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PubMed1993

Cytoplasmic oxytocin and vasopressin gene transcripts decline postpartum in the hypothalamus of the lactating rat.

Oxytocin (OT) and vasopressin (AVP) gene expression are enhanced in the rat hypothalamus in late gestation and during the second and third weeks of lactation. We report that during the first 3 postpartum days, OT and AVP cytoplasmic mRNAs in the supraoptic and paraventricular nuclei of lactating rats decreased dramatically, reaching less than one fifth of peak gestational levels by day 2 postpartum. Differences in the temporal pattern of OT and AVP expression were observed in the supraoptic and paraventricular nuclei from days 4-10 of lactation. We also compared OT and AVP cytoplasmic mRNAs isolated from the hypothalamus of day 3 lactating rats to cohorts that had litters removed at the time of parturition. Lactating rats had significantly lower OT and AVP cytoplasmic mRNA levels than their nonlactating cohorts. We further compared OT and AVP cytoplasmic mRNAs in the hypothalamus of day 12 lactating rats that had been ovariectomized or sham ovariectomized on day 3 of lactation. Ovariectomized day 12 lactating animals had significantly lower OT and AVP cytoplasmic mRNA levels than their intact cohorts. These data refute the hypothesis that lactation is characterized by persistently elevated hypothalamic cytoplasmic OT and AVP mRNAs produced as a result of continuous stimulation by suckling and suggest that ovarian steroids may exert a modulatory effect on hypothalamic OT and AVP expression during early lactation.

Animals↗
PubMed1998

Epitope tag mapping of the extracellular and cytoplasmic domains of the rat parathyroid hormone (PTH)/PTH-related peptide receptor.

The PTH/PTH-related peptide (PTHrP) receptor is predicted to span the plasma membrane seven times with an amino-terminal extracellular extension and a cytoplasmic carboxyl-terminal tail. To assess this prediction, we inserted 10- or 9-amino acid epitope tags from c-myc or hemophilus influenza hemaglutinin (HA), which are recognized by the monoclonal antibodies 9E10 and 12Ca5, respectively, in different extracellular and cytoplasmic regions of the receptor and examined the immunoreactivity of the epitopes in intact and permeabilized cells. The data show that the epitopes were well tolerated when introduced into the E2 region of the extracellular amino-terminus (E2-myc and E2-HA), in the first extracellular loop (EL1), in the second and third cytoplasmic loops (CL2c and CL3), or in the carboxyl-terminal tail (T-myc). Receptors tagged at these locations were well expressed, bound PTH with high affinity, and increased cAMP accumulation with a good efficiency. Receptors tagged in the second and third extracellular loops (EL2c and EL3c) or the first cytoplasmic loop (CL1c) bound the PTH radioligand with a low affinity, stimulated cAMP accumulation with a low efficiency, and had low expression levels. The receptors tagged on presumed extracellular regions, E2-myc, E2-HA, EL1, EL2c, and EL3c, were readily detected on the surface of intact cells with the monoclonal antibody against the epitope tag. In contrast, receptors tagged with the c-myc epitope in the cytoplasmic loops (CL1c, CL2c, and CL3) or in the carboxyl-terminal tail (T-myc) did not show any 9E10 binding in intact cells. These receptors, however, were well expressed on the cell surface, as detected by the binding of the monoclonal antibody, 12Ca5, to the HA tag that was introduced into the E2 region of these constructs. The c-myc epitopes, however, became accessible after permeabilization of the cell membrane. In conclusion, these data provide experimental evidence for the sidedness of the extracellular and cytoplasmic domains of the PTH/PTHrP receptor.

Amino Acid Sequence↗
PubMed1987

Selective increase in cytoplasmic calcium by anesthetic in lymphocytes from malignant hyperthermia-susceptible pigs.

Anesthetic-induced malignant hyperthermia in pigs and humans is characterized by muscle rigidity and rapid, often fatal, increases in body temperature. A defect in Ca2+ homeostasis has been suspected as underlying the disease, based on the preventive effect of dantrolene sodium, an agent thought to reduce Ca2+ levels in the cytoplasm. We describe here direct measurements of cytoplasmic ionized Ca2+ levels in lymphocytes from seven normal and 12 malignant hyperthermia-susceptible pigs, using the fluorescent indicator quin2. No differences in the concentration of cytoplasmic ionized Ca2+ were found in cells from malignant hyperthermia-susceptible pigs (160 +/- 10 nM) relative to the controls (150 +/- 10 nM). However, addition of halothane in vitro caused a significant increase (to 270 +/- 30 nM) in lymphocytes from malignant hyperthermia-susceptible pigs, but not from normal pigs (180 +/- 10 nM). The halothane-mediated increase in cytoplasmic ionized Ca2+ required extracellular Ca2+. It is suggested that general anesthetics such as halothane increase the permeability of the cell surface to Ca2+, and that this increase may, on its own or indirectly, increase the cytoplasmic level of ionized Ca2+ during a malignant hyperthermia crisis. The detection of a halothane-dependent increase in cytoplasmic ionized Ca2+ selectively in malignant hyperthermia-susceptible pigs could be the basis for a noninvasive test for malignant hyperthermia.

Aminoquinolines↗
PubMed1994

Functions of the cytoplasmic domain of the beta PS integrin subunit during Drosophila development.

Integrins constitute a family of membrane-spanning, heterodimeric proteins that mediate adhesive interactions between cells and surrounding extracellular matrices (or other cells) and participate in signal transduction. We are interested in assessing integrin functions in the context of developing Drosophila melanogaster. This report, using mutants of the beta PS subunit encoded by the myospheroid (mys) locus, analyzes the relationships between integrin protein structure and developmental functions in an intact organism. As a first step in this analysis, we demonstrated the ability of a fragment of wild-type mys genomic DNA, introduced into the germ line in a P-element vector P[mys+], to rescue phenotypes attributed to lack of (or defects in) the endogenous beta PS during several discrete morphogenetic events. We then produced in vitro a series of modifications of the wild-type P[mys+] transposon, which encode beta PS derivatives with mutations within the small and highly conserved cytoplasmic domain. In vivo analysis of these mutant transposons led to the following conclusions. (1) The cytoplasmic tail of beta PS is essential for all developmental functions of the protein that were assayed. (2) An intron at a conserved position in the DNA sequence encoding the cytoplasmic tail is thought to participate in important alternative splicing events in vertebrate beta integrin subunit genes, but is not required for the developmental functions of the mys gene assayed here. (3) Phosphorylation on two conserved tyrosines found in the C terminus of the beta PS cytoplasmic tail is not necessary for the tested developmental functions. (4) Four highly conserved amino acid residues found in the N-terminal portion of the cytoplasmic tail are important but not critical for the developmental functions of beta PS; furthermore, the efficiencies with which these mutant proteins function during different morphogenetic processes vary greatly, strongly suggesting that the cytoplasmic interactions involving PS integrins are developmentally modulated.

Amino Acid Sequence↗
PubMed1991

Cytoplasmic localization during storage and translation of the mRNAs of transition protein 1 and protamine 1, two translationally regulated transcripts of the mammalian testis.

During spermatogenesis in mammals, the transcripts of transition protein 1 (TP 1) and protamine 1 (Prm 1) are under translational regulation. Following their transcription in round spermatids, the mRNAs for TP 1 and Prm 1 are stored in the cytoplasm from 3-7 days before being translated towards the end of spermatogenesis. To test the hypothesis that the inactivation or activation of transcripts during spermiogenesis could be mediated by mRNA compartmentalization in the cytoplasm of spermatids, light and electron microscopy were used to localize, by in situ hybridization, the cellular and subcellular sites of stored and translated mRNAs for these two testis-specific transcripts. During early spermiogenesis (before step 7) nuclear transcripts of both TP 1 and Prm 1 were seen. After step 7 the TP 1 and Prm 1 mRNAs were only detected in the cytoplasm. Throughout spermiogenesis the cytoplasmic mRNAs were not localized to any membrane-bound organelles such as the endoplasmic reticulum or mitochondria or to non-membrane-bound structures such as the chromatoid body. These studies demonstrate that the translational arrest of the TP 1 and Prm 1 mRNAs is not primarily controlled by compartmentalized storage in the cytoplasm of spermatids. Moreover, when translation of these mRNAs occurs in elongated spermatids, the mRNAs are present throughout the cytoplasm.

Animals↗
PubMed1993

The MHC class II-associated invariant chain contains two endosomal targeting signals within its cytoplasmic tail.

The oligomeric complex formed by major histocompatibility complex (MHC) class II alpha and beta chains and invariant chain (Ii) assembles in the endoplasmic reticulum and is then transported via the Golgi complex to compartments of the endocytic pathway. When Ii alone is expressed in CV1 cells it is sorted to endosomes. The Ii cytoplasmic tail has been found to be essential for targeting to these compartments. In order to characterize further the signals responsible for endosomal targeting, we have deleted various segments of the cytoplasmic tail. The Ii mutants were transiently expressed and the cellular location of the proteins was analyzed biochemically and morphologically. The cytoplasmic tail of Ii was found to contain two endosomal targeting sequences within its cytoplasmic tail; one targeting sequence was present within amino acid residues 12-29 and deletion of this segment revealed the presence of a second endosomal targeting sequence, located within the first 11 amino acid residues. The presence of a leucine-isoleucine pair at positions 7 and 8 within this sequence was found to be essential for endosomal targeting. In addition, the presence of this L-I motif lead to accumulation of Ii molecules in large endosomal vacuoles containing lysosomal marker proteins. Both wild type Ii and Ii mutant molecules containing only one endosomal targeting sequence were rapidly internalized from the plasma membrane. When the Ii cytoplasmic tail was fused to the membrane-spanning region of neuraminidase, a resident plasma membrane protein, the resulting chimera (INA) was found in endocytic compartments containing lysosomal marker proteins. Thus the cytoplasmic tail of Ii is sufficient for targeting to the endocytic/lysosomal pathway.

Amino Acid Sequence↗
PubMed1996

A novel cytoplasmic dynein heavy chain: expression of DHC1b in mammalian ciliated epithelial cells.

Organisms that have cilia or flagella express over a dozen dynein heavy chain genes. Of these heavy chain genes, most appear to encode axonemal dyneins, one encodes conventional cytoplasmic dynein (MAP1C or DHC1a), and one, here referred to as DHC1b, encodes an unclassified heavy chain. Previous analysis of sea urchin DHC1b (Gibbons et al. (1994) Mol. Biol. Cell 5, 57-70) indicated that this isoform is either an axonemal dynein with an unusual protein sequence or a cytoplasmic dynein whose expression increases during ciliogenesis. In the present study, we examined the expression of DHC1b in rat tissues. The DHC1b gene is expressed in all tissues examined, including unciliated liver and heart cells. In contrast, rat axonemal dyneins are only expressed in tissues that produce cilia or flagella. In cultured rat tracheal epithelial (RTE) cells, DHC1b is expressed in undifferentiated cells and increases in expression during ciliogenesis. In contrast, the expression of conventional cytoplasmic dynein, DHC1a, does not change during RTE differentiation and axonemal dynein is not expressed until after differentiation commences. In order to examine the expression of DHC1b protein, we produced an isoform-specific antibody to a synthetic peptide derived from the rat DHC1b sequence. The antibody demonstrated that DHC1b is a relatively minor component of partially purified cytoplasmic dynein. Indirect immunofluorescence microscopy revealed that DHC1b is not detected in cilia and remains in the cytoplasm of ciliated RTE cells, often accumulating at the apical ends of the cells. These results suggest that DHC1b is a cytoplasmic dynein that may participate in intracellular trafficking in polarized cells.

Amino Acid Sequence↗
PubMed1999

Substitution of flight muscle-specific actin by human (beta)-cytoplasmic actin in the indirect flight muscle of Drosophila.

The human (beta)-cytoplasmic actin differs by only 15 amino acids from Act88F actin which is the only actin expressed in the indirect flight muscle (IFM) of Drosophila melanogaster. To test the structural and functional significance of this difference, we ectopically expressed (beta)-cytoplasmic actin in the IFM of Drosophila that lack endogenous Act88F. When expression of the heterologous actin was regulated by approximately 1.5 kb of the 5' promoter region of the Act88F gene, little (beta)-cytoplasmic actin accumulated in the IFM of the flightless transformants. Including Act88F-specific 5' and 3' untranslated regions (UTRs) yielded transformants that expressed wild-type amounts of (beta)-cytoplasmic actin. Despite the assembly of (beta)-cytoplasmic actin containing thin filaments to which endogenous myosin crossbridges attached, sarcomere organization was deficient, leaving the transformants flightless. Rather than affecting primarily actin-myosin interactions, our findings suggest that the (beta)-cytoplasmic actin isoform is not competent to interact with other actin-binding proteins in the IFM that are involved in the organization of functional myofibrils.

Actins↗
PubMed1990

Cytoplasmic pool of histone H1 in mammalian cells.

Two types of cell populations, nondividing mouse liver cells and exponentially growing Friend erythroleukemia cells, were studied for the presence of a histone H1 pool in the cytoplasm. Purified cytoplasmic fractions were extracted with 5% perchloric acid and the resulting protein preparation was characterized by two types of electrophoresis, gel filtration, peptide mapping, ELISA and immunoblotting. The occurrence of significant quantities of H1 in isolated cytoplasmic fractions was confirmed by indirect immunofluorescence on whole cells. The existence of a cytoplasmic pool of H1 contrasts with the lack of detectable amounts of core histones in the cytoplasm. This indicates that the observed H1 pool is not just a reflection of its cytoplasmic synthesis but probably has some functional significance.

Animals↗
PubMed2004

Cytoplasmic immunoreactivity for thyroid transcription factor-1 in hepatocellular carcinoma: a comparative immunohistochemical analysis of four commercial antibodies using a tissue array technique.

To evaluate the consistency of cytoplasmic immunoreactivity in hepatocellular carcinoma (HCC), we performed immunohistochemical stains for 4 commercial anti-thyroid transcription factor (TTF)-1 antibodies (DAKO, Zymed, Novocastra, Santa Cruz Biotechnology [see text]) on 77 HCCs and 334 nonhepatic epithelial tumors. The HCC cases were submitted for hepatocvte antigen immunohistochemical stain. Heat-induced epitope retrieval (HIER) methods were used: with DAKO Target Retrieval Solution, the positive rates of cytoplasmic TTF-1 in HCC for DAKO, Zymed, Santa Cruz, and Novocastra antibodies were 58% (45), 14% (11), 6% (5), and 0% (0), respectively; with EDTA buffer, the positive rates increased to 70% (54), 40% (31), 69% (53), and 0% (0), respectively. Immunoreactivity for the DAKO anti-TTF-1 antibody generally correlated with that for hepatocyte antigen. Among nonhepatic tumors, 2 of 6 ovarian mucinous carcinomas and 2 of 11 pancreatic adenocarcinomas showed cytoplasmic reactivity for the DAKO antibody; 28 cases showed nonspecific cytoplasmic staining for the Santa Cruz antibody with EDTA HIER. Zymed and Novocastra antibodies did not produce cytoplasmic staining in nonhepatic tumors. Owing to the staining variation, we do not consider TTF-1 a reliable marker to distinguish HCC. In general, the Novocastra antibody with EDTA HIER is superior for its consistent nuclear positivity and absence of erratic cytoplasmic staining.

Biomarkers, Tumor↗
PubMed2006

Role of the cytoplasmic domain in Anabaena sensory rhodopsin photocycling: vectoriality of Schiff base deprotonation.

Light-induced electric signals in intact E. coli cells generated by heterologously expressed full-length and C-terminally truncated versions of Anabaena sensory rhodopsin (ASR) demonstrate that the charge movements within the membrane-embedded part of the molecule are stringently controlled by the cytoplasmic domain. In particular, truncation inverts the direction of proton movement during Schiff base deprotonation from outward to cytoplasmic. Truncation also alters faster charge movements that occur before Schiff base deprotonation. Asp(217) as previously shown by FTIR serves as a proton acceptor in the truncated ASR but not in the full-length version, and its mutation to Asn restores the natural outward direction of proton movement. Introduction of a potential negative charge (Ser(86) to Asp) on the cytoplasmic side favors a cytoplasmic direction of proton release from the Schiff base. In contrast, mutation of the counterion Asp(75) to Glu reverses the photocurrent to the outward direction in the truncated pigment, and in both truncated and full-length versions accelerates Schiff base deprotonation more than 10-fold. The communication between the cytoplasmic domain and the membrane-embedded photoactive site of ASR demonstrated here is likely to derive from the receptor's use of a cytoplasmic protein for signal transduction, as has been suggested previously from binding studies.

Anabaena↗
PubMed2001

Translocation of MRE11 from the nucleus to the cytoplasm as a mechanism of radiosensitization by heat.

Zhu, W-G., Seno, J. D., Beck, B. D. and Dynlacht, J. R. Translocation of MRE11 from the Nucleus to the Cytoplasm as a Mechanism of Radiosensitization by Heat. Radiat. Res. 156, 95-102 (2001).Hyperthermia sensitizes mammalian cells to ionizing radiation, presumably by inhibiting the repair of radiation-induced double-strand breaks (DSBs). However, the mechanism by which heat inhibits DSB repair is unclear. The nuclear protein MRE11 is a component of a multi-protein complex involved in nonhomologous end joining (NHEJ) of radiation-induced DSBs. Using one-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blotting, we found that MRE11 is translocated from the nucleus to the cytoplasm when human U-1 melanoma or HeLa cells are heated for 15 min at 45.5 degrees C or when cells are heated after irradiation with 12 Gy of X rays. No such translocation is observed in unheated irradiated cells. The kinetics of migration of MRE11 to the cytoplasm was dependent upon whether the heated cells were irradiated, while the magnitude of redistribution of MRE11 was dependent upon post-treatment incubation time at 37 degrees C. Cytoplasmic MRE11 content reached a maximum 2-4 h after heating; the increase was not due to new protein synthesis. Partial recovery of nuclear MRE11 content was observed when heated cells or heated irradiated cells were incubated for up to 7 h at 37 degrees C after treatment. Western blotting results showing translocation of MRE11 from the nucleus to the cytoplasm after heating and irradiation were confirmed using confocal microscopy and immunofluorescence staining of fixed cells. Our data suggest that radiosensitization by heat may be caused, at least in part, by translocation of the DNA repair protein MRE11 from the nucleus to the cytoplasm.

Active Transport, Cell Nucleus↗
PubMed2003

Speciation of small molecules and inorganic ions in salmon egg cell cytoplasm by surfactant-mediated HPLC/ICP-MS.

The speciation of diverse elements in salmon egg cell cytoplasm was performed by a surfactant-mediated HPLC/ICP-MS hyphenated system. In the present experiment, an ODS column coated with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), which is a zwitterionic bile acid derivative, was employed as a surfactant-mediated separation column, and ICP-MS was used as an element-selective detector. The present surfactant-mediated HPLC allowed us to separate large and small molecules within 10 min; large molecules, such as proteins, were eluted within 2.5 min, while small molecules were eluted after 2.5 min, but within 10 min. In the present experiment, Fe, Cu, and Zn in egg cell cytoplasm were observed mostly in species with large molecular weights, indicating that these elements are contained as metalloproteins or metalloenzymes in egg cell cytoplasm. On the contrary, it was found that P, S, Mo, and halogens in egg cell cytoplasm were contained as small molecules or inorganic ions. The major species of P in egg cell cytoplasm was identified as the phosphate ion (PO4(3-)). Molybdenum, Cl, and Br in egg cell cytoplasm were molybdate (MoO4(2-), chloride (Cl-), and bromide (Br-) ions, respectively.

Animals↗
PubMed1984

Development of methods to measure activity of polysomes and cytoplasmic enzymes from bovine skeletal muscle in in vitro protein-synthesis assays.

A cell-free, protein-synthesis system containing components from bovine skeletal muscle was developed as prerequisite to attempts to learn whether polysomes or cytoplasmic enzymes limit rate of muscle protein synthesis in the current population of domestic animals. Amino acid incorporation into trichloroacetic acid-precipitable protein was optimal at pH 7.5 and, if Tris buffer was used, at K+ and Mg2+ concentrations of 40 mM and 4 mM, respectively. Optimal concentrations of compounds that provide energy for amino acid incorporation were 1 mM ATP, .2 mM GTP, 20 micrograms creatine phosphokinase/ml and 20 mM creatine phosphate; .03 mM of each of the 20 amino acids was required for assays lasting up to 60 min. Neither rate of tRNA acylation nor availability of aminoacyl-tRNA was rate-limiting in the cell-free system established; hence, the cytoplasmic enzyme fraction from bovine skeletal muscle contains ample tRNA and has aminoacyl-tRNA synthases that are sufficiently active to form aminoacyl-tRNA faster than these compounds are used to form polypeptides in the cell-free system developed. Reinitiation of ribosomes onto new mRNA occurred very slowly, if at all, in the protein-synthesis system developed. Cytoplasmic enzymes were rate-limiting whenever cytoplasmic enzyme protein to polysomal protein ratios were 3.2:1 or lower in the cell-free system. Polysomes were rate-limiting whenever cytoplasmic enzyme protein to polysomal protein ratios were 400:1 or higher in the cell-free system. These ratios define the conditions needed to assay activity of cytoplasmic enzymes or polysomes from different animals quantitatively.

Adenosine Triphosphate↗
PubMed1989

In vitro studies of interactions between frequent and unique mRNAs and cytoplasmic factors from brain tissue of several species of wild timber voles of northern Eurasia, Clethrionomys glareolus, Clethrionomys frater and Clethrionomys gapperi: a new criticism to a modern molecular-genetic concept of biological evolution.

As a result of the complex comparative neurochemical study of the translation machinery functioning in the brain cells of three conventionally "phylogenetically related" species of wild timber voles (Clethrionomys glareolus, Clethrionomys frater and Clethrionomys gapperi), it has been found that the cytoplasm of brain cells of the latter contain an oligonucleotide (oligoribonucleotide) factor(s) with mol. weight below 1.0 KD which is able completely and highly selectively to inhibit the translation directed by mRNA which are species-specific templates and which were isolated from analogical tissue (brain) of "closely related" organisms. This phenomenon was found for the first time using special Cell-Free Translation Systems (CFTS) of very different variants of their composition consisting of the following main components: Post-Mitochondrial Supernatant (PMS), total cytoplasmic poly(A)+ mRNA or a species-specific poly(A)+ mRNA isolated form the PMS by affinity chromatography on the columns with the anti-mRNA1-FAB-(CNBr)-Sepharose, or purified 9S or 11S globin or histone specific mRNAs, respectively, and, finally a few samples of the CFTS used contain the additions of high or low molecular weight cytosolic compounds isolated from S150 fraction by ultrafiltration on Diaflo UM2 membrane with an exclusion limit of 1.0 KD. All CFTs components listed were isolated separately from the brain tissue of each organism studied. A new complex way for construction and using of the CFTS leads to an adequately documented conclusion which suggested the existence of special, so far uncharacterized in detail, cytoplasmic oligoribonucleotide factor(s) for efficient blocking for the cytoplasmic expression of "evolutionally renovated part" of genome; i.e., these factors seem to be sufficiently powerful suppressors of the translation of every mRNA template if the latter is not usual for the cell type containing the cytoplasmic suppressors mentioned in the case of a "so-called" newly found (perhaps, due to spontaneous but nonlethal mutagenesis) genes expression at the level of mRNA functioning in the cytoplasm. All findings and ideas of the paper are under discussion.

Animals↗
PubMed1992

Cell cycle-dependent localization of immunoreactive basic fibroblast growth factor to cytoplasm and nucleus of isolated ovine fetal growth plate chondrocytes.

Basic fibroblast growth factor (basic FGF) is a potent mitogen for chondrocytes in vitro and is present in developing cartilage in vivo. Studies of intracellular basic FGF localization in other cell types have revealed a transient nuclear presence. We have examined ovine fetal growth plate chondrocytes for the presence of intracellular basic FGF by immunocytochemistry. Chondrocytes were isolated from the proximal tibial growth plate of lamb fetuses between 75 and 80 days' gestation using collagenase, and were cultured in monolayer before use between passages 3 and 6. In non-synchronized cell cultures 58 +/- 6% of cells (mean +/- s.d., n = 3) demonstrated cytoplasmic staining for immunoreactive basic FGF. Of these cells, 18 +/- 3% also exhibited strong nuclear staining. Chondrocytes were growth-restricted and restarted into the cell cycle with 2% (vol/vol) fetal calf serum. The timing of S phase was followed by nuclear labelling of nuclei with [3H] thymidine followed by autoradiography, or by the incorporation of [3H] thymidine into trichloroacetic acid-precipitable DNA in parallel cultures. A cytoplasmic presence of immunoreactive basic FGF did not appear, following immunocytochemistry, until the second half of G1 with 97% of cells immunopositive 2 hr prior to S phase. Nuclear staining for basic FGF appeared 2 hr before peak nuclear labelling index, and 56% of cell nuclei were immunopositive. Following entry into S phase cytoplasmic and nuclear basic FGF immunostaining rapidly disappeared. When these experiments were repeated with or without the presence of anti-basic FGF antibody or heparin, the presence of the antibody significantly reduced peak [3H] thymidine incorporation into DNA during S phase while exposure to heparin increased this. However, the proportion of cells demonstrating cytoplasmic or nuclear staining for immunoreactive basic FGF, and the time of onset of staining, were unaltered. Incubation of cells with suramin blocked subsequent DNA synthesis and no intracellular basic FGF was visualized. Cell-conditioned culture medium, extracellular matrix and cytoplasm from synchronized cultures of chondrocytes were taken at time points throughout the cell cycle and assessed for basic FGF content by radioimmunoassay. Basic FGF was detectable in each compartment and steadily rose throughout the second half of G1 to reach maximum values around the S phase. The accumulation of basic FGF in medium, matrix and cytoplasm was blocked by the presence of cycloheximide.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1998

Variances of direct genetic effects, maternal genetic effects, and cytoplasmic inheritance effects for milk yield, fat yield, and fat percentage.

Milk yield, fat yield, and fat percentage during the first three lactations were studied using New York Holsteins that were milked twice daily over a 305-d, mature equivalent lactation. Those data were used to estimate variances from direct and maternal genetic effects, cytoplasmic effects, sire by herd interaction, and cow permanent environmental effects. Cytoplasmic line was traced to the last female ancestor using DHI records from 1950 through 1991. Records were 138,869 lactations of 68,063 cows calving from 1980 through 1991. Ten random samples were based on herd code. Samples averaged 4926 dams and 2026 cytoplasmic lines. Model also included herd-year-seasons as fixed effects and genetic covariance for direct-maternal effects. Mean estimates of the effects of maternal genetic variances and direct-maternal covariances, as fractions of phenotypic variances, were 0.008 and 0.007 for milk yield, 0.010 and 0.010 for fat yield, and 0.006 and 0.025 for fat percentage, respectively. Average fractions of variance from cytoplasmic line were 0.011, 0.008, and 0.009 for milk yield, fat yield, and fat percentage. Removal of maternal genetic effects and covariance for maternal direct effects from the model increased the fraction of direct genetic variance by 0.014, 0.021, and 0.046 for milk yield, fat yield, and fat percentage; little change in the fraction was due to cytoplasmic line. Exclusion of cytoplasmic effects from the model increased the ratio of additive direct genetic variance to phenotypic variance by less than 2%. Similarly, when sire by herd interaction was excluded, the ratio of direct genetic variance to phenotypic variance increased 1% or less.

Animals↗
PubMed2006

Cytoplasmic expression of p27(kip1) is associated with a favourable prognosis in colorectal cancer patients.

AIM: To evaluate the prognostic significance of p27(kip1) in colorectal cancer patients. METHODS: Cytoplasmic and nuclear p27(kip1) expression was evaluated in 418 colorectal cancers using tissue microarrays. Data were associated with known patient and tumor variables and long-term patient outcomes, providing further insight into the mechanisms by which p27(kip1) may influence tumor development. RESULTS: Nuclear and cytoplasmic p27(kip1) expressions were detected in 59% and 19% of tumors respectively. Cytoplasmic p27(kip1) was almost invariably associated with positive nuclear p27(kip1) expression. Neither case correlated with known clinical or pathological variables, including tumor stage, grade or extramural vascular invasion. Furthermore, nuclear p27(kip1) expression had no impact on survival. However, we identified a significant correlation between expression of cytoplasmic p27(kip1) and longer disease-specific survival times. On multivariate analysis, TNM stage and extramural vascular invasion were highly significant independent prognostic factors, with positive cytoplasmic p27 expression showing a trend towards improved patient survival (P = 0.059). CONCLUSION: These findings support the recent evidence that cytoplasmic p27(kip1) has a distinct and important biological role that can influence tumor outcome.

Aged↗