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Biomedical subjects

D Beach

Publications and source records attributed to D Beach.

At least 19 recordsLinked to original sources
PubMed2001

Phosphorylation of gamma-tubulin regulates microtubule organization in budding yeast.

gamma-Tubulin is essential for microtubule nucleation in yeast and other organisms; whether this protein is regulated in vivo has not been explored. We show that the budding yeast gamma-tubulin (Tub4p) is phosphorylated in vivo. Hyperphosphorylated Tub4p isoforms are restricted to G1. A conserved tyrosine near the carboxy terminus (Tyr445) is required for phosphorylation in vivo. A point mutation, Tyr445 to Asp, causes cells to arrest prior to anaphase. The frequency of new microtubules appearing in the SPB region and the number of microtubules are increased in tub4-Y445D cells, suggesting this mutation promotes microtubule assembly. These data suggest that modification of gamma-tubulin is important for controlling microtubule number, thereby influencing microtubule organization and function during the yeast cell cycle.

Alleles↗
PubMed2001

Stimulated healing of recalcitrant wounds by topical application of enriched cell culture medium: a clinical report.

This study was designed to test the efficacy of enriched cell culture medium as a wound dressing. The rationale was to create within the wound space an optimal microenvironment, conducive to cellular proliferation, vascular granulation tissue formation, and epithelialization. This study was performed on various wounds that failed to respond to previous conventional treatments.A total of 288 wounds were within the inclusion criteria, with only contaminated and neoplastic wounds excluded. Most of the patients (80 percent) were ambulatory, and the wounds were examined by the attending physician once every 7 to 14 days at an outpatient clinic. The remaining 20 percent of patients were admitted to the study while hospitalized. Cell culture medium MCDB, supplemented with insulin, thyroxin, and growth hormone, was gelled. The gel was self-applied once a day to freshly washed wounds, covered with a gauze pad, and anchored with netting. Healing started 7 to 14 days after the initiation of treatment with enriched cell culture medium. However, the criterion for success of the treatment was determined on complete wound closure, which was achieved in 189 of 288 wounds (65.6 percent). Wound closure was correlated with the initial wound volume, stage, and origin. The average time required for closure of wounds caused by systemic pathologies (n = 181) and those based on regional status (n = 107) were 12.0 and 4.4 weeks, respectively, compared with 290 and 10.3 weeks of the previous conventional treatment. In 19 extensive wounds, when vascularized granulation tissue was established, a successful surgical closure was attained. Most wounds of patients who did not continue the enriched cell culture medium treatment (34.4 percent) manifested reduced wound volume, ranging from 11 to 98 percent of initial volume. Discontinuation of treatment was associated with difficulties in reaching the clinic for the weekly examination, rather than for reasons directly related to the treatment itself, and occurred significantly earlier during the treatment period.Thus, enriched cell culture medium was effective in stimulating wound healing in recalcitrant wounds. The healing was rapid with minimum scarring and pain. No side effects or allergic reactions were reported or observed.

Administration, Topical↗
PubMed2000

An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.

In a diverse group of organisms that includes Caenorhabditis elegans, Drosophila, planaria, hydra, trypanosomes, fungi and plants, the introduction of double-stranded RNAs inhibits gene expression in a sequence-specific manner. These responses, called RNA interference or post-transcriptional gene silencing, may provide anti-viral defence, modulate transposition or regulate gene expression. We have taken a biochemical approach towards elucidating the mechanisms underlying this genetic phenomenon. Here we show that 'loss-of-function' phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs. This coincides with a marked reduction in the level of cognate cellular messenger RNAs. Extracts of transfected cells contain a nuclease activity that specifically degrades exogenous transcripts homologous to transfected double-stranded RNA. This enzyme contains an essential RNA component. After partial purification, the sequence-specific nuclease co-fractionates with a discrete, approximately 25-nucleotide RNA species which may confer specificity to the enzyme through homology to the substrate mRNAs.

Animals↗
PubMed1999

RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides.

S100/calgranulin polypeptides are present at sites of inflammation, likely released by inflammatory cells targeted to such loci by a range of environmental cues. We report here that receptor for AGE (RAGE) is a central cell surface receptor for EN-RAGE (extracellular newly identified RAGE-binding protein) and related members of the S100/calgranulin superfamily. Interaction of EN-RAGEs with cellular RAGE on endothelium, mononuclear phagocytes, and lymphocytes triggers cellular activation, with generation of key proinflammatory mediators. Blockade of EN-RAGE/RAGE quenches delayed-type hypersensitivity and inflammatory colitis in murine models by arresting activation of central signaling pathways and expression of inflammatory gene mediators. These data highlight a novel paradigm in inflammation and identify roles for EN-RAGEs and RAGE in chronic cellular activation and tissue injury.

Amino Acid Sequence↗
PubMed1999

A rate limiting function of cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2.

The cdc25A phosphatase removes inhibitory phosphates from threonine-14 and tyrosine-15 of cyclin dependent kinase-2 (cdk2) in vitro, and it is therefore widely assumed that cdc25A positively regulates cyclin E- and A-associated cdk2 activity at the G1 to S phase transition of the mammalian cell division cycle. Human cdc25A was introduced into mouse NIH3T3 fibroblasts co-expressing a form of the colony-stimulating factor-1 (CSF-1) receptor that is partially defective in transducing mitogenic signals. Cdc25A enabled these cells to form colonies in semisolid medium containing serum plus human recombinant CSF-1 in a manner reminiscent of cells rescued by c-myc. However, cdc25A-rescued cells could not proliferate in chemically defined medium containing CSF-1 and continued to require c-myc function for S phase entry. When contact-inhibited cells overexpressing cdc25A were dispersed and stimulated to synchronously enter the cell division cycle, they entered S phase 2-3 h earlier than their parental untransfected counterparts. Shortening of G1 phase temporally correlated with more rapid degradation of the cdk inhibitor p27Kip1 and with premature activation of cyclin A-dependent cdk2. Paradoxically, tyrosine phosphorylation of cdk2 increased considerably as cells entered S phase, and cdc25A overexpression potentiated rather than diminished this effect. At face value, these results are inconsistent with the hypothesis that cdc25A acts directly on cdk2 to activate its S phase promoting function.

3T3 Cells↗
PubMed1999

An integrated approach for increasing the survival of autologous fat grafts in the treatment of contour defects.

Autologous fat grafting as a technique to correct soft-tissue defects is a controversial subject. The high percentage of fat resorption and the resulting need for additional grafting considerably reduce the value of this method. The purpose of this study was to evaluate the clinical application of tissue-culturing methodology in the handling of the lipocyte aspirate in an endeavor to improve the survival rate and therefore the take of the grafted lipocytes. The method consists of syringe aspiration of the lipocytes from the donor site, isolation of intact lipocytes by gentle centrifugation, suspension of the aspirate in an enriched cell culture medium, and injection of the cell suspension into preformed subdermal tunnels. A number of media were tested and shown to prolong the survival of lipocytes ex vivo using fluorescent acridine orange stain. Implementing the integrated cell culture techniques increased the lipocytes' viability, as indicated in clinical evaluation in which the amount of graft take ranged between 50 and 90 percent. The results of 15 patients with varied types of cases who were operated on using this new methodology show that the tissue defect was filled and remained so in postoperative follow-ups of 6 to 24 months. A three-dimensional CAT scan-aided evaluation method was developed and used in one of four case histories presented herein.

Adipose Tissue↗
PubMed1999

Novel hyperactive mitogen to endothelial cells: human decidual NKG5.

PROBLEM: The purpose of this study was the isolation and characterization of decidual extract proteins that exhibit mitogenicity on endothelial cells. METHOD OF STUDY: A partially purified extract (F1 fraction) was obtained from human decidua of the first trimester of pregnancy. F1 was separated by heparin-sepharose column and showed significant mitogenicity on bovine brain capillary endothelial (BBCE) cells in vitro, using methylene blue stain nuclear assay. Sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed four fractions with MW less than 30 KDa. RESULTS: Mitogenic fraction, E11-12 (eluted at 1.5 M NaCl) was identified as basic fibroblast growth factor (bFGF). Vascular endothelial growth factor (VEGF) and acidic (a)FGF were not identified as one of the mitogenic fractions. However, fractions E5-6, and E7-8 induced statistically significant mitogenicity at concentration of 1 pg/mL, which is 10(3) times lower than bFGF. Sequencing of E5-6 fraction identified NKG5 protein, a putative secreted polypeptide derived from human natural killer (NK) cells and activated T cells of unknown function. CONCLUSION: This work demonstrates that NKG5 stimulates mitogenicity of endothelial cells and may be involved in angiogenesis.

Angiogenesis Inducing Agents↗
PubMed1999

Direct observation of microtubule-f-actin interaction in cell free lysates.

Coordinated interplay of the microtubule and actin cytoskeletons has long been known to be crucial for many cellular processes including cell migration and cytokinesis. However, interactions between these two systems have been difficult to document by conventional approaches, for a variety of technical reasons. Here the distribution of f-actin and microtubules were analyzed in the absence of fixation using Xenopus egg extracts as an in vitro source of microtubules and f-actin, demembranated Xenopus sperm to nucleate microtubule asters, fluorescent phalloidin as a probe for f-actin, and fluorescent tubulin as a probe for microtubules. F-actin consistently colocalized in a lengthwise manner with microtubules of asters subjected to extensive washing in flow chambers. F-actin-microtubule association was heterogenous within a given aster, such that f-actin is most abundant toward the distal (plus) ends of microtubules, and microtubules heavily labeled with f-actin are found in close proximity to microtubules devoid of f-actin. However, this distribution changed over time, in that 5 minute asters had more f-actin in their interiors than did 15 minute asters. Microtubule association with f-actin was correlated with microtubule bending and kinking, while elimination of f-actin resulted in straighter microtubules, indicating that the in vitro interaction between f-actin and microtubules is functionally significant. F-actin was also found to associate in a lengthwise fashion with microtubules in asters centrifuged through 30% sucrose, and microtubules alone (i.e. microtubules not seeded from demembranated sperm) centrifuged through sucrose, indicating that the association cannot be explained by flow-induced trapping and alignment of f-actin by aster microtubules. Further, cosedimentation analysis revealed that microtubule-f-actin association could be reconstituted from microtubules assembled from purified brain tubulin and f-actin assembled from purified muscle actin in the presence, but not the absence, of Xenopus oocyte microtubule binding proteins. The results provide direct evidence for an association between microtubules and f-actin in vitro, indicate that this interaction is mediated by one or more microtubule binding proteins, and suggest that this interaction may be responsible for the mutual regulation of the microtubule and actomyosin cytoskeletons observed in vivo.

Actins↗
PubMed1999

Improved vitality of experimental random dorsal skin flaps in rats treated with enriched cell culture medium.

A defined, serum-free cell culture medium supplemented with nonsteroidal anabolic hormones, insulin, thyroxin, and growth hormone was found to accelerate wound healing by stimulating vascularized granulation tissue formation, epithelialization, and angiogenesis. The aim of this work was to study the effect of cell culture medium on the survival rate of cephalically based random dorsal skin flaps in an animal model. A total of 77 Sprague-Dawley rats were randomized into five treatment groups: pharmacologic delay with cell culture medium, flap enhancement with cell culture medium, surgical delay, biological delay with saline, and control. Statistically significant differences in distal flap necrosis were found among all groups (p<0.003). The rats treated with cell culture medium before flap elevation showed a significant increase in flap viability: a survival rate of 83 percent, compared with the control group, which demonstrated a survival rate of only 58 percent (p<0.0001). The surgical delay and the groups treated with cell culture medium yielded similar results with no significant difference between them. This study indicates that preoperative injection of cell culture medium may play a role in decreasing skin flap necrosis.

Animals↗
PubMed1998

p53-independent role of MDM2 in TGF-beta1 resistance.

Transforming growth factor-beta (TGF-beta) inhibits cell proliferation, and acquisition of TGF-beta resistance has been linked to tumorigenesis. A genetic screen was performed to identify complementary DNAs that abrogated TGF-beta sensitivity in mink lung epithelial cells. Ectopic expression of murine double minute 2 rescued TGF-beta-induced growth arrest in a p53-independent manner by interference with retinoblastoma susceptibility gene product (Rb)/E2F function. In human breast tumor cells, increased MDM2 expression levels correlated with TGF-beta resistance. Thus, MDM2 may confer TGF-beta resistance in a subset of tumors and may promote tumorigenesis by interference with two independent tumor suppressors, p53 and Rb.

Animals↗
PubMed1998

Myc activates telomerase.

Telomere maintenance has been proposed as an essential prerequisite to human tumor development. The telomerase enzyme is itself a marker for tumor cells, but the genetic alterations that activate the enzyme during neoplastic transformation have remained a mystery. Here, we show that Myc induces telomerase in both normal human mammary epithelial cells (HMECs) and normal human diploid fibroblasts. Myc increases expression of hEST2 (hTRT/TP2), the limiting subunit of telomerase, and both Myc and hEST2 can extend the life span of HMECs. The ability of Myc to activate telomerase may contribute to its ability to promote tumor formation.

Cell Division↗
PubMed1998

Enhancing the survival of aspirated human fat injected into nude mice.

Injection of aspirated fat is now the most commonly used technique for the filling of depressed areas. Partial absorption of the injected fat is the main limitation of this procedure. Cariel T.M. is an enriched serum-free cell culture medium, its ability to enhance the survival of human aspirated fat grafts was investigated in the nude mouse model. A volume of 0.75-cc Cariel preprocessed fat was injected under the scalp skin of 16 nude mice in the experimental group, and the same volume of saline preprocessed fat was injected to 15 control group of mice. Significant maintenance of the weight, 46 percent in the experimental group compared with 29 percent in the control group (p < 0.008), and the volume, 44 percent in the experimental group compared with 31 percent in the control group (p < 0.026), was observed, after 15 weeks, in this newly used model. It seems that addition of the nutrients enriched with anabolic hormones enabled the survival and take of more adipose cells in the graft.

Adipose Tissue↗
PubMed1997

Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.

Oncogenic ras can transform most immortal rodent cells to a tumorigenic state. However, transformation of primary cells by ras requires either a cooperating oncogene or the inactivation of tumor suppressors such as p53 or p16. Here we show that expression of oncogenic ras in primary human or rodent cells results in a permanent G1 arrest. The arrest induced by ras is accompanied by accumulation of p53 and p16, and is phenotypically indistinguishable from cellular senescence. Inactivation of either p53 or p16 prevents ras-induced arrest in rodent cells, and E1A achieves a similar effect in human cells. These observations suggest that the onset of cellular senescence does not simply reflect the accumulation of cell divisions, but can be prematurely activated in response to an oncogenic stimulus. Negation of ras-induced senescence may be relevant during multistep tumorigenesis.

Animals↗
PubMed1997

Prospective evaluation of three smoking interventions in 205 recovering alcoholics: one-year results of Project SCRAP-Tobacco.

A total of 205 (113 male, 92 female) nonhospitalized recovering alcoholics with > 3 months of continuous abstinence from alcohol and drugs and relatively heavy tobacco dependence (Fagerstrom Tolerance Questionnaire score = 7.7; mean number of cigarettes per day, 26.8; mean number of years smoked, 24.4) were randomized to standard treatment (ST) American Lung Association quit program plus nicotine anonymous meetings (n = 70), behavioral counseling plus physical exercise (BEX; n = 72), or behavioral counseling plus nicotine gum (BNIC; n = 63). A 3 x 4 repeated measures design was used to evaluate the effectiveness of the interventions on smoking outcome at baseline, posttreatment, and 6- and 12-month follow-ups. Self-reported smoking status was verified with biochemical and informant report. Verified self-report indicated that significantly more smokers in BEX quit by posttreatment (60%) than in either BNIC (52%) or ST (31%), chi 2 (2, N = 205) = 17.85, p < .01, but not at the 6-month (29%, 27%, and 21%, respectively) or 12-month (27%, 27%, and 26%, respectively) follow-up. Only 4% (7 of 188) relapsed to alcohol or drugs. Alcohol relapse did not differ by treatment group or smoking status. Length of alcohol abstinence was not associated with smoking cessation outcome.

Adult↗
PubMed1997

The Cdc2 protein kinase controls Cdc10/Sct1 complex formation.

In the fission yeast Schizosaccharomyces pombe, the execution of Start requires the activity of the Cdc2 protein kinase and the Cdc10/Sct1 transcription complex. The loss of any of these genes leads to G1 arrest and activation of the mating pathway under appropriate conditions. We have undertaken a genetic and biochemical analysis of these genes and their protein products to elucidate the molecular mechanism that governs the regulation of Start. We demonstrate that serine-196 of Cdc10 is phosphorylated in vivo and provide evidence that suggests that phosphorylation of this residue is required for Cdc10 function. Substitution of serine-196 of Cdc10 with alanine (Cdc10 S196A) leads to inactivation of Cdc10. We show that Cdc10 S196A is incapable of associating with Sct1 to form a heteromeric complex, whereas substitution of this serine with aspartic acid (S196D) restores DNA-binding activity by allowing Cdc10 to associate with Sct1. Furthermore, we demonstrate that Cdc2 activity is required for the formation of the heteromeric Sct1/Cdc10 transcription complex and that the Cdc10 S196D mutation alleviates this requirement. We thus provide biochemical evidence to demonstrate one mechanism by which the Cdc2 protein kinase may regulate Start in the fission yeast cell cycle.

CDC2 Protein Kinase↗
PubMed1997

Ran1 functions to control the Cdc10/Sct1 complex through Puc1.

We have undertaken a biochemical analysis of the regulation of the G1/S-phase transition and commitment to the cell cycle in the fission yeast Schizosaccharomyces pombe. The execution of Start requires the activity of the Cdc2 protein kinase and the Sct1/Cdc10 transcription complex. Progression through G1 also requires the Ran1 protein kinase whose inactivation leads to activation of the meiotic pathway under conditions normally inhibitory to this process. We have found that in addition to Cdc2, Sct1/Cdc10 complex formation requires Ran1. We demonstrate that the Puc1 cyclin associates with Ran1 and Cdc10 in vivo and that the Ran1 protein kinase functions to control the association between Puc1 and Cdc10. In addition, we present evidence that the phosphorylation state of Cdc10 is altered upon inactivation of Ran1. These results provide biochemical evidence that demonstrate one mechanism by which the Ran1 protein kinase serves to control cell fate through Cdc10 and Puc1.

Cell Cycle↗