Expression and folding of an antibody fragment selected in vivo for high expression levels in Escherichia coli cytoplasm

In this review, we summarize some of our results on folding and directed evolution of an antibody fragment in Escherichia coli cytoplasm. We will also discuss some attempts to construct other antibodies active in this cellular compartment.     

Human and mouse alpha-synuclein genes: comparative genomic sequence analysis and identification of a novel gene regulatory element

The human alpha-synuclein gene (SNCA) encodes a presynaptic nerve terminal protein that was originally identified as a precursor of the non-beta-amyloid component of Alzheimer's disease plaques. More recently, mutations in SNCA have been identified in some cases of familial Parkinson's disease, presenting numerous new areas of investigation for this important disease. Molecular studies would benefit from detailed information about the long-range sequence context of SNCA. To that end, we have established the complete genomic sequence of the chromosomal regions containing the human and mouse alpha-synuclein genes, with the objective of using the resulting sequence information to identify conserved regions of biological importance through comparative sequence analysis. These efforts have yielded approximately 146 and approximately 119 kb of high-accuracy human and mouse genomic sequence, respectively, revealing the precise genetic architecture of the alpha-synuclein gene in both species. A simple repeat element upstream of SNCA/Snca has been identified and shown to be necessary for normal expression in transient transfection assays using a luciferase reporter construct. Together, these studies provide valuable data that should facilitate more detailed analysis of this medically important gene.     

De novo 13q partial duplication identified by cytogenetic, biochemical and molecular approaches

A 3.5-month-old female infant manifesting dysmorphic facies, developmental delay and failure to thrive was referred for cytogenetic evaluation. Peripheral lymphocytes revealed three chromosomally distinct cell lines: 46,XX/46,XX,10p+/47,XX,10p+,+mar. Dermal fibroblasts revealed only the 46,XX,10p+cell line. High resolution G-, R-, and Q-banding suggested that the extra chromosomal material (10p+) represented a duplication of the segment 13q14----13qter. Parental karyotypes were normal. As absolute identification of de novo chromosomal abnormalities, based solely on cytogenetic studies, is sometimes difficult, both biochemical and molecular approaches were undertaken to elucidate this abnormality in more detail. Dosage effects were examined using esterase D (localized to 13q14.1) and the DNA probes p1E8 and p9A7 (localized to 13q22 and 13q31/32, respectively). These studies suggested the presence of only 2 copies of esterase D, but 3 copies of both DNA probes, allowing identification of the breakpoint at 13q14.2.     

Analphoid 3qter markers

Two cases of marker chromosomes derived from a non-centromeric location were studied to determine the characteristics of these markers with respect to the presence of functional centromeres and whether an associated phenotype could be described. The markers were characterized by fluorescence in situ hybridization and centromeric protein studies. Assessments were done to identify clinical features. Case 1 is a girl referred at age 1.5 years with swirly areas of hyperpigmentation, bilateral preauricular pits, hypotonia, developmental delay, and seizures. Case 2 is a male first evaluated as a newborn and then later during the first year of life. He had streaky hypopigmentation, right preauricular pit, accessory nipples, postaxial polydactyly, asymmetric cerebral ventricles, duplicated right kidney, a right pulmonary artery stenosis, and seizures. Mosaicism for an extra marker from the 3qter region was present in both cases. Both markers had a constriction near one end and were C-band negative. Centromeric protein studies indicated absence of CENP-B, presence of CENP-C (data for case 1 only), and presence of CENP-E. Marker chromosomes were thus identified with a chromosomal origin far from their usual centromeric region and yet appeared to have functional centromeres. These two cases did not permit a specific clinical phenotype to be ascribed to the presence of tetrasomy for 3q26.2 approximately 3q27.2-->3qter.     

Detailed immunoautoradiographic mapping of enkephalinase (EC 3.4.24.11) in rat central nervous system: comparison with enkephalins and substance P

The metallopeptidase enkephalinase known to participate in the inactivation of endogenous enkephalins and, possibly, other neuropeptides such as tachykinins, was visualized by autoradiography using a [125I]iodinated monoclonal antibody. A detailed mapping of the enzyme in rat brain and spinal cord was established on 10-micron serial sections prepared in a frontal plane as well as a few sections in a sagittal plane. On adjacent sections, and for the purpose of comparison, substance P-like and enkephalin-like immunoreactivities were also visualized by autoradiography using a 125I-monoclonal antibody and a polyclonal antibody detected by a secondary 125I-anti-rabbit antibody respectively. Histological structures were identified on adjacent Nissl-stained sections. Using the highly sensitive 125I-probe, enkephalinase immunoreactivity was found to be distributed in a markedly heterogeneous manner in all areas of the central nervous system. Immunoreactivity was undetectable in white matter areas, for example the corpus callosum or fornix, and had a laminar pattern in, for example, the cerebral cortex or hippocampal formation. Hence, although immunodetection was not performed at the cellular level, a major neuronal localization of the peptidase is suggested. The latter is consistent with the detection of a strong immunoreactivity in a pathway linking the striatum to the globus pallidum, the entopeduncular nucleus and the substantia nigra, as well as with a series of biochemical and lesion data. The strong immunoreactivity also present in choroid plexuses and ependymal cells as well as in the intermediate lobe and in scattered cells of the anterior lobe of the pituitary suggests that populations of glial and endocrine cells also express the peptidase. The highest density of enkephalinase immunoreactivity was observed in basal ganglia and limbic areas (caudate putamen, globus pallidus, nucleus accumbens, olfactory tubercles) as well as in areas involved in pain control mechanisms (superficial layers of the spinal nucleus of the trigeminal nerve or of the dorsal horn of the spinal cord) which also display the highest immunoreactivities for both enkephalins and substance P (except in globus pallidus for the latter). These localizations account for the opioid-like analgesic and motor effects of enkephalinase inhibitors inasmuch as a selective or predominant participation of the peptidase in enkephalin inactivation is assumed. A number of other areas appear richly endowed in both enkephalinase and enkephalins whereas substance P is hardly detectable. This is particularly the case for the olfactory bulb, bed nucleus of the accessory olfactory tract, the cerebellum (where enkephalinase mainly occurs in the molecular layer) and the hippocampal formation (namely in the molecular layer of the dentate gyrus).(ABSTRACT TRUNCATED AT 400 WORDS)     

Beta 2-microglobulin amyloidosis (AB2M) in patients undergoing long-term hemodialysis. A new type of amyloid

Amyloidosis has been increasingly recognized in association with renal failure and chronic hemodialysis. This report describes three patients who had long-term hemodialysis (between 7-18 years), in whom deposits developed of a new type of amyloid of beta 2-microglobulin origin. Beta 2-microglobulin amyloid (AB2M) was found in multiple organs, i.e., bone, subendocardium, gastrointestinal blood vessels, tongue, and carpal tunnel connective tissue. AB2M displayed characteristic amyloid features on conventional light and polarized microscopic examination after congo red staining. However immunostaining with anti-amyloid A protein, kappa, and lambda antisera were negative. The studied material reacted positively with beta 2-microglobulin antisera, identifying AB2M in all three cases. Ultrastructural study revealed an unusual curvi-linear fibrillar configuration. AB2M appears to be a new subtype of systemic amyloidosis secondary to renal failure and long-term hemodialysis.     

Effect of compounds which disrupt proton gradients on secretion of neurosecretory proteins from PC12 pheochromocytoma cells

Treatment of PC12 cells with chroquine (10-50 microM) obliterated the low intragranular pH, as detected by Acridine Orange fluorescence, and depleted the cells of dopamine and norepinephrine. However, these concentrations of chloroquine did not prevent the release of the newly synthesized proteins which normally undergo stimulus-coupled secretion with the catecholamines. Higher concentrations of chloroquine (200 microM) and ammonium chloride (10 and 25 mM) inhibited the release of most of these proteins. This inhibition did not result from alterations in protein synthesis, since the profile of proteins synthesized was not substantially altered. Nor did the inhibition result from degradation of the neurosecretory proteins, since prelabeled proteins were capable of undergoing stimulated secretion from chloroquine-treated cells, as from normal cells. The findings indicated that the inhibition was at the step of packaging of the proteins into the neurosecretory granules. While release of the major secretory proteins, including chromogranin B, was inhibited with 200 microM chloroquine, chromogranin A was secreted upon stimulation of these cells. The results of this study indicate that an acidic intragranular pH is not a requirement for the packaging and secretion of neurosecretory proteins. Higher concentrations of chloroquine had a differential effect on the regulated secretion of different neurosecretory proteins.