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Biomedical subjects

H Gainer

Publications and source records attributed to H Gainer.

At least 19 recordsLinked to original sources
PubMed1995

Expression of neurofilament proteins during development of the nervous system in the squid Loligo pealei.

The squid nervous system includes various brain ganglia, optic lobes (the visual center), and the stellate ganglia, the system of giant motor fibers responsible for rapid jet-propelled escape behavior. The large caliber of giant fibers is due, in part, to the accumulation of squid-specific neurofilaments (NFs) made up of a heavily phosphorylated NF 220 protein together with NF 70 and NF 60 subunits. Using antibodies prepared against known peptide sequences in these proteins, together with a mammalian-derived antibody that specifically recognizes phosphorylated squid NF 220, we studied the localization of NFs in adult tissues and during neural development. Immunoblot and immunohistochemical analyses showed that NFs were present in adult neural tissues, primarily in selected fibers, with giant axons showing the most robust expression. After the first neurons differentiated at stage 22, immunoblots showed NF 60- and NF 70-immunoreactive proteins at all stages. The NF 220 subunit, however, was not detected in immunoblots at any developmental stage. Phosphorylated NF 220 immunoreactivity, although absent in immunoblots, was first seen in selected fibers of the stellate ganglia at stage 25, increasing thereafter in all giant fibers until hatching (stage 30). The stellate ganglion is the first neural tissue to acquire a mature neurofilament complement (i.e., phosphorylated NF 220), shortly before the onset of jet-propelled escape behavior. The temporal pattern of expression of the NFs during development resembled that seen in vertebrates; i.e., the smaller NFs appeared before the larger subunit in most neural tissues. In the squid, the expression pattern seems to depend upon the post-transcriptional regulation of a single gene rather than upon transcriptional regulation of three independent genes as in vertebrates.

Animals↗
PubMed1995

Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures.

Voltage- and ligand-activated channels in embryonic neurons containing luteinizing hormone-releasing hormone (LHRH) were studied by patch-pipette, whole-cell current and voltage clamp techniques. LHRH neurons were maintained in explant cultures derived from olfactory pit regions of embryonic mice. Cells were marked intracellularly with Lucifer yellow following recording. Sixty-two cells were unequivocally identified as LHRH neurons by Lucifer yellow and LHRH immunocytochemistry. The cultured LHRH neurons had resting potentials around -50 mV, exhibited spontaneous discharges generated by intrinsic and/or synaptic activities and contained a time-dependent inward rectifier (Iir). Voltage clamp analysis of ionic currents in the LHRH neuron soma revealed a tetrodotoxin-sensitive Na+ current (INa) and two major types of K+ currents, a transient current (IA), a delayed rectifier current (IK) and low- and high-voltage-activated Ca2+ currents. Spontaneous depolarizing synaptic potentials and depolarizations induced by direct application of gamma-aminobutyrate were both inhibited by picrotoxin or bicuculline, demonstrating the presence of functional gamma-aminobutyrate type A synapses on these neurons. Responses to glutamate were found in LHRH neurons in older cultures. Thus, embryonic LHRH neurons not yet positioned in their postnatal environment in the forebrain contained a highly differentiated repertoire of voltage- and ligand-gated channels.

Action Potentials↗
PubMed1994

Identification of endogenously phosphorylated KSP sites in the high-molecular-weight rat neurofilament protein.

The high-molecular-weight neurofilament protein (NF-H) is highly phosphorylated in vivo, with estimates as high as 16-51 mol of Pi/mol of protein. Most of the phosphorylation sites are thought to be located on Ser residues in multiple KSP repeats, in the carboxy-terminal tail region of the molecule. Because the extent and site-specific patterns of tail domain phosphorylation are believed to modulate neurofilament structure and function, it becomes essential to identify the endogenous sites of phosphorylation. In this study, we have used selective proteolytic cleavage procedures, Pi determinations, microsequencing, and mass-spectral analysis to determine the endogenously phosphorylated sites in the NF-H tail isolated from rat spinal cord. Twenty Ser residues in NF-H carboxy-terminal tail were analyzed; nine of these, all located in KSP repeats, were phosphorylated. No detectable phosphorylation could be identified in any of the 11 "non-KSP" Ser residues that were examined. KSPXKX, KSPXXX, and KSPXXK motifs were found to be phosphorylated. In addition, a 27-kDa KSP-rich domain, containing 43 virtually uninterrupted KSPXXX repeats, was isolated from the tail domain and found to contain between 30 and 35 mol of Pi/mol of protein. This domain appeared to be highly resistant to endoproteinase Glu-C digestion, although it contains a large number of glutamate residues. It could be proteolyzed, however, after dephosphorylation. This suggests that phosphorylation of the tail domain may contribute to neurofilament stability in vivo. A neuronal-derived protein kinase that specifically phosphorylates only KSPXKX motifs in neurofilaments has been reported. The presence of extensively phosphorylated KSPXXX repeats in NF-H in vivo suggests the existence of yet another, unidentified kinase(s) with specificity for KSPXXX motifs.

Alkaline Phosphatase↗
PubMed1992

A high-molecular-weight squid neurofilament protein contains a lamin-like rod domain and a tail domain with Lys-Ser-Pro repeats.

Previous studies have shown that two low molecular-weight neurofilament (NF) proteins (NF-60 and NF-70) from the squid Loligo pealei are translated from mRNAs that are splice variants of a single squid NF gene. In this study, we report the isolation and characterization of cDNA clones encoding a high-molecular-weight squid NF protein (NF-220), the mRNA of which derives from the same squid NF gene. All three proteins are identical in their amino-terminal and lamin-like rod domains but differ in their carboxyl-terminal tail regions. In contrast to the short tail domains of NF-60 and NF-70, the NF-220 protein has a longer tail domain containing an acidic cluster of amino acids immediately followed by repeated copies of the sequence motif Lys-Ser-Pro. The Lys-Ser-Pro domain is similar to that of mammalian medium NF (NF-M) and high NF (NF-H) proteins, where the serines are highly phosphorylated. Except for these Lys-Ser-Pro motifs, there is surprisingly little structural similarity between the squid NF-220 protein and mammalian NF-M and NF-H proteins. Furthermore, the location of introns in squid NF-220 protein shows that it is more closely related to nuclear lamins and type III intermediate-filament proteins than to vertebrate NF proteins.

Amino Acid Sequence↗
PubMed1992

Ultrastructural immunolocalization of rat oxytocin-neurophysin in transgenic mice expressing the rat oxytocin gene.

Cell-specific expression of the rat oxytocin (OT)-neurophysin transgene in mice was achieved using a construct containing both OT and vasopressin genes (Young III, W.S., Reynolds, K., Shepard, E.A., Gainer, H. and Castel, M., Cell-specific expression of the rat oxytocin gene in transgenic mice, J. Neuroendocrinol., 2 (1990) 1-9). The present study describes the distribution of the protein products of these genes in various regions of the cell, and determines whether the transgenic rat and endogenous mouse OT-neurophysins are colocalized within the same neurosecretory granules. Two monoclonal antibodies against OT-neurophysins were used: PS38 which can react with both rat and mouse OT-neurophysin (pan-specific), and PS67 which is specific for rat OT-neurophysin only. Various approaches to double immunolabeling at the ultrastructural level were employed; these included: (1) pre-embedding immunoperoxidase followed by post-embedding immunogold; (2) post-embedding immunolabeling using gold particles of different sizes; and (3) labeling of consecutive ultrathin sections with different antibodies. Results from each of these approaches showed that both in the transgenic mouse and in the rat (used as control), immunocytochemical labeling for both PS38 and PS67 occurred in the same OT-ergic neurosecretory granules. In the control mouse, only PS38 elicited labeling. Hence, it may be concluded that the protein and peptide products of the transgene and the endogenous gene for OT-neurophysin are being processed similarly in the cell and finally concentrated together in the same neurosecretory granules.

Animals↗
PubMed1992

Identification and developmental expression of a novel low molecular weight neuronal intermediate filament protein expressed in Xenopus laevis.

Xenopus laevis is a valuable model system for the study of vertebrate neuroembryogenesis. However, very few well-characterized nervous system-specific molecular markers are available for studies in this organism. We screened a X. laevis adult brain cDNA library using a cDNA probe for mouse low molecular weight neurofilament protein (NF-L) in order to identify neuron-specific intermediate filament proteins. Clones for two distinct neuron-specific intermediate filament proteins were isolated and sequenced. One of these encoded for a Xenopus NF-L (XNF-L) and the other for a novel neuron-specific Xenopus intermediate filament protein (XNIF) that was present earlier and more abundantly than XNF-L during development. XNIF contained a central rod domain with multiple sequence features characteristic of IF proteins. The XNF-L was very similar to mouse NF-L, with a 77% sequence identity in the rod domain and the presence of a polyglutamic acid region in the tail domain, characteristic of type IV neurofilament proteins. In contrast, XNIF showed only 60% identity to mouse NF-L in the rod domain and lacked the glutamic acid-rich sequence in the tail domain. XNIF also had a very low (approximately 38%) sequence identity in the head and tail domains as compared to NF-L and other neurofilament proteins (45% identity to the head domain of alpha-internexin). In the adult frog, XNIF mRNA is detected by Northern blots only within the nervous system and by in situ hybridization histochemistry exclusively in neurons, particularly in the medullary reticular system and spinal cord. Antisera raised against the unique tail region of XNIF detected a single distinct 60 kDa band in Western blots of nervous system cytoskeletal preparations, and this XNIF immunoreactivity was concentrated in axons in the PNS and in small perikarya in the dorsal root ganglion. In contrast, NF-L immunoreactivity was principally in the large perikarya in the dorsal root ganglion. In development, XNIF mRNA appears more abundant than XNF-L mRNA in all premetamorphic stages examined. XNIF mRNA is first detectable at stage 24 (26 hr), whereas stable expression of XNF-L is at stage 35/36 (50 hr). XNIF immunoreactivity is detectable within the cement gland, within many neuronal cell bodies and axon tracts within the developing nervous system, and within all cellular layers of the developing retina. The availability of these two distinct neuron-specific intermediate filament proteins, with different temporal and spatial expression patterns, should provide new markers as well as targets for functional perturbation in the developing X. laevis nervous system.

Amino Acid Sequence↗
PubMed1991

Inhibition of axonal development after injection of neurofilament antibodies into a Xenopus laevis embryo.

The ability to target specific cytoskeletal components in axons for disruption within intact developing embryos would provide a valuable tool for studying neuronal development. Neurofilaments are an attractive target for such an approach, because they are neuron specific and are expressed late in embryogenesis principally beginning during axon outgrowth. No pharmacological agents are currently available that disrupt neurofilaments without also affecting general development. One approach that has been used successfully to affect proteins in vivo is to inject specific antibodies into living cells. We employed this approach in Xenopus laevis embryos by injecting two antibodies directed against the middle molecular weight neurofilament protein (NF-M) into a single blastomere of a two-cell stage embryo. Injected antibodies could be detected for as long as 3.5 days in cells descended from the injected blastomere. Only cell bodies of neurons descended from anti-NF-M-injected blastomeres contained abnormal accumulations of intermediate filament proteins, and peripheral nerve development was unilaterally retarded in these neurofilament antibody-injected tadpoles. Such accumulations and peripheral nerve defects were not seen in neurons derived from uninjected blastomeres or from blastomeres injected with control antibodies. These data demonstrate the usefulness of specific antibodies to perturb neuronal development in intact frog embryos and, in addition, suggest a role for neurofilaments in axon elongation.

Animals↗
PubMed1991

NF-L and peripherin immunoreactivities define distinct classes of rat sensory ganglion cells.

Double immunofluorescence studies using antibodies against NF-L and peripherin revealed three distinct subpopulations of neurons in rat dorsal root ganglia (DRG). In the adult rat, 46% of the DRG neurons were small and peripherin-positive (NF-L-negative), and 48% were large and NF-L-positive (peripherin-negative). About 6% were both peripherin- and NF-L-positive. All of the DRG neurons reacted with antibodies to NF-M and nonphosphorylation-dependent or phosphorylation-independent antibodies to NF-H. The neuropeptides were predominantly found in the peripherin-positive small cell population. Eighty-seven percent of the peripherin-positive small cell population contained substance P immunoreactivity, while 43% of this cell population contained CGRP. In contrast, only 18-24% of the NF-L-positive large-cell population contained neuropeptides, and these were primarily in a smaller sized subpopulation. Similar patterns of antigen representation were observed in neonatal (PN2) DRG cell populations. Tissue cultures of sensory ganglion cells from PN2 DRG, in serum-free medium, stably maintained exclusively peripherin-positive neurons, with about 5% of these containing coexistent NF-L immunoreactivity. Very high levels of neuropeptide gene expression were exhibited by these postnatal neurons in culture.

Aging↗
PubMed1991

Distribution of Na+, K(+)-ATPase alpha-subunit isoforms in rat pituitary.

The distributions of alpha-subunit isoforms of the Na+,K(+)-ATPase in rat pituitary were determined by immunoblotting and immunohistochemistry. Immunoreactivity for all three forms is present in the neural lobe, whereas the anterior lobe contains only alpha 1 and alpha 2. Most areas of the intermediate lobe exhibit faint immunoreactivity for only alpha 1, but thin strands of cells which stain strongly for all three isoforms are also present in this lobe. The previously reported ouabain inhibitable Na+,K(+)-ATPase activity in the neural lobe is consistent with the presence of both alpha 2 and alpha 3 subunits.

Animals↗
PubMed1991

Bombesin-like peptides induce Ca2(+)-activated K+ conductance increases in mouse fibroblasts.

Peptide receptor-activated membrane currents were studied in two mouse fibroblast cell lines, Swiss and Balb/c 3T3 cells, using a patch-electrode voltage-clamp technique. About 50% of the Swiss 3T3 cells examined responded to bombesin (Bn; 10(-9) to 10(-6) M), either by inducing outward current flow or inward current flow at the membrane holding potential (Vh) of -60 mV. The outward current type was more common (approximately 70%) than the inward current type (30%). The Bn-induced outward current (IBn) was reversed as the Vh was held to more negative than -90 mV (avg reversal potential, Erev = -82 mV). This Erev was closer to the equilibrium potential for K+ and shifted by altering the extracellular-to-intracellular K+ concentration ratio, in a Nernst-like relationship. The chance of recording this type of IBn was greatly reduced when K+ conductance blockers were present in the bathing solution (i.e., tetraethylammonium, Ba2+) or in the pipette solution (i.e., Cs). It was also reduced by recording with the pipette containing 5-10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Application of Ca2+ ionophore A23187 (5 microM) induced a similar membrane current with conductance increase. Thus the outward IBn in Swiss 3T3 cells appears to be induced by the intracellular Ca2(+)-dependent K+ conductance increase. Applications of bradykinin (Bk), arginine vasopressin (AVP), neuromedin B (NmB), and gastrin releasing peptide (GRP) to Swiss 3T3 cells also induced receptor-activated currents similar to IBn. Balb/c 3T3 cells rarely generated outward currents in response to Bn, GRP, and NmB but did not respond to both AVP and Bk with outward current flows.

Animals↗
PubMed1991

Whole cell current analyses of pancreatic acinar AR42J cells. I. Voltage- and Ca(2+)-activated currents.

Voltage- and Ca(2+)-activated whole cell currents were studied in AR42J cells, a clonal cell line derived from rat pancreatic acinar cells, using a patch electrode voltage-clamp technique. Four kinds of ionic currents were identified by their ionic dependencies, pharmacological properties, and kinetic parameters: 1) an outward current flow due mainly to a voltage-dependent K(+)-conductance increase, 2) an initial transient inward current due to an Na(+)-conductance increase, 3) transient and long-duration inward current due to a Ca(2+)-conductance increase, and 4) a slowly activating inward current that persists over the duration of the depolarizing pulse and deactivates slowly upon repolarization, producing a slow inward tail current. The slow inward tail current was particularly robust and was interpreted as due to a Ca(2+)-activated Cl(-)-conductance increase, since 1) the generation of this current was blocked by removing the extracellular Ca2+, applying Ca(2+)-channel blockers (Cd2+, nifedipine), or by lowering the intracellular Ca2+ concentration [( Ca2+]i) with EGTA; and 2) the reversal potential (Erev) of the slow inward tail current was close to 0 mV in the control condition (152 mM [Cl-]o/154 mM [Cl-]i), and changes of the [Cl-]o/[Cl )i ratio shifted the Erev toward the predicted Cl- equilibrium potential.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1991

Whole cell current analyses of pancreatic acinar AR42J cells. II. CCK and receptor-activated membrane currents.

Receptor currents generated in response to the application of sulfated octa-cholecystokinin (CCK) and various pancreatic secretagogues were studied in AR42J cells, a rat pancreatic acinar cell line. A whole cell configuration of the patch electrode voltage-clamp technique was utilized in these studies. Over 60% of the cells examined responded to 10(-6) M CCK by generating inward current at a membrane holding potential of -70 mV, accompanied by an increased membrane conductance. The CCK-induced receptor current (ICCK) was typically inactivated within 30 s in the presence of the ligand. The reversal potential (Erev) of the ICCK was approximately 0 mV when recorded in control bathing solution with a pipette containing 154 mM Cl-. When Cl- concentration in the bathing solution or in the recording pipette was modified, the Erev of the ICCK shifted toward the predicted Cl- potential. Na+ and K+ did not show significant contributions to the Erev of ICCK. Hence, the ICCK involves an increased Cl- conductance. This increased Cl- conductance appears to be due to an increase in intracellular Ca2+, since the ICCK could not be recorded from cells using pipettes containing 5 mM EGTA. Application of a Ca2+ ionophore A23187 (5 microM) to cells in control saline induced a similar inward current flow. The source of the Ca2+ involved in the ICCK appears to be mainly intracellular, since the ICCK could be recorded under conditions designed to completely block the voltage-dependent Ca2+ entry. These included exposure of the cells to a Ca2(+)-free saline for a brief period or the presence of 500 microM Cd2+ in the control bathing solution.

Animals↗
PubMed1991

Maintenance of LHRH and oxytocin neurons in slice explants cultured in serum-free media: effects of tetrodotoxin on gene expression.

A variety of neuroendocrine cells survive and express specific neuropeptide genes for long periods of time in slice explant cultures in the presence of serum. However, before use of these slice explant cultures as experimental models for physiological and pharmacological studies on the regulation of neuropeptide gene expression, it is first necessary to evaluate their characteristics in defined (e.g. serum free) media and to control for the spontaneous electrical and synaptic activity of neurons in these cultures. In this study, brain slices from postnatal day 4 rats were cultured in serum-containing media (SCM) for 12 days to allow thinning, and then maintained in a serum-free, defined media (SFM) for 6 days. Culture slices transferred to SFM appeared healthy and numerous neuroendocrine neurons containing messenger RNA (mRNA) encoding for LHRH and magnocellular neurons containing mRNA encoding for oxytocin (OT) were detected using in situ hybridization histochemistry (ISHH). Each of these neuronal subtypes robustly produced their appropriate gene products as determined by immunocytochemical analysis. Abundant magnocellular OT neurons were found in cultures grown in either SCM or SFM. In contrast, magnocellular vasopressin (VP) neurons were rarely detected under these conditions. Inhibition of spontaneous electrical and synaptic activity in these slice explant cultures was effectively achieved by incubation for the last 2.5 days of culture in the presence of tetrodotoxin (TTX; 10(-6) M). Densitometric single cell analyses after ISHH was performed on both LHRH and OT cells. Comparisons of the density values (corresponding to mRNA levels), from these slice explants, found that: (1) cellular LHRH mRNA levels decreased in the absence of serum, whereas cellular OT mRNA levels did not significantly change under these conditions; (2) the presence of TTX in the media resulted in an overall decrease in cellular LHRH mRNA values in both SCM and SFM, and (3) the OT neurons in these slice cultures appear to be composed of a heterogeneous population, with one cell subtype responding to TTX with an increase in cellular OT mRNA levels. These data show that factors in serum and spontaneous electrical activity can differentially influence mRNA levels of LHRH cells and magnocellular OT neurons in culture.

Action Potentials↗
PubMed1990

Phosphorylation-dependent epitopes on neurofilament proteins and neurofilament densities differ in axons in the corticospinal and primary sensory dorsal column tracts in the rat spinal cord.

The highest molecular weight neurofilament protein (NF-H) is multiply phosphorylated at epitopes which can be distinguished by specific monoclonal antibodies on Western blots. Eight characterized antibodies were used in immunocytochemistry to examine the tissue distributions of phosphorylated variants of NF-H in axons of the adult rat spinal cord. The most striking difference in staining was found between axons in the cuneate tract and those in the neighboring dorsal corticospinal tract. Axons in the cuneate tract reacted intensely with antibodies to phosphorylated epitopes of NF-H and poorly with antibodies to dephosphorylated epitopes of NF-H, whereas the reverse was the case for the axons of the dorsal corticospinal tract. These differences showed that systematic variations in the phosphorylation of NF-H in long-tract axons in the central nervous system occur as a function of cell type. When the cytoskeletons of these axons were compared by electron microscopy, the neurofilaments of the cuneate fibers were seen to be more abundant and formed a latticework, more compactly organized than the neurofilaments of the dorsal corticospinal axons. By comparison, the dorsal corticospinal axons were relatively richer in microtubules than the cuneate axons. Although the cuneate fiber tract contained many more large (greater than 2.0 microns 2 in cross section) axons than did the dorsal corticospinal tract, these differences in cytoskeletal organization were apparent even when myelinated axons of similar sizes (0.4 micron 2 to 2.0 microns 2) were compared. In addition, the number of neurofilaments in cuneate axons in the 0.4 to 2.0 microns 2 size range was significantly better correlated with axon size than was the case for this size range of dorsal corticospinal axons. Thus, the differences seen in the organization of the neurofilament latticework and the phosphorylation of NF-H between axons found in these two tracts both appeared to be correlated with cell type, and were independent of length or caliber of the axons.

Animals↗
PubMed1990

Characterization of the distinctive neurofilament subunits of the soma and axon initial segments in the squid stellate ganglion.

The stellate ganglion, which gives rise to the giant axons of the squid, was dissected into two parts, one containing primarily cell bodies and the other axon initial segments. A neurofilament protein-enriched extract of each was prepared and compared biochemically and immunochemically with an axoplasmic neurofilament preparation and with the glial sheath that surrounds the axons. Both parts of the ganglion lacked the 220 kDa subunit of axoplasmic neurofilaments (NFs). However, they did contain a protein of about 190 kDa that reacted with the Pruss anti-intermediate filament antibody (aIFA; Pruss et al.: Cell 27:419-428, 1981), but not with a phosphorylation-dependent NF antibody (Cohen et al.: J Neurosci 7: 2056-2074, 1987). Dephosphorylation of the axoplasmic NF220 yielded a product that comigrated on two-dimensional (2D) gel electrophoresis with the 190 kDa ganglion protein, suggesting that the latter represented the incompletely phosphorylated precursor of NF220. The major low molecular weight aIFA-reactive species in the ganglion preparations was a polypeptide of about 65 kDa. A relatively small quantity of that polypeptide was also found in axoplasm and it comigrated in 2D gels with an aIFA-reactive polypeptide from the glial sheath. These results indicate that the site of modification of the 190 kDa NF precursor to the 220 kDa axonal form is probably at the point where the axon initial segments leave the ganglion, which is several mm distal to its site of synthesis in the cell body. Furthermore, the filament network of the axoplasm and possibly the cell bodies includes a glial-like intermediate filament protein in addition to the NF protein subunits.

Animals↗
PubMed1990

Structure of mouse vasopressin and oxytocin genes.

Mouse vasopressin (VP) and oxytocin (OT) genes were isolated from a genomic library and the nucleotide sequences of the two genes were determined. The two genes have similar three exon structures and a high similarity in the part of exon 1 encoding vasopressin or oxytocin nonapeptide and in exon 2 encoding the central core of neurophysin. They are linked together in a tail to tail orientation separated by a short 3.5 kb intergenic sequence and are transcribed from opposite strands. Both genes have a single transcription initiation site downstream from a TATA-like sequence and a single polyadenylated transcript of about 760 bp for the vasopressin mRNA and about 700 bp for the oxytocin mRNA.

Amino Acid Sequence↗