Molecular characterization of two human autoantigens: unique cDNAs encoding 95- and 160-kD proteins of a putative family in the Golgi complex.

Serum autoantibodies from a patient with autoantibodies directed against the Golgi complex were used to screen clones from a HepG2 lambda Zap cDNA library. Three related clones, designated SY2, SY10, and SY11, encoding two distinct polypeptides were purified for further analysis. Antibodies affinity purified by adsorption to the lambda Zap-cloned recombinant proteins and antibodies from NZW rabbits immunized with purified recombinant proteins reproduced Golgi staining and bound two different proteins, 95 and 160 kD, from whole cell extracts. The SY11 protein was provisionally named golgin-95 and the SY2/SY10 protein was named golgin-160. The deduced amino acid sequence of the cDNA clone of SY2 and SY11 represented 58.7- and 70-kD proteins of 568 and 620 amino acids. The in vitro translation products of SY2 and SY11 cDNAs migrated in SDS-PAGE at 65 and 95 kD, respectively. The in vitro translated proteins were immunoprecipitated by human anti-Golgi serum or immune rabbit serum, but not by normal human serum or preimmune rabbit serum. Features of the cDNA suggested that SY11 was a full-length clone encoding golgin-95 but SY2 and SY10 together encoded a partial sequence of golgin-160. Analysis of the SY11 recombinant protein identified a leucine zipper spanning positions 419-455, a glutamic acid-rich tract spanning positions 322-333, and a proline-rich tract spanning positions 67-73. A search of the SwissProt data bank indicated sequence similarity of SY11 to human restin, the heavy chain of kinesin, and the heavy chain of myosin. SY2 shared sequence similarity with the heavy chain of myosin, the USO1 transport protein from yeast, and the 150-kD cytoplasmic dynein-associated polypeptide. Sequence analysis demonstrated that golgin-95 and golgin-160 share 43% sequence similarity and, therefore, may be functionally related proteins.

A functional in vitro model for studies of intracellular motility in digitonin-permeabilized erythrophores.

Phase contrast cine results demonstrate that erythrophores maintain saltatory particle motion for hours after permeabilization with 0.001% digitonin in a cytoskeletal stabilizing solution at 23 degrees C. High voltage electron microscopy (HVEM) studies reveal that cytoskeletal elements are retained intact, except in immediate subplasmalemmal regions where the plasma membrane is punctured by digitonin. During digitonin treatments, cells are permeable to ions, small molecules, and antibodies. We find that motion is Ca2+ and ATP-sensitive, and optimal in PIPES buffer (pH 7.2 containing 1 mM Mg2+/ATP and EGTA-CA2+ (10(-7) M Ca2+) at 37 degrees C. Experiments testing the inhibitory effects of vanadate (0.4-10 microM), ouabain (100-600 microM), N-ethyl maleimide, and the cytochalasins B and D indicate that a dyneinlike ATPase may provide the motive force for driving saltatory pigment motion in erythropores.

Alterations in chromatin conformation are accompanied by reorganization of nonchromatin domains that contain U-snRNP protein p28 and nuclear protein p107.

The intranuclear distribution of nuclear matrix-associated protein p107 and the 28-kD Sm antigen of U-snRNPs have been studied using double-label immunofluorescence and immunoperoxidase electron microscopy. In interphase nuclei of HeLa cells, Novikoff hepatoma cells, and rat kangaroo kidney cells, p107 was confined to discrete interchromatin domains. The domains had an irregular contour, with an average diameter of 1-1.5 micron. Each domain appeared to be composed of interconnected granules. The Sm antigen colocalized and appeared concentrated in these domains but also showed some general nucleoplasmic distribution. During mitosis, the interchromatin domains disassembled such that the Sm portion redistributed to the perichromosomal and spindle regions and the p107 component redistributed throughout the mitotic cytoplasm. During anaphase, p107 assembled into discrete clusters throughout the mitotic cytoplasm. The Sm antigen was not a component of these clusters. Double-label immunofluorescence with anti-p107 and the anti-DNA tight-binding protein, AhNa1, showed that the extranuclear p107 domains assumed an interchromatin localization only after the chromosomes had decondensed. The correlation between chromosome decondensation and the occurrence of p107 within interchromatin domains was also observed during chicken erythrocyte nuclear reactivation. We propose that the discrete interchromatin domains that contain p107 and p28 may be important for processing and splicing of RNA and that their structural assembly within nuclei is sensitive to the presence of the transcriptionally active conformation of chromatin.

cDNA cloning and characterization of a novel nucleolar protein.

In an initial study of anti-nuclear antibodies in the chronic inflammatory bladder disease interstitial cystitis, we reported that 7% of interstitial cystitis patients studied had autoantibodies to the nucleolus. We now report that, using an autoimmune serum from a patient with interstitial cystitis, we have identified and partially characterized a novel protein with an M(r) of approximately 55 kDa (hereafter referred to as No55) localized to the granular component of the nucleolus. No55 was initially characterized by diffuse nucleolar immunofluorescence staining in interphase cells and by Western blotting as a 55-kDa doublet on whole-cell extracts. During mitosis, No55 was associated with chromosomes and appeared in prenucleolar bodies during telophase, but it did not colocalize with p80-coilin in coiled bodies. Immunoelectron microscopy revealed that No55 was localized uniformly throughout the granular component of the nucleolus compared with a more peripheral localization of nucleolar granular component protein B23. On segregation of the nucleolus with actinomycin D, No55 remained with the granular component of the segregated nucleolus, whereas protein B23 was found predominantly in the nucleoplasm. Finally, a cDNA expression library was screened with the human autoantibody against No55, and a 2.4-kb insert was isolated, subcloned to homogeneity, and then sequenced. Analysis of this sequence showed an open reading frame of approximately 1.3 kb coding for 437 amino acids with a predicted molecular weight of 50 kDa. A search of the gene sequence database indicated homology with SC65, a rat synaptonemal complex protein. Therefore, on the basis of molecular weight, nucleolar sublocalization, response to actinomycin D, and cDNA sequence determination, No55 is a novel protein of the interphase nucleolus.

Nucleologenesis: U3 snRNA-containing prenucleolar bodies move to sites of active pre-rRNA transcription after mitosis.

We have investigated the distribution of U3 snRNA and rRNA in HeLa cells and normal rat kidney cells during interphase and mitosis. U3 snRNA, known to be involved in pre-rRNA processing, was detected in nucleoli and coiled bodies during interphase, whereas rRNA was distributed in the nucleoli and throughout the cytoplasm. By comparison, ribosomal protein S6 was detected in nucleoli, coiled bodies, and in the cytoplasm. During nucleologenesis, pre-rRNA was observed in newly forming nucleoli during late telophase but not in prenucleolar bodies (PNBs), whereas U3 snRNA was detected in forming nucleoli and PNBs. Similar findings to those reported here for the localization of U3 snRNA have been reported previously for the U3 small nuclear ribonucleoprotein fibrillarin. These results suggest that components involved in pre-rRNA processing localize to discrete PNBs at the end of mitosis. The nucleolus is formed at specific telophase domains (nucleolar organizing regions) and the PNBs, containing factors essential for pre-rRNA processing, are recruited to these sites of rRNA transcription and processing.

Formation of nuclear bodies in hepatocytes of estrogen-treated roosters.

As a model for cellular growth and stimulation without accompanying proliferation, we have examined the induction and formation of nuclear bodies (NBs) in hepatocytes of estrogen-treated roosters. Four-week-old roosters were injected with a single intramuscular dose of estradiol and then killed at time points of 8 h, 48 h, and 4 wk post-injection. For immunofluorescence analyses, livers were excised and isolated hepatocyte nuclei were fixed and then labeled with antibody to the coiled body-specific protein, p80-coilin. In control animals (no estradiol) or in animals 8 h post-injection, each hepatocyte nucleus contained an average of 1.0 coiled body (CB), which appeared randomly distributed in the nucleoplasm. At 48 h post-injection, there were an average of 2.7 CBs/nucleus and many of these appeared to be in contact with the nucleolus. Pairs of CBs were also observed. By 4 wk post-injection an average of 1.5 CBs/nucleus were detected, with no apparent relationship to the nucleolus observed. By serial-section electron microscopy of intact livers, two different types of round NBs were observed, sometimes in close proximity to each other and to the expanded interchromatin granule region in maximally stimulated cells. One type of NB was a classical CB that averaged 0.35 microns in diameter and the other NB type was ring shaped, averaged 0.25 microns in diameter, was composed of a fibrous shell surrounding a hollow interior, and appeared as a simple NB when sectioned tangentially through its outer shell. Immunoelectron microscopy revealed that CBs were the only class of NBs that contained p80-coilin. From these data, we conclude that CBs proliferate in response to estrogen stimulation, possibly arising from the nucleolar surface and then increasing in number by replicative division.

Intranucleolar localization of human proliferating cell nucleolar antigen p120.

The human proliferation-associated nucleolar antigen p120 was localized to substructures within HeLa cell nucleoli by immunofluorescence and immunoelectron microscopy of cells whose nucleoli were segregated by drug treatment or extracted with nucleases. By indirect immunofluorescence, protein p120 was localized diffusely throughout all interphase nucleoli. However, high resolution immunoelectron microscopy demonstrated that protein p120 staining delineated a network of 20-30-nm diameter beaded fibrils distributed throughout the nucleolus. This distribution was unique compared to that of the nucleolar proteins p145, RNA polymerase I, or B23 which were examined simultaneously. Drug-induced segregation of nucleoli by actinomycin D or dichlorobenzimidazole riboside, followed by immunoelectron microscopy, indicated that protein p120 was concentrated at the periphery of the granular region in segregated nucleoli. In situ nuclease digestion of cells with DNase I and/or RNase A did not release p120 from the nucleolus. Instead, p120 immunoreactivity was retained within phase-dense residual nucleoli. These results provide evidence that protein p120 is associated with, and in fact delineates, a network of fibrils which is retained in the nucleolar residue fraction of proliferating cells.

Effect of differentiation on the expression of a nucleolar antigen with a molecular weight of 145,000 in HL-60 cells.

Mr 145,000 nucleolar protein antigen (p145) is associated with growing cells (R. L. Ochs et al., J. Cell Biol., 101: 211a, 1985) and has been found in a broad range of human cancers (J. W. Freeman et al., Cancer Res., 46: 3593-3598, 1986). In this study the presence of nucleolar antigen p145 was examined in the human promyelocytic tumor cell line HL-60 which was induced to differentiate by retinoic acid. Differentiation was monitored by morphological changes, [3H]thymidine accumulation, the ability of cells to reduce nitroblue tetrazolium, and cell number. The monoclonal antibody to nucleolar antigen p145 produced bright immunofluorescence in all cycling interphase HL-60 cells; during mitosis only diffuse staining was detected. Nucleolar antigen p145 in HL-60 cells was undetectable after 132 h of treatment with retinoic acid. The absence of nucleolar antigen p145 was associated with an 81% decline in thymidine accumulation and apparent inactivation of ribosomal and nonribosomal DNA transcription as observed by electron microscopy. The loss in expression of the antigen also correlated with increased nitroblue tetrazolium-positive cells, appearance of morphologically distinct myeloid cells, and termination of cell proliferation. These data indicate that the expression of nucleolar antigen p145 occurred in cycling HL-60 cells but not in terminally differentiated noncycling HL-60 cells.

Mechanism for the inhibitory and stimulatory actions of proteins on the activity of phospholipase A2.

The influence of proteins on phospholipase A2 was found to depend strongly on the enzyme assay system. We have used three different systems to measure phospholipase A2 which represent the different assay conditions used by a number of previous investigators. Two distinct stimulatory and two distinct inhibitory effects of proteins were observed. (1) A number of proteins - such as albumin, gamma-globulin and lysozyme - were found to inhibit phospholipase A2 activity only at very low substrate concentrations. This 'substrate depletion' was recently proposed as the mode of action for lipocortin. We therefore suggest that substrate depletion is not sufficiently specific to serve as a physiological regulatory mechanism and that the observed inhibition by lipocortin and other proteins more recently reported to mimic it are unlikely to be of physiological significance. (2) Use of liposomes at higher concentrations led to a nonlinear time-course. In this assay system, albumin (and other protein) stimulation can be accounted for as relief of product inhibition. (3) With high concentrations of phospholipids in the presence of cholate (mixed micelles), the behavior of proteins in the assay was complex. The assay time-course appeared linear in the absence of added protein, but at concentrations of added albumin up to 1 mg/ml, stimulation of phospholipase A2 activity was observed. Concentrations greater than this led to diminution of enzyme activity to the original activity. No effect whatever was observed when lysozyme was substituted for albumin. Since this biphasic result was not observed with liposomes, we suggest that the product whose inhibition is being relieved is the lysophosphatidylcholine, and not the free fatty acid. The inhibitory effect at high albumin concentrations is probably the result of removal of free fatty acids from the micelle: fatty acids are known to cause stimulation of phospholipase A2 by providing a negative charge to the lipid/water interface. (4) A different type of phospholipase A2 stimulation was apparent with melittin. This was found to be more specific than generally believed: we found no melittin stimulation of pancreatic phospholipase A2, yet confirmed a several-fold stimulation of bee venom phospholipase A2. We also found that high (millimolar) concentrations of calcium suppressed the melittin stimulation of bee venom phospholipase A2, and that a cationic detergent mimicked the stimulation by melittin. (5) We conclude that the effects of proteins on phospholipase A2 studied here can all be explained by proteins binding to substrate or product rather than enzyme-protein interactions.

Aminooxyacetate inhibits gluconeogenesis by isolated chicken hepatocytes.

Although the pathway for glucose synthesis from lactate in avian liver is not thought to involve transamination steps, inhibitors of transamination (aminooxyacetate and L-2-amino-4-methoxy-trans-3-butenoic acid) block lactate gluconeogenesis by isolated chicken hepatocytes. Inhibition of glucose synthesis from lactate by aminooxyacetate is accompanied by a large increase in the lactate-to-pyruvate ratio. Oleate largely relieves inhibition by aminooxyacetate and lowers the lactate-to-pyruvate ratio. In parallel studies with rat hepatocytes, oleate did not overcome aminooxyacetate inhibition of glucose synthesis. The ratios of lactate used to glucose formed were greater than 2 with both rat and chicken hepatocytes, were increased by aminooxyacetate, and were restored toward 2 by oleate. Thus, in the absence of oleate, lactate is oxidized to provide the energy needed to meet the metabolic demand of chicken hepatocytes. Excess cytosolic reducing equivalents generated by the oxidation of lactate to pyruvate are transferred from the cytosol to the mitosol by the malate-aspartate shuttle. Aminooxyacetate inhibits the shuttle and, consequently, glucose synthesis for want of pyruvate.

Vasopressin stimulates pyruvate utilization through a Ca(2+)-dependent mechanism and lactate formation by a protein kinase C-dependent mechanism in isolated rat hepatocytes.

Vasopressin stimulates lactate production by hepatocytes from fed rats, an effect which has been attributed exclusively to Ca2+ activation of glycogenolysis. We provide evidence here for two further actions of vasopressin which affect lactate formation by rat hepatocytes. In the presence of 50 mM glucose, vasopressin inhibited lactate production by hepatocytes. The inhibition was relieved by the presence of alpha-cyano-4-hydroxycinnamate (alpha-CHC), which blocks mitochondrial pyruvate transport. This suggests that vasopressin stimulates pyruvate utilization in the presence of a high concentration of glucose. Epidermal growth factor (EGF), which also increases lactate formation by hepatocytes, did not similarly decrease lactate accumulation in the presence of high glucose, suggesting no stimulation of lactate and pyruvate utilization by this hormone. In cells depleted of Ca2+, vasopressin also stimulated lactate formation. Although vasopressin did not cause the apparent translocation of protein kinase C between cell spaces, phospholipase C treatment of hepatocytes did duplicate vasopressin stimulation of lactate formation, provided fatty acid oxidation was suppressed by the simultaneous presence of the inhibitor palmixorate. We conclude that three actions of vasopressin affect lactate and pyruvate formation: the calcium-linked activations of glycogenolysis and mitochondrial pyruvate utilization, and a stimulation of glycolysis likely mediated by protein kinase C.

Mechanism for the oleate stimulation of gluconeogenesis from dihydroxyacetone by hepatocytes from fasted rats.

Oleate stimulates glucose production and concomitantly decreases lactate and pyruvate production by rat hepatocyte suspensions incubated with dihydroxyacetone as substrate. The actions of oleate could be blocked by D-(+)dodecanoylcarnitine, which inhibits transport of the fatty acid into the mitochondria and the subsequent oxidation. beta-Hydroxybutyrate, but not acetoacetate, also stimulated glucose synthesis and inhibited lactate and pyruvate production. Furthermore, both beta-hydroxybutyrate and oleate stimulated oxygen consumption to the same extent. This suggests that oleate stimulates glucose production by the provision of energy subsequent to mitochondrial beta-oxidation of the fatty acids. The content of ATP itself did not appear to be responsible for the effects of oleate. Crossover analysis of the gluconeogenic intermediates implicated a site of oleate action between fructose 1,6-bisphosphate and fructose 6-phosphate, suggesting phosphofructokinase and/or fructose-bisphosphatase as possible regulatory sites. Coupled with the finding that intracellular citrate accumulates upon addition of oleate or beta-hydroxybutyrate, but not acetoacetate, the results suggest that citrate inhibition of phosphofructokinase accounts for the redirection of carbon flow from lactate and pyruvate formation and towards that of glucose.

Purification of an 86-70 kDa nuclear DNA-associated protein complex.

In the course of studies on nucleolar antigens, monoclonal antibodies were developed, one of which recognized an 86 kDa antigen as shown by analysis of nuclear extracts from HeLa or Namalwa cells. Immunofluorescence studies on HeLa cells showed a nucleoplasmic and phase-dependent nucleolar localization of the monoclonal antibody was decreased after digestion with DNAase I but not with RNAase A. For purification, the antigen was released from nuclei by digestion with DNAase I and then purified by chromatography on DEAE cellulose, phosphocellulose and antibody-Sepharose affinity chromatography. Interestingly, the immunoaffinity purified product contained two polypeptide chains; the immunoreactive polypeptide had an Mr of 86 000 and a pI of 6.0. The complex also contained a 70 kDa, pI 6.5 nonantigenic polypeptide in a 1:1 ratio. The overall purification of the complex was 5700-fold. Both polypeptides contained approx. 15 mol% glutamic acid and the 70 kDa polypeptide contained approx. 15 mol% serine.

Distinct cleavage products of nuclear proteins in apoptosis and necrosis revealed by autoantibody probes.

A central mechanism in apoptosis is the activation of proteases of the caspase (cysteine aspartases) family. Protease activation has also been implicated in necrosis, but its role in this cell death process and the identity of the proteases involved and their substrates, are unknown. Using human autoantibodies to well characterized cellular proteins as detecting probes in immunoblotting, we observed that a defined and somewhat similar set of nuclear proteins, including poly (ADP-ribose) polymerase (PARP) and DNA topoisomerase I (Topo I), were selectively cleaved during both apoptosis and necrosis of cultured cells induced by various stimuli. The resulting cleavage products were distinctively different in the two cell death pathways. In contrast to apoptosis, the cleavages of PARP and Topo I during necrosis were not blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). These findings suggest that different proteases act in apoptosis and necrosis, and that although both cell death processes result in selective cleavage of almost identical cellular proteins, they can be distinguished immunochemically on the basis of their cleavage products.

Nuclear remodeling in response to steroid hormone action.

Steroid and similar hormones comprise the broadest class of gene regulatory agents known, spanning vertebrates through the lower animals, and even fungi. Not unexpectedly, therefore, steroid receptors belong to an evolutionarily highly conserved family of proteins. After complexing with their cognate ligands, receptors interact with hormone response elements on target genes and modulate transcription. These actions are multifaceted and only partly understood, and include large-scale changes in the structure and molecular composition of the affected cell nuclei. This chapter examines steroid hormone action and the resultant nuclear remodeling from the following perspectives: (1) Where are the receptors located? (2) Which nuclear domains are most affected? (3) Are there extended or permanent nuclear changes? (4) What is the role of coiled bodies and similar structures in this regard? To address these and related questions, information is drawn from several sources, including vertebrates, insects, and malignant tissues. Entirely new data are presented as well as a review of the literature.

The role of microtubules in cell shape and pigment distribution in spreading erythrophores.

In order to investigate the role of microtubules in determining overall shape of the cell and distribution of pigment granules, a correlative whole cell electron microscopic and immunofluorescence light microscopic study was done of microtubule distribution during spreading of cultured erythrophores from scales of the squirrel fish, Holocentrus. After dissociation from the scale and immediately after attachment, erythrophores are round with long thin processes containing bundles of microtubules with associated pigment granules. In time these processes attach, and elongate, and cytoplasmic ground substance fills areas between microtubule bundles, preceding the appearance of microtubules in these areas of the cell. By 8 h cells are fully spread (star-shaped), prominent microtubule bundles have disappeared, and microtubules (along with pigment granules) are more evenly dispersed throughout the cell. After 24 h cells have increased in size, in number of microtubules, and in the length of preexisting tubules. The sequence of events in normal spreading is altered if cytochalasin D, cycloheximide, or nocodazole is included in the culture medium. Cytochalasin D, a microfilament-disrupting drug, prevents attachment and spreading, but allows elongation to occur. Cycloheximide, a protein synthesis inhibitor, allows for attachment and elongation, but no spreading. Nocodazole, a microtubule-disrupting drug, allows for attachment and spreading, but an irregular cell outline is the result. Even though spreading can occur without them, it is concluded that a normal number and distribution of microtubules are required for the development of normal cell shape and pigment distribution.

The effect of harmine on the localization of the nucleolar proteins C23 and B23.

In this study we show that harmine treatment (10 mg/l for 2 or 24 h) of PtK2 cells had a marked effect on the localization of the nucleolar phosphoproteins C23 and B23. C23 was localized with silver staining in the fibrillar areas of completely segregated nucleoli. B23 was localized mainly on the periphery of the nucleoli with the aid of immunofluorescence.

A pathologic basis for Kleine-Levin syndrome.

A patient with Kleine-Levin syndrome, typical except that onset was at 39 years of age, died during a symptomatic period. Autopsy disclosed recent and old lesions in the medial thalamus involving intralaminar, medial, and some dorsal nuclei as well as the pulvinar. Despite massive microglial infiltration, there was minimal neuronal loss. The hypothalamus was not involved. The findings suggest a viral cause for Kleine-Levin syndrome.

Brainstem auditory evoked responses in leukodystrophies.

Brainstem auditory evoked responses (BAERs) were recorded in seven patients with Pelizaeus-Merzbacher leukodystrophy (PMD), two with adrenoleukodystrophy (ALD), and one with metachromatic leukodystrophy (MLD). BAERs were altered in all patients, and the alterations were severe in 9 of the 10 patients. A patient with ALD who as yet had no neurologic symptoms showed only minimal abnormality of the BAERs, consisting of prolongation of the latency of wave V. In the remaining nine patients, only wave I generated in the extramedullary portion of the eighth nerve was recorded with or without a wave II. Subsequent components (III through VII) were absent. No abnormality of BAERs was observed in the 10 known carriers of PMD. The combination of BAERs and EEG is helpful in differentiating leukodystrophy from progressive gray matter diseases.

Does head-turning during a seizure have lateralizing or localizing significance?

Forced lateralized head-turning, occurring as the first clinical sign in 106 epileptic seizures in 43 patients, was recorded on videotape simultaneously with the EEG. Forty-five ictal EEGs were obtained with stereotaxically implanted intracerebral electrodes. Forced head-turning was seen with seizures that had a frontal, temporal, unilateral diffuse, or a generalized onset in the EEG. Ipsilateral was as common as contralateral head-turning in all groups, including the seizures with frontal lobe onset. Initial head-turning in a seizure has no localizing or lateralizing significance.