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Biomedical subjects

R J Schmidt

Publications and source records attributed to R J Schmidt.

At least 37 records · Page 2Linked to original sources
PubMed1998

Biocompatibility of potential wound management products: hydrogen peroxide generation by fungal chitin/chitosans and their effects on the proliferation of murine L929 fibroblasts in culture.

Agaricus bisporus, Fusarium graminearum, Phycomyces blakesleeanus, unbleached and bleached, Rhizomucor miehei, and Rhizopus oryzae were examined as sources of fungal chitin/chitosan. The nitrogen content of the alkalitreated mycelia/sporangiophores obtained after optimization of culture conditions, and of similarly treated A. bisporus stipes, was 2.87, 1.29, 6.27, 6.50, 4.80, and 4.95% w/w, respectively, which relates to an estimated chitin content of 42, 19, 91, 94, 70, and 72%, respectively. The hydrogen peroxide (H2O2)-generating ability of the treated fungal materials after 8 h at pH 7.4 and 37 degrees C decreased in the order R. oryzae > P. blakesleeanus unbleached approximately R. miehi > F. graminearum > A. bisporus > P. blakesleeanus bleached. This did not correlate with estimated chitin content. The effect of these fungal materials on the rate of proliferation of murine L929 fibroblasts in culture also was examined. Both pro- and antiproliferant effects were observed. Significant (P < .05) proproliferant effects were observed on day 6 with R. miehei, R. oryzae, and P. blakesleeanus (unbleached and bleached) at 0.01% w/v. The greatest antiproliferant effect was observed with R. oryzae at 0.05% w/v on day 6 (-63% relative to the control, P < .05; cell viability, 95%). In contrast, A. bisporus failed to affect cell yield significantly at either 0.01 or 0.05% w/v. Addition of catalase to cultures containing R. oryzae or R. miehei at 0.05% w/v failed to abolish the antiproliferant effect on day 3, instead producing a small but significant (P < .05) increase in the effect. Catalase also failed to affect significantly the antiproliferant effect of F. graminearum at 0.05% w/v, but did abolish the proproliferant effect of P. blakesleeanus (unbleached and bleached) on day 3. Overall, our results suggest that the H2O2 being generated by the fungal materials modulates cell proliferation but that this effect is superimposed upon a H2O2-independent antiproliferant effect manifesting itself at the higher concentrations of fungal material. The antiproliferant effect was not attributable to Ca2+, Mg2+, or Fe2+ depletion although chelation of Fe2+ did correlate with H2O2-generating ability. Only P. blakesleeanus appears to lack this antiproliferant activity while retaining H2O2-generating activity. These results may aid the selection of fungal chitin/chitosan for further evaluation as a potential wound management material.

Animals↗
PubMed1998

Fertility and contraception in end-stage renal disease.

The hormonal aberrations that occur with end-stage renal disease (ESRD) are presented in this review in relation to fertility and conception among women on dialysis. The imbalance in gonadotropin production in dialysis-dependent men and women is characterized by elevations in luteinizing hormone (LH). In women dialysis patients, the normal estradiol-stimulated LH surge does not occur, resulting in anovulation. In men dialysis patients spermatogenesis is impaired, and low testosterone levels cause elevated LH. Infertility in those with ESRD is a culmination of many factors, including impotence and loss of libido, anovulation, and an altered hormonal milieu. Despite these inhibitors of conception, women on dialysis can conceive; pregnancy has been reported in 1% to 7% of women on dialysis in survey studies. The influence of dialysis mode (hemodialysis v peritoneal dialysis), recombinant human erythropoietin (EPO), and dialysis adequacy on the likelihood of conception among patients of either sex on dialysis is unknown. Reduced sexual activity and interest has consistently been reported in the ESRD population. The reasons for this are complex and likely involve the effects of comorbid illnesses, overall health status, body image factors, and hormonal alterations. Nephrologists rarely discuss conception and contraception with their women dialysis patients. Greater attention to these issues is needed.

Adult↗
PubMed1998

The blooming of grass flower development.

The past half decade has provided a wealth of information concerning the molecular and genetic control of floral organ and meristem identity in dicotyledonous plants. Comparatively little is understood about these processes in grass species in spite of the importance that these species play in human agriculture. The isolation of grass genes that are homologous to dicot floral homeotic genes in combination with recent advances in reverse genetic technology and improvements in cereal transformation opens the door for understanding molecular mechanisms of grass flower development. Such information will also focus attention on the evolutionary relationships between grass and dicot flowers and the degree to which the developmental pathways leading to reproductive organ development in divergent angiosperms have utilized conserved mechanisms.

Cloning, Molecular↗
PubMed1998

An endosperm-specific DOF protein from barley, highly conserved in wheat, binds to and activates transcription from the prolamin-box of a native B-hordein promoter in barley endosperm.

A cDNA encoding a DNA-binding protein of the DOF class of transcription factors was isolated from a barley endosperm library. The deduced amino acid sequence for the corresponding protein is 94% identical through the DOF domain to the prolamin-box (P-box) binding factor PBF from maize. The gene encoding the barley PBF (BPBF) maps to chromosome 7H, and its expression is restricted to the endosperm where it precedes that of the hordein genes. The BPBF expressed in bacteria as a GST-fusion binds a P-box 5'-TGTAAAG-3' containing oligonucleotide derived from the promoter region of an Hor2 gene. Binding was prevented when the P-box motif was mutated to 5'-TGTAgAc-3'. A P-box binding activity, present in barley and wheat endosperm nuclei, interacted similarly to BPBF with this synthetic oligonucleotide, and the binding was abolished by 1,10-phenanthroline. Transient expression experiments in developing barley endosperms demonstrate that BPBF transactivates transcription from the P-box element of a native Hor2 promoter and that direct binding of BPBF to its target site is essential for transactivation since mutations in the DOF DNA-binding domain or in the P-box motif of this promoter abolished both binding and transactivation. Evidence was also obtained for the presence in wheat of a Pbf homologue having similar DNA-binding properties to that of BPBF. These results strongly implicate this endosperm-specific DOF protein from barley as an important activator of hordein gene expression and suggest the evolutionary conservation of the Pbf gene function among small grain cereals.

Amino Acid Sequence↗
PubMed1998

Early referral and its impact on emergent first dialyses, health care costs, and outcome.

Early referral (ER) to nephrologists of patients with chronic renal failure was assessed for its impact on the incidence of emergent first dialyses and choice of dialysis modality (hemodialysis [HD] or peritoneal dialysis [PD]), and survival. We reviewed events preceding first dialyses of 238 patients with end-stage renal disease (ESRD) starting dialysis between January 1990 and April 1997, with follow-up extending through November 1997. Patients referred more than 1 month before needing dialysis (early referral [ER]) were compared with patients presenting within 30 days of needing dialysis (late referral [LR]). The need for emergent HD was significantly less among ER (29%) as compared with LR (90%) (P < 0.0001). Initial modality chosen was similar among ER patients (59% for HD v 41% for PD), a finding that contrasts with national percentages, which approximate 85% and 15%, respectively. Whereas most patients had not changed modality at 4 months, significantly more had changed from HD to PD (36 of 160 or 23%) than from PD to HD (7 of 78 or 9%) (P < 0.0001). Despite starting out on HD, ER and LR patients were amenable to ultimately changing to PD. ER and LR groups had similar numbers of Medicaid patients and patients living 1 hour or more distant to tertiary medical care. Furthermore, no difference was observed in the incidence of emergent HD when ER and LR living more than 1 hour away were compared. LR was not associated with lack of insurance or distance from referral site, although these patients more often required emergent HD, with its higher attendant medical care costs. Controlling for age and cause of ESRD, there was no statistically significant difference in long-term survival when ER patients were compared with LR patients or when patients who had received emergent HD were compared with those who had not. Despite the lack of difference in long-term survival, the financial costs of emergent HD alone merit greater promotion of ER and the psychosocial preparation and modality choice it allows.

Adult↗
PubMed1997

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2.

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5'-TGTAAAG-3') found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

Amino Acid Sequence↗
PubMed1997

Gynecologic and reproductive issues in women on dialysis.

Most women on dialysis are amenorrheic and do not ovulate, but little information about menstrual patterns in women on dialysis exists, especially since the introduction and use of recombinant human erythropoietin, a therapy that may improve sexual interest and function. In this study, women who were < or = 55 years of age at the start of dialysis (n = 76) completed questionnaires and form the study group. Women older than 55 years at the start of dialysis did not complete the entire questionnaire (n = 115), but their medication records were reviewed for estrogen replacement therapy. The questionnaire asked about pregnancies, menstrual periods (regularity, frequency, duration, character of flow, menopause), and menopause before beginning dialysis and currently. Women also responded to questions about sexual activity, use of birth control, contraception counseling by physicians, yearly Papanicolaou smears, and mammograms. Demographic data (age, race, age at the time dialysis started, mode of dialysis, use of recombinant human erythropoietin, and history of renal transplant) were also obtained through the questionnaires. Fifty-nine percent of the 76 women who completed the study were white and had been on dialysis a median of 3 years (range, 0.1 to 18 years). The median age was 43 years, 68% were on hemodialysis, 90% were receiving recombinant human erythropoietin, and 70% had been pregnant (a total of 179 pregnancies; four pregnancies in four women occurred after the start of dialysis). Significantly more women were menstruating before dialysis started than currently (63% v 42%; P < 0.025), but the difference could be explained by patient age: currently menstruating women were younger (37 +/- 9 v 46 +/- 11 years; P = 0.0002). More women reported menstrual regularity before beginning dialysis (75% v 42% currently; P < 0.005), but there were no differences in number of days between or number of days of menstruation before beginning dialysis and currently. Menstrual flow was reported as heavier currently by more women (64% heavy flow with clots v 38% before dialysis started; P < 0.05). The median age at menopause was 47 years; 28% of the women were postmenopausal. Fifty percent of the women were sexually active, but only 36% used birth control. Discussions between the women and their nephrologist about possible pregnancy and contraception were reported by only 13% of women. Sixty-three percent of the women reported having yearly Papanicolaou smears and 73% had had a mammogram. Only 5% of the 113 women who were older than 55 years when they began dialysis were receiving estrogen replacement therapy. Amenorrhea was reported in this study by a smaller proportion of women than in studies conducted before the introduction of recombinant human erythropoietin. The possibility that erythropoietin may restore normal hormonal cyclic function in women with end-stage renal disease requires further study. Nephrologists as well as primary care physicians and gynecologists need to focus more on the gynecologic concerns of women on dialysis, including the potential for pregnancy. The effects of estrogen replacement on atherosclerosis and osteoporosis, and consideration of such therapy in women on dialysis warrants attention.

Adolescent↗
PubMed1997

The antioxidant activity of Chinese herbs for eczema and of placebo herbs--I.

A standardized mixture of Chinese herbs has recently been demonstrated to be an effective treatment for chronic atopic eczema in placebo controlled trials in the UK. Aqueous decoctions of this formulated mixture (PSE 222), the placebo mixture, and their component herbs were examined for antioxidant activity to determine whether antioxidant activity could account for the anti-eczema activity. Two measures of antioxidant activity were employed: the DPPH assay for non-specific hydrogen atom (or electron) donating activity and a superoxide scavenging assay. Antioxidant activity was detected in some components of both the active and placebo mixtures, but the formulated active mixture (PSE 222) was significantly more effective than the formulated placebo mixture. Further studies are needed to elucidate the in vivo significance of this result.

Antioxidants↗
PubMed1997

Phosphorylation of Opaque2 changes diurnally and impacts its DNA binding activity.

In the maize endosperm, the Opaque2 (O2) basic leucine zipper transcriptional activator regulates the expression of a subset of the zein seed storage protein gene family. Immunodetection of wild-type or mutant O2 polypeptides fractionated by SDS-PAGE resolved a closely spaced doublet migrating in the 68- to 72-kD range, whereas by using isoelectric focusing, seven to nine isoforms were detected for each allele. Phosphatase treatment simplified the protein patterns to a single band corresponding to the nonphosphorylated component. In vivo and in vitro labeling confirmed that O2 can be phosphorylated. In protein gel blots probed with DNA, only the nonphosphorylated and hypophosphorylated O2 polypeptides were able to bind an oligonucleotide containing the O2 binding sequence. Upon in situ dephosphorylation of the focused isoforms by phosphatase treatment of the isoelectric focusing filter, the hyperphosphorylated forms acquired DNA binding activity. The ratio among the various isoforms remained constant throughout the developmental stages of endosperm growth but changed from daytime to nighttime, with a significant increase of the hyperphosphorylated forms during the night period. These results indicate that O2 exists in vivo as a pool of differently phosphorylated polypeptides and demonstrate that O2 DNA binding activity is modulated by a phosphorylation/dephosphorylation mechanism that appears to be influenced by environmental conditions.

Circadian Rhythm↗
PubMed1996

Diversification of C-function activity in maize flower development.

The Arabidopsis gene AGAMOUS is required for male and female reproductive organ development and for floral determinacy. Reverse genetics allowed the isolation of a transposon-induced mutation in ZAG1, the maize homolog of AGAMOUS. ZAG1 mutants exhibited a loss of determinacy, but the identity of reproductive organs was largely unaffected. This suggested a redundancy in maize sex organ specification that led to the identification and cloning of a second AGAMOUS homolog, ZMM2, that has a pattern of expression distinct from that of ZAG1. C-function organ identity in maize (as defined by the A, B, C model of floral organ development) may therefore be orchestrated by two closely related genes, ZAG1 and ZMM2, with overlapping but nonidentical activities.

Alleles↗
PubMed1996

Characterization of the maize OHP1 gene: evidence of gene copy variability among inbreds.

The maize proteins Opaque-2 (O2) and OHP1 are members of the bZIP class of transcriptional regulatory proteins. The genomic organizations of the O2 and OHP1 genes are conserved in terms of the number and placement of introns which suggests a common evolutionary origin. The organization of O2 and OHP1 contrasts with the maize bZIP protein gene OBF1, which lacks introns, suggesting that there exist at least two evolutionary paths for bZIP genes in maize. The OHP1 gene is located on chromosome 1L and a duplicate gene, OHP2, is located on 5S. Interestingly, the OHP1 gene, but not OHP2, has been amplified within recent history. Two out of ten maize inbreds analyzed contain multiple linked copies of OHP1, and in one of these lines at least two copies of the OHP1 genes are expressed. This variation in copy number among inbreds may prove useful in understanding the role of OHP1 in maize growth and development and yield insights into the evolution of this group of bZIP DNA binding proteins.

Amino Acid Sequence↗
PubMed1996

Development of highly potent inhibitors of Ras farnesyltransferase possessing cellular and in vivo activity.

Analogs of CVFM (a known nonsubstrate farnesyltransferase (FT) inhibitor derived from a CA1A2X sequence where C is cysteine, A is an aliphatic residue, and X is any residue) were prepared where phenylalanine was replaced by (Z)-dehydrophenylalanine, 2-aminoindan-2-carboxylate, 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Tic), and indoline-2-carboxylate. The greatest improvement in FT inhibitory potency was observed for the Tic derivative (IC50 = 1 nM); however, this compound was ineffective in blocking oncogenic Ras-induced transformation of NIH-3T3 fibroblast cells. A compound was prepared in which both the Cys-Val methyleneamine isostere and the Tic replacement were incorporated. This derivative inhibited FT with an IC50 of 0.6 nM and inhibited anchorage-independent growth of stably transformed NIH-3T3 fibroblast cells by 50% at 5 microM. Replacing the A1 side chain of this derivative with a tert-butyl group and replacing the X position with glutamine led to a derivative with an IC50 of 2.8 nM and an EC50 of 0.19 microM, a 26-fold improvement over (S*,R*)-N-[[2-[N-(2-amino-3-mercaptopropyl)-L-valyl]-1,2,3,4- tetrahydro-3-isoquinolinyl]carbonyl]-L-methionine. This derivative, (S*,R*)-N-[[2-[N-(2-amino-3-mercaptopropyl)-L-tert-leucyl]-1,2,3,4 - tetrahydro-3-isoquinolinyl]-carbonyl]-L-glutamine, was evaluated in vivo along with (S*,R*)-N-[[2-[N-(2-amino-3- mercaptopropyl)-L-tert-leucyl]-1,2,3,4-tetrahydro-3- isoquinolinyl]carbonyl]-L-methionine methyl ester for antitumor activity in an athymic mouse model implanted ip with H-ras-transformed rat-1 tumor cells. When administered by injection twice a day at 45 mg/kg for 11 consecutive days, both compounds showed prolonged survival time (T/C = 142-145%), thus demonstrating efficacy against ras oncogene-containing tumors in vivo.

3T3 Cells↗
PubMed1996

Characterization of the molecular defect in a null allele of the opaque-2 locus of maize.

The molecular defect in an opaque-2 (o2) mutant, previously characterized as a null allele, has been identified as containing an insertion of the transposable element of the Bergamo (Bg) family. Restriction mapping and partial sequence analysis of the Bg in the o2 null allele indicates that this element is distinct from the previously described Bg as well as the defective Bg (rbg) of the o2m(r) allele. It is, however, inserted at the same site in O2 as the rbg of o2m(r) and can transpose when Bg is present. This study shows that, depending on genetic background, this allele may not behave as a stable null which could dramatically influence the conclusions drawn from experiments based on this particular mutant.

Alleles↗
PubMed1995

Farnesyl diphosphate-based inhibitors of Ras farnesyl protein transferase.

The rational design, synthesis, and biological activity of farnesyl diphosphate (FPP)-based inhibitors of the enzyme Ras farnesyl protein transferase (FPT) is described. Compound 3, wherein a beta-carboxylic phosphonic acid type pyrophosphate (PP) surrogate is connected to the hydrophobic farnesyl group by an amide linker, was found to be a potent (I50(FPT) = 75 nM) and selective inhibitor of FPT, as evidenced by its inferior activity against squalene synthetase (I50(SS) = 516 microM) and mevalonate kinase (I50(MK) = > 200 microM). A systematic structure-activity relationship study involving modifications of the farnesyl group, the amide linker, and the PP surrogate of 3 was undertaken. Both the carboxylic and phosphonic acid groups of the beta-carboxylic phosphonic acid PP surrogate are essential for activity, since deletion of either group results in 50-2600-fold loss in activity (6-9, I50 = 4.6-220 microM). The farnesyl group also displays very stringent requirements and does not tolerate one carbon homologation (12, I50 = 17.7 microM), substitution by a dodecyl fragment (14, I50 = 9 microM), or introduction of an extra methyl group at the allylic position (18, I50 = 55 microM). Modifications around the amide linker group of 3 were more forgiving, as evidenced by the activity of N-methyl analog (21, I50 = 0.53 microM), the one carbon atom shorter farnesoic acid-derived retroamide analog (32, I50 = 250 nM), and the exact retroamide analog (49, I50 = 50 nM). FPP analogs such as 3, 32, and 49 are novel, potent, selective, small-sized, nonpeptidic inhibitors of FPT that may find utility as antitumor agents.

Alkyl and Aryl Transferases↗
PubMed1995

Bisubstrate inhibitors of farnesyltransferase: a novel class of specific inhibitors of ras transformed cells.

We describe the biological properties of a new class of potent farnesyltransferase (FT) inhibitors designed as bisubstrate analog inhibitors. These inhibitors incorporate the structural motifs of both farnesyl pyrophosphate and the CAAX tetrapeptide, the two substrates of the reaction catalyzed by FT. Both the phosphinate inhibitor, BMS-185878, and the phosphonate inhibitor, BMS-184467, exhibited higher in vitro FT selectivity than some of the previously reported CVFM peptidomimentics and benzodiazepine analogs. Xenopus oocyte maturation induced by microinjected oncogenic Ras proteins was blocked by coinjected BMS-184467 and BMS-185878. However, both inhibitors showed poor cell activity presumably because of the doubly charged nature of the compounds. Thus, masking the charge on the carboxylate ion markedly improved the cell permeability of BMS-185878, leading to BMS-186511, the methyl carboxyl ester prodrug. BMS-186511 inhibited FT activity in whole cells as determined by inhibition of p21 Ras protein processing, inhibition of farnesylation of proteins including Ras and the accumulation of unfarnesylated Ras proteins in the cytosolic fraction. While the cellular effects of these bisubstrate analog inhibitors had no significant effect on growth of untransformed NIH3T3 cells, they produced pronounced inhibition of Ras transformed cell growth. Both the anchorage dependent and independent growth of ras transformed cells were severely curtailed by micromolar concentrations of BMS-186511. We also found that both H-ras and K-ras transformed cells are affected by this bisubstrate inhibitor. However, K-ras transformed cells appear to be less sensitive. The inhibition of FT activity in cells and the ensuing inhibition of ras transformed cell growth is further manifested in distinct morphological changes in cells. Cells flattened, became less refractile and grew in contact inhibited monolayer. Moreover, the highly diffused character of the actin cytoskeleton in the ras transformed cells was dramatically reverted to an organized network of stress cables crisscrossing the entire cells upon treatment with BMS-186511. All of these effects of BMS-186511 are limited to ras transformed cells that utilize farnesylated Ras, but are not seen in transformed cells that use geranylgeranyl Ras or myristoyl Ras. Significantly, these FT inhibitors did not produce any signs of gross cytotoxicity in untransformed, ras transformed cells or other oncogene transformed cells.

3T3 Cells↗
PubMed1995

Phosphinyl acid-based bisubstrate analog inhibitors of Ras farnesyl protein transferase.

The rational design, synthesis, and biological activity of phosphonyl- and phosphinyl-linked bisubstrate analog inhibitors of the enzyme Ras farnesyl protein transferase (FPT) are described. The design strategy for these bisubstrate inhibitors involved connection of the critical binding components of the two substrates of FPT (ras protein and farnesyl pyrophosphate, FPP) through a phosphonyl- or phosphinyl-bearing linker. Compound 14, the first example in this series, was found to be a potent FPT inhibitor (I50 = 60 nM). A further 15-fold enhancement in activity was observed upon replacement of the VLS tripeptide sequence in 14 with VVM (15, I50 = 6 nM). The phosphinic acid analog 16 (I50 = 6 nM) was equiactive to phosphonic acid 15. Compounds 14-16 afforded 1000-fold selectivity for FPT against the closely related enzyme geranylgeranyl protein transferase type I, GGT-I [14, I50(GGT-I) = 59 microM; 15 I50(GGT-I) = 10 microM; 16 I50(GGT-I) = 21 microM]. Methyl and POM ester prodrugs 17-19 were prepared and evaluated in whole cell assays and appear to block ras-induced cell transformation, as well as colony formation in soft agar. A distinctive feature of this novel class of potent and selective bisubstrate FPT inhibitors is that they are non-sulfhydryl in nature.

3T3 Cells↗