43K Protein Research Articles

Comparison of acellular and whole-cell pertussis-component diphtheria-tetanus-pertussis vaccines in infants

In a multicenter, double-blind, randomized, longitudinal study, 252 children received licensed Lederle diphtheria-tetanus toxoids and pertussis vaccine adsorbed (DTP) at 2, 4, and 6 months of age, and 245 children received a DTP vaccine with the Lederle/Takeda acellular pertussis component (APDT) at the same ages. Both groups of children received APDT vaccine at 18 months of age. After each of the first three immunizations, APDT vaccine recipients had fewer local and systemic reactions than did DTP vaccinees. Reactions after the 18-month APDT vaccination were minimal in severity regardless of the vaccine previously received. Antibody responses to lymphocytosis-promoting factor and agglutinogens were more pronounced in DTP recipients; however, APDT recipients had a better serologic response to filamentous hemagglutinin, and responses to the 69K protein were equivalent. This APDT vaccine produces fewer reactions than the standard whole-cell DTP vaccine. The protective significance of the serologic responses to the APDT vaccine is unknown, but the greater response to filamentous hemagglutinin and equivalent response to the 69K protein compared with those to DTP vaccine seem promising.

Enteric adenovirus type 40: Complementation of the E4 defect in Ad2 d1808

The enteric adenovirus type 40 cannot be passaged in HeLa cells, but will grow productively in cells that express the E1B region of adenovirus types 2 or 5. Even in such permissive cells, the lytic cycle is prolonged, there is an abnormal pattern of E1B early gene expression and a failure to switch off host cell functions, suggesting that other gene functions might be impaired in Ad40. For Ad2, E4 ORF 6 and ORF 3 proteins are known to have an essential role in progressing from the early to the late phase of lytic infection and the shutoff of host functions requires an interaction between the E4 ORF 6 34K protein and the E1B 55K protein. To test whether E4 functions of Ad40 are impaired, complementation tests have been made between Ad40 and the E4 deletion mutant Ad2 d1808, which lacks all but ORF 1 of the E4 region. In HeLa and Vero cells, Ad40 complements d1808 to levels equivalent to an Ad2 wild-type infection, as demonstrated by measuring virion packaged DNA, virus titration, and viral protein synthesis. Surprisingly, Ad2 d1808 fails to reciprocally complement Ad40. The results show that Ad40 produces functional E4 ORF 6 and/or ORF 3 activity, and that their expression precedes DNA replication.

Serine-specific phosphorylation of nicotinic receptor associated 43K protein

In Torpedo marmorata electroplaque, an extrinsic membrane protein of apparent mass 43,000 daltons colocalizes with the cytoplasmic face of the nicotinic acetylcholine receptor (AChR) in approximately 1:1 stoichiometry. We show that this 43K protein can be phosphorylated in vitro by endogenous protein kinases present in AChR-rich membranes. The extent of 43K protein phosphorylation exceeds that of the subunits of the AChR, well-established substrates for enzymatic phosphorylation. We demonstrate that significant 43K phosphoprotein exists in vivo. The kinetics of phosphate incorporation mediated by endogenous kinases differed significantly from those of the AChR subunits, suggesting that different phosphorylation cascades are involved. Use of specific inhibitors of a variety of protein kinases indicated that endogenous cAMP-dependent protein kinase catalyzes phosphorylation of the 43K protein in vitro. All of the phosphate incorporated into 43K protein was accounted for by phosphoserine (0.65 mol/mol of 43K protein). Potential structural and functional consequences of 43K protein phosphorylation are discussed.

Subcellular distribution of the major immediate early proteins of human cytomegalovirus changes during infection.

The 72K immediate early (IE) 1 protein of human cytomegalovirus and the 68K protein, also encoded by the IE1 gene, were detected by immunoelectron microscopy using monoclonal antibodies specific for the 68K or 72K proteins. Early after infection, both proteins were localized to the nucleus, but with different localization patterns. Late after infection, both of the proteins decreased markedly in the nucleus, in which nucleocapsids appeared. Simultaneously, the 68K protein became diffusely distributed in the cytoplasm and the plasma membrane, whereas the 72K protein was distributed in the nuclear envelope and the plasma membrane. Both proteins were also observed in the coating membrane of the extracellular dense bodies.

17D yellow fever vaccine virus envelope protein expressed by recombinant baculovirus is antigenically indistinguishable from authentic viral protein

We have constructed a recombinant baculovirus containing cloned DNA encoding the membrane and envelope (E) proteins of 17D yellow fever vaccine virus. Spodoptera frugiperda cells infected with this recombinant baculovirus produced a 66K protein which corresponded to the estimated size of the protein encoded by the cloned inserted DNA, and a 54K protein with the same molecular size as that of the authentic 17D yellow fever virus E protein. This recombinant 54K protein was labile, producing E protein-specific breakdown products (45K to 36K). Indirect immunofluorescence, using a panel of E protein-specific monoclonal antibodies, showed that the recombinant protein was presented both inside as well as on the surface of cells and was antigenically indistinguishable from the E protein of 17D yellow fever vaccine virus.

The use of beta-galactosidase fusion proteins encoding the early region 1 transforming proteins of adenovirus type 12 to examine the humoral response in tumour-bearing animals

Sera from 26 rats bearing tumours induced by wild-type (wt) and mutant human adenovirus type 12 (Ad12), or by cells transformed with these viruses, were analysed for antibodies against the early region 1 (E1) transforming proteins. Six Ad12-beta-galactosidase fusion proteins encoding different regions of the Ad12 E1 proteins were constructed. The sera from the tumour-bearing animals reacted most strongly with the fusion protein encoding the N terminus of the E1A protein. Tumour-bearing rats exposed to the E1B 54K and 19K proteins showed strong reactions with the N terminus of the 54K protein and the C terminus of the 19K protein. Monospecific polyclonal antisera were raised against five of the fusion proteins by immunization of rats and rabbits; these sera cross-reacted with the purified native protein. No antibodies could be obtained which recognized a fusion protein containing amino acids 136 to 268 of the 54K protein. The fusion proteins were also used to purify monospecific antisera from tumour-bearer sera using affinity chromatography.

Molecular cloning, partial purification, and characterization of a haemin‐binding lipoprotein from Haemophilus influenzae type b

A library of genomic DNA fragments from Haemophilus influenzae type b (Hib) DL42 was constructed in plasmid pBR322, transformed into Escherichia coli strain RR1, and screened for recombinant clones with haemin-binding activity by plating onto haemin-containing agar. Expression of haemin-binding activity by clones correlated with the expression of a protein with an apparent molecular weight of 51,000 (51K) that was also recognized by anti-Hib strain DL42 serum in immunoblots. One recombinant clone, designated pHM2, with the smallest DNA insert (3.62 kb) was characterized further. Ethanol inhibition of expression of pHM2 in minicells revealed that the 51K protein was the result of a processing event involving a larger precursor. E. coli RR1(pHM2) adsorbed haemin in liquid suspensions as well as from solid media. Subcloning of a 2.6 kb fragment of pHM2 into a shuttle vector permitted the construction of a recombinant Hib clone, DL42(pHM1002), which overexpressed the 51K haemin-binding protein. This 51K protein appears to be peripherally associated with the inner, and possibly outer, membranes of Hib. Affinity chromatography on haemin-agarose was utilized to purify the haemin-binding protein from both E. coli RR1(pHM2) and Hib DL42(pHM1002) to near homogeneity. The use of the antibiotic globomycin in a minicell expression system and radioimmunoprecipitation analysis of Hib proteins intrinsically radiolabelled with [3H]-palmitate indicated that the 51K haemin-binding protein is a lipoprotein.

A nuclear factor that interacts with metal responsive elements of a human metallothionein gene

Metallothioneins (MTs) are low molecular weight heavy metal- binding proteins which are known to play a major role in heavy metal detoxification and understanding of their regulatory mechanism is toxicologically important. Expression of MT genes is induced by heavy metals and metal responsive elements (MREs) upstream of MT genes are essential for the transcriptional activation. By several types of mobility shift assay with 32P-labeled oligonucleotide probes, we detected HeLa cell nuclear as well as cytoplasmic factors that bind to MRE sequences of human MTII A (hMTIIA) gene. One of the nuclear factors, which gives stronger signal than others, was further characterized. Competition experiments showed that the nuclear factor (named MREBP) specifically recognizes MREs of hMTII A gene. EDTA abolished the binding of MREBP to MRE, suggesting that a divalent cation(s) is required for the complex formation. Also in blotting experiments with HeLa nuclear extract and the [ 32P]MRE probes an EDTA-sensitive 95k protein band, which possibly represents MREBP, was detected.

The origin of a sperm maturation-associated antigen, 54K sialoglycoprotein; an immunohistochemical study with monoclonal antibody MC81 in the mouse.

We have reported several aspects of a mouse sperm maturation-associated antigen, a 54, 000 dalton (54K) sialoglycoprotein, of which epitope can be masked by sialic acid residues against a monoclonal antibody T21 (35-38). However, the producing cell of the 54K sialoglycoprotein has long been unclear. This report documents the origin of the 54K sialoglycoprotein based on morphological evidence using avidin-biotin peroxidase complex (ABC) immunohistochemistry and indirect immunoperoxidase-electron microscopy with a recently established monoclonal antibody MC81. Immunoblots of Nonidet P-40 extract of cauda epididymidis sperm after one-dimensional, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that MC81 recognized a major protein of 54, 000 dalton (54K). The epitope for MC81 antibody was suggested to be present on a sugar chain of the 54K protein when assessed by periodate treatment. Immunoblots of sperm extracts separated by two-dimensional SDS-PAGE indicated that a major spot of 54K dalton was in acidic range less than 5 in the isoelectric focusing point. These results suggest that the antigen of MC81 is identical to that of T21. Immunohistochemistry with MC81 revealed that principal cells located at the distal caput to corpus epididymidis were immunoreactive. The region where the principal cells were first recognized by MC81 was more proximal than the region where luminal sperm were first stained. In addition, MC81 exclusively recognized the Golgi apparatus area at the supranuclear region of the immunoreactive principal cells. Sialidase-treatment prior to primary antibody (MC81) increased of the immunostaining intensity in all immunochemical studies examined. Together with the result of the previous part of this study, it is strongly suggested that the 54K sialoglycoprotein can be secreted by the principal cells at the distal caput to corpus epididymidis, with the epitope for MC81 masked by sialic acids.

A coat subunit of Golgi-derived non-clathrin-coated vesicles with homology to the clathrin-coated vesicle coat protein β-adaptin

Four high-molecular-weight proteins form the main subunits of the coat of Golgi-derived (non-clathrin) coated vesicles. One of these coat proteins, beta-COP, is identical to a Golgi-associated protein of relative mass 110,000 (110K) that shares homology with the adaptin proteins of clathrin-coated vesicles. This connection, and the comparable molecular weights of the coat proteins of Golgi-derived and clathrin-coated vesicles, indicates that they may be structurally related. The identification of beta-COP as the 110K protein explains the blocking of secretion by the drug brefeldin A.

Inoculation Study of Alzheimer Buffy Coat into Hamster Brain

Buffy coats from 4 members of a family with Alzheimer''s disease and 2 genetically unrelated healthy subjects were inoculated into the hamster brain. Eighteen months after the inoculation, the brains were studied by immunostaining with antibodies against neurofilaments, tau, ubiquitin, and paired-helical filaments (PHF) as well as by electron microscopy. Vacuolar or spongioform degeneration was minimum. Intense positive staining by antibodies against neurofilament 200K protein was demonstrated in neuronal perikarya of the lower brain stem nucleus in the hamster inoculated with the buffy coats from the 2 patients with Alzheimer''s disease and 2 children of the patient, which was not observed in the hamster brain inoculated with the buffy coat from the control subjects. Electron microscopy revealed intracytoplasmic accumulation of 10-nm neurofilament-like fibers. The accumulated neurofilaments were positively stained by antitau and antiubiquitin, but not by anti-PHF. The neurons containing massive intracytoplasmic neurofilament accumulation resembled ballooned neurons in some degenerative diseases including Alzheimer''s disease. The results indicate that the buffy coat obtained from the patients and members of a family with Alzheimer''s disease contains a factor which produces intracytoplasmic accumulation of neurofilament-like fibers in neurons of the lower brain stem nuclei.

Expression of plant virus genes in animal cells: high-level synthesis of cowpea mosaic virus B-RNA-encoded proteins with baculovirus expression vectors

The baculovirus expression system has been used to produce non-structural proteins encoded by bottom-component RNA (B-RNA) of cowpea mosaic virus (CPMV). For this, cDNAs containing the 60K, 87K, 110K and 170K protein coding sequences were each provided with an ATG start codon and the cDNA containing the 60K coding sequence with a TAA stop codon immediately downstream of the coding sequence. Recombinant baculoviruses were retrieved which harboured the modified B-cDNA sequences under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Upon infection of Spodoptera frugiperda cells with these recombinant baculoviruses, proteins were produced which were indistinguishable from the viral proteins found in CPMV-infected plants as judged by their migration in polyacrylamide gels and their reactivity with CPMV-specific antisera. Specific processing of CPMV polyproteins in cells infected with the 110K- and 170K-encoding baculovirus recombinants proved that the CPMV-encoded 24K protease activity contained in these polyproteins is active in these cells. Approximately 10% of the 110K protein was processed into 87K and 24K proteins and the 170K protein almost completely into the 110K, 87K, 84K, 60K and 24K polypeptides. In S. frugiperda cells infected by recombinant AcNPVs harbouring the 87K or 110K coding sequences, the CPMV-specific proteins amounted to 10 to 20% of the total cellular protein content, whereas in cells infected by recombinants encoding the 60K and 170K polypeptides the amounts of CPMV-specific proteins synthesized were much lower. Northern blot analysis indicated that the low-level synthesis of the 60K and 170K polypeptides was not due to inferior transcription of the cloned genes but was probably the result of inefficient translation of the RNAs derived from these constructs. It is concluded that plant virus genes can be efficiently expressed in an animal cell expression system to yield proteins that are structurally and, in at least one case (24K protein), functionally identical to the authentic plant virus proteins.

Developmental expression of the 43K and 58K postsynaptic membrane proteins and nicotinic acetylcholine receptors in Torpedo electrocytes

The expression of the postsynaptic 43-kDa and 58-kDa proteins and actin during development of the Torpedo marmorata electric organ was compared to that of nicotinic acetylcholine receptors (AChRs). Western blot analysis demonstrates that AChRs and proteins of 43 kDa (43K protein) and 58 kDa (58K protein) are all present prior to synaptogenesis. Subsequently, levels of all 3 synaptic proteins increase dramatically during differentiation and innervation of electrocytes. In contrast, actin is present in relatively high concentrations at early times and decreases thereafter. The equimolar ratio of AChRs and the 43K protein found in the adult electric organ is established early in development. Furthermore, the AChR and 43K protein share a common postsynaptic localization in electrocytes following synapse formation. Aggregates of the AChR that form at the ventral pole of the oval-shaped electrocytes prior to innervation, however, show no detectable immunofluorescence staining with anti-43K monoclonal antibodies. Therefore, in some cases, aggregation of AChRs occurs without the 43K protein.

The postsynaptic 43k protein clusters muscle nicotinic acetylcholine receptors in xenopus oocytes

Nicotinic acetylcholine receptors (AChRs) are localized at high concentrations in the postsynaptic membrane of the neuromuscular junction. A peripheral membrane protein of M r 43,000 (43K protein) is closely associated with AChRs and has been proposed to anchor receptors at postsynaptic sites. We have used the Xenopus oocyte expression system to test the idea that the 43K protein clusters AChRs. Mouse muscle AChRs expressed in oocytes after injection of RNA encoding receptor subunits are uniformly distributed in the surface membrane. Co-injection of AChR RNA and RNA encoding the mouse muscle 43K protein causes AChRs to form clusters of 0.5–1.5 μm diameter. AChR clustering is not a consequence of increased receptor expression in the surface membrane or nonspecific clustering of all membrane proteins. The 43K protein is colocalized with AChRs in clusters when the two proteins are expressed together and forms clusters of similar size even in the absence of AChRs. These results provide direct evidence that the 43K protein causes clustering of AChRs and suggest that regulation of 43K protein clustering may be a key step in neuromuscular synaptogenesis.

Biochemical and immunological studies of proteins from polydnavirus Chelonus sp. near curvimaculatus

Polydnavirus from Chelonus sp. curvimaculatus (CcV) was purified and 18 structural polypeptides associated with CcV were identified by silver staining. Antibodies were raised against CcV protein and used in testing for ovary and in stung eggs. It was also established that the virus does not enter during the first 5 s of oviposition. Furthermore, no degradation of the virus proteins was Detected inside the egg within 2 h after oviposition. The glycoprotein nature of virus proteins was also determined by concanavalin A/horseradish peroxidase staining. The amino acid compositions of the most highly abundant peptides (41K, 33K, 21K, 17K and 13K) were determined, as was the N-terminal amino acid sequence of the 41K protein. The latter did not show similarity with any reported protein sequences.

Nucleotide sequence and genomic organization of melon necrotic spot virus

Cloned cDNA copies of the genomic RNA of melon necrotic spot virus (MNSV) have been sequenced and the sizes and locations of predicted viral proteins have been deduced. The genome consists of at least 4262 nucleotides and the positive strand contains three to five open reading frames (ORFs) which may be expressed. The 5' proximal ORF encodes a 29K protein (p29) and terminates with an amber codon which may be read through to produce an 89K protein (p89). Two small centrally located ORFs each encode a 7K protein (p7A and p7B). As p7A is in frame with p7B, readthrough of the amber codon terminating p7A may occur, producing a 14K protein (p14). The 3' proximal ORF encodes the 42K coat protein. The genomic organization of MNSV, its probable translation strategy and the amino acid sequences of its putative proteins closely resemble those of known carmoviruses, suggesting that MNSV should be classified as a member of the carmovirus group. Unusual properties of the putative MNSV replicase (p89) suggest that MNSV should be classified in a new virus supergroup with several other viruses sharing these properties.

Cardiac myocyte gap junctions: evidence for a major connexon protein with an apparent relative molecular mass of 70,000

It is widely accepted that there is a family of gap junction connexon proteins, their distribution appearing to vary with tissue type and species. In cardiac tissues the major junctional channel component identified is a 43K (K = 10(3) Mr) polypeptide. Using a gap junction isolation protocol in which low temperatures are maintained, and which is detergent-free, we have identified a second gap junction-related protein in cardiac tissues with an apparent relative molecular mass of 70,000. Antibodies raised to three synthetic peptides matching portions of the 43K gap junction protein cDNA sequence cross-react with the 70K protein, but biochemical studies indicate that these proteins are distinct from one another. The structures that contain the 70K protein are susceptible to fragmentation at warm temperatures, and by electron microscopy this can be correlated with loss of 'minidomains' within the junctional plaque. Using a gap junction enriched-fraction prepared from purified isolated adult myocytes we show that both the 43K and 70K gap junction proteins are present in ventricular cardiac myocytes. In such preparations, and those from whole heart, the 70K protein appears to be the major gap junction-related protein present.

Differential expression of heat shock proteins in Drosophila embryonic cells following metal ion exposure

Drosophila embryonic cells were exposed to a number of metal ions that have been previously reported to act as teratogens in mammalian systems, including some known to induce heat shock (stress) proteins in a variety of model systems. This study examined the effects of these ions both on differentiation of muscles and neurons and on the induction of heat shock proteins. Metals such as arsenate, cadmium, and mercury all inhibited neuron and/or muscle differentiation in Drosophila embryonic cultures, while they also induced the entire set of heat shock proteins. Two metal ions, nickel and zinc, were shown to induce only the 22-and 23-K proteins, a pattern similar to that seen in “classical” teratogens reported previously. None of the metals tested induced only the 26-and 27-K proteins. These results suggest that there exist different regulatory mechanisms responsible for the heat shock response.

A Progesterone-Modulated, Low-Molecular-Weight Protein from the Uterus of the Sheep is Associated with Crystalline Inclusion Bodies in Uterine Epithelium and Embryonic Trophectoderm1

A protein of Mr 14,000 (14K protein) has been identified in the medium after culturing sheep conceptuses in vitro. However, it was also a component of uterine flushes of nonpregnant ewes during the luteal phase of the estrous cycle. The 14K protein, which consists of several isoforms, was purified from conceptus culture medium by a simple three-step procedure, involving ammonium sulfate precipitation, high-performance anion-exchange chromatography, and gel filtration on Sephacryl S-200. Western blotting procedures with an anti-14K antiserum showed that endometrium from ovariectomized ewes began to release the 14K protein after 6 to 14 days of progesterone treatment. Immunostaining of tissue from Day 16 pregnant ewes and from ewes treated with progesterone for 14 or 30 days showed the protein to be confined to the surface and upper glandular epithelium of the endometrium and to be absent from the deep glands. It was also present in trophectoderm of Day 16 conceptuses. Immunogold labeling in conjunction with electron microscopy revealed that within trophectoderm the 14K protein was localized to large, membrane-bound rhomboidal or needle-shaped crystal structures, and it seems likely that the protein was accumulated by the conceptus as a result of uptake of uterine histotroph. In the uterine epithelium, immunogold label was again most strongly concentrated over crystal-like structures but was also uniformly present over the cytoplasm and nucleoplasm. It was absent over mitochondria, over lipid droplets, and over non-epithelial types of endometrial cell. Such a cellular distribution for a secretory protein is novel, and it remains unclear whether the protein is synthesized by these cells.

Identification of a 32K plasma membrane protein that binds to the myristylated amino-terminal sequence of p60v-src

The transforming protein of Rous sarcoma virus, p60v-src, is a myristylated membrane-bound phosphoprotein. Interaction of p60v-src with the plasma membrane is essential for transforming activity, and is mediated by association with a membrane-bound Src receptor protein. Evidence for the existence of an Src receptor is based on the ability of a myristylated peptide containing the N-terminal Src sequence to inhibit binding of p60v-src to plasma membranes in vitro: binding of p60v-src to a plasma membrane receptor is therefore mediated by N-terminal Src sequences. Here we report that a myristyl-Src peptide, but not the corresponding non-myristylated peptide, can be specifically crosslinked to a plasma membrane protein of relative molecular mass 32,000 (Mr32K). The 32K protein represents an Src-binding protein in the plasma membrane that is likely to be a component of the myristyl-Src receptor, and which could be involved in cellular transformation.