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PubMed1970

Regulation of polyribosome formation and protein synthesis in the uterus. Effect of administration of graded doses of oestradiol-17-beta and actinomycin D on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of polyribosomes.

1. The effects of graded doses of oestradiol-17beta and actinomycin D, administered separately or together, on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of uterine polyribosomes are described. Preparations of polyribosomes isolated from uteri of ovariectomized adult rats were determined for cytoplasmic concentration in vivo and assayed for [(14)C]leucine-incorporation activity in the cell-free system, exactly as described by Teng & Hamilton (1967b). 2. A minimal dose of 10mug of oestradiol-17beta administered for 10h was found to increase, by about 100%, both the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of the polyribosomes. A minimal dose of 250mug of actinomycin D administered for 10h was found to inhibit, by about 50%, the incorporation activity in vitro of the polyribosomes. All doses of the inhibitor administered for 10h failed to alter the cytoplasmic concentration in vivo of the polyribosomes. 3. A dose of 10mug of oestradiol-17beta restored to the control value the inhibitory effect of a dose of either 50 or 125mug of actinomycin D on the activity in vitro of the polyribosomes, at 10h after treatment with the inhibitor and the hormone. In these experiments, there was an increase of 60-100% in the cytoplasmic concentration in vivo of the polyribosomes. 4. A dose of 125mug of actinomycin D, administered to animals along with 10mug of oestradiol-17beta for 6-36h, abolished the hormone-induced enhancement of the incorporation activity in vitro, but did not prevent an increase of about 200% in the cytoplasmic concentration in vivo of the polyribosomes. However, treatment with 750mug of the inhibitor abolished both stimulatory effects of the hormone. 5. The results reported indicate that the stimulatory effects of oestradiol-17beta in vivo on the number and activity of the cytoplasmic polyribosomes in the uterus of the ovariectomized rat have different sensitivities to actinomycin D, but the primary molecular mechanisms responsible for the results are unknown. The major conclusion drawn is that the formation and appearance in the cytoplasm of newly formed polyribosomes are important features of the early action of oestrogen in the uterus.

Amino Acids↗
PubMed2001

The peroxisomal proliferator clofibrate enhances the hepatic cytoplasmic movement of fatty acids in rats.

The role of cytosolic fatty acid binding protein (FABP) in cellular fatty acid metabolism remains poorly defined. The intracellular movement of fatty acids is thought to be facilitated through codiffusion with FABP. Peroxisomal proliferators like clofibrate induce FABP and may stimulate fatty acid use by increasing cytoplasmic diffusion rates. Our aim was to determine if induction of FABP by clofibrate increases the cytoplasmic transport of a fluorescent fatty acid NBD-stearate. Male rats were fed clofibrate for 9 days to increase the hepatic concentration of FABP. Hepatocytes were isolated using collagenase perfusion. Cytosolic FABP was measured by enzyme-linked immunosorbent assay (ELISA) using a specific antibody to hepatic FABP. Two-dimensional laser photobleaching (FRAP) was used to measure the cytoplasmic movement of NBD-stearate in hepatocytes. Cytoplasmic transport of NBD-stearate occurred by isotropic diffusion with no evidence for convective or directed transport. Treatment with clofibrate increased FABP levels, the fraction of NBD-stearate found in cytosol, and the observed cytoplasmic diffusion rate. The increase in cytoplasmic movement exceeded that predicted from FABP binding changes suggesting that clofibrate also enhances fatty acid diffusion within intracellular membranes. Nonspecific effects of clofibrate on cytoplasmic viscosity or pore size were not observed. These data suggest that clofibrate treatment induces cytosolic FABP and stimulates the intracellular movement of fatty acids by 2 distinct mechanisms. Soluble proteins like FABP promote the cytoplasmic transport of fatty acids by increasing diffusion. These data further support a role for intracellular binding proteins in facilitating the intracellular movements of fatty acids.

4-Chloro-7-nitrobenzofurazan↗
PubMed1970

Characterization of RNAs that do and do not migrate between cytoplasm and nucleus.

We have investigated the nature of the RNA that moves from cytoplasm to nucleus against a concentration gradient in Amoeba proteus. We find that: In the presence of actinomycin D an unlabeled nucleus grafted into a [(3)H]RNA cytoplasm acquires RNAs with sedimentation constants 30 S, 19 S, and 4-6S that are not related to the general population of cytoplasmic ribosomal and transfer RNAs. RNAs of sedimentation constants 39 S and 16 S may also enter the nucleus from the cytoplasm, but not in the presence of actinomycin D. Nuclei were transplanted from [(3)H]RNA cells through several unlabeled cytoplasms to dilute out migrating [(3)H]RNA. This resulted in the 4-6 S [(3)H]RNA being retained as the predominant labeled material of the nucleus and establishes that a substantial portion of 4-6S nuclear RNA does not leave the interphase nucleus. We conclude that nuclear RNAs may be classified in a new way: (1) RNA that is to become cytoplasmic RNA and presumably moves only from nucleus to cytoplasm; (2) RNA that migrates back and forth between nucleus and cytoplasm; and (3) RNA that does not leave the nucleus during interphase.

Amoeba↗
PubMed1978

Mammalian cytoplasmic actins are the products of at least two genes and differ in primary structure in at least 25 identified positions from skeletal muscle actins.

Muscle and cytoplasmic actins from several species have been compared by extensive fingerprint analysis and by partial amino acid sequence determination with the known amino acid sequence of rabbit muscle actin. Although complete sequences have not been established, the following characteristics are apparent. (a) Cytoplasmic actins are the products of two different genes. The difference seen in isoelectric focusing studies is probably determined only by the nature of the three amino-terminal acidic residues. (b) Mammalian cytoplasmic actins are exceedingly similar and perhaps identical. (c) Cytoplasmic actins may differ by at least 25 amino acid replacement from rabbit muscle actin. These replacements have been identified for calf thymus actin; however, other cytoplasmic actins show the same replacements. (d) The replacements always involve-except for the first five residues-neutral amino acid residues. (e) The replacements are not randomly distributed. Residues 18-75 are constant whereas residues 2-18 and 259-298 show many substitutions. (f) The main component of smooth muscle actin from chicken gizzard shows the charge characteristics found at the amino terminus of the less acidic cytoplasmic actin species. In the rest of the polypeptide chain, gizzard actin resembles skeletal muscle actin, although two substitutions of the cytoplasmic type have been identified. (g) Heart muscle actin is very similar to skeletal muscle actin. Only two amino acid replacements have been found; they are of the cytoplasmic type. (h) Skeletal muscle actins from chicken and beef have not shown a replacement.

Actins↗
PubMed1995

Residues throughout the cytoplasmic domain affect the internalization efficiency of P-selectin.

The cytoplasmic domains of many membrane proteins have short sequences, usually including a tyrosine or a di-leucine, that function as sorting signals. P-selectin is an adhesion receptor for leukocytes that is expressed on activated platelets and endothelial cells. Its 35-residue cytoplasmic domain contains signals for sorting into regulated secretory granules, for endocytosis, and for movement from endosomes to lysosomes. The domain has a membrane-distal sequence, YGVFTNAAF, that resembles some tyrosine-based signals. We studied the effects of deletions and mutations in the cytoplasmic tail of human P-selectin on its internalization in clathrin-coated pits of transfected Chinese hamster ovary cells. Mutations and deletions in the putative tyrosine-based motif did not clearly implicate these residues as critical components of a short internalization signal. Indeed, a construct containing a truncated 18-residue cytoplasmic domain with a single substitution (K761A/H773Stop) was internalized nearly three times as fast as wild-type P-selectin; this construct contained no di-leucine, tyrosine, or other known sorting motif. Substitution of residues throughout the cytoplasmic domain affected the internalization rate of P-selectin. Furthermore, the cytoplasmic domain of P-selectin mediated faster internalization when attached to the extracellular and transmembrane domains of the low density lipoprotein receptor than when attached to the corresponding domains of P-selectin. Thus, we were unable to identify a short internalization signal in the cytoplasmic tail of P-selectin. Residues throughout the cytoplasmic domain, and perhaps the transmembrane sequence to which the domain is attached, affect the efficiency of internalization.

Amino Acid Sequence↗
PubMed2000

The human lysyl-tRNA synthetase gene encodes both the cytoplasmic and mitochondrial enzymes by means of an unusual alternative splicing of the primary transcript.

Two cDNAs encoding human lysyl-tRNA synthetase have been identified. One encodes the cytoplasmic form of the enzyme identified previously. The second cDNA contains the same sequence but with a 180-bp insertion at the 5'-end of the mRNA. This results in a predicted protein whose carboxyl 576 amino acids are identical to those of the cytoplasmic enzyme but with a different amino terminus of 49 amino acids that contains a putative mitochondrial targeting sequence. Expression of the two lysyl-tRNA synthetase-green fluorescent protein gene fusions in a human cell line confirmed that the cytoplasmic form was targeted to the cytoplasm and the mitochondrial form to mitochondria. The genomic lysyl-tRNA synthetase gene consisted of 15 exons. The two isoforms were created by alternative splicing of the first three exons of the gene. The cytoplasmic form was created by splicing exon 1 to exon 3. The inclusion of exon 2 between exons 1 and 3 produced an mRNA encoding the mitochondrial isoform with an additional upstream small open reading frame, consisting mainly of a portion of the 5' coding region of the cytoplasmic isoform. This is the first example of mitochondrial targeting sequence being encoded on the second exon of a gene. Ribonuclease protection analysis showed that the mRNA encoding the cytoplasmic isoform makes up approximately 70%, and the mitochondrial isoform approximately 30%, of the mature transcripts from the lysyl-tRNA synthetase gene. The mitochondrial form of the enzyme, purified after expression in Escherichia coli, aminoacylated in vitro transcripts corresponding to both the cytoplasmic and mitochondrial tRNA(Lys), despite the difference in the discriminator base sequence in the acceptor stems of these tRNAs.

Alternative Splicing↗
PubMed2000

alpha 2 integrin subunit cytoplasmic domain-dependent cellular migration requires p38 MAPK.

The alpha(2) integrin subunit cytoplasmic domain uniquely supported epidermal growth factor (EGF)-stimulated migration on type I collagen. p38 MAP kinase- and phosphatidylinositol 3-kinase-specific inhibitors, but not a MEK-specific inhibitor, eliminated EGF-stimulated and unstimulated alpha(2)-cytoplasmic domain-dependent migration. Following adhesion to collagenous matrices, cells expressing the full-length alpha(2) integrin subunit, but not cells expressing a chimeric alpha(2) integrin subunit in which the alpha(2)-cytoplasmic domain was replaced by the cytoplasmic domain of the alpha(1)-subunit, exhibited sustained and robust phosphorylation of p38 MAP kinase. Expression of dominant negative p38 MAP kinase inhibited alpha(2)-cytoplasmic domain-dependent, EGF-stimulated migration as well as unstimulated migration on collagen. Expression of constitutively active Rac1(Val-12) augmented p38 MAP kinase activation and alpha(2)-cytoplasmic domain-dependent migration. It also rescued the ability of cells expressing the alpha(1)-cytoplasmic domain to activate p38 MAPK and to migrate. These results suggest that the alpha(2) integrin cytoplasmic domain uniquely stimulates the p38 MAP kinase pathway that is required for unstimulated and EGF-stimulated migration on type I collagen.

Animals↗
PubMed2004

The interaction of protein kinase C isozymes alpha, iota, and theta with the cytoplasmic domain of L-selectin is modulated by phosphorylation of the receptor.

The leukocyte adhesion molecule L-selectin has an important role in the initial steps of leukocyte extravasation during inflammation and lymphocyte homing. Its cytoplasmic domain is involved in signal transduction after L-selectin cross-linking and in the regulation of receptor binding activity in response to intracellular signals. However, the signaling events occurring at the level of the receptor are largely unknown. This study therefore addressed the question of whether protein kinases associate with the cytoplasmic domain of the receptor and mediate its phosphorylation. Using a glutathione S-transferase fusion protein of the L-selectin cytoplasmic domain, we isolated a kinase activity from cellular extracts of the human leukemic Jurkat T-cell line that phosphorylated L-selectin on serine residues. This kinase showed characteristics of the protein kinase C (PKC) family. Moreover, the Ca(2+)-independent PKC isozymes theta and iota were found associated with the cytoplasmic domain of L-selectin. Pseudosubstrate inhibitors of these isozymes abolished phosphorylation of the cytoplasmic domain, demonstrating that these kinases are responsible for the phosphorylation. Analysis of proteins specifically bound to the phosphorylated cytoplasmic tail of L-selectin revealed that PKCalpha and -theta are strongly associated with the phosphorylated cytoplasmic domain of L-selectin. Binding of these isozymes to L-selectin was also found in intact cells after phorbol ester treatment inducing serine phosphorylation of the receptor. Furthermore, stimulation of Jurkat T-cells by CD3 cross-linking induced association of PKCalpha and -theta with L-selectin, indicating a role of these kinases in the regulation of L-selectin through the T-cell receptor complex. The phosphorylation-regulated association of PKC isozymes with the cytoplasmic domain of L-selectin indicates an important role of this kinase family in L-selectin signal transduction.

Amino Acid Sequence↗
PubMed1987

Fluorescence ratio imaging microscopy: temporal and spatial measurements of cytoplasmic pH.

Fluorescence ratio imaging microscopy (Tanasugarn, L., P. McNeil, G. Reynolds, and D. L. Taylor, 1984, J. Cell Biol., 98:717-724) has been used to measure the spatial variations in cytoplasmic pH of individual quiescent and nonquiescent Swiss 3T3 cells. Fundamental issues of ratio imaging that permit precise and accurate temporal and spatial measurements have been addressed including: excitation light levels, lamp operation, intracellular probe concentrations, methods of threshold selection, photobleaching, and spatial signal-to-noise ratio measurements. Subcellular measurements can be measured accurately (less than 3% coefficient of variation) in an area of 3.65 microns 2 with the present imaging system. Quiescent Swiss 3T3 cells have a measured cytoplasmic pH of 7.09 (0.01 SEM), whereas nonquiescent cells have a pH of 7.35 (0.01 SEM) in the presence of bicarbonate buffer. A unimodal distribution of mean cytoplasmic pH in both quiescent and nonquiescent cells was identified from populations of cells measured on a cell by cell basis. Therefore, unlike earlier studies based on cell population averages, it can be stated that cells in each population exhibit a narrow range of cytoplasmic pH. However, the mean cytoplasmic pH can change based on the physiological state of the cells. In addition, there appears to be little, if any, spatial variation in cytoplasmic pH in either quiescent or nonquiescent Swiss 3T3 cells. The pH within the nucleus was always the same as the surrounding cytoplasm. These values will serve as a reference point for investigating the role of temporal and spatial variations in cytoplasmic pH in a variety of cellular processes including growth control and cell movement.

Animals↗
PubMed1992

Cytoplasmic dynein participates in the centrosomal localization of the Golgi complex.

The localization of the Golgi complex depends upon the integrity of the microtubule apparatus. At interphase, the Golgi has a restricted pericentriolar localization. During mitosis, it fragments into small vesicles that are dispersed throughout the cytoplasm until telophase, when they again coalesce near the centrosome. These observations have suggested that the Golgi complex utilizes a dynein-like motor to mediate its transport from the cell periphery towards the minus ends of microtubules, located at the centrosome. We utilized semi-intact cells to study the interaction of the Golgi complex with the microtubule apparatus. We show here that Golgi complexes can enter semi-intact cells and associate stably with cytoplasmic constituents. Stable association, termed here "Golgi capture," requires ATP hydrolysis and intact microtubules, and occurs maximally at physiological temperature in the presence of added cytosolic proteins. Once translocated into the semi-intact cell cytoplasm, exogenous Golgi complexes display a distribution similar to endogenous Golgi complexes, near the microtubule-organizing center. The process of Golgi capture requires cytoplasmic tubulin, and is abolished if cytoplasmic dynein is immunodepleted from the cytosol. Cytoplasmic dynein, prepared from CHO cell cytosol, restores Golgi capture activity to reactions carried out with dynein immuno-depleted cytosol. These results indicate that cytoplasmic dynein can interact with isolated Golgi complexes, and participate in their accumulation near the centrosomes of semi-intact, recipient cells. Thus, cytoplasmic dynein appears to play a role in determining the subcellular localization of the Golgi complex.

Adenosine Triphosphate↗
PubMed1993

Determinants of the translational mobility of a small solute in cell cytoplasm.

The purposes of this study were: (a) to measure the translational mobility of a small solute in cell cytoplasm; (b) to define quantitatively the factors that determine solute translation; and (c) to compare and contrast solute rotation and translation. A small fluorescent probe, 2,7-bis-(2-carboxyethyl)-5-(and 6-)-carboxyfluorescein (BCECF), was introduced into the cytoplasm of Swiss 3T3 fibroblasts. BCECF translation was measured by fluorescence recovery after photo-bleaching; rotation was measured by Fourier transform polarization microscopy. Diffusion coefficients relative to those in water (D/D0) were determined by comparing mobility in cytoplasm with mobility in standard solutions of known viscosity. At isosmotic cell volume, the relative diffusion coefficients for BCECF translation and rotation in cytoplasm were 0.27 +/- 0.01 (SEM, n = 24, 23 degrees C) and 0.78 +/- 0.03 (n = 4), respectively. As cell volume increased from 0.33 to 2 times isosmotic volume, the relative translational diffusion coefficient increased from 0.047 to 0.32, while the relative rotational diffusion coefficient remained constant. The factors determining BCECF translation were evaluated by comparing rotation and translation in cytoplasm, and in artificial solutions containing dextrans (mobile barriers) and agarose gels (immobile barriers). It was concluded that the hindrance of BCECF translation in cytoplasm could be quantitatively attributed to three independent factors: (a) fluid-phase cytoplasmic viscosity is 28% greater than the viscosity of water (factor 1 = 0.78); (b) 19% of BCECF is transiently bound to intracellular components of low mobility (factor 2 = 0.81); and most importantly, (c) translation of unbound BCECF is hindered 2.5-fold by collisions with cell solids comprising 13% of isosmotic cell volume (factor 3 = 0.40). The product of the 3 factors is 0.25 +/- 0.03, in good agreement with the measured D/D0 of 0.27 +/- 0.01. These results provide the first measurement of the translational mobility of a small solute in cell cytoplasm and define quantitatively the factors that slow solute translation.

3T3 Cells↗
PubMed1993

Interchangeable alpha chain cytoplasmic domains play a positive role in control of cell adhesion mediated by VLA-4, a beta 1 integrin.

Integrins can exist in a range of functional states, depending on the cell type and its state of activation. Although the mechanism that controls activity is unknown, it has been suggested that for some integrins, alpha chain cytoplasmic domains may exert either a negative effect or no effect on adhesion function. To address this issue for VLA-4 (an alpha 4 beta 1 heterodimer), we constructed an alpha 4 cytoplasmic deletion mutant and chimeric alpha chains composed of the extracellular domains of alpha 4 and the cytoplasmic domains of alpha 2, alpha 4, or alpha 5. Upon stable transfection of wild-type alpha 4, VLA-4 heterodimer was obtained that mediated (a) poor adhesion to CS1 peptide, fibronectin, or vascular cell adhesion molecule 1 (VCAM-1) (in K562 cells); (b) poor adhesion to CS1 peptide but moderate adhesion to VCAM-1 (in MIP101 cells); and (c) moderate adhesion to both CS1 peptide and VCAM-1 (in PMWK cells). Chimeric alpha 4 constructs and wild-type alpha 4 yielded similar results in these cell lines. In contrast, truncation of the alpha 4 cytoplasmic domain (after the conserved GFFKR motif) caused an almost complete loss of adhesive activity in all three cell lines. Thus, several interchangeable alpha chain cytoplasmic domains play a fundamentally positive role in determining the state of constitutive activity for VLA-4. The alpha chain cytoplasmic domain is also required for agonist-stimulated adhesion, since phorbol ester stimulated the cell adhesion mediated by wild-type and chimeric alpha chains, but not by the cytoplasmic deletion mutant. The inactivity of both wild-type VLA-4 (in K562 cells), and truncated VLA-4 (in all three cell lines) was overcome by the addition of a stimulatory anti-beta 1 monoclonal antibody. Thus, the alpha cytoplasmic domain-dependent cellular mechanism controlling both constitutive and agonist-stimulated VLA-4 activity could be bypassed by external manipulation of the integrin.

Cell Adhesion↗
PubMed1992

HTS1 encodes both the cytoplasmic and mitochondrial histidyl-tRNA synthetase of Saccharomyces cerevisiae: mutations alter the specificity of compartmentation.

Genetic and biochemical evidence shows that a single nuclear gene HTS1 encodes both the mitochondrial and cytoplasmic histidyl-tRNA synthetases (Hts). The gene specifies two messages, one with two in-frame ATGs (-60 and +1) and another with only the downstream ATG (+1). We have made a new set of mutations that enables us to express only the mitochondrial or the cytoplasmic form and compared the subcellular distribution of the Hts1 protein in these mutants and wild type, using an antibody that interacts with both the mitochondrial and cytoplasmic Hts1 as well as Hts1::LacZ fusions. Mutations in the upstream ATG (-60) or frameshift mutations in the presequence affect only the mitochondrial enzyme and not the cytoplasmic enzyme. Mutations in the downstream ATG (+1 ATG to ATC) destroy the function of the cytosolic enzyme, but do not affect the function of the mitochondrial enzyme. Overexpression of this construct restores cytoplasmic function. Cells expressing a truncated form of Hts containing a deletion of the first 20 amino-terminal residues (Htsc) produce a functional cytoplasmic enzyme, which does not provide mitochondrial function. Overexpression of this truncated cytoplasmic protein provides mitochondrial function and produces detectable levels of the synthetase in the mitochondrion. These experiments suggest that Hts1 contains two domains that together allow efficient localization of Htsm to the mitochondrion: an amino-terminal presequence in the mitochondrial precursor that is likely cleaved upon delivery to the mitochondrion and a second amino-terminal sequence (residues 21-53) present in both the precursor and the cytoplasmic form. Neither one by itself is sufficient to act as an efficient mitochondrial targeting signal. Using our antibody we have been able to detect a protein of increased molecular mass that corresponds to that of the predicted precursor. Taken together these studies show that the specificity of compartmentation of the Hts protein depends upon both the primary sequence and the concentration of the protein in the cell.

Base Sequence↗
PubMed2004

The correlation between cytoplasmic overexpression of epidermal growth factor receptor and tumor aggressiveness: poor prognosis in patients with pancreatic ductal adenocarcinoma.

OBJECTIVES: Recent studies have shown that some growth factor receptors with tyrosine kinase activity, eg, the epidermal growth factor receptor (EGFr) and the c-erbB-2 (HER-2) oncoprotein, are associated with aggressive biologic behavior of various cancer cell types. We examined the clinicopathological significance of the expression and localization of EGFr and HER-2 in both invasive and intraductal components of ductal adenocarcinomas of the pancreas. METHODS: Tissue samples from 76 archival cases of pancreatic ductal adenocarcinoma were immunohistochemically analyzed for both membrane and cytoplasmic overexpression of EGFr and HER-2 oncoprotein. The rate of incidence between the invasive and intraductal components was analyzed and then their correlation with tumor differentiation and patient prognosis was analyzed. RESULTS: Cytoplasmic EGFr overexpression was more frequent in invasive components (47 of 76, 62%) than in intraductal components (19 of 76, 25%), while membrane EGFr overexpression was more frequent in intraductal components (41 of 76, 54%) than in invasive components (11 of 76, 14%). The membrane HER-2 overexpression was also more frequent in intraductal components (15 of 76, 20%) than in invasive components (2 of 76, 3%), but the incidence of cytoplasmic HER-2 overexpression did not differ between intraductal components (12 of 76, 16%) and invasive components (8 of 76, 11%). The cytoplasmic EGFr overexpression in invasive components was more frequent in grade 3 group (32 of 33, 97%) than in grade 2 (15 of 32, 47%) and grade 1 groups (0 of 10, 0%) (P < 0.001). Patients with adenocarcinoma with cytoplasmic EGFr overexpression showed shorter overall survival than those with adenocarcinoma without cytoplasmic EGFr overexpression (P = 0.02). CONCLUSION: It is suggested that the cytoplasmic overexpression of EGFr plays a significant role in the progression of pancreatic ductal adenocarcinoma, especially in the invasion and acquisition of aggressive clinical behavior. Both membrane and cytoplasmic expression of HER-2 showed no significant correlation between tumor differentiation and poor survival.

Aged↗
PubMed1976

Epidermal cytoplasmic antibodies: incidence and type in normal persons and patients with melanoma.

Antibodies to epidermal cytoplasmic antigens were present in 25 to 34% of 32 normal persons and in 34 to 50% of 53 patients with melanoma. There were at least two different types of cytoplasmic antibodies. The most common reacted to cytoplasmic antigens present only in the upper layers of the epidermis. This type of antibody occurred with equal frequency in patients with melanoma and in normal persons. The increased incidence of cytoplasmic antibodies in melanoma was due to antibodies to cytoplasmic antigens present throughout the epidermis. These antibodies were 2 1/2 times more common in patients with melanoma than in normal persons. The presence of different cytoplasmic antigens in distinct strata of the epidermis suggests they result from epidermal cell differentiation. The epidermal cytoplasmic antigens present throughout the epidermis appear to partially cross-react with cytoplasmic antigens in melanoma cells.

Antibodies↗
PubMed1998

The 5D4 antibody (anti-cyclin D1/D2) related antigen: cytoplasmic staining is correlated to the progression of gastric cancer.

In order to clarify the relationship between cyclin D1 and D2 (CD1/CD2) overexpression and progression, 191 gastric cancer cases (81 early and 110 advanced cancers) were investigated using the 5D4 monoclonal antibody for both CD1/CD2 in immunohistochemistry. 5D4 immunoreactivity was noted in 68 (35.6%) cases, staining being restricted to the nucleus in 27 (14.1%) cases, the cytoplasm in 34 (17.8%) cases, and its presence in both the nucleus and cytoplasm in seven (3.7%). Cases demonstrating cytoplasmic positivity, including both positive cases, were significantly more frequent in advanced cancers (P = 0.010), those having lymph node metastasis (P = 0.004) and cases showing cancer invasion of vessels (P = 0.009), although no relation to histological malignant grading was apparent. In contrast, cases of nuclear positivity behaved no differently from 5D4-negative cases. Statistics showed a trend where survival in patients was worse in the cytoplasm-positive cases than the cytoplasm-negative group. However, multivariate analysis revealed no independent statistical significance in the cytoplasmic positivity of prognosis. Additional studies using DCS-6 antibody for CD1 and C-17 antibody for CD2, suggest that nuclear staining of 5D4 indicates the presence of CD1 but cytoplasmic staining is derived from an antigen that is related to CD2. In conclusion, the present results indicate that the accumulation of CD2 in the cytoplasm may play some role in the progression of gastric cancers but not prognosis; however, CD1 overexpression is not linked to either.

Cell Nucleus↗
PubMed1984

Total cytoplasmic calcium in relaxed and maximally contracted rabbit portal vein smooth muscle.

The concentration of total cytoplasmic Ca in vascular smooth muscle was measured by electron probe microanalysis of strips of rabbit portal anterior mesenteric vein that were rapidly frozen either when relaxed or during a maintained (30 min) maximal contraction stimulated with high K and noradrenaline. Strips were also frozen and analysed after incubation in Ca-free, high-Mg2+ solution. Probe diameters of 0.1-0.2 micron and 1.0-1.5 micron were used to measure, respectively, cytoplasmic and cellular (including stored) Ca. There was a highly significant increase (P less than 0.0005) in cytoplasmic Ca of 1.0 +/- 0.2 (S.D.) mmol Ca/kg dry wt. from 0.8 +/- 0.2 (S.E. of mean) mmol/kg dry wt. (n = 262 spectra, six animals) to 1.8 +/- 0.2 (S.E. of mean) mmol Ca/kg dry wt. (n = 296 spectra, six animals), during maximal contraction. This increase is greater than can be accounted for by Ca binding to calmodulin and to myosin, suggesting the presence of other Ca-binding proteins in smooth muscle. A small amount (0.4-0.6 mmol/kg dry wt.) of cytoplasmic Ca remained after incubation in Ca-free, high-Mg2+ EGTA solution. This tightly bound, cytoplasmic Ca is insufficient to account for the total amount of divalent cation known to be bound to F-actin. We conclude that Mg is the major inexchangeably bound cation in F-actin in smooth as in striated muscle. In the contracted muscles, the cellular Ca concentration, measured with the large probes that include Ca stored in the sarcoplasmic reticulum (s.r.), was 3.2 +/- 0.3 (S.E. of mean) mmol Ca/kg dry wt. (n = 93), significantly higher than the cytoplasmic Ca concentration measured with small probes. This value of cellular Ca is probably an underestimate, as the large-diameter probes did not cover all of the peripheral s.r. The cellular Ca (measured with large probes) was highest in the contracted and lower in the relaxed tissue, and was significantly reduced in the muscles incubated in Ca-free solution. In contracted muscle, cytoplasmic Mg significantly decreased and mitochondrial Mg increased. In 0 Ca, high-Mg2+ solution, the cytoplasmic Mg increased significantly. Mitochondrial Ca did not significantly change during a maintained contraction, but was significantly lower (0.0 +/- 0.2 (S.E. of mean) mmol Ca/kg dry wt.) after incubation in Ca-free, high-Mg2+ solution than in the relaxed tissue (1.6 +/- 0.2 mmol Ca/kg dry wt.) in normal Ca-containing solution.

Animals↗
PubMed2001

Cytoplasmic filament-deficient mutant of Treponema denticola has pleiotropic defects.

In Treponema denticola, a ribbon-like structure of cytoplasmic filaments spans the cytoplasm at all stages of the cell division process. Insertional inactivation was used as a first step to determine the function of the cytoplasmic filaments. A suicide plasmid was constructed that contained part of cfpA and a nonpolar erythromycin resistance cassette (ermF and ermAM) inserted near the beginning of the gene. The plasmid was electroporated into T. denticola, and double-crossover recombinants which had the chromosomal copy of cfpA insertionally inactivated were selected. Immunoblotting and electron microscopy confirmed the lack of cytoplasmic filaments. The mutant was further analyzed by dark-field microscopy to determine cell morphology and by the binding of two fluorescent dyes to DNA to assess the distribution of cellular nucleic acids. The cytoplasmic filament protein-deficient mutant exhibited pleiotropic defects, including highly condensed chromosomal DNA, compared to the homogeneous distribution of the DNA throughout the cytoplasm in a wild-type cell. Moreover, chains of cells are formed by the cytoplasmic filament-deficient mutant, and those cells show reduced spreading in agarose, which may be due to the abnormal cell length. The chains of cells and the highly condensed chromosomal DNA suggest that the cytoplasmic filaments may be involved in chromosome structure, segregation, or the cell division process in Treponema.

Bacterial Proteins↗