Long anchor using Flo1 protein enhances reactivity of cell surface-displayed glucoamylase to polymer substrates

We investigated the influence of anchor length on the reactivity to polymer substrate of enzyme displayed on yeast cell surfaces. Using various lengths [42, 102, 146, 318, 428, and 1,326 amino acids (aa)] of the C-terminal region of the Saccharomyces cerevisiae Flo1 protein (Flo1p), which plays a major role in yeast flocculation, six display systems with various anchor lengths were constructed. In these systems, the target protein was displayed on the yeast cell surface under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis ( UPR-ICL). Cell-surface display of Rhizopus oryzae glucoamylase by these systems was induced and confirmed in all systems by immunofluorescence microscopy and immunoblotting. Flow-cytometer measurement of the fluorescence intensity of immunofluorescence-labeled yeast cells displaying glucoamylase indicated that glucoamylase displayed with longer anchors, especially those of 428 and 1,326 aa in length, had higher reactivity to antibodies. The reactivity of starch to displayed glucoamylase, which was evaluated by plate assay, increased with anchor length, as did the cell growth-rate in starch-containing medium. These results indicate that cell-surface display systems using 428- and 1,326-aa length anchors of Flo1p are effective for the display of enzymes on the outer surface of yeast cells.     

New human myelodysplastic cell line,TER-3: G-CSF specific downregulation of Ca2+/calmodulin-dependent protein kinase IV

We have established a new hematopoietic cell line from a patient with myelodysplastic syndrome (MDS), which was refractory anemia with excess blasts (RAEB). This cell line, designated TER-3, depends on several cytokines for long-term survival and growth, and requires interleukin-3 (IL-3) for continuous growth. Cytochemical analysis revealed that TER-3 cells are weakly dianisidine positive and nonspecific esterase positive, but peroxidase negative. The surface marker profile shows that the TER-3 cells are strongly positive for myeloid, lymphoid, and megakaryocytic antigens such as CD15, CD19, and CD61, and negative for some common multilineage antigens such as CD13, CD33, and CD34. Thus, this cell line has a multilineage phenotype, suggesting that the transformation event occurred in multipotent stem cells. Dianisidine- and nonspecific esterase-positive TER-3 cells increase with granulocyte-colony stimulating factor (G-CSF) rather than with IL-3. These results suggest that the cell line is useful for understanding the mechanism underlying G-CSF-associated hematopoietic cell differentiation and activation in the patient with MDS.     

Characterization of berberine transport into Coptis japonica cells and the involvement of ABC protein

Cultured Coptis japonica cells are able to take up berberine, a benzylisoquinoline alkaloid, from the medium and transport it exclusively into the vacuoles. Uptake activity depends on the growth phase of the cultured cells whereas the culture medium had no effect on uptake. Treatment with several inhibitors suggested that berberine uptake depended on the ATP level. Some inhibitors of P-glycoprotein, an ABC transporter involved in multiple drug resistance in cancer cells, strongly inhibited berberine uptake, whereas a specific inhibitor for glutathione biosynthesis and vacuolar ATPase, bafilomycin A1, had little effect. Vanadate-induced ATP trap experiments to detect ABC proteins expressed in C. japonica cells showed that three membrane proteins of between 120 and 150 kDa were photolabelled with 8-azido-[alpha-32P] ATP. Two revealed the same photoaffinity-labelling pattern as P-glycoprotein, and the interaction of these proteins with berberine was also demonstrated. These results suggest that ABC proteins of the MDR-type are involved in the uptake of berberine from the medium.     

Molecular basis for exaggerated sensitivity to mexiletine in the cardiac isoform of the fast Na channel

Cardiac sodium channels have been shown to have a higher sensitivity to local anesthetic agents, such as lidocaine, than the sodium channels of other tissues. To examine if this is also true for mexiletine, we have systematically measured mexiletine sensitivity of the Na channel isoforms, rH1, (mu)1, and rBII, which were transiently expressed in human embryonic kidney (HEK) 293 cells. We confirmed that the cardiac isoform rH1 exhibited the highest sensitivity among the three tested channel isoforms. In rH1, (mu)1, and rBII, the respective IC(50) values were 62, 294, and 308 microM mexiletine, in regard to tonic block, and 18, 54, and 268 microM mexiletine, in relation to use (8 Hz)-dependent block. The relatively high drug sensitivity of rH1 was an invariant finding, irrespective of channel state or whether channels were subjected to infrequent or frequent depolarizing stimuli. Mutating specific amino acids in the skeletal muscle isoform (mu)1 (namely, (mu)1-I433V and (mu)1-S251A) to those of the cardiac isoform at putative binding sites for local anesthetic agents revealed that only one of the point mutations ((mu)1-S251A) has relevance to the high cardiac drug sensitivity, because mexiletine produced significantly more use-dependent and tonic block in (mu)1-S251A than wild-type (mu)1.     

Cold-induced mitochondrial uncoupling and expression of chicken UCP and ANT mRNA in chicken skeletal muscle

Although bird species studied thus far have no distinct brown adipose tissue (BAT) or a related thermogenic tissue, there is now strong evidence that non-shivering mechanisms in birds may play an important role during cold exposure. Recently, increased expression of the duckling homolog of the avian uncoupling protein (avUCP) was demonstrated in cold-acclimated ducklings [Raimbault et al., Biochem. J. 353 (2001) 441-444]. Among the mitochondrial anion carriers, roles for the ATP/ADP antiporter (ANT) as well as UCP variants in thermogenesis are proposed. The present experiments were conducted (i) to examine the effects of cold acclimation on the fatty acid-induced uncoupling of oxidative phosphorylation in skeletal muscle mitochondria and (ii) to clone the cDNA of UCP and ANT homologs from chicken skeletal muscle and study differences compared to controls in expression levels of their mRNAs in the skeletal muscle of cold-acclimated chickens. The results obtained here show that suppression of palmitate-induced uncoupling by carboxyatractylate was greater in the subsarcolemmal skeletal muscle mitochondria from cold-acclimated chickens than that for control birds. An increase in mRNA levels of avANT and, to lesser degree, of avUCP in the skeletal muscle of cold-acclimated chickens was also found. Taken together, the present studies on cold-acclimated chickens suggest that the simultaneous increments in levels of avANT and avUCP mRNA expression may be involved in the regulation of thermogenesis in skeletal muscle.     

The role of SmpB protein in trans-translation

The function of SmpB protein in the trans-translation system was evaluated using the well-defined cell-free translation system consisting of purified ribosome, alanyl-tRNA synthetase and elongation factors. The analysis showed that SmpB protein enhances alanine-accepting activity of tmRNA and that SmpB protein and tmRNA are sufficient to complete the trans-translation process in the presence of translational components. Moreover, SmpB is indispensable in the addition of tag-peptide onto ribosomes by tmRNA. In particular, the A-site binding of tmRNA is inhibited in the absence of SmpB.     

The Xq22 inversion breakpoint interrupted a novel Ras-like GTPase gene in a patient with Duchenne muscular dystrophy and profound mental retardation

A male patient with profound mental retardation, athetosis, nystagmus, and severe congenital hypotonia (Duchenne muscular dystrophy [DMD]) was previously shown to carry a pericentric inversion of the X chromosome, 46,Y,inv(X)(p21.2q22.2). His mother carried this inversion on one X allele. The patient's condition was originally misdiagnosed as cerebral palsy, and only later was it diagnosed as DMD. Because the DMD gene is located at Xp21.2, which is one breakpoint of the inv(X), and because its defects are rarely associated with severe mental retardation, the other clinical features of this patient were deemed likely to be associated with the opposite breakpoint at Xq22. Our precise molecular-cytogenetic characterization of both breakpoints revealed three catastrophic genetic events that had probably influenced neuromuscular and cognitive development: deletion of part of the DMD gene at Xp21.2, duplication of the human proteolipid protein gene (PLP) at Xq22.2, and disruption of a novel gene. The latter sequence, showing a high degree of homology to the Sec4 gene of yeast, encoded a putative small guanine-protein, Ras-like GTPase that we have termed "RLGP." Immunocytochemistry located RLGP at mitochondria. We speculate that disruption of RLGP was responsible for the patient's profound mental retardation.