Collagen receptors mediate early events in the attachment of epithelial (MDCK) cells

Summary Madin-Darby canine kidney (MDCK) cells kept in suspension culture for 12–15 hr displayed high-affinity binding sites for¹²⁵I-lathyritic (soluble) collagen (120,000/cell,K _D =30nm) and preferred collagens types I and IV over laminin or fibronectin as substrates during the first hour of attachment. On the other hand, after 4 hr, attachment to all four substrates was equally efficient. Upon challenge with a collagen substrate, the high-affinity sites were rapidly recruited on it (T1/2=6 min). Their occupancy by soluble collagen triggered the exocytosis of a second large population of low-affinity collagen binding sites that included laminin and seems to be involved in a second cell-attachment mechanism. These results are compatible with a twostep model of MDCK cell attachment to the substrate: first, via high-affinity collagen binding sites, and second, via laminin of cellular origin.     

Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (K_i) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.     

THE EFFECT OF GROWTH ILLUMINATION ON THE PIGMENTATION OF A MARINE DINOFLAGELLATE12

Amphidinium klebsii cultures grown under different light intensities exhibited similar chlorophyll a content per cell. Among the accessory pigments, chlorophyll c concentration decreased slightly in cells exposed to increasing light intensities up to 0.129 ly/min. The concentration of the 2 major xanthophylls present in A. klebsii cells–peridinin and diadinoxanthin–however, varied according to the light background of the cells. Some biochemical pathways in the formation of peridinin in dinoflagellates are discussed.     

Whole-cell recording of neuroblastoma x glioma cells during downregulation of a major substrate, 80K/MARCKS,of protein kinase C

Summary Differentiated neuroblastoma cells exhibit both the delayed rectifier potassium current (I _K) and the M-current (I _M). The present study was designed to determine the roles of protein kinase C (PKC) and of the calmodulin-binding protein 80K/MARCKS, a prominent substrate for PKC and possible regulator of these currents. Neuroblastoma x glioma (NG108-15) hybrid cells transfected with m1 muscarinic receptors were grown with 1% fetal bovine serum (FBS) without the prostaglandin E₁ (PGE₁) and isobutylmethylxanthine (IBMX) usually added in preparation for electrophysiological studies. Under these conditions, the usual pleomorphism was largely abolished, leaving two populations of small cells with stellate and spherically symmetrical geometries. Whole-cell patch clamping indicated that the two cell types had identical electrophysiological properties, displaying: I _k, a small current through a T-like Ca²⁺ channel, and no M-current.Stimulation with carbachol shifted the distribution of cells to a more stellate morphology within 24 hr and later (after 48 hr) reduced the PKC substrate 80K/MARCKS by 22±7%. In contrast to the stimulation of I _k observed with cardiac cells, PKC activation produced only a small inhibition of I _k, which was independent of carbachol pretreatment. Thus, PKC and 80K/MARCKS can be dissociated from the regulation of I _k in neuroblastoma cells.Supported in part by research grants from the National Institutes of Health (DK-40145 and EY-08343) and from the U.K. Medical Research Council.We thank Dr. Peter J. Parker for his generous gift of PKC, and Yvonne Vallis for her skillful assistance with the cultures and harvesting of the NG108-15 transfected cells.     

Photosynthetic preparation and characterization of 13C-labeled carbohydrates in agmenellum quadruplicatum

The blue-green alga Agmenellum quadruplicatum (strain PR6) has been used to prepare photobiosynthetically ¹³C-labeled d-glucose, 2-O-(α-d-glucopyranosyl)-glyceric acid (glucosylglycerate), 2-hydroxy-1-(hydroxymethyl)ethyl α-d-gluco-pyranoside (glucosylglycerol), and α-d-glueopyranosyl β-d-fructofuranoside (sucrose). When grown to a cell density of 4.4 g.L^-1 (dry weight) under nitrate-nitrogen limiting growth conditions for 120 h, the algal cells contained 38% of the dry-cell weight as(1 → 4)-α-d-glucan (amylose). About 1% of the dry-cell weight was glucosylglycerol, glucosylglycerate, and sucrose. Glutamate was obtained, together with carbohydrates of low molecular weight, when the cells were extracted with chloroform-methanol; d-glucose was recovered from the extracted cells by acid hydrolysis of the starch. The algae were grown by using 20 mol% [¹³C] carbon dioxide for preparation of labeled carbohydrates and for cellular component identification by whole-cell n.m.r. spectroscopy.     

Alcohol:NAD oxidoreductase in brain of rats from a colony fed dilute ethanol for many generations.

—Alcohol:NAD oxidoreductase (EC 1.1.1.1) was studied in brain cortex, hypothalamus, cerebellum and midbrain of adult and immature rats, and in the whole encephalon of neonatal rats. The rats used in this study were (i) from a colony which has been given 12% (v/v) aqueous ethanol as the only fluid for 54 generations (‘E.F.’ rats); (ii) rats removed from this colony after the forty-eighth generation and thereafter fed water instead of the alcohol solution (‘E.F./H₂O’ rats); and (iii) normal rats. Enzyme activity in the 20,000 g supernatant of tissue homogenates was measured by the method of Raskin and Sokoloff. Activity was found to be highest in neonatal rat brain and to decrease as the age increased. Activity in the hypothalamus of adult E.F. rats was significantly higher than that found in the same region of adult E.F./H₂O rats. Immature rat cerebellum alcohol:NAD oxidoreductase activity was higher both in ‘E.F.’ and ‘E.F./H₂O’ suggesting a possible genetic change be involved in this CNS region. It may be concluded that, with the exception of neonatal rats, ethanol consumption induces an increase in rat CNS alcohol :NAD oxidoreductase activity.