Determinants of Slow Walking Speed in Ambulatory Patients Undergoing Maintenance Hemodialysis

Walking ability is significantly lower in hemodialysis patients compared to healthy people. Decreased walking ability characterized by slow walking speed is associated with adverse clinical events, but determinants of decreased walking speed in hemodialysis patients are unknown. The purpose of this study was to identify factors associated with slow walking speed in ambulatory hemodialysis patients. Subjects were 122 outpatients (64 men, 58 women; mean age, 68 years) undergoing hemodialysis. Clinical characteristics including comorbidities, motor function (strength, flexibility, and balance), and maximum walking speed (MWS) were measured and compared across sex-specific tertiles of MWS. Univariate and multivariate logistic regression analyses were performed to examine whether clinical characteristics and motor function could discriminate between the lowest, middle, and highest tertiles of MWS. Significant and common factors that discriminated the lowest and highest tertiles of MWS from other categories were presence of cardiac disease (lowest: odds ratio [OR] = 3.33, 95% confidence interval [CI] = 1.26–8.83, P<0.05; highest: OR = 2.84, 95% CI = 1.18–6.84, P<0.05), leg strength (OR = 0.62, 95% CI = 0.40–0.95, P<0.05; OR = 0.57, 95% CI = 0.39–0.82, P<0.01), and standing balance (OR = 0.76, 95% CI = 0.63–0.92, P<0.01; OR = 0.81, 95% CI = 0.68–0.97, P<0.05). History of fracture (OR = 3.35, 95% CI = 1.08–10.38; P<0.05) was a significant factor only in the lowest tertile. Cardiac disease, history of fracture, decreased leg strength, and poor standing balance were independently associated with slow walking speed in ambulatory hemodialysis patients. These findings provide useful data for planning effective therapeutic regimens to prevent decreases in walking ability in ambulatory hemodialysis patients.     

Early development of the laboratory-reared flounder,Pleuronichthys cornutus

Fertilized eggs ofPleuronichthys cornutus were obtained by both artificial fertilization and natural spawning of laboratory-reared fish. The present paper describes in detail the early development of the fish and the rearing methods employed to provide basic information for mass production of this species. Eggs and sperm for artificial fertilization were obtained from adult fish caught in the Ariake Sound, Kyushu in November and December of 1984. Their maturation was successfully induced by intermuscular injection of pituitary homogenate of the silver carp,Hypophthalmichthys molitrix. Fertilized eggs were also obtained in 1985 by natural spawning of a broodstock kept in a tank for a year. Hatched larvae were fed successively with rotifers,Artemia nauplii and the harpacticoid copepod,Tigriopus japonicus and reared for 80 days. Ten thousand young fish of about 33 mm TL were obtained in 1984 and 1985 with the survival rate of about 17%. Ten developmental stages were defined on the basis of the morphological characteristics: A) newly hatched to 4 day old larvae, 2.7 to 4.1 mm TL (2.6 to 3.9 mmNL), yolk sac present; B) 4 to 16 day old larvae, 3.8 to 5.9 mm (3.6 to 5.6 mm), yolk resorbed, actively feeding on rotifers; C) 15 to 30 day old larvae, 6.3 to 8.3 mm (6.0 to 7.9 mm), notochord straight, hypural fin ray visible; D) 24 to 40 day old larvae, 6.7 to 9.2 mm (6.4 to 8.8 mm), caudal notochord upturned (45°); E) 28 to 45 day old larvae, 7.9 to 10.8 mm (7.5 to 10.3 mm), caudal notochord upturned (45°–90°); F) 32 to 50 day old larvae, 10.8 to 15.7 mm (8.8 to 12.8 mm BL), eyes symmetrical; G) 35 to 66 day old larvae, 13.4 to 20.0 mm (10.9 to 16.3 mm), eyes asymmetrical, but left eye not visible from the right side; H) 40 to 75 day old larvae, 13.8 to 26.2 mm (11.3 to 21.4 mm), the upper edge of left eye visible over top of the head from the right side; I) 46 to 89 day old larvae, 20.1 to 27.4 mm (16.4 to 22.4mm), left eye on the edge of the head and pupil visible from the right side; and J) juveniles of 51 day old or over, 23.6 mm or more (19.3 mm or more), metamorphosis completed. One to three inflections were found for relative growth of total length, eye diameter, upper jaw length, preanal length, and distance between the base of the pectoral fin and the anus against the notochord length or body length. Two inflections were found for body length (or notochord length)-body weight relationship. Most inflections appeared at the stages of D, F and J, corresponding to the body length of 8, 9–12 and 18–22 mm respectively.     

Light Activation of NADP-Malate Dehydrogenase in Guard Cell Protoplasts from Vicia faba L

Light-induced swelling of guard cell protoplasts (GCP) from Vicia faba was accompanied by increases in content of K⁺ and malate. DCMU inhibited the increase of K⁺ and malate, and consequently swelling.

Effect of light on the activity of selected enzymes that take part in malate formation was studied. When isolated GCP were illuminated, NADP-malate dehydrogenase (NADP-MDH) was activated, and the activity reached a maximum within 5 minutes. The enzyme activity underwent 5- to 6-fold increase in the light. Upon turning off the light, the enzyme was inactivated in 5 minutes NAD-MDH and phosphoenolpyruvate carboxylase (PEPC) were not influenced by light. The rapid light activation of NADP-MDH was inhibited by DCMU, suggesting that the enzyme was activated by reductants from the linear electron transport in chloroplasts. An enzyme localization study by differential centrifugation indicates that NADP-MDH is located in the chloroplasts, NAD-MDH in the cytosol and mitochondria, and PEPC in the cytosol. After light activation, the activity of NADP-MDH in guard cells was 10 times that in mesophyll cells on a chlorophyll basis. The physiological significance of light-dependent activation of NADP-MDH in guard cells is discussed in relation to stomatal movement.

     

Expression of a xylanase gene of Bacillus pumilus in Escherichia coli and Bacillus subtilis

Summary A hybrid plasmid, pOXN29 (10.4 Mdal), coding the xylanase (xynA) and -xylosidase (xynB) genes of Bacillus pumilus IPO was constructed by the ligation of pBR322 and a 7.7 Mdal PstI-fragment of chromosomal DNA as reported in our previous paper (Panbangred et al. 1983). A deletion plasmid of pOXN29, pOXN293 (9.2 Mdal), which contains xynA and xynB, was ligated with pUB110 at an EcoRI site, and used to transform B. subtilis MI111. Two selected clones of B. subtilis as xylanase hyper-producers contained plasmids pOXW11 (4.2 Mdal) and pOXW12 (4.0 Mdal), both consisting of only pUB110, xynA, and its flanking regions, as the result of spontaneous deletion. These B. subtilis clones produced 2.7–3.0 times as much xylanase as B. pumilus. Escherichia coli and B. subtilis clones harbouring the hybrid plasmids synthesized xylanase and -xylosidase constitutively, whereas both enzymes were induced by xylose in B. pumilus.Xylanase synthesized by B. subtilis harbouring pOXW11 or pOXW12 was excreted into the medium like that of B. pumilus IPO, but xylanase synthesized in E. coli harbouring pOXN29, 293 or pOXW1 coding xynA was intracellular. In a previous investigation (Panbangred et al. 1983), xylanase was found to be located in the cytoplasm, not the periplasm nor the membrane fraction in E. coli cells harbouring pOXN29 derivatives. In spite of the abnormal location of xylanase synthesized in E. coli, the signal peptide was processed in the same way as in B. pumilus, with the same molecular weight and the same amino terminal sequences of xylanase prepared from E. coli cells and B. pumilus culture fluid.     

Sulfated proteoglycans synthesized by Neuro 2a neuroblastoma cells: Comparison between cells with and without ganglioside-induced neurites

Mouse neuroblastoma Neuro 2a cells are known to extend neurite-like processes in response to gangliosides added to the culture medium. We compared the structural features of proteoglycans (PG) synthesized by conventional Neuro 2a cells with those of neurite-bearing cells. Two different proteoglycans labeled with [³⁵S]sulfate, namely, chondroitin sulfate proteoglycan (CS-PG) and heparan sulfate proteoglycan (HS-PG), were found both in the cell layer and in the culture medium of the conventional cells. CS-PG isolated from the cell layer had a K_av value of 0.38 on Sepharose CL-6B, and had CS side chains with Mr of 27,000. HS-PG in the cell layer was slightly larger (K_av of 0.33) in terms of hydrodynamic size than CS-PG, and the apparent Mr of the heparan sulfate side chains was 10,000. The structural parameters of CS-PG and HS-PG isolated from the medium were almost identical to those of the PGs in the cell layer. In addition to these PGs, single-chain HS, with an average Mr of 2,500, was observed only in the cell layer and this component was the major sulfated component in the cell layers of both control and ganglioside treated cells. The neurite-bearing cells also synthesized both CS-PG and HS-PG which were very similar in hydrodynamic size to those synthesized by the conventional cells, but the size of HS side chains was greater. Radioactivity, as³⁵S, of each sulfated component from the gangliosideteated culture seemed to be slightly less than that of the corresponding component from the control culture. These findings indicate that the marked morphological change in Neuro 2a cells, induced by gangliosides is not accompanied by major changes in the synthesis of PGs.     

A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene

The mechanisms that control the wound-induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5′-deleted promoters showed that a positive element involved in the response to wounding was located between −307 and −99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from −296 to −283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5′ end to −289 with a disrupted Box 1 was fused to a reporter gene for β-glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2(−289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from −296 to −283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP-1, that bound specifically to the Box 1 sequence was identified. The putative TFHP-1 protein contains a basic region and leucine zipper (bZip) motif and a helix—loop—helix (HLH) motif. The mRNA for TFHP-1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP-1 suppressed the expression of prxC2 (−529)/GUS.     

Expression and Promoter Activity of Genes for Isozymes of Horseradish Peroxidase

The expression and promoter activity of genes for isozymes ofhorseradish peroxidase, namely, prxCla, prxClb, prxC2 and prxC3,were studied. Organ-specific expression of these genes in horseradishplants was examined by Northern blot analysis. The group ofprxCl genes was expressed mostly in stems, while prxC2 and prxC3were expressed to a greater extent in roots. Hardly any expressionof any of the genes was detected in leaves. In transient-expressionassays with tobacco protoplasts, about 500 bp of the 5'-noncodingregions of each of the genes, ligated to the gene for ß-glucuronidase(GUS), exhibited significant promoter activity. In particular,the fragments extending from the initiation codon of the prxC2gene to –529 bp and –1 kbp supported high levelsof GUS activity, which were 4.4 and 11.4 times respectively,the activity observed under control of the 35S promoter fromcauliflower mosaic virus (CaMV). Conserved enhancer sequencesof human genes were found in the 5'-flanking region of prxC2,and deletion of the regions that contained the enhancer sequencesreduced the GUS activity. High levels of GUS activity were observedin transgenic tobacco plants that contained 1 kbp of the 5'flanking region of prxC2 fused to the GUS gene. GUS activitywas diminished when deletion from the 5' end extended as faras the CAAT box. No significant organ-specific expression ofGUS was observed with any such deletion. (Received April 15, 1992; Accepted September 11, 1992)     

Methylation analysis of extracellular polysaccharides from suspension-cultured cells of Nicotiana tabacum

The soluble extracellular polysaccharides (ECP) of suspension-cultured tobacco cells were fractionated by DEAE-Sephadex CC into seven sub-fractions. Su     

Localization of laminin in nephritic glomeruli as revealed by a quick-freezing and deep-etching method with immunohistochemistry

Summary The three-dimensional localization of laminin in rat glomeruli at the chronic phase of Masugi nephritis was investigated by a quick-freezing and deep-etching method combined with immunohistochemistry. Light-microscopically, laminin was localized in increased mesangial matrix and thickened glomerular basement membrane. The quick-freezing and deep-etching method revealed that the increased mesangial matrix, which was newly formed in axial portions and areas of mesangial interposition, was composed of fine fibrillar networks. They were revealed with the 3,3-diaminobenzidine tetrahydrochloride (DAB) reaction products of peroxidase-labelled secondary antibody following anti-laminin antibody. However, these reaction products were not uniformly distributed in the newly formed matrix. Although the fibrils organizing lamina densa were also immunostained with anti-laminin antibody, the fibrils connected to mesangial cells, podocytes and endothelial cells had smaller amounts of DAB reaction products for laminin. These results indicate that one of the components of fibrils in the mesangial matrix and lamina densa is laminin, which is heterogeneously distributed in the newly formed matrix.