Isolation of apical plasma membrane in rabbit gallbladder epithelium by Percoll density gradient centrifugation

The apical membranes of rabbit gallbladder epithelial cells were isolated by treating the homogenate with Ca2+ or Mg2+ and centrifuging the suspension in Percoll gradient. In this way brush-border membranes were obtained with enrichment factors ranging between 10 and 20 and yields of 15-30%. A second method is described with which membranes were isolated, without any preliminary treatment, first by differential centrifugation, then with Percoll gradient; the final membrane enrichment was over 15, however the yield was very low (3%). Many possible enzymatic markers of the apical plasma membrane were investigated: L-gamma-glutamyltransferase, alkaline phosphatase, leucine aminopeptidase, sucrase. The first appears to be that of choice. Apical membrane fraction could be also evidenced by autofluorescence or by labeling with Lotus tetragonolobus lectin. Preliminary experiments showed that apical plasma membranes isolated in this way form vesicles.     

Surface charges of the membrane and cell adhesion substances determine the structural integrity of hair bundles from the inner ear of fish

Summary The hairs (stereocilia = stereovilli) of sensory cells from the inner ear of vertebrates are interconnected by several types of connectors, whose role is unknown. They appear to stabilize the hair bundle mechanically, and may be directly involved in mechano-electric transduction. Our transmission electron-microscopical investigation of sensory epithelia from two species of fish (Rutilus rutilus, Scardinius erythrophthalmus, both Leuciscidae) has shown that not only the connectors but also the surface charges of the membrane are important factors for determining the shape of the hair bundle and the spatial interrelation of the stereovilli. A reduction of the ionic strength in the medium leads to an increase in distance between the stereovilli. This may be the result of an extension of the spread of the surface potential of the membrane at low ionic strength. The connectors are not broken by the increase in distance between the stereovilli. They are EDTA (ethylene-diamine-tetra-acetic-acid) resistant as are some cell adhesion molecules such as N-CAM (nerve-cell adhesion molecule) and protein A from Dictyostelium discoideum. The connectors do not prevent polycation-induced fusion of adjacent stereovillar membranes.     

Observations on the germinability in vitro of pollen from Camellia japonica L. (Theaceae)

Pollen grains from samples of Camellia japonica living in soil (A) and in greenhouse (B) were collected daily from just-opened anthers. The pollen was sown in various liquid media and incubated at 28 C. Higher germinative ability of A in comparison with B was observed and related to the relative humidity which is higher in soil than in greenhouse. This phenomenon recurs even if the composition of the culture medium is changed. However the composition of the medium greatly influences the percentage of germination of both A and B notwithstanding the environmental conditions. The pollen collected from anthers dehiscing in the first day of the anthesis has a high germinative ability which suddenly decreases in the ripe pollen of the following days.     

Trivalent behavior during prophase I in male mice heterozygous for three Robertsonian translocations: an electron-microscopic study

A synaptonemal complex (SC) analysis was carried out in male mice heterozygous (CHT/+) for three Robertsonian translocations. All pachytene preparations studied showed the presence of three trivalents. At early pachytene, the nonhomologous centromeric regions of the acrocentric chromosomes were unpaired. Heterosynapsis subsequently took place with complete pairing of the trivalents. Association between one of the three trivalents and the sex vesicle was observed in 30.4% of the nuclei. Association between the unpaired regions of two trivalents was present in 14.4% of the cells, suggesting that the relationship between unpaired regions of structural rearrangements and the X-Y bivalent may simply reflect the tendency of unpaired regions to establish end-to-end associations or heterosynapses among them, which are usually resolved during the pachytene stage of prophase I. Since the sex bivalent always has unpaired regions, these associations often affect the sex chromosomes.     

The human endonexin II (ENX2) gene is located at 4q28----q32

A relatively recently identified family of structurally similar Ca2(+)-dependent phospholipid binding proteins is called the annexin gene family. At least seven genes are known, although their exact functions are unclear. The endonexin II gene (ENX2), one member of the gene family, is assigned to 4q28----q32 using both Southern transfer analysis of human x rodent somatic cell hybrid DNAs and in situ chromosome hybridization. One of the lipocortin II genes, another annexin, had previously been assigned to the long arm of chromosome 4.     

On the trends in protein molecular evolution: Amino acid composition

Data on the amino acid composition of proteins having various functions from organisms representing different evolutionary levels (83 superfamilies) are used in order to elucidate the trends in protein molecular evolution. The interconnections evolutionary rate (rate of mutation acceptance) — amino acid composition, and evolutionary level of the organism — amino acid composition (in case of proteins of the same or very similar function) are studied. The amino acid compositions of proteins performing jointly an evolutionarily old functions are also juxtaposed. The mean contemporary protein composition is used as a basis for comparison. The obtained results are evidence in favour of the existence of a trend for an increase of the special amino acids (Met, Ile, Gln, His, Lys, Asn, Phe, Tyr, Trp, Cys) at the expense of the usual ones (Thr, Pro, Ala, Ser, Arg, Gly, Leu, Val, Glu, Asp). The tests of statistical significance of the obtained results (comparison of the mean compositions of proteins from low evolutionary level organisms with that of all sequenced proteins; comparison of the mean contemporary protein composition with that obtained after simulation of the evolutionary process) confirm and universalize the observed trend. The above results direct the attention to the concept of a smaller number of amino acids in the ancient proteins and respectively simpler genetic code. A fluctuation around the initial primitive level is suggested to explain the conservatism of proteins of the same function in evolutionarily low level organisms. The observed trend could be applied for designing new proteins.     

Enkephalin regulation of L-valine transport in rabbit ileum

We studied the influence on ionic basal transport (Na+ and Cl-) and L-valine transport of two enkephalins which are not metabolized and act in delta and mu receptors respectively. Transports have been indirectly determined measuring the transepithelial electric potential and the short circuit current. DADLE does not significantly influence ion and amino-acid transport, while DAGO alters both of them in the presence of the myenteric plexus (muscle layers present) or inhibits only L-valine transport in the absence of the plexus (muscle layers removed).     

Hydrocortisone regulates arylsulfatase A (cerebroside-3-sulfate-3-sulfohydrolase) by decreasing the quantity of the enzyme in cultures of cells dissociated from embryonic mouse cerebra

Previous work from our laboratory (Biochem. J. 219:689–697 (1984) had shown that hydrocortisone stimulated the net accumulation of the myelin-specific sulfolipid in cultures of cells dissociated from embryonic mouse cerebra. This accumulation caused by hydrocortisone was shown to be due to a decrease of sulfolipid degradation by arylsulfatase A (ASA) and not due to a stimulation of its synthesis by a sulfotransferase. Both ASA activity and the turnover of sulfolipid were decreased by hydrocortisone to 60–62% of untreated cells. In current work the same decrease in enzyme activity was obtained and enzyme linked immunosorbent assays demonstrate that hydrocortisone decreased the number of ASA protein molecules to 61% of untreated cells [(-)hydrocorcortisone 0.31±0.06 ng ASA/g protein; (+)hydrocortisone: 0.18±0.04 ng ASA/g protein]. This decrease in the number of ASA molecules correlates well with the decrease in both the enzyme activity and the sulfolipid turnover, which suggests that the major mode of inhibition of ASA activity by hydrocortisone involves a decrease in the concentration of ASA in the cells rather than some other mechanism of inhibition.The material in this paper has been included in a dissertation submitted by A.J.M. in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Temple University.     

The cytotaxonomy ofEmilia spp. (Asteraceae: Senecioneae) occurring in Brazil

Analysis of several populations in a large part of the distribution area of the genusEmilia in Brazil has revealed only two species: the diploidE. sonchifolia and the tetraploidE. fosbergii. The more widely reportedE. coccinea was not found. They show a karyotype constancy in morphology and chromosome number (2n = 10 and 2n = 20, respectively), C-banding pattern and number of secondary constrictions. Some indications were found thatE. fosbergii may be an allopolyploid and that its ancestors had different genome sizes.     

The control of root, vegetative shoot and flower morphogenesis in tobacco thin cell-layer explants (TCLs)

Thin cell-layer explants (TCLs) have been proposed as favorable tissues for the study of root, vegetative shoot and flower formation. We tested the effects of pH, light quality, light quantity, and IBA and kinetin concentrations on the morphogenesis of TCLs cultured individually on a liquid medium. Alterations of the amounts of exogenously supplied IBA and kinetin were sufficient to induce the formation of roots, vegetative shoots and flowers on TCLs cultured on otherwise identical media. The type and number of organs formed were sensitive to the intensity of light (55, 75, 100 and 120 muEinsteins m-2 sec-1) under which TCLs were grown. Evidence was obtained that the effects of light on TCL morphogenesis were associated with photochemical degradation of IBA in the medium. Evaluation of the organogenesis that occurred in TCLs cultured on a medium containing a range of IBA and kinetin concentrations showed that the number and type of organs formed, and overall growth, were dependent upon the initial concentrations of auxin and cytokinin. We have developed the TCL culture system into a sensitive and reproducible bioassay for the study of morphogenesis. The advantages of using the TCL morphogenesis bioassay for the identification and study of molecules (e.g. cell wall oligosaccharides) that may regulate morphogenesis are discussed.