Cell cycle arrest and release in starfish oocytes and eggs

A neural net-based, semiautomated, interactive computerized cell analysis system (The PAPNET system, Neuromedical Systems, Suffern, NY) was used to examine cells from 138 esophageal smears obtained by lavage, brushings, or balloon from as many patients. From each smear, trained human observers examined 128 cell images selected by the machine. Abnormal cells were identified in all 35 patients with cancer, whether esophageal, gastric, oral, or metastatic. Further, in 11 smears, the displayed images allowed the recognition of effects of radiotherapy and, in 14 smears, the diagnosis of a specific tumor type, such as squamous cell carcinoma (8 patients) or adenocarcinoma (6 patients). In 3 additional cases, the diagnosis of "carcinoma, not further specified," was established. One case of esophageal carcinoma in situ, not previously recognized on a smear or in the biopsy specimen, and one case of gastric adenocarcinoma, not recognized in the smear, were identified in PAPNET-generated images. The possible application of the apparatus to the triage of smears and population screening for esophageal and gastric carcinoma precursors is discussed.     

Morphological differences in nuclear materials released from hamster sperm heads at an early stage of incorporation into immature oocytes, mature oocytes, or fertilized eggs

To elucidate the effects of ooplasmic factors on the early morphological changes in hamster sperm heads within the ooplasm, immature ovarian oocytes at the germinal vesicle stage (GV oocytes), ovulated fully mature oocytes, and fertilized eggs at anaphase II or the pronuclear stage (PN eggs) were examined in detail 15-30 min after insemination or reinsemination. Thin-sectioning studies demonstrated distinct materials released from the sperm nucleus over the entire postacrosomal nuclear surface immediately after disappearance of the sperm nuclear envelope. The release occurred in all of the oocytes and eggs prior to or even in the absence of subsequent chromatin decondensation. Depending upon the stage of the penetrated oocyte or egg, however, the materials varied in morphology: several hemispherical projections of amorphous material within mature oocytes; a number of electron-dense globules within GV oocytes and PN eggs; and both forms within eggs at anaphase II-telophase II. These observations and the fact that only the release of the amorphous material was accompanied by sperm chromatin decondensation indicate that this release was the initial process of chromatin decondensation, whereas the release of the globules resulted from a deficiency or lack of ooplasmic factors affecting the sperm nucleus. Restriction of the release in both forms of material to the late meiotic phase suggests changes in the factors associated with progression of meiosis. To approach an understanding of the mechanism of successful decondensation of sperm chromatin, the ooplasmic factors considered responsible for the stage-dependent release of nuclear materials are discussed.     

Characterization of the interactions between human cdc25C, cdks, cyclins and cdk-cyclin complexes

We have overexpressed and purified human dual-specificity phosphatase cdc25C from a prokaryotic expression system at high levels and in a soluble, active form, and have studied and quantified its potential to interact with cdks, cyclins and preformed cdk-cyclin complexes by fluorescence spectroscopy and size-exclusion chromatography. Our data indicate that human cdc25C forms stable complexes, through hydrophobic contacts, with cdk and cyclin monomers, as well as with preformed cdk-cyclin complexes. In vitro, cdc25C interacts with cyclin monomers with high affinity, with tenfold less affinity with cdks, and with intermediate affinity with cdk-cyclin complexes. Moreover, changes observed in the intrinsic fluorescence of cdks, cyclins and cdk-cyclin complexes upon interaction with cdc25C are indicative of concomitant conformational changes within cdks and cyclins. From our results, we propose that in vitro, in the presence of monomeric cdks and cyclins, cdc25C forms stable ternary complexes, first through a high affinity interaction with a cyclin, which may then help target cdc25C towards a cdk. We discuss the biological relevance of our results and propose that a similar, two-step mechanism of interaction between cdc25C and cdk-cyclin complexes may occur in vivo.     

Mitotic cyclins and cyclin-dependent kinases in melanocytic lesions

Recent evidence has implicated cyclins and cyclin-dependent kinases in the evolution and progression of various malignancies. We studied the immunohistochemical expression of cyclin A, cyclin B, and cyclin-dependent kinase p34cdc2 in a broad spectrum of benign and malignant melanocytic lesions. Formalin-embedded, parrafin-fixed tissue sections from 66 malignant melanomas (MM) and 60 benign nevi were examined for the expression of these cell-cycle proteins. The results were compared with the standard proliferative marker Ki-67 and mitotic index. MM showed significantly higher immunoreactivity for cyclin A, cyclin B, p34cdc2, and Ki-67 compared with benign nevi. Cyclin A, p34cdc2, and Ki-67 displayed strong co-expression in MM. Overexpression of cyclin A and p34cdc2 correlated with histological type, mitotic activity, Ki-67 index, tumor thickness, Clark's level, and clinical outcome in MM. In invasive MM, increased immunostaining of cyclin A and Ki-67 were associated with decreased patient survival. These findings indicate potential roles of mitotic cyclins and cyclin-dependent kinases in the pathogenesis and progression of malignant melanoma.     

Clinical implications of cyclins, cyclin-dependent kinases, RB and E2F1 in squamous-cell lung carcinoma

OBJECTIVE: To develop, implement, and assess the outcomes of a system for providing pharmaceutical care to medical progressive care patients. METHODS: A system for providing pharmaceutical care was developed and implemented for an 8-week period beginning in June 1995. Both patient care outcomes and drug therapy cost change from the intervention period were compared with those of an 8-week baseline period. Variables compared included unit length of stay, hospital length of stay, transfers to the intensive care unit, readmissions, and adverse drug reactions requiring treatment. Differences between periods for these variables were assessed by using chi 2 tests and t-tests with alpha set at p less than 0.05. The clinical significance of the interventions were assessed independently by four physicians: two intensivists and two internists. The total drug therapy cost change from the intervention period was calculated as follows: total cost avoidance from individual recommendations subtracted from the total cost incurred from individual recommendations. RESULTS: The pharmacist evaluated 152 patients during the intervention period. A total of 235 pharmacotherapy recommendations were made on 103 patients, of whom 86.4% were accepted. Significantly fewer adverse drug reactions (ADRs) received treatment during the intervention period (p = 0.027). The mean unit length of stay was lower during the intervention period (4.8 +/- 3.7 d) than during the baseline period (6.0 +/- 5.6 d); however, this difference was not significant (p = 0.053). Individual physician assessment of the pharmacists' recommendations revealed that 75.8% were considered somewhat significant, significant, or very significant. The total drug therapy cost change from the intervention period was -$6534.53. The projected annual drug therapy cost reduction from this study is $42,474.45. CONCLUSIONS: The provision of pharmaceutical care to medical progressive care patients was associated with a substantial decrease in drug therapy cost and a decrease in the number of ADRs that required treatment.     

The major soluble tubulins are found in mega dalton (MDa) fractions in fully-grown oocytes and eggs but not in brain of the frog, Rana pipiens

During apoptosis, one of the first membrane changes that can be detected is exposure of phosphatidylserine residues at the outer plasma membrane leaflet, while early apoptosis is also accompanied by changes in the cytoskeletal organization. In this study we investigated the relationship between these two phenomena during olomoucine- and roscovitin-induced apoptosis in human lung cancer and neuroblastoma cell lines. Loss of membrane asymmetry was detected by biotin-labeled or FITC-labeled annexin V binding to negatively charged phosphatidylserine, while cytoskeletal components were visualized by immunocytochemistry. The apoptotic, annexin V-positive, cells were analyzed by flow cytometry, confocal scanning laser microscopy, and Western blotting. We report that cytokeratin and vimentin aggregation in early apoptosis occurs simultaneously with phosphatidylserine exposure and chromatin condensation. In contrast to these intermediate filament proteins, which were disassembled and proteolytically cleaved in early apoptosis, microfilaments and microtubuli were not proteolytically degraded but were found to be present as aggregated filaments in the apoptotic bodies. We also show that loss of membrane asymmetry and cytokeratin aggregation are independent processes, since N-ethylmaleimide-induced phosphatidylserine exposure does not cause cytokeratin disassembly. Vice versa, phorbol 12-myristate 13-acetate-induced cytokeratin filament aggregation does not result in phosphatidylserine exposure.     

Expression of cyclins D1 and E in human colon adenocarcinomas

Cyclins D1 and E play critical roles in the progression of cells through the G1 phase of the cell cycle. Amplification and/or overexpression of the cyclin D1 gene and aberrant expression of cyclin E have been described in several forms of human cancer. In the present study, we examined the expression of these two genes by Western, Northern and Southern blot analyses in a series of primary human colon carcinomas of various stages and degrees of differentiation and in paired adjacent normal mucosa samples, and also in a series of human colon carcinoma cell lines. About 50% of the colon carcinomas displayed a two to five fold increase in the expression of cyclin D1 mRNA and protein, when compared with the paired normal mucosa samples. Six out of eight carcinomas examined showed a four to nine fold increase in cyclin E mRNA and about 50% of the carcinomas displayed a two to three fold increase in cyclin E protein. Low molecular weight cyclin E-related proteins were observed in four out of ten carcinomas. These changes in cyclins D1 and E occurred in both early and late stage tumors. Three of the six cell lines examined displayed a high expression of cyclin D1 mRNA and protein. A very high level of cyclin E mRNA expression was seen in HCT116 cells and this was associated with the presence of low molecular weight cyclin E-related proteins. None of the primary colon carcinomas nor the six cell lines examined displayed amplification of either the cyclin D1 or cyclin E genes. Thus, an aberrant expression of both cyclins D1 and E occurs in a significant fraction of human colon carcinomas.     

Trichlorfon exposure, spindle aberrations and nondisjunction in mammalian oocytes

Consumption of trichlorfon-poisoned fish by women in a small Hungarian village has been associated with trisomy resulting from an error of meiosis II in oogenesis. We therefore examined mouse oocytes exposed for 3 h during fertilization to 50 microg/ml trichlorfon. Spindle morphology was not visibly altered by the pesticide. Chromosomes segregated normally at anaphase II with no induction of aneuploidy. However, formation of a spindle was disturbed in many oocytes resuming meiosis I in the presence of trichlorfon. In spite of the spindle aberrations and the failure of bivalents to align properly at the equator, oocytes did not become meiotically arrested but progressed to metaphase II. At this stage, spindles were highly abnormal, and chromosomes were often totally unaligned, unattached or dispersed on the elongated and disorganized spindle. By causing spindle aberrations and influencing chromosome congression, trichlorfon appears, therefore, to predispose mammalian oocytes to random chromosome segregation, especially when they undergo a first division and develop to metaphase II during exposure. This is the first case in which environmentally induced human trisomy can be correlated with spindle aberrations induced by chemical exposure. Our observations suggest that oocytes may not possess a checkpoint sensing displacement of chromosomes from the equator at meiosis I and may therefore be prone to nondisjunction.     

Milk, eggs and peanuts: food allergies in children

True food allergies are much less prevalent than is generally believed. They are more common in infants and children under age three than in older children and adults. Infant colic generally is not caused by a food allergy. In infants, urticaria, eczema or gastrointestinal bleeding may be due to foods such as milk and eggs, but clinical tolerance usually develops within a few years. Peanuts, tree nuts, seafood and seeds, as well as milk and eggs, can cause anaphylaxis in highly allergic children, and reexposure to such foods presents the risk of life-threatening reactions. Immediate-reacting allergy skin tests and in vitro IgE antibody tests can be used to screen for food allergy. Only food challenge, however, can confirm a reaction to a particular food. Management of food allergy, once the initial symptoms are confirmed, consists of avoidance of specific foods, sometimes for a lifetime. All children at risk for food anaphylaxis should be identified, and their parents or caretakers should be prepared to administer epinephrine before taking the child to the emergency room.     

Expression of G1 cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors in androgen-induced prostate proliferation in castrated rats

Androgen induces prostate cell proliferation in the castrated rat. We hypothesized that G1 cyclins, cyclin-dependent kinases (cdk), and cdk inhibitors mediate this cellular response to mitogenic signals. In this study, induction of cyclins D1, D2, D3, E, and cdks 2, 4, and 6 expression was observed at various time points during testosterone replacement in the ventral prostate of castrated rats. The induction followed prostate epithelium proliferation, which peaked at 48 h and decreased at 120 h during the treatment. The study of cyclin/cdk complex formation revealed that more cyclin D1/cdk4 and cyclin D1/cdk6 complexes were formed at 48 h than at 120 h of treatment, but cyclin D1/cdk2 complexes remained the same. Furthermore, both hyperphosphorylated and hypophosphorylated forms of Rb were detected at 48 h, but only the hypophosphorylated form was detected at 120 h of treatment. p21Cip1, which was very abundant in the ventral prostate of castrated and intact rats, was not detected when the prostate started proliferation and increased gradually as proliferation decreased during the androgen treatment. Meanwhile, p27Kip1 dramatically increased after androgen treatment, and the induction levels were less at the peak of prostate proliferation and higher when proliferation was low. The results presented here suggest that expression of G1 cyclins and their related kinases and kinase inhibitors are well regulated after androgen replacement in the ventral prostate of castrated rats. The cooperation between these cell cycle regulators leads to a well-controlled prostate regeneration.     

On the existence of polyadenylated histone mRNA in Xenopus laevis oocytes

In a variety of systems, histone mRNA has been shown to lack poly(A) (Adesnik and Darnell, 1972; Grunstein et al., 1973). We have found, however, that in Xenopus laevis oocytes, poly (A)-containing mRNA codes for histones, in a wheat germ cell-free system, based on the following criteria: first, co-migration with authentic X. laevis oocyte histones on polyacrylamide gels; second, no detectable incorporation of tryptophan; third, differential incorporation of lysine and methionine into histone fraction H2A; fourth, resistance of histone fraction H2A to cleavage with cyanogen bromide; and fifth, correspondence of tryptic peptide maps of partially purified cell-free products with authentic X. laevis oocyte histone. RNA which directs the synthesis of histones in the cell-free system is retained on oligo(dT)-cellulose, even after denaturation in 80% DMSO at 70 degrees C, thereby demonstrating the covalent attachment of polyadenylic acid sequences to the mRNA. Poly (A)- RNA (7S-14S fraction) was also found to code for histones using the same criteria. We discuss the significance of the finding that X. laevis oocytes contain two classes of histone mRNA as well as the potential developmental implications of this observation.     

The geriatric gravida: multifetal pregnancy reduction, donor eggs, and aggressive infertility treatments

OBJECTIVE: Recent technologic advances and societal acceptance have dramatically increased the use of donor eggs for infertile couples who require assisted reproductive technologies. Now many "older" couples can access assisted reproductive technologies to achieve pregnancies. We sought to evaluate the changing pattern of patients referred for multifetal pregnancy reduction as a result of donor eggs and age factors in aggressive infertility treatment. STUDY DESIGN: Patients undergoing multifetal pregnancy reduction from 1986 to 1996 were included and categorized by year groupings, age, and the use of donor eggs. RESULTS: A total of 523 patients were referred for and underwent multifetal pregnancy reduction. Before 1994, only 4 of 226 (1.8%) had received donor eggs, whereas in 1994 to 1996, 29 of 297 (9.8%) had received donor eggs (chi 2 = 12.6, p < 0.001). Eight of 9 patients aged > or = 45 years undergoing multifetal pregnancy reduction received donor eggs. There were no patients aged > or = 45 years before 1994 but 9 in 1994 to 1996. Four of 9 patients aged > or = 45 years with multifetal pregnancies chose reduction to singleton gestation. The proportions of patients aged > or = 40 years have increased from 0% to 11% in the last 8 years. CONCLUSIONS: The availability of donor eggs has dramatically increased the use of assisted reproductive technologies and subsequent use of multifetal pregnancy reduction in older patients. Older patients are more inclined to want reduction to singleton gestation; they cite parental demands, financial issues, and their ability to parent in their 60s and 70s as reasons for reduction to singleton gestation.     

Polarographic determination of robenidine residues in chicken tissues, eggs, litter, soil, and plant tissue

Polarographic residue methods have been developed for determining robenidine (Robenz), 1,3-bis[p-chlorobenzylidene)amino]-guanidine monohydrochloride, in chicken tissues, eggs, litter, soil, and plants. The compound is extracted from chicken fat, skin, muscle, liver, and eggs with ethyl acetate; from blood with acetone; from plant tissue, litter, and kidney with acidic acetone; and from soil with basic methanol. After extraction by high-speed blending or overnight shaking, the extract is cleaned up by evaporation, solvent partition and/or elution from CG-50 ion exchange resin. Robenidine is quantitated by differential cathode ray polarography, using acidic aqueous methanol or acetic acid (1+1) supporting electrolyte. Recoveries ranged from 64 to 125% with an average overall recovery of 90%. The validated sensitivity is 0.1 ppm for chicken tissues, soil, and plants, 0.01 ppm for eggs, and 1 ppm for litter.     

n-3 fatty acid-enriched eggs, lipids, and Western diet: time for change

Laparoscopic cholecystectomy is an integral part of the general surgery operative inventory. The conventional approach may need to be modified to fit different clinical situations. We present the first reported case of a laparoscopic cholecystectomy combined with a ventral hernia repair.     

Differential expression of G1 cyclins and cyclin-dependent kinase inhibitors in normal and transformed melanocytes

The myocardial concentration of many cardioactive drugs has been identified as an important determinant of their short-term effects in previous studies. Although sotalol is frequently administered via short-term intravenous injection, no previous studies had sought to correlate its uptake by the heart with its various effects. We determined the time course of short-term uptake of d,l-sotalol by human myocardium in vivo and investigated the relation between myocardial content of sotalol and the short-term hemodynamic, electrocardiographic, and electrophysiologic effects of the drug. Sixteen patients received a 20-mg intravenous bolus of sotalol at the time of diagnostic cardiac catheterization. Myocardial content of d- and l-sotalol (by using a paired transcoronary sampling technique) and the short-term hemodynamic and electrophysiologic effects of the drug were determined < or = 20 min after injection. Myocardial accumulation of sotalol was not enantioselective, proceeded very rapidly (maximal at 0.74 +/- 0.10 min, representing 2.05 +/- 0.45% of the total injected dose), and was not significantly influenced by left ventricular systolic function or the extent of coronary artery disease. Approximately one third of peak myocardial content was still present 17.5 min after sotalol administration. Maximal effects of the drug (reduction in spontaneous heart rate, p < 0.005; reduction in maximal rate of LV pressure increase (LV+dP/dtmax, p < 0.005); and prolongation of PR intervals, p < 0.02) were delayed by approximately 10 min relative to maximal myocardial sotalol content. The significant prolongation of AH intervals (p < 0.01) and atrioventricular nodal effective refractory periods (p < 0.0002) that was observed was also maximal 10 min after administration of sotalol. Thus a consistent delay between myocardial sotalol content and the short-term effects of the drug was observed. In conclusion, the accumulation of both d- and l-sotalol by the human myocardium is more rapid than that of any other agent studied to date, with considerable hysteresis between myocardial drug uptake and subsequent cardiac effects.