Lectins in the hemolymph of Japanese horseshoe crab, Tachypleus tridentatus

The hemolymph of the Japanese horsehoe crab, Tachypleus tridentatus contains lectins which agglutinate mammalian erythrocytes. Affinity chromatographic purification of the lectins using bovine submaxillary gland mucin-conjugated Sepharose resulted in the separation of the lectins into four fractions; one major and three minor lectins. Protein subunits revealed by polyacrylamide gel electrophoresis and the immunoprecipitin line of these lectins against antiserum to crude lectins were unique to each fraction. The activities of all the lectins were optimal at pH values between 6 and 8, and were destroyed by heating at 60°C. Calcium chloride augumented the activities of three lectins, but the major lectin was not influenced by the salt. Bovine erythrocytes were not agglutinated by any of the lectins and comparative agglutination titers for other erythrocytes from various sources were different among these lectins. The activities of all the lectins were inhibited by N-acetylamino sugars. They were more effectively inhibited by glycoproteins which contain sialic acid.     

A potential prognostic marker and therapeutic target: SPOCK1 promotes the proliferation,metastasis, and apoptosis of pancreatic ductal adenocarcinoma cells

Pancreatic ductal adenocarcinoma (PDAC), a common malignancy originated from the digestive system worldwide, has a poor clinical outcome. SPOCK1 is a widely investigated member of the Ca²⁺-binding proteoglycan family and functions as an essential driver in several cancers. However, the complex regulatory role of SPOCK1 in PDAC is unclear. Bioinformatics analysis predicted an interrelationship between increased SPOCK1 expression and the clinical characteristics of patients with PDAC. The SPOCK1 expression levels in fresh tissue samples were confirmed, and SPOCK1 expression was then knocked down by lentivirus-mediated short hairpin RNA. Cell proliferation, metastasis, and apoptosis were detected through Cell Counting Kit-8, colony formation assays, invasion and migration assays, flow cytometric analysis, quantitative real-time polymerase chain reaction, and Western blot experiment. On the basis of the Cancer Genome Atlas database, we found a significantly higher level of SPOCK1 in PDAC than in adjacent nontumor tissues. Patients with PDAC with high SPOCK1 expression exhibited shorter overall survival time, as well as disease-free survival time. The knockdown of SPOCK1 significantly decreased the proliferation and metastasis of PCNA-1 and MIA PaCa-2 cells. Moreover, the knockdown of SPOCK1 led to cell cycle arrest in G0/G1 phase and increased the proportion of apoptotic PDAC cells by regulating members of the caspase and Bcl-2 families. Our data proved that SPOCK1 is a critical regulator of tumor proliferation and metastasis in PDAC cells. Therefore, SPOCK1 might be a potential prognostic and therapeutic target molecule in PDAC.     

The leg exocrine system inSerritermes serrifer (Hagen, 1858), phylogenetic implications (Isoptera: Serritermitidae)

Summary The leg exocrine gland was examined in two species of Neotropical termites. Scanning microscopy studies showed a set of pores on the ventral surface of the first and second tarsomeres in all legs ofSerritermes serrifer. InHeterotermes tenuis these pores are present on a sunken plate in all castes. To date, this gland has been observed only in Rhinotermitid species. The presence of leg exocrine gland provides additional evidence supporting a close phylogenetic relationship between the Serritermitidae and Rhinotermitidae.     

Structural and functional characteristics of primary cell cultures derived from the adrenal gland tissue of neonatal mallard ducklings (Anas platyrhynchos)

Summary Primary cell cultures were prepared from the adrenal glands of one-day-old mallard ducklings (Anas platyrhynchos). The cells attached equally well to uncoated plastic and glass surfaces and on surfaces that had been coated with collagen. The phase of logarithmic growth occurred between the second and the fourth day, and the cells became confluent between the fifth and the sixth day. Staining with Sudan black B and toluidine blue and viewing fixed preparations by transmission electron microscopy indicated that the cultures consisted mostly of steroidogenic cells. A smaller population of chromaffin cells was also present. Scanning electron microscopy showed that most of the cells had long filopodia, and some cells had numerous surface blebs that were interpreted as exocytotic vesicles. When incubated in Krebs-Henseleit buffer containing 1–24 ACTH the cultured cells released three corticosteroids, namely, corticosterone, aldosterone and deoxycorticosterone. These responses occurred within 15 min of exposure to medium containing 1–24 ACTH and continued throughout a 60-min period of continuous stimulation. The minimally effective concentration of 1–24 ACTH was 0.078 ng per ml (0.0234 nM) and, as the concentration was increased up to 10 ng per ml (2.99 nM), the total output of each hormone during the 60-min incubation period increased significantly according to the following semi-logarithmic relationship: Y=a+b log X, where Y=the total output of hormone, X=the concentration of 1–24 ACTH in the medium, and a=the total output of hormone when the medium contained 1.0 ng of 1–24 ACTH per ml. The total outputs of each hormone in the presence of a maximally effective concentration of 1–24 ACTH, however, were low compared to the responses of similarly stimulated tissue slices taken from the neonatal duckling. It is concluded that most of the cells comprising the confluent cultures were derived from steroidogenic cells in the neonatal adrenal. These cells appeared to retain corticotropin receptors during the course of developing into confluent monolayers, but their diminished steroidogenic capacity to respond when stimulated maximally suggests that some generational changes may have occurred.This work was supported by grants to James Cronshaw and W.N. Holmes from the University of California Committee on Research and the National Science Foundation (DIR-8820923), Washington, DC, USA     

Unidirectional uptake of l-alanine and l-serine by system ASC in the basolateral membrane of cat salivary epithelium. Selective inhibition of rapid alanine metabolism by aminooxyacetate

Unidirectional influx of l-alanine and l-serine across the basolateral membrane of cat salivary epithelium was investigated in perfused submandibular glands using a rapid (<30 s) paired-tracer dilution technique. (Bustamante, J.C., Mann, G.E. and Yudilevich, D.L. (1981) J. Physiol. 313, 65–79). High uptakes were measured for both neutral amino acids, however, only the uptake of l-alanine was followed by an immediate tracer efflux into the circulation. Aminooxyacetate, an inhibitor of alanine aminotransferase, completely inhibited this efflux and glandular pyruvate production. These results demonstrate that in assessing unidirectional amino acid uptake, the influence of metabolism should be considered even during short intervals of measurement.     

Restricted distribution of laminin α1 chain in normal adult mouse tissues

The distribution of laminin α1 chain in adult mouse tissue was determined by immunofluorescence using monoclonal antibody 200, reacting with the globular carboxyterminus E3 fragment of α1 chain. Strong reactivity was noted only in a few tissues. Reactivity was restricted to epithelial basement membranes. Expression was noted in several epithelial basement membranes of the urinary tract, and male and female reproductive organs. In addition, expression was seen in some parts of the nervous system. Expression was seen in pia mater which surrounds the brain, and in the extracellular matrices covering the vitreous chamber and the lens of the eye. Staining was seen in the adrenal gland cortex, with strongest staining in the zona glomerulosa. Staining was negative in all other studied epithelial basement membranes, such as the lung (trachea or lung epithelium), epidermis, and all parts of the gastrointestinal tract (liver, gut) except for weak staining in the ventricle and Brunner’s glands. No expression was seen in basement membranes of fat, Schwann, or endothelial cells in any studied parts of the body. Both small- and large-size vessel walls were negative both in endothelial basement membranes and blood vessel walls, with the exception of some larger brain blood vessels in locations where epithelial cells have invaginated. Neither smooth muscle, myocardium or striated muscle expressed α1 chain. We conclude that α1-containing heterotrimers including laminin-1 (α1β1γ1) have a very restricted tissue distribution.     

SecB-Mediated Protein Export Need Not Occur via Kinetic Partitioning

In Escherichia coli, the cytosolic chaperone SecB is responsible for the selective entry of a subset of precursor proteins into the Sec pathway. In vitro, SecB binds to a variety of unfolded substrates without apparent sequence specificity, but not native proteins. Selectivity has therefore been suggested to occur by kinetic partitioning of substrates between protein folding and SecB association. Evidence for kinetic partitioning is based on earlier observations that SecB blocks the refolding of the precursor form of maltose-binding protein (preMBP)⁵ and slow-folding maltose-binding protein (MBP) mutants, but not faster-folding mature wild-type MBP. In order to quantitatively validate the kinetic partitioning model, we have independently measured each of the rate constants involved in the interaction of SecB with refolding preMBP (a physiological substrate of SecB) and mature MBP. The measured rate constants correctly predict substrate folding kinetics over a wide range of SecB, MBP, and preMBP concentrations. Analysis of the data reveals that, for many substrates, kinetic partitioning is unlikely to be responsible for SecB-mediated protein export. Instead, the ability of SecB-bound substrates to continue folding while bound to SecB and their ability to interact with other components of the secretory machinery such as SecA may be key opposing determinants that inhibit and promote protein export, respectively.     

Long noncoding RNA DLEU1 aggravates pancreatic ductal adenocarcinoma carcinogenesis via the miR-381/CXCR4 axis

Recent evidence has highlighted that long noncoding RNAs (lncRNA) are associated with many diseases, particularly cancer. However, current understanding of the lncRNA deleted in lymphocytic leukemia 1 (DLEU1) in pancreatic ductal adenocarcinoma (PDAC) remains limited. Our studies indicated that the DLEU1 expression level was upregulated in PDAC tissue samples compared with adjacent normal tissue. Moreover, the aberrant overexpression of DLEU1 indicated poor prognosis of patients with PDAC. Loss-of-function experiments revealed that DLEU1 knockdown inhibited the proliferation, migration, and invasion of PDAC cells in vitro and decreased tumor growth in vivo. Bioinformatics analysis predicted that miR-381 potentially targeted the DLEU1 3′-untranslated region (UTR), suggesting an interaction between miR-381 and DLEU1. Furthermore, miR-381 also targeted the chemokine receptor-4 (CXCR4) messenger RNA 3′-UTR, which was validated by luciferase reporter assay. Taken together, our study demonstrated the oncogenic role of DLEU1 in clinical PDAC specimens and cellular experiments, showing the potential involvement of DLEU1/miR-381/CXCR4 pathway. These results provide novel insight into PDAC tumorigenesis.