Janos M. Varga

Gangliosides of normal and neoplastic human melanocytes

The major ganglioside component isolated from diploid human melanocytes is sialosyllactosylceramide (GM3 86-91% of total sialic acid). The corresponding disialo derivative (GD3) is found as a minor component (2-6% of total sialic acid) in the membranes of these cells. In human melanoma cells, grown in tissue culture, GD3 is the predominant ganglioside component (48-63% of total sialic acid). Withdrawal of TPA from the culture medium of normal melanocytes or addition of TPA to the medium of melanoma cells had no significant effect on GM3/GD3 ratios. We conclude that the difference between the composition of gangliosides is related to the normal vs transformed phenotypes of melanocytes.

The fate of Staphylococcal exfoliatin in newborn and adult mice.

The distribution and excretion of the staphylococcal exfoliatin was investigated following in vivo administration of highly purified 125I‐labelled exfoliatin fractions to adult and newborn mice. Adult mice excrete approximately one‐third of a test dose by 3 hours as compared to a fifteenth of a test dose excreted by newborn mice. Accordingly, blood tracer radioactivity reaches a relatively higher peak and shows a slower decline in newborns than in adults. The urine of adult mice contains considerable biologically active exfoliating material. Both nephrectomized and carbon tetrachloride‐poisoned adult mice injected with exfoliatin develop generalized exfoliation whereas comparable doses in untreated controls have no effect. On the other hand, subtotal hepatectomy, followed by injection of exfoliatin, does not lead to exfoliation. We conclude that renal immaturity is a critical factor responsible for the susceptibility of neonates to generalized staphylococcal scalded skin syndrome.

Inhibition of transplantable melanoma tumor development in mice by prophylactic administration of Ca-ascorbate

Hemicalcium ascorbate (Ca-Asc, 51 mM, 1% wt/vol), added to the drinking water, had the following effects in DBA/2 mice inoculated with 10(5) S91 (Cloudman) melanoma cells: 1) it delayed the appearance of visible tumors by 2-4 weeks; 2) it increased the survival rate at three months after tumor challenge by 12-50%; 3) it had no significant effect on the rate of tumor growth once the size of the tumors had reached 10 mm3; 4) the inhibition was maximal when the treatment with Ca-Asc was started at least one week prior to the inoculation of cells 5) when free ascorbic acid was used instead of Ca-Asc, the animals consumed 50% less water, they became dehydrated and the treatment was less effective; 6) Ca++ (51 mM) alone had no significant inhibitory effect.--Since Ca Asc (1 mM) was not toxic to S91 melanoma cells in vitro, we suggest that prophylactic treatment of the animals with Ca-Asc inhibited tumor development by increasing the resistance of the host.

Quantitative amino acid compositions at the picomole level using fluorescence scanning

Abstract Simple techniques are described for quantitating dansyl amino acids by direct fluoresence scanning and by photo-copying and densitometry. This technique is accurate between 1.0 to 10 × 10 −12 moles with a lower limit of detection around 1 × 10 −14 moles. It is possible to obtain amino acid compositions on very small quantities of peptides and it seems likely that this method will be useful for automatic peptide sequenators which use subtractive dansyl-Edman degradation as a detection procedure.

Synthesis of a hormonally active conjugate of α-MSH, ferritin, and fluorescein

A conjugate composed of α-MSH, ferritin, and fluorescein has been synthesized. The conjugate is biologically active: It is one-tenth as active as free α-MSH in darkening frog skin and one-third as active as free α-MSH in stimulating tyrosinase activity in cultivated Cloudman melanoma cells. Binding of the conjugate to cultivated Cloudman melanoma cells was specific; binding was inhibited by at least 80% with either 5 × 10−5m α- or β-MSH. An average affinity constant of 1.3 × 109 liters/mol with approximately 1 to 4 × 104 sites/cell was calculated. The half-life of the conjugate-receptor complex is 58 min. The binding of the conjugate to cultivated Cloudman melanoma cells can be visualized by fluorescence microscopy. A patchy, perinuclear distribution of the conjugate, similar to that of FITC-β-MSH, was observed. Electron microscopic examination revealed the conjugate to be at the cell surface.

Structural assembly of the signaling competent ERK2–RSK1 heterodimeric protein kinase complex

Significance Signaling pathways often use kinase cascades, but structural characterization of catalytic complexes between heterodimeric kinase pairs has been elusive. For MAPK–MAPKAPK binary complexes, a high-affinity “docking” interaction holds kinase domains proximal within a tethered complex. This heterodimer provided a unique opportunity to shed light on kinase domain–domain contacts that play a role in the assembly of the transient catalytic complex. Starting out from a new precatalytic extracellular signal regulated kinase 2–ribosomal S6 kinase 1 (ERK2–RSK1) crystallographic complex, where the activation loop of the downstream kinase (RSK1) faced the enzymes (ERK2) catalytic site, we used molecular dynamics simulation to show how the catalytic ERK2–RSK1 complex forms. Our findings reveal the dynamic process through which transient, physiologically relevant kinase heterodimers form in a prototypical kinase cascade. Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase–kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzymes (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 “docking” groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they “readjust,” whereas generic kinase domain surface contacts bring them into a catalytically competent state.

ANP(1-28), BNP(1-32) and CNP(1-22) increase the severity of picrotoxin-kindled seizure syndrome in rats.

The administration of rANP(1-28) in doses of 1.0 and 2.0 nmol (but not 0.2 nmol) into the lateral cerebral ventricle (i.c.v) of rats preliminarily kindled with picrotoxin resulted in an increase of the severity of picrotoxin-kindled convulsions 24 hrs after injection of the peptide. I.c.v. injection of pBNP(1-32) also resulted in a proepileptic effect when it was applied in the same doses with a similar time course; the increased seizure severity was observed 48 hrs after injection of pBNP in a dose of 2 nmol. I.c.v. administration of CNP(1-22) in a dose of 2 nmol induced an increase in the severity of picrotoxin-kindled convulsions 24 and 48 hrs after application of the peptide. None of the peptides influenced the seizure syndrome immediately after the injections. It is presumed that the delayed proepileptic properties of the three natriuretic peptides could be caused by some of their stable fragments which accumulate during their metabolism.

Expression of idiotype 315(Id315) and I-A antigens in MOPC-315 tumor cells grown in vivo.

Abstract MOPC-315 murine myeloma grown intraperitoneally as ascites cells in BALB/cJ mice were removed at successive days after transplantation. They were stained for Id 315 and I-A markers by indirect immunofluorescence techniques by means of FITC conjugates. Flow cytometry (FCM) measurements on cell surface markers were correlated with the phase of the cell cycle by quantitating cellular DNA of cells stained with propidium iodide. Variations in cell size due to cell growth were determined by low-angle light scattering. FCM data on the two cell surface markers were normalized for unit cell surface and cell volume. Cells grew rapidly in the early days (5–7) of tumor growth. No significant variation in the expression of surface markers was observed during this period. Parallel with a slowdown in cell growth, the expression of Id 315 increased about threefold between the 7th and 9th days. The increase in the Id 315 marker was dependent on the cell cycle, with G 1 cells having the highest density. No cell cycle dependence was observed for the I-A marker.

Antibody Combining Regions

An immune serum usually consists of a very heterogeneous population of immunoglobulins (Ig’s) the appearance of which in the serum has been induced by antigen. The antigen-ligating function of the Ig molecule (see Figure 1) is confined to the combining regions, which are two symmetrical areas at the solvent-exposed ends of the Fab arms of the Y-shaped Ig molecules. The combining region is situated in the variable (V-region) domain, a compact region consisting of the N-terminal half of the light (L) chain and the N-terminal quarter of the heavy (H) chain that is linked by sulfhydryl bonds. Between the areas of this domain occupied by the L- and H-chain V regions is a cleft exposed to the solvent. Antigens have been shown to bind in, or close to, this cleft (Amzel et al., 1974). An induced antibody population is said to be specific because it usually binds most strongly to the immunizing antigen and with lesser binding energies to certain compounds that resemble the immunogen in structure. Heteroclitic antibodies may be induced that bind more strongly to some determinant other than the immunogen (Makela, 1965). In general, antibody populations show a high degree of specificity, in that they are able to discriminate among chemical compounds differing by as little as a single functional group, between stereoisomers, or between two proteins differing by as little as a single amino acid residue (Reichlin, 1974).