Heparin potentiates the action of plasma membrane-associated growth stimulatory activity

Plasma membranes prepared from mouse liver have been previously shown to contain growth stimulatory activity as determined with cultured mouse fibroblasts. This growth stimulatory activity, termed plasma membrane-associated growth stimulatory activity (PMGA), is highly mitogenic in the presence of platelet-poor plasma. We now demonstrate that the growth stimulatory action of PMGA is dramatically enhanced by the addition of heparin. The half-maximal effect of heparin was observed at 1-3 micrograms/ml. The synergistic effect was seen in two distinct assays; the stimulation of DNA synthesis in quiescent cells, and an increase of cell number over a 3-day culture period. Heparin, by itself, does not have any measurable influence on the growth of fibroblasts. The action of heparin is not unique to this glycosaminoglycan, as several other highly sulfated polysaccharides, including dextran sulfate, pentosan polysulfate, and fucoidan, also exhibited the highly synergistic effect. Among other glycosaminoglycans examined, chondroitin sulfate B and heparan sulfate had a small, but significant, effect on enhancing the growth stimulatory action of PMGA. Chondroitin sulfate A, chondroitin sulfate C, hyaluronic acid dextran, and poly-L-glutamic acid, however, had no detectable effect. Further experiments suggested that the effect of heparin is twofold, namely, both a potentiation of growth stimulatory activity and a protection of PMGA activity. The data presented here suggest that the association of various cell surface components, such as PMGA and specific proteoglycans, can modulate the growth potential of a cell.     

A pyrene fluorescence technique and microchamber for measurement of oxygen consumption of single isolated axons

Pyrene fluorescence is quenched by oxygen in an inverse and linear manner related to the partial pressure of O2 in solution. We have developed a microchamber for measuring QO2 of a single isolated axon, monitoring the change in fluorescence of a pyrene probe. The probe consists of a Spectra/Por dialysis hollow fiber filled with 2.5 mM pyrene in paraffin oil. The probe is inserted into a 1-mm-i.d. 2-cm-long quartz capillary tube with a freshly isolated crayfish medial giant axon. The capillary is mounted in an apparatus that forms an air- and water-tight seal except for a 0.2-mm-i.d. stainless steel tube at both ends permitting the exchange of solutions. An Olympus inverted microscope, equipped with epifluorescence optics and a 150-W xenon lamp, is used to view the preparation, generate the excitation light, and monitor the emitted fluorescence with a photomultiplier tube placed in the microscope TV port. A dichroic filter unit is utilized to select an excitation wavelength of 350 nm and collect emitted light above 420 nm. The signal is amplified with a Keithley 480 picoammeter and recorded on a strip chart. QO2 of isolated axons was 552 +/- 70 X 10(-6) mol O2/liter tissue X min. Following sequential treatment with 2 mM ouabain and 2 mM NaCN, QO2 decreased by 22 and 82%, respectively. These data are consistent with QO2 measurements of whole nerve cord made with a Clark electrode O2 monitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carboxypeptidase Y digestion of band 3, the anion transport protein of human erythrocyte membranes

The exposure of the carboxyl-terminal of the Band 3 protein of human erythrocyte membranes in intact cells and membrane preparations to proteolytic digestion was determined. Carboxypeptidase Y digestion of purified Band 3 in the presence of non-ionic detergent released amino acids from the carboxyl-terminal of Band 3. The release of amino acids was very pH dependent, digestion being most extensive at pH 3, with limited digestion at pH 6 or above. The 55,000 dalton carboxyl-terminal fragment of Band 3, generated by mild trypsin digestion of ghost membranes, had the same carboxyl-terminal sequence as intact Band 3, based on carboxypeptidase Y digestion. Treatment of intact cells with trypsin or carboxypeptidase Y did not release any amino acids from the carboxyl-terminal of Band 3. In contrast, carboxypeptidase Y readily digested the carboxyl-terminal of Band 3 in ghosts that were stripped of extrinsic membrane proteins by alkali or high salt. This was shown by a decrease in the molecular weight of a carboxyl-terminal fragment of Band 3 after carboxypeptidase Y digestion of stripped ghost membranes. No such decrease was observed after carboxypeptidase Y treatment of intact cells. In addition, Band 3 purified from carboxypeptidase Y-treated stripped ghost membranes had a different carboxyl-terminal sequence from intact Band 3. Cleavage of the carboxyl-terminal of Band 3 was also observed when non-stripped ghosts or inside-out vesicles were treated with carboxypeptidase Y. However, the digestion was less extensive. These results suggest that the carboxyl-terminal of Band 3 may be protected from digestion by its association with extrinsic membrane proteins. We conclude, therefore, that the carboxyl-terminal of Band 3 is located on the cytoplasmic side of the red cell membrane. Since the amino-terminal of Band 3 is also located on the cytoplasmic side of the erythrocyte membrane, the Band 3 polypeptide crosses the membrane an even number of times. A model for the folding of Band 3 in the erythrocyte membrane is presented.     

Modification of galactose and N-acetylgalactosamine residues by oxidation of C-6 hydroxyls to the aldehydes followed by reductive amination: model systems and antifreeze glycoproteins

Amino acids and peptides have been attached to the C-6 hydroxyls of the galactose and the N-acetylgalactosamine by first oxidizing the C-6 hydroxyls to the aldehydes by galactose oxidase in the presence of small amounts of catalase, followed by reductive amination (-amino group) in the presence of cyanoborohydride. The activity of oxidized antifreeze glycoprotein was >70% of the original, and considerable activity has been retained with some substitutions on reductive amination using cyanoborohydride. The following were some activities retained (as compared with the oxidized antifreeze glycoprotein): Gly, 64; (Gly)₂, 88; (Gly)₃, 82; (Gly)₄, 70; Gly-Gly-NH₂, 44, Gly-Glu, 13; Gly-Leu, 40; Gly-Tyr, 57; Gly-Gly-Leu, 50; Gly-Gly-Phe, 30; and Gly-Gly-Val, 35. On amino acid analysis of acid hydrolysates, some release of the amino acid attached by amination occurred; e.g., Gly-Tyr gave 0.26 Gly and 0.49 Tyr per disaccharide.     

Interaction of (Na + K + 2 Cl) cotransport and the Na/K pump in cultured chick cardiac myocytes

We have recently reported the presence of an electroneutral (Na + K + 2 Cl) cotransport mechanism that is bumetanide-sensitive and maintains Cl_i above its electrochemical equilibrium in cultured chick heart cells. In steady state, (Na + K + 2 Cl) cotransport is inwardly directed and so contributes to the Na influx that must be counterbalanced by the activity of the Na/K pump to maintain Na_i homeostasis. We now show that manipulating (Na + K + 2 Cl) cotransport by restoring Cl_o to a Cl-free solution indirectly influences Na/K pump activity because the bumetanide-sensitive recovery of a _infNa ^supi to its control level and the accompanying hyperpolarization could be blocked by 10^–4M ouabain. In another protocol, when the Na/K pump was reactivated by restoring K_o (from 0.5 mM to 5.4 mM) and removing ouabain, the recovery of a_Na was attenuated by 10^–4M bumetanide. The relatively slow rate of ouabain dissociation coupled with the activation of Na influx by (Na + K + 2 Cl) cotransport clearly establishes the interaction of these transport mechanisms in regulating Na_i. Although (Na + K + 2 Cl) cotransport is electroneutral, secondary consequences of its activity can indirectly affect the electrophysiological properties of cardiac cells.     

Guanine tracts enhance sequence directed DNA bends.

Synthetic DNA fragments were constructed to determine the effect of G tracts, in conjunction with periodically spaced A tracts, on DNA bends. Relative length measurements showed that the G tracts spaced at the half helical turn enhanced the DNA bend. When the G tract was interrupted with a thymine or shortened to one or two guanines, the relative lengths decreased. If the G tract was replaced with either an A tract or a T tract, the bend was cancelled. Replacement with a C tract decreased the relative length to that of a thymine interruption suggesting that bend enhancement due to G tracts requires A tracts on the same strand.     

The resolution of aneuploid DNA stem lines by flow cytometry: limitations imposed by the coefficient of variation and the percentage of aneuploid nuclei

Factors important in the resolution of cell sub-populations with differing DNA contents were investigated using an EPICS C flow cytometer. Software is available for the EPICS C which permits data from any two histograms to be superimposed or added together before display. Samples of fresh and archival thyroid tissue, stained with propidium iodide, were analysed on the flow cytometer and the peak channel number noted. The photomultiplier (PMT) voltage was increased and the sample analysed again producing a second histogram with a higher peak channel number. The two histograms were added together to simulate a cell suspension with two sub-populations with a different DNA content. By systematically altering the PMT voltage and the number of nuclei included in each analysis, it was possible to examine the importance of DNA index and the percentage of tumor cells with an aneuploid DNA content for both fresh and paraffin-embedded thyroid nuclei. The crucial importance of achieving a low coefficient of variation (CV) was demonstrated and consequently the reservations that pertain when archival material is studied, particularly in tumours where DNA aneuploidy is frequently expressed with a low DNA index.     

On human speech,syntax, and language

The evolution of human speech and syntax, which appear to be the defining characteristics of modern human beings, is discussed. Speech depends on the morphology of the mouth, tongue, and larynx which yield the human «vocal tract», and neural mechanisms that facilitate the perception of speech and make possible the control of the articulatory gestures that underly speech. The neural mechanisms that underly human syntax may have derived by means of the Darwinian process of preadaption from the structures of the brain that first evolved to facilitate speech motor control. Recent data consistent with this theory are presented; deficits in the comprehension of syntax of normal aged people are correlated with a slowdown in speech rate.