Carcinoembryonic antigens: alternative splicing accounts for the multiple mRNAs that code for novel members of the carcinoembryonic antigen family.

The recent cloning of complete cDNAs encoding carcinoembryonic antigen (CEA) and non-specific cross-reacting antigen has revealed the existence of a new gene family belonging to the immunoglobulin gene superfamily. We have reported the isolation of a partial CEA cDNA and of L-cell transfectant cell lines that express human antigens cross-reactive with commercial antibodies directed to native CEA (Kamarck, M., J. Elting, J. Hart, S. Goebel, P. M. M. Rae, J. Nedwin, and T. Barnett. 1987. Proc. Natl. Acad. Sci. USA. 84:5350-5354). In this study, we describe the identification and cloning of 3.9-, 3.7-, 2.2-, and 1.8-kb cDNAs and a 23-kb genomic transcription unit, which code for new members of the CEA gene family. DNA sequence analysis of these cloned DNAs establishes the existence of a set of four alternatively spliced mRNAs which are expressed in several tumor cell lines, in human fetal liver, and in L-cell transfectants. Deduced amino acid sequences of the encoded isoantigens show extensive similarity to CEA and nonspecific cross-reacting antigens, but in addition demonstrate transmembrane and cytoplasmic domains. We designate members of this antigen family transmembrane CEAs. The transmembrane CEA isoantigens share general structural characteristics with members of the immunoglobulin gene superfamily and can be specifically compared to the cell adhesion molecules, N-CAM (neural cell adhesion molecule) and MAG (myelin-associated glycoprotein).

Effects of cesalin on the ultrastructure and biological properties of cultured mammalian cells.

Cesalin rapidly inhibits the incorporation of uridine and thymidine into KB, MCF-7, HBL-100, and HTC cells but has no measurable effect on AlAb cells. Protein syntheiss is inhibited only after the effect on DNA and RNA is observed. After 12 to 48 hr, the cells in cultures containing cesalin increasingly lose adhesion to the flask surface and float in the medium. Both the inhibition of nucleotide incorporation and the inhibition of the cell growth, used as assays for cytotoxicity, show a varying sensitivity of these cell lines to cesalin, with AlAb cells being the most resistant and KB cells being the most sensitive. The ultrastructural changes induced by cesalin in KB cells demonstrate alteration in the nucleolus, increase in rough endoplasmic reticulum, extensive blebbing of the plasma membrane, and invagination of the nuclear membrane. The blebbing of the plasma membrane decreases after 24 hr with the appearance of a highly disorganized nuclear structure and numerous vacuoles containing insoluble fragments.

Nonspecific cross-reacting antigen 50/90 is elevated in patients with breast, lung, and colon cancer.

A total of 22 genes have been identified in the carcinoembryonic antigen (CEA) gene family. The protein products of this family are highly homologous and include CEA, biliary glycoprotein, nonspecific cross-reacting antigen 50/90 (NCA 50/90), NCA 95, and pregnancy-specific beta-glycoprotein. We used a monoclonal antibody with high affinity to develop a specific enzyme-linked immunosorbent assay (ELISA) method for NCA 50/90 in serum and plasma. Our calibrators were based on affinity-purified recombinant protein from a baculovirus expression system. No significant reactivity with purified CEA, recombinant NCA 95, or recombinant biliary glycoprotein was found by Western blot analysis or in the ELISA method. Only 1 of 15 sera from pregnant women (chorionic gonadotropin > 1000 ng/ml) was positive in the NCA 50/90 ELISA, suggesting that this method does not detect pregnancy-specific glycoprotein. A cutoff value of 18 ng/ml was established based on the 95% value of serum and plasma from 147 healthy volunteers. Only 3 of 31 serum and plasma samples from patients with clinically inactive breast cancer were elevated above the cutoff value, but 44% of 136 samples from patients with clinically active breast cancer were positive. NCA 50/90 measurements were elevated in 7 of 25 patients with active breast cancer whose CEA and CA 15-3 values were below cutoff, and NCA 50/90 values do not correlate with CEA in breast cancer. In addition, we found sensitivities of 70, 39, and 42% for lung cancer, colon cancer, and leukemia, respectively. The sensitivity for non-small cell lung cancer was 85%, however, compared to 50% for small cell lung cancer. Serum from leukemia patients showed an overall sensitivity of 43%, but 71% (10 of 14) sera from patients with chronic myelogenous leukemia were positive compared to, for example, chronic lymphocytic leukemia where 0 of 7 sera had NCA 50/90 values above the cutoff. These studies suggest that NCA 50/90 may have clinical utility in the management of patients with a variety of cancers.

Selective localization of the parathyroid secretory protein-I/adrenal medulla chromogranin A protein family in a wide variety of endocrine cells of the rat.

Secretory protein-I (SP-I) of parathyroid glands and chromogranin A ( CGA ) of adrenal medullary chromaffin cells are chemically similar if not identical proteins. Both proteins are contained within secretory granules and appear to be cosecreted with granule contents, for example, in the parathyroid with PTH and in the adrenal with epinephrine and dopamine beta-hydroxylase. Antisera to bovine SP-I and porcine CGA , together with antisera to a variety of peptide hormones, were used in an immunofluorescence study of rat tissues in order to determine the probable distribution and cellular localization of these proteins. In addition to their previously demonstrated presence in parathyroid and adrenal cells, the SP-I/ CGA protein family was detected in cells of the thyroid that contained calcitonin and often SRIF but not thyroglobulin; in cells of the anterior pituitary staining for the alpha-subunit of TSH/FSH/LH but not in cells staining for GH, PRL, ACTH, or beta-endorphin; in pancreatic islet cells staining for SRIF and pancreatic polypeptide-related peptides, but not for insulin or glucagon; in the celiac and mesenteric ganglia in cells some of which contained SRIF; and in the gastric antrum in cells containing SRIF, but not gastrin. SP-I/ CGA was not detected in cells of the liver, kidney, parotid gland, or acinar pancreas or in the intermediate or posterior lobes of the pituitary. These results suggest that this protein family enjoys a widespread but highly restricted distribution in many different endocrine-peptide cells of the rat, many that are believed to be of the APUD cell series. The possibility is raised that SP-I/ CGA plays some physiological role in the secretory process or exerts an effect of its own in the periphery after secretion.

SPECT evaluation of lumbar spondylolysis and spondylolisthesis.

Fifty patients with back pain and radiologically diagnosed spondylolysis were evaluated by a single-photon emission computed tomography (SPECT bone scanning). These patients were separated into three groups according to the degree of spondylolisthesis accompanying the spondylolysis. The data obtained from the study indicate that in acute spondylolysis, the SPECT scan is positive at the pars interarticularis. As the spondylolysis becomes chronic, the SPECT scan tends to revert toward normal even though healing of the spondylolysis has not occurred. As spondylolisthesis develops and progresses, the SPECT scan again becomes positive. The positivity, however, is more anterior and more diffuse. The authors propose that SPECT scanning in spondylolysis is not a positive or negative process, but rather varies with the time and stability of the spondylolytic spine.

SPECT evaluation of unilateral spondylolysis.

Three patients with unilateral spondylolysis were evaluated with SPECT and Lumbar CT. All three cases had positive scans. However, the SPECT images were unique to each case. Therefore, the interpretation of the SPECT activity was undertaken in terms of the known natural history of both unilateral and bilateral spondylolysis.

Impaction grafting: preliminary report of a new method for exchange femoral arthroplasty.

ABSTRACTGie et al described a method for femoral component revision using compressed morselized cancellous allograft and a cemented collarless polished taper stem. We have reviewed our first 37 exchanges performed in this manner. Results after 2 to 5 years have reflected few complications, a majority of satisfactory results, evidence of graft incorporation, and no mechanical loosening. Further study is necessary; however, these preliminary findings give rise to cautious optimism.

A possible case of pre-Columbian treponematosis from New York State.

This paper presents evidence for what is believed to be the first reported case of pre-Columbian treponematosis in northeastern North America. The temporal position of the skeletal material, salvaged from a site in New York State, is determined on the basis of archeological evidence.

Unilateral frame distraction: proximal tibial valgus osteotomy for medial gonarthritis.

An undertreated group has emerged in modern orthopaedic surgery--patients with degenerative disease of the medial compartment of the knee who are young and active, who may have undergone multiple arthroscopic procedures, and who have been advised to "take it easy and wait for a total knee replacement." If the knee is in varus, unilateral frame distraction with upper tibial corticotomy is advocated to achieve valgus alignment. The procedure is straightforward, with few complications in our first 100 cases, with almost universal success. Even if it is believed that all tibial osteotomies will eventually fail and require a total knee replacement, this method, by which bone stock is actually enhanced, length is maintained, and the ligamentous structures about the knee are undamaged, is superior to methods previously described.

Characterization of two new members of the pregnancy-specific beta 1-glycoprotein family from the myeloid cell line KG-1 and suggestion of two distinct classes of transcription unit.

Pregnancy-specific beta 1-glycoproteins (PSGs) represent a large group (approximately 12-15) of proteins, related to members of the carcinoembryonic antigen family, that are abundant in placental tissue and in the sera of pregnant women. We describe the isolation and characterization of two additional PSG cDNAs, PSG9 and PSG10, whose transcripts are largely expressed in placental tissue and to a lesser extent in some other cell types, including myeloid cells differentiated to granulocytes. PSG9 and PSG10 are representatives of two distinct classes of PSG protein that have N-termini with or without the Arg-Gly-Asp motif implicated in adhesion. In addition to this distinction at the amino acid level, our analysis of several PSG cDNAs suggests that the transcription units encoding these proteins may be further distinguished in their 3' untranslated sequences, thus suggesting possibilities for transcriptional regulation of the two major protein classes.

Carcinoembryonic antigen family: characterization of cDNAs coding for NCA and CEA and suggestion of nonrandom sequence variation in their conserved loop-domains.

We have isolated cDNAs for carcinoembryonic antigen (CEA) and for a normal cross-reacting antigen (NCA) and report here their nucleotide and derived amino acid sequences. Our data show that both the CEA and NCA polypeptides are organized into extracellular domains, some with cysteine-linked loops, that share extensive sequence homology (approximately 78% overall) with each other and appear similar to immunoglobulin superfamily members. A major difference between the two apoproteins is the presence of a single loop-domain in NCA compared to three tandemly repeated loop-domains in CEA. Sequence comparisons between the extracellular domains of CEA and NCA show that the N-terminal and adjacent loop domains of each apoprotein have high homology (85-90%) to each other, while comparison of loop-domain regions reveals a possible nonrandom distribution of base changes and altered amino acids near certain cysteine residues that are inferred to be involved in forming disulfide loops. Both apoproteins show high identity in their hydrophobic C-termini that are reminiscent of the type of transmembrane tails seen in proteins that potentiate signal transduction. These findings, coupled with distinct expression profiles of CEA and NCA mRNAs, suggest that these apoproteins may function as unique cell-surface molecules mediating cell-specific interactions in normal and neoplastic cells.

Co-localization of parathyroid hormone and secretory protein-I in bovine parathyroid glands: a double immunocytochemical study at the electron microscopical level.

Secretory protein-I (SP-I), also known as chromogranin A, is an acidic glycoprotein of unknown function that is found in large amount in the secretory granules of all endocrine and neuroendocrine cells, but not in exocrine or epithelial cells. It is cosecreted with parathyroid hormone (PTH) and by immunocytochemical staining has been reported to exist in the same subcellular structures of the gland. In the present study we have used the colloidal gold-double immunocytochemical technique at the ultrastructural level to precisely define the locales of SP-I and PTH in the bovine parathyroid cell. SP-I and PTH were co-localized to the same secretory granules. The patterns for both gold labels were diffuse ones throughout the granule and suggested that there was a general association of SP-I and PTH. There was little or no localization of the SP-I at the secretory granule membrane. The results support the concept that SP-I is responsible for stabilization of PTH within the secretory granules.

Identification of an additional negative regulatory region for p53 sequence-specific DNA binding.

The DNA binding activity of p53 is crucial for its tumor suppressor function and is subject to tight regulation. Previous studies revealed that the inhibitory function of the p53 C terminus is implicated in the latent, low affinity sequence-specific DNA binding activity of p53 in the uninduced state. Sequence-specific DNA binding of p53 has been shown to be activated by several posttranslational modifications and interacting proteins that target predominantly the C terminus. Moreover, several authors have shown that synthetic peptides corresponding to p53 C-terminal sequences activate p53 sequence-specific DNA binding. In an effort to identify the interaction site of p53 with these activating peptides we assessed complex formation between p53 deletion constructs and C-terminal activating peptides by peptide affinity precipitation. This study revealed that two distal regions of the p53 molecule contribute synergistically to the interaction with activating C-terminal peptides: amino acids 80-93 and 364-393. The C-terminal residues 364-393 are already well characterized as having negative regulatory function. DNA binding analyses with these deletion constructs reveal a comparable negative regulatory activity for residues 80-93, defining this region as a previously unidentified negative regulatory domain of p53. Furthermore, synthetic peptides spanning this newly identified proline-rich negative regulatory region (residues 80-93) are able to activate p53 sequence-specific DNA binding in vitro. We suggest that both negative regulatory regions, residues 80-93 and 364-393, contribute cooperatively to the maintenance of the latent, low-affinity DNA binding conformation of p53.

A peptide sequence on carcinoembryonic antigen binds to a 80kD protein on Kupffer cells.

Clearance of carcinoembryonic antigen (CEA) from the circulation is by binding to Kupffer cells in the liver. We have shown that CEA binding to Kupffer cells occurs via a peptide sequence YPELPK representing amino acids 107-112 of the CEA sequence. This peptide sequence is located in the region between the N-terminal and the first immunoglobulin like loop domain. Using native CEA and peptides containing this sequence complexed with a heterobifunctional crosslinking agent and ligand blotting with biotinylated CEA and NCA we have shown binding to an 80kD protein on the Kupffer cell surface. This binding protein may be important in the development of hepatic metastases.