Carbohydrate metabolism during cold-induced sweetening of potato tubers

The aim of this work was to discover the effects of lowering the temperature from 25° to 2° on the metabolism of glucose [U-¹⁴C] by tubers of Solanum tuberosum. Isotope was applied to tubers via a 50-μl hole made with a capillary pipette. Tubers were incubated for 2 hr, the pulse; then the glucose- [U-¹⁴C] was replaced with glucose, and incubation was continued for 18 hr, the chase. The detailed distribution of ¹⁴C was determined at the end of the pulse and at the end of the chase at 2°, and compared with those found at 25°. Lowering the temperature reduced the proportion of metabolized ¹⁴C that entered the respiratory pathways. At 2°, but not at 25°, hexose phosphates were the most heavily labelled fraction after the pulse: during the chase at 2° much of this label was metabolized to sucrose. We conclude that lowering the temperature preferentially restricts glycolysis and diverts hexose phosphates to sucrose. We suggest that this is an important cause of cold-inducing sweetening of the tubers and is due to cold-lability of key glycolytic enzymes.     

Immunogenic properties of hamster tumours of herpesvirus hominis aetiology

Summary Cells derived from HSV-induced tumour lines were attenuated by X-irradiation (15,000 rads) and used to immunize groups of hamsters prior to challenge with homologous tumour cells. The results indicate that the three HSV tumours studied possess a weak transplantation antigen(s). Some cross-immunity between these tumours was observed, although the rejection antigen(s) were distinct from those of a SV40-induced hamster tumour line.Bacillus Calmétte-Guérin (BCG) inoculated in admixture with X-irradiated tumour cells or given 7 days prior to immunization with X-irradiated tumour cells increased host immunocompetence to subsequent tumour cell challenge. Thus, immunization with BCG was shown to induce a higher level of immunity than immunization with attenuated tumour cells alone, as demonstrated on re-challenge of hamsters with homologous tumour cells.     

Hydrogen bonding requirements for the insulin-sensitive sugar transport system of rat adipocytes

(1) The $\text{t}\text{1}\text{2}$ for 1.3 mM D-allose uptake and efflux in insulin-stimulated adipocytes is 1.7 ± 0.1 min. In the absence of insulin mediated uptake of D-allose is virtually eliminated and the uptake rate ($\text{t}\text{1}\text{2}\text{= 75.8 ± 4.99}\text{min}$) is near that calculated for nonmediated transport. The kinetic parameters for D-allose zero-trans uptake in insulin-treated cells are K_zt^oi = 271.3 ± 34.2 mM, V_zt^oi = 1.15 ± 0.12 mM · s^?1. (2) A kinetic analysis of the single-gate transporter (carrier) model interacting with two substrates (or substrate plus inhibitor) is presented. The analysis shows that the heteroexchange rates for two substrates interacting with the transporter are not unique and can be calculated from the kinetic parameters for each sugar acting alone with the transporter. This means that the equations for substrate analogue inhibition of the transport of a low affinity substrate such as D-allose can be simplified. It is shown that for the single gate transporter the K_i for a substrate analogue inhibitor should equal the equilibrium exchange K_m for this analogue. (3) Analogues substituted at C-1 show a fused pyranose ring is accepted by the transporter. 1-Deoxy-D-glucose is transported but has low affinity for the transporter. High affinity can be restored by replacing a fluorine in the β-position at C-1. The K_i for $\text{d}\text{-glucose}\text{= 8.62}\text{mM}$; the K_i for $\text{β-}\text{fluoro-}\text{d}\text{-glucose}\text{= 6.87}\text{mM}$. Replacing the ring oxygen also results in a marked reduction in affinity. The K_i for $\text{5-}\text{thio-}\text{d}\text{-glucose}\text{= 42.1}\text{mM}$. (4) A hydroxyl in the gluco configuration at C-2 is not required as 2-deoxy-d-galactose (K_i = 20.75 mM) has a slightly higher affinity than d-galactose (K_i = 24.49 mM). A hydroxyl in the manno configuration at C-2 interferes with transport as d-talose (K_i = 35.4 mM) has a lower affinity than d-galactose. (5) d-Allose (K_m = 271.3 mM) and 3-deoxy-d-glucose (K_i = 40.31 mM) have low affinity but high affinity is restored by substituting a fluorine in the gluco configuration at C-3. The K_i for $\text{3-}\text{fluoro-}\text{d}\text{-glucose}\text{= 7.97}\text{mM}$. (6) Analogues modified at C-4 and C-6 do not show large losses in affinity. However, 6-deoxy-d-glucose (K_i = 11.08 mM) has lower affinity than d-glucose and 6-deoxy-d-galactose K_i = 33.97 mM) has lower affinity than d-galactose. Fluorine substitution at C-6 of d-galactose restores high affinity. The K_i for $\text{6-}\text{fluoro-}\text{d}\text{-galactose}\text{= 6.67}\text{mM}$. Removal of the C-5 hydroxymethyl group results in a large affinity loss. The K_i$\text{d}\text{-xylose}\text{= 45.5}\text{mM}$. The K_i for $\text{l}\text{-arabinose}\text{= 49.69}\text{mM}$. (7) These results indicate that the important hydrogen bonding positions involved in sugar interaction with the insulin-stimulated adipocytes transporter are the ring oxygen, C-1 and C-3. There may be a weaker hydrogen bond to C-6. Sugar hydroxyls in non-gluco configurations may sterically hinder transport.     

Modification of the cytotoxicity of aflatoxin B1 in rat liver cells by steroids

The addition of steroids with aflatoxin B1 (AFB1) to rat liver cells in culture has been shown to increase the toxin's inhibitory action on growth and protein synthesis. In contrast the inhibition of RNA synthesis by AFB1 was unaffected. The steroid potentiates the direct action of AFB1 at initiation of translation.     

Experimental evaluation of the effective dielectric constant of proteins

Chemical modifications that alter the net charge of residues in reduction-oxidation proteins influence the redox potential of the protein by changing the electrostatic potential at the redox center. If the locations of the modified charges are known, the shift in redox potential may be used to determine the effective dielectric constant for the interactions between the redox center and modified residues. From the shift in redox potential upon charge neutralization of specific lysines in the hemoprotein cytochrome c, an effective dielectric constant of approximately 50 is calculated for the electrostatic interaction between the modified lysines and heme iron in the native protein.     

Chiroptical and stoichiometric evidence of a specific,primary dimerisation process in alginate gelation

The stoichiometry of calcium-ion chelation to alginate chains has been investigated by circular dichroism (c.d.), and by equilibrium dialysis in the presence of various concentrations of sodium chloride. C.d. intensity in the carboxylate π → π * spectral region increases linearly with calcium-ion concentration up to a level equivalent to half the total poly-L-guluronate stoichiometric requirement, and thereafter shows little further change. Similarly, the level of bound calcium resistant to displacement by swamping concentrations of sodium ions is equivalent to half the stoichiometric requirement of poly-L-guluronate chain-sequences alone. In terms of the previously developed “egg-box” model of co-operative junction-zone formation in alginate gelation, these results are interpreted as showing that the primary mechanism of interchain association is by dimerisation of poly-L-guluronate chain-segments in a regular, buckled, two-fold conformation related to that characterized for the free acid in the solid state, with tight interchain chelation of calcium to the carboxylate groups on the interior faces of the dimer (i.e., half the carboxylate residues of the participating chain-sequences). This interpretation is entirely consistent with previous evidence from electron microscopy, and offers a simple rationalisation of experimental results from competitive-ion binding studies.     

Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture

Our object was to characterize the morphological changes occurring in pre- and postsynaptic elements during their initial contact and subsequent maturation into typical synaptic profiles. Neurons from superior cervical ganglia (SCG) of perinatal rats were freed of their supporting cells and established as isolated cells in culture. To these were added explants of embryonic rat thoracic spinal cord to allow interaction between outgrowing cord neurites and the isolated autonomic neurons. Time of initial contact was assessed by light microscopy; at timed intervals thereafter, cultures were fixed for electron microscopy. Upon contact, growth cone filopodia became extensively applied to the SCG neuronal plasmalemma and manifested numerous punctate regions in which the apposing plasma membranes were separated by only 7-10 nm. The Golgi apparatus of the target neuron hypertrophied, and its production of coated vesicles increased. Similar vesicles were seen in continuity with the SCG plasmalemma near the close contact site; their apparent contribution of a region of postsynaptic membrane with undercoating was considered to be the first definitive sign of synapse formation. Tracer work with peroxidase and ferritin confirmed that the traffic of coated vesicles within the neuronal soma is largely from Golgi region to somal surface. Subsequent to the appearance of postsynaptic density, the form and content of the growth cone was altered by the loss of filopodia and the appearance of synaptic vesicles which gradually became clustered opposite the postsynaptic density. As the synapse matured, synaptic vesicles increased in number, cleft width and content increased, presynaptic density appeared, branched membranous reticulum became greatly diminished, and most lysosomal structures disappeared. Coated vesicles continued to be associated with the postsynaptic membrane at all stages of maturation. The incorporation of Golgi-derived vesicles into discrete regions of the cell membrane could provide the mechanism for confining specific characteristics of the neuronal membrane to the synaptic region.     

Linear structure of the oligosaccharide chains in alpha1-protease inhibitor isolated from human plasma.

Two glycopeptides present in equal amounts were isolated from a pronase digest of alpha1-protease inhibitor of human plasma by gel filtration on Sephadex G-50 and chromatography on DEAE-cellulose. The carbohydrate side chains in both glycopeptides are linked through asparaginyl residues. The glycopeptides were digested sequentially with specific glycosidases; and after each step, the released sugars as well as the composition of the residual peptides were determined. The linear structures of these glycopeptides deduced from these data are shown below. Based on the total carbohydrate content of the intact protein and with these structural data, it is postulated that 4 oligosaccharide units are attached to 1 molecule of the protein; 2 of these were represented as in Equation 1, the other 2 as in Equation 2.