfa (Medicago sativa L.) cell suspension cultures accumulated high concentrations of the pterocarpan phytoalexin medicarpin, reaching a maximum within 24 hours after exposure to an elicitor preparation from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. This was preceded by increases in the extractable activities of the isoflavonoid biosynthetic enzymes l-phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate coenzyme A-ligase, chalcone synthase, chalcone isomerase, and isoflavone O-methyltransferase. Pectic polysaccharides were weak elicitors of phenylalanine ammonia-lyase activity but did not induce medicarpin accumulation, whereas reduced glutathione was totally inactive as an elicitor in this system. The fungal cell wall extract was a weak elicitor of the lignin biosynthetic enzymes, caffeic acid O-methyltransferase and coniferyl alcohol dehydrogenase, but did not induce appreciable increases in the activities of the hydrolytic enzymes chitinase and 1,3-beta-d-glucanase. The results are discussed in relation to the activation of isoflavonoid biosynthesis in other legumes and the development of the alfalfa cell culture system as a model for studying the enzymology and molecular biology of plant defense expression.

Inhibition of heat shock (stress) protein induction by deuterium oxide and glycerol: additional support for the abnormal protein hypothesis of induction.

The patterns of radioactively labeled proteins from cultured chicken embryo cells stressed in the presence of either D2O or glycerol were analyzed by using one-dimensional polyacrylamide gel electrophoresis. These hyperthermic protectors blocked the induction of stress proteins during a 1-hour heat shock at 44 degrees C. The inhibitory effect of glycerol but not D2O on the induction of heat shock proteins could be overcome by increased temperature. By using transcriptional run-on assays of isolated nuclei and cDNA probes to detect hsp70- and hsp88-specific RNA transcripts, it was shown that the D2O and glycerol blocks occurred at or before transcriptional activation of the hsp70 and hsp88 genes. After heat-stressed cells were returned to 37 degrees C and the protectors were removed, heat shock proteins were inducible by a second heating. This result and the fact that the chemical stressor sodium arsenite induced stress proteins in glycerol medium indicated that the treatments did not irreversibly inhibit the induction pathways and that the stress response could be triggered even in the presence of glycerol by a stressor other than heat. In principle then, cells incurring thermal damage during a 1-hour heat shock at 44 degrees C in D2O or glycerol medium should be competent to respond by inducing heat shock proteins during a subsequent recovery period at 37 degrees C in normal medium. We found that heat shock proteins were not induced in recovering cells, suggesting that glycerol and D2O protected heat-sensitive targets from thermal damage. Evidence that the heat-sensitive target(s) is likely to be a protein(s) is summarized. During heat shocks of up to 3 hours duration, neither D2O nor glycerol significantly altered hsp23 gene activity, a constitutively expressed chicken heat shock gene whose RNA transcripts and protein products are induced by stabilization (increased half-life). During a 2-hour heat shock, glycerol treatment blocked the heat-induced stabilization of hsp23 RNA and proteins; however, D2O treatment only blocked RNA transcript stabilization, effectively uncoupling the hsp23 protein stabilization pathway from hsp23 RNA stabilization and transcriptional activation of hsp70 and hsp88 genes.

Innovative concepts in public-private coordination of benefits for retirees.

Benefits--in the broadest and most literal sense of the word--can be coordinated between Medicare and employer group health plans. What that may entail, however, ranges from (1) researching complementary plans' effect on Medicare utilization and costs; to (2) incorporating those findings into rate setting for the Medicare capitation payment that would be administered by the EGHPs; to (3) some potential cost-containing redesign of complementary plans that would be acceptable to employees/retirees and their unions and would involve improvements in information provided to consumers; to (4) some consideration given to allowing savings achieved through capitation to become part of a trust that would prefund the EGHP retiree plan.

Inducible repair of phosphotriesters in Escherichia coli.

Extracts from Escherichia coli cells induced for the adaptive response have been prepared that are capable of repairing O6-methylguanine, O4-methylthymine, and the phosphotriesters produced on the DNA backbone by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The phosphotriesters are repaired by a methyltransferase distinct from the one that demethylates O6-methylguanine. We propose that this increased capacity to repair phosphotriesters accounts for much of the increased resistance to MNNG toxicity seen in cultures induced for the adaptive response.

Age at first coitus and choice of contraceptive method: preliminary report on a study of factors related to cervical neoplasia.

PIP: The incidence of cancer of the uterine cervix appears to be species-specific, thus confounding extrapolation of results from studies with experimental animals. In humans, the incidence varies more by social groups than does any other primary cancer. In a recent study, Puerto Ricans were found to have a 25 times higher risk of cervical cancer than Jews. Blacks had 4 times the risk of non-Jewish Caucasians. Age at 1st coitus (AFC) and number of coital partners have been related to increased risks. Upper socioeconomic level women have had contraceptive pills prescribed more often than others. Initial interviews and recordings of gynecologic results have been completed by the Institute for Survey Research for 25,000 American women attending large clinics and group health centers in 9 continental cities and 3 cities in Puerto Rico. Data concerning contraceptive, sexual, and reproductive history have been completed for almost 12,000. Years of education and current age have had a significant effect (p less than .001) on AFC. Gravida was also significantly related to AFC (p less than .001). The AFC of North American blacks was lower than others. Women who chose to use the pill were found to have a higher prevalence of cytologic dysplasia at the time of choice than other women. Data provide no evidence to suggest that the widespread use of oral contraceptives since the mid-1960s has been followed by increasing rates of carcinoma in situ. Other epidemiological research has failed to reveal retrospective associations between oral contraceptive use and abnormal cervical cytology. Present results are consistent with this pattern.^ieng