Burkitt's lymphoma - a human tumor model system for immunological studies.

Burkitt's lymphoma occurs mainly in parts of tropical Africa and has attracted the attention of experimental workers due to its epidemiological and clinical features, which indicate a viral etiology and a host immune response to the tumor. As a result of virological studies, Epstein-Barr virus (EBV) DNA has been demonstrated in almost all tested biopsies of African BL. This contrasts to the absence of EBV in all, or almost all, of the non-African Burkitt's lymphoma-like tumors, even though the number of tested tumors in this group is small, and to the lack of EBV in all other types of lymphoma or leukemia. Immunological studies have revealed the presence of antibodies to different EBV-associated antigens in all African patients with Burkitt's lymphoma. However the antibodies are not specific for Burkitt's lymphoma but are found in most adults all over the world, although at lower levels. They cannot therefore serve diagnostic purposes, but they can give prognostic information and occasionally give clues to the mechanisms behind late tumor recurrences, and possibly guide so-called immunotherapy. Burkitt's lymphoma patients contrast to appropriate control groups where some of the persons are anti-EBV seronegative, and this, together with the presence of EBV in Burkitt's lymphoma biopsies and the absence of EBV in other lymphomas, even though the cell type involved may be infectable by EBV in vitro and the tumor may arise in an EBV-carrying person, favors an etiological role in EBV in Burkitt's lymphoma and speaks against the "passenger" hypothesis, according to which EBV is picked up by the Burkitt's lymphoma cell which happens to be particularly suitable for EBV persistence. To explain the geographical distribution, a cofactor, such as certain forms of malaria, has been implied.


Apoproteins of human serum high density lipoproteins. Isolation and characterization of the peptides of Sephadex fraction V from normal subjects and patients with abeta-lipoproteinemia.

1. Sephadex fraction V, obtained from human serum high density lipoprotein apoprotein (HDL apoprotein) of normal subjects and of patients with abetalipoproteinemia, was resolved by DEAE-cellulose ion exchange column chromatography into several fractions which were defined in terms of amino acid composition, NH2- and COOH-terminsls, sialic acid content, immunologic and electrophoretic properties, and in vitro activation of purified lipoprotein lipase from rat adipose tissue. 2. Fraction V of HDL apoprotein of both normal and abetalipoproteinemic subjects was found to contain polypeptides corresponding to apolipoproteins C-I, C-II, C-III-1, and C-III-2, which had been described previously in very low-density lipoproteins (VLDL). The content of apo C-III-1 in abetalipoproteinemia-HDL was very low, whereas the percentage, by weight, of apo C-I was about twice as high as that in the normal subjects studied. Furthermore, both normal and abetalipoproteinemia-HDL apoprotein contained a previously unreported peptide which had a molecular weight of about 7 000 and electrophoretic, chemical, and immunological properties distinct from those of the known C apolipoproteins. Of all of the peptides comprising fraction V, only apo C-II activated a purified preparation of rat adipose tissue lipoprotein lipase. This was the case for both normal and abetalipoproteinemic subjects.


Isolation and characterization of the major apolipoprotein from chicken high density lipoproteins.

High density lipoproteins were isolated from plasma of white Leghorn hens by ultracentrifugal flotation between densities 1.063 and 1.210 g/ml. After delipidation, the lipid-free proteins were fractionated by chromatography on Sephadex G-150 in urea; one major apolipoprotein was isolated and characterized. From its chemical, physical and immunochemical properties, the major apoprotein from hen high-density lipoproteins has characteristics similar to the major apoprotein of human high density lipoproteins, apoA-I. Thus the hen protein has been designated hen apoA-I. Hen apoA-I has a molecular weight of approximately 28 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Its calculated molecular weight from its 234 constituent amino acids is 26 674. Hen apoA-I differed from its human counterpart by containing isoleucine. Treatment of hen apoA-I with carboxypeptidase A yielded a COOH-terminal sequence of Leu-Val-Ala-Gln. Automatic Edman degradation of the apoprotein gave an NH2-terminal sequence of Asp-Glu-Pro-Gln-Pro-Glu-Leu. Hen apoA-I had a circular dichroic spectrum typical of alpha-helical structures; the calculated helicity was 90%. Goat antisera prepared to hen apoA-I formed precipitin lines of complete identity to the hen apoprotein but lines of only partial identity to human apoA-I. These studies show that the major apoprotein from hen and human high-density lipoproteins have similar properties to each other suggesting a common physiologic function.


Rabbit alveolar macrophage collagenase: evidence of polymeric forms.

Collagenase harvested in vitro from rabbit alveolar macrophages eluted in gel chromatography corresponding to apparent molecular weights of 45 000, 85 000, and 165 000. Reversible changes from one molecular weight to another in low salt concentration and predominance of the 45 000 species in salt concentrations above 1.0 M (NaCl, KCl) suggest that the higher molecular weights represent polymeric forms of collagenase.


Change in ATP-pyrophosphohydrolase activity during spherule formation of Physarum polycephalum.

The activity of Ca2+-dependent ATP pyrophosphohydrolase was found to fluctuate during spherule formation of the acellular slime mold Physarum polycephalum under starving incubation. The enzyme activity increased up to 16-fold at the 3rd day of the starvation, then decreased drastically to less than its original level. Column chromatography of the enzyme preparation suggested that the increase in the activity was due to de novo synthesis of a new isozyme. Cycloheximide inhibited the synthesis. The two isozymes were different in their Ca2+ sensitivity, the new one being less sensitive.


Effect of prostaglandin E on the adenyl cyclase-cyclic AMP system and gluconeogenesis in rat renal cortical slices.

Prostaglandin E was found to increase the formation of cyclic acdenosine 3',5'-monophosphate (cyclic AMP) by renal cortical slices. This increased release of cyclic AMP was not influenced by the absence of Ca2+ in the incubating media. The enhanced production of cyclic AMP was probably mediated by stimulation of membrane-bound adenylate cyclase activity. An increase in adenyl cyclase activity was observed with increasing concentrations of prostaglandin E. Furthermore, prostaglandin E augmented glucose production from alpha-ketoglutarate. This effect on gluconeogenesis was abolished by the removal of Ca2+ from the incubating medium. These effects are similar to those described for parathyroid hormone and suggest that the renal cortex is a prostaglandin-dependent system. Prostaglandin E decreased cyclic AMP production and glucose production (from alpha-ketoglutarate) in response to submaximal doses of parathyroid hormone, suggesting that prostaglandin may be important in modulating the intracelluar action of parathyroid hormone in the kidney cortex.


Localization of cell groups sensitive to parathyroid hormone and calcitonin in rat skeletal tissue.

The level of cyclic AMP in various fractions of rat skeletal tissue was measured after in vitro or in vivo administration of parathyroid hormone and calcitonin. Incubations of bone fractions prepared from young (5 weeks of age thyroparathyroidectomized rats revealed that both parathyroid hormone and calcitonin increased the cyclic AMP level in fractions of epiphysis, metaphysis and marrow cells. Cyclic AMP accumulation in incubated perisoteum and diaphysis were induced solely by parathyroid hormone. In in vivo experiments the cyclic AMP level in the tibia of the thyroparathyroidectomized rat was increased by infusion of either parathyroid hormone or calcitonin, and the simultaneous administration of each maximally effective dose of the two hormones exhibited an additive effect. Within 2 min, parathyroid hormone infusion caused an elevation of cyclic AMP content in periosteum and metaphysis. Rapid increase of cyclic AMP in the metaphysis was also induced by calcitonin, and the effect of the two hormones on cyclic AMP accumulation in this fraction was additive. Small but significant increase of cyclic AMP in the diaphysis was detected at 5 min after the administration of parathyroid hormone. Calcitonin infusion did not show any consistent effects on periosteum and diaphysis.


Dietary and hormonal regulation of some enzyme activities associated with gluconeogenesis in rabbit liver.

1. Starvation increases the activity of cytosolic P-enolpyruvate carboxkinase in rabbit liver some 4-5 fold but does not alter the activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase or glucose-6-phosphatase.2. Alloxan-induced diabetes increases the activities of cytosolic P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase approx. 6-, 2- and 2-fold, respectively. Again the activity of mitochondrial P-enolpyruvate carboxykinase is not altered. 3. Administration of mannoheptulose rapidly increases blood glucose levels and also causes a significant increase in cytosolic P-enolpyruvate carboyxkinase activity within 4 h. The activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase are not affected. 4. Administration of hydrocortisone also increases blood glucose levels and the activities of cytosolic P-enolpyruvate carboxykinase and glucose-6-phosphatase are significantly increased within 12h. Again, mitochondrial P-enolpyruvate carboxykinase and fructose-1,6-diphosphatase activities remain unaffected. 5. The observations that (A) the activity of cytosolic P-enolpyruvate carboxykinase responds to more situations conducive to gluconeogenesis than do the activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase, and (B) cytosolic P-enolpyruvate carboxykinase activity is rapidly adaptive under appropriate circumstances, suggests that this particular enzyme's activity plays an important role in the regulation of gluconeogenesis in rabbits.


Regulation by insulin of gluconeogenesis in isolated rat hepatocytes.

Insulin (10nM) completely suppressed the stimulation of gluconeogenesis from 2 mM lactate by low concentrations of glucagon (less than or equal to 0.1 nM) or cyclic AMP (less than or equal to 10 muM), but it had no effect on the basal rate of gluconeogenesis in hepatocyctes from fed rats. The effectiveness of insulin diminished as the concentration of these agonists increased, but insulin was able to suppress by 40% the stimulation by a maximally effective concentration of epinephrine (1 muM). The response to glucagon, epinephrine, or insulin was not dependent upon protein synthesis as cycloheximide did not alter their effects. Insulin also suppressed the stimulation by isoproterenol of cyclic GMP. These data are the first demonstration of insulin antagonism to the stimulation of gluconeogenesis by catecholamines. Insulin reduced cyclic AMP levels which had been elevated by low concentrations of glucagon or by 1 muM epinephrine. This supports the hypothesis that the action of insulin to inhibit gluconeogenesis is mediated by the lowering of cyclic AMP levels. However, evidence is presented which indicates that insulin is able to suppress the stimulation of gluconeogenesis by glucagon or epinephrine under conditions where either the agonists or insulin had no measurable effect on cyclic AMP levels. Insulin reduced the glucagon stimulation of gluconeogenesis whether or not extracellular Ca2+ were present, even though insulin only lowered cyclic AMP levels in their presence. Insulin also reduced the stimulation by epinephrine plus propranolol where no significant changes in cyclic AMP were observed without or with insulin. In addition, insulin suppressed gluconeogenesis in cells that had been preincubated with epinephrine for 20 min, even though the cyclic AMP levels had returned to near basal values and were unaffected by insulin. Thus insulin may not need to lower cyclic AMP levels in order to suppress gluconeogenesis.


Effect of adenosine 3',5'-cyclic monophosphate derivatives on alpha-amylase release, protein kinase and cyclic nucleotide phosphodiesterase activity from rat parotid tissue.

Several 8-substituted derivatives of cyclic AMP were tested for their effects on alpha-amylase release. None of the 8-substituted compounds were more active than N6,O2-dibutyryl- or N6-monobutyryl adenosine 3',5'-monophosphate in causing alpha-amylase release. The rat parotid was found to contain a high (105 muM) and a low (1.15 muM) Km cyclic AMP phosphodiesterase activity. All of the 8-substituted cyclic AMP compounds inhibited the hydrolysis of 1 muM cyclic AMP. However, there was only a partial correlation between the ability to cause alpha-amylase release and inhibit cyclic AMP hydrolysis. Extracts of parotid tissue contained a cyclic AMP-dependent protein kinase activity. None of the compounds were as effective as cyclic AMP in activating the protein kinase. As in the case of inhibition of cyclic AMP hydrolysis, the ability of the 8-substituted cyclic AMP compounds to increase protein kinase activity did not correlate with their effects on alpha-amylase release. It is concluded that factors in addition to the in vitro inhibition of cyclic AMP hydrolysis and activation of protein kinase are important in determining the net result of the 8-substituted cyclic AMP compounds on parotid gland function. These additional factors might include differences in the rate of uptake and differences in rats of conversion to compounds with modified activity.


An EPR study of myeloperoxidase in human granulocytes.

1. EPR spectra of human granulocytes (4 - 10(8) cells per ml) show an intense high-spin ferric heme signal with rhombic symmetry (gx = 6.90 and gy = 5.07) for the heme group. These g-values are identical to those of partially purified myeloperoxidase and thus the signal is derived from ferric myeloperoxidase. In chicken granulocytes, which contain little or no myeloperoxidase, only an axial type of heme iron signal, weak in intensity, can be detected at g = 6.0. 2. Upon phagocytosis of latex particles by human granulocytes the high-spin heme signal with rhombic symmetry is slowly converted into a signal with axial symmetry (gx = gy = 6.0), showing that the EPR signals of myeloperoxidase in the intact cell can be used to study the involvement of the enzyme in metabolic changes during phagocytosis.


Cyclic AMP formation and morphology of myocardial cells isolated from adult heart: effect of Ca2+ and Mg2+.

Adult rat heart cells were isolated by perfusion with a calcium-free phosphate buffer containing collagenase. Optimal conditions gave a high proportion of elongated cells. Isoprenaline increased cydic AMP content linearly, with ED50 (dose effective in 50% of the population) about 10(-7) M. Ca2+ made the cells spherical, and it nearly abolished cyclic AMP response as did lack of Mg2+.


Nervonic acid biosynthesis by erucyl-CoA elongation in normal and quaking mouse brain microsomes. Elongation of other unsaturated fatty acyl-CoAs (mono and poly-unsaturated).

Biosynthesis of nervonic acid by enzymatic elongation of erucyl-CoA has been studied in mouse brain microsomes. The substrate and cofactor requirements have been measured. Malonyl-CoA and reduced nicotine-adenine-dinucleotide phosphate are required, but not FMN, FAD or NADH. The effect of protein concentration, incubation time, ATP and CoA has been determined; the reaction products were checked by gas-liquid chromatography with automatic counting of the eluate. Very little activity was found in hydroxylated fatty acids. In the presence of phosphotransacetylase (which impedes the de novo microsomal system), the main reaction product was nervonic acid. It is concluded that nervonic acid is biosynthesised by elongation using a two-carbon unit from malonyl-CoA. The same enzyme biosynthesises saturated and mono-unsaturated very long chain fatty acids. The elongation capacity of "quaking" microsomes is reduced to 30% of the normal value with both erucyl-CoA and behenyl-CoA. Elongation of trans isomer (brassidyl-CoA) and poly-unsaturated homologue (clupanodonyl-CoA) was compared to elongation of erucyl-CoA in both normal and mutant mice. Both unsaturated acyl-CoAs are elongated under the same conditions as erucyl-CoA in brain: the poly-unsaturated acyl-CoA is elongated more actively than the mono-unsaturated acyl-CoA in the mutant.


Characterization of the Ehrlich ascites tumor plasma lipoproteins.

1. The lipoproteins of the Ehrlich ascites tumor plasma were separated into 3 distinct fractions, very low density, low density and high density lipoproteins by preparative ultracentrifugation combined with agarose column chromatography. 2. High density lipoproteins contained 74% of the total protein in the lipoproteins. By contrast, most of the lipids were present in the very low density lipoprotein fraction. 3. The fatty acid compositions of the cholesteryl esters were appreciably different in the very low, low and high density lipoproteins, whereas phospholipid and triacylglycerol fatty acid compositions were quite similar in the 3 lipoprotein fractions. 4. Very low and high density apoprotein electrophoretic patterns on sodium dodecyl sulfate-acrylamide gels were similar to those observed in the corresponding lipoprotein fractions obtained from other mammalian species. The low density fraction, however, contained 7 apoprotein bands, and 32% of the low density apoprotein was soluble in tetramethyl urea. 5. The average molecular weights as determined by analytical ultracentrifugation were 2-10(7) (very low density), 6-10(6) (low density) and 4.4-10(5) (high density).


High performance preparative column chromatography of lipids using a new porous silica, Iatrobeads. I. Separation of molecular species of sphingoglycolipids.

Good preparative separation of neutral glycolipids of human erythrocytes was achieved by column chromatography using the totally porous silica spheres, Iatrobeads. The solvent flow through the Iatrobeads column was rapid under atmospheric pressure, and so elution bands migrated with minimal diffusion in the column. Ceramide dihexoside and ceramide trihexoside were both separated into two fractions of different molecular species on the Iatrobeads column. Two ceramide tetrasaccharides, globoside I and paragloboside, were also clearly separated on the Iatrobeads column. Chemical analysis showed that separation of these molecular species was due to differences in their fatty acid and long chain base compositions. Sphingadienine and phytosphingosine were found as minor components of long chain bases in human erythrocyte glycolipids.


Characterisation of the small molecular weight apolipoproteins from pig plasma very low density lipoprotein.

The small molecular weight apolipoproteins of pig very low density lipoprotein were investigated following their separation by gel filtration and ion exchange chromatography. Gel filtration through Sephadex G-200 in 6 M urea, produced essentially the same elution profile to that obtained after filtration of human very low density apolipoprotein. However, separation of the pig Sephadex fraction corresponding to human C proteins on DEAE-cellulose columns revealed the presence of only one major peptide and minor quantities of several others. Some properties of three apparent homogeneous fractions and one heterogeneous DEAE fraction were investigated. Unlike human apoprotein CII apoprotein, none of the pig peptides studied activated cow's milk lipase and sialic acid was not detected in any of the three purified C peptides of pig VLDL. The amino acid compositions of the pig peptides were different to those reported for human C apoproteins. The carboxy terminal residue of the major pig C peptide was shown to be serine. The differences so far revealed between pig and human C peptides need further investigation especially since this animal is regarded as a suitable model for investigating human lipoprotein metabolism and the development of atherosclerosis.


Random cleavage of superhelical SV 40 DNA S1 nuclease.

SV40 DNA FO I is randomly cleaved by S1 nuclease both at moderate (50 mM) and higher salt concentrations (250 mM NaC1). Full length linear S1 cleavage products of SV40 DNA when digested with various restriction endonucleases revealed fragments that were electrophoretically indistinguishable from the products found after digestion of superhelical SV40 DNA FO I with the corresponding enzyme. Concordingly, when the linear S1 generated duplexes were melted and renatured, circular duplexes were formed in addition to complex larger structures. This indicated that cleavage must have occurred at different sites. The double-strand-cleaving activity present in S1 nuclease preparations requires circular DNA as a substrate, as linear SV40 DNA is not cleaved. With regard to these properties S1 nuclease resembles some of the complex type I restriction nucleases from Escherichia coli which also cleave SV40 DNA only once, and, completely at random.


The fragments from the 28 S ribosomal RNA of Galleria mellonella with unesterified uridine at the 3'-termini.

The three 3'-ends of the 28 S galleria RNA are all unesterified uridines. The two 18-S products due to the primary nick of the 28 S RNA have a similar 3'-terminal dinucleotide: G-Uoh. All of the seven species of secondary products from the Galleria 28 S RNA were suggested to have unesterified uridines, in common, at the 3'-ends. These results raise the possibility that the enzyme concerned in generating the primary and secondary nicks is a uracil-specific and 5'-phosphate-forming phosphodiesterase. The esterase, in association with the higher structure of the rRNA molecule in the larger subunit of the ribosome, probably determines the sites for these nicks in the 28 S rRNA. It is proposed also that the same enzyme can be responsible for cleaving the 28-S and 5.8-S rRNAs from their immediate precursor molecules.


G1 specific increases in cyclic AMP levels and protein kinase activity in Chinese hamster ovary cells.

Chinese hamster ovary cells were synchronized by selective detachment of cells in mitosis. The adenosine 3':5'-cyclic monophosphate (cyclic AMP) intracellular concentrations and cyclic AMP-dependent protein kinase activities were measured as these cells traversed G1 phase and entered S phase. Protein kinase activity, assayed in the presence or absence of saturating exogenous cyclic AMP in the reaction mixture, was lowest in early G1 phase (2 h after mitosis), increased 2-fold (plus exogenous cyclic AMP in reaction mixture) or 3.5-fold (minus cyclic AMP in reaction mixture) to maximum values in mid to late G1 phase (4-5 h after mitosis), and then decreased as cells entered S phase. Intracellular cyclic AMP concentrations were minimal 1 h after mitosis, increased 5-fold to maximum levels at 4-6 after mitosis, and decreased as cells entered S phase. Similar to the fluctuations in intracellular cyclic AMP, the cyclic AMP-dependent protein kinase activity ratio increased more than 40% in late G1 or early S phase. Puromycin (either 10 mug/ml or 50 mug/ml) administered 1 h after mitosis inhibited cyclic AMP-dependent protein kinase activity up to 50% by 5 h after mitosis, while similar treatment (10 mug/ml) had no effect on the increase in cyclic AMP formation. These data demonstrate that: (1) total protein kinase activity changed during G1 phase and this increase was dependent on new protein synthesis; (2) the increased intracellular concentrations of cyclic AMP were not dependent on new protein synthesis; and (3) the activation of cyclic AMP-dependent protein kinase was temporally coordinated with increased intracellular concentration of cycli AMP as Chinese hamster ovary cells traversed G1 phase and entered S phase. These results suggest that cyclic AMP acts during G1 phase to regulate the activation of cyclic AMP-dependent protein kinase.


Membrane studies of Streptococcus pyogenes and its L-form growing in hypertonic and physiologically isotonic media. An electron spin resonance spectroscopy approach.

Electron spin resonance spectroscopy (ESR) was used to compare the lipid organization, thermal stability and the physical state of the membrane of a human pathogen, Streptococcus pyogenes and its osmotically fragile L-form with this same L-form now adapted to grow under physiologically isotonic conditions (physiological L-form). Comparison of the hyperfine splittings of a derivative of 5-ketostearic acid spin label, I(12, 3), after incorporation into the membrane, revealed that the lipid chain rigidity of these membranes is in the order physiological L-form greater than osmotically fragile L-form greater than streptococcus. The signal intensity (of the center magnetic field line) versus temperature analysis showed two transitions for these membranes. The first with melting points of 45, 26 and 36 degrees C and second transition at 70, 63 and 60 degrees C for the physiological L-form, osmotically fragile L-form and streptococcal membranes, respectively. This same order of membrane lipid chain rigidity was seen from the cooperativities obtained for each of these systems from analysis based on the expression for an n-order reaction. The I(12, 3) and other probes with the paramagnetic group close to the methyl end of the molecule suggested that this difference in lipid chain rigidity between these organisms resides in the environment closer to the lipid head group region rather than in the hydrophobic lipid core. Another major finding was the binding of I(12, 3) at two or more different sites in each of the membranes examined. This change in lipid chain rigidity now provides an explanation to account for the survival of a previously osmotically fragile L-form in physiologically isotonic media by focusing on changes in the physical nature of its membrane. In so doing, it adds to and reinforces the speculation of the potential survival in vivo and involvement in pathogenesis of osmotically fragile aberrant forms of bacteria.


[EPR spectra of silkworm eggs].

ESR spectra of the native grain of the silkworm have been studied in the course of embryo formation, during a diapause, and during embryo development after the diapause. It is shown that the nature of ESR spectra of the grain is not determined by the metabolic processes, but by the presence of pigments in it and other stationary biological structures having developed pi-systems and unpaired electrones. The latter are mainly found in the envelope and may give the ESR spectra, with various g-factors. A dependence of the ESR spectra integral intensity of the grain on the denotype is discovered.


Parameters in the construction of an immobilized dual enzyme catalyst.

The glucose oxidase and catalase activities immobilized to the gamma-aminopropyltriethoxysilane derivative of nickel-impregnated silica alumina was controlled by several factors. The most important of these was enzyme concentration. In constructing the dual immobilized enzyme catalyst, competition between the two enzymes for available binding sites was observed. The order of addition of the various reactants during immobilization was also important. Higher glucose oxidase activities were immobilized when glutaraldehyde was added concurrently with enzyme, while maximal coupling of catalase occurred if glutaraldehyde was first added to react with the amino derivative of the silica alumina support, excess reagent washed away, and then the catalase added. Bovine serum albumin, which aids in the crosslinking of glucose oxidase, hindered the coupling of the enzyme to the support particles.


[Effect of lyophilization on the free-radical state of animal cells].

The process of lyophylisation carried out very carefully under strictly defined conditions does not in inself bring about the changes in the parameters of the ESR signal with g approximately equal to 2.00 observed in animal cells. The free radicals responsible for the ESR signal observed under usual experimental conditions, result from a contact of the lyophylised specimen with moisture at an optimum water content of about 10%.


Electrolytic regeneration of the reduced from the oxidized form of immobilized NAD.

A covalently bound adduct of nicotinamide adenine dinucleotide (NAD) with alginic acid has been found to be enzymatically active and to undergo electrochemical oxidation or reduction without significant loss of its enzymatic activity. The preparation of the adduct itself (from NAD+, alginic acid, and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate) is also accomplished with substantially complete retention of enzymatic activity. This adduct has been converted from the oxidized to the reduced form by controlled potential electrolysis using mercury and stainless-steel electrodes. This electrolytically produced NADH complex could be oxidized again to the enzymatically active NAD+ complex by enzymatic reaction with the proton acceptor, 2,6-dichlorophenol indophenol, as catalyzed by diaphorase. Using this electrolytic method with immobilized NAD, it is now possible to carry out redox reactions in which NADH is enzymatically oxidized to NAD+, with the simultaneous electrolytic regeneration of the reduced form, NADH, from the oxidized form, NAD+, produced in the enzymatic reaction.


Pilot plant production of glucose with glucoamylase immobilized to porous silica.

Glucoamylase was immobilized to porous silica and its kinetics and stability were observed with acid- and alpha-amylase-hydrolyzed dextrin as feed. The enzyme was found to be extremely stable in both laboratory and pilot plant operations. When the feed had been previously only lightly hydrolyzed, pore diffusion limitation caused appreciable decreases in glucose production rate. The severity of starch hydrolysis to dextrin markedly affected ultimate glucose yields. The diffusional gradients present in the carrier pores caused the immobilized enzyme to yield lower glucose concentrations than the free enzyme at similar feed conditions.


[The effect of extracardiac nerves on the intensity of glycogen metabolism in cardiac muscle].

The left vagus was cut in rats on the neck; this was followed by a reduction of the glycogen content in the myocardium and by an increase in the intensity of its metabolism (by the glucose-I-C14 incorporation). Sympathetic denervation of the heart led to the increase in the glycogen level and a fall of the intensity of its metabolism in the cardiac muscle.


[Changes in the sensitivity of developing sea urchin embryos to prostaglandin F2alpha and cyclic 3',5'-adenosine monophosphate].

It was shown that at various stages of the first mitotic cycles the developing ova of Strongylocentrotus intermedius displayed a different sensitivity to toxic concentrations of prostaglandin F2alpha and to cyclic 3',5'-adenosinmonophosphate (cAMP). The differences in the sensitivity to each of these substances were regular and similar in character and depended on the time lapse after the completion of division next in turn. There were revealed two periods of increased sensitivity of the ova--the first one--during the 20th minute after the completion of each division--to both substances, and the second one--during the 40th minute--to cAMP, and during the 50th minute--to prostaglandin F2alpha.


[Ultrastructural study of a Masson's "humid meningioma"].

The authors report the ultrastructural analysis of a Masson's humic meningioma. The tumor presents at the electronic microscopic examination every sign of secretive hyperactivity and does not show any degenerative characteristics. The existence of an important extracellular material rich in pre-collagenic fibres accounts for lacunar texture of this neoplasia at optic microscopic investigation; the role played by tumoral cells in the elaboration of the material is taken into account and seems to corroborate histogenic ecto-mesenchymatous theory of arachnoidal cells.


Prosthetic restoration in congenital lower limb deficiency. A case study.

We presented a synopsis of a young girl with congenitally deficient limb who underwent seven corrective surgical procedures and fabrication of a specially designed and constructed prosthesis. The surgical procedures are detailed as in the total rehabilitation approach. We have provided plastic laminated orthoses and prostheses to many patients with congenitally deficient upper and lower limbs. An additional paper will detail our experience with a series of plastic laminated orthoses and prostheses in patients with congentally deficient upper and lower extremities.


[Epidemiology in the study of the role of viruses in human cancers].

An epidemiological approach is very useful in the study of the role of viruses in the development of human tumors, if properly integrated with the experimental approach and laboratory studies. Certain epidemiological characteristics of human tumours favour a viral etiology, e.g. space and time clustering, vertical and horizontal transmission of a tumour risk, succession in space or in time of viral induced benign lesions and of malignant tumours. In the framework of the association between the EB herpesvirus and both Burkitt's lymphoma and nasopharyngeal carcinoma, we conducted an epidemiological survey of the EBV infection in populations at different risk for EBV associated diseases. This survey showed that, in Uganda, the EBV infection was much earlier and heavier than in Hong Kong or Singapore. A hypothesis that early infection is related to BL risk is discussed as well as the value of epidemiology in both prospective studies and in prevention of virally induced tumours. The IARC prospective sero-epidemiological study on Burkitt's lymphoma and EBV in Uganda is given as an exemplar.


The influence of motor nerve tetanization on the effect of organophosphorus cholinesterase inhibitors on neuromuscular transmission.

1. The changes in the activity of synaptic cholinesterases (ChE) of the ant. tibial muscle in the anaesthetized cat were detected by recording the changes of the blocking activity of acetylcholine (ACh) and comparing them with the blocking activity of carbaminoylcholine (carbachol) injected intra-arterially or intravenously. After the administration of organophosphorus inhibitors (OPI) of ChE the ACh blocking dose diminished 500 to 2000-fold but the carbachol blocking dose did not change. In 4-6 h after the injection of OPI the ACh blocking dose increased again 8 to 15-fold, but the dose of carbachol still remained unchanged. The transmission of high frequency impulses improved after OPI in parallel with the decrease of the ACh blocking activity. Thus the synaptic ChE is partly restored in a few hours after its irreversible inhibition with OPI. 2. Tetanization of the motor nerve (50-60 Hz, 10 min), started simultaneously with the intravenous injection of OPI (armine Gd-42), diminished the impairment of neuromuscular transmission. On the side of tetanization the ACh blocking action was less pronounced and the transmission of high frequency impulses better than on the control side. Thus the tetanization produced some protection of synaptic ChE against inhibition by OPI. The protective effect of tetanization was absent when the tetanization was performed before the injection of OPI or was started 10-20 min after the injection of OPI. 3. The protective effect of tetanization was also observed on the isolated phrenic nerve diaphragm preparation of the rat. 4. The possible mechanisms of the protective effect of tetanization are discussed.


[DNA form of the genome of oncornaviruses].

The oncornaviruses share several biological, biochemical, and structural properties that distinguish them from other RNA viruses. Rous sarcoma virus, a representative of avian oncornaviruses, manifests two major functions: cell transformation and virus replication. Both functions are independent, and are expressed separately in defective viruses. The virus has a 60-70S RNA, composed of 35 S subunits. It appears that each subunit of viral RNA carries the same genetic information and, furthermore, that 10% of this information is coding for cell transformation. Without this 10% the virus can still replicate. The RNA genome is expressed when the virus penetrates a sensitive cell, provided the virus carries an active reverse transcriptase. Virus-transformed cells possess a DNA form of the viral genome. The viral DNA is infectious, and can lead, upon transfection of permissive cells, to the production of a new viral progeny. We have established that the minimum molecular weight of the viral DNA is 6 X 10(6) daltons, and that this DNA is covalently bound to the chromosomal DNA. Also by examining the frequency of transfection events in the DNA-treated cells we suggest that transfection is a single-hit phenomenon. It follows that viral DNA of 6 X 10(6) daltons carries all the genetic information to code for the virus production. It seems likely that the transfection mechanism giving rise to transforming viruses is different from that producing nontransforming segments. In general terms, the experimental data indicate that certain RNAs and DNAs could accomplish malignant transformation if they are able to penetrate into the cell and give rise to the DNA forms which could be integrated into the cellular genome.


The effect of edrophonium on erythrocyte acetylcholinesterase and neuromuscular function in the rat.

1. The relation between the concentration of edrophonium in plasma, inhibition of red cell acetylcholinesterase, and neuromuscular transmission was studied in the rat. 2. In both in vivo and in vitro conditions, red cell acetylcholinesterase activity was predictably related to the concentration of the quaternary amine. 3. After low doses of edrophonium (1.0 mumol/kg) there was a significant correlation between the monophasic potentiation of twitch tension and the plasma concentration of the drug. In contrast, with higher doses of edrophonium (4.0 or 10.0 mumol/kg) a biphasic potentiation of twitch tension was observed; this was only correlated with the plasma concentration of the drug during the secondary decline in neuromuscular facilitation. Subsequent recovery of normal neuromuscular transmission invariably occurred at a constant plasma concentration of edrophonium.


[Host-leukemogenic virus relation: genetic control of the appearance of leukemia].

A complex genetic system controls the susceptibility to virus induced leukemia. A good example is provided by AKR mice in which several genes acting in association are involved. The control is demonstrable at several levels: a) virus expression, b) virus replication, c) antitumor immune response. In addition one or two other genes could be involved. The "Leukemia-permissive" genotype would be probably rarely present and the AKR provides the model of a mice in which all genetic locus bear the permissive alleles.


Hypothalamo-pituitary adrenocortical function in the rat after treatment with betamethasone.

1 Hypothalamo-pituitary-adrenocortical (HPA) activity was suppressed in rats treated with betamethasone. 2 Recovery of normal HPA function occurred after corticosteroid withdrawl. 3 Although corticotrophin release was rapidly restored to its basal rate there was a delay in the return of the normal adrenocorticotrophic response to stress and normal adrenocortical function was evident only after the plasma adrenocorticotrophic hormone had reached 'supra-normal' levels. 4 The physiological significance and possible clinical relevance of the results are discussed.


A genetic model of narcolepsy.

An analysis of recently published family history data on rapid eye movement (REM) narcolepsy was undertaken to determine the goodness-of-fit of a multifactorial (MF) model of inheritance. The analysis revealed that a two-threshold MF model can successfully account for the prevalence of REM narcolepsy and other disorders of excessive sleep (DES) observed in the first-degree relatives of the REM narcoleptic probands studied.


Oculodento-digital dysplasia: a rare syndrome.

A rare syndrome that can be diagnosed radiographically has been reviewed. A characteristic physiognomy, variable ophthalmologic anomalies and relatively specific dental and digital defects provide the diagnostic features. No hereditary component was identified and the chromosomal pattern was normal. The history, appearance and characteristic radiographic changes may, as in this case, allow the radiologist to provide a diagnosis not previously appreciated.


Sterol synthesis by myelinating cultures of mouse spinal cord.

We studied the control of sterol synthesis during myelination of central nervous system in culture. Explants of fetal mouse spinal cord were cultured for 3-30 days in vitro (DIV). Myelination was visible by electron micriscopy at 3 DIV, and by bright-field light microscopy beginning at 6 DIV. All explants were heavily myelinated by 15 DIV. Specific activity of 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNP) increased rapidly until 15 DIV. The rate of incorporation of [1-14C]acetate into sterol was greatest at 12 DIV, more than double that at 4 and 22 DIV. Specific activity of the mevalonate synthesizing enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) was greatest at 11 DIV, threefold greater than at 6 and 30 DIV. Previous studies in fibroblasts and other non-neural tissues had shown that deprivation of exogenous lipid caused a rapid increase in HMG CoA reductase activity. In contrast, when the spinal cord explants were incubated for 24 h in a lipid-deficient medium, there was either a marked decrease in specific activity of HMG CoA reductase (at 6, 15 and 30 DIV), or no change in enzyme activity (at 11 DIV).


Hippocampal theta rhythm. II. Depth profiles in the freely moving rabbit.

Depth profiles of hippocampal theta rhythm were investigated in the freely moving rabbit during three behavioral conditions: REM sleep, voluntary movement, and during sensory stimulation applied to the motionless animal. Profiles were found to be the same in all three conditions. Dorsoventral microelectrode penetration of the dorsal hippocampus revealed an approximately uniform amplitude of theta rhythm in strata oriens and pyramidal of CA1. Further microelectrode advancement revealed a sharp reversal of phase and a coincident null in amplitude in the proximal stratum radiatum. There was also a peak of theta rhythm amplitude which occurred in the molecular layer of the dorsal blade of the dentate gyrus. These data imply that in the rabbit, as in the freely moving rat, there are two generators of theta rhythm in the dorsal hippocampus, one in the dentate gyrus and the other in the overlying CA1 layer. The data also indicate the existence of a species difference in generating systems between the rabbit and the rat.


Glucocorticoid binding to receptor-like proteins in rat brain and pituitary: ontogenetic and experimentally induced changes.

Cytosol binding of [3H]corticosterone and [3H]dexamethasone was measured in various brain areas and the pituitary of perfused rats 12 h and 72 h, respectively, after adrenalectomy (ADX). A considerable regional heterogeneity was found 12 h post ADX representing differences of the normal cytosol binding capacities between various areas. When the second phase of the adrenalectomy-induced increase in binding capacity was allowed to develop (72 h post ADX), the cytosol binding of all regions increased to various extents. The highest percentage increases were found in those areas with the highest glucocorticoid binding capacity, namely the hippocampus and the septum. The ontogeny of the cytosol glucocorticoid binding macromolecules was investigated in the hippocampus, hypothalamus and pituitary using [3H]corticosterone and [3H]dexamethasone. The concentration of corticosterone binding sites is lowest in all three areas around day one and then increases by a factor of 2-3 reaching adult levels around day 32. For [3H]dexamethasone a similar pattern was observed in the hippocampus and hypothalamus. In the pituitary, however, the concentration of binding sites was slightly higher at day 1 than at any later developmental stage. Interruption of the fimbria in 3-day-old rats did not affect the development of the binding sites in the hippocampus. In an attempt to interfere with the normal glucocorticoid binding of the hippocampus as well as with the postadrenalectomy increase of the cytosol binding sites, bilateral transection of the fimbria was performed either 3 days or 80 days before ADX. In neither case did fimbria transection prevent the increase of the binding sites. The intrinsic (12 h post ADX) cytosol binding capacity of the hippocampus was also not affected by this lesion.


Interactions in vivo and in vitro of corticoids and progesterone with cell nuclei and soluble macromolecules from rat brain regions and pituitary.

Adrenalectomized-ovariectomized (ADX-OVX) rats were given tail vein infusions of [3H]corticosterone, dexamethasone, cortisol, deoxycorticosterone or progesterone in doses around 10 nmoles/kg body weight. After a 30-60 min uptake period, cell nuclei were isolated from 9 brain regions and pituitary. Patterns of cell nuclear retention of [3H]corticosterone and [3H]dexamethasone differed: the former steroid was highest in hippocampus and septum and low in pituitary; the latter steroid was highest in pituitary and more uniformly distributed in the brain. The other 3H steroids showed very little cell nuclear labeling in vivo. In contrast, in vitro cytosol binding in hippocampus for [3H]progesterone, cortisol, deoxycorticosterone, and dexamethasone was 40-60% of that observed for [3H]corticosterone. The specificity of cell nuclear binding in slices of hippocampus in vitro was similar to that observed for cytosol binding. Reasons for the selectivity of in vivo cell nuclear labeling remain to be discovered but the selectivity does not appear to be an intrinsic feature of the receptors themselves. The pattern of in vivo labeling by [3H]corticosterone and [3H]dexamethasone differs from the in vivo distribution of [3H]estradiol in ADX-OVX rats using the same dissection procedure and this demonstrates the regional differentiation within brain of steroid hormone uptake and 'receptor' processes.


[Feline leukemogenic and sarcomatogenic viruses].

Feline Leukemia and sarcoma viruses (FeLV, FeSV) are RNA viruses, belonging to the Oncornavirus group. They possess common morphological, biochemical and immunological properties, with viruses of leukemia and sarcoma already known in others mammalian and avian species. FeLV is widely present in feline population and its transmission is by horizontal way. FeLV and FeSV are oncogenic for others species, i.e., dog and some non-human primates but as far as we know, the human receptivity is not established. A correlation between humoral antibody directed against membran new-antigens and regression of tumors induced by FeSV, led to actual investigations on vaccination.


Mode of C-type virus production by bone cells of C3H/Fg mice.

Electron microscopy of bone from the distal ends of femurs of six 3-month-old male C3H/Fg mice revealed varying modes and quantities of C-type virus production by cells in the preosteoblast to osteocyte developmental gradient. Production occurred by budding from the cell surface, either from very short pedicles or from longer microvillous-like processes. Although apparently absent in preosteoblasts, limited numbers of budding viruses (90-110 nM) were seen in 6 of 53 osteoblasts examined. Examination of 200 osteocytes revealed evidence of virus production in all but eight. In these cells, budding occurred from the cell body and its processes with release of viruses into the pericellular lacunar space. Budding from the longer microvillous-like processes sometimes resulted in binding together of viruses in bead-like fashion by thin strands of limiting membrane. In a few cases budding was also seen on the cell processes within the canaliculi. The quantities of viruses present within the lacunae usually increased with increasing age of the osteocytes, indicating continual virus production. Most of the viruses observed, however, were pre C-types along with lesser numbers of mature C particles. Thus, factors contributing to the production of mature C particles may be diminished in osteocytes of mice at this age.


Demonstration of cyclic AMP in bone cells by immuno-histochemical methods.

By utilizing immunohistochemical procedures it was possible to locate bone cells that contained adenosine 3'5'-monophosphate (cyclic AMP) in frozen, undecalcified cat jaw sections. An immunoglobulin-enzyme bridge method was found to be superior to other techniques, including immunofluorescence. Differences in staining pattern were found between bone cells of kittens and young adult cats, indicating different metabolic levels related to cellular maturity, and different intracellular compartmentalization of cyclic AMP in the two age groups.


Cellular localization of cyclic AMP in periodontal tissues during experimental tooth movement in cats.

Using an immune-histochemical method, cyclic AMP was localized in cells of periodontal tissues in orthodontically-treated cats. Sixteen cats were treated for periods ranging from 1 hour to 4 weeks. Fresh, frozen, undecalcified 6 mu sections of the tissues were incubated with rabbit anti-cyclic AMP antibodies, followed by sequential incubations with sheep anti-rabbit IgG, rabbit anti-peroxidase IgG and horseradish peroxidase. In the final step, the peroxidase was demonstrated by the diaminobenzidine (DAB) method. It was found that the number of intensely stained cells increased within a short time in areas in which bone resorption or apposition occurred later. However, differences in the pattern of cellular activation were found to exist between areas of compression and tension. The alveolar osteocytes appeared to be affected to only a slight degree by the applications of mechanical forces. These results indicate: (a) that our immune-histochemical method was useful in following the cellular distribution of cyclic AMP during bone remodeling; and (b) that mechanical forces may affect only a small part of the bone cell population and therefore cannot be regarded as an efficient means to bring about extensive bone remodeling.


Assembly of complex III into newly developing mitochondrial membranes.

Yeast cells grown anaerobically in 0.02% linoleic acid were transferred to air in the presence of 0.02% elaidic acid. At varying times Arrhenius plots were made of QH2-cytochrome c reductase activities in isolated mitochondria. A transition temperature of 8.2 degrees C at 0.5 h was characteristic of linoleate; at 3 h the transition temperature was increased to 24 degrees C characteristic of elaidate. At early times the enzyme was associated with anaerobic promitochondrial membranes; at later states the newly synthesized enzyme was associated with newly developed elaidate membranes.


3':5'-Cyclic-AMP phosphodiesterase activities in white and brown adipose tissues of cold-acclimated rats.

3':5'-Cyclic-AMP phosphodiesterase (PDE) (EC 3.1.4.17) activity was measured in interscapular brown adipose tissue (BAT) and in white epididymal adipose tissue of rats acclimated to constant or fluctuating cold. Experiments were carried out on isolated adipocytes or tissue homogenates. In brown or white adipose tissue or isolated adipocyte homogenates, two different apparent Km values were found according to the substrate (cAMP) concentration. The low Km was at about 10(-6) M and the high one at about 10(-4) M. The apparent V of the high Km enzyme was about 10-fold higher than the V of the low Km enzyme. Cold acclimation to constant or fluctuating cold did not modify appreciably the Km or V values. For low substrate concentrations (10(-6)-10(-8) M), the specific activity of PDE expressed per milligram of protein was decreased in BAT adipocytes of the two groups of cold-acclimated rats, compared to controls. Inversely, it was increased in total tissue homogenates. These variations were smaller in fluctuating cold than in constant cold-acclimate rats. They could, in part, induce the increases in lipolysis and in blood flow observed in the BAT of cold-acclimated rats.


Inhibition of lymphocyte transformation: effect of soy bean trypsin inhibitor and synthetic anti-proteases.

Soybean trypsin inhibitor (SBI) was found to inhibit transformation of human lymphocytes induced by mitogens (leucoagglutinin, concanavalin A, NaIO4) or in mixed lymphocyte reaction (MLR). SBI covalently cross-linked to Sepharose beads inhibited the MLR and mitogen stimulation virtually completely. We have confirmed the work of others which showed that the synthetic anti-proteases epilson-aminocaproic acid and tosyl-L-lysyl-chloromethane (TLCK) also inhibited mitogen-induced blastogenesis and we have shown that phenylmethylsulfonylfluride was effective also; all of these agents were found to inhibit the MLR as well. SBI and TLCK were most inhibitory when added along with mitogen or when mixing allogeneic cells in a MLR; significant decrease in inhibition was noted when TLCK was added 1 h after mitogen. These data support the hypothesis that protease action at a cell surface is an essential early event common to all types of lymphocyte transformation.


On the monomeric structure and proposed regulatory properties of phosphoenolpyruvate carboxykinase of Escherichia coli.

Phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating)) (EC 4.1.1.49) has been purified to homogeneity from Escherichia coli. The enzyme shows the same molecular weight (ca. 65000) either by sedimentation equilibrium under nondenaturing conditions or by polyacrylamide gel electrophoresis in the presence of detergent, indicating that the enzyme has a monomeric structure. We have confirmed the previous observation that NADH is an inhibitor of this enzyme, but we have failed to detect the previously reported appearance of homotropic cooperativity with respect to substrate binding the presence of this inhibitor. Lack of such homotropic interactions is in harmony with our conclusion that the enzymes is a monomer. Replacement of Mg2+ by Mn2+ in the assay medium lowers the Km for phosphoenolpyruvate by an order of magnitude, but does not affect the characteristics of inhibition by NADH.


Plasma lipoprotein profile and composition in White Carneau and Show Racer breeds of pigeons.

Plasma lipoprotein profile and composition in atherosclerosis-susceptible White Carneau and atherosclerosis-resistant Show Racer pigeons were investigated while consuming a regular pigeon chow diet free of cholesterol. Plasma was studied by analytical and preparative ultracentrifugation and paper electrophoresis. Lipid composition of each lipoprotein was determined by combined TLC-GLC techniques. The major plasma lipoprotein of both breeds was high density lipoprotein (HDL) with some low density lipoprotein (LDL) and no very low density lipoprotein (VLDL). Cholesterol was mainly found in the HDL in both breeds (71.7%), and no difference was noticed in the total cholesterol content of whole plasma or in various lipoproteins. The LDL fraction in White Carneaux showed a significantly lower (P less than 0.05) percentage of cholesterol esters compared with Show Racers (58.63 +/- 4.9 in White Carneaux vs. 72.12 +/- 2.1 in Show Racers). In LDL, the percentage of the triglyceride concentration in White Carneaux was significantly lower (P less than 0.01) than that of Show Racers while the percentage of protein content in White Carneaux was higher than in Show Racers. No significant differences were observed in fatty acid composition of steryl esters phospholipids, and triglycerides in the lipoprotein fractions of the two breeds. These studies show important differences in the cholesterol esters, protein, and triglyceride content of LDL in the atherosclerosis-susceptible breed of pigeons.


The carbohydrate composition of human apo low density lipoprotein from normal and type II hyperlipoproteinemic subjects.

The carbohydrate composition of the apoprotein from low density lipoprotein (LDL) of normal (average LDL cholesterol, 122 mg/100 ml) and type II hyperlipoproteinemic (average LDL cholesterol, 236 mg/100 ml) males was studied using gas-liquid chromatographic analysis of the methul glycoside derivatives. All samples containing detectable sinking pre-beta-lipoprotein were excluded from the study. The apo LDL from both groups of subjects contained mannose, galactose, N-acetyl-glucosamine and N-acetylneuraminic acid. Glucose and fucose were not found while trace quantities of galactosamine were detected. Although the quantities of galactose and N-acetylglycosamine were the same in the two groups, lower quantities of mannose (p less than or equal to 0.01) and N-acetylneuraminic acid (p less than or equal to 0.05) were found in the type II patients as opposed to normal subjects.


Androgen and estrogen binding in rat skeletal and perineal muscles.

Specific in vitro binding of [3H]testosterone (T), 5ALPHA[3H]dihydrotestosterone (DHT), and [3H[estradiol (E2) was demonstrated in the 30 000 X g supernatant (cytosol) of thigh muscles (TM) and of the levator ani - bulbocavernosus muscle complex (LA-BC) by gel filtration through Sephadex G-25 columns. In TM cytosol, T and E2 [are bound with high affinity  (Ka = 1.1 X 10(9) M-1, and 2.3 X 10(9) M-1 respectively) whereas DHT binding is of lower affinity (Ka = 5.0 X 10(7) M-1).] In LA-BC cytosol, T, E2, and DHT are bound with high affinity (Ka = 1.9 X 10(9) M-1, 0.3 X 10(9) M-1, and 0.5 X 10(9) M-1, respectively). Competition experiments suggest that the binding of the three hormones (T, E2, and DHT) is due to different proteins. In addition to TM and LA-BC, T and E2 binding was found in other muscles of male and female rats, including gastrocnemius, the pectoralis, diaphragm, and heart.


Evidence of respiratory tract infection induced by equine herpesvirus, type 2, in the horse.

Five horses were experimentally exposed to equine herpesvirus 2 strain LK. Two young foals developed chronic pharyngitis (98 and 232 days, respectively). Growth characteristics, cytopathic effects (CPE), inclusion body formation, ether sensitivity, and immunofluorescent analysis indicated that the virus recovered from infected animals was a herpesvirus serologically identical with, or at least antigenically related to EHV-2 strain LK. No significant complement-fixing (CF) or virus-neutralizing (VN) antibody responses were observed in adult horses while both foals demonstrated a rise in CF antibody titer. One of the two foals demonstrated a rise in VN antibody only. The results suggest that EHV-2 virus induced chronic pharyngitis, primarily the result of lymphoid proliferation, with no overt clinical signs.


Enzymic comparisons of the inorganic sulfur metabolism in autotrophic and heterotrophic Thiobacillus ferrooxidans.

Activities of enzymes which mediate the oxidation of thiosulfate to sulfate and the assimilation of sulfate to sulfide were assayed in various cell-free fractions of Thiobacillus ferrooxidans grown autotrophically on either ferrous iron or thiosulfate or heterotrophically on glucose. There was no activity of the thiosulfate-oxidizing enzyme in extracts of bacteria grown with ferrous iron. Comparable activities for ATP-sulfurylase (EC 2.7.7.4), ADP-sulfurylase (EC 2.7.7.5), and adenylate kinase (EC 2.7.4.3) were found in the bacteria grown autotrophically with either Fe2+ or S2O32- or heterotrophically with glucose.


Purification of Oidiodendron kalrai proteases.

Oidiodendron kalrai yeast-phase cells demonstrate proteolytic activity. Some of the proteolytic enzymes of the crude extract were purified by a combination of ammonium sulfate precipitation, Sephadex G-200, and diethylaminoethyl (DEAE) cellulose column chromatography. At least six proteins exhibiting a range of proteolytic activities could be identified by these procedures. Purity of the enzyme fractions obtained from the DEAE-cellulose columns was tested by running polyacrylamide gels.


Lissencephaly.

The first reported case of lissencephaly resulting from a consanguinous union strengthens the supposition that in some cases, it is transmitted as an autosomal recessive trait. Comparison of this case with a sporadically occurring case of lissencephaly, with different cortical morphology, suggests that lissencephaly may be an example of either varying gene expressivity or genetic heterogeneity. Lissencephaly and pachygyria may eventually be shown to be due to different causes, some inherited, some acquired. The classical examples of lissencephaly are different morphologically from a case in which antenatal cytomegalovirus infection had produced a small smooth brain. This suggests that antenatal viral infections are destructive rather than teratogenic.


Peripheral neuropathy in oxalosis. A case report with electron microscopic observations.

A 61 year old man had chronic renal failure because of oxaluria and renal calculi. Two years before death, while on hemodialysis, he developed severe progressive peripheral neuropathy. At autopsy calcium oxalate crystals were found in the peripheral nerves and other tissues. Nerve lesions included segmental demyelination, axonal degeneration and crystalline deposits within the myelin sheath. Ultrastructurally there were foci of osmiophilic granular material within myelin lamellae and endoneurium, and pleomorphic lamellar bodies in the perinuclear Schwann cell cytoplasm. It is probable that chronic hemodialysis favors the deposition of oxalate in the Schwann cells and the development of neuropathy in patients with primary hyperoxaluria and renal failure.


Ultrastructural classification of pituitary adenomas.

Forty-eight surgically removed pituitary adenomas have been investigated by electron microscopy. Distinct differences in fine structural appearances permitted their separation into 5 classes: 1) Growth hormone, (GH)-cell tumors; 2) Prolactin cell tumors; 3) Mixed adenomas composed of GH and prolactin cells; 4) Adrenocorticotrophic-Melanocyte stimulating hormone (ACTH-MSH) cell tumors; 5) Undifferentiated cell adenomas. Densely and sparsely granulated tumors were distinguished within classes 1, 2 and 4. Although these two forms appeared to represent well defined entities, they may be variants of the same tumor differing only in pace of hormone production and/or release. Number and size of secretory granules varied considerably among tumors composed of the same cell type indicating that pituitary tumor classification cannot be based solely on granule morphology. This classification takes into account morphogenesis with emphasis on clinical features and structure-function relationship.


A normal adult and fetal lung antigen present at different quantitative levels in different histologic types of human lung cancer.

Rabbits were immunized with extract of either an adenocarcinoma of the lung, or one of three oat cell carcinomas of the lung. Unabsorbed and variously absorbed antisera, and various extracts, were analyzed by double and radial immunodiffusion. The adenocarcinomas-antisera identified an antigen subsequently quantitated at high concentration in seven adenocarcinomas of the  lung, but a low concentration in five normal adult and four fetal lungs. Three of four epidermoid carcinomas of the lung contained the antigen at low concentration, however only trace amounts were detected in four of five oat cell carcinomas of the lung. The oat cell carcinoma-antisera failed to identify a significant antigen. Quantitatively, the above antigen appeared to distinguish adenocarcinomas and to a lesser extent epidermoid carcinomas from oat cell carcinomas of the lung, and to be consistent with the concept that oat cell carcinomas arise from a different cell type than the other lung tumors.


Signet-ring cell carcinoma of the breast. The mucinous variant of infiltrating lobular carcinoma?

Five cases of the type of mammary carcinoma that has been designated "signet-ring cell carcinoma" are presented. This tumor is characterized by the presence of numerous cells containing intracellular mucin, without large amounts of extracellular mucin as is seen in colloid (gelatinous, mucinous) carcinoma of the breast. Although such cells may be seen in many mammary carcinomas, they are never as frequent as in the variant described. Ultrastructurally, the most characteristic finding is the presence of numerous intracellular lumina containing material which appears to represent the mucin identified with the light microscope. This finding differs from that in colloid carcinoma, in which the scantier intracellular mucin occurs in the form of intracytoplasmic membrane-bound vesicles. The five tumors in the present series were all associated with either in situ lobular carcinoma or a "sinus catarrh"-like pattern of nodal metastases, or both. On the basis of these light and electron microscopic data, the signet-ring cell carcinoma is suggested as a variant of infiltrating lobular carcinoma, clinically and pathologically distinct from colloid carcinoma.


Congenital anomalies among children born of mothers receiving chemotherapy for gestational trophoblastic neoplasms.

Fifty-eight women who conceived following successful chemotherapy of gestational trophoblastic neoplasms subsequently became pregnant a total of 96 times. Seventy-eight of these 96 pregnancies or 81% terminated in live-born infants. Fifteen of the 96 or 16% ended in abortion and three or 3% terminated with stillborn infants. Among the live-born and stillborn infants there were three with congenital malformations classified as major. While this incidence of congenital malformations does not appear to be increased over that expected, the numbers are too small to perceive a two-fold increase in the expected incidence. Suggestions are made for improvements in the quality of these data.


Growth hormone deficiency in children with brain tumors.

Nine children with brain tumors are described who have received various combinations of treatment, including surgery, radiotherapy, and chemotherapy. Many of the children were noted to be of short stature. Endocrine assessment was carried out from 2 to 10 years after treatment. The combined results of insulin tolerance and Bovril stimulation tests show an impaired growth hormone response in six of the nine children. Bone age is retarded in all cases, and the present height is below the 10th percentile in five of the six. The cause of this growth hormone deficiency is obscure, but further studies are in progress.


The epidemiology of avian lymphoid leukosis.

Avian lymphoid leukosis can be induced by lymphoid leukosis viruses belonging to Subgroups A, B, C, and D. The endogenous virus of the chicken (Rous-associated virus type 0) belongs to Subgroup E and has little, if any, potential for inducing lymphoid leukosis. Nearly all chicken flocks are infected with Subgroup A lymphoid leukosis virus. This virus can be transmitted from dam to offspring or by contact with infected birds. Early infection, either by congential means, or soon after hatching, leads to the highest incidence of lymphoid leukosis. Maternal antibody or genetic resistance to infection delays or prevents infection, leading to a lower incidence of disease. In flocks segregating for genetic resistance to infection, continued infection is maintained through dynamic interactions between genetic resistance, acquired or maternal antibody ,and virus infection. Expression of endogenous viral information is controlled by dominant genes, but spontaneously produced Rous-associated virus type 0 can spread through a susceptible flock and be transmitted like an exogenous virus.


The epidemiology and virology of C-type virus-associated hematological cancers and related diseases in wild mice.

In several different populations of wild mice, observed over a 35-month period in laboratory geriatric colonies, a direct correlation was found between the prevalence and titer of spleen complement-fixing gs (p30) antigen and C-type particles in newly trapped healthy mice and a predilection to lymphoma and a hind leg paralytic disease upon aging. Other studies have established the indigenous C-type virus as the essential etiological determinant of both diseases in wild mice. An increased incidence of breast carcinomas, hepatomas, and pulmonary adenomas associated with C-type virus also occurred in the lymphoma-paralysis-prone colony as compared with the tumor-resistant colonies.


Biology of feline leukemia virus in the natural environment.

The feline leukemia virus (FeLV) was discovered in 1964 in a cluster of cats with lymphosarcoma. The observed clustering of cases of feline lymphosarcoma suggested that FeLV was an infectious agent for cats. The development of a simple immunofluorescent test for FeLV permitted a seroepidemiological study to be undertaken on the distribution of the virus in cats living in their natural environment. Over 2000 cats were tested, and the results showed conclusively that FeLV is an infectious agent for cats. This finding has now been independently confirmed using three different test procedures. After the infectious nature of FeLV was discovered, a simple FeLV test and removal program was devised to control the spread of the virus in the natural environment. The spread of FeLV was controlled in 45 households by removing the FeLV-infected cats, while in 25 households, where the infected cats were left in contact with the uninfected cats, 12% of the uninfected cats became infected. The ultimate control of FeLV awaits the development of an effective FeLV vaccine, which now seems feasible since we have already experimentally immunized some cats with attenuated FeLV. Although FeLV is infectious for cats there is no evidence that FeLV can infect humans.


Immune response to oncornaviruses and tumor-associated antigens in the chicken.

Two principal virus-directed antigens have been identified on the surface of oncornavirus-infected chick embryo cells. One is identical with the major virus type-specific envelope antigen, which is a glycoprotein with a molecular weight of 85,000. The 2nd antigen (tumor-specific cell surface antigen) is specific for transformed cells, i.e., absent from productively infected but nontransformed cells. This antigen has been identified as a glycoprotein with a molecular weight of 100,000 and was not found in the mature virion. Remarkably, both antigens induce humoral as well as cellular immunity in the chicken. It could be shown that cells can be killed in cytotoxic assays via either the tumor-specific cell surface antigen or the virus envelope glycoprotein alone.


Physiopathology of human and virus-induced murine leukemias.

The authors describe a coherent model for differentiated leukemias derived from physiopathological studies on Friend leukemia. In Friend leukemia, Friend virus induces permanent differentiation of erythropoietin-responsive cells. This erythropoietic proliferation and maturation is accompanied by a marked cell loss and provokes enlargement of the stem cell compartment. The so-called leukemic cells have a limited proliferation capacity and may not be truly malignant as opposed to blastic cells in acute leukemias. Clinical, hematological, and physiopathological data that are presently available in chronic granulocytic leukemia, polycythemia vera, and the erythroblastic component of erythroleukemia are compatible with the Friend physiopathological model. It is suggested that these differentiated leukemias initiate from an uncontrolled differentiation of a committed cell compartment, which stimulates proliferation of the stem cell compartment. The disease would be due to a proliferation and accumulation of "subnormal" cells characterized by a shorter mean life-span than the normal differentiated cell population. Although limited, the data available suggest that the physiopathology of acute leukemias is clearly distinguishable from that of differentiated leukemias; several immunological and therapeutic applications of this model are outlined.


An approach to C-type virus immunoprevention of spontaneously occurring tumors in laboratory mice.

A review of our current progress in C-type virus vaccine research is presented. This includes the findings of C-type virus or its antigen expressions in every naturally occurring tumor of two strains of "low-incidence" laboratory mice, the BALB/cCr mouse and the NIH Swiss mouse. Vaccine preparation methods are described including the inactivation of C-type virus infectivity with optimal maintenance of the antigen titers of at least two of the polypeptides of the C-type virus, gp69/71 and p30. The cell-mediated immune response of the mouse to C-type virus vaccines, as measured by a footpad assay for delayed-type hypersensitivity and an in vitro lymphocyte transformation assay, is described. Studies with two murine C-type viruses (Rauscher leukemia and Gross leukemia) a simian C-type virus, and an avian C-type virus (avian myeloblastosis virus) showed that the cell-mediated immune response of the animal includes type-specific, group-specific, and interspecies-specific reactivity. The mouse gave a cell-mediated immune response to at least one of the polypeptides of the C-type virus, the gp69/71, whether this polypeptide was presented to the immune system of the mouse as whole virus, Tween-ether-treated virus, or a purified polypeptide. One measure of the effectiveness of the C-type virus vaccines was provided by immunization of the mouse with Rauscher leukemia virus preparation that induced resistance to challenge with both live Rauscher leukemia virus and a naturally occurring BALB/c leukemia virus. Evidence is presented that the C-type virus can act as an effective transplantation antigen in syngeneic tumor cell lines resulting in the immunogenicity and loss of tumorigenicity of these cell lines. An approach to the viral immunoprevention of spontaneously occurring tumors is discussed.


Epstein-Barr virus-malaria interaction models for Burkitt's lymphoma: implications for preventive trials.

Infections with both Epstein-Barr virus (EBV) and malaria have been implicated as causal factors in the pathogenesis of Burkitt's lymphoma (BL). Proposed trials of preventive measures for both infections are receiving serious consideration as possible means of establishing a causal relationship with BL. In this paper we examine certain models for the interaction of EBV and malaria in the induction of BL, and also review the aims of the longitudinal, population-based study being conducted in the West Nile District of Uganda. Given existing knowledge, the outcome of preventive trials, even for the most simple interaction models, is unpredictable and, under certain circumstances, trials of an EBV vaccine could actually increase the incidence of BL. It is suggested that trials of an EBV vaccine at this time would be premature and should be delayed at least until the results from the West Nile prospective study are clear.


Experimental models for DNA-RNA viral interactions: a brief review.

Evidences that demonstrate or imply the occurrence of DNA-RNA viral interactions in man and animals are reviewed. The concurrent presence of two dissimilar oncogenic viruses influenced responses both in vivo and in vitro. Cell-mediated immune responses to Marek's disease herpesvirus were altered in specific-pathogen-free chickens inoculated as embryos with an avian leukosis virus. The possible roles of immunological factors in the pathogenesis and control of tumor development associated with DNA-RNA viral interactions remain to be investigated.


Biochemical approaches to detection of Epstein-Barr virus in human tumors.

The application of biochemical studies for the detection of Epstein-Barr virus (EBV)-DNA in human tumor cells is discussed. These studies resulted in the consistent demonstration of viral nucleic acid in African Burkitt's lymphoma biopsies and in epithelial tumor cells of nasopharyngeal carcinomas. The viral DNA resides within those cells regularly in multiple copies per cell. Besides these tumors our group detected significant concentrations of EBV-DNA in a German lymphoma patient revealing histological characteristics of Burkitt's lymphoma. Moreover, virus DNA was also found in a patient suffering from immunoblastic lymphadenopathy. More than 50 additional B-cell lymphomas and more than 40 biopsies from patients with Hodgkin's disease did not contain detectable amounts of EBV-DNA when tested by nucleic acid hybridization. A tentative scheme of EBV-induced pathogenesis is discussed.


Natural mechanisms of controlling lymphotropic herpesvirus infection (Marek's disease) in the chicken.

Four categories of natural resistance phenomena against Mareks disease in chickens, i.e., maternal antibody, age resistance, genetic resistance, and natural immunization, are described. In nature, these forms of resistance probably act in concert through comlex interrelationships. The mechanisms are not well understood but include humoral immunity (maternal antibody), cell-mediated immunity, and possibly other mechanisms. The possible role of newly discovered tumor-specific antigens in cell-mediated immunity against Marek's disease lymphomas is discussed.


Immune response of monkeys to lymphotrophic herpesvirus antigens.

Herpesvirus saimiri induces leukemia and/or malignant lymphoma when inoculated into different species of nonhuman primates including marmosets and owl monkeys. No malignant disease, however, has been recognized in the natural host for this virus, the squirrel monkey, although a high percentage of these monkeys are chronically infected with H. saimiri. Furthermore, one species of marmosets, as well as capuchin and some owl monkeys, fail to develop lymphoma following experimental virus infection but developed a chronic infection similar to that noted in the natural host. The availability of susceptible and resistant species made it possible to attempt to delineate those humoral and cellular immune parameters that might correlate both with tumor induction and resistance. The findings from these investigations are reviewed and discussed in relation to vaccine studies in this system as a model for a human herpesvirus vaccine.


Preventive vaccination against Herpesvirus saimiri-induced neoplasia.

In this paper we discuss the use of Herpesvirus saimiri as a model for the development of vaccines against herpesvirus-induced neoplasia in primates. Attempts at protection against the oncogenicity of H. saimiri have centered on the inactivation of virus by heat and formalin, the production of temperature-sensitive H. saimiri mutants, and the attenuation of the virus. Each of these approaches has provided information of use in the development of vaccines that may possibly be used in man.


Vaccination of nonhuman primates with killed oncogenic herpesviruses.

Killed herpesvirus cancer vaccines were prepared by inactivation of oncogenic herpesviruses (Herpesvirus saimiri and Herpesvirus ateles) with heat and formaldehyde. The killed vaccines, which are not free of viral nucleic acid, were safe in 121 vaccinated monkeys of 4 different species. Some of these monkeys have now been under observation for 2 years. The vaccines induced high titers of specific humoral antibodies in all vaccinated monkeys. The killed vaccines proved to be efficient against the i.m. challenge with cell-free oncogenic herpesviruses. The challenged animals remained clinically well without signs of an infection and have now been under observation for 1 year, while the nonvaccinated control monkeys died of malignant lymphoma 34 to 52 days after inoculation. The protection against H. saimiri could be passively transmitted with the serum of the vaccinated monkeys. The vaccination did not prevent but delayed tumor development after tumor cell transplantation. The resistance against the oropharyngeal route of infection remains to be determined.


Experimental infection of squirrel and marmoset monkeys with attenuated Herpesvirus saimiri.

Herpesvirus saimiri (HVS) was propagated in vero cells for 3 passages at 39 degrees and cloned 3 times at 34 degrees. This virus was inoculated into cotton-topped marmoset and squirrel monkeys; all inoculated monkeys became infected as HVS was reisolated after their circulating lymphocytes were cultured with vero cells and measurable levels of antiviral antibodies developed that were measured by immunofluorescence and/or neutralization tests. None of the inoculated monkeys developed any signs of overt disease and all inoculated monkeys have survived 9 to 14 months postinoculation. The attenuated virus appears to be genetically stable as virus isolated from an infected marmoset was passed 3 times in vitro and then inoculated into other marmosets, which became infected and remained clinically well. Marmosets latently infected with attenuated HVS were not protected when challenged with a large dose (770 plaque-forming units) of oncogenic HVS, although these marmosets survived about 3 times longer than did inoculated control marmosets.


Implications of a vaccine for the prevention of Epstein-Barr virus infection: ethical and logistic considerations.

Reasons are given for considering that there is sufficiently substantial indirect and circumstantial evidence linking Epstein-Barr (EB) virus to African Burkitt's lymphoma (BL) and nasopharyngeal carcinoma to call for a dynamic new approach to establish a causal role for the virus in these human cancers. It would seem that the only way to do this would be to develop a vaccine, vaccinate a population at risk in a high-tumor-incidence area, and subsequently follow the population for a consequential decrease in tumor incidence. Recent developments in the control of animal herpesvirus-induced malignant tumors by vaccines free of viral nucleic acid make it possible to envisage that a similar vaccine could be developed against EB virus without undue difficulty. Experiments showing the tumor-inducing ability of EB virus in South American subhuman primates have provided an in vivo laboratory system in which to test the safety and efficacy of the vaccine. Trial of the vaccine in human populations could be carried out by testing its ability to protect those at risk from primary EB virus infection accompanied by infectious mononucleosis. Although in world terms BL is not a major health problem, nevertheless African BL provides uniquely favorable conditions in which to test for a causative role for EB virus: high incidence areas are known, the peak tumor incidence is at the age of 5 or 6, and the effects of vaccination on tumor incidence could be assessed within a decade. Should a carcinogenic role for EB virus be demonstrated in African BL, a much longer term program would be called for to extend the vaccine control of infection to areas where EB virus is implicated in the induction of nasopharyngeal carcinoma. Although a high incidence of this tumor is confined to populations of Southern Chinese origin, the very large numbers of such people and the frequency of the tumor among them make this a substantial world health problem and, therefore, worth the cost and effort necessary to develop a vaccine giving life-long immunity and to conduct a program that will take more than a generation to give positive results.


Pilot studies with human interferon in Herpesvirus saimiri-induced lymphoma in owl monkeys.

The nature of Herpesvirus saimiri-induced disease in owl monkeys is described with emphasis on those biological parameters useful in monitoring the disease. These parameters are lymphocyte response to general mitogens, lymphocyte-infective centers, and antibody to virus-associated early antigen. Human interferon was used in treating owl monkeys with virus-induced leukemia. In 2 animals evidence was obtained that suggested a positive antileukemic effect.


Natural immunity in the oncornavirus-infected mouse.

BALB/cfC3H females neonatally infected with mammary tumor virus (MTV) possess spleen cells capable of significant cytotoxic activity against target MTV-induced mammary tumor cells in microcytotoxicity assay. Spleen cells from supposedly MTV-free BALB/c females are also specifically reactive, as a result of horizontal transmission of MTV antigens. No quantitative differences in reactivity exist between females not susceptible to the development of mammary tumors (BALB/c) and those that may develop or already have tumors (multiparous BALB/cfC3H). However, there are qualitative differences in response between those at risk and those not at risk.


Correlation of in vitro and in vivo studies of antigens relevant to the control of murine breast cancer.

The specific immune response of C3H [mammary tumor virus (MTV)] (MTV+) and C3Hf sublines (MTV- or milk-MTV-) to mammary tumors of C3H origin was measured in vitro by the ability of lymphocytes derived from immunized animals to destroy 3H-proline prelabeled target cells after 36 hr of incubation in vitro (lymphocyte:target ratio, 400:1). Primary cytotoxic responses were obtained both in C3H and C3Hf mice and were mediated mainly by T-lymphocytes (Thy.1-positive cells). The degree of cross-reactivity between different C3H mammary tumors showed wide ranges and actually depended on the amounts of MTV-related antigens expressed in the tumor cells. An inverse relationship between MTV-related and H2 histocompatibility antigens was observed. Thy.1.2 antigen (theta-C3H) was also detected on the surface of mammary tumor cells. Both C3H and C3Hf recognized cross-reacting and noncross-reacting antigens in the tumor cells, although the magnitude of the response in the MTV+ mice was lower than in the C3Hf sublines. Soluble antigens could be extracted by 3 M KCl treatment of the tumor cells and could be used as immunogens (eliciting cytotoxic responses against mammary tumor cells), or as stimulators for thymidine uptake (blast transformation in vitro) for specifically immune T-lymphocytes. Attempts to modify spontaneous tumor development in C3H and C3HfA virgin female mice by immunization with formalinized MTV or with soluble antigens extracted from C3H mammary tumors, although still in progress, showed a moderate preventive effect (especially in the C3HfA) immunized with MTV and an acceleration of tumor appearance both in C3H and C3HfA mice immunized with the soluble antigens extracted from C3H mammary tumors. This last set of results, although preliminary, indicates that a better understanding of the immunological events in this system is essential for the design of experiments on prophylaxis of tumor development.


Implications of humoral antibody in mice and humans to breast tumor and mouse mammary tumor virus-associated antigens.

As a part of a program directed toward the elucidation of the role of viruses in mouse and human breast cancer, a variety of immunological techniques were applied to a study of the humoral immune response of mice and of humans to their breast tumors. Tumor-bearing mice were found to produce antibodies against a complex array of tumor cell-associated antigens, including mouse mammary tumor virus (MMTV), components, heterophile and Forssman-like antigens, embryonic antigens, and possibly other tumor-associated antigens. Mice bearing MMTV-positive tumors had high titer antibodies against both viral and heterophile antigens. Tumor-free mice, whether of high or low mammary cancer strains, were remarkably free of antibodies that could label MMTV particles, although some sera contained antibodies to viral components. Patients with breast cancer also had antibodies against a variety of antigens associated with their own and homologous breast cancer cells. These antibodies reacted with heterophile, embryonic, and other tumor-associated antigens, some of which appeared to be viral. Sera of some patients with breast cancer gave positive immunofluorescence reactions with mouse mammary tumor cells grown in tissue culture and producing MMTV. Most of these reactions were due to heterophile antibodies in the sera, but a small number of sera contained antibodies apparently directed specifically toward MMTV particles, as determined by immunoperoxidase electron microscopy. Although human-mouse cross-reactions must be interpreted with caution, these data suggest that a virus putatively associated with human breast cancer is antigenically related to MMTV.


Biological considerations of tumor-specific and virus-associated antigens of human breast cancers.

In vivo and in vitro studies bearing on tumor-specific and viral-associated antigenicity of human breast carcinomas were reviewed with particular attention to the following clinical considerations: (a) breast carcinomas arise in a nonrandom fashion; (b) in situ carcinomas precede invasive breast carcinomas; (c) invasive breast carcinomas behave in a heterogeneous fashion. Microscopically demonstrable lymphoreticuloendothelial responses, skin window tests, and leukocyte migration tests all indicate that tumor-specific antigenicity develops in assoication with the early phases of mammary carcinogenesis. Such antigenicity is maximally expressed in in situ carcinomas without associated invasive breast cancer and minimally in invasive breast cancers with metastases. Immunogenic breast cancer tissues commonly contain a protein component the antigenic and physicochemical properties of which are similar to those of a protein component of murine mammary tumor virus. Advances in our understanding and control of human mammary carcinogenesis and biological behavior are dependent on the clinicopathological characterization of individual patients and their breast tissues as well as on the analytical procedures used.


Further immunization studies with mammary tumor virus.

A single i.m. dose of formalin-inactivated murine mammary tumor virus greatly reduces viral expression and mammary tumorigenesis in Af (tumor incidence, 39%) and RIIIf (tumor incidence, 11%) mice, which carry only  endogenous, gamete-transmitted virus. In C57BL mice, 1 mug of vaccine in Freund's complete adjuvant protects against later challenge with RIII virus.


Human cervical cancer as a venereal disease.

The operation of venereal factors in human cervical cancer is suggested by demographic and epidemiological data. Recent evidence supporting the genital herpesvirus hypothesis is consistent with this notion. A controlled epidemiological investigation has been undertaken on the role of the male coital partner in cervical carcinogenesis. An answer is sought to the question, "Is the risk of developing cervical cancer increased among the wives of men who at some other time were married to other women who developed cervical cancer?" With one-quarter of the study completed, a total of 14 marital clusters have been identified in which 2 or more wives of the same men developed cervical carcinoma. The expected number of such clusters is estimated to be 4.


Model systems for cervical cancer.

The evidence suggesting that a venereally transmitted virus and/or hormonal factors are involved in the etiology of cervical cancer is reviewed. The animal models used to test the oncogenic potential of these two types of agents are also discussed.


A search for herpes simplex virus type 2 markers in cervical carcinoma.

Carcinoma of the uterine cervix is now clearly recognized as the second most common malignant disease of women in the United States. Epidemiological studies provided the first suggestive evidence that an infectious, venereally transmitted agent was involved in this disease in either a causal or a casual fashion. Later cytohistopathological, virological, and seroepidemiological studies confirmed this observation and identified the suspect agent as herpes simplex virus type 2. Recently, several laboratories have directed their energies towards establishing lines of direct evidence linking herpes simplex virus type 2 etiologically with human cervical carcinoma. Some of these approaches have involved attempts to detect infectious virus, viral components, or virus-specific modifications in neoplastic cervical tissues. Results obtained utilizing human tissues will be reviewed and discussed.


Experimental evidence for the oncogenic potential of herpes simplex virus.

The data presented in this review reveal the transforming potential and oncogenic capability of herpes simplex viruses in cultured cells and in experimental animals, respectively. Unfortunately, the role of these agents in human cancer, especially cervical, cannot be unequivocally established through the indirect approaches used, but circumstantial evidence that these viruses represent potential oncogens in the human population is mounting.


Herpesvirus type 2-related antigens and their relevance to humoral and cell-mediated immunity in patients with cervical cancer.

The central theme of this communication is the interaction of herpes simplex virus type 2 with its host. In addition to the productive infection, we are confronted by latency and, as suggested by recent studies, by cancer. The possible mechanisms of latency and the role it may play as a precursor of carcinogenesis are discussed. If virus is to coexist with its host, a defined level of molecular interaction between host and viral gene products must exist. The association of AG-4 with active tumor growth and its identification as a minor virion protein, also exposed on the surface of the infected cell, open new vistas in the understanding of the role virus-host cell interactions may play in tumor growth. The modulation of the host immune response by the results of this interaction may play a significant role in cancer control. In these terms, the observation that antibody to AG-4 is a macroglobulin and that, therefore, immunity to AG-4 may be T-cell independent, should be given further consideration.


Studies on herpes simplex virus and cancer.

Virus-induced polypeptides of cells infected by herpes simplex virus (HSV) types 1 and 2 were investigated by analysis on polyacrylamide gels and by determination of their antigenicity. Some polypeptides, VP154 and VP134, had immunological reactivity common to both virus types, while others (VP175 and VP123) were type specific. Only the glycosylated polypeptides were able to induce neutralizing antibody. The expression of viral genetic information was studied in newborn mice infected with wild-type and ts mutant viruses; some mutants had become attenuated and had lost pathogenicity for newborn mice while others had not. From induction experiments in HSV=transformed hamster cells, it appears that detection of enhanced replication of ts mutants in human cancer cells would be an indication of resident HSV genetic information. Sera obtained from cancer patients were examined for antibodies to early proteins synthesized in HSV-infected cells. The method used was an indirect radioimmune precipitation test followed by polyacrylamide gel electrophoretic analysis of immune precipitates. Cervical cancer patients had sera with a higher reactivity to early nonstructural polypeptides than to breast cancer patients or to matched healthy women. In contrast to the results with early polypeptides, little difference was detectable between the matched sera in their reactivity with a major capsid polypeptide, which is synthesized late in the infectious cycle.


Induction of syncytia by the bovine C-type leukemia virus.

Bovine buffy coat cells infected with the bovine leukemia virus (BLV) induce syncytia formation in human diploid embryonic lung cells as well as in monolayer cell cultures of bovine, simian, ovine, bat, and caprine origin, but not in mouse fibroblast cells, normall rat kidney cells, or RSV-transformed rat cells. Syncytia were not observed in diploid embryonic lung cells inoculated with bovine buffy coat cells free of BLV. The syncytia-induction effect is associated with the synthesis of complete BLV by the buffy coat cells and is independent of whether these cells are viable, disrupted, normal, or malignant. Cell-free preparations of BLV and density gradient-purified virus also induce syncytia when added directly to diploid embryonic lung cells and to bovine, bat, and caprine monolayer cell cultures. Ether treatment, ultraviolet light irradiation, heating, freezing, and thawing destroy the syncytia-inducing activity of BLV. This activity is also neutralized when the virus is incubated with sera containing antibodies to BLV, but not when incubated with sera free of these antibodies or reference serum for the foamy-like bovine syncytial virus. Several other lines of evidence rule out the possibility that this virus or other bovine viruses are responsible for the syncytia-inducing phenomenon described here. BLV antigen was consistently detected by the immunofluorescent test in the syncytia-positive monolayer indicator cultures. However, syncytia formation was not necessarily associated with BLV production by the indicator cells. The ability to induce syncytia in monolayer cultures of nontransformed cells distinguishes BLV from all the known C-type luekemia viruses.


Development of an in vitro infectivity assay for the C-type bovine leukemia virus.

The ability of the bovine C-type leukemia virus to induce syncytia formation in monolayer cell cultures has been used to develop a specific and simple infectivity assay for the virus. Using bovine embryonic spleen cells or human diploid embryonic lung cells as indicator cells, the results of the assay can be evaluated in 4 to 6 or 6 to 8 days, respectively. Pretreatment of the indicator cells with DEAE-dextran greatly increases the sensitivity of the assay. The assay is quantitative and can be applied as a direct method for the identification of bovine C-type leukemia virus-infected animals; it also provides a simple, and sensitive procedure for the detection and titration of virus-neutralizing antibodies.