Identification of the posttranslational modifications of bovine lens alpha B-crystallins by mass spectrometry.

A combination of mass spectrometric techniques has been used to investigate the amino acid sequence and post-translational modifications of alpha B-crystallin isolated from bovine lenses by gel filtration chromatography and reversed-phase high performance liquid chromatography. Chromatographic fractions were analyzed by electrospray ionization mass spectrometry to determine the homogeneity and molecular weights of proteins in the fractions. The alpha B-crystallin primary gene product, its mono- and diphosphorylated forms, its N- and C-terminal truncated forms, as well as other lens proteins unrelated to the alpha B-crystallins were identified by their molecular weights. Detailed information about the sites of phosphorylation, as well as evidence supporting reassignment of Asn to Asp at position 80, was obtained by analyzing proteolytic digests of these proteins by fast atom bombardment mass spectrometry. Results of this investigation indicate that alpha B-crystallin is phosphorylated in vivo at Ser 45, Ser 59, and either Ser 19 or 21. From the specificity of phosphorylation of alpha-crystallins, it appears that there may be two different kinases responsible for their phosphorylation.     

Packaging and unpackaging the sea urchin sperm genome.

Two species of histones in sea urchin sperm (Sp H1 and Sp H2B) are chimeric molecules whose highly basic amino-terminal domains are dephosphorylated at the last stage of sperm cell differentiation, and rephosphorylated immediately following fertilization. The phosphorylated regions consist largely of repeating tetrapeptides with two basic residues flanking Ser-Pro residues ('SPKK' motifs) and are predicted to have beta-turn secondary structures. Alteration of the charge and structure of the SPKK sites may play a role in the unusually dense DNA packaging of the mature sperm chromatin. The motif resembles the target site of cell-cycle-associated cdc2 kinases and is found in several other proteins whose nucleic acid affinities may be altered during the cell cycle.     

Peptidergic transmission in the brain. VI. Behavioral consequences of central activation.

Having described a peptidergic transmitter system in the rat brain, we now begin to evaluate its behavioral function. We stimulated cell bodies in the medial amygdaloid nucleus (AME) with indwelling bilateral electrodes. These cell bodies contain a vasopressin-like peptide and send fibers to the hippocampus where the peptide is released upon stimulation. There the peptide inhibits hippocampal output in the awake rat just as it does in the anesthetized rat and in the rat brain slice. The stimulation reorganizes behavior with the same latency and duration as the hippocampal effect. For about 15-20 minutes after the brief stimulus, rats remain motionless with eyes wide open. This "freezing" state is punctuated by episodes of exploratory behavior. The stimulus appears to have a positive affective quality. Review of the literature in light of the present results indicates a probable role for this peptidergic system in the generation of sexual behavior in male rats.     

A hypothetical model for the peptide binding domain of hsp70 based on the peptide binding domain of HLA

The sequences of the peptide binding domains of 33 70 kd heat shock proteins (hsp70) have been aligned and a consensus secondary structure has been deduced. Individual members showed no significant deviation from the consensus, which showed a beta 4 alpha motif repeated twice, followed by two further helices and a terminus rich in Pro and Gly. The repeated motif could be aligned with the secondary structure of the functionally equivalent peptide binding domain of human leucocyte antigen (HLA) class I maintaining equivalent residues in structurally important positions in the two families and a model was built based on this alignment. The interaction of this domain with the ATP domain is considered. The overall model is shown to be consistent with the properties of products of chymotryptic cleavage.     

Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution.

U2 auxiliary factor (U2AF) is a non-snRNP protein required for the binding of U2 snRNP to the pre-mRNA branch site. Purified U2AF comprises two polypeptides of 65 and 35 kd. We have performed biochemical complementation and immunological assays to characterize U2AF in greater detail. First, we use an extract lacking only U2AF activity to show that U2AF is an essential splicing factor. Second, we show that all U2AF activity in vitro resides in the 65 kd U2AF polypeptide. Third, based upon both immunological and functional criteria, we show that U2AF is evolutionarily conserved. Most significantly, a Drosophila melanogaster nuclear extract contains proteins that are antigenically related to both human U2AF polypeptides and can substitute for human U2AF in vitro. Finally, we show that U2AF has an unexpected intranuclear distribution. Although diffusely present throughout the nucleoplasm, U2AF is also concentrated in a small number (between one and five) of nuclear 'centers.' This localization differs strikingly from that reported for snRNP antigens and splicing factors. Our data, in conjunction with those in the accompanying paper [Carmo-Fonseca et al. (1991) EMBO J., 10, 195-206.], suggest that these centers represent novel aspects of nuclear organization.     

Enzyme-linked immunosorbent assays for detecting antibodies to Shiga-like toxin I,Shiga-like toxin II,andEscherichia coli O157:H7 lipopolysaccharide in human serum

Shiga-like toxin-producingEscherichia coli O157:H7 are important causes of bloody diarrhea and hemolytic uremic syndrome. To facilitate the epidemiologic study of these organisms, we developed enzyme-linked immunosorbent assays (ELISAs) for antibodies to Shiga-like toxin I (SLT I), Shiga-like toxin II (SLT II), andE. coli O157 lipopolysaccharide (LPS). We tested serum samples from 83 patients in two outbreaks ofE. coli O157:H7 diarrhea and from 66 well persons. Forty-three patients (52%) had at least one serum sample positive for anti-O157 LPS antibodies; among 26 culture-confirmed patients, 24 (92%) had at least one positive serum sample. Two (3%) of 66 control sera had positive anti-O157 LPS titers. ELISA results for SLT I and II were compared with those of HeLa cell cytotoxicity neutralization assays on both patient and control sera. Neutralization assays detected anti-SLT I antibodies in at least one serum sample from each of 17 (20%) patients and 7 (10.6%) controls, while 16 (19%) patients and 7 controls had positive titers by anti-SLT I ELISA. Although all serum samples, including control sera, showed nonspecific neutralization of SLT II, no antibody titers to SLT II were detected by either neutralization or ELISA. These results indicate that ELISAs for SLT I and SLT II antibodies are comparable to HeLa cell cytotoxicity neutralization assays. Both the ELISAs and neutralization assays are insensitive in detecting infected patients. However, the ELISA for antibodies toE. coli O157 LPS is both sensitive and specific, and may be more useful than assays for antitoxic antibodies in detecting persons withE. coli O157:H7 infection.     

Aggregation of macrophages and fibroblasts is inhibited by a monoclonal antibody to the hyaluronate receptor

To examine the role of the hyaluronate receptor in cell to cell adhesion, we have employed the K-3 monoclonal antibody (MAb) which specifically binds to the hyaluronate receptor and blocks its ability to interact with hyaluronate. In the first set of experiments, we investigated the spontaneous aggregation of SV-3T3 cells, which involves two distinct mechanisms, one of which is dependent upon the presence of divalent cation and the other is independent. The divalent cation-independent aggregation was found to be completely inhibited by both intact and Fab fragments of the K-3 MAb. In contrast, the K-3 MAb had no effect on the divalent cation-dependent aggregation of cells. In a second set of experiments, we examined alveolar macrophages. The presence of hyaluronate receptors on alveolar macrophages was demonstrated by the fact that detergent extracts of these cells could bind [3H]hyaluronate, and this binding was blocked by the K-3 MAb. Immunoblot analysis of alveolar macrophages showed that the hyaluronate receptor had a Mr of 99,500, which is considerably larger than the 85,000 Mr for that on BHK cells. When hyaluronate was added to suspensions of alveolar macrophages, the cells were induced to aggregate. This effect was inhibited by the K-3 MAb, suggesting that the hyaluronate-induced aggregation was mediated by the receptor.     

Expression of human nerve growth factor receptor on cells derived from all three germ layers

Nerve growth factor (NGF) is a protein which promotes the survival and differentiation of neuronal cells in vitro and plays an important role in neuronal development. In this study, we have examined the expression of the receptor for NGF (NGFR) in human neuronal and nonneuronal cells, both in tissue culture and in vivo. In addition to cell lines derived from neuroblastoma, astrocytoma, and melanoma, all of which share a common neuroectodermal origin, NGFR was detected in a number of cultured cells of mesenchymal, epithelial, and hematopoietic derivation. Immunohistochemical analysis showed that NGFR is expressed in several nonneural human tissues, and the cell types in which NGFR was found include derivatives from all three germ layers. Thus, our findings demonstrate that NGFR is much more widely expressed in human cells and tissues than was previously thought.     

Differential sensitivities to gamma radiation of human bladder and testicular tumour cell lines

Gamma radiation sensitivities of continuous cell lines from nine human tumours were measured, comparing four derived from transitional cell carcinomas of the bladder with five from non-seminomatous germ cell tumours of the testis. The testicular cells were significantly more radiosensitive than the bladder cells, corresponding to the response to therapy of these tumour types in patients. These observations indicate that radiosensitivity is retained in vitro and is an inherent property of the testicular tumour cells. These gamma radiation sensitivities were compared with those of SV40-transformed fibroblasts derived from a normal individual and one with the heritable disease, ataxia-telangiectasia (A-T). The bladder cells had gamma radiation sensitivities similar to that of the SV40-transformed normal line. The testicular cells were hypersensitive to gamma radiation, although not as sensitive as the SV40-transformed A-T line. A-T cells, unlike those derived from normal individuals, continue to synthesize DNA at a normal rate following radiation exposure, prompting a comparison of the kinetics of DNA synthesis in three bladder and three testicular tumour cell lines. One of the bladder and two testicular lines showed a reduced inhibition when compared to the other tumour cell lines and the SV40-transformed normal line. Thus there was no clear association between DNA synthesis inhibition and radiosensitivity.     

The glutamine residues reactive in transglutaminase-catalyzed cross-linking of involucrin

The protein involucrin, synthesized by human keratinocytes, contains 585 amino acids, largely in the form of 10 amino acid repeats, each containing glutamines in 3 conserved positions. Involucrin is a substrate for the keratinocyte transglutaminase and is labeled by the cosubstrate amine, glycine ethyl ester. Study of tryptic peptides of involucrin shows that a single glutamine (residue 496), located 89 residues from the C-terminal end, is preferentially labeled by the enzyme. Additional glutamine residues become reactive when the molecule is fragmented. The C-terminal end, isolated as a cyanogen bromide fragment of 275 residues, is labeled equally at 2 glutamine residues. The polypeptide containing residues 148 to 280 accepts practically no amine while in intact involucrin but as a free fragment is labeled at multiple glutamine residues. It is concluded that the C-terminal and N-terminal ends of the protein are directive influences in that they suppress the reactivity of a number of glutamine residues in the intact molecule, leaving one glutamine highly preferred by the transglutaminase.