Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.     

Immunoaffinity fractionation of high-density lipoprotein subclasses 2 and 3 using anti-apolipoprotein A-I and A-II immunosorbent gels

High-density lipoprotein (HDL) subclasses 2 and 3 prepared by density gradient ultracentrifugation have been further fractionated by immunoaffinity chromatography using antibody affinity gels targetting the major HDL apolipoproteins, A-I and A-II. Fractions containing A-I without A-II (AI w/o AII) and A-I with A-II (AI w AII) were isolated from both density ranges. Whereas there were similar concentrations of the major subfraction (HDL3(AI w AII] in both males and females, the remaining subfractions were present in higher concentrations in females as compared to males, in the order HDL3 (AI w/o AII) less than HDL2(AI w AII) less than HDL2(AI w/o AII). The difference was most marked for HDL2 (AI w/o AII), where plasma concentrations in females were almost 3-fold greater than in males. Compositional analyses indicated that the plasma concentrations of the fractions, rather than their compositions, were the major determinants of male-female differences in HDL levels. In contrast, fractions defined by similar apolipoprotein criteria and isolated from different density subclasses (i.e., HDL2(AI w/o AII) vs. HDL3(AI w/o AII) and HDL2(AI w AII) vs. HDL3(AI w AII] showed major compositional differences. This is suggestive of distinct lipoprotein particles.     

New variety and nomenclatural changes in neotropical Gaultheria (ericaceae)

Gaultheria sclerophylla var.hirsuta Luteyn from Ecuador is described, illustrated, and discussed, and the following new combinations are validated:Gaultheria buxifolia var.elassantha (A. C. Smith) Luteyn,G. buxifolia var.secunda (E. J. Remy) Luteyn,G. lanigera var. rufolanata (Sleumer) Luteyn,G. strigosa var.revoluta (A. C. Smith) Luteyn,G. eriophylla var.mucronata (E. J. Remy) Luteyn,G. alnifolia var.grata (A. C. Smith) Luteyn,G. serrata (Vell. Conc.) Sleumer ex Kinoshita-Gouvêa.     

Evidence for chromosomal replicons as units of sister chromatid exchanges

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 m with a peak value around 0.3 to 0.6 m regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments. A concentration of units around 0.4 to 1.0 m was found for L/S SCD segment size distributions regardless of CP treatment with an apparent bimodal profile. Our in vivo data support the existence of a subunit organization of chromosomal replication with a basic functional unit being 0.3 to 0.6 m in size. In addition, we have found that this chromosomal unit of replication or chromosomal replicon does not seem to be functionally perturbed by the mutagen CP. We also found that small SCD segments of 0.4 to 0.7 m in length were involved in the formation of an SCE, suggesting that both spontaneous and CP-induced SCEs occur between chromosomal replicons. These findings provide direct cytogenetic evidence to support a replicon cluster/chromosomal replicon model for SCE formation.     

Type II collagen defects in the chondrodysplasias. I. Spondyloepiphyseal dysplasias.

The spondyloepiphyseal dysplasias (SEDs) and spondyloepimetaphyseal dysplasias (SEMDs) are a heterogeneous group of skeletal dysplasias (dwarfing disorders) characterized by abnormal epiphyses, with and without varying degrees of metaphyseal irregularities, flattened vertebral bodies, and myopia. To better define the underlying cause of these disorders, we have analyzed the collagens from costal cartilage from several of these patients, using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) of intact chains and cyanogen bromide (CNBr) peptides and amino acid analysis. In almost all of the patients in this study group, the type II collagen exhibited a slower electrophoretic mobility when compared with that in normal controls. The mobility of many, but not all, of the CNBr peptides was also retarded. Peptides near the amino terminus were almost always altered, while the mobility of peptides close to the carboxyl terminus were normal in all but the severely affected cases. Analysis of the CNBr peptides on an HPLC sieving column confirmed that the electrophoretically abnormal peptides were of a higher molecular weight than were control peptides. Amino acid analysis indicated that the abnormal collagens have a higher ratio of hydroxylysine to lysine than does control collagen, suggesting that overmodification may be involved in the altered mobility. Our results are consistent with a defect in the collagen helix that results in overmodification of the molecule from that point toward the amino terminus. We propose that some forms of SED and SEMD are associated with abnormalities in type II collagen that results in delayed helix formation and consequent overmodification of the collagen. Cases of SED fit onto a continuous spectrum of clinical severity that correlates positively with both the extent of alteration and the proximity of the defect to the carboxyl terminus.     

Beta-thalassemia mutations in Indonesia and their linkage to beta haplotypes.

A total of 72 chromosomes from 36 Indonesian patients, 23 with beta-thalassemia major and 13 with Hb E-beta-thalassemia, were analyzed by specific oligonucleotide hybridization after DNA amplification. Thirteen had the beta E mutation (codon 26 GAG----AAG). Of the 59-beta-thalassemic chromosomes, 32 were of the variant IVS-1 nt5 (G----C). Seven had the mutation IVS-2 nt654 (C----T), one had the mutation codon 41/42 (deletion CTTT), and one had the mutation codon 17 (AAG----TAG). Another six with the mutation IVS-1 nt1 (G----T), one with the mutation IVS-1 nt1 (G----A), four with the mutation codon 15 (TGG----TAG), one with a mutation codon 30 (AGG----ACG), and one with a mutation codon 35 (deletion C) were first identified by direct sequencing of a patient's genomic DNA followed by further hybridizing other patients' DNA with the appropriate oligonucleotide probes. Five did not carry the common mutations previously described in Asian populations. The four most prevalent mutations encountered made up 83% of the total number of beta-thalassemic chromosomes studied. The most common mutation, IVS-1 nt5 (G----C), was mostly associated with two different haplotypes.     

Alternative mating tactics in the Great Plains toad

Male Great Plains toads, Bufo cognatus, exhibit two mating tactics. At any time, most males give advertisement calls to attract females, while other males, ‘satellites’, remain silent and station themselves near callers in an attempt to intercept females. Females are usually capable of moving through choruses undetected by males. Those females detected by males can avoid being clasped by quickly darting away; but if clasped, can detach the male by inflating. Females initiated amplexus with callers; subsequently calling males mated with 92% of the females and satellites mated with the remaining 8%. Toads employing the satellite tactic associated with males that had longer calls and that were larger, although call duration was the stronger predictor. The percentage of males exhibiting satellite behaviour during a sampling period varied from 0 to 57%. Several hypotheses explaining satellite frequency were explored, including variation in male density, male quality, and operational sex ratio. Of the variables considered, only two aspects of male density (namely, the number of unmated males per m of shoreline and number of calling males per m shoreline) affected satellite frequency. High male density may cause at least some calling males to lose vocal conspicuousness, thereby favouring satellite behaviour. However, male density accounted for only 17% of the observed variation in satellite frequency. This, plus the fact that up to 74% of marked males switched between tactics, suggests that factors such as higher energetic demands of calling may affect satellite frequency.     

Binding of actinomycin D to [d(ATCGAT)]2: NMR evidence of multiple complexes

Actinomycin D (actD) binds to the oligonucleotide [d(ATCGAT)]2 with a hypochromatic and red-shifted visible absorbance band compared to free drug and a CD spectrum with double negative bands at 460 and 385 nm. These spectral features are similar to those of the actD-[d(ATGCAT)]2 complex, while actD-[d(AT)5]2 gives spectra similar to those of free drug. Upon dilution or raising the temperature, the spectral characteristics accompanying complex formation disappear in the actD-[(ATCGAT)]2 sample but remain in the actD-[d(ATGCAT)]2 complex under the same experimental conditions. These results suggest that (a) sequence-specific binding of actD occurs with [d(ATCGAT)]2 but not with [d(AT)5]2, (b) the binding is not as strong as with [d(ATGCAT)]2, and (c) actD binds [d(ATCGAT)]2 with the same mechanism as it binds [d(ATGCAT)]2, i.e., by intercalation. From NMR spectra of the actD-[d(ATCGAT)]2 complex, three types of signals can be detected below 20 degrees C, one major and two minor ones. At higher temperatures, exchange between the two minor ones becomes fast enough that only one type of minor signal was seen. Partial resonance assignments were made by using 2D nuclear Overhauser effect (NOE) and 2D homonuclear Hartmann-Hahn (HOHAHA) experiments. Proton chemical shift changes of the major complex are consistent with actD chromophore ring intercalation between hexamer base pairs. Data from NOE-detected dipolar interactions between actD and [d(ATCGAT)]2 protons were interpreted in terms of a major complex with the actD chromophore ring system intercalated at the CG position and minor complexes with the drug intercalated off center at the GA positions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including alpha-(2----8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis alpha-(2----8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3200 pmol of [3H]TTX-binding sites/mg of protein and a single polypeptide of approximately 285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. We further describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.     

The immunocytochemical localization of superoxide dismutase in the enterocytes of the avian intestine: The effect of vitamin D3

Summary Both light microscopical and electron microscopical immunocytochemical techniques were utilized to localize CuZnsuperoxide dismutase (SOD) in the duodenum of normal, rachitic and vitamin-D₃-replete chicks. This enzyme catalyses the dismutation of the superoxide anion, a toxic free radical generated during the normal aerobic metabolism of most respiring cells. Light microscopy showed no SOD activity associated with the duodenal enterocytes of normal and rachitic chicks. However, in rachitic animals subsequently treated with vitamin D, i.e. vitamin-D-replete chicks, intense immunoreactivity for the enzyme was seen in association with the apical border of the duodenal absorptive cells. Immunostaining for SOD was not seen in goblet cells. With electron microscopy, immunostaining for SOD activity was identified in association with the apical microvilli and, to a lesser degree, with the terminal web, a well as in association with both lysosomes and peroxisomes. From this report it appears that there is a physiological relationship between vitamin D, SOD and the intestinal absorptive cell. However, the precise relationship must await further clarification.