A mutation in the extracellular cysteine-rich repeat region of the beta3 subunit activates integrins alphaIIbbeta3 and alphaVbeta3

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, alphaIIbbeta3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of alphaIIbbeta3. One clone (AM-1) expressed constitutively active alphaIIbbeta3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antialphaIIbbeta3 antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of beta3(T562N) was responsible for receptor activation. In fact, T562N also activated alphaVbeta3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated alphaIIbbeta3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of beta3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to alphaIIbbeta3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125(FAK), indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of beta3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.     

A case of tooth autotransplantation after long-term cryopreservation using a programmed freezer with a magnetic field

This case report describes the treatment of a skeletal Class III malocclusion with autotransplantation of a cryopreserved tooth. To gain an esthetic facial profile and good occlusion, extraction of bimaxillary premolars and surgical therapy were chosen. The patient had chronic apical periodontitis on the lower left first molar. Although she did not feel any pain in that region, the tooth was considered to have a poor prognosis. Therefore, we cryopreserved the extracted premolars to prepare for autotransplantation in the lower first molar area because the tooth would probably need to be removed in the future. The teeth were frozen by a programmed freezer with a magnetic field (CAS freezer) that was developed for tissue cryopreservation and were cryopreserved in −150°C deep freezer. After 1.5 years of presurgical orthodontic treatment, bilateral sagittal split ramus osteotomy was performed for mandible setback. Improvement of the facial profile and the occlusion were achieved in the retention phase. Six years after the initial visit, the patient had pain on the lower left first molar, and discharge of pus was observed, so we extracted the lower left first molar and autotransplanted the cryopreserved premolar. Three years later, healthy periodontium was observed at the autotransplanted tooth. This case report suggests that long-term cryopreservation of teeth by a CAS freezer is useful for later autotransplantation, and this can be a viable technique to replace missing teeth.     

Effects of insulin, dexamethasone and glucagon on the production of apolipoprotein A-I in cultured rat hepatocytes

Cultured rat hepatocytes were used to study the effects of hormones on the production of apo A-I. In addition, we compared these effects with the production of albumin. Hepatocytes were isolated from normal adult rat livers and cultured in MEM, as nearly confluent monolayers. In the absence of hormones, apo A-I and albumin accumulated in the culture medium almost linearly for periods up to 24 h. The rates of accumulation of apo A-1 and albumin in the medium were 22 ng/mg cell protein per h and 1.2 μg/mg cell protein per h, respectively. During the incubations the cellular contents of apo A-1 remained constant.

Insulin stimulated the production of albumin at concentrations over 10⁻¹⁰ M, but inhibited the production of apo A-I at concentrations over 10⁻⁸ M. Dexamethasone showed no significant effects on albumin production but stimulated apo A-1 production at concentrations over 10⁻⁶ M. Glucagon inhibited the production of albumin and apo A-I dose-dependently at concentrations over 10⁻¹⁰ M. Thus, the production of albumin and apo A-1 are presumably controlled by different regulatory mechanisms.     

Laparoscopy-assisted and open distal gastrectomies for early gastric cancer at a general hospital in Japan

BACKGROUND/AIMS: At general hospitals in Japan, laparoscopic surgery for early gastric cancer is not yet popular. The benefits and feasibility of this procedure remain to be established. The aim of this study was to evaluate the surgical outcome of laparoscopy-assisted distal gastrectomy (LADG) in comparison with open distal gastrectomy (ODG) in a general hospital. METHODOLOGY: We performed LADG in 20 patients with early gastric cancer between 2000 and 2001. Clinicopathologic data, blood analyses, clinical course and financial cost of treating patients with LADG were compared with 22 patients treated with ODG between 1998 and 1999. RESULTS: All patients were treated successfully by LADG. Neither reduced operative curability nor increased complications were found with this procedure. Although LADG required a significantly longer operation time than ODG, blood loss was lower in LADG than in ODG. The leukocyte count on day 1 and day 3, and serum C-reactive protein levels on day 1 were significantly lower after LADG than after ODG. There was no significant difference between LADG and ODG in the period and volume of analgesics required. High body temperature continued longer after ODG than after LADG. The first walking, passage of flatus and oral diet initiation were significantly earlier in patients with LADG than in those with ODG. LADG required a significantly shorter hospital stay and less total hospital charge than ODG. CONCLUSIONS: Laparoscopy-assisted distal gastrectomy offered faster recovery of gastrointestinal function, a shorter hospital stay, and consequently less financial cost when compared with open surgery. Therefore, LADG may be a safe and recommendable procedure for patients with early gastric cancer at general hospitals in Japan.     

Serum levels and pharmacodynamics of methotrexate and its metabolite 7-hydroxy methotrexate in Japanese patients with rheumatoid arthritis treated with 2-mg capsule of methotrexate three times per week

Methotrexate (MTX) is the first-choice drug for rheumatoid arthritis (RA); however, the pharmacodynamics of MTX in Japanese patients with RA treated legitimately according to the government recommended dosage, 6 mg per week, are unknown. Methotrexate and its metabolite, 7-hydroxy MTX (7-OH MTX), were measured in sera of 16 outpatients with active RA in the first week of MTX treatment and 4–12 weeks after the introduction at 0, 1, 2, 4, and 8 h after administration of the first and the third 2-mg capsule, followed by sampling at 48, 96, and 168 h. The mean maximal serum drug concentration (mean C_max) of MTX attained at 1–2 h after ingestion of the first capsule was 0.215 and 0.252 μM, respectively, in the first and the follow-up week. The mean C_max after ingestion of the third capsule was 0.223 μM and 0.357 μM. The mean C_max of 7-OH MTX was 0.0334 and 0.0289 μM for the first capsule, and 0.0495 and 0.0672 μM for the third capsule. The results indicate that MTX does not accumulate or deposit in the body of Japanese patients with RA when treated with 6 mg per week, and pharmacodynamics of MTX are comparable to those in overseas patients treated with 7.5 mg per week.     

Diagnosis and assessment of monkeypox virus (MPXV) infection by quantitative PCR assay: differentiation of Congo Basin and West African MPXV strains

Human monkeypox, an infectious disease caused by monkeypox virus (MPXV), is endemic to western and central Africa. A LightCycler quantitative PCR (LC-qPCR) system was developed for the diagnosis of this disease, targeting the A-type inclusion body gene (ATI gene) of MPXV. One naive monkey was infected with MPXV Zr-599 (Congo Basin strain) and one with MPXV Liberia (West African strain). Another three monkeys were immunized with smallpox vaccine on 0, 3, or 7 days, respectively, before infection with MPXV Zr-599. Peripheral blood cell (PBC) and throat swab (TS) specimens were serially collected. The LC-qPCR was validated for the diagnosis of monkeypox using virus isolation. Sequencing of the partial ATI gene revealed the insertion of a unique 453-nucleotide residue in the West African strains but not in the Congo Basin strains. Specific reverse primers for Congo Basin and West African strains were designed based on the unique sequence insertion. The LC-qPCR detected the MPXV genome, but not those of the other orthopoxviruses tested nor the varicella-zoster virus. Both the sensitivity and specificity of the LC-qPCR were over 90% in comparison to virus isolation when TS specimens were tested. Fourteen of the 15 virus isolation-positive PBC specimens showed positive reactions in the assay. Further, most PBC specimens collected from symptomatic monkeys in the later stage of illness showed positive reactions in the assay but negative reaction in virus isolation. It was possible to differentiate between these two groups with the LC-qPCR. Thus, the newly developed LC-qPCR is a useful and reliable diagnostic tool for MPXV infection.     

An Immunohistochemical Study of Matrix Components in Early‐Stage Vascular Canals Within Mandibular Condylar Cartilage in Midterm Human Fetuses

Matrix components of vascular canals (VCs) in human fetal mandibular condylar cartilage (15–16 weeks of gestation) were analyzed by immunohistochemistry. Prevascular canals (PVCs), consisting of spindle‐shaped cells without capillary invasion, were observed within the cartilage. Intense immunoreactivity for collagen type I, weak immunoreactivity for aggrecan and tenascin‐C, weak hyaluronan (HA) staining, and abundant argyrophilic fibers in PVCs indicated that they contain noncartilaginous fibrous connective tissues that was different from those in the perichondrium/periosteum. These structural and immunohistochemical features of PVCs are different from those of previously reported cartilage canals of the long bone. Capillaries entered the VCs from the periosteum and ascended through VCs. Following capillary invasion, loose connective tissue had formed in the lower part of VCs, and immunoreactivity for collagen types I and III, tenascin‐C, and HA staining was evident in the matrix of loose connective tissue. No chondroclasts or osteogenic cells were seen at the front of capillary invasion, although small, mononuclear tartrate‐resistant acid phosphatase (TRAP)‐positive cells were present. Meanwhile, TRAP‐positive, multinucleated chondroclasts and flattened, osteoblast‐like cells were observed in the loose connective tissue at the lower part of VCs. These results may indicate slow progress of endochondral ossification in human fetal mandibular condyle. Further, unique matrix components in PVCs/VCs, which were different from those in cartilage canals in long bone, may reflect the difference of speed of endochondral ossification in cartilage canals and human fetal mandibular condyles. Anat Rec, 298:1560–1571, 2015. © 2015 Wiley Periodicals, Inc.