Effects of RBT-1 on preconditioning response biomarkers in patients undergoing coronary artery bypass graft or heart valve surgery: a multicentre, double-blind, randomised, placebo-controlled phase 2 trial.

Background: RBT-1 is a combination drug of stannic protoporfin (SnPP) and iron sucrose (FeS) that elicits a preconditioning response through activation of antioxidant, anti-inflammatory, and iron-scavenging pathways, as measured by heme oxygenase-1 (HO-1), interleukin-10 (IL-10), and ferritin, respectively. Our primary aim was to determine whether RBT-1 administered before surgery would safely and effectively elicit a preconditioning response in patients undergoing cardiac surgery. Methods: This phase 2, double-blind, randomised, placebo-controlled, parallel-group, adaptive trial, conducted in 19 centres across the USA, Canada, and Australia, enrolled patients scheduled to undergo non-emergent coronary artery bypass graft (CABG) and/or heart valve surgery with cardiopulmonary bypass. Patients were randomised (1:1:1) to receive either a single intravenous infusion of high-dose RBT-1 (90 mg SnPP/240 mg FeS), low-dose RBT-1 (45 mg SnPP/240 mg FeS), or placebo within 24-48 h before surgery. The primary outcome was a preoperative preconditioning response, measured by a composite of plasma HO-1, IL-10, and ferritin. Safety was assessed by adverse events and laboratory parameters. Prespecified adaptive criteria permitted early stopping and enrichment. This trial is registered with ClinicalTrials.gov, NCT04564833. Findings: Between Aug 4, 2021, and Nov 9, 2022, of 135 patients who were enrolled and randomly allocated to a study group (46 high-dose, 45 low-dose, 44 placebo), 132 (98%) were included in the primary analysis (46 high-dose, 42 low-dose, 44 placebo). At interim, the trial proceeded to full enrollment without enrichment. RBT-1 led to a greater preconditioning response than did placebo at high-dose (geometric least squares mean [GLSM] ratio, 3.58; 95% CI, 2.91-4.41; p < 0.0001) and low-dose (GLSM ratio, 2.62; 95% CI, 2.11-3.24; p < 0.0001). RBT-1 was generally well tolerated by patients. The primary drug-related adverse event was dose-dependent photosensitivity, observed in 12 (26%) of 46 patients treated with high-dose RBT-1 and in six (13%) of 45 patients treated with low-dose RBT-1 (safety population). Interpretation: RBT-1 demonstrated a statistically significant cytoprotective preconditioning response and a manageable safety profile. Further research is needed. A phase 3 trial is planned. Funding: Renibus Therapeutics, Inc.

The Effects of Peroxisome Proliferator-Activated Receptor-Delta Modulator ASP1128 in Patients at Risk for Acute Kidney Injury Following Cardiac Surgery.

Introduction: Peroxisome proliferator-activated receptor δ (PPARδ) plays a central role in modulating mitochondrial function in ischemia-reperfusion injury. The novel PPARδ modulator, ASP1128, was evaluated. Methods: A randomized, double-blind, placebo-controlled, biomarker assignment-driven, multicenter study was performed in adult patients at risk for acute kidney injury (AKI) following cardiac surgery, examining efficacy and safety of a 3-day, once-daily intravenous dose of 100 mg ASP1128 versus placebo (1:1). AKI risk was based on clinical characteristics and postoperative urinary biomarker (TIMP2)•(IGFBP7). The primary end point was the proportion of patients with AKI based on serum creatinine within 72 hours postsurgery (AKI-SCr72h). Secondary endpoints included the composite end point of major adverse kidney events (MAKE: death, renal replacement therapy, and/or ≥25% reduction of estimated glomerular filtration rate [eGFR]) at days 30 and 90). Results: A total of 150 patients were randomized and received study medication (81 placebo, 69 ASP1128). Rates of AKI-SCr72h were 21.0% and 24.6% in the placebo and ASP1128 arms, respectively (P = 0.595). Rates of moderate/severe AKI (stage 2/3 AKI-SCr and/or stage 3 AKI-urinary output criteria) within 72 hours postsurgery were 19.8% and 23.2%, respectively (P = 0.609). MAKE occurred within 30 days in 11.1% and 13.0% in the placebo and ASP1128 arms (P = 0.717), respectively; and within 90 days in 9.9% and 15.9% in the placebo and ASP1128 arms (P = 0.266), respectively. No safety issues were identified with ASP1128 treatment, but rates of postoperative atrial fibrillation were lower (11.6%) than in the placebo group (29.6%). Conclusion: ASP1128 was safe and well-tolerated in patients at risk for AKI following cardiac surgery, but it did not show efficacy in renal endpoints.

Impacts of Coastal Development on the Ecology of Tidal Creek Ecosystems of the US Southeast including Consequences to Humans.

Upland areas of southeastern U.S. tidal creek watersheds are popular locations for development, and they form part of the estuarine ecosystem characterized by high economic and ecological value. The primary objective of this work was to define the relationships between coastal development, with its concomitant land use changes and associated increases in nonpoint source pollution loading, and the ecological condition of tidal creek ecosystems including related consequences to human populations and coastal communities. Nineteen tidal creek systems, located along the southeastern United States coast from southern North Carolina to southern Georgia, were sampled during summer, 2005 and 2006. Within each system, creeks were divided into two primary segments based upon tidal zoning: intertidal (i.e., shallow, narrow headwater sections) and subtidal (i.e., deeper and wider sections) and then watersheds were delineated for each segment. Relationships between coastal development, concomitant land use changes, nonpoint source pollution loading, the ecological condition of tidal creek ecosystems, and the potential impacts to human populations and coastal communities were evaluated. In particular, relationships were identified between the amount of impervious cover (indicator of coastal development) and a range of exposure and response measures including increased chemical contamination of the sediments, increased pathogens in the water, increased nitrate/nitrite levels, increased salinity range, decreased biological productivity of the macrobenthos, alterations to the food web, increased flooding potential, and increased human risk of exposure to pathogens and harmful chemicals. The integrity of tidal creeks, particularly the headwaters or intertidally-dominated sections, were impaired by increases in nonpoint source pollution associated with sprawling urbanization (i.e., increases in impervious cover). This finding suggests these habitats are valuable early warning sentinels of ensuing ecological impacts and potential public health and flooding risk from sprawling coastal development. Results also validate the use of a conceptual model with impervious cover thresholds for tidal creek systems in the southeast region.

Phenotypic analysis of Paf1/RNA polymerase II complex mutations reveals connections to cell cycle regulation, protein synthesis, and lipid and nucleic acid metabolism.

Paf1 is an RNA polymerase II-associated protein in yeast, which defines a complex that is distinct from the Srb/Mediator holoenzyme. The Paf1 complex, which also contains Ctr9, Cdc73, Hpr1, Ccr4, Rtf1 and Leo1, is required for full expression of a subset of yeast genes, particularly those responsive to signals from the Pkc1/MAP kinase cascade. We have extensively characterized the pleiotropic phenotypes of deletion mutants for factors present in the Paf1 complex, identifying more than a dozen new phenotypes, and, in some cases, establishing possible molecular explanations for the growth defects. For example, paf1 Delta causes sensitivity to hydroxyurea; this phenotype correlates with a reduction in RNR1 transcript abundance and is suppressed by over-expression of RNR1. In contrast, the resistance of paf1 Delta cells to the transcription elongation inhibitors 6-azauracil and mycophenolic acid correlates with its ability to derepress the IMD2 transcript. We tested the hypothesis that Paf1 communicates with some promoters through the DNA-binding factors Swi4, Mbp1 or Rlm1. The phenotypes of mutations in Paf1 complex components are exacerbated in the swi4 Delta background, suggesting that the complex acts in a pathway parallel to that controlled by Swi4. Conversely, the fact that mbp1 Delta and rlm1 Delta mutations do not enhance the phenotypes suggests that the Paf1 complex may function in the same regulatory pathway(s) with Mbp1 and Rlm1.

Early development of the gubernaculum and cremaster sac in estrogen receptor knockout mice.

AIM exogenous estrogen causes gubernacular atrophy and cryptorchidism in fetal rodents. Mice with an estrogen receptor-alpha (ERalpha) disrupted gene mutation (alphaERKO) were studied to determine whether ablation of endogenous estrogen action, through ERalpha, had an effect on gubernacular development. Serial sagittal sections were made of the pelvis in fetal and day 7 postnatal wild-type and alphaERKO mice with the estrogen receptor-alpha "knockout" gene mutation. Wild-type (n = 24), heterozygote (n = 13) and alphaERKO mice (n = 12) were sacrificed at 16, 17 and 18 days fetal life and at 7 days postnatally. The size of the gubernaculum, cremaster muscle, cremaster sac, and the width of the sac at both ends in day 7 mice were quantitated by computer analysis. Visually and statistically the ERKO mice could not be separated from the wild-type mice during fetal life. At day 7 postnatally, a thicker cremaster sac was noted morphologically, and also a statistically significant difference was seen in the width of the cremaster sac at the sac's tip. Sac area, cremaster muscle area and the width of the sac at the sac's end did not differ significantly. Overall there is minimal phenotypic change observed in the alphaERKO mouse compared to wild-type at the early developmental stages investigated. However, at postnatal day 7, there is a difference in the width of the cremasteric sac tip. This suggests that the effect of ERalpha, and thus signaling on the developing gubernaculum, occurs late in development. Alternatively, an action from the recently discovered ERbeta may be involved. Exploration of a betaERKO and the double knock-out alphaERKO/betaERKO mouse should be informative in evaluating the effect of endogenous estrogens in gubernacular development.

Modifications of testosterone-dependent behaviors by estrogen receptor-alpha gene disruption in male mice.

The role of the a form of estrogen receptor (ER alpha) gene expression in the regulation of testosterone-dependent male reproductive behaviors was investigated using ER knockout mice (ERKO), which are specifically deficient in functional ER alpha, but not ER beta, gene expression. Previous studies in gonadally intact ERKO mice revealed that male aggressive behavior was greatly reduced by the lack of a functional ER alpha gene. In the present study the almost complete suppression of male-typical offensive attacks was further confirmed in ERKO mice that had been singly housed since weaning. Regarding aggression, it was also found that ER alpha gene disruption virtually abolished the propensity to initiate offensive attacks, even though ERKO mice could elicit attacks from resident C57BL/6J mice as wild-type (WT) and heterozygous littermates. Daily injection of testosterone propionate (TP) was completely ineffective in inducing aggressive behavior in gonadectomized ERKO mice, whereas it successfully restored aggression in WT mice. In contrast, male sexual behaviors, mounts and intromissions, were induced by daily injection of TP in both gonadectomized ERKO and WT mice. In addition to TP, dihydrotestosterone propionate (DHTP) was also effective in restoring mounts in ERKO mice, although DHTP was much more potent in WT mice than in ERKO mice. Neither TP nor DHTP, however, ever induced ejaculation in ERKO mice. These results together with previous findings in gonadally intact ERKO mice suggest that ER alpha may be responsible for the regulation by testosterone of consummatory, but not motivational, aspects of male sexual behavior. Finally, ERKO male mice retrieved newborn pups placed in their home cage with similar latencies to males of the two other genotypes. During parental behavior tests, however, a higher percentage of ERKO mice (70%) showed infanticide compared with WT mice (35%). The latter result was interpreted as showing that ER alpha activation by testosterone during the perinatal period may exert a suppressive effect on testosterone-inducible infanticide in adulthood. With respect to three major testosterone-dependent behavioral systems reflecting masculinization, these findings demonstrate three different types of effects due to ER alpha gene disruption.

Modulation of estrogen receptor levels in mouse uterus by protein kinase C isoenzymes.

We have recently shown that protein kinase C (PKC) modifies estrogen receptor (ER) binding and modulates the responsiveness to estrogens in a clonal osteoblast-like cell line stably transfected with the ER. The purpose of the present study was to determine whether the interaction observed between the ER and PKC signaling in these cells occurs in additional estrogen target organs, such as the uterus. When uteri were incubated for 2 h with increasing concentrations of a kinase inhibitor (H7), ER binding was enhanced in a dose-dependent manner. Stimulation of PKC with phorbol ester reduced PKC activity levels, but increased ER binding. Interestingly, the changes in binding appeared to be due primarily to alterations in cytosolic ER levels, as binding in the nuclear fraction was minimally enhanced. When levels of ER messenger RNA were evaluated by Northern blot analysis, no differences were observed among the H7- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated and untreated groups. Western blot analysis, however, demonstrated that levels of ER cytosolic protein in the H7-, TPA-, and staurosporine-treated groups were increased relative to those in the untreated controls. When uteri were incubated with diethylstilbestrol in the presence of either H7 or TPA, no change in cytosolic ER levels was found, suggesting that only unoccupied ERs are responsive to modulation by PKC. Western blotting of the various PKC isoforms indicated that although PKC alpha, -beta1, -betaII, -delta, and -zeta are expressed in the uterus, only PKC alpha and -beta1 are translocated from the soluble to the particulate fraction and then degraded after phorbol ester stimulation. Hence, one or both of these latter PKC isoforms may regulate cytosolic ER levels. Collectively, these data indicate that PKC may play an important role in the modulation of uterine ER levels and that PKC may exert its effect on the ER at some posttranscriptional or posttranslational step. Finally, our results show that an ER-PKC interaction occurs in a whole organ such as the uterus and that this interaction may be important in the regulation of the ER activity in a variety of estrogen-responsive tissues.

Using tracking data to find complications that physicians miss: the case of renal failure in cardiac surgery.

BACKGROUND: Health Data Research, Inc (HDR) develops, manages, and maintains clinical registries from physicians and hospitals, including the Merged Cardiac Registry. Quarterly reports indicate data that are inconsistent, out of range, or outside the norms found in other medical centers. CASE STUDY: In reports on cardiac surgery patients, HDR noted that for the 1992-1996 period, 3 of the 30 contributing centers experienced a significant increase in the incidence of moderate and severe renal failure. One of these three contributors gave HDR access to its detailed clinical database, and HDR ruled out most of the suspected causes for this increase in renal failure. A risk model for renal failure identified 20% of the patients to be at high risk. HDR then isolated a fast-track protocol as the culprit. One of the 30 contributing centers found that the protocol was associated with significant decreases in the intensive care unit (ICU) and hospital lengths of stay. However, as the severity of renal failure increased, charges, average length of stay, transfusions, and ICU times increased. At one of the three sites, after protocol changes were instituted in mid-1995 for the high-risk patients, the rate of renal failure reverted to below baseline levels. SUMMARY AND CONCLUSIONS: Analysis of run charts led to protocol changes for patients at high risk of renal failure, while retaining the positive outcomes associated with rapid extubation and shorter ICU stays for the remaining 80% of the patients.

Notch activity influences the alphabeta versus gammadelta T cell lineage decision.

The choice between the alphabeta or gammadelta T cell fates is influenced by the production of functional, in-frame rearrangements of the TCR genes, but the mechanism that controls the lineage choice is not known. Here, we show that T cells that are heterozygous for a mutation of the Notch1 gene are more likely to develop as gammadelta T cells than as alphabeta T cells, implying that reduced Notch activity favors the gammadelta T cell fate over the alphabeta T cell fate. A constitutively activated form of Notch produces a reciprocal phenotype and induces thymocytes that have functional gammadeltaTCR gene rearrangements to adopt the alphabeta T cell fate. Our data indicate that Notch acts together with the newly formed T cell antigen receptor to direct the alphabeta versus gammadelta T cell lineage decision.

Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility.

The reproductive system of male mice homozygous for a mutation in the estrogen receptor (ER) gene (ER knock-out; ERKO) appears normal at the anatomical level. However, these males are infertile, indicating an essential role for ER-mediated processes in the regulation of male reproduction. Adult ERKO male mice have significantly fewer epididymal sperm than heterozygous or wild-type males. Although spermatogenesis is occurring in some seminiferous tubules of 3- to 5-month-old ERKO males, other tubules either have a dilated lumen and a disorganized seminiferous epithelium with few spermatogenic cells or lack a lumen and contain mainly Sertoli cells. There are no obvious differences in seminiferous tubules at 10 days of age between wild-type and ERKO mice, but the lumen in ERKO males is dilated in all seminiferous tubules by 20 days. However, spermatogenesis progresses and similar numbers of sperm are present in the cauda epididymis of ERKO and wild-type males until 10 weeks of age. Disruption of spermatogenesis and degeneration of the seminiferous tubules become apparent after 10 weeks in the caudal pole of the testis and progresses in a wave to the cranial pole by 6 months. However, the seminal vesicles, coagulating glands, prostate, and epididymis do not appear to be altered morphologically in ERKO mice. Serum testosterone levels are somewhat elevated, but LH and FSH levels are not significantly different from those in wild-type males. Sperm from 8- to 16-week-old mice have reduced motility and are ineffective at fertilizing eggs in vitro. In addition, ERKO males housed overnight with hormone-primed wild-type females produce significantly fewer copulatory plugs than do heterozygous or wild-type males. These results suggest that estrogen action is required for fertility in male mice and that the mutation of the ER in ERKO males leads to reduced mating frequency, low sperm numbers, and defective sperm function.

Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor.

Past studies have shown that epidermal growth factor (EGF) is able to mimic the uterotropic effects of estrogen in the rodent. These studies have suggested a "cross-talk" model in which EGF receptor (EGF-R) signaling results in activation of nuclear estrogen receptor (ER) and its target genes in an estrogen-independent manner. Furthermore, in vitro studies have indicated the requirement for ER in this mechanism. To verify the requirement for ER in an in vivo system, EGF effects were studied in the uteri of ER knockout (ERKO) mice, which lack functional ER. The EGF-R levels, autophosphorylation, and c-fos induction were observed at equivalent levels in both genotypes indicating that removal of ER did not disrupt the EGF responses. Induction of DNA synthesis and the progesterone receptor gene in the uterus were measured after EGF treatment of both ERKO and wild-type animals. Wild-type mice showed increases of 4.3-fold in DNA synthesis, as well as an increase in PR mRNA after EGF treatment. However, these responses were absent in ERKO mice, confirming that the estrogen-like effects of EGF in the mouse uterus do indeed require the ER. These data conclusively demonstrate the coupling of EGF and ER signaling pathways in the rodent reproductive tract.

Morphometric study of the gubernaculum in male estrogen receptor mutant mice.

To determine role of estrogen receptors in testicular descent, a morphometric study of the testis and structures derived from the gubernaculum was made in sexually mature male mice having an estrogen receptor disrupted gene mutation (ERKO). Macroscopic dissections and sagittal serial sections were made of the pelvis of four wild-type mice, four mice heterozygous for the ERKO mutation, and four homozygous ERKO males. By external morphological examination the testes appeared to be descended in all three genotypes. All mice had development of a cremaster sac, which is derived from the gubernaculum, but this was twice as large in wild-type mice than in both the heterozygote or homozygote ERKO groups. The cause for the smaller cremaster sac appeared to be excessive development of the cremaster muscle in ERKO mice. The thickened muscle was associated with postmortem retraction of the testes into the inguinal canal or abdomen. Spermatogenesis and testicular volume were deficient in homozygous ERKO mice at this age. This study demonstrates that estrogen has a previously unknown role in masculine sexual development of the gubernaculum and the structures derived from it, such as the cremaster muscle.

Estrogen receptor gene disruption: molecular characterization and experimental and clinical phenotypes.

The estrogen receptor (ER) is thought to play a crucial role in the regulation of many life processes, including development, reproduction and normal physiology. Because there have been no known mutations of the estrogen receptor in normal tissue of humans and animals, its presence and tissue distribution is thought to be essential for survival. Using the techniques of homologous recombination, we have disrupted the ER gene and have produced a line of transgenic mice possessing the altered ER gene (ERKO). The mouse ER gene was disrupted by inserting a 1.8 kb PGK-Neomycin sequence into exon 2, approximately 280 bp downstream of the transcription start codon. The correct targeting of the disruption was demonstrated by Southern blot analysis and PCR. Western blot analysis of uterine preparations from ERKO females showed no detectable ER protein. Heterozygotes had one half the level of ER protein compared to wild-type animals. Estrogen insensitivity was confirmed using estrogen agonists, estradiol, hydroxy tamoxifen, diethylstilbestrol treatment for 3 days which resulted in a 3-4-fold increase in uterine wet weight and vaginal cornification in wild-type females, while ERKO mice were totally unresponsive. These data were further supported by the failure of estrogen or EGF treatment to induce DNA synthesis in uterine tissue of similarly treated mice. Lactoferrin, an estrogen-responsive gene in the uterus, was also assayed by Northern blot. Wild-type mice treated with a single estradiol injection showed a 350-fold induction in lactoferrin mRNA. while ERKO females showed no detectable response. Both male and female animals survive to adulthood with normal gross external phenotypes. As expected, females are infertile and demonstrate hypoplastic uteri and hyperemic ovaries with no apparent corpora lutea. Males are also infertile, with atrophy of the testes and seminiferous tubule dysmorphogenesis. Although the reproductive capabilities have been altered with a dramatic effect on the gonads, prenatal development of the reproductive tracts of both sexes appear to be independent of an ER-mediated response. Analysis of the mammary glands of the ERKO females at 4 months of age showed a primitive ductal rudiment rather than the fully developed ductal tree seen in wild-type siblings. Also absent were the terminal end buds seen during normal ductal morphogenesis. Both sexes show a decrease in skeletal bone density, supporting a direct role for ER action in bone. A single patient is described who is homozygous for a point mutation in the human ER gene at codon 157. The mutation produces a truncation of the ER protein and results in estrogen insensitivity syndrome. Most significant of the clinical findings are effects on skeletal bone density and retarded bone age. Findings from the patient and mice suggest that the absence of functional ER is not lethal. Mutation in the ER gene is present in the human population. Further characterization of the mice and identification of additional patients will be required to more fully understand the consequences of ER gene mutations.

Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene.

We employed homologous recombination in mouse embryonic stem cells to disrupt the estrogen receptor (ER) gene. Subsequently generated mice that are homozygous for the gene disruption, termed ERKO, possess no demonstrable wild-type ER by Western blot analysis. However, the presence of residual high affinity binding, as detected by [3H]estradiol binding assays and sucrose gradients in uterine extracts from ERKO females prompted further investigation of transcription and translation products from the disrupted ER gene. Analysis of ERKO uterine messenger RNA (mRNA) by reverse transcriptase-polymerase chain reaction demonstrated that although no full-length wild-type ER mRNA was present, two smaller transcripts, labeled E1 and E2, were identified and partially sequenced. Both ERKO transcripts are splicing variants that result in the disrupting NEO sequence being partially or completely removed from the mRNA. In the ERKO-E2 variant, this results in a frame shift and the creation of at least two stop codons downstream. In the ERKO-E1 variant, the ER reading frame is preserved and encodes for a smaller mutant ER that could be the source of the residual estradiol binding. When this mutant form is overexpressed and characterized in vitro, it results in a smaller protein of the predicted size that possesses significantly reduced estrogen-dependent transcriptional activity compared with that of the wild-type ER. Despite residual amounts of an impaired ER variant, estrogen insensitivity in the female ERKOs was confirmed by the failure of estrogen treatment to induce known uterine markers of estrogen action, such as increased DNA synthesis, and transcription of the progesterone receptor, lactoferrin, and glucose-6-phosphate dehydrogenase genes. Furthermore, serum levels of estradiol in the ERKO female are more than 10-fold higher than those in the wild type, consistent with a syndrome of hormone insensitivity.

DET1, a negative regulator of light-mediated development and gene expression in arabidopsis, encodes a novel nuclear-localized protein.

The mechanisms by which plants integrate light signals to modify endogenous developmental programs are largely unknown. One candidate for a signal transduction component that may integrate light with developmental pathways is the Arabidopsis DET1 gene product. Here we report the positional cloning of the DET1 locus and show that DET1 is a unique nuclear-localized protein. An analysis of a number of det1 mutants indicates that mutants with partial DET1 activity develop as light-grown plants in the dark. det1 null mutants share this phenotype, but also display severe defects in temporal and spatial regulation of gene expression. These results suggest that DET1 acts in the nucleus to control the cell type-specific expression of light-regulated promoters.

A Role for Cytokinins in De-Etiolation in Arabidopsis (det Mutants Have an Altered Response to Cytokinins).

When grown in the absence of light, Arabidopsis thaliana deetiolated (det) mutants develop many of the characteristics of light-grown plants, including the development of leaves and chloroplasts, the inhibition of hypocotyl growth elongation, and elevated expression levels of light-regulated genes. We show here that dark-grown wild-type seedlings exhibit similar phenotypic traits if any one of a variety of cytokinins are present in the growth medium. We further show that the striking phenotype of det mutants is unlikely to be caused by different levels of cytokinins in these mutants. The three major Arabidopsis cytokinins, zeatin, zeatin riboside, and isopentenyladenosine, accumulate to similar levels in wild-type seedlings grown in either the light or the dark. There is no consistently different pattern for the levels of these cytokinins in wild-type versus det1 or det2 mutants. However, det1 and det2 have an altered response to cytokinin in a detached leaf senescence assay and in tissue culture experiments. A model is proposed in which light and cytokinins act independently or sequentially through common signal transduction intermediates such as DET1 and DET2 to control the downstream light-regulated responses.

Endogenous protein kinase-C activation in osteoblast-like cells modulates responsiveness to estrogen and estrogen receptor levels.

The osteoblast-like osteosarcoma cell line ROS 17/2.8, which expresses very low levels of estrogen receptor (ER), was stably transfected with the mouse ER in order to more easily evaluate the physiological role of estrogens in bone cell homeostasis. These transfected ROS.SMER 14 cells are highly responsive to estrogenic stimulation at subconfluence, but become refractory to estrogenic stimulation when postconfluency is reached. The purpose of these studies was to determine the mechanisms underlying this loss of responsiveness in these ER stably transfected cells at postconfluence. When proliferative capacity was evaluated by bromodeoxyuridine immunocytochemistry, approximately 70% of the subconfluent cells were actively dividing, whereas none of the postconfluent cells underwent division. Subconfluent cells were found to contain 2500-3000 ER-binding sites/cell, whereas the ER in postconfluent cells was low and often undetectable. Steady state ER mRNA levels were not significantly modified by postconfluency. ER protein levels were also unaffected by confluency status. Since protein kinase-C (PKC) has been reported to influence cell proliferation and steroid hormone receptor binding, PKC activity was measured in sub- and postconfluent cells. Calcium-dependent PKC activity was approximately about 2-fold higher in postconfluent compared to subconfluent cells, whereas no differences were discerned in calcium-independent PKC activity. In an effort to examine the role of PKC in greater detail, postconfluent cells were treated with PKC inhibitors (H-7 or staurosporine) or with the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate) to down-regulate PKC activity, and changes in ER were evaluated. Inhibition or down-regulation of the PKC activity in postconfluent cells enhanced ER-binding capacity in a dose-dependent manner and estrogen responsiveness of an exogenous reporter gene and of the endogenous alkaline phosphatase, representing an endogenous estrogen-stimulated gene. These data indicate that there is an interaction between the PKC and ER signaling systems in bone cells and that this interaction may be influenced by the proliferative and/or differentiative state of the cells, resulting in modulation of hormone responsiveness.

Regulation of gene expression by light.

The past year has seen significant advances in the biochemical, genetic and molecular dissection of the light signal transduction and developmental pathways that lead to photoregulated gene expression in higher plants. A major part of recent research has focused on the assignment of biological functions to the various photoreceptors, the genetic dissection of the photoreceptor action pathways, and the identification of the cis-acting sequences and trans-acting factors that regulate the downstream light-regulated genes.