Therapeutic effect of Bushen Huoxue recipe on autoimmune premature ovarian failure mice established by immunization with recombinant porcine zona pellucida 4 antigen.

OBJECTIVE: To investigate the efficacy and mechanism of Bushen Huoxue Recipe (, BHR) in the treatment of murine autoimmune premature ovarian failure (POF). METHODS: The recombinant porcine zona pellucida 4 (pZP4) was expressed in E. coli BL21 (DE3) strain within prokaryotic plasmid pET28a (+), purified by Ni-affinity chromatography and verified by Western blot. Murine autoimmune POF model was established by immunization with pZP4 of female BALB/c mice. Fifty POF mice were randomly divided into 5 groups, which were respectively given low (3.75 mg/kg), moderate (7.5 mg/kg), and high dose (15.0 mg/kg) of BHR by gastrogavage once daily for 20 days, with 17-ß-estradiol (0.13 mg/kg) and normal saline as positive and negative control. Estrous cycles were analyzed through vaginal smears, serum estradiol (E) levels, and anti-pZP4 antibody titers were detected by ELISA. The proliferative responses in vitro of spleen lymphocytes to pZP4 antigen restimulation were measured by [(3)H]-thymidine incorporation, and the histomorphology changes of ovary were evaluated by optical microscope. RESULTS: The purified pZP4 was visible as a single lane with 14.4 kD in SDS-PAGE and Western blot. The murine POF model with lengthening estrous cycles, decreased levels of serum E2, high titers of serum anti-pZP4 antibody, and reduced ovarian follicles and corpus lutea were established by immunization with recombinant pZP4. Treatment with moderate and high dosage BHR significantly increased ovarian follicles and reduced the proliferation of spleen lymphocytes to the pZP4 antigen of POF mice (P <0.05). However, only the high dosage BHR administration significantly improved the estrous cycles, elevated the serum E levels (P <0.01), and decreased the serum anti-pZP4 antibody titers of model mice P<0.05). CONCLUSIONS: The recombinant pZP4 could evoke the antigen-specific immune response in mice and induce the autoimmune ovarian injury. It has been demonstrated that BHR was able to increase the serum E levels and protect ovarian functions from the autoimmune injury in murine POF model.

Cloning and characterization of SCART1, a novel scavenger receptor cysteine-rich type I transmembrane molecule.

We have cloned and characterized a novel murine transmembrane molecule, mSCART1 belonging to the scavenger receptor cysteine-rich superfamily. The cDNA encodes a polypeptide chain of 989 amino acids, organized as a type I transmembrane protein that contains eight extracellular SRCR domains followed by a transmembrane region and a cytoplasmic domain. The cytoplasmic domain contains two putative src kinase consensus substrate sequences, three additional potential phosphorylation sites, and two potential internalization motifs. Two possible secreted forms that lack the transmembrane region arise by alternative splicing. The murine SCART1 gene maps to chromosome 7 band F5 and the analysis of the genomic organization showed that the gene spans 12.86 kb and contains 14 exons. Quantitative real-time PCR analyses on murine tissues showed high mSCART1 mRNA expression in the lymph node, the trachea, and the lung, and low expression was found in the thymus, the spleen, the skin, and in tissues throughout the gastrointestinal tract. Comparative studies of the domain organization as well as the cytoplasmic domain of mSCART1 with the other members of the SRCR superfamily show that mSCART1 is highly related to the WC1 family of the SRCR superfamily. Finally, a novel human scavenger receptor cysteine-rich molecule with high homology to mSCART1 was identified by searching in the human genomic databases using the mSCART1 cDNA sequence.

The F-box protein TIR1 is an auxin receptor.

The plant hormone auxin regulates diverse aspects of plant growth and development. Recent studies indicate that auxin acts by promoting the degradation of the Aux/IAA transcriptional repressors through the action of the ubiquitin protein ligase SCF(TIR1). The nature of the signalling cascade that leads to this effect is not known. However, recent studies indicate that the auxin receptor and other signalling components involved in this response are soluble factors. Using an in vitro pull-down assay, we demonstrate that the interaction between transport inhibitor response 1 (TIR1) and Aux/IAA proteins does not require stable modification of either protein. Instead auxin promotes the Aux/IAA-SCF(TIR1) interaction by binding directly to SCF(TIR1). We further show that the loss of TIR1 and three related F-box proteins eliminates saturable auxin binding in plant extracts. Finally, TIR1 synthesized in insect cells binds Aux/IAA proteins in an auxin-dependent manner. Together, these results indicate that TIR1 is an auxin receptor that mediates Aux/IAA degradation and auxin-regulated transcription.

Identification, cloning, and functional characterization of a murine lipoxin A4 receptor homologue gene.

To identify additional members of the murine N-formyl-Met-Leu-Phe peptide receptor family (fMLF-R), a mouse macrophage cDNA library was screened using the open reading frame of murine N-formyl peptide receptor. Four individual hybridizing cDNA clones were maintained through tertiary screening. One cDNA clone was a truncated, polyadenylated version of the previously described murine-fMLF-R. The other three cDNA clones varied in length, but contained identical open reading frame sequences. One clone, 8C10, was selected for further study and shared 70% sequence identity with murine-fMLF-R and 89% sequence identity with murine lipoxin A4 receptor cDNA. When placed into the pcDNA-3 expression vector and cotransfected with Galpha16 cDNA into COS-1 cells, 8C10 cDNA induced the production of inositol-1,4,5-triphosphate when concentrations of 1-1600 nM lipoxin A4 (LXA4) were tested as ligands. Northern blot analysis of murine organs indicated that the 8C10 message is present in lung, spleen, and adipose tissue. Moreover, mice treated with LPS demonstrated increased expression of 8C10 message in spleen and adipose tissue, while showing a slight reduction in lung. We have also characterized the 8C10 structural gene from a 129Sv/J genomic library and have determined its size to be >6.1 kb in length and comprised of two exons separated by a 4.8-kb intron. Collectively, these data indicate that this homologue receptor is closely related to the murine LXA4 receptor and functionally responds to LXA4 as a ligand.

Vitellogenin-derived yolk proteins of white perch, Morone americana: purification, characterization, and vitellogenin-receptor binding.

The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a approximately 20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, beta'-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the beta'-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.

Cloning and characterization of a receptor-like protein kinase gene associated with senescence.

Senescence-associated genes are up-regulated during plant senescence and many have been implicated in encoding enzymes involved in the metabolism of senescing tissues. Using the differential display technique, we identified a SAG in bean (Phaseolus vulgaris) leaf that was exclusively expressed during senescence and was designated senescence-associated receptor-like kinase (SARK). The deduced SARK polypeptide consists of a signal peptide, a leucine-rich repeat in the extracellular region, a single membrane-spanning domain, and the characteristic serine/threonine protein kinase domain. The mRNA level for SARK increased prior to the loss of chlorophyll and the decrease of chlorophyll a/b-binding protein mRNA. Detached mature bean leaves, which senesce at an accelerated rate compared with leaves on intact plants, showed a similar temporal pattern of SARK message accumulation. Light and cytokinin, which delayed the initiation of leaf senescence, also delayed SARK gene expression; in contrast, darkness and ethylene, which accelerated senescence, advanced the initial appearance of the SARK transcript. SARK protein accumulation exhibited a temporal pattern similar to that of its mRNA. A possible role for SARK in the regulation of leaf senescence was considered.

Identification of the melatonin-binding site MT3 as the quinone reductase 2.

The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt(1) and MT(2), the existence of a novel putative melatonin receptor, MT(3), was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt(1) and MT(2). In this report, we present the purification of MT(3) from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR(2), EC ). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT(3) to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR(2). After transfection of CHO-K1 cells with the human QR(2) gene, not only did the QR(2) enzymatic activity appear, but also the melatonin-binding sites with MT(3) characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data from MT(3) binding and QR(2) enzymatic activity obtained from samples of Syrian hamster kidney or QR(2)-overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding site MT(3) as the quinone reductase QR(2) and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.

Identification of a receptor for reg (regenerating gene) protein, a pancreatic beta-cell regeneration factor.

Reg (regenerating gene) was isolated as a gene specifically expressed in regenerating islets (Terazono, K., Yamamoto, H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y., and Okamoto, H. (1988) J. Biol. Chem. 263, 2111-2114). Rat and human Reg gene products, Reg/REG proteins, have been demonstrated to stimulate islet beta-cell growth in vitro and in vivo and to ameliorate experimental diabetes. In the present study, we isolated a cDNA for the Reg protein receptor from a rat islet cDNA library. The cDNA encoded a cell surface 919-amino acid protein, and the cells into which the cDNA had been introduced bound Reg protein with high affinity. When the cDNA was introduced into RINm5F cells, a pancreatic beta-cell line that shows Reg-dependent growth, the transformants exhibited significant increases in the incorporation of 5'-bromo-2'-deoxyuridine as well as in the cell numbers in response to Reg protein. A homology search revealed that the cDNA is a homologue to a human multiple exostoses-like gene, the function of which has hitherto been unknown. These results strongly suggest that the receptor is encoded by the exostoses-like gene and mediates a growth signal of Reg protein for beta-cell regeneration.

Molecular cloning and characterization of a novel retinoic acid-inducible gene that encodes a putative G protein-coupled receptor.

The effects of retinoids such as all-trans-retinoic acid (ATRA) on cell growth, differentiation, and apoptosis are thought to be mediated by nuclear retinoid receptors, which are involved in ligand-dependent transcriptional activation of target genes. Using differential display, we identified the cDNA of a novel gene, designated retinoic acid-inducible gene 1 (RAIG1), which was induced by ATRA in the squamous carcinoma cell line UMSCC-22B. Two RAIG1 transcripts of 2.4 and 6.8 kilobase pairs, respectively, have the same ORF that encodes a 357-amino acid polypeptide. RAIG1 mRNA is expressed at high level in fetal and adult lung tissues. Induction of RAIG1 expression by ATRA is rapid (within 2 h) and dose-dependent in the range between 1 nM to 1 microM. The constitutive RAIG1 mRNA levels, which were low in three of five head and neck and four of six lung cancer cell lines, increased after ATRA treatment in most cell lines. The deduced RAIG1 protein sequence contains seven transmembrane domains, characteristic of G protein-coupled receptors. A fusion protein of RAIG1 and the green fluorescent protein was localized in the cell surface membrane and perinuclear vesicles in transiently transfected cells. RAIG1 was mapped to chromosome 12p12. 3-p13. Our results provide novel evidence for a possible interaction between retinoid and G protein signaling pathways.

Molecular identification of a novel family of human Ig superfamily members that possess immunoreceptor tyrosine-based inhibition motifs and homology to the mouse gp49B1 inhibitory receptor.

The co-cross-linking of gp49B1, a member of the Ig superfamily containing immunoreceptor tyrosine-based inhibition motifs, with the high affinity Fc receptor for IgE on mouse bone marrow culture-derived mast cells inhibits IgE-dependent exocytosis and lipid mediator generation. We now describe the cloning of human cDNAs homologous to the mouse gp49 family. A human monocyte cDNA library was probed with the mouse gp49A cDNA, which is 97% identical with mouse gp49B1, to obtain a homologous partial cDNA that was then used to identify and clone full-length cDNAs from monocyte and human lung cDNA libraries. The 1.6-kbp cDNA, HM18, predicts a 49-kDa type 1 integral membrane protein that, like mouse gp49B1, contains two extracellular C2 type Ig superfamily domains and two consensus immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic domain. ALIGN analysis of the amino acid sequence of the extracellular domains showed that HM18 belongs to a family that includes mouse gp49, the bovine Fc receptor for IgG2, the human myeloid Fc receptor for IgA, and the human NK cell inhibitory receptors. The gene encoding HM18, in common with the genes for the human Fc receptor for IgA and the human NK cell inhibitory receptors, was localized to chromosome 19q13.4. Two other closely related cDNAs, each with four C2 Ig superfamily domains, were characterized. Transcripts for these novel Ig superfamily members were identified in peripheral blood monocytes, the THP-1 monocytic cell line, human lung, human lung mast cells, and NK cells. The data suggest that HM18 is a novel mononuclear cell inhibitory receptor homologous to mouse gp49B1.

Cloning and functional analysis of a polymorphic variant of the ovine Mel 1a melatonin receptor.

We have isolated a novel variant of the Mel 1a melatonin receptor from an ovine PT cDNA library. Relative to the reported sequence for the Mel 1a melatonin receptor there are 8 changes in the DNA sequence. Only 3 of these result in amino acid substitutions, one in extracellular loop 3 and two in the carboxy-terminal tail. We have designated the novel variant of the sheep Mel 1a receptor Mel 1a(beta), and correspondingly the previously reported variant Mel 1a(alpha). As minor changes in the primary amino acid sequence of G-protein-coupled receptors can influence their functional characteristics we have accordingly characterized this novel variant of the Mel 1a melatonin receptor. This melatonin receptor displays high affinity binding and inhibits the cAMP second messenger pathway in transfected L-cells demonstrating that this receptor is fully functional. PCR analysis shows Mel 1a(beta) is present in several breeds of sheep and suggests that the Mel 1a(beta) receptor was established early in the evolution of the sheep species.

NOR-2 (neuron-derived orphan receptor), a brain zinc finger protein, is highly induced during liver regeneration.

Zinc-finger proteins are involved in several cellular processes. Some of these proteins are implicated in the primary cellular response in regenerating liver and mitogen-stimulated cells. Using a rat cDNA brain library, we have isolated a clone designated NOR-2, encoding a protein containing two zinc-finger motifs and whose expression is highly induced during G0/G1 transition. We analysed the expression of NOR-2 mRNAs during early growth in regenerating liver and in both insulin-stimulated H4-II cells and pheochromocytoma-derived cell line PC12 treated by NGF. In these systems, there is an early, rapid and transient accumulation of NOR-2 mRNAs. The induction of NOR-2 mRNAs does not require de novo protein synthesis, since it is not prevented by cycloheximide treatment. Mobility shift assays show that NOR-2 protein binds to NBRE, a target sequence for r-NGFI-B family. Structurally, NOR-2 is closely related to the recently identified NOR-1 factor. Therefore, like NOR-1, NOR-2 belongs to the r-NGFI-B sub-family of nuclear receptors superfamily.

Calcium sensing receptor: molecular cloning in rat and localization to nerve terminals.

We have molecularly cloned a calcium sensing receptor (CaSR) from a rat striatal cDNA library. Rat CaSR displays 92% overall homology to its bovine counterpart with seven putative transmembrane domains characteristic of the superfamily of guanine nucleotide-binding proteins and significant homology with the metabotropic glutamate receptors. Northern blot analysis reveals two transcripts in thyroid, kidney, lung, ileum, and pituitary. In brain highest regional expression of the RNA occurs in the hypothalamus and the corpus striatum. Immunohistochemistry reveals discrete punctate localizations throughout the brain that appear to be associated with nerve terminals. No staining is evident in cell bodies of neurons or glia. Cerebral arteries display an intense network of CaSR immunoreactive fibers associated with vessel innervation. CaSR on nerve terminal membranes may regulate neurotransmitter disposition in response to Ca2+ levels in the synaptic space.

The Neisseria meningitidis haemoglobin receptor: its role in iron utilization and virulence.

The Neisseria meningitidis haemoglobin receptor gene, hmbR, was cloned by complementation in a porphyrin-requiring Escherichia coli mutant. hmbR encodes an 89.5 kDa outer membrane protein which shares amino acid homology with the TonB-dependent receptors of Gram-negative bacteria. HmbR had the highest similarity to Neisseria transferrin and lactoferrin receptors. The utilization of haemoglobin as an iron source required internalization of the haemin moiety by the cell. The mechanism of haemin internalization via the haemoglobin receptor was TonB-dependent in E. coli. A N. meningitidis hmbR mutant was unable to use haemoglobin but could still use haemin as a sole iron source. The existence of a second N. meningitidis receptor gene, specific for haemin, was shown by the isolation of cosmids which did not hybridize with the hmbR probe, but which were able to complement an E. coli hemA aroB mutant on haemin-supplemented plates. The N. meningitidis hmbR mutant was attenuated in an infant rat model for meningococcal infection, indicating that haemoglobin utilization is important for N. meningitidis virulence.

A novel receptor-type protein tyrosine phosphatase with a single catalytic domain is specifically expressed in mouse brain.

Protein tyrosine phosphatases (PTPases) are important regulatory proteins that, together with protein tyrosine kinases, determine the phosphotyrosine levels in cell signalling proteins. By PCR amplification of mouse brain cDNA fragments encoding the catalytic domains of these enzymes, we identified three novel members of the PTPase gene family. Northern-blot analysis showed that two of these novel clones represent brain-specific PTPases, whereas the third originates from a large-sized mRNA that is more ubiquitously expressed. A full-length cDNA encoding one of the brain-specific PTPases, PTP-SL, was isolated. Sequence analysis revealed a transmembrane PTPase containing a single catalytic phosphatase domain that has 45% homology to a rat cytoplasmic brain-specific PTPase named STEP. This suggests a role for PTP-SL in cell-cell signalling processes in the brain.

The 30-kilodalton protein present in purified fusicoccin receptor preparations is a 14-3-3-like protein.

We have recently reported on the purification of the fusicoccin (FC) receptor from corn (Zea mays L.) and its identification by photoaffinity labeling (P. Aducci, A. Ballio, V. Fogliano, M.R. Fullone, M. Marra, N. Proietti [1993] Eur J Biochem 214: 339-345). Pure preparations of FC receptors, obtained under nondenaturing conditions, showed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis two doublets of proteins with apparent molecular masses of 30 and 90 kD. In the present paper we describe the isolation and identification of the primary structure of the 30-kD doublet proteins. Sequencing studies of peptides resulting from the digestion of the 30-kD protein showed a full identity with a 14-3-3-like protein from corn, named GF14. The 14-3-3 family is a class of proteins that is widely distributed in eukaryotes and is known to play various regulatory roles. The 30-kD protein has been immunologically identified by specific antibodies prepared against a synthetic peptide based on the determined amino acid sequence. A similar protein is recognized in partially purified FC receptor preparations from bean and spinach leaves.

ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons.

The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes, are thought to be involved in the actin filament/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoprecipitation studies using antimoesin mAb and cultured baby hamster kidney (BHK) cells metabolically labeled with [35S]methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140-kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb that recognized the 140-kD membrane protein. We next cloned a cDNA encoding the 140-kD membrane protein and identified it as CD44, a broadly distributed cell surface glycoprotein. Immunoprecipitation with various anti-CD44 mAbs showed that ezrin and radixin, as well as moesin, are associated with CD44, not only in BHK cells, but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CD44 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CD44 and actin-based cytoskeletons.

Interaction of the peroxisome-proliferator-activated receptor and retinoid X receptor.

The rat peroxisome-proliferator-activated receptor (PPAR) was expressed in insect cells and was shown to bind to a cognate PPAR response element (PPRE) from the acyl-CoA oxidase gene. Upon purification, PPAR was no longer able to bind DNA, although binding could be restored by addition of insect cell extracts. We investigated whether the retinoid X receptor (RXR) could supplement for this accessory activity. The rat RXR alpha cDNA was cloned and it was found that addition of in vitro-translated RXR alpha to purified PPAR facilitated binding of PPAR to a PPRE. Furthermore, an additional activity, which appeared to be distinct from rRXR alpha, was found in COS cell nuclear extracts that enabled binding of PPAR to a PPRE. Transient expression of RXR alpha in CHO cells was found to be essential for the response of a chloramphenicol acetyltransferase reporter construct containing PPREs to activators of PPAR. These results raise the possibility of convergence of the PPAR and retinoid-dependent signaling pathways on promoters containing PPRE-like responsive elements.

Epitope mapping reveals conserved regions of an auxin-binding protein.

There is now good evidence that maize (Zea mays) auxin-binding protein (ABP) functions as a receptor. We have synthesized sequential overlapping hexapeptides to map the epitopes recognized by a number of antisera to ABP. Only a few regions of the protein are recognized, and these are shown to be exposed on the surface. Three epitopes predominate, and these are clustered around, but do not include, the glycosylation site. A comparison is made between these maps of sera against purified ABP, maps of sera raised against recombinant maize ABP expressed in Escherichia coli and computer antigenicity predictions. Our anti-(maize ABP) serum recognizes ABP counterparts in other plant species. We have used immunoblotting to affinity-purify the immunoglobulins which cross-react from the antiserum. Epitope mapping of these immunoglobulins suggests that two of the three predominant epitopes may be conserved in both monocotyledonous and dicotyledonous plants. The possible functional significance of these conserved epitopes is discussed.

Identification and characterization of receptors for tumor necrosis factor-alpha in the brain.

Specific receptors for murine TNF have been identified in homogenates of rodent brain. These receptors are saturable and bind TNF with sufficient affinity to ensure occupancy by cytokine elaborated during infection. 125I-mTNF was detected in four specific complexes of Mr 130,000, 90,000, 66,000 and 60,000 after affinity labeling. Solubilization of brain membranes into detergent increased binding capacity 4-fold which indicates the presence of latent receptors for mTNF in the brain. Specific binding was greatest in the brainstem, least in the cerebellum and was also detected in the cortex, thalamus and basal ganglia.