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Spinal muscular atrophy (SMA) is a frequent autosomal recessive disease in human characterized by degeneration of motor neurons of the spinal cord. The genomic region containing the defective gene (5q13) is particularly unstable and prone to large-scale deletions whose characterization led to the identification of the survival motor neuron (SMN) gene, the SMA determining gene encoding a hitherto unknown protein. As an initial step toward the generation of a murine model for SMA, we identified and characterized a full-length murine Smn cDNA. The coding sequence of the mouse Smn gene was found to be 82% identical, at the amino acid level, with the human SMN coding sequence. The Smn locus was mapped to the segment of mouse chromosome 13 exhibiting conservation of synteny with human chromosome 5q11-q23, which contains the SMN gene. However, no evidence for a duplication of the Smn gene was found in the mouse, suggesting that the duplication reported in human is a recent evolutionary event.

Mutations of the survival motor neurone gene (SMN) are associated with spinal muscular atrophy (SMA), a frequent lethal autosomal recessive disorder. In spite of this, no phenotype-genotype correlation was observed, since the SMN gene is lacking in the majority of patients affected with either the severe form (type I) or the milder forms (types II and III). Here, we show that the gene encoding p44, a subunit of the basal transcription factor TFIIH, is duplicated in the SMA region and that the p44 gene products (p44t and p44c) differ by three amino acid changes. Gene analysis of a total of 94 unrelated SMA patients revealed that the p44t gene is involved in large-scale deletions associated with Werdnig-Hoffmann disease (type I). The TFIIH polypeptide composition as well as transcription and DNA repair activities are normal in patients lacking the p44t gene on both mutant chromosomes, suggesting that the p44t gene is not critical for the development of SMA.

The survival motor neuron (SMN) gene was lacking in 6/12 patients with arthrogryposis multiplex congenita (AMC) associated with spinal muscular atrophy (SMA). Neither point mutation in the SMN gene nor evidence for linkage to chromosome 5q13 were found in the other patients. Hitherto, arthrogryposis was regarded as an exclusion criterion in SMA. Our data strongly suggest that AMC of neurogenic origin is genetically heterogeneous, with a subgroup being allelic to SMA. Absence or interruption of the SMN gene in the AMC-SMA association will make the diagnosis easier and genetic counselling will now become feasible.

Spinal muscular atrophies (SMA) are characterized by degeneration of the anterior horn cells of the spinal cord and represent the second most common fatal autosomal-recessive disorder after cystic fibrosis. We have previously identified the survival motor neurone gene (SMN), a SMA-determining gene in the 5q13 region encoding a hitherto unknown protein. In this report, we describe the organization and structure of SMN. The gene is approximately equal to 20 kb in length and consists of nine exons. Sequence data of the 5' end of the gene show that the dinucleotide repeat C272 is close to several putative binding sites for transcription factors, which will help to characterize the regulation of the SMN and CBCD541 gene expression. The availability of the human SMN and its highly homologous counterpart (CBCD541) gene structures and exon-intron boundaries will hopefully speed up the characterization of SMN gene mutations in SMA.

The most common pediatric surgical procedures performed in the United States today are tonsillectomies and adenoidectomies (T&A). Surgical team members must be highly trained and efficient to ensure optimal patient outcomes, reduce surgical costs, and decrease the risk and potential complications inherent in T&A procedures. The authors review current surgical indications for T&A procedures; recommended preoperative, intraoperative, and postoperative patient care; and the management of potential complications.

Spinal muscular atrophy (SMA) is a frequent autosomal recessive disease characterized by degeneration of the motor neurons of the spinal cord causing proximal paralysis with muscle atrophy. The region on chromosome 5q13 encompassing the disease gene is particularly unstable and prone to large-scale deletions whose characterization recently led to the identification of the survival motor neuron (SMN) gene. We now present a genetic analysis of 54 unrelated Spanish SMA families that has revealed a 4-basepair (bp) deletion (AGAG) in exon 3 of SMN in four unrelated patients. This deletion, which results in a frameshift and a premature stop codon, occurs on the same haplotype background, suggesting that a single mutational event is involved in the four families. The other patients showed either deletions of the SMN gene (49/54) or a gene conversion event changing SMN exon 7 into its highly homologous copy (cBCD541, 1/54). This observation gives strong support to the view that mutations of the SMN gene are responsible for the SMA phenotype as it is the first frameshift mutation reported in SMA.

Diagnosis of ectopic acromegaly was made in a 21-year-old female patient who 3 years before had undergone a right pneumectomy for a disseminated bronchial carcinoid. Plasma growth hormone-releasing hormone (GHRH) concentrations were markedly elevated (6440 ng/l; normal value < 100 ng/l), as were serum GH (187 micrograms/l; normal < 5 micrograms/l) and plasma insulin-like growth factor I (IGF-I) levels (6.7 U/ml; normal < 2 U/ml). Retrospective immunohistochemical examination of the carcinoid tumor was positive for GHRH and the tumoral content of GHRH was 2130 ng/g wet weight. Subcutaneous treatment with octreotide was begun and first resulted in a profound inhibition of GH hypersecretion, normalization of plasma IGF-I and only partial reduction of GHRH concentrations. However, the initial dose of 3 x 100 micrograms had to be increased gradually to 4 x 750 micrograms because of a progressive deterioration of the hormonal control. After 15 months of intermittent therapy, octreotide was administered by continuous sc infusion. This treatment improved compliance, allowed the daily dose of octreotide to be reduced to 1500 micrograms and normalized serum GH levels. A near-normalization of the plasma IGF-I concentrations was also obtained, whereas the suppression of plasma GHRH concentrations remained incomplete. Despite favorable evolution of the endocrine parameters, intramedullar metastases were diagnosed and required radiation therapy. This observation emphasizes the superiority of continuous over intermittent administration of octreotide in the treatment of ectopic acromegaly. It also shows that the somatostatin analog acts more at the pituitary level to inhibit GH secretion than at the site of the neuroendocrine tumor.

Microcapsules were prepared from human serum albumin (HSA) by interfacial cross-linking with terephthaloyl chloride (TC). TC concentrations were increased from 0.5 to 5% w/v, while pH (9.8) and reaction time (30 min) were kept constant. Fourier transform infrared (FT-IR) spectra of lyophilized microcapsules were compared. Correlations were established with microcapsule morphology and size. The results were compared with those of previous studies exploring pH or reaction time and with those of parallel determinations of microcapsule free amino groups. With 0.5% TC, decreases of the ester-assigned 1724-cm-1 band area and of the carboxylate-assigned 1394-cm-1 band area were observed compared with pure HSA. This phenomenon was attributed to a removal of contaminating lipids of HSA. Increasing TC concentration resulted in a progressive increase of the band areas at 1724 cm-1 (esters) and 1795 cm-1 (anhydrides), in a further decrease of the 1394-cm-1 band area (carboxylates), and in marked alterations of teh 1340-1080-cm-1 region. These changes, which revealed the progressive acylation of hydroxy and carboxylate groups of HSA, were accompanied by an increase of the 1624-cm-1 band area (beta-sheet), reflecting interchain H-bonding due to cross-linking. As observed in the previous studies of pH and reaction time, important spectral changes corresponded to low values of -NH2 content, to a decrease in microcapsule mean size (from > 30 to < 15 microns), and to modifications of the membrane surface (made rough).

cDNAs prepared from total RNA extracted from plaques of multiple sclerosis were amplified by the polymerase chain reaction. The 11-bp degenerate primers used were derived from conserved sequences of reverse transcriptase. Amplified cDNAs were fractionated according to size by electrophoresis in polyacrylamide gels under denaturing conditions. cDNAs of the proper size were cloned, grouped according to the sequence of their insert by differential hybridization, and sequenced. Six cDNAs were isolated and found to belong to new members of two groups of human endogenous retroviruses: the group related to ERV9 and that related to HERVK10 and HUMMTV. These sequences were expressed in all human organs tested, including normal white matter of brain. The approach described in this article is a powerful tool with which to isolate new members of the reverse transcriptase gene family.

Spinal muscular atrophy (SMA) is a common fatal autosomal recessive disorder characterized by degeneration of lower motor neurons, leading to progressive paralysis with muscular atrophy. The gene for SMA has been mapped to chromosome 5q13, where large-scale deletions have been reported. We describe here the inverted duplication of a 500 kb element in normal chromosomes and narrow the critical region to 140 kb within the telomeric region. This interval contains a 20 kb gene encoding a novel protein of 294 amino acids. An highly homologous gene is present in the centromeric element of 95% of controls. The telomeric gene is either lacking or interrupted in 226 of 229 patients, and patients retaining this gene (3 of 229) carry either a point mutation (Y272C) or short deletions in the consensus splice sites of introns 6 and 7. These data suggest that this gene, termed the survival motor neuron (SMN) gene, is an SMA-determining gene.

Ingestion is foreign body lodgment in the esophagus, whereas aspiration is lodgment of foreign bodies in the larynx, trachea, or bronchi. Foreign bodies constitute an emergency when actual or potential airway obstruction occurs, when there is an esophageal perforation, or when disc batteries are ingested and lodged in the esophagus. A ventilating bronchoscope of appropriate size is essential to the removal of a foreign body in the airway or esophagus. Successful management of foreign bodies in the upper aerodigestive tracts of children is directly proportional to the time and effort spent in preoperative preparation.

Spinal muscular atrophies (SMAs) represent the second most common fatal autosomal recessive disorder after cystic fibrosis. Childhood spinal muscular atrophies are divided into severe (type I) and mild forms (types II and III). By a combination of genetic and physical mapping, a yeast artificial chromosome contig of the 5q13 region spanning the disease locus was constructed that showed the presence of low copy repeats in this region. Allele segregation was analyzed at the closest genetic loci detected by markers C212 and C272 in 201 SMA families. Inherited and de novo deletions were observed in nine unrelated SMA patients. Moreover, deletions were strongly suggested in at least 18 percent of SMA type I patients by the observation of marked heterozygosity deficiency for the loci studied. These results indicate that deletion events are statistically associated with the severe form of spinal muscular atrophy.

The gene for autosomal recessive forms of spinal muscular atrophy (SMA) has recently been mapped to chromosome 5q13, within a 4-cM region between the blocks D5S465/D5S125 and MAP-1B/D5S112. We identified two new highly polymorphic microsatellite DNA markers--namely, AFM265wf5 (D5S629) and AFM281yh9 (D5S637)--which are the closest markers to the SMA locus. Multilocus analysis by the location-score method was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for SMA is between locus D5S629 and the block D5S637/D5S351/MAP-1B/D5S112/D5S357. Genetic analysis of inbred SMA families, based on homozygosity by descent and physical mapping using mega-YACs, gave additional information for the loci order as follows: cen-D5S6-D5S125/D5S465-D5S435-D5S629-SMA-+ ++D5S637-D5S351-MAP-1B/D5S112-D5S357- D5S39-tel. These data give the direction for bidirectional walking in order to clone this interval and isolate the SMA gene.

Microcapsules were prepared from human serum albumin (HSA) through interfacial cross-linking with terephthaloyl chloride (TC). Reaction times were increased from 2 to 60 min, while pH (9.8) and TC concentration (2.5% w/v) were kept constant. Fourier-transform infrared (FT-IR) spectra of lyophilized microcapsules were compared. Correlations were established with microcapsule morphology and size, as had been done in a previous study exploring the effect of increasing pH values. Microcapsules obtained after 2 min had to be considered separately. Minor alterations were observed in the spectrum as compared with pure HSA. They consisted of a decrease of the ester-assigned 1724-cm-1 band and of the carboxylate-assigned 1394-cm-1 band, attributed to a removal of contaminating lipids of HSA, and an increase of the 1624-cm-1 band, attributed to interchain H bonding following acylation of the NH2 groups. Prolonging the reaction time resulted in a progressive increase of the bands at 1724 (esters), 1795 (anhydrides), and 1624 cm-1 (beta-sheet), in a further decrease of the 1394-cm-1 band (carboxylates), and in marked alterations of the 1340-1080-cm-1 region. These important changes, which appeared after 5 min, reflect the progressive acylation of the hydroxy and carboxylate groups of HSA. As in the previous series of pH-based assays, important spectral changes were shown to correspond to a decrease in microcapsule mean size (from 32 to < 15 microns) and in important modifications of the membrane surface, made rough.

Spinal muscular atrophies (SMA) represent the second most common fatal autosomal recessive disorder after cystic fibrosis. Childhood SMAs are divided into severe (type I) and mild forms (types II and III). By a combination of genetic and physical mapping, a YAC contig of the 5q13 region spanning the disease locus was constructed that showed the presence of low copy-repeats in this region. Allele segregation was analyzed at the closest genetic loci detected by markers C212 and C272 in 201 SMA families. Inherited and de novo deletions were observed in 10 SMA patients. Moreover, deletions were strongly suggested in at least 18% of SMA type I patients by the observation of marked heterozygosity deficiency for the loci studied. These results indicate that deletion events are statistically associated with the severe form of SMA.

Inbred strains of mice differ greatly in their susceptibility to the demyelinating disease caused by Theiler's Murine Encephalomyelitis Virus. In this murine disease, which is an animal model for the study of multiple sclerosis, demyelination depends on the persistent infection of the central nervous system. Previous studies identified a locus in the H-2D region of the major histocompatibility complex which controls susceptibility to the persistent infection, and also showed that other loci are involved. In order to identify these loci, we screened the genome of a set of backcross animals with a combination of polymorphic microsatellites and restriction enzymes sites. We now show that viral persistence is also controlled by a locus close to Ifg on chromosome 10 and possibly by a locus near Mbp on chromosome 18.

Acylation-stimulating protein (ASP) is the most potent recognized stimulant of triacylglycerol synthesis in human adipocytes. However, its mechanism(s) of action have not yet been elucidated in detail. The present study examines the effects of ASP on membrane transport of glucose and fatty acids in cultured human skin fibroblasts. The data demonstrate that ASP stimulated carrier-mediated glucose transport in a time- and concentration-dependent manner, an effect that was greater than and independent of that observed with insulin. ASP increased the Vmax for glucose transport with no change in the transport Km, suggesting that ASP might result in increased sugar transporters in the plasma membrane. This finding was supported by quantitative Western blot analyses using a monoclonal antibody (G3) that demonstrated an increase in the plasma membrane content of the glucose transporter (Glut 1) with a concomitant decrease in the glucose transporter content of an intracellular membrane fraction. By contrast, ASP had no effect on specific membrane transport of fatty acids. Maximal effects of ASP on triacylglycerol synthesis were demonstrated at saturating levels of both glucose and oleate. Comparable stimulation by ASP in the absence of glucose (with or without pyruvate) was also demonstrated, although the absolute rates of triacylglycerol synthesis were substantially lower. Finally, it was shown that ASP increased the apparent Vmax for triglyceride synthesis without changing its Km. Since ASP will act in the absence of extracellular glucose, ASP must have additional actions, independent of its effect on specific membrane transport of glucose by which it accelerates intracellular triglyceride synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

A new human brain cDNA molecule was mapped by in situ hybridization to the 11q21-q23.1 region of the human genome, probably to the 11q22 band. An EcoRI restriction site and a (GT)n repeat element within the gene were shown to be polymorphic. Both polymorphisms were readily studied by the polymerase chain reaction. A two-allele polymorphism was described for the EcoRI restriction site, whereas four different alleles were detected for the second genetic marker. The observed heterozygosities were 37% and 42% for the former and the latter polymorphism, respectively. The combined heterozygosity index was estimated to be 0.56. These new genetic markers will be useful for linkage analysis of neurogenetic diseases that have been mapped to this chromosomal region.

The effect of 2-acetylaminofluorene (2-AAF) on the intracellular free Ca2+ ([Ca2+]i) and viability of isolated rat hepatocytes has been investigated using the fluorescent probes quin 2 and propidium iodide respectively. At the highest concentration tested (224 microM), 2-AAF produces an elevation of [Ca2+]i which shows a biphasic profile. A small initial increase is observed during the first 5 min; this is followed by a considerable rise which reaches up to 2.5 times the control value at 15 min. These changes in intracellular calcium are not accompanied by detectable alterations in cell viability. In order to determine the mechanisms by which this effect of 2-AAF takes place, three calcium antagonists, namely verapamil, TMB-8 (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate) and ruthenium red (RuR), have been used. The results suggest that the first phase is dependent upon internal Ca2+ store mobilization, while the second phase seems to be related to Ca2+ entry from the extracellular space. The data obtained with RuR further indicate that mitochondria may be involved in the perturbation of calcium homeostasis caused by 2-AAF. In addition, in the experiments involving antagonists, no consistent pattern emerges that suggests a close relationship between intracellular Ca2+ levels and cell viability. The present study provides further information on the mechanisms by which these well-known hepatotoxin 2-AAF may interact with liver cells. It also shows that when these cells are exposed to a toxin, short-term changes in [Ca2+]i may not be accompanied by loss of cell viability, and conversely, that changes in cell viability may occur without alterations in [Ca2+]i.

Antiestrogens are widely used in the management of hormonally responsive breast cancer in both adjuvant and palliative settings, and are currently being evaluated as chemopreventive agents. The classical mechanism of action of these drugs involves inhibition of estrogen-stimulated neoplastic cell proliferation by blockade of estrogen receptors present on breast cancer cells. This paper reviews recent clinical and laboratory data that suggest that the commonly used antiestrogen tamoxifen also acts to reduce serum IGF-I levels. Estrogens appear to play a permissive role in growth hormone (GH) release by the pituitary gland and GH is known to stimulate IGF-I expression by hepatocytes. It is therefore possible that blockade of estrogen receptors in the hypothalamic-pituitary axis by tamoxifen interferes with GH release, leading to reduced hepatic IGF-I expression. In view of results suggesting that IGF-I is a more potent mitogen than estradiol for breast cancer cells and data demonstrating a positive correlation between estrogen receptor level and IGF-I receptor level of breast cancer cells, the IGF-I lowering effect of tamoxifen may contribute to the cytostatic activity of the drug. The interrelationships between steroid hormone physiology and IGF-I physiology may have relevance to a variety of commonly used treatments for hormonally responsive cancers.

Fourier transform infrared (FT-IR) spectroscopic studies were performed on microcapsules prepared through interfacial cross-linking of human serum albumin (HSA) with terephthaloylchloride at various pH values (5.9 to 11). Correlations were established with morphology and size of microcapsules. Increasing polycondensation pH resulted notably in a progressive increase of peaks at 1795 and 1724 cm-1, assigned to anhydride and ester; respectively, in a decrease of the carboxylate-assigned 1394 cm-1 peak, and in alterations of the 1340-1080-cm-1 region. These spectral changes were most pronounced from pH 9 and were shown to correspond to smaller-sized microcapsules (mean size decreased from 30-40 microns to less than 15 microns) with rough surfaces. Further soaking of highly cross-linked microcapsules in a pH 7.5 buffer resulted in the disappearance of the 1795 cm-1 peak, with a concurrent increase of the 1394 cm-1 peak and a decrease of the 1724 cm-1 peak. These changes, attributed to complete breaking of anhydride and partial hydrolysis of esters, were accompanied by an unwrinkling of the microcapsule membrane, then made smooth, and a significant increase in size. Treating microcapsules with hydroxylamine under alkaline conditions allowed complete reversal of the spectral alterations assigned to anhydride and ester formation. A comparable (slightly higher) increase in size was observed with microcapsules which exhibited smooth surfaces and a low density.

The polymerase chain reaction was used, in a blind study, to look for retroviral sequences in DNA extracted from the peripheral blood mononuclear cells of 11 patients with the spinal form of multiple sclerosis (MS). Control subjects consisted of 7 patients with other neurological diseases and 5 healthy blood donors. Three sets of oligonucleotides were used. They could detect all known human oncoretroviruses, lentiviruses, or spumaretroviruses. The primers recognized conserved sequences in the long terminal repeats of the proviral DNA. Control experiments showed that the primers crossreacted within the human immunodeficiency virus or human T-cell lymphotropic virus group and that they provided the expected level of sensitivity. Therefore the assay could have detected not only known human retroviruses but also new related members. In spite of this, no retroviral sequences were detected in either the MS or the control specimen.

High responder lines of Hartley guinea-pigs were sensitized by repeated inhalations of toluene diisocyanate (TDI). After 3 weeks, we demonstrated a degree of TDI substitution of the serum-albumin-enriched fraction (AEF) and we ascertained the sensitization of the most exposed animals using PCA methodology. Fourier transform infrared spectroscopy (FT-IR), used to investigate conformational changes in AEF, highlighted the structural modifications of the native protein conformation. Such crucial changes may support, at least in part, the relationship between TDI exposure and triggering of hypersensitivity reactions.

Certain redox properties of bacteriochlorophyll alpha were used to probe the structure of several light-harvesting pigment-protein complexes or holochromes. To attribute redox properties unequivocally to a given holochrome, we worked with purified holochromes. We developed purification procedures for the B880 holochromes from Rhodospirillum rubrum, Rhodopseudomonas sphaeroides and Ectothiorhodospira sp. and for the B800-850 holochromes from the latter two species. In all these holochromes, bacteriochlorophyll alpha could be oxidized by ferricyanide as witnessed by the bleaching of their near-infrared absorption bands. However, only in B880 holochromes was this oxidation reversible. Another important difference between the B800-850 and the B880 holochromes is that oxidation of the latter gives rise to a g = 2.0025 electron paramagnetic resonance (EPR) signal with linewidth varying, according to species, from 0.37 mT to 0.48 mT. Both the reversible EPR signal and absorption changes titrate with a midpoint redox potential (pH 8.0) of approximately 570 mV. Linewidth narrowing can be interpreted by delocalization of the free electron spin over approximately 12 bacteriochlorophyll molecules. While the B880 holochromes from the three species considered had indistinguishable redox properties, the B800-850 holochromes differed from one another by their circular dichroic spectra and by the relative ease of oxidation of their 800-nm and 850-nm bands. This indicates that, contrary to the B880 holochromes, the B800-850 holochromes may not form a homogeneous class.

In the presence of physiological levels of Na+ (10 mM), K+ (150 mM), and Mg2+ (2 mM), dephosphorylated neurofilaments contained two Ca2+ specific binding sites with Kd = 11 microM per unit consisting of eight low, three middle, and three high molecular subunits, as well as 46 sites with Kd = 620 microM. Only one class of 126 sites with Kd = 740 microM was detected per unit of untreated neurofilaments. A chymotryptic fraction enriched in the alpha-helical domains of neurofilament subunits contained one high-affinity Ca2+-binding site (Kd = 3.6 microM) per domain fragment of approximately 32 kDa. This site may correspond to a region in coil 2b of the alpha-helical domain, which resembles the I-II Ca2+-binding site in intestinal Ca2+-binding protein. Homopolymeric filaments composed of the low or middle molecular weight subunits contained low-affinity Ca2+-binding sites with Kd = 37 microM and 24 microM, respectively, while the Kd values for the low-affinity sites in heteropolymeric filaments were 8-10-fold higher. Competitive binding studies, using the chymotryptic fraction to assay the high-affinity Ca2+-binding sites and 22Na+ to monitor binding to the phosphate-containing low-affinity sites, yielded Kd values for Al3+ of 0.01 microM and 4 microM, respectively. This suggests that the accumulation of Al3+ in neurons may be due in part to its binding to neurofilaments.

Exhaustive in vitro dephosphorylation of porcine neurofilaments (NFs) by alkaline or acid phosphatase did not cause a dissociation of the 210-kD (NF-H), 160-kD (NF-M), or 70-kD (NF-L) subunits and had no effect on the reassembly of NFs from urea or guanidine solution. Electron microscopy revealed that the NFs reassembled from isolated or dephosphorylated subunits had similar morphologies. Phosphatase treatment caused significant increases in the mobilities of NF-M and NF-H on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the subunits underwent marked conformational changes after dephosphorylation. Chemical phosphate analysis showed that as isolated NF-H, NF-M, and NF-L contained about 22, 11, and 3 mol phosphate/mol polypeptide, respectively. The corresponding values for the three subunits from alkaline phosphatase-treated NFs were about 8, 6, and 2 mol phosphate/mol polypeptide, respectively. These results indicate the occurrence of a class of phosphate moieties that is not accessible to exogenous phosphatases.

The calcium-binding properties of untreated and in vitro dephosphorylated neurofilaments were determined by partition centrifugation. Scatchard plot analysis of the binding data indicated that each type of neurofilament contained both high and low affinity calcium-binding sites. The number of calcium-binding sites per unit consisting of three 140,000, three 107,000 and eight 62,000 molecular weight subunits was 4 sites with Kd = 4.1 microM and 126 sites with Kd = 293 microM for untreated neurofilaments. The in vitro dephosphorylated neurofilaments contained 8 sites with Kd = 15 microM and 54 sites with Kd = 332 microM, per unit.