Distribution of Calcium and Boron in the Pectin Fraction of Tomato Leaf Cell Wall

Calcium was present in the pectin fraction of tomato leaf cellwall in association with pectin constituents and with a pectin-proteincomplex. Boron deficiency induced a decrease in the amount ofCa associated with pectin constituents. Most of the boron inthe pectin fraction obtained by pectinase treatment was in afree form. These results suggest that boron plays an importantrole in Ca metabolism in the cell wall. ¹Present address: Institute of Applied Biochemistry, Yagi MemorialPark, Mitake, Gifu, Japan. (Received July 31, 1985; Accepted February 18, 1986)     

The laser method for efficient introduction of foreign DNA into cultured cells

A minute hole upon a cultured cell, perforated with a finely focused laser beam, was found to repair itself within a short period of time. The procedure constitutes a new way of introducing exogenous gene materials dissolved in medium into cells. The 'laser-aided' DNA transfection is better than the existing methods because it allows the treatment of a large number of cells in a shorter time, and an improved success rate.     

Morphological analysis of neovascularization at early stages of rat splenic autografts in comparison with tumor angiogenesis

Summary This study was undertaken to reveal the neovascularization at early stages of splenic autografts three-dimensionally, to illustrate the differences between it and tumor angiogenesis, and to establish its origin. Early vascular formation after transplantation of the rat spleen or Waker tumor into the major omentum was examined by using a video macroscope, vascular casting methods and the organ culture technique. A complex vascular network layer (vascular cortex) was first formed beneath the capsule of an autograft; later, vascular buds grew from this network toward the necrotic center. They anastomosed and changed into a form resembling with-ered twigs (vascular medulla). Tumor angiogenesis did not present such morphological features and was characterized by capillary loop formation with a columnar vertex resembling an inverted V. This fundamental structure did not change throughout angiogenesis except for dilation and irregularity of vascular diameter. The organ culture technique demonstrated that the preliminary vasculature was formed in splenic autografts by regeneration of preexisting vessels in the graft and not by invading capillaries. Transmission electron microscopy showed that the cells present had characteristics of sinus endothelial cells. These results suggest that preexisting sinus endothelial cells rearrange themselves after devascularization and reconstruct a new vasculature that an-astomoses with the penetrating capillaries. This mechanism establishes vascular circulation at an early stage, and accelerates regeneration of the splenic autograft before complete necrosis.     

Development of artificial restriction DNA cutter composed of Ce(Iv)/EDTA and PNA

An artificial restriction enzyme, which we developed recently by combining Ce(IV)/EDTA and peptide nucleic acids, was used for PCR-free construction of a fusion protein. The fusion protein was successfully expressed in mammalian cells. This artificial DNA cutter can be also applied to site-selective scission of huge DNAs. Promising features of this novel tool were concretely evidenced.     

Anesthetic effects of a three-drugs mixture —comparison of administrative routes and antagonistic effects of atipamezole in mice—

The anesthetic mixture of medetomidine (MED), midazolam (MID) and butorphanol (BUT) produced anesthetic duration of around 40 minutes (min) in ICR mice. We reported that this anesthetic mixture produced almost the same anesthetic effects in both male and female BALB/c and C57BL/6J strains. Intraperitoneal (IP) administration of drugs has been widely used in mice. However, various injectable routes of the anesthetic mixture may cause different anesthetic effects. First, we examined effects of the anesthetic mixture by subcutaneous (SC) and intravenous (IV) injection compared to IP injection. After injection of the anesthetic mixture, administration of atipamezole (ATI) induced mice recovery from anesthesia. Secondly, we examined how different dosage and optimum injection timing of ATI affected mice recovery from anesthesia. We used an anesthetic score to measure anesthetic duration and a pulse oximeter to monitor vital signs under anesthesia. Usually, drugs from SC injection work more weakly than IP or IV injection. However, we found no significant differences of anesthetic duration among the three different injection routes. Antagonistic effects of ATI (0.3 mg/kg and 1.5 mg/kg) worked equally when administered at 30 min after injection of the anesthetic mixture. Antagonistic effects of ATI (1.5 mg/kg) were stronger than ATI (0.3 mg/kg) at 10 min after injection of the anesthetic mixture. The anesthetic mixture is a useful drug to induce nearly the same anesthetic effects by different injection routes and has an antagonist of ATI which helps mice quickly recover from anesthesia. These results may contribute to the welfare of laboratory animals.     

Hypothermia-induced tyrosine phosphorylation of SIRPα in the brain

Signal regulatory protein α (SIRPα) is a neuronal membrane protein that undergoes tyrosine phosphorylation in the brain of mice in response to forced swim (FS) stress in cold water, and this response is implicated in regulation of depression-like behavior in the FS test. We now show that subjection of mice to the FS in warm (37 °C) water does not induce the tyrosine phosphorylation of SIRPα in the brain. The rectal temperature (T(rec) ) of mice was reduced to 27° to 30 °C by performance of the FS for 10 min in cold water, whereas it was not affected by the same treatment in warm water. The level of tyrosine phosphorylation of SIRPα in the brain was increased by administration of ethanol or picrotoxin, starvation, or cooling after anesthesia, all of which also induced hypothermia. Furthermore, the tyrosine phosphorylation of SIRPα in cultured hippocampal neurons was induced by lowering the temperature of the culture medium. CD47, a ligand of SIRPα, as well as Src family kinases or SH2 domain-containing protein phosphatase 2 (Shp2), might be important for the basal and the hypothermia-induced tyrosine phosphorylation of SIRPα. Hypothermia is therefore likely an important determinant of both the behavioral immobility and tyrosine phosphorylation of SIRPα observed in the FS test.     

ADAP regulates cell cycle progression of T cells via control of cyclin E and Cdk2 expression through two distinct CARMA1-dependent signaling pathways

Adhesion and degranulation-promoting adapter protein (ADAP) is a multifunctional scaffold that regulates T cell receptor-mediated activation of integrins via association with the SKAP55 adapter and the NF-κB pathway through interactions with both the CARMA1 adapter and serine/threonine kinase transforming growth factor β-activated kinase 1 (TAK1). ADAP-deficient T cells exhibit impaired proliferation following T cell receptor stimulation, but the contribution of these distinct functions of ADAP to this defect is not known. We demonstrate that loss of ADAP results in a G₁-S transition block in cell cycle progression following T cell activation due to impaired accumulation of cyclin-dependent kinase 2 (Cdk2) and cyclin E. The CARMA1-binding site in ADAP is critical for mitogen-activated protein (MAP) kinase kinase 7 (MKK7) phosphorylation and recruitment to the protein kinase C θ (PKCθ) signalosome and subsequent c-Jun kinase (JNK)-mediated Cdk2 induction. Cyclin E expression following T cell receptor stimulation of ADAP-deficient T cells is transient and associated with enhanced cyclin E ubiquitination. Both the CARMA1- and TAK1-binding sites in ADAP are critical for restraining cyclin E ubiquitination and turnover independently of ADAP-dependent JNK activation. T cell receptor-mediated proliferation was most dramatically impaired by the loss of ADAP interactions with CARMA1 or TAK1 rather than SKAP55. Thus, ADAP coordinates distinct CARMA1-dependent control of key cell cycle proteins in T cells.     

Ras regulates neuronal polarity via the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway

The establishment of a polarized morphology is an essential event in the differentiation of neurons into a single axon and dendrites. We previously showed that glycogen synthase kinase-3beta (GSK-3beta) is critical for specifying axon/dendrite fate by the regulation of the phosphorylation of collapsin response mediator protein-2 (CRMP-2). Here, we found that the overexpression of the small GTPase Ras induced the formation of multiple axons in cultured hippocampal neurons, whereas the ectopic expression of the dominant negative form of Ras inhibited the formation of axons. Inhibition of phosphatidylinositol-3-kinase (PI3-kinase) or extracellular signal-related kinase (ERK) kinase (MEK) suppressed the Ras-induced formation of multiple axons. The expression of the constitutively active form of PI3-kinase or Akt (also called protein kinase B) induced the formation of multiple axons. The overexpression of Ras prevented the phosphorylation of CRMP-2 by GSK-3beta. Taken together, these results suggest that Ras plays critical roles in establishing neuronal polarity upstream of the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway and mitogen-activated protein kinase cascade.     

Mucinous tubular and spindle cell carcinoma of the kidney: Report of a case with imprint cytologic features

BACKGROUND: Mucinous tubular and spindle cell carcinoma of the kidney is a newly established subtype in the World Health Organization classification. The tumor has a good prognosis, and its diagnosis is clinically important. However, there are no reports of its cytologic features. Here we report the cytologic findings of this rare tumor. CASE: A 68-year-old female had left-sided abdominal pain, fatigue and hematuria. A large mass in the left kidney was removed. Histologically, the tumor was compatible with mucinous tubular and spindle cell carcinoma. Imprint cytologic examination revealed a large amount of tumor cells arranged in tubular, sheetlike and spindle structures. Some tumor cells showed clear cytoplasm. Nuclear atypia was mild, with occasional distinctive nucleoli. CONCLUSION: The cytologic features of mucinous tubular and spindle cell carcinoma have a varied appearance. It should be included in the differential diagnosis to avoid overtreatment.