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H Segawa

Publications and source records attributed to H Segawa.

At least 55 records · Page 3Linked to original sources

Regulation of PiT-1, a sodium-dependent phosphate co-transporter in rat parathyroid glands.

A cDNA encoding an Na+-Pi co-transporter, termed rat PiT-1, has now been isolated from rat parathyroid. Expression of rat PiT-1 in Xenopus oocytes revealed that it possesses Na+-dependent Pi co-transport activity. The amount of PiT-1 mRNA in the parathyroid of vitamin D-deficient rats was reduced compared with that in normal animals, and increased markedly after administration of 1,25-dihydroxyvitamin D3. Furthermore, the abundance of PiT-1 mRNA in the parathyroid was much greater in rats fed a low-Pi diet than in those fed a high-Pi diet. Thus, rat PiT-1 may contribute to the effects of Pi and vitamin D on parathyroid function.

Animals↗

Expression and activity of chimeric molecules between human UDP-galactose transporter and CMP-sialic acid transporter.

Human UDP-galactose transporter (hUGT1) and CMP-sialic acid transporter (hCST) are related Golgi proteins with eight putative transmembrane helices predicted by computer analysis. We constructed chimeric molecules in which segments of various lengths from the C- or N-terminus of hUGT1 were replaced by corresponding portions of hCST. The chimeras were transiently expressed in UGT-deficient mutant Lec8 cells, and their UGT activity was assessed by the binding of GS-II lectin to the transfected cells. The replacement of either the N- or C-terminal cytoplasmic segment by that of hCST did not affect the expression or activity of hUGT1. A chimera in which the eighth helix and the C-terminal tail were replaced also retained the UGT activity, indicating that this helix is not involved in the determination of substrate specificity. In contrast, three types of chimeras, in which the first helix, the first and the second helices, and a segment from the seventh helix to the C-terminus were replaced, respectively, were expressed very infrequently in the transfected cells, and had no UGT activity. They are likely folded incorrectly and degraded by a quality-control system, since the amounts of their mRNAs were normal and the proteins were mainly localized in the ER. The first and the seventh helices are important for the stability of the transporter protein.

Animals↗

Xenon has greater inhibitory effects on spinal dorsal horn neurons than nitrous oxide in spinal cord transected cats.

UNLABELLED: Xenon (Xe) suppresses wide dynamic range neurons in cat spinal cord to a similar extent as nitrous oxide (N2O). The antinociceptive action of N2O involves the descending inhibitory system. To clarify whether the descending inhibitory system is also involved in the antinociceptive action of Xe, we compared the effects of Xe on the spinal cord dorsal horn neurons with those of N2O in spinal cord-transected cats anesthetized with alpha-chloralose and urethane. We investigated the change of wide dynamic range neuron responses to touch and pinch by both anesthetics. Seventy percent Xe significantly suppressed both touch- and pinch-evoked responses in all 12 neurons. In contrast, 70% N2O did not show significant suppression in touch- and pinch-evoked responses. These results suggest that the antinociceptive action of Xe might not be mediated by the descending inhibitory system, but instead may be produced by the direct effect on spinal dorsal horn neurons. IMPLICATIONS: Xenon (Xe) is an inert gas with anesthetic properties. We examined the antinociceptive effects of Xe and nitrous oxide (N2O) in spinal cord-transected cats. Our studies indicate that Xe has a direct antinociceptive action on the spinal cord that is greater than that of N2O.

Anesthetics, Inhalation↗

New aneurysm clip and applier for narrow spaces: technical note.

OBJECTIVE: Aneurysm surgery carries considerable risk to the patient, in part because the surgical field can be so constricted that accurate placement of the aneurysm clip is impeded. In fact, most aneurysm clip appliers are so bulky that they can impair the surgeon's view, if the field is tight. We describe a system of aneurysm clips and appliers that have a very low profile and consequently allow better visualization of the operative field at critical moments during surgery. INSTRUMENTATION: A new system of clips for aneurysm surgery was developed to overcome some of the deficiencies of conventional sets. The new clip blades are opened from inside the spring, so the clip blades are clearly seen as they are applied across the base of the aneurysm. In addition, these clips can be angled in any desired direction in the applier, which makes their application easier. A third advantage is that the clip applier is used as a clip remover should adjustment or reapplication of the clip be necessary. RESULTS: Thirty aneurysms were obliterated with the new clips without any complications attributable to the clips. The new instruments allowed the safe application of clips to aneurysms in deep, confined, and anatomically complex spaces, such as aneurysms of the basilar tip and anterior communicating artery. CONCLUSION: The newly described system of aneurysm clips and appliers appears to have significant advantages over other currently available systems.

Animals↗

Infection after total knee arthroplasty. A retrospective study of the treatment of eighty-one infections.

BACKGROUND: The clinical presentation of an infection at the site of a total knee arthroplasty can be used as a guide to treatment, including the decision as to whether the prosthesis should be retained or removed. We reviewed the results of treatment of infection after total knee arthroplasty to evaluate the effectiveness of four treatment protocols based on the clinical setting of the infection. METHODS: We retrospectively evaluated the results of treatment of eighty-one infections in seventy-six consecutive patients who either had an infection after a total knee arthroplasty or had multiple positive intraoperative cultures of specimens of periprosthetic tissue obtained during a revision total knee arthroplasty performed because of presumed aseptic loosening. The patients were managed according to one of four protocols. Five infections in five patients who had positive intraoperative cultures were treated with antibiotic therapy alone. Twenty-three early postoperative infections in twenty-one patients were treated with débridement, antibiotic therapy, and retention of the prosthesis. Twenty-nine late chronic infections in twenty-eight patients were treated with a delayed-exchange arthroplasty after a course of antibiotics. Seven acute hematogenous infections in six patients were treated with débridement, antibiotic therapy, and retention of the prosthesis. Seventeen infections in seventeen patients were not treated according to one of the four protocols. Sixteen late chronic infections were treated either with an arthrodesis (five infections) or with débridement, antibiotic therapy, and retention of the prosthesis (eleven infections). One acute hematogenous infection was treated with resection arthroplasty because of life-threatening sepsis. RESULTS: The mean duration of follow-up was 4.0 years (range, 0.3 to 14.0 years). Eleven patients who had an arthrodesis, a resection arthroplasty, or an above-the-knee amputation after less than two years of follow-up were included in the study as individuals who had a failure of treatment. In the group of patients who were managed according to protocol, the initial course of treatment was successful for all five infections that were diagnosed on the basis of positive intraoperative cultures, five of the ten deep early infections, all thirteen superficial early infections, twenty-four of the twenty-nine late chronic infections, and five of the seven acute hematogenous infections. Only one of eleven prostheses in patients who had a late chronic infection that was not treated according to protocol was successfully retained after débridement. CONCLUSIONS: Our treatment protocols, which were based on the clinical setting of the infection, were successful for most patients. A major factor associated with treatment failure was a compromised immune status. Bone loss and necrosis of the soft tissues around the joint also complicated the treatment of these infections.

Aged↗

Identification of three isoforms for the Na+-dependent phosphate cotransporter (NaPi-2) in rat kidney.

We have isolated three unique NaPi-2-related protein cDNAs (NaPi-2alpha, NaPi-2beta, and NaPi-2gamma) from a rat kidney library. NaPi-2alpha cDNA encodes 337 amino acids which have high homology to the N-terminal half of NaPi-2 containing 3 transmembrane domains. NaPi-2beta encodes 327 amino acids which are identical to the N-terminal region of NaPi-2 containing 4 transmembrane domains, whereas the 146 amino acids in the C-terminal region are completely different. In contrast, NaPi-2gamma encodes 268 amino acids which are identical to the C-terminal half of NaPi-2. An analysis of phage and cosmid clones indicated that the three related proteins were produced by alternative splicing in the NaPi-2 gene. In a rabbit reticulocyte lysate system, NaPi-2 alpha, beta, and gamma were found to be 36, 36, and 29 kDa amino acid polypeptides, respectively. NaPi-2alpha and NaPi-2gamma were glycosylated and revealed to be 45- and 35-kDa proteins, respectively. In isolated brush-border membrane vesicles, an N-terminal antibody was reacted with 45- and 40-kDa, and a C-terminal antibody was reacted with 37-kDa protein. The sizes of these proteins corresponded to those in glycosylated forms. A functional analysis demonstrated that NaPi-2gamma and -2alpha markedly inhibited NaPi-2 activity in Xenopus oocytes. The results suggest that these short isoforms may function as a dominant negative inhibitor of the full-length transporter.

Amino Acid Sequence↗

Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98).

A cDNA was isolated from rat C6 glioma cells by expression cloning which encodes a novel Na+-independent neutral amino acid transporter designated LAT1. For functional expression in Xenopus oocytes, LAT1 required the heavy chain of 4F2 cell surface antigen (CD98), a type II membrane glycoprotein. When co-expressed with 4F2 heavy chain, LAT1 transported neutral amino acids with branched or aromatic side chains and did not accept basic amino acids or acidic amino acids. The transport via LAT1 was Na+-independent and sensitive to a system L-specific inhibitor 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. These functional properties correspond to those of the classically characterized amino acid transport system L, a major nutrient transporter. In in vitro translation, LAT1 was shown to be a nonglycosylated membrane protein consistent with the property of 4F2 light chain, suggesting LAT1 is at least one of the proteins formerly referred to as 4F2 light chain. LAT1 exhibits relatively low but significant amino acid sequence similarity to mammalian cationic amino acid transporters and amino acid permeases of bacteria and yeasts, indicating LAT1 is a new member of the APC superfamily. Because of highly regulated nature and high level of expression in tumor cell lines, LAT1 is thought to be up-regulated to support the high protein synthesis for cell growth and cell activation. The cloning of LAT1 is expected to facilitate the research on the protein-protein interaction in the transporter field and to provide a clue to the search for still unidentified transporters.

Amino Acid Sequence↗

Effects of dietary Pi on the renal Na+-dependent Pi transporter NaPi-2 in thyroparathyroidectomized rats.

Dietary Pi and parathyroid hormone (PTH) are two most important physiological and pathophysiological regulators of Pi re-absorption in the renal proximal tubule. Effects of dietary Pi on Na+/Pi co-transporter NaPi-2 were investigated in thyroparathyroidectomized (TPTX) rats. NaPi-2 protein and mRNA in the kidney cortex of TPTX rats were increased approximately 3.8- and 2.4-fold in amount respectively compared with those in the sham-operated animals. Administration of PTH to the TPTX rats resulted in a decrease in the amount of NaPi-2 protein, but not in the abundance of NaPi-2 mRNA. Deprivation of dietary Pi in the TPTX rats did not affect the amount of NaPi-2 mRNA and protein. In the Pi-deprived TPTX rats, feeding of a high-Pi diet resulted in marked decreases in Pi transport activity and the amount of NaPi-2 protein in the superficial nephrons. Immunohistochemical analysis demonstrated that administration of PTH to TPTX rats resulted in a decrease in NaPi-2 immunoreactivity from both superficial and juxtamedullary nephrons within 4 h. Switching TPTX animals from a low-Pi diet to the high-Pi diet decreased NaPi-2 immunoreactivity from superficial nephrons, but not from juxtamedullary nephrons, within 4 h. These results suggest that dietary Pi could regulate the amount of NaPi-2 protein in the superficial nephrons in a PTH-independent manner.

Animals↗

Regulation of type II renal Na+-dependent inorganic phosphate transporters by 1,25-dihydroxyvitamin D3. Identification of a vitamin D-responsive element in the human NAPi-3 gene.

Vitamin D is an important regulator of phosphate homeostasis. The effects of vitamin D on the expression of renal Na+-dependent inorganic phosphate (Pi) transporters (types I and II) were investigated. In vitamin D-deficient rats, the amounts of type II Na+-dependent Pi transporter (NaPi-2) protein and mRNA were decreased in the juxtamedullary kidney cortex, but not in the superficial cortex, compared with control rats. The administration of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to vitamin D-deficient rats increased the initial rate of Pi uptake as well as the amounts of NaPi-2 mRNA and protein in the juxtamedullary cortex. The transcriptional activity of a luciferase reporter plasmid containing the promoter region of the human type II Na+-dependent Pi transporter NaPi-3 gene was increased markedly by 1,25-(OH)2D3 in COS-7 cells expressing the human vitamin D receptor. A deletion and mutation analysis of the NaPi-3 gene promoter identified the vitamin D-responsive element as the sequence 5'-GGGGCAGCAAGGGCA-3' nucleotides -1977 to -1963 relative to the transcription start site. This element bound a heterodimer of the vitamin D receptor and retinoid X receptor, and it enhanced the basal transcriptional activity of the promoter of the herpes simplex virus thymidine kinase gene in an orientation-independent manner. Thus, one mechanism by which vitamin D regulates Pi homeostasis is through the modulation of the expression of type II Na+-dependent Pi transporter genes in the juxtamedullary kidney cortex.

Animals↗

Regulation of the PepT1 peptide transporter in the rat small intestine in response to 5-fluorouracil-induced injury.

BACKGROUND & AIMS: The oligopeptide transport system of the small intestine is resistant to mucosal injury. The mechanism of this resistance was investigated by examining the activity level and expression of the peptide transporter PepT1 in the intestine of rats treated with 5-fluorouracil. METHODS: The expression and localization of PepT1 were examined by immunoblot analysis of brush border membrane vesicles and immunohistochemical analysis of intestinal sections with PepT1-specific rabbit polyclonal antibodies. Also, Northern blot analysis was used for the expression of PepT1 messenger RNA (mRNA). RESULTS: Although the amounts of sucrase and an Na+-dependent glucose transporter protein in intestinal vesicles decreased markedly after 5-fluorouracil treatment, the amount of PepT1 protein remained largely unaffected. Immunohistochemical analysis also showed that the PepT1 immunoreactivity level was preserved in the brush border membrane of the remaining villi of 5-fluorouracil-treated rats. Levels of amino acid, glucose, and phosphate transporter mRNAs were profoundly depressed in 5-fluorouracil-treated animals, whereas the level of PepT1 mRNA conversely increased. CONCLUSIONS: The resistance of intestinal peptide transport to tissue injury may be attributable to increased synthesis of PepT1 rather than to a change in the kinetic properties of the residual absorbing cells.

Animals↗

The roles of individual cysteine residues of Sendai virus fusion protein in intracellular transport.

The role of intramolecular disulfide bonds in the fusion (F) protein of Sendai virus was studied. The 10 cysteine residues were changed to serine residues using site-directed mutagenesis. None of the cysteine mutant F proteins reacted with a monoclonal antibody specific for the mature conformation of the F protein, but eight of ten mutants reacted with an immature conformation-specific monoclonal antibody. The transport of these mutant proteins to the cell surface was drastically reduced. All of the cysteine mutant F proteins remained sensitive to endoglycosidase H (endo H) for 3 h after their synthesis. Moreover, cell surface transport of the hemagglutinin-neuraminidase (HN) protein co-expressed with each of these cysteine mutant F proteins was also reduced. These results suggest that all cysteine residues participate in the formation of intramolecular disulfide bonds, that co-translational disulfide bond formation is crucial to the correct folding and intracellular transport of the F protein, and that interaction of the F and HN proteins takes place intracellulary.

Animals↗

Isoflurane and sevoflurane augment norepinephrine responses to surgical noxious stimulation in humans.

BACKGROUND: Suppression of hypertensive response to noxious stimulation by volatile anesthetics may be a result of suppression of the stimulation-induced norepinephrine response or that of the cardiovascular response to catecholamines, or both. The suppression of the cardiovascular response is established, but that of norepinephrine response has not been confirmed. The authors hypothesized that the suppression of cardiovascular response but not that of norepinephrine response plays a major role in suppressing the noxious stimulation-induced hypertensive response by volatile anesthetics. METHODS: Forty healthy donors for living-related liver transplantation were allocated to four groups: receiving 1.2% (end-tidal) isoflurane in oxygen and nitrogen, 2.0% isoflurane, 1.7% sevoflurane, or 2.8% sevoflurane. The intraoperative plasma norepinephrine and epinephrine concentrations, arterial blood pressure and pulse rate were measured for the first 15 min of surgery and were compared with the preoperative values. RESULTS: Norepinephrine and epinephrine concentrations both increased intraoperatively in all four groups. The values of maximum increase and area under the concentration-versus-time curve of norepinephrine were greater in the high dose groups of both anesthetics. The intraoperative blood pressure did not differ by different doses of anesthetics, and the degree of increase of blood pressure was not proportional to the plasma catecholamine concentrations. CONCLUSION: The effects of isoflurane and sevoflurane on the surgical noxious stimulation-induced norepinephrine response were inversely proportional to the dose. The suppression of noxious stimulation-induced blood pressure response by anesthetics that were studied may be the result of suppression of the responses of vascular smooth muscle and myocardium to catecholamines.

Adult↗

Symptomatic tonsillar ectopia.

OBJECTIVE: To determine if slight descent of the cerebellar tonsils (< 5 mm below the foramen magnum; tonsillar ectopia) may cause surgically treatable symptomatology. METHODS: A consecutive series of nine symptomatic patients with tonsillar ectopia seen between December 1990 and March 1993 are reported on. The same number of age and sex matched controls were selected at random from outpatients. Twelve asymptomatic subjects with tonsillar ectopia were found among 5000 people between January 1991 and March 1996. Diagnosis of tonsillar ectopia was based on midsagittal MRI. RESULTS: Patients presented mainly with chronic intractable occipital dull pain, vertigo, and dysequilibrium. In all patients MRI showed normal brain structure except for tonsillar ectopia (-2.9 (SD 0.8) mm), which has historically been thought to be of no clinical relevance. In the control group the tonsilar position was +2.1 (SD 2.8) mm (p<0.01). Neurotologically abnormal findings were detected with a monaural speech integration test (100%), eye tracking test (56%), optokinetic nystagmus test (89%), and visual suppression test (67%) which strongly suggested a CNS lesion. In accordance with the results of MRI and precise neurotological examination, posterior fossa decompression surgery was carried out, followed by improvement of preoperative symptoms and less severity of neurotological abnormalities in all patients. CONCLUSION: Tonsillar ectopia could cause neurological symptoms in small populations, which were surgically treatable. Neurotological assessment was necessary to verify the aetiological relation between tonsillar ectopia and various symptoms.

Adult↗

Molecular cloning and hormonal regulation of PiT-1, a sodium-dependent phosphate cotransporter from rat parathyroid glands.

The extracellular concentration of inorganic phosphate (Pi) is an important determinant of parathyroid cell function. The effects of Pi may be mediated through specific molecules in the parathyroid cell membrane, one candidate molecule for which would be a Na+-dependent Pi cotransporter. A complementary DNA encoding a Na+-Pi cotransporter, termed rat PiT-1, has now been isolated from rat parathyroid. The 2890-bp complementary DNA encodes a protein of 681 amino acids that shows sequence identities of 97% and 93% with the type III Na+-Pi cotransporters mouse PiT-1 and human PiT-1, respectively. Expression of rat PiT-1 in Xenopus oocytes revealed that it possesses Na+-dependent Pi cotransport activity. PiT-1 messenger RNA (mRNA) is widely distributed in rat tissues and is most abundant in brain, bone, and small intestine. The amount of PiT-1 mRNA in the parathyroid of vitamin D-deficient rats was reduced compared with that in normal animals and increased markedly after administration of 1,25-dihydroxyvitamin D3. Furthermore, the abundance of PiT-1 mRNA in the parathyroid was much greater in rats fed a low-Pi diet than in those fed a high-Pi diet. Thus, rat PiT-1 may contribute to the effects of Pi and vitamin D on parathyroid function.

Amino Acid Sequence↗

Cloning, functional expression and dietary regulation of the mouse neutral and basic amino acid transporter (NBAT).

The Na+-independent dibasic and neutral amino acid transporter NBAT is among the least hydrophobic of mammalian amino acid transporters. The transporter contains one to four transmembrane domains and induces amino acid transport activity via a b0,+-like system when expressed in Xenopus oocytes. However, the physiological role of NBAT remains unclear. Complementary DNA clones encoding mouse NBAT have now been isolated. The expression of mouse NBAT in Xenopus oocytes also induced an obligatory amino acid exchange activity similar to that of the b0,+-like system. The amount of NBAT mRNA in mouse kidney increased during postnatal development, consistent with the increase in renal cystine and dibasic transport activity. Dietary aspartate induced a marked increase in cystine transport via the b0,+ system in mouse ileum. A high-aspartate diet also increased the amount of NBAT mRNA in mouse ileum. In the ileum of mice fed on the aspartate diet, the extent of cystine transport was further increased by preloading brush border membrane vesicles with lysine. Hybrid depletion of NBAT mRNA from ileal polyadenylated RNA revealed that the increase in cystine transport activity induced by the high-aspartate diet, as measured in Xenopus oocytes, was attributable to NBAT. These results demonstrate that mouse NBAT has an important role in cystine transport.

Amino Acid Sequence↗

Relative contributions of Na+-dependent phosphate co-transporters to phosphate transport in mouse kidney: RNase H-mediated hybrid depletion analysis.

Reabsorption of Pi in the proximal tubule of the kidney is an important determinant of Pi homoeostasis. At least three types (types I-III) of high-affinity Na+-dependent Pi co-transporters have been identified in mammalian kidneys. The relative roles of these three types of Na+/Pi co-transporters in Pi transport in mouse kidney cortex have now been investigated by RNase H-mediated hybrid depletion. Whereas isolated brush-border membrane vesicles showed the presence of two kinetically distinct Na+/Pi co-transport systems (high Km-low Vmax and low Km-high Vmax), Xenopus oocytes, microinjected with polyadenylated [poly(A)+] RNA from mouse kidney cortex, showed only the high-affinity Pi uptake system. Kidney poly(A)+ RNA was incubated in vitro with antisense oligonucleotides corresponding to Npt-1 (type I), NaPi -7 (type II) or Glvr-1 (type III) Na+/Pi co-transporter mRNAs, and then with RNase H. Injection of such treated RNA preparations into Xenopus oocytes revealed that an NaPi-7 antisense oligonucleotide that resulted in complete degradation of NaPi-7 mRNA (as revealed by Northern blot analysis), also induced complete inhibition of Pi uptake. Degradation of Npt-1 or Glvr-1 mRNAs induced by corresponding antisense oligonucleotides had no effect on Pi transport, which was subsequently measured in oocytes. These results indicate that the type II Na+/Pi co-transporter NaPi-7 mediated most Na+-dependent Pi transport in mouse kidney cortex.

Animals↗

Gene structure and functional analysis of the human Na+/phosphate co-transporter.

Three lambda phage clones encompassing the Na+/phosphate co-transporter (NaPi-3) gene and its 5' flanking region were isolated from a human genomic DNA library. The gene comprises 13 exons and 12 introns and spans approx. 14 kb. All exon-intron junctions conform to the GT/AG rule. The major transcription-initiation site was determined by primer-extension analysis and is an adenosine residue 57 bp upstream of the 3' end of the first exon. There is a typical TATA box 28 bp upstream of the major transcription-initiation site and various cis-acting elements, including a cAMP-responsive element, AP-1, AP-2 and SP-1 sites in the 5' flanking region. This region also contains three direct-repeat-like sequences that resemble the consensus binding sequence for members of the steroid-thyroid hormone receptor superfamily, including vitamin D. Deletion analysis suggests that the region from nt-2409 to nt-1259 in the 5' flanking region may be involved in kidney-specific gene expression. Vitamin D responsiveness of the NaPi-3 promoter was also detected in COS-7 cells co-transfected with a human vitamin D receptor expression vector. The presence of the three vitamin D receptor- responsive elements in the NaPi-3 promoter may be important in mediating the enhanced expression of the gene by 1,25-dihydroxyvitamin D3.

Amino Acid Sequence↗