ABSTRACT
OBJECTIVE: Cytomegalovirus (CMV) is the most common cause of congenital infection and can cause neurodevelopmental disabilities, although a majority of patients are asymptomatic. Biomarkers associated with disease severity would be desirable to distinguish asymptomatic from mildly symptomatic patients who may benefit from antiviral treatment. MicroRNAs (miRNAs) are noncoding RNAs that may have the potential to serve as biomarkers. STUDY DESIGN: Thirteen infants with congenital CMV infection were enrolled, and plasma levels of 11 human- and 3 CMV-encoded miRNAs were quantitated by real-time PCR. Plasma levels of miRNAs and their associations with clinical features were evaluated. RESULTS: The levels of miR-183-5p and miR-210-3p were significantly higher in patients with congenital CMV infection than in control infants, whereas no significant associations between levels of miRNAs and clinical features of congenital CMV infection were observed. CONCLUSION: Plasma miRNAs could be associated with the pathogenesis of congenital CMV infection and could be used as disease biomarkers.
Subject(s)
Cytomegalovirus Infections/congenital , Cytomegalovirus/genetics , MicroRNAs/blood , Antiviral Agents , Biomarkers/blood , Cytomegalovirus Infections/blood , DNA, Viral/urine , Female , Humans , Infant , Infant, Newborn , Male , Real-Time Polymerase Chain ReactionABSTRACT
The precise measurement of forces is one way to obtain deep insight into the fundamental interactions present in nature. In the context of neutral antimatter, the gravitational interaction is of high interest, potentially revealing new forces that violate the weak equivalence principle. Here we report on a successful extension of a tool from atom optics--the moiré deflectometer--for a measurement of the acceleration of slow antiprotons. The setup consists of two identical transmission gratings and a spatially resolving emulsion detector for antiproton annihilations. Absolute referencing of the observed antimatter pattern with a photon pattern experiencing no deflection allows the direct inference of forces present. The concept is also straightforwardly applicable to antihydrogen measurements as pursued by the AEgIS collaboration. The combination of these very different techniques from high energy and atomic physics opens a very promising route to the direct detection of the gravitational acceleration of neutral antimatter.
ABSTRACT
BACKGROUND: NY-ESO-1 antibodies are specifically observed in patients with NY-ESO-1-expressing tumours. We analysed whether the NY-ESO-1 humoral immune response is a useful tumour marker of gastric cancer. METHODS: Sera from 363 gastric cancer patients were screened by enzyme-linked immunosorbent assay (ELISA) to detect NY-ESO-1 antibodies. Serial serum samples were obtained from 25 NY-ESO-1 antibody-positive patients, including 16 patients with curative resection and 9 patients who received chemotherapy alone. RESULTS: NY-ESO-1 antibodies were detected in 3.4% of stage I, 4.4% of stage II, 25.3% of stage III, and 20.0% of stage IV patients. The frequency of antibody positivity increased with disease progression. When the NY-ESO-1 antibody was used in combination with carcinoembryonic antigen and CA19-9 to detect gastric cancer, information gains of 11.2% in stages III and IV, and 5.8% in all patients were observed. The NY-ESO-1 immune response levels of the patients without recurrence fell below the cutoff level after surgery. Two of the patients with recurrence displayed incomplete decreases. The nine patients who received chemotherapy alone continued to display NY-ESO-1 immune responses. CONCLUSION: When combined with conventional tumour markers, the NY-ESO-1 humoral immune response could be a useful tumour marker for detecting advanced gastric cancer and inferring the post-treatment tumour load in seropositive patients.
Subject(s)
Antibodies, Neoplasm/blood , Antigens, Neoplasm/immunology , Biomarkers, Tumor/blood , Membrane Proteins/immunology , Stomach Neoplasms/immunology , Aged , Antigens, Tumor-Associated, Carbohydrate/analysis , Carcinoembryonic Antigen/analysis , Disease Progression , Female , Humans , Immunity, Humoral , Male , Middle Aged , Prognosis , Stomach Neoplasms/diagnosis , Stomach Neoplasms/pathology , Tumor BurdenABSTRACT
AIM: To evaluate the prognosis of the patients who had visiting nurse service and discuss the place of death (life at the terminal stage). To determine the roles of visiting nurses in providing the patients at a terminal stage with their desirable life till death. METHODS: A total of 180 patients, who were registered for their home healthcare service in our Shonan Kamakura General Hospital and died between January 2000 and February 2001, were subjected to the study. All the subjects were classified into 3 groups according to the places of their death, 1) death at home, 2) death in the hospital and 3) death upon arrival after the admission to the hospital. Moreover, the following items were also surveyed and analyzed: 1) diagnosis (name of diseases), 2) cause of death, 3) age, 4) family structure, 5) whether their primary care physicians explained the prognosis and possible expected conditions to the patients and their family before hand, and 6) how the visiting nurses interact with the patients and their family members. RESULTS: Sixty-six patients died at home, 105 in the hospital and 9 upon arrival at the hospital. During this survey period, there were a total of 5,274 and 5,574 visits by primary care physicians and visiting nurses, respectively. The patients who died at home were more often observed in the patients whose primary care physicians explained their conditions to them and whose visiting nurses closely related to them. Moreover, the patients with malignant tumor also more often died at home. On the contrary, there were very few patients with chronic diseases, with whom death at home was accepted and agreed before hand, and there were some cases with chronic diseases who died inside of the ambulance transported on the way to the hospital after a sudden change in their conditions. DISCUSSION AND CONCLUSION: In order to have the patients live their desirable life till their death, it is required for the caretakers to prepare their mind for the day of the patient's death in addition to the patient's own wishes. For the patients with malignant tumor, it is easy to predict their prognosis, thus the caretakers can get prepared for the day of the patient's death. On the contrary, in case of the patients with chronic diseases, it is more difficult for the caretakers to experience an indefinite time with the patients since their prognosis is generally longer but the sudden change in their conditions may give the caretakers a high anxiety. Thus, it is essential for the visiting nurses to play a role as a mediator to interact between the patients and their family members, and their primary care physicians, and to establish a trustful relationship with the patients while their conditions are still stable. Moreover, similar to the malignant patients, the visiting nurses should explain the situations to the patients with chronic diseases, that they can choose the place of their own death and specific medical treatment at emergency and can decide the detail for their terminal stage with their family members. Thus, it was considered to be very important that the visiting nurses should frequently confirm these issues with the patients and their family according to their conditions.
Subject(s)
Attitude to Death , Community Health Nursing , Home Care Services, Hospital-Based , Aged , Aged, 80 and over , Female , Humans , Male , Terminally IllABSTRACT
The crystal structures of chitosan acid salts were studied by X-ray diffraction measurements on a fiber diagram and a new procedure to obtain an anhydrous polymorph of chitosan was found. The salts prepared by immersing a chitosan into a mixture of acid solution and isopropanol were classified into two types (Types I and II) depending on their conformation. Molecular conformation of the Type I salt retains the extended 2-fold helical structure of the original chitosan, but that of Type II salt is a twisted 2-fold helix. All the Type II salts changed to the anhydrous "Annealed" polymorph of chitosan when soaking in 75% aqueous isopropanol, but when the Type I salts were immersed in the solution, they returned to the hydrated "Tendon" polymorph which is that of the original chitosan. The strange transformation observed in Type II salt may be related to the stability of the molecular conformation of chitosan in the salt.
Subject(s)
Chitin/chemistry , Acids , Carbohydrate Conformation , Chitin/analogs & derivatives , Chitin/classification , Chitin/isolation & purification , Chitosan , Crystallization , Crystallography, X-Ray , SaltsABSTRACT
The CRMP (collapsin response mediator protein) family is thought to play key roles in growth cone guidance during neural development. The four members (CRMP1-4) identified to date have been demonstrated to form hetero-multimeric structures through mutual associations. In this study, we cloned a novel member of this family, which we call CRMP5, by the yeast two-hybrid method. This protein shares relatively low amino acid identity with the other CRMP members (49-50%) and also with dihydropyrimidinase (51%), whereas CRMP1-4 exhibit higher identity with each other (68-75%), suggesting that CRMP5 might be categorized into a third subfamily. The mouse CRMP5 gene was located at chromosome 5 B1. Northern blot and in situ hybridization analyses indicated that CRMP5 is expressed throughout the nervous system similarly to the other members (especially CRMP1 and CRMP4) with the expression peak in the first postnatal week. Association experiments using the yeast two-hybrid method and co-immunoprecipitation showed that CRMP5 interacts with dihydropyrimidinase and all the CRMPs including itself, except for CRMP1, although the expression profile almost overlaps with that of CRMP1 during development. These results suggest that CRMP complexes in the developing nervous system are classifiable into two populations that contain either CRMP1 or CRMP5. This indicates that different complexes may have distinct functions in shaping the neural networks.
Subject(s)
Nerve Tissue Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , DNA, Complementary , Mice , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , RNA, Messenger/genetics , Sequence Homology, Amino Acid , Two-Hybrid System TechniquesSubject(s)
Avian Proteins , Brain Mapping , Oncogene Proteins , Retina/embryology , Superior Colliculi/embryology , Viral Proteins , Animals , Body Patterning , DNA-Binding Proteins/physiology , Ephrin-A2 , Ephrin-A5 , Forkhead Transcription Factors , Membrane Proteins/physiology , Mice , Mice, Knockout , Proto-Oncogene Proteins/physiology , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/physiology , Transcription Factors/physiologyABSTRACT
To evaluate an association of ischemic stroke and Chlamydia pneumoniae (C. pneumoniae) infection, we investigated specific antibodies to C. pneumoniae by ELISA in sera of 91 patients (male 53, female 38, mean age 73 years) with acute ischemic stroke. We divided the patients under clinical diagnosis into three groups, those with cardiac embolism (E group, n = 35), atherothrombotic infarction (A group, n = 29) and lacunar infarction (L group, n = 27). Carotid ultrasound examination was performed in 79 patients. The active infection, including acute primary infection, reinfection and chronic active infection, was defined by IgG or IgA index being > or = 3.00, while inactive infection, including previous and chronic inactive infection, was defined by IgG index being > or = 1.10 and < 3.00, and IgA index being < 3.00. We found that 20.7% of A group, 2.9% of E group, and 7.4% of L group had indexes suggesting an active infection (A vs. E. p = 0.040, A vs. L, p = 0.254, L vs. E, p = 0.575: Fisher's exact test). The IgG indexes of A group (mean, 1.50) were higher than those of E group (mean, 1.35) and those of L group (mean, 0.93, A vs. L, p = 0.049: unpaired t-test). The patients with carotid plaque (n = 7) had higher IgG indexes (mean, 2.28) than those without carotid plaque (n = 72, mean, 1.11, p = 0.002: unpaired t-test). These data support the association of acute atherothrombotic infarction with chronic active C. pneumoniae infection.
Subject(s)
Antibodies, Bacterial/blood , Chlamydophila Infections/immunology , Chlamydophila pneumoniae/immunology , Stroke/microbiology , Acute Disease , Aged , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , MaleABSTRACT
We report on a patient who developed primary position upbeat nystagmus (ppUBN) due to a unilateral medial medullary infarction. On oculography, the slow phases of the nystagmus sometimes had an exponentially decreasing velocity waveform, indicating that the nystagmus was due to impairment of the vertical position-to-velocity neural integrator. On magnetic resonance imaging, the lesion was caudal to the vestibular nuclei and to the most rostral of the perihypoglossal nuclei, the nucleus intercalatus, a structure that was also involved in a previously reported case of ppUBN due to a unilateral medullary lesion. On the basis of these imaging and oculographic observations, we propose that a unilateral lesion of the nucleus intercalatus is sufficient to cause ppUBN and that the nucleus intercalatus is a part of the vertical position-to-velocity neural integrator in the human ocular-motor system.
Subject(s)
Cerebral Infarction/physiopathology , Medulla Oblongata/blood supply , Nystagmus, Physiologic/physiology , Cerebral Infarction/diagnosis , Electrooculography , Humans , Magnetic Resonance Imaging , Male , Middle AgedABSTRACT
The crystal structures of chitosan-L- and D-lactate salts were studied by X-ray diffraction measurements on fiber diagrams. In each lactate, chitosan took on a different crystalline polymorph depending on the preparation temperature. At low temperature, they gave a similar fiber pattern to that of the type II salt which has been found to be one of the two forms of chitosan acid salts in which the backbone chitosan molecules take on an eight-fold helix. At high temperature, however, the fiber pattern was that of the type I salt, another form of chitosan salt in which the backbone chains apparently retain the 21 symmetry of chitosan itself. The high-temperature polymorph of the L-lactate was a monoclinic (pseudoorthorhombic) unit cell whose lattice parameters were a=10.51, b=10.85, c(fiber axis)=10.34 Å and γ=90°. That of the D-lactate was also a monoclinic cell having parameters a=11.20, b=11.60, c(fiber axis)=10.38 Å and γ=93.0°. Their unit cell volumes coupled with their observed density values indicate that two chains of chitosan lactate were accommodated in each unit cell, that the L-lactate was an anhydrous crystal, but that the D-lactate was hydrated. The preparation temperature at which the salt changed from type II to type I was different between the D- and L-lactate, suggesting that these acids had different affinity to the chitosan molecule. When chitosan powder was suspended in a racemic lactic acid solution, the resultant solution always showed a minus sign for the rotation angle, indicating that D-lactic acid had higher affinity to chitosan than the L-isomer.
ABSTRACT
Mutations in the STA gene at the Xq28 locus have been found in patients with X-linked Emery-Dreifuss muscular dystrophy (EDMD). This gene encodes a hitherto unknown protein named 'emerin'. To elucidate the subcellular localization of emerin, we raised two antisera against synthetic peptide fragments predicted from emerin cDNA. Using both antisera, we found positive nuclear membrane staining in skeletal, cardiac and smooth muscles in the normal controls and in patients with neuromuscular diseases other than EDMD. In contrast, a deficiency in immunofluorescent staining of skeletal and cardiac muscle from EDMD patients was observed. A 34 kD protein is immunoreactive with the antisera--the protein is equivalent to that predicted for emerin. Together, our findings suggest the specific deficiency of emerin in the nuclear membrane of muscle cells in patients with EDMD.
Subject(s)
Membrane Proteins/deficiency , Muscular Dystrophies/metabolism , Nuclear Envelope/metabolism , Thymopoietins/deficiency , Adolescent , Adult , Amino Acid Sequence , Base Sequence , DNA , Fluorescent Antibody Technique, Indirect , Humans , Immunoblotting , Male , Molecular Sequence Data , Muscles/cytology , Muscles/metabolism , Muscular Dystrophy, Emery-Dreifuss , Mutation , Nuclear Proteins , Subcellular FractionsABSTRACT
We presented a case of critical illness polyneuropathy after bacterial peritonitis. A 62-year-old male was received an emergency colectomy because of perforation of the sigmoid colon five days after the endoscopic polypectomy. He developed sepsis from peritonitis after operation in spite of the antibiotics therapy. On 15-th hospital days he developed muscle weakness and numbness of all limbs. He needed an artificial ventilator due to respiratory failure. Hematological and blood chemical findings showed a leukocytosis and metabolic acidosis with renal dysfunction because of sepsis. Serum anti-Campylobacter antibody was negative. Serial CSF examinations failed to show any abnormalities including albuminocytologic dissociation. Electrophysiological studies revealed a primary axonal degeneration, mainly in the motor, but also in the sensory nerve. Compound muscle and sensory action potentials were not elicited or markedly reduced without conduction velocity prolongation. Microscopic findings of the left sural nerve biopsy showed a primary axonal degeneration without evidence of inflammation. His prognosis was poor and three months later, he still required ventilatory assistance. Because of these clinical findings this patient was thought to have a critical illness polyneuropathy after excluding various etiologies of polyneuropathies. This case suggests that sepsis may be one of a cause of primary axonal polyneuropathy. The certain mechanism of this disease is still unknown. However cytokine, tumor necrotic factor(TNF) and/or Platelet activating factor(PAF) that secreted during sepsis may have an important role for the primary axonal degeneration.
Subject(s)
Intestinal Perforation/complications , Peripheral Nervous System Diseases/etiology , Peritonitis/etiology , Sigmoid Diseases/complications , Critical Illness , Humans , Male , Middle AgedABSTRACT
We studied the changes of frontal and parietal somatosensory evoked potentials (SEPs) in the awake state versus different stages of sleep in 10 normal adult subjects. Frontal and parietal SEP components were affected differentially as sleep stages progressed. In general, the amplitudes of frontal components, notably P22, were increased in sleep, whereas the amplitudes of parietal components were decreased in sleep. A sensitive waveform change from the awake state to sleep was present in the frontal response, where a subtle notched negativity, termed "N40," was present only in the awake state and quickly dissipated in all stages of sleep, including stage 1. The amplitude changes from the awake state to stage 3/4 sleep were neither linear nor parallel among SEP components. The most discordant changes occurred in stage 3/4. The amplitudes for the frontal N18-P22-N30 complex and parietal N20-P26-N32 complex increased from stage 2 to stage 3/4, while those for frontal N30-fP40 and parietal N32-pP40 decreased. In contrast to these divergent amplitude changes, the latencies of all components except P14 and frontal N18 showed progressive prolongation from the awake state to slow-wave sleep. The SEP waveforms and latencies in REM sleep approximated those in the awake state, although amplitudes for frontal peaks still remained slightly higher and amplitudes for parietal peaks slightly lower. We postulate that interactions of excitatory and inhibitory phenomena are responsible for the component-dependent and sleep-stage-dependent amplitude enhancement or depression in sleep.
Subject(s)
Evoked Potentials, Somatosensory , Frontal Lobe/physiology , Parietal Lobe/physiology , Sleep Stages/physiology , Adult , Electroencephalography , Humans , Male , Middle Aged , Reference Values , Sleep, REM/physiology , Wakefulness/physiologyABSTRACT
Studies were made on the fate of implanted material during bone induction. Mixtures of 1 mg of crude bone morphogenetic protein (BMP), or bovine serum albumin as a control, and 1.5 mg of bovine collagen, were pressed into discs and implanted under the fascia of the rectus abdominus muscle of rats. The tissues with implants were fixed 7, 10, and 14 days later and examined histologically. On day 7 after implantation, the implant was surrounded and invaded by alkaline phosphatase-positive cells. New bone and cartilage were seen at the periphery of the implant. In the regions of calcified cartilage and bone, these osteogenic matrices were intermixed with the implant. The mineral deposits were seen by electron microscopy not only on the osteogenic matrices but also on the implanted collagen. On day 14, the bone had spread to the center of the implant. No osteogenesis or chondrogenesis was seen in control implants. It was concluded that the calcification occurred on the implanted collagen during bone induction, and that it was related to successive bone formation and remodeling.
Subject(s)
Collagen , Osseointegration , Prostheses and Implants , Proteins/pharmacology , Alkaline Phosphatase/metabolism , Animals , Bone Morphogenetic Proteins , Calcium/metabolism , Cartilage/cytology , Cartilage/ultrastructure , Cattle , Collagen/pharmacology , Drug Carriers , Histocytochemistry , Male , Microscopy, Electron , Proteins/isolation & purification , Rats , Rats, Sprague-DawleyABSTRACT
To determine the characteristics and clinical course of patients with subacute thyroiditis (SAT) without typical signs and symptoms, clinical and pathologic records of the patients were reviewed and compared with those of patients with typical SAT. During the past 10 years, 11 of 105 patients with SAT did not have enough typical features for a diagnosis of SAT (atypical SAT). They included one male and 10 females with the average age of 50 years. The incidence of neck pain as the initial symptom in the patients with atypical SAT was low (18%) compared with that in the patients with typical SAT (69%). Of the patients with atypical SAT, 42% had elevated thyroid hormone levels and 46% had suppressed TSH levels, whereas the percentages in the patients with typical SAT were 76% and 85% respectively. Nine of 11 patients were misdiagnosed as having papillary carcinoma by physical examination, and 4 by ultrasonography. However, aspiration cytology could make a precise diagnosis of SAT in 4 patients at the time of clinical diagnosis and 3 other patients after reevaluation. Eight patients were admitted to the hospital under the diagnosis of thyroid cancer and 4 underwent surgical resection. Differential diagnosis between atypical SAT and papillary carcinoma is important, and aspiration cytology could be a conclusive diagnostic tool to avoid unnecessary treatment.
Subject(s)
Thyroiditis, Subacute/diagnosis , Adult , Biopsy, Needle , Carcinoma, Papillary/diagnosis , Diagnosis, Differential , Female , Humans , Male , Middle Aged , Retrospective Studies , Thyroid Gland/pathology , Thyroid Neoplasms/diagnosis , Thyroiditis, Subacute/physiopathologyABSTRACT
A new microcrystalline boehmite (tentatively named PT-A) was synthesized as an efficient phosphate adsorbent to replace aluminum hydroxide gel. The characteristic structure of PT-A was examined by nitrogen adsorption/desorption, X-ray diffraction, deviation microscopy, and scanning electron microscopy to establish a pore structural model of PT-A. With this model structure, the details of the mechanism of interaction between PT-A and phosphate in the presence of bovine serum albumin (BSA) are discussed. PT-A is a spherical particle with a diameter of approximately 100 microns and a porous surface structure, and its inside is packed with boehmite microcrystals (crystallite size, 2 nm). PT-A has three types of pores in its structure: a micropore with a narrow size-distribution, a mesopore with a broad size-distribution, and a macropore (radii of pores are 0.7, 1-20, and approximately 300 nm, respectively). When phosphate was incubated with PT-A in human gastric and intestinal juices or in an aqueous solution containing BSA, the amounts of phosphate adsorbed by PT-A were not affected by the presence of proteins. The nitrogen adsorption/desorption isotherms and energy dispersive X-ray analyses demonstrated that phosphate could diffuse to the smaller tunnels freely even if the external surface of PT-A was covered with BSA. It was also demonstrated that the main site of adsorption for phosphate was in micropores of PT-A, whereas BSA was adsorbed only to the external surface and none entered inside smaller tunnels consisting of micro- and mesopores.
Subject(s)
Aluminum Hydroxide/chemistry , Aluminum Oxide/chemistry , Phosphates/chemistry , Adsorption , Gastric Juice/chemistry , Humans , Ligands , Microscopy, Electron, Scanning , Nitrogen/chemistry , Particle Size , Porosity , Serum Albumin, Bovine , Thermodynamics , X-Ray DiffractionABSTRACT
Modification of function of the glucose transporter by nucleotides was studied by using liposomes reconstituted with the human erythrocyte glucose transporter. ADP enclosed in the liposomes inhibited the uptake of D-glucose and nicotinamide in a dose-dependent manner, but other enclosed nucleotides (ATP, AMP, CDP, GDP, UDP) showed no effect on the uptake of both. Only intraliposomal ADP was effective, and extra-liposomal ADP was not, under our experimental conditions. Intraliposomal ADP did not change Km, but decreased Vmax to approximately one-third of control for uptake of both D-glucose and nicotinamide. However, the binding and the affinity of cytochalasin B to the reconstituted liposomes were not affected by intraliposomal ADP. The uptake of uridine was not changed in the presence of ADP, indicating that the nucleoside transporter co-existing in the liposomal membranes is not regulated by ADP. Human erythrocytes whose intracellular ATP was decreased by Ca2+ ionophore A23187 also showed decreased uptake of 2-deoxy-D-glucose and nicotinamide. This phenomenon was very similar to that found in the liposomes. These findings suggest the possibility that the function of the glucose transporter is directly and negatively modified by an increased concentration of intracellular ADP.
Subject(s)
Adenosine Diphosphate/pharmacology , Erythrocyte Membrane/metabolism , Monosaccharide Transport Proteins/blood , Adenosine Monophosphate/pharmacology , Adenosine Triphosphate/pharmacology , Cytochalasin B/metabolism , Glucose/metabolism , Humans , Kinetics , Liposomes , Models, Biological , Monosaccharide Transport Proteins/drug effects , Monosaccharide Transport Proteins/isolation & purification , Niacinamide/metabolism , Uridine/metabolismABSTRACT
The water-soluble fraction containing bone-inductive activity was purified from guanidine-hydrochloride extracts of bovine demineralized bone. The purification steps include ultrafiltration, dialysis, affinity chromatography on heparin-Sepharose and gel chromatography on Sephacryl S-200. Combination of these steps was proven to be an effective and rapid method for the purification of this protein. Subcutaneous implantation of the water-soluble protein with type I collagen was carried out in the thorax of rats. When alkaline phosphatase activity and calcium content in implants were used as indices for purification, the water-soluble bone-inductive protein was purified > 600-fold according to the enzyme activity and 64-fold according to the calcium content. A morphological examination revealed that many chondrocyte and osteoblast cells were seen in the location of the implanted material. Sodium dodecyl sulfate/gel electrophoresis of the protein produced in this way under non-reducing conditions revealed four protein bands of 18, 16, 14 and 11 kDa. None of the separated bands had any biological activity. This result suggests that the water-soluble bone-inductive activity depends on an associated form of various proteins in the range of 18 to 11 kDa.
Subject(s)
Bone Matrix/chemistry , Proteins/isolation & purification , Alkaline Phosphatase/metabolism , Animals , Bone Development/drug effects , Bone Morphogenetic Proteins , Bone and Bones/drug effects , Bone and Bones/enzymology , Bone and Bones/metabolism , Calcium/metabolism , Cattle , Chromatography, Affinity , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Male , Proteins/pharmacology , Rats , Rats, Wistar , UltrafiltrationABSTRACT
A kinetic study of the uptake of nicotinamide by reconstituted liposomes containing the human erythrocyte glucose transporter, compared with that of D-glucose, demonstrated that the Km and Vmax. values were almost the same for each compound, and that the uptake of D-glucose was competitively inhibited by nicotinamide. At 20 mM concentration, 2-deoxy-D-glucose, 3-O-methyl-D-glucose and 4,6-O-ethylidene-D-glucose all caused 50% inhibition of nicotinamide uptake, but L-glucose and nicotinic acid were not inhibitory. Similar results were obtained for the uptake of D-glucose. Cytochalasin B binding to the liposomes was inhibited in a dose-dependent manner by either nicotinamide or D-glucose. Antibody for glucose transporter detected in band 4.5 by SDS/PAGE inhibited the uptake of D-glucose and nicotinamide. A possible uptake of nicotinamide by nucleoside transporter was excluded. In human erythrocytes, cytochalasin B binding was inhibited dose-dependently by either nicotinamide or D-glucose, and cytochalasin B depressed the uptake of both nicotinamide and 2-deoxy-D-glucose. These findings were well reproduced in the reconstituted liposomes. The very close similarities between uptake of nicotinamide and D-glucose suggest that the glucose transporter plays a direct role in transport of nicotinamide, which is structurally quite different from monosaccharides, and thus that the transporter is probably multifunctional.