bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for splicing new basement membrane into old during glomerular development in newborn rat kidneys.
Authors: Abrahamson D R , Perry E W
Published: May 2004

Tannic acid in glutaraldehyde fixatives greatly enhanced the visualization of two developmentally and morphologically distinct stages in glomerular basement membrane (GBM) formation in newborn rat kidneys. First, in early stage glomeruli, double basement membranes between endothelial cells and podocytes were present and, in certain areas, appeared to be fusing. Second, in maturing stage glomeruli, elaborate loops and outpockets of basement membrane projected into epithelial, but not endothelial, sides of capillary walls. When Lowicryl thin sections from newborn rat kidneys were sequentially labeled with rabbit anti-laminin IgG and anti-rabbit IgG-colloidal gold, gold bound across the full width of all GBMs, including double basement membranes and outpockets. The same distribution was obtained when sections from rats that received intravenous injections of rabbit anti-laminin IgG 1 h before fixation were labeled directly with anti-rabbit IgG-colloidal gold. When kidneys were fixed 4 d after anti-laminin IgG injection, however, loops beneath the podocytes in maturing glomeruli were usually unlabeled and lengths of unlabeled GBM were interspersed with labeled lengths. In additional experiments, rabbit anti-laminin IgG was intravenously injected into newborn rats and, 4-14 d later, rats were re-injected with sheep anti-laminin IgG. Sections were then doubly labeled with anti-rabbit and anti-sheep IgG coupled to 10 and 5 nm colloidal gold, respectively. Sheep IgG occurred alone in outpockets of maturing glomeruli and also in lengths of GBM flanked by lengths containing rabbit IgG. These results indicate that, after fusion of double basement membranes, new segments of GBM appear beneath developing podocytes and are subsequently spliced into existing GBM. This splicing provides the additional GBM necessary for expanding glomerular capillaries.
Lymphoma Thy-1 glycoprotein is linked to the cytoskeleton via a 4.1-like protein.
Authors: Bourguignon L Y , Suchard S J , Kalomiris E L
Published: May 2004

In this study we have found that the phosphoprotein doublet of 68,000 and 65,000 daltons (68/65 kD) in mouse T-lymphoma cells shares several structural and functional similarities with erythrocyte band 4.1. Our evidence for identifying the 68/65-kD doublet as a lymphoma 4.1-like protein is as follows: it displays an immunological cross-reactivity with anti-erythrocyte band 4.1 antibody; it exhibits a Svedberg unit of sedimentation coefficient of 4 S; it is phosphorylated in the presence of phorbol ester (phorbol-12-O-tetradecanoylphorbol-13-acetate) and its phosphorylation requires Ca2+; it is phosphorylated primarily at serine residues; and it can bind directly to fodrin (a spectrin-like actin-binding protein). In addition, this lymphoma 4.1-like protein can be both colocalized and coisolated with the major T-lymphocyte-specific glycoprotein, Thy-1 (gp 25). Therefore, all of these results strongly suggest that the lymphoma 4.1-like protein (68/65-kD doublet) may play a pivotal role in linking the Thy-1 (gp 25) glycoprotein to fodrin which, in turn, binds to the actin filaments that are responsible for recruiting Thy-1 antigens into cap structures.
Acidic and basic hair/nail ("hard") keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to "soft" keratins.
Authors: Lynch M H , O'Guin W M , Hardy C , Mak L , Sun T T
Published: May 2004

Although numerous hair proteins have been studied biochemically and many have been sequenced, relatively little is known about their in situ distribution and differential expression in the hair follicle. To study this problem, we have prepared several mouse monoclonal antibodies that recognize different classes of human hair proteins. Our AE14 antibody recognizes a group of 10-25K hair proteins which most likely corresponds to the high sulfur proteins, our AE12 and AE13 antibodies define a doublet of 44K/46K proteins which are relatively acidic and correspond to the type I low sulfur keratins, and our previously described AE3 antibody recognizes a triplet of 56K/59K/60K proteins which are relatively basic and correspond to the type II low sulfur keratins. Using these and other immunological probes, we demonstrate the following. The acidic 44K/46K and basic 56-60K hair keratins appear coordinately in upper corticle and cuticle cells. The 10-25K, AE14-reactive antigens are expressed only later in more matured corticle cells that are in the upper elongation zone, but these antigens are absent from cuticle cells. The 10-nm filaments of the inner root sheath cells fail to react with any of our monoclonal antibodies and are therefore immunologically distinguishable from the cortex and cuticle filaments. Nail plate contains 10-20% soft keratins in addition to large amounts of hair keratins; these soft keratins have been identified as the 50K/58K and 48K/56K keratin pairs. Taken together, these results suggest that the precursor cells of hair cortex and nail plate share a major pathway of epithelial differentiation, and that the acidic 44K/46K and basic 56-60K hard keratins represent a co-expressed keratin pair which can serve as a marker for hair/nail-type epithelial differentiation.
Alteration of the cytoplasmic domain of the membrane-spanning glycoprotein p62 of Semliki Forest virus does not affect its polar distribution in established lines of Madin-Darby canine kidney cells.
Authors: Roman L M , Garoff H
Published: May 2004

Expression of the Semliki Forest virus p62/E2 protein was studied in the polarized epithelial cell line Madin-Darby canine kidney (MDCK). After infection this transmembrane protein, together with the other spike subunit E1, accumulates at the basolateral surface of MDCK cells (Fuller, S. D., C.-H. von Bonsdorff, and K. Simons, 1985, EMBO (Eur. Mol. Biol. Organ.) J., 4:2475-2485). The cDNAs encoding truncated forms of the protein were used to stably transform MDCK cells to examine the role of subunit oligomerization (E1-E2) and the cytoplasmic domain of p62/E2 in directed transport to the basolateral surface. The biochemical characteristics and polarity of the expressed proteins were studied using cell monolayers grown on nitrocellulose filters. A wild-type form of p62/E2, in the absence of E1, and two forms having either 15 or 3 of the wild-type 31-amino acid carboxyl cytoplasmic domain were all localized to the basolateral surface. These results indicate that the cytoplasmic domain of E2 does not contain the information essential for directed transport to the plasma membrane, and imply that this information resides in either the lumenal and/or membrane-spanning segments of this transmembrane protein.
Protein translocation across the yeast microsomal membrane is stimulated by a soluble factor.
Authors: Waters M G , Chirico W J , Blobel G
Published: May 2004

We have found that a soluble activity present in the postribosomal supernatant fraction of Saccharomyces cerevisiae stimulates posttranslational translocation of yeast prepro-alpha-factor across yeast microsomal membranes. Stimulation of translocation is not due to a nonspecific affect on ATP levels. The activity is likely to be due to protein(s) as it is destroyed by N-ethylmaleimide, protease, or heat treatment but not by incubation with RNase. Its apparent sedimentation coefficient is approximately 9.6 S.
Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion.
Authors: Humphries M J , Akiyama S K , Komoriya A , Olden K , Yamada K M
Published: May 2004

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH-terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.
A novel cadherin cell adhesion molecule: its expression patterns associated with implantation and organogenesis of mouse embryos.
Authors: Nose A , Takeichi M
Published: May 2004

The Ca2+-dependent cell adhesion molecules, termed cadherins, were previously divided into two subclasses, E- and N-types, with different adhesive specificity. In this study, we identified a novel class of cadherin, termed P-cadherin, using a visceral endoderm cell line PSA5-E. This cadherin was a 118,000-D glycoprotein and distinct from E- and N-cadherins in immunological specificity and molecular mass. In accord with these findings, cells with P-cadherin did not cross-adhere with cells with E-cadherin. P-Cadherin first appeared in developing mouse embryos in the extraembryonic ectoderm and the visceral endoderm at the egg cylinder stage and later was expressed in various tissues. The placenta and the uterine decidua most abundantly expressed this cadherin. The expression of P-cadherin was transient in many tissues, and its permanent expression was limited to certain tissues such as the epidermis, the mesothelium, and the corneal endothelium. When the tissue distribution of P-cadherin was compared with that of E-cadherin, we found that: each cadherin displayed a unique spatio-temporal pattern of expression; P-cadherin was co-expressed with E-cadherin in local regions of various tissues; and onset or termination of expression of P-cadherin was closely associated with connection or segregation of cell layers, as found with other cadherins. These results suggested that differential expression of multiple classes of cadherins play a role in implantation and morphogenesis of embryos by providing cells with heterogenous adhesive specificity.
Distinct molecular interactions mediate neuronal process outgrowth on non-neuronal cell surfaces and extracellular matrices.
Authors: Tomaselli K J , Reichardt L F , Bixby J L
Published: May 2004

We have compared neurite outgrowth on extracellular matrix (ECM) constituents to outgrowth on glial and muscle cell surfaces. Embryonic chick ciliary ganglion (CG) neurons regenerate neurites rapidly on surfaces coated with laminin (LN), fibronectin (FN), conditioned media (CM) from several non-neuronal cell types that secrete LN, and on intact extracellular matrices. Neurite outgrowth on all of these substrates is blocked by two monoclonal antibodies, CSAT and JG22, that prevent the adhesion of many cells, including neurons, to the ECM constituents LN, FN, and collagen. Neurite outgrowth is inhibited even on mixed LN/poly-D-lysine substrates where neuronal attachment is independent of LN. Therefore, neuronal process outgrowth on extracellular matrices requires the function of neuronal cell surface molecules recognized by these antibodies. The surfaces of cultured astrocytes, Schwann cells, and skeletal myotubes also promote rapid process outgrowth from CG neurons. Neurite outgrowth on these surfaces, though, is not prevented by CSAT or JG22 antibodies. In addition, antibodies to a LN/proteoglycan complex that block neurite outgrowth on several LN-containing CM factors and on an ECM extract failed to inhibit cell surface-stimulated neurite outgrowth. After extraction with a nonionic detergent, Schwann cells and myotubes continue to support rapid neurite outgrowth. However, the activity associated with the detergent insoluble residue is blocked by CSAT and JG22 antibodies. Detergent extraction of astrocytes, in contrast, removes all neurite-promoting activity. These results provide evidence for at least two types of neuronal interactions with cells that promote neurite outgrowth. One involves adhesive proteins present in the ECM and ECM receptors on neurons. The second is mediated through detergent-extractable macromolecules present on non-neuronal cell surfaces and different, uncharacterized receptor(s) on neurons. Schwann cells and skeletal myotubes appear to promote neurite outgrowth by both mechanisms.
Myelin instability and oligodendrocyte metabolism in myelin-deficient mutant mice.
Authors: Matthieu J M , Roch J M , Omlin F X , Rambaldi I , Almazan G , Braun P E
Published: May 2004

During the active phase of myelination in myelin-deficient mutant mice (mld), myelin basic protein (MBP) synthesis is defective and the myelin lamellae are uncompacted. In these mutants, we found a fast metabolism of the myelin-associated glycoprotein (MAG) and of sulfatides, and the presence of cholesterol esters and a degradation product of MAG, dMAG, indicating that mld myelin was unstable. The increased synthesis of MAG and Wolfgram protein, two proteins present in uncompacted myelin sheath and paranodal loops, was demonstrated by high levels of messengers. Simultaneously, we found an accumulation of inclusion bodies, vacuoles, and rough endoplasmic reticulum in mld oligodendrocytes. This material was heavily immunostained for MAG. Furthermore, the developmental change between the two molecular forms of MAG (p72MAG/p67MAG) was delayed in mld mice. In 85-d-old mld mice, the MBP content increased and myelin lamellae became better compacted. In these mutants, dMAG was absent and MAG mRNAs were found in normal amounts. Furthermore, the fine structure of mld oligodendrocytes was normal and the MAG immunostaining was similar to age-matched controls. These results support a functional role for MBP in maintaining the metabolic stability and the compact structure of myelin. Furthermore, in the absence of MBP and myelin compaction, the regulation of the synthesis of at least two membrane proteins related to myelin cannot proceed.
Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells.
Authors: Rapraeger A , Jalkanen M , Bernfield M
Published: May 2004

The cell surface proteoglycan on normal murine mammary gland mouse mammary epithelial cells consists of an ectodomain bearing heparan and chondroitin sulfate chains and a lipophilic domain that is presumed to be intercalated into the plasma membrane. Because the ectodomain binds to matrix components produced by stromal cells with specificity and high affinity, we have proposed that the cell surface proteoglycan is a matrix receptor that binds epithelial cells to their underlying basement membrane. We now show that the proteoglycan surrounds cells grown in subconfluent or newly confluent monolayers, but becomes restricted to the basolateral surface of cells that have been confluent for a week or more; Triton X-100 extraction distinguishes three fractions of cell surface proteoglycan: a fraction released by detergent and presumed to be free in the membrane, a fraction bound via a salt-labile linkage, and a nonextractable fraction; the latter two fractions co-localize with actin filament bundles at the basal cell surface; and when proteoglycans at the apical cell surface are cross-linked by antibodies, they initially assimilate into detergent-resistant, immobile clusters that are subsequently aggregated by the cytoskeleton. These findings suggest that the proteoglycan, initially present on the entire surface and free in the plane of the membrane, becomes sequestered at the basolateral cell surface and bound to the actin-rich cytoskeleton as the cells become polarized in vitro. Binding of matrix components may cross-link proteoglycans at the basal cell surface and cause them to associate with the actin cytoskeleton, providing a mechanism by which the cell surface proteoglycan acts as a matrix receptor to stabilize the morphology of epithelial sheets.
Focal adhesion sites and the removal of substratum-bound fibronectin.
Authors: Grinnell F
Published: May 2004

Fibronectin was not removed from the substratum beneath focal adhesion sites when fibroblasts spread in serum-free medium on adsorbed fibronectin substrata, or when fibroblasts spread in serum-containing medium on covalently cross-linked fibronectin substrata. Under these conditions, there was colocalization between 140-kD fibronectin receptors and focal adhesion sites. It was concluded that removal of adsorbed fibronectin from beneath focal adhesion sites was a mechanical process that required serum. The effect of serum was nonspecific since serum could be replaced by equivalent concentrations of serum albumin, ovalbumin, or gamma globulins. Quantitative measurements indicated that the presence of proteins in the incubation medium weakens the interaction of fibronectin with the substratum, thereby allowing the adsorbed protein to be removed from the substratum at sites of high stress. After removing fibronectin from the substratum, cells reorganized this material into patches and fibrils beneath cells, and the reorganized fibronectin colocalized with fibronectin receptors. Some of the patches of fibronectin were phagocytosed. The fibronectin fibrils were observed to be in register with actin filament bundles and sometimes translocated to the upper cell surfaces. It is proposed that removal of fibronectin from beneath focal adhesion sites is an example of how cells can modify their extracellular matrices through contractile activity.
The effect of neutrophil migration on epithelial permeability.
Authors: Milks L C , Conyers G P , Cramer E B
Published: May 2004

To reach an inflammatory lesion, neutrophils must frequently traverse the epithelium of an infected organ. Whether the actual migration of neutrophils alters the epithelial permeability is unknown. Through the use of an in vitro model system it was possible to directly determine the effect of neutrophil emigration on the transepithelial electrical resistance of the monolayer. Human neutrophils (5 X 10(6) cells/ml) were placed in the upper compartment of a combined chemotaxis/resistance chamber and stimulated for 40 min by a gradient of 10(-7) M n-formyl-methionyl-leucyl-phenylalanine to traverse a confluent monolayer of canine kidney epithelial cells grown on micropore filters. Neither the chemoattractant alone (10(-5)-10(-9) M) nor the accumulation of an average of eight neutrophils per millimeter of epithelium lowered the transepithelial electrical resistance. However, under certain conditions the migration of neutrophils temporarily increased the permeability of the monolayer. The resistance fell approximately 48% within 5 min if the migratory cells were stimulated to reverse their migration across the same monolayer. As re-migration continued, the resistance returned to its initial levels within 60 min. Doubling the initial neutrophil concentration to 10 X 10(6) cells/ml resulted in the accumulation of an average of 66 neutrophils per millimeter of epithelium and an average fall in resistance of 46% (r = 0.98; P less than 0.001) in 40 min. If the resistance had fallen less than 45%, removal of the neutrophils remaining in the upper compartment resulted in a return of the transepithelial electrical resistance to its initial level within 65 min. However, when the fall was greater than 45%, the resistance only recovered to 23.5% of its initial levels within the same time frame. Thus, these results suggest that the integrity of an epithelium can, under certain conditions, be affected by the emigration of neutrophils, but that this effect is either completely or partially reversible within 65 min.
Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments.
Authors: Pollard T D
Published: May 2004

I measured the rate of elongation at the barbed and pointed ends of actin filaments by electron microscopy with Limulus sperm acrosomal processes as nuclei. With improvements in the mechanics of the assay, it was possible to measure growth rates from 0.05 to 280 s-1. At 22 degrees C in 1 mM MgCl2, 10 mM imidazole (pH 7), 0.2 mM ATP with 1 mM EGTA or 50 microM CaCl2 or with EGTA and 50 mM KCl, the elongation rates at both ends have a linear dependence on the ATP-actin concentration from the critical concentration to 20 microM. Consequently, over a wide range of subunit addition rates, the rate constants for association and dissociation of ATP-actin are constant. This shows that the nucleotide composition at or near the end of the growing filament is either the same over this range of growth rates or has no detectable effect on the rate constants. Under conditions where polymerization is fastest (MgCl2 + KCl + EGTA) the rate constants have these values: (table; see text) Compared with ATP-actin, ADP-actin associates slower at both ends, dissociates faster from the barbed end, but dissociates slower from the pointed end. Taking into account the events at both ends, these constants and a simple Oosawa-type model account for the complex three-phase dependence of the rate of polymerization in bulk samples on the concentration of ATP-actin monomers observed by Carlier, M.-F., D. Pantaloni, and E. D. Korn (1985, J. Biol. Chem., 260:6565-6571). These constants can also be used to predict the reactions at steady state in ATP. There will be slow subunit flux from the barbed end to the pointed end. There will also be minor fluctuations in length at the barbed end due to occasional rapid dissociation of strings of ADP subunits but the pointed end will be relatively stable.
Reactivated melanophore motility: differential regulation and nucleotide requirements of bidirectional pigment granule transport.
Authors: Rozdzial M M , Haimo L T
Published: May 2004

To study the molecular basis for organized pigment granule transport, procedures were developed to lyse melanophores of Tilapia mossambica under conditions in which pigment granule movements could be reactivated. Gentle lysis of the melanophores resulted in a permeabilized cell model, which, in the absence of exogenous ATP, could undergo multiple rounds of pigment granule aggregation and dispersion when sequentially challenged with epinephrine and cAMP. Both directions of transport required ATP, since aggregation or dispersion in melanophores depleted of nucleotides could be reactivated only upon addition of MgATP or MgATP plus cAMP, respectively. Differences between the nucleotide sensitivities for aggregation and dispersion were demonstrated by observations that aggregation had a lower apparent Km for ATP than did dispersion and could be initiated at a lower ATP concentration. Moreover, aggregation could be initiated by ADP, but only dispersion could be reactivated by the thiophosphate ATP analog, ATP gamma S. The direction of pigment transport was determined solely by cAMP, since pigment granules undergoing dispersion reaggregated when cAMP was removed, and those undergoing aggregation dispersed when cAMP was added. These results provide evidence that pigment granule motility may be based on two distinct mechanisms that are differentially activated and regulated to produce bidirectional movements.
Immunocytochemical studies of endothelial cells in vivo. I. The presence of desmin only, or of desmin plus vimentin, or vimentin only, in the endothelial cells of different capillaries of the adult chicken.
Authors: Fujimoto T , Singer S J
Published: May 2004

It is currently believed that the intermediate filaments of endothelial cells contain vimentin subunits exclusively. This inference, however, is derived from studies of only a few types of endothelial cells. By double indirect immunofluorescence and immunoelectron microscopy, we have now examined the endothelial cells of the micro- and macrovasculature of a variety of tissues and organs of adult chicken in vivo for their content of desmin and vimentin. Endothelial cells of the peritubular capillary in the renal cortex, the hepatic sinusoid, and the splenic sinusoid were found to contain only desmin; those of the exocrine pancreas capillary contained both desmin and vimentin; and the endothelial cells of the macrovasculatures and of all the other microvasculatures examined, including the vasa recta of the renal medulla, contained only vimentin. Such heterogeneity suggests that different types of adult chicken endothelial cells may have different embryological origins. To the extent that desmin and vimentin intermediate filaments may be functionally distinct, these results also suggest that different capillary endothelial cells may have different functional properties.
A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation.
Authors: Skalli O , Ropraz P , Trzeciak A , Benzonana G , Gillessen D , Gabbiani G
Published: May 2004

A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha-smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm-1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha-smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti-desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin-negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.
Cell accumulation in the junctional region of denervated muscle.
Authors: Connor E A , McMahan U J
Published: May 2004

If skeletal muscles are denervated, the number of mononucleated cells in the connective tissue between muscle fibers increases. Since interstitial cells might remodel extracellular matrix, and since extracellular matrix in nerve and muscle plays a direct role in reinnervation of the sites of the original neuromuscular junctions, we sought to determine whether interstitial cell accumulation differs between junctional and extrajunctional regions of denervated muscle. We found in muscles from frog and rat that the increase in interstitial cell number was severalfold (14-fold for frog, sevenfold for rat) greater in the vicinity of junctional sites than in extrajunctional regions. Characteristics of the response at the junctional sites of frog muscles are as follows. During chronic denervation, the accumulation of interstitial cells begins within 1 wk and it is maximal by 3 wk. Reinnervation 1-2 wk after nerve damage prevents the maximal accumulation. Processes of the cells form a multilayered veil around muscle fibers but make little, if any, contact with the muscle cell or its basal lamina sheath. The results of additional experiments indicate that the accumulated cells do not originate from terminal Schwann cells or from muscle satellite cells. Most likely the cells are derived from fibroblasts that normally occupy the space between muscle fibers and are known to make and degrade extracellular matrix components.
Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion.
Authors: Primakoff P , Hyatt H , Tredick-Kline J
Published: May 2004

Sperm-egg plasma membrane fusion during fertilization was studied using guinea pig gametes and mAbs to sperm surface antigens. The mAb, PH-30, strongly inhibited sperm-egg fusion in a concentration-dependent fashion. When zona-free eggs were inseminated with acrosome-reacted sperm preincubated in saturating (140 micrograms/ml) PH-30 mAb, the percent of eggs showing fusion was reduced 75%. The average number of sperm fused per egg was also reduced by 75%. In contrast a control mAb, PH-1, preincubated with sperm at 400 micrograms/ml, caused no inhibition. The PH-30 and PH-1 mAbs apparently recognize the same antigen but bind to two different determinants. Both mAbs immunoprecipitated the same two 125I-labeled polypeptides with Mr 60,000 (60 kD) and Mr 44,000 (44 kD). Boiling a detergent extract of sperm severely reduced the binding of PH-30 but had essentially no effect on the binding of PH-1, indicating that the two mAbs recognize different epitopes. Immunoelectron microscopy revealed that PH-30 mAb binding was restricted to the sperm posterior head surface and was absent from the equatorial region. The PH-30 and PH-1 mAbs did not bind to sperm from the testis, the caput, or the corpus epididymis. PH-30 mAb binding was first detectable on sperm from the proximal cauda epididymis, i.e., sperm at the developmental stage where fertilization competence appears. After purification by mAb affinity chromatography, the PH-30 protein retained antigenic activity, binding both the PH-30 and PH-1 mAbs. The purified protein showed two polypeptide bands of 60 and 44 kD on reducing SDS PAGE. The two polypeptides migrated further (to approximately 49 kD and approximately 33 kD) on nonreducing SDS PAGE, showing that they do not contain interchain disulfide bonds, but probably have intrachain disulfides. 44 kD appears not to be a proteolytic fragment of 60 kD because V8 protease digestion patterns did not reveal related peptide patterns from the 44- and 60-kD bands. In the absence of detergent, the purified protein precipitates, suggesting that either 60 or 44 kD could be an integral membrane polypeptide.
Acetylated alpha-tubulin in Physarum: immunological characterization of the isotype and its usage in particular microtubular organelles.
Authors: Sasse R , Glyn M C , Birkett C R , Gull K
Published: May 2004

We have used monoclonal antibodies specific for acetylated and unacetylated alpha-tubulin to characterize the acetylated alpha-tubulin isotype of Physarum polycephalum, its expression in the life cycle, and its localization in particular microtubular organelles. We have used the monoclonal antibody 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) as the probe for acetylated alpha-tubulin and have provided a biochemical characterization of the monoclonal antibody KMP-1 as a probe for unacetylated tubulin in Physarum. Concomitant use of these two probes has allowed us to characterize the acetylated alpha-tubulin of Physarum as the alpha 3 isotype. We have detected this acetylated alpha 3 tubulin isotype in both the flagellate and in the myxameba, but not in the plasmodium. In the flagellate, acetylated tubulin is present in both the flagellar axonemes and in an extensive array of cytoplasmic microtubules. The extensive arrangement of acetylated cytoplasmic microtubules and the flagellar axonemes are elaborated during the myxameba-flagellate transformation. In the myxameba, acetylated tubulin is not present in the cytoplasmic microtubules nor in the mitotic spindle microtubules, but is associated with the two centrioles of this cell. These findings, taken together with the apparent absence of acetylated alpha-tubulin in the ephemeral microtubules of the plasmodium suggest a natural correspondence between the presence of acetylated alpha-tubulin and microtubule organelles that are intrinsically stable or cross-linked.
Transport of a fluorescent macromolecule via endosomes to the vacuole in Saccharomyces cerevisiae.
Authors: Makarow M , Nevalainen L T
Published: May 2004

Fluorescein isothiocyanate-conjugated dextran (FITC-dextran) is internalized by endocytosis into the lysosome-like vacuoles of Saccharomyces cerevisiae (Makarow, M., 1985, EMBO (Eur. Mol. Biol. Organ.) J. 4:1861-1866). Here we show that under energy depletion conditions FITC-dextran accumulated in a cytoplasmic compartment, from which it could be chased to the vacuole when the energy block was removed. The internal pH of the intermediate compartment under energy depletion was determined by fluorometry to be 5.8. The pH could be raised by the lysosomotropic agent ammonium chloride, the protonophore carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (CCCP) and the ATPase inhibitors dicyclohexylcarbodiimide (DCCD) and sodium vanadate. The pH of the vacuole was found to be 6.5. It was raised by ammonium chloride, CCCP, and DCCD, but not with sodium vanadate. Efrapeptin had no effect on the internal pH of either compartment. By dissecting the endocytic pathway, two portions of the route leading to the vacuole could be studied separately. The internalization of FITC-dextran from the extracellular fluid to the intermediate compartment followed linear kinetics, was independent of energy, and occurred at temperatures of between 15 degrees and 37 degrees C. Transfer of the marker from the intermediate compartment to the vacuole required energy, took place at temperatures between 19 degrees and 37 degrees C, and had a half-time of 7 min at 37 degrees C. Transport of the marker from the exterior of the cell to the vacuole did not require acidic pH values in the intermediate compartment or the vacuole. We suggest that the cytoplasmic compartment revealed by FITC-dextran, under energy depletion, represents the equivalent of the endosomes of mammalian cells.
Characterization of endocytic compartments using the horseradish peroxidase-diaminobenzidine density shift technique.
Authors: Ajioka R S , Kaplan J
Published: May 2004

We have employed a modification of the horseradish peroxidase (HRP)-diaminobenzidine density shift technique of Courtoy et al. (J. Cell Biol., 1984, 98:870-876) to examine the biochemical properties of the endosome. This organelle is involved in receptor recycling and the sorting of internalized receptor ligand complexes. Transferrin covalently bound to HRP was used to place peroxidase activity specifically within the endosome. The peroxidase-catalyzed polymerization of diaminobenzidine within these vesicles causes an increase in buoyant density, thus allowing them to be separated from other membranes. Using this technique we demonstrate that 125I-low density lipoprotein, 131I-epidermal growth factor, and Tf-HRP are internalized into the same endosome. We discovered that the diaminobenzidine reaction product "cross-links" the lumen of the vesicle, rendering vesicular components detergent insoluble. Furthermore, the reaction inactivates enzymatic activities associated with the endosome. Thus, the diaminobenzidine density shift procedure has limited usefulness in studies designed to isolate endosomal constituents. Nonetheless, we have found that the inactivation of enzymatic activities is confined to those endosomes that contain peroxidase. This selectivity allows us to define endosome-specific activities.
Acetylcholine receptor clustering and nuclear movement in muscle fibers in culture.
Authors: Englander L L , Rubin L L
Published: May 2004

We have studied the formation of acetylcholine receptor (AChR) clusters and the behavior of myonuclei in rat and chick skeletal muscle cells grown in cell culture. These cells were treated with a factor derived from Torpedo electric extracellular matrix, which causes a large increase in their number of AChR clusters. We found that these clusters were located preferentially in membrane regions above myonuclei. This cluster-nucleus colocalization is explained by our finding that most of the nuclei near clusters remain relatively stationary, while most of those away from clusters are able to translocate throughout the myotube. In some cases, clusters clearly formed first, then nuclei migrated underneath and became immobilized. If clustered AChRs later dispersed, their associated nuclei resumed moving. These results suggest that AChR clustering initiates an extensive cytoskeletal rearrangement that causes the subcluster localization of organelles, potentially providing a stable source of newly synthesized AChRs for insertion into the cluster.
U1 precursors: variant 3' flanking sequences are transcribed in human cells.
Authors: Patton J G , Wieben E D
Published: May 2004

Using RNase protection and oligonucleotide hybridization experiments, we have shown that U1 precursors are derived by transcription of 3' flanking sequences. A labeled SP6 transcript of one of the true U1 genes (pD2) was able to protect a subset of the 3' flanking sequences present in HeLa cytoplasmic U1 RNA. However, not all U1 precursors were protected using this probe, suggesting that variant U1 precursor 3' tail sequences are expressed in HeLa cells. This conclusion has been confirmed by hybridization of HeLa RNA samples with specific oligonucleotide probes representing variant U1 3' flanking sequences. Interestingly, these variant tail sequences contain the putative Sm antigen binding site, A(U)3-6G. The conservation of this flanking sequence through evolution suggests a possible functional role for these precursor tails in ordering protein binding to U1 RNA.
Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site-specific antibodies.
Authors: De Lemos-Chiarandini C , Frey A B , Sabatini D D , Kreibich G
Published: May 2004

Fifteen peptides, ranging in length from 6 to 31 amino acids and corresponding in sequence to portions of the major phenobarbital-inducible form of rat liver cytochrome P-450 (P-450 PB-4), were previously synthesized chemically and used to prepare site-specific rabbit antibodies (Frey, A. B., D.J. Waxman, and G. Kreibich, 1985, J. Biol. Chem., 260:15253-15265). The antipeptide antibodies were affinity purified using Sepharose resins derivatized with the respective peptides and 14 preparations were obtained that in an ELISA assay showed affinities to immobilized P-450 judged to be adequate for binding studies on intact rat liver microsomes. The binding of these antibodies to rough microsomes from the livers of phenobarbital treated rats was assessed using 125I-labeled IgG and by immunoelectron microscopy employing protein A-gold as a marker. It was found that many of the antibodies bound to the cytoplasmic surface of the membrane but none bound to the luminal face of ruptured or inverted microsomal vesicles or to contaminating membranes of other organelles present in the preparations. These observations eliminate previously proposed models for the transmembrane disposition of P-450 that postulate the existence of multiple transmembrane domains and the exposure of several polar segments of the polypeptide on the luminal side of the membrane. The fact that an antibody raised to the first 31 residues of P-450 bound well to the purified P-450 but very poorly to rough microsomes, whereas an antibody to a peptide comprising residues 24-38 showed relatively strong binding to intact microsomes, is consistent with the proposal that the amino terminal segment of P-450 extending approximately to residue 20 is embedded in the phospholipid bilayer and the immediately following segment is exposed on the cytoplasmic surface of the membrane. All these results favor a model in which the cytochrome P-450 molecule is largely exposed on the cytoplasmic surface of the endoplasmic reticulum membrane to which it is anchored by its short amino terminal hydrophobic segment.
Microtubule-acting drugs lead to the nonpolarized delivery of the influenza hemagglutinin to the cell surface of polarized Madin-Darby canine kidney cells.
Authors: Rindler M J , Ivanov I E , Sabatini D D
Published: May 2004

The synchronized directed transfer of the envelope glycoproteins of the influenza and vesicular stomatitis viruses from the Golgi apparatus to the apical and basolateral surfaces, respectively, of polarized Madin-Darby canine kidney (MDCK) cells can be achieved using temperature-sensitive mutant viruses and appropriate temperature shift protocols (Rindler, M. J., I. E. Ivanov, H. Plesken, and D. D. Sabatini, 1985, J. Cell Biol., 100:136-151). The microtubule-depolymerizing agents colchicine and nocodazole, as well as the microtubule assembly-promoting drug taxol, were found to interfere with the normal polarized delivery and exclusive segregation of hemagglutinin (HA) to the apical surface but not with the delivery and initial accumulation of G on the basolateral surface. Immunofluorescence analysis of permeabilized monolayers of influenza-infected MDCK cells treated with the microtubule-acting drugs demonstrated the presence of substantial amounts of HA protein on both the apical and basolateral surfaces. Moreover, in cells infected with the wild-type influenza virus, particles budded from both surfaces. Viral counts in electron micrographs showed that approximately 40% of the released viral particles accumulated in the intercellular spaces or were trapped between the cell and monolayer and the collagen support as compared to less than 1% on the basolateral surface of untreated infected cells. The effect of the microtubule inhibitors was not a result of a rapid redistribution of glycoprotein molecules initially delivered to the apical surface since a redistribution was not observed when the inhibitors were added to the cells after the HA was permitted to reach the apical surface at the permissive temperature and the synthesis of new HA was inhibited with cycloheximide. The altered segregation of the HA protein that occurs may result from the dispersal of the Golgi apparatus induced by the inhibitors or from the disruption of putative microtubules containing tracks that could direct vesicles from the trans Golgi apparatus to the cell surface. Since the vesicular stomatitis virus G protein is basolaterally segregated even when the Golgi elements are dispersed and hypothetical tracks disrupted, it appears that the two viral envelope glycoproteins are segregated by fundamentally different mechanisms and that the apical surface may be incapable of accepting vesicles carrying the G protein.
Phasic release of newly synthesized secretory proteins in the unstimulated rat exocrine pancreas.
Authors: Arvan P , Castle J D
Published: May 2004

Pancreatic lobules from fasted rats secrete pulse-labeled proteins in two phases comprising 15 and 85% of basal output, respectively. The first (0-6.5 h) is initially (less than or equal to 0.5 h) unstimulated by secretagogues, probably represents vesicular traffic of Golgi and post-Golgi origin (including condensing vaculoles/immature granules), and notably contains two groups of polypeptides with distinct release rates: zymogens (t1/2 approximately 2.4 h) and minor nonzymogens plus one unique zymogen (t1/2 approximately 1 h). The second phase (peak at 9-10 h) is stimulable, probably represents basal granule exocytosis (t1/2 approximately 5 h), and contains zymogens exclusively. Newly synthesized proteins released in both phases appear asynchronously, reiterating their asynchronous transport through intracellular compartments. Zymogens in both phases are secreted apically. The sorting of first from second phase zymogen release does not appear to be carrier-mediated, although the sorting of zymogens from other secretory proteins may use this process. Finally, data are presented that suggest that both secretory phases are subject to physiologic regulation.
Dynamic and stable populations of microtubules in cells.
Authors: Schulze E , Kirschner M
Published: May 2004

Using a new immunocytochemical technique, we have visualized the spatial arrangement of those microtubules in cells that are stable to biotin-tubulin incorporation after microinjection. Cells fixed at various periods of time after injection were exposed to antibody to biotinylated tubulin and several layers of secondary antibodies; these layers prevented reaction of biotin-containing microtubules with antitubulin antibodies. The microtubules that had not incorporated biotin-tubulin could then be stained with anti-tubulin and a fluorescent secondary antibody. In BSC1 cells, most microtubules in the cell exchange with a half-time of 10 min. A separate population of microtubules can be detected, using the above techniques, that are stable to exchange for 1 h or more; these have a characteristic pericentrosomal spatial arrangement as compared to the majority of dynamic microtubules. Unlike the dynamic microtubules, most of the stable microtubules are nongrowing. The average BSC-1 cell contains approximately 700 microtubules: approximately 500 growing at 4 micron min-1, 100 shrinking at approximately 20 micron min-1, and approximately 100 that are relatively more stable to exchange. The potential significance of these stable microtubules is discussed.