bluuebunny/crossref_metadata_2025 huggingface dataset:
A distinct signal peptidase for prolipoprotein in escherichia coli
Authors: Tokunaga Masao , Loranger Judith M. , Wu Henry C.
Published: December 2004

We have previously demonstrated the modification and processing of Escherichia coli prolipoprotein (Braun's) in vitro (Tokunaga M, Tokunaga H. Wu HC: Proc Natl Acad Sci USA 79:2255, 1982). Using this in vitro assay of prolipoprotein signal peptidase and globomycin selection, we have isolated and partially characterized an E coli mutant which contained a higher level of prolipoprotein signal peptidase activity. In contrast, the procoat protein signal peptidase activity was not increased in this mutant as compared to the wild‐type strain. Furthermore, E coli strains containing cloned procoat protein signal peptidase gene were found to contain elevated levels of procoat protein signal peptidase, but normal levels of prolipoprotein signal peptidase. These two signal peptidase activities were also found to exhibit different stabilities during storage at 4°C. Thus biochemical, immunological, and genetic evidence clearly indicate that prolipoprotein signal peptidase is distinct from procoat protein signal peptidase in E coli.
ATP‐released large subunits participate in the assembly of RuBP carboxylase
Authors: Milos Patrice , Roy Harry
Published: December 2004

Preincubation of 35S‐methionine‐labeled chloroplast extracts with ATP at 0°C potentiates the subsequent assembly of labeled large subunits into RuBPCase. This is correlated with the dissociation of newly synthesized large subunits from the 29S large subunit binding protein complex. These released large subunits then assemble into RuBPCase in a second, nucleotide‐stimulated reaction. The data demonstrate that the 29S complex can play an active role in the assembly of RuBPCase.
Adaptive reorganization of protein and lipid components in chloroplast membranes as associated with herbicide binding
Authors: Mattoo Autar K. , St. John Judith B. , Wergin William P.
Published: December 2004

Cultivation of Spirodela oligorrhiza (Kurtz) Hegelm on a sublethal dose of atrazine results in a higher linolenic to linoleic acid ratio in the thylakoid membrane lipids, less starch, more osmiophilic globules, and a reduced stroma lamellar system. Also, the grana become randomly oriented and contain more numerous and elongated lamellae. These alterations in the lipid composition and ultrastructure of the chloroplast resemble those previously observed in triazine‐resistant weed biotypes and in chloroplasts developed under low light. Thylakoid membranes from atrazine‐adapted plants revealed an additional high‐affinity binding constant for [14C]‐diuron but the number of diuron binding sites actually decreased by 20 times compared to controls. The 32,000‐dalton membrane protein of the chloroplast is synthesized actively, but its breakdown appears decreased compared to control plants. The adaptive reorganization of thylakoid components may be a compensatory mechanism for maintenance of a functional interaction of the proteins and lipids of the photosystem II complex.
Fate of surface immunoglobulin during induction of lymphocyte proliferation
Authors: Ramanadham Madduri , Gollapudi Sastry V. S. , Kern Milton
Published: December 2004

The modulation of immunoglobulin on the surface of rabbit B lymphocytes by goat antibodies with specificity for rabbit surface membrane immunoglobulin or by such goat antibodies covalently linked to Sepharose was studied in relation to the proliferative response to these agents. Although the induction of DNA synthesis was greater in the presence of Sepharose‐linked antibody than in the presence of free antibody, modulation of surface membrane immunoglobulin was induced with free but not with Sepharose‐linked antibody. Thus, in the presence of free antibody the surface membrane immunoglobulin content of cells was rapidly decreased and remained at a low level throughout the culture period, whereas the surface immunoglobulin content of cells incubated with Sepharose antibody was essentially unaltered. The surface immunoglobulin lost from cells incubated with free goat antibodies reappeared slowly upon further incubation in culture medium devoid of antibody, and such reappearance of rabbit surface membrane immunoglobulin was inhibited by puromycin. Upon culture with Sepharose‐linked antibody the surface membrane immunoglobulin content of B cells was unaffected by puromycin. This result was interpreted as indicating that surface membrane immunoglobulin loss followed by reappearance does not occur. Lastly, the linkage of surface membrane immunoglobulin to cytoskeletal elements induced by free antibody was not induced by Sepharose‐linked antibody as judged from differences in detergent solubilization characteristics. Possible mechanisms to account for these differences in surface membrane immunoglobulin modulation as they relate to the proliferative response are considered.
Correlation of structure and function of chloroplast membranes at the supramolecular level
Authors: Staehelin L. Andrew , DeWit Marcia
Published: December 2004

Freeze‐fracture electron microscopy has revealed that different size classes of intramembrane particles of chloroplast membranes are nonrandomly distributed between appressed grana and nonappressed stroma membrane regions. It is now generally assumed that thylakoid membranes contain five major functional complexes, each of which can give rise to an intramembrane particle of a defined size. These are the photosystem II complex, the photosystem I complex, the cytochrome f/b6 complex, the chlorophyll a/b light‐harvesting complex, and the CF0‐CF1 ATP synthetase complex. By mapping the distribution of the different categories of intramembrane particles, information on the lateral organization of functional membrane units of thylakoid membranes can be determined. In this review, we present a brief summary of the evidence supporting the correlation of specific categories of intramembrane particles with known biochemical entities. In addition, we discuss studies showing that ions and phosphorylation of the membrane adhesion factor, the chlorophyll a/b light‐harvesting, complex, can affect the lateral organization of chloroplast membrane components and thereby regulate membrane function.
Molecular species of epidermal growth factor carrying immunosuppressive activity
Authors: Koch J. H. , Fifis T. , Bender V. J. , Moss B. A.
Published: December 2004

The suppression of antibody formation to sheep red cells in mice by partially purified fractions of mouse submaxillary gland [7] was shown to be caused by epidermal growth factor (EGF). Purification of EGF by the method of Savage and Cohen [11] resolved three components referred to as EGF a, EGF b, and EGF c. All three induced premature eye opening in neonatal mice, but only EGF a (identified as EGF1‐53 ) had full immunosuppressive activity. EGF c was shown by micropeptide mapping of chymotryptic and thermolytic digests and aminoterminal analysis to differ from EGF a only by the presence of β‐aspartyl instead of an asparaginyl residue. EGF b differed from EGF a in that it lacked the N‐terminal asparagine. EGF shortened enzymatically at its carboxy terminal by two or five amino acids did not have any immunosuppressive activity. These findings‐suggest that, in contrast to some other biological effects of EGF, intact amino and carboxy terminals are required for the expression of immunosuppressive activity.
Stimulation of glycosaminoglycan production in murine tumors
Authors: Knudson Warren , Biswas Chitra , Toole Bryan P.
Published: December 2004

Three types of murine tumors, B‐16 melanoma, A‐10 carcinoma, and S‐180 sarcoma, were shown to contain elevated glycosaminoglycan (GAG) concentrations in vivo as compared to normal muscle or subcutaneous tissue. Hyaluronate was especially concentrated in the A‐10 carcinoma, which contained approximately six times more hyaluronate than subcutaneous tissue and 18 times more than muscle. In all three tumors, chondroitin sulfates, especially chondroitin‐4‐sulfate, were present in higher concentrations than in the. normal tissues. In culture, however, all three tumor cell lines produced less than 5% as much GAG as mouse fibroblasts, when measured by incorporation of [3H] acetate or by chemical analysis. Varying the culture passage number or the medium composition, ie, glucose, serum, and insulin concentrations, had little effect on GAG synthesis by the tumor cells. The low GAG levels in the tumor cell cultures were not due to hyaluronidase activity in their media. In an attempt to mimic possible host‐tumor cell interactions that could account for the elevated GAG levels in vivo, tumor cells were cocultured with fibroblasts, but no stimulation above the amount made by the tumor cells alone plus that by the fibroblasts alone was observed. Conditioned media from the tumor cells, either dialyzed or not against fresh complete medium, had no effect on fibroblast GAG synthesis. Tumor extracts, however, were found to stimulate synthesis of hyaluronate by fibroblasts. Stimulation by extracts of A‐10 carcinoma was greater than and additive to that of serum. The above results strongly suggest that GAG production in these tumors is in pail regulated by host‐tumor interactions.
Enzymatic characteristics of pp60v‐src isolated from vanadium‐treated transformed cells
Authors: Collett Marc S. , Belzer Susan K. , Kamp Lisa E.
Published: December 2004

The transforming protein of Rous sarcoma virus (RSV) typically appears as a single phosphorylated polypeptide designated pp60v‐src, pp60v‐src possesses a protein kinase activity specific for tyrosine residues on select protein substrates. Treatment of RSV‐transformed cells with vanadium ions resulted in the appearance of an electrophoretic variant of pp60v‐src and was paralleled by a significant increase in the src kinase specific activity in purified enzyme preparations. Both the normal (standard) src kinase and the src kinase preparations obtained from vanadium‐treated cells exhibited similar optimal activity profiles for MgCl2, KCl, and pH. Furthermore, their site specificities of phosphorylation of the substrates casein and vinculin were the same. The reaction kinetic profile of the standard src kinase showed a nonlinear pattern, while the vanadium enzyme exhibited conventional linear Michaelis‐Menten kinetics. These results are discussed with respect to the possible functional regulation of pp60v‐src activity by a vanadium‐sensitive protein phosphatase activity.
A 38 base pair insertion in the proα2(I) collagen gene of a patient with Marfan syndrome
Authors: Henke Elizabeth , Leader Mark , Tajima Shingo , Pinnell Sheldon , Kaufman Russel
Published: December 2004

Abnormalities in type I collagen have been recognized in a number of connective tissue disorders. In the Marfan syndrome, an autosomal dominant condition producing a generalized abnormality in connective tissue, no consistent abnormality has been identified, although one individual has been found to have an elongated proα2(I) collagen chain [Byers et al, Proc Natl Acad Sci USA 78:7745, 1981]. To determine the nature of the alteration in the gene that produced this abnormality, we studied the proα2(I) gene from this individual by genomic blotting and gene cloning. Genomic mapping studies detected no abnormalities. However, analysis of the cloned segment of the proα2(I) collagen gene from the Marfan individual indicates that the gene contains a 38 base pair insertion in an intron near the collagenase cleavage site. Although the relationship of this insertion to the protein abnormality is unclear, it may be a useful marker for the diagnosis of the Marfan syndrome.
Phospholipid and Ca++ dependency of phorbol ester receptors
Authors: König Bernhard , Di Nitto Patricia A. , Blumberg Peter M.
Published: December 2004

The phospholipid and Ca++ dependency of a partially purified phorbol ester apo‐receptor from the soluble fraction of mouse brain homogenates was studied. This apo‐receptor is believed to be identical with the Ca++ and phospholipid‐dependent protein kinase C. Binding of phorbol esters to the receptor/kinase C was shown to be entirely dependent on phospholipids. The negatively charged phospholipids phosphatidylserine, phosphatidylinositol, and phosphatidic acid all fully reconstituted binding. The neutral phospholipids were inactive. Among active phospholipids and mixtures of phospholipids, substantial differences ( > 100‐fold) were observed in the amounts required to achieve reconstitution. Although Ca++ was not required for reconstitution of binding activity, it dramatically (up to 100‐fold) increased the potency of phospholipids for reconstitution. The phospholipids not only permitted reconstitution of the apo‐receptor but also played a major role in determining the binding characteristics of the complex. The KD values of [3H]phorbol 12,13–dibutyrate were in the range of 0.8 nM for the complex with phosphatidylserine to 30 nM for the complex with diolcoyl‐phosphatidic acid. Like the binding affinity, the stimulation of protein kinase C activity by phorbol esters was dependent on the phospholipid into which the receptor/kinase C was reconstituted. The importance of the lipid domain for controlling the receptor/kinase C activity and for modulation of cellular sensitivity to phorbol esters is discussed.
Basement membrane component changes in skeletal muscle transplants undergoing regeneration or rejection
Authors: Gulati Adarsh K.
Published: December 2004

The basement membrane of myofibers plays an important role during orderly regeneration of skeletal muscle after injury. In this report, changes in various basement membrane components were analyzed in skeletal muscle grafts undergoing regeneration (autografts) or immune rejection (allografts). The immunofluorescence technique using specific antibodies against laminin, types IV and V collagen, heparan sulfate proteoglycan, fibronectin, in combination with binding of concanavalin A (ConA) was used to monitor basement membranes. In normal muscle, these components were localized in the pericellular region of myofiber corresponding to its basement membrane, After transplantation, the majority of myofibers underwent degeneration as a result of is chemic injury, followed by regeneration from precursor myosatellite cells. Various components of basement membrane zone disappeared from the degenerating myofibers, leaving behind some unidentifiable component that still bound ConA. A new basement membrane appeared around the regenerated myotubes which persisted during maturation of the regenerating muscle, In rejected skeletal muscles, the immunoreactivity of various components persisted even after the disappearance of myotubes and myofiber cytoplasm. In addition, an accumulation of fibronectin was seen throughout the rejected muscle with the onset of immune rejection. These results demonstrate that the major basement membrane components disappear and reappear sequentially during myofiber degeneration and regeneration. Such a turnover is not seen in rejected skeletal muscles. Thus, the myofiber basement membrane is not a static structure as previously thought but one which changes chemically during degeneration and regeneration. This feature of basement membrane may be important in the orderly regeneration of skeletal muscle after injury.
Enzymes converting procollagens to collagens
Authors: Peltonen L. , Halila R. , Ryhänen L.
Published: December 2004

Conversion from procollagen to collagen is a specific process that is a requirement for proper alignment of collagen molecules to form functional fibers. This process is catalyzed by at least three structurally and functionally distinct enzymes cleaving collagen types I–III. The cleavage processes possibly taking place in the more recently discovered collagen types are not known to any extent at this time.

Two amino‐terminal proteinases, one cleaving type I and type II procollagens and the other cleaving type III procollagen, have been purified close to homogeneity, and the more unspecific activity of carboxy‐terminal proteinase has been isolated from several tissues. In our experimental model, however, cleavage of the carboxy‐terminal propeptides of types I and III procollagen is differently affected by lysine. This suggests the presence of at least two distinct enzymes for the removal of carboxyl‐terminal propeptides.

The regulation of the reaction process from procollagen to collagen is not well known at present. The importance of the phenomenon in terms of fibril formation, however, is demonstrated by several elegant studies in vitro; and certain genetic disorders in which this process is defective demonstrate the significance in vivo. Moreover, the factors shown to effect the cleavage process may be potentially beneficial in the treatment of the pathological processes with abnormal collagen accumulation such as fibrosis.

In this paper we briefly review the current knowledge of the converting enzymes, including some very recent findings of our laboratory as well as the evidence presented for the biological significance of the conversion process.
Human keratinocytes that have not terminally differentiated synthesize laminin and fibronectin but deposit only fibronectin in the pericellular matrix
Authors: Clark Richard A. F. , Nielsen Larry D. , Howell Samuel E. , Folkvord Joy M.
Published: December 2004

Fibronectin and laminin production by human keratinocytes cultured in serum‐free, low‐calcium medium without a fibroblast feeder layer were examined by several techniques. By indirect immunofluorescence, fibronectin but not laminin appeared as short radial fibrils between the cells and the substratum, and in the pericellular matrix. Synthesis of fibronectin and laminin by 7‐day keratinocyte cultures was determined by 18 hr 35S‐methionine metabolic labeling followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Fibronectin accounted for 2.9% of total synthesized protein, 26.5% of fluid phase protein secretion, and 4.3% of deposited ECM protein. In contrast, only 0.1% of the total synthesized protein was laminin, little (6.3%) of this product was secreted, and none of this product was deposited in the ECM. Our results indicate that human keratinocytes under culture conditions that prevent terminal differentiation in vitro can synthesize, secrete, and deposit fibronectin in the extracellular matrix. Although these cells synthesize laminin, they secrete very little and deposit no detectable laminin in the matrix under these culture conditions. From these data we believe that fibronectin may play an important role in the interaction of epidermal cells with connective tissue matrix during wound healing or morphogenesis in in vivo situations in which the epidermis is not terminally differentiated.
Immunochemical and biochemical characterization of a glioma‐associated extracellular matrix glycoprotein
Authors: Bourdon Mario A. , Matthews Thomas J. , Pizzo Salvatore V. , Bigner Darell D.
Published: December 2004

A novel human glioma‐associated extracellular matrix (ECM) glycoprotein has been identified by murine monoclonal antibody 81C6. The glycoprotein, designated GMEM, is expressed in the ECM of glioma and mesenchymal cell cultures, in the perivascular matrix of endothelial proliferations of human gliomas, and in the stroma of human glioma xenografts in athyrnic mice, where it has been used as a target antigen for monoclonal antibody tumor localization and radioimaging. We report here on the immunochemical and biochemical characterization of GMEM. Polyacrylamide gel analysis of immunoprecipitated [3H]‐leucine‐ and [3H]‐glucosamine‐labeled ECM from the human glioma cell line U‐251MG has shown that GMEM is a high‐molecular‐weight macromolecule (Mr ∼ 1,000,000) composed of Mr ∼ 230,000 disulfide‐bonded glycoprotein subunits. Immunoprecipitation, immunoblot, and one‐dimensional peptide map analysis have shown that GMEM is distinct from human fibroblast and plasma fibronectin. These results support previous immunohistology and absorption analysis findings, indicating that GMEM is distinct from fibronectin, laminin, and glycosaminoglycans secreted by U‐251MG.
Pteridine formation during lectin‐induced lymphocyte activation
Authors: Ziegler Irmgard
Published: December 2004

After iodine oxidation, biopterin, 6‐hydroxymethylpterin, and 6‐formylpterin were identified in mouse spleen lymphocytes by means of reverse‐phase HPLC, Crithidia assay, and oxidative degradation. Concanavalin A activation induces a 30‐fold increase in the pteridine amounts; biopterin as well as the sum of the carbinol and the aldehyde attain levels of 6–8 × 10−12 mol/106 cells. The most rapid increase occurs during the first 24 hr. Thus, pteridine accumulation precedes the period of lymphocyte proliferation; maximum DNA synthesis was found after 72 hr. Biopterin remains largely inside the cells, whereas 6‐hydroxymethylpterin and 6‐formylpterin were found in the supernatant if the stimulated cells were subsequently incubated in a phosphate buffered salt solution (PBS). Isoxanthopterin was found in the PBS supernatant of control cells that previously were kept in medium alone rather than subjected to lectin stimulation. Only minimal amounts were found inside these cells, and this pterin was absent from the stimulated lymphocytes. The early increase in cellular pteridines and their differential release may well provide the basis for their modulating effect on interleukin‐2 activity (Ziegler I, et al: Lymphokine Research 3:284, 1984).
Cell surface galactosyltransferase: Immunochemical localization
Authors: Shaper Nancy L. , Mann Paul L. , Shaper Joel H.
Published: December 2004

A cell surface UDP‐galactose:N‐acetylglucosamine galactosyltransferase (GT) has been directly localized on bovine cells in tissue culture by immunohistochemical techniques. A conventional rabbit heteroantiserum was prepared against an affinity‐purified soluble form of GT from bovine milk, and a monospecific IgG fraction was isolated by affinity chromatography on a GT adsorbent. As demonstrated by indirect immunofluorescence, antigen to this antibody is present on the surface of all three bovine cell lines tested. It was uniformly distributed over the exposed membrane surface of fixed cells. Exposure of living cells to the anti‐GT antibody resulted in its time‐dependent aggregation in the plane of the membrane. Antigen (GT) was released from the membrane surface by trypsin digestion, and its reappearance required protein synthesis, since cycloheximide effectively prevented repopulation of the cell surface.
Stoichiometric binding of diacylglycerol to the phorbol ester receptor
Authors: König Bernhard , DiNitto Patricia A. , Blumberg Peter M.
Published: December 2004

The major phorbol ester receptor is the Ca++‐activated, phospholipid‐dependent protein kinase C. Diacylglycerol stimulates protein kinase C in a fashion similar to the phorbol esters. Likewise, it inhibits phorbol ester binding competitively. Both results suggest that diacylglycerol is the/an endogenous phorbol ester analogue. Alternatively, the diacylglycerol might simply be acting to modify the phospholipid environment of the protein. If diacylglycerol were indeed functioning as an analogue, it should interact with the receptor stoichiometrically. This interaction can be quantitated by measuring the perturbation in apparent diacylglycerol binding affinity as a function of the ratio of diacylglycerol to receptor. We report here that 1,2‐dioleoylglycerol interacts with the receptor with the predicted stoichiometry.
Ankyrin and synapsin: Spectrin‐binding proteins associated with brain membranes
Authors: Bennett Vann , Baines Anthony J. , Davis Jonathan Q.
Published: December 2004

Brain membranes contain an actin‐binding protein closely related in structure and function to erythrocyte spectrin. The proteins that attach brain spectrin to membranes are not established, but, by analogy with the erythrocyte membrane, may include ankyrin and protein 4.1. In support of this idea, proteins closely related to ankyrin and 4.1 have been purifed from brain and have been demonstrated to associate with brain spectrin. Brain ankyrin binds with high affinity to the spectrin beta subunit at the midregion of spectrin tetramers. Brain ankyrin also has binding sites for the cytoplasmic domain of the erythrocyte anion channel (band 3), as well as for tubulin. Ankyrins from brain and erythrocytes have a similar domain structure with protease‐resistant domains of Mr = 72,000 that contain spectrin‐binding activity, and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the union channel. Brain ankyrin is present at about 100 pmol/mg membrane protein, or about twice the number of copies of spectrin beta chains. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes, and it has the potential to attach microtubules to membranes as well as to interconnect microtubules with spectrin‐associated act in filaments.

Another spectrin‐binding protein has been purified from brain membranes, and this protein cross‐reacts with erythrocyte 4.1. Brain 4.1 is identical to the membrane protein synapsin, which is one of the brain's major substrates for cAMP‐dependent and Ca/calmodulin‐dependent protein kinases with equivalent physical properties, immunological cross‐reaction, and peptide maps. Synapsin (4.1) is present at about 60 pmol/mg membrane protein, and thus is a logical candidate to regulate certain protein linkages involving spectrin.
Comparative analysis of p21 proteins from various cell types by two‐dimensional gel electrophoresis
Authors: Feuerstein Nili , Ali Iqbal Unnisa
Published: December 2004

The products of the ras gene family arc related proteins at a molecular weight of 21 kDa, designated p21. In the present study we used two‐dimensional gel electrophoresis to compare p21 proteins from five different normal and malignant cell lines. Using a known protein (3H‐labeled translation initiation factor [elF‐4D]) as a standard internal marker for isoelectric point (pI), we show that p2l proteins from various cells differ only slightly in molecular weight (21–24 kDa) but express a wide variety in charge (pl 4.8 to 7) that could only be detected by the use of two‐dimensional gel electrophoresis. p21 in NIH/3T3 cells was expressed as a single protein, which migrated at 21 kDa and pl 5.1. This peptide, which is probably the product of the normal cellular ras gene, was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIH/3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p21 peptides. Four additional p21‐associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten murine sarcoma virus‐transformed NIH/3T3 cells. Transformation of cells with Harvey murine sarcoma virus was found to be associated with prominent expression of two major pairs of p21‐associated proteins, one at 21 kDa (pl, 5.2 and 5.3) and the other at 23 kDa (pl, 5.1 and 5.2). In HL‐60 leukemic cells there was an additional, more acidic form (pl 5.0) of p21, which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p21 and slight charge modifications associated with malignancy should call for the use of 2‐D gel electrophoresis as an important tool in future studies involving p21 proteins.
Functional ion channel formation by mouse macrophage IgG Fc receptor triggered by specific ligands
Authors: Young John Ding‐E , Unkeless Jay C. , Cohn Zanvil A.
Published: December 2004

The mouse macrophage Fcγ 2b/γ 1R has previously been purified with the aid of the monoclonal antibody 2.4G2. That this FcγR functions as a ligand‐dependent ion channel is supported by the following evidence: (a) Employing [3H]tetraphenylphosphonium ([3H]Ph4P+) as a probe for membrane potential changes in intact cells, we found a biphasic change in membrane potential following treatment with immune complexes, monoclonal antibody 2.4G2 IgG and 2.4G2 Fab‐Sephadex particles. We observed an immediate depolarization followed by prolonged hyperpolarization. (b) [3H]Ph4P+ uptake experiments with plasma membrane vesicles prepared from J774 macrophages showed that binding of ligands to the FcR led to transmembrane monovalent cation flow. (c) Similar [3H]Ph4P+ uptake experiments were done with phospholipid vesicles containing purified and reconstituted Fcγ 2b/γR. Following challenge with specific ligands, transmembrane monovalent cation flow was observed. (d) Purified FcR was reconstituted into planar lipid bilayers; exposure to ligands led to transient bilayer conductance increase. The conductance change was resolved into single channel events.

Quin‐2 measurements showed an increase of free cytosolic calcium levels in macrophages following exposure of cells to different ligands of the FcR. An optimal range of calcium was found to be required for phagocytosis, below and above which inhibition of ingestion occurred.
Nerve growth factor biosynthesis: Isolation and characterization of a guinea pig prostate kallikrein
Authors: Dunbar Joan C. , Bradshaw Ralph A.
Published: December 2004

Guinea pig prostate contains one major soluble esteropeptidase activity. The protein has been purified and characterized and found to be a glycoprotein comprised of a single polypeptide chain. The molecular weight of the deglycosylated protein is approximately 26,000. The esteropeptidase has a similar Km for lysine and arginine synthetic substrates, although the Vmax for arginine is much greater than that for lysine. Amino‐terminal sequence analysis has also revealed a marked degree of homology to mouse γ‐nerve growth factor (NGF) and the kallikrein family of serine proteases. In contrast to γ‐NGF, however, the guinea pig enzyme does not appear to form stable complexes with β‐NGF.
Aluminum ions stimulate mitosis in murine cells in tissue culture
Authors: Jones Trevor R. , Antonetti Donna L. , Reid Ted W.
Published: December 2004

Addition of aluminum to the culture medium of Nakano mouse lens epithelial (NMLE) cells and Swiss 3T3K cells induced both 3H‐thymidine incorporation and mitosis. This is in contrast to other metal ions such as vanadium, which, at concentrations high enough to increase 3H‐thymidine incorporation, actually inhibits mitosis (Jones and Reid, J Cell Physiol 121:199, 1984 [1]). Aluminum concentrations between 20 μM and 50 μM were most effective. The 3T3 cells respond to aluminum with a 7.6‐fold increase, and NMLE cells respond with a 21‐fold increase in 3H‐thymidine incorporation. DNA synthesis in NMLE cells was also found to be synergistically stimulated by aluminum and low concentrations of insulin (4.5 × 10−8 M). A 3.25‐hr incubation with 50 μM aluminum was sufficient to induce 50% of maximum 3H‐thymidine incorporation during the 40‐hr assay. Aluminum‐stimulated 3H‐thymidine incorporation is inhibited by hydroxyurea, and aluminum causes an increase in cell number. Also, by sedimentation equilibrium analysis of the product of aluminum‐stimulated DNA synthesis it was found that a single copy of DNA was synthesized following addition of aluminum to quiescent cells. These facts indicate that aluminum induces both S‐phase DNA synthesis and mitosis. However, only 48% of the NMLE cells found to be labeled with DNA went on to divide. In contrast, although only a small percentage of 3T3 cells were found to be labeled after aluminum treatment, all of these cells appeared to go through mitosis.
Mechanisms of cytoskeletal regulation: Functional and antigenic diversity in human erythrocyte and brain beta spectrin
Authors: Harris Alan S. , Anderson John P. , Yurchenco Peter D. , Green L. A. David , Ainger Kevin J. , Morrow Jon S.
Published: December 2004

A study of human erythrocyte and brain spectrin with particular emphasis on the beta subunits revealed a structural homology but functional dissimilarity between these two molecules. Six monoclonal antibodies raised to human erythrocyte beta spectrin identify three of the four proteolytically defined domains of erythrocyte beta spectrin. Five of these monoclonal antibodies cross‐react with human brain spectrin. None of a previously identified set of alpha erythrocyte spectrin monoclonal antibodies [Yurchenco et al: J Biol Chem 257:9102, 1982] reacted with brain spectrin. A domain map generated by limited tryptic digestion shows that brain spectrin is composed of proteolytically resistant domains analogous to erythrocyte spectrin, but the brain protein is more basic. The binding of brain spectrin to erythrocyte ankyrin, both in solution and on erythrocyte IOVs, yielded an association constant approximately 100 times weaker than for erythrocyte spectrin. The binding of azido‐calmodulin under native conditions was specific for the erythrocyte beta subunit but was not calcium dependent. In contrast, azido‐calmodulin bound only to the alpha subunit of brain spectrin in a calcium‐dependent manner. The similarity of structure but modified functional character‐istics of the brain and erythrocyte beta spectrins suggest that these proteins serve different cellular roles.
Association of spectrin with desmin intermediate filaments
Authors: Langley Robert C. , Cohen Carl M.
Published: December 2004

The association of erythrocyte spectrin with desmin filaments was investigated using two in vitro assays. The ability of spectrin to promote the interaction of desmin filaments with membranes was investigated by electron microscopy of desmin filament‐erythrocyte inside‐out vesicle preparations. Desmin filaments bound to erythrocyte inside‐out vesicles in a spectrin‐dependent manner, demonstrating that spectrin is capable of mediating the association of desmin filaments with plasma membranes. A quantitative sedimentation assay was used to demonstrate the direct association of spectrin with desmin filaments in vitro. When increasing concentrations of spectrin were incubated with desmin filaments, spectrin cosedimented with desmin filaments in a concentration‐dependent manner. At near saturation the spectrin:desmin molar ratio in the sedimented complex was 1:230. Our results suggest that, in addition to its well characterized associations with actin, spectrin functions to mediate the association of intermediate filaments with plasma membranes. It might be that nonerythrocyte spectrins share erythrocyte spectrin's ability to bind to intermediate filaments and function in nonerythroid cells to promote the interaction of intermediate filaments with actin filaments and/or the plasma membrane.
One‐ and two‐dimensional nmr spectral analysis of the consequences of single amino acid replacements in proteins
Authors: Markley John L. , Croll David H. , Krishnamoorthi R. , Ortiz‐Polo Gilberto , Westler William M. , Bogard W. C. , Laskowski M.
Published: December 2004

The traditional approach of using homologous sequences to elucidate the role of specific amino acid residues in protein structure and function becomes more meaningful as the number of differences is minimized, with the limit being alteration of a single residue. For small proteins in solution, NMR spectroscopy offers a means of obtaining detailed information about each residue and its response to a given change in the protein sequence. Extraction of this information has been aided by recent progress in spectrometer technology (higher magnetic fields, more sensitive signal detection, more sophisticated computers) and experimental strategies (new NMR pulse sequences including multiple‐quantum and two‐dimensional NMR methods). The set of avian ovomucoid third domains, which consists of the third domain proper plus a short leader (connecting peptide) and has a maximum of 56 amino acid residues, offers an attractive system for developing experimental methods for investigating sequence‐structure and structure‐function relationships in proteins. Our NMR results provide examples of sequence effects on pKa′ values, average conformation, and internal motion of amino acid side chains.
Phenotypic differences in subclones and long‐term cultures of clonally derived rat bone cell lines
Authors: Bellows Carlton G. , Sodek Jaro , Yao Kam‐Ling , Aubin Jane E.
Published: December 2004

Previous studies with clonally derived populations of cells have shown that cells released from embryonic rat calvaria by enzymatic digestion are heterogeneous with respect to their hormone responsiveness, morphology, and production of matrix components [Aubin JE et al; J. Cell Biol 92:452, 1982].Several of these clonal populations have been used to study the effects of long‐term culture and inter‐ and intraclonal cell heterogeneity. During continuous subculture, marked changes in collagen synthesis were observed in two clonal populations. Both of these clones were originally responsive to parathyroid hormone (PTH) and synthesized primarily type I collagen with small amounts of type III and V collagens, although one clone (RCJ 3.2) had a fibroblastic morphology whereas the second clone (RCB 2.2) displayed a more polygonal shape. Following routine subculture over 3 yr, clone RCB 2.2 was found to synthesize exclusively αl(I)‐trimer and not other interstitial collagens. When the same cells were maintained at confluence for 1‐2 wk, however, they also synthesized type III collagen. Whereas RCJ 3.2 did not show such dramatic changes in collagen synthesis after long‐term subculture, two subclones derived from RCJ 3.2 were found to synthesize almost exclusively either type III collagen (RCJ 3.2.4.1) or type V collagen (RCJ 3.2.4.4). Immunocytochemical staining indicated that both subpopulations also produced type IV collagen, laminin, and basement membrane proteoglycan, proteins that are typically synthesized by epithelial cells. The differences in collagen expression by the various clonal cell populations were accompanied by qualitative and quantitative differences in other secreted proteins and differences in cell morphology. The results demonstrate both the inter‐ and intraclonal heterogeneity of connective tissue cells and their diverse potentiality with respect to extracellular matrix synthesis.
3′,5′‐Cyclic adenosine monophosphate as an intracellular second messenger of luteinizing hormone: Application of the forskolin criteria
Authors: Adashi Eli Y. , Resnick Carol E.
Published: December 2004

The role of adenosine 3′,5′‐cyclic monophosphate (cAMP) as an intracellular second messenger of luteinizing hormone (LH) was reinvestigated in vitro with diterpene forskolin, a highly specific activator of adenylate cyclase. Treatment of cultured testicular cells from adult hypophysectomized rats with increasing concentrations (10−7–10−4 M) of forskolin produced dose‐dependent increments in cAMP and testosterone accumulation. Concomitant blockade of cAMP‐phosphodiesterase activity with 3‐isobutyl‐1‐methyl‐xanthine (10−4 M) resulted in significant (P < 0.05) enhancement of the forskolin effect for all but the 10−4 M forskolin dose. Potency evaluation as judged by half‐maximal stimulation of testosterone accumulation revealed median effective doses (mean ± SE) of 1.25 ± 0.2 × 10−5, 1.7 ± 0.5 × 10−5, and 2.5 ± 0.4 × 10−10 M for forskolin, N6, O2′‐dibutyryl cAMP (Bt2cAMP), and human chorionic gonadotropin (hCG), respectively. Examination of the time requirements of forskolin disclosed time‐dependent increments in the accumulation of extracellular cAMP and testosterone, the earliest significant (P < 0.05) increases being noted by 6 hr of treatment. In comparison, a minimal time requirement of ⩽ 12hr was noted for hCG‐ and choleragen‐stimulated androgen biosynthesis, whereas the apparent onset of action of Bt2cAMP was delayed to the 24‐hr time point. Although 10−7 M of forskolin by itself did not alter the accumulation of testosterone, its addition resulted in substantial amplification of the hCG effect, producing a 4.6‐fold reduction in the median effective dose (ED50) of hCG. Moreover, concurrent treatment with this functionally inert dose of forskolin rendered steroidogenically inert doses of hCG (eg, 10−11 or 3 × 10−11M) steroidogenically potent. However, combined treatment with maximally stimulatory doses of Bt2cAMP (10−4 M) and one of several testicular cell agonists [forskolin (10−4 M), choleragen (10−9 M) or hCG (10−9 M)] did not prove additive. Taken together, our findings indicate that forskolin, like LH, is capable of stimulating testicular cAMP generation as well as androgen biosynthesis and that a functionally inert low dose of forskolin can significantly amplify LH hormonal action. Inasmuch as forskolin‐stimulated and forskolin‐amplified hormonal action are acceptable as novel criteria of cAMP dependence, our observations provide new evidence in keeping with the notion that cAMP may be in intracellular second messenger of LH.
Inactivation of human plasma serine proteinase inhibitors (serpins) by limited proteolysis of the reactive site loop with snake venom and bacterial metalloproteinases
Authors: Kress Lawrence F.
Published: December 2004

Human plasma serine proteinase inhibitors (serpins) gradually lost activity when incubated with catalytic amounts of snake venom or bacterial metalloproteinases. Electrophoretic analyses indicated that antithrombin III, C1‐inhibitor, and α2‐antiplasmin had been converted by limited proteolysis into modified species which retained inhibitory activity. Further proteolytic attack resulted in the formation of inactivated inhibitors; α1‐proteinase inhibitor (α1‐antitrypsin) and α1‐antichymotrypsin were also enzymatically inactivated, but active intermediates were not detected. Sequence analyses indicated that the initial, noninactivating cleavage occurred in the amino‐terminal region of the inhibitors. Inactivation resulted in all cases from the limited proteolysis of a single bond near, but not at, the reactive site bond in the carboxy‐terminal region of the inhibitors. The results indicate that the serpins have two regions which are susceptible to limited proteolysis—one near the amino‐terminal end and another in the exposed reactive site loop of the inhibitor.
Identification of the chick neural retina cell surface N‐acetylgalactosaminyltransferase using monoclonal antibodies
Authors: Balsamo Janne , Pratt Rodney S. , Emmerling Mark R. , Grunwald Gerald B. , Lilien Jack
Published: December 2004

Intact embryonic chick neural retina cells have at their surface an N‐acetylgalactosaminyltransferase which catalyzes the incorporation of N‐acetylgalactosamine from UDP‐N‐acetylgalactosamine into endogenous macromolecular acceptors. The enzyme along with its endogenous acceptors can be isolated as a particulate complex following treatment of membrane‐enriched fractions with Triton X‐100. In this paper we report on two separate fusions generating monoclonal antibodies: one using as immunogen the particulate complex and the second using as immunogen a soluble N‐acetylgalactosaminyltransferase found in tissue‐culture‐conditioned medium which lacks endogenous acceptor activity. Antibodies from both fusions recognize an antigen which is tightly associated with the particulate transferase/acceptor complex and a soluble antigen having N‐acetyl‐galactosaminyltransferase activity toward exogenously added acceptors. The antibodies recognize a component of ca Mr 220,000, which shows N‐acetylgalactosaminyltransferase activity after SDS‐gel electrophoresis and transfer to nitrocellulose. This component comigrates on two‐dimensional gel electrophoresis with an iodinatable cell surface component whose presence at the cell surface correlates with endogenous transferase activity. We conclude that the antibodies recognize the transferase enzyme itself. Immunohistochemical analysis shows that the enzyme is initially localized throughout the embryonic neural retina in a pattern indicative of a cell surface disposition but becomes restricted to the outer plexiform layer and to outer segments in the adult.
A monoclonal antibody reactive with an activated ras protein expressing valine at position 12
Authors: Carney W. P. , Hamer P. , Petit D. , Rabin H. , Cooper G. , Lefebvre M. , Wolfe H.
Published: December 2004

Activated ras transforming genes have been described in a variety of neoplasms and encode 21,000‐Dalton (p21) proteins with amino acid substitutions at positions 12, 13, and 61. In this report we describe a monoclonal antibody designated DWP that reacts. Specifically with synthetic dodecapeptides containing valine at position 12, to a lesser extent with peptides containing cysteine at position 12 and not with peptides containing glycine, arginine, serine, aspartic acid, glutamic acid or alanine at the same position. Western blot and immunoperoxidase studies showed that DWP specifically reacts with activated rasH or rasK proteins in NIH cells transformed by DNA from the human carcinoma cells that encode valine at position 12. DWP did not react with normal p21s encoding glycine at position 12, nor with activated p21s encoding aspartic acid, glutamic acid, arginine, serine, or cysteine at position 12. A survey of human tumor cell lines demonstrated that DWP reacted with the human bladder carcinoma cell line T24 but not with human tumor cell lines previously shown td contain other activating mutations at positions 12 or 61. DWP and perhaps additional antibodies that specifically react with alterations at positions 12 or 61 of the ras protein may be valuable in determining the presence and frequency of activated ras proteins in human malignancy.
Evolution in the structure and function of aspartic proteases
Authors: Tang Jordan , Wong Ricky N. S.
Published: December 2004

Aspartic proteases (EC3.4.23) are a group of proteolytic enzymes of the pepsin family that share the same catalytic apparatus and usually function in acid solutions. This latter aspect limits the function of aspartic proteases to some specific locations in different organisms; thus the occurrence of aspartic proteases is less abundant than other groups of proteases, such as serine proteases. The best known sources of aspartic proteases are stomach (for pepsin, gastricsin. and chymosin). lysosomes (for cathepsins D and E), kidney (for renin), yeast granules, and fungi (for secreted proteases such as rhizopuspepsin, penicillopepsin, and endothiapepsin). These aspartic proteases have been extensively studied for their structure and function relationships and have been the topics of several reviews or monographs (Tang: Acid Proteases, Structure, Function and Biology. New York: Plenum Press, 1977; Tang: J Mol Cell Biochem 26:93–109, 1979; Kostka: Aspartic Proteinases and Their Inhibitors. Berlin: Walter de Gruyter, 1985). All mammalian aspartic proteases are synthesized as zymogens and are subsequently activated to active proteases. Although a zymogen for a fungal aspartic protease has not been found, the cDNA structure of rhizopuspepsin suggests the presence of a “pro” enzyme (Wong et al: Fed Proc 44:2725, 1985). It is probable that other fungal aspartic proteases are also synthesized as zymogens.

It is the aim of this article to summarize the major models of structure‐function relationships of aspartic proteases and their zymogens with emphasis on more recent findings. Attempts will also be made to relate these models to other aspartic proteases.