Application of long-range 13C,H nuclear spin-spin coupling constants in the study of imidazole tautomerism in L-histidine, and related compounds.

Three-bond 13C,H nuclear spin-spin coupling constants can be used to estimate the relative populations of the two possible tautomers of the imidazole ring in compounds containing this moiety. The mole fraction, XII, of the 1-H tautomer (i.e. the tautomer having a proton bound to the nitrogen atom at position 1 of the ring, where the ring is numbered according to IUPAC-IUB convention) can be calculated from the following equation: XII congruent to 1.705--0.164 3J(C5,H2)obs. Estimates of the predominant tautomer in L-histamine (both the neutral molecule and the monocation), and the dipeptide L-histidyl-L-glutamic acid indicate that the 1-H form is favored in basic aqueous solution. The results are compared with those obtained by other methods, and show that the present technique can provide a simple, rapid means of determining tautomeric equilibrium in imidazole derivatives.


Effects of acetylcholine on potassium-induced changes of water and sodium uptakes in cerebral cortex slices from the rat.

The increases in uptakes of water and of sodium ions that occur in rat brain cortex slices when they are incubated in a physiological saline-glucose medium in presence of a high concentration of potassium ions (105 muequiv./ml) are abolished by acetylcholine in presence of eserine but not by choline. Acetylcholine is effective at 20 micron but its optimal effect occurs at about 0.7 micron. Its action is suppressed by atropine and not by d-tubocurarine. The potassium-induced change of permeability of brain cell membranes to sodium ions occurs at a site different from the tetrodotoxin-sensitive channel of sodium entry, because the suppressive effects of acetylcholine and tetrodotoxin are apparently independent of each other. The acetylcholine effect does not occur in the absence of calcium ions from the incubation medium. It is suggested that the increase of cell calcium ions, brought about by high concentrations of potassium ions in the incubation medium, induces an increase of glial permeability to sodium ions, with a resultant change in the sodium gradient, and that this increase is suppressed by acetylcholine.


Effects of acetylcholine on potassium-induced changes of GABA and taurine uptakes and release in cerebral cortex slices from the rat.

Acetylcholine, in presence of eserine, has little or no effect on the potassium-ion-suppressed concentrative uptakes of GABA and taurine by rat brain cortex slices in contrast with its effect on those of L-glutamate, L-aspartate, and glycine. Potassium ions at a concentration of 30 muequiv./ml in the incubation medium has a marked suppressive effect on the uptakes of GABA and taurine when there is no apparent change in the sodium ion content of the brain tissue. It is concluded that some factor, besides the change in sodium gradient, operates in the mechanism of potassium suppression of GABA and taurine uptakes. Acetylcholine diminishes the potassium-evoked release of endogenous GABA and taurine from brain slices. Its action is Ca2+ dependent and is diminished by atropine. Acetylcholine does not affect the potassium-accelerated release of GABA from brain slices previously loaded with this amino acid. The differences in uptake and release phenomena exhibited by GABA and taurine from those of L-glutamine and L-aspartate may be due to differences between the mechanisms, as well as the sites, of cerebral uptake and release of these two groups of amino acids.


Actions of prednisolone acetate, pregnenolone-16alpha-carbonitrile, and triamcinolone upon drug resistance and metabolism: a comparative study.

In female rats, pretreatment with prednisolone acetate diminished the duration of zoxazolamine-induced paralysis as well as its plasma concentrations and increased significantly the in vitro metabolism of zoxazolamine and ethylmorphine. These actions of prednisolone were compared with those of equimolar amounts of pregnenolone-16alpha-carbonitrile (PCN) (a known microsomal enzyme inducer) and of triamcinolone (an agent that reduces the sensitivity of the body to drugs although not via microsomal enzyme induction). Prednisolone proved to be a strong drug-metabolizing enzyme inducer but it was less potent than PCN.


Depression by magnesium ion of neuronal excitability in tissue cultures of central nervous system.

Magnesium ion (Mg2+) in concentrations of 5-10 micron has been used to block synaptic transmission in in vitro preparations of central nervous system tissue. We have used explants of mouse cerebellum grown in culture to test whether significant levels of direct depression of neuronal membrane excitability could occur in such experiments. Using thresholds for antidromic activation and iontophoretically applied glutamate as tests of excitability, potent depressions of membrane excitability were found using 5-10 micron Mg2+. The additive depressant effects of increased Ca2+ concentrations provided further evidence that the effects were largely on membrane excitability and not on transmitter release mechanisms.


Effects of tricyclic antidepressants on the content and metabolism of dopamine in the rat striatum.

The effects of tricyclic antidepressants: imipramine (IMI), desmethylimipramine (DMI), and amitriptyline (AMI) on the levels and metabolism of dopamine (DA) in rat striatum were investigated. All three antidepressant drugs produced at 60 min a marked increase in striatal DA content. The DA-enhancing effect of DMI was dose dependent until a ceiling (about 140% increase) was reached. The time-course study of DMI (10 mg/kg) revealed a biphasic effect: an initial accumulation of DA peaking at 60 min followed by a decline in levels to about 20% of controls at 3 h after administration. In DMI-treated rats, homovanilic acid concentration was decreased to 58% at 60 min, but increased to 118% at 3 h, whereas 3,4-dihydroxyphenylacetic acid levels were unchanged at 60 min, but significantly suppressed at 3-h time interval. The observed effects of tricyclic anti-depressants on striatal DA may be relevant to the beneficial effect of these compounds in Parkinson's disease.


Effect of dipyridamole and 6-(2-hydroxy-5-nitro)-benzylthioguanosine on low-frequency-stimulated relaxation in the guinea pig taenia coli.

Isolated guinea pig taenia coli responded to electrical field stimulation at 0.3 s-1 with relaxation which was potentiated from 6 to 230% in the presence of 1-1.5 micron dipyridamole. However, no potentiation was seen in the presence of 10 micron 6-(2-hydroxy-5-nitro)-benzylthioguanosine (HNBTG), another more selective inhibitor of adenosine uptake. Both drugs effectively potentiated the relaxant response to low doses of added adenosine. When complete frequency-response curves were recorded in the range 0.1 to 5 s-1, neither dipyridamole nor HNBTG caused any shift of the curves, although a statistically significant increase in relaxation was again seen in the presence of dipyridamole at 0.3 s-1. Thus, the response of taenia coli to transmural stimulation is not modified by all concentrations of inhibitors of adenosine uptake and the limited effect seen with dipyridamole at 0.3 s-1 may be based on another unknown mechanism. No evidence for or against the purinergic nerve hypothesis can be derived from our experiments.


Dynamic elasticity of human mitral valve chorade tendinease.

Chordae tendineae are under constant dynamic stress and, therefore, measurements of static properties alone cannot provide a complete analysis of their mechanical response under stress. This study investigated the dynamic viscoelastic properties of human mitral valve chordae tendineae. The tissue was subjected to sinusoidal strain variations over a frequency range of 0.42 to 6.68 Hz. At a fixed strain, the storage modulus, E', was found to be independent of applied frequency but varied inversely with chordal thickness. Values in the order of 10(8) dyn cm-2 (1 dyn=10 muN) were found. E' also increased with strain level. The phase lag, phi, between stressing function and response was found to be small (0.2-0.058 rad) and decreased with frequency. Values of phi at each frequency were found to be independent of chordal size and strain level. This property would enable a smooth and even closure of the valve and provide a more rapid response at elevated heart rates. The loss modulus, E'', was found to be 18 to 50 times smaller than E'. This implied an almost complete recovery on removal of any stress on the tissue. E'', which also decreased with frequency, was found to be smaller for the larger chordae.


Effect of folic acid deficiency on pregnant rats and their offspring.

Two groups of 63-day-old female Wistar rats were fed a folic acid deficient diet, based on 20% of vitamin-free casein and containing 1% of succinylsulfathiazole, for 5 weeks (group A) and 9 weeks (group B) before being bred, and the same diet was continued through pregnancy and lactation. Three out of eleven (21.3%) and three out of seven (42.9%) rats in groups A and B, respectively, resorbed completely, while no control rat resorbed. No pups from group B survived to weaning. Both groups (A and B) showed depressed feed consumption (although the effect in group A rats was small) and weight gains and increased formiminoglutamic acid excretion in the urine during gestation, and low serum folic acid by the end of lactation. A study of blood components in group A rats revealed leucopenia, granulocytopenia, and increased reticulocyte count. While no congenital deformities were observed in pups from deficient dams, group A and group B dams in contrast to controls produced smaller sized litters with lower birth weights and poor survival rate. Surviving pups from group A dams had decreased weaning weights with significantly lower brain weights and brain DNA per gram of tissue.


Digestibility and nutrient value of palmitoyl-L-lysine derivatives in weanling rats.

alpha-N, epsilon-N-Dipalmitoyl-L-lysine, alpha-N-monopalmitoyl-L-lysine, and epsilon-N-monopalmitoyl-L-lysine were compared with 18% casein and lysine HCl as sources of lysine in the diets of weanling rats. The lysine derivatives and lysine HCl were added to a basal diet containing 14.1% of an amino acid mixture with no lysine. Weight gain, food intake, plasma lysine, and urine and fecal lysine were determined. Animals fed the basal and lysine derivative diets lost weight over the 10-day study period. They had lower food intake, urine and plasma lysine content, and a higher fecal lysine content than animals fed the other diets. alpha-N-Monopalmitoyl-L-lysine was absorbed whereas the epsilon-N-monopalmitoyl-L-lysine and the alpha-N, epsilon-N-dipalmitoyl compounds were not.


Species variation with respect to the metabolism and excretion of d-amphetamine and d,l-N-hydroxyamphetamine succinate.

The metabolism of amphetamine and N-hydroxyamphetamine was studied in the rat and mouse. Extensive reduction of N-hydroxyamphetamine to amphetamine occurred in both species. In addition, para-hydroxylation of amphetamine was a common metabolic route, although it was more predominant in the rat. No appreciable difference in the 24-h excretion of amphetamine and p-hydroxyamphetamine in either species was seen after substitution of N-hydroxyamphetamine for amphetamine.


Red blood cell catechol O-methyltransferase activity in thyroid dysfunction.

Using a modification of the method of Mannl et al. (Mannl, H.F.K., Hempel, K., and Kubler, W. 1972. Catechol O-methyltransferase in human erythrocytes. Arch. Pharmacol. 272, 265-276), we have measured the activity of catechol O-methyltransferase (COMT) (EC 2.1.1.6) in red blood cells (RBC) of patients with hyperthyroidism and hypothyroidism to establish whether thyroid dysfunction is associated with alterations in catecholamine catabolism. The activity of COMT averaged 4.4+/-0.54 nmol/ml RBC per hour of incubation (mean+/-SEM) in euthyroid subjects compared to 4.76+/-0.64 nmol/ml RBC per hour of incubation in hyperthyroidism and 4.42+/-0.81 nmol/ml RBC per hour of incubation in hypothyroidism; these values are not significantly different. There were no significant differences observed in urinary excretion of vanillylmandelic acid, epinephrine, and norepinephrine among the three groups. These data are compatible with the possibility that thyroid status has little influence on the degradation of circulating catecholamines and suggest that hypothyroidism, with its attendant elevations in serum norepinephrine concentration, may be related to a compensatory noradrenergic response.


Inhibition of hypoxic pulmonary vasoconstriction by dipyridamole is not platelet mediated.

Dipyridamole, which is known to alter platelet function, has also been shown to reduce hypoxic pulmonary vasoconstriction. This latter effect could result from dipyridamole either acting on a platelet-mediated system, or acting directly on pulmonary vascular smooth muscle. To investigate these two possibilities, normal dogs were compared with dogs rendered thrombocytopenic by a platelet antiserum. Compared with the hypoxic pressor response before drug treatment, the hypoxic response following dipyridamole was only 32% as great in the normal dogs and only 38% as great in the thrombocytopenic dogs. Thus, dipyridamole was no less effective in reducing the hypoxic pressor response in the virtual absence of platelets. This supports a direct effect of dipyridamole on pulmonary vascular smooth muscle, which could be mediated by an increase in adenosine levels.


The role of coronary flow and pressure as determinants of myocardial oxygen consumption in the presence or absence of vasomotor tone.

When coronary flow was kept constant and coronary pressure increased by angiotensin or decreased by dipyridamole, myocardial oxygen consumption (MVO2) remained unchanged. When vasomotor tone was abolished by dipyridamole or was intact, changing coronary flow resulted in a change in MVO2 in the same direction as flow; this change in MVO2 was in part the result of a significant alteration in coronary oxygen extraction. These results suggest that coronary flow, but not coronary pressure is an important determinant of MVO2 both in the presence or absence of vasomotor tone.


Reserpine-induced supersensitivity to the activation of cardiac glycogen phosphorylase by calcium.

Rats receiving reserpine (2.5 mg/kg, 24 h before sacrifice) developed a supersensitivity to the phosphorylase activating effect of both noradrenaline and Ca2+. Supersensitivity to the inotropic and chronotropic effects of the agonists did not develop with this dose of reserpine. It is concluded that reserpine induced supersensitivity, as well as being dose and species dependent, is also dependent on the response measured. It is concluded further that the supersensitivity developed to noradrenaline may be due to an alteration in the responding system (in this case the phosphorylase activating system) to calcium.


Endocrine effects of the pineal gland and neutralization of circulating melatonin and N-acetylserotonin.

Adrenocortical function was studied in rats after shampinealectomy, pinealectomy, or immunization against melatonin and N-acetylserotonin (NAS). Pineals were stimulated to increase melatonin synthesis by blinding or exposure to 23 h of darkness daily. Blinding elevated morning corticosterone levels without altering the corticosterone response to novelty stimulation. Among blinded animals, pinealectomy partially reversed and immunization completely reversed the elevations in morning steroid levels. Exposure to short daily photoperiods flattened the diurnal corticosterone rhythm. Pinealectomy did not affect morning corticosterone levels but reduced evening corticosterone levels. More importantly, immunization resulted in reduced corticosterone levels throughout the diurnal cycle. These findings suggest that melatonin and (or) NAS may be involved in the regulation of resting diurnal adrenocortical function.


Blood and plasma volume changes in dogs after sympathectomy with 6-hydroxydopamine.

The effect of chemical sympathectomy with 6-hydroxydopamine (6-OHDA) on blood volume was studied in unanesthetized splenectomized and nonsplenectomized dogs. A significant increase in total blood volume essentially accounted for by a marked plasma volume expansion was found in both groups after 6-OHDA treatment (50 mg/kg). The red cell volume in the nonsplenectomized dogs was significantly reduced 3 and 7 days after sympathectomy but returned to normal after 15 days. In contrast, the red cell volume was unchanged in the splenectomized dogs after sympathectomy. The blood volume changes were accompanied in both groups by significant decreases in hematocrit in plasma sodium and potassium and in serum protein concentrations while serum calcium concentrations were only slightly increased. These results confirm that the inhibition of adrenergic tone directly influences blood volume. They also indicate that an increase in blood volume constitutes an important compensatory mechanism for the long-term maintenance of adequate blood pressure levels after chemical sympathectomy by 6-OHDA.


Response of oculomotor neurons to eye muscle stretch.

Neurons within the ventral portion of the oculomotor nucleus respond to stretch of extraocular muscles. Response latencies range from 30 to 170 ms. Antidromically identified extraocular motoneurons with this input have slowly conducting axons in the range of 30 to 50 m/s. Only responses to stretch of muscles other than those innervated by these motoneurons were recorded. Responses to stretch of several muscles from both eyes are common.


Measurement of blood oxygen saturation using a multichannel fiberoptic oximeter-densitometer.

Design principles and performance of a new fiberoptic oximeter-densitometer for measurement of blood oxygen saturation are described. This instrument is capable of performing measurements using either an intravascular catheter, flow-through cuvette, or earpiece. The operations of the flow-through cuvette and earpiece are based on the principles of light transmission, that of the catheter on the principles of hemoreflection. The system incorporates five interference filters permitting the selection of a particular wavelength or combination of wavelengths to perform different modes of operation. Estimates using both catheter and cuvette appeared to be independent of variations over a wide range in hematocrit and flow rate. In the range from 20 to 100% saturation the standard deviation of the differences between the in vitro estimates by the catheter and (or) cuvette and the Van Slyke analyses were 1.62 and 1.73%, respectively. In 53 children with congenital heart disease (22 cyanotic) values calculated from readings by the earpiece were related to values of arterial oxygen saturation as measured by American Optical Company reflection oximeter at the time of cardiac catheterization. In the range from 50 to 100% saturation, the regression line between the two techniques was linear and the standard deviation of the differences was 2.89% (3.12% in cyanotic children alone). The method provides a high degree of compensation for variations in skin pigmentation.


Rat intestinal and renal calcium-binding proteins.

The effect of vitamin D activity on the major renal Ca-binding protein has been compared with that on the intestinal Ca-binding protein. Using a method based on Ca-binding capacity, these proteins were measured in vitamin D deficient rats after vitamin D treatment for varying periods up to 5 days. Since P deficiency has been shown to stimulate synthesis of the active metabolite 1,25-dihydroxycholecalciferol, a similar experiment was done on rats fed a P-deficient diet for periods up to 21 days. The renal Ca-binding protein was unchanged by vitamin D treatment to vitamin D deficient rats and was only slightly increased (50%) by phosphate deficiency. By comparison, the intestinal protein was increased twofold by vitamin D treatment and fivefold by phosphate deficiency. Results indicate that vitamin D activity has no direct effect on the major renal Ca-binding protein.


Plasma and tissue levels of dietary radiotocopherols in the harp seal, Phoca groenlandica.

Plasma and tissue levels of dietary radiotracer-labeled tocopherols were examined in the harp seal, Phoca groenlandica. The d-alpha form showed much higher plasma maxima and retention than d,l-gamma-tocopherol. Concentration peaks occurred somewhat later but were greater when the vitamin was fed with Tween 80 than with herring and corn oil respectively. Greater plasma levels occurred with the use of herring oil as compared with corn oil. Seals previously maintained on a vitamin E deficient diet showed greater plasma maxima than seals not so deprived. Plasma and tissues generally showed maximum levels within 24 h postingestion. Tissue levels of labelled dietary alpha-tocopherol were examined by biopsy and postmortem studies in selected tissues of vitamin E deprived seals.


Effect of neuronal uptake inhibitors on the adrenergic-neuron blockade produced by guanethidine in rabbit vas deferens.

The effect of neuronal-uptake inhibitors on the guanethidine-induced inhibition of responses of rabbit vas deferens to transmural stimulation was investigated. The twitch and sustained responses were inhibited by about 70% by 1.2 X 10(-5) M guanethidine. Prior or subsequent exposure to the noradrenaline-uptake inhibitors, 3.3 X 10(-5) M cocaine, 10(-5)M nisoxetine, and 10(-7)M desipramine prevented or reversed these effects of guanethidine. The depressant effects of guanethidine were not modified by the serotonin-uptake inhibitor, 10(-6)M fluoxetine. The uptake of 5 X 10(-8)M (-)-[3H]noradrenaline by rabbit vas deferens was significantly reduced by guanethidine, cocaine, nisoxetine, and desipramine, but not by fluoxetine. It was concluded that guanethidine inhibited both the twitch and sustained responses of the vas by producing adrenergic-neuron blockade and that these effects were dependent on continued transport into adrenergic neurons.


Biochemical reactions involved in pancreatic enzyme secretion. 2. Inhibitory effects of tetracaine and atropine.

Pancreatic enzyme secretion induced by urecholine or cholecystokinin-pancreozymin (CCK-PZ) is inhibited by tetracaine. If the pancreatic tissue is preincubated with tetracaine then washed out, the subsequent response to CCK-PZ is not affected while that to urecholine is impaired. In contrast with atropine, tetracaine loses its inhibitory potency once secretion has been initiated by urecholine before the addition of the local anaesthetic. Calcium efflux studies have shown that addition of tetracaine in vitro is associated with release of calcium in the incubation medium. This effect on calcium efflux might explain partly the inhibitory action of the drug.


GABA and glycine actions on spinal motoneurons.

Applied microiontophoretically in the spinal cord of cats, glycine is consistently more powerful than gamma-aminobutyric acid (GABA) in raising the membrane conductance of lumbosacral motoneurons (mean ratio of equipotent iontophoretic currents tested on same cells is 5.6:1). This is the reverse of the situation in cerebral cortex. The effect of glycine is well maintained during applications lasting about 1 min, but that of GABA, after an early peak, drops to a much lower plateau (mean plateau-over-peak ratio is 0.23). The reversal potentials for the action of GABA and glycine are initially similar but they behave differently during a prolonged application; that for glycine usually remains constant or becomes more negative whereas that for GABA tends to shift in the positive direction. Various explanations of these phenomena are considered. It is suggested that a single process, electrogenic uptake of GABA, may account for both desensitization (by removing GABA from its site of action) and the positive shift in GABA reversal potential (became uptake is probably associated with an influx of Na+).


Bicuculline, benzyl penicillin, and inhibitory amino acids in the spinal cord of the cat.

Bicuculline methochloride (BMC), applied by microiontophoresis, tends to depolarize spinal motoneurons and lower their input resistance. With approximately equal iontophoretic currents of gamma-aminobutyric acid (GABA) and BMC, there is an almost equal chance of observing no change, a potentiation, or a depression of the GABA-evoked conductance increase. A block of the GABA action is seen consistently only when the iontophoretic current of BMC is at least double that of GABA. Under these conditions BMC can selectively antagonize GABA without blocking the effects of glycine, though the latter can also be blocked by larger amounts of BMC. BMC also regularly eliminates the usual apparent desensitization to GABA. This may be due to depression of GABA uptake by BMC, which would also account for its potentiating action at lower relative doses. Comparable effects are observed with iontophoretic applications of benzyl penicillin (BP); but even large doses of BP produce no definite change in membrane properties or in conductance increase evoked by GABA or glycine.


Increased circulating plasma catecholamines and plasma renin activity in dogs after chemical sympathectomy with 6-hydroxydopamine.

Circulating plasma catecholamines, plasma renin activity, and other variables were measured in unanesthetized dogs before and after chemical sympathectomy with 6-hydroxydopamine (6-OHDA, 50 mg/kg). Chemical sympathectomy resulted in an immediate fall in mean arterial blood pressure and a delayed reduction in heart rate. Significant increases in plasma glucose and lactate concentrations, circulating plasma catecholamines, and plasma renin activity were found 24 h after 6-OHDA treatment. Circulating catecholamine levels decreased rapidly as time elapsed after sympathectomy and were half the initial values after 2 weeks. Plasma renin activity remained elevated during the 1st week after 6-OHDA treatment and returned to control levels during the 2nd week. Significant correlations were found between circulating catecholamines and heart rate mean arterial pressure, and plasma glucose and lactate concentrations. A significant correlation was also found between plasma renin activity and the mean arterial blood pressure. These results confirm that the adrenal medulla increases its catecholamine secretion rate into the circulation to compensate for the loss of adrenergic innervation after 6-OHDA treatment. They also indicate that the renin-angiotensin system represents another important compensatory mechanism for circulatory homeostasis in sympathectomized animals.


Inhibition of cerebral cortical neurones by a 5-hydroxytryptaminergic pathway from median raphé nucleus.

Recent observations made in our laboratory have shown that metergoline is a selective 5-hydroxytryptamine (5-HT) antagonist in the cerebral cortex. Fluoxetine is a reportedly selective 5-HT neuronal uptake blocker. In the present investigation these drugs have been used to examine the existence of a putative 5-HT input to the cerebral cortex. In rats anaesthetized with a mixture of methoxyflurane, nitrous oxide, and oxygen, stimulation of the median raphé nucleus or of the ipsilateral cortical surface and iontophoretically applied 5-HT decreased the firing of the deep cerebral cortical neurones. Metergoline antagonized the 5-HT-induced depression and reduced the duration of inhibition produced by raphé nucleus stimulation. These results suggest that the cerebral cortical neurones are inhibited by an ascending 5-HT-containing pathway.


The effects of alcohol upon cooling rates of humans immersed in 7.5 degrees C water.

The effects of the ingestion of a moderate dose of alcohol, a known vasodilator, upon the cooling rate of four human subjects immersed in 7.5 degrees C water for periods of time ranging from 38 to 75 min is reported. In three of the cases the cooling rate was retarded, in no case was it accelerated. This result, while lacking statistical significance, has not previously been reported. The difficulties attendant upon such research are noted.


The effect on fungal growth of some 2,5-dihydroxy-1,4-benzoquinones.

It has been shown that 2,5-dihydroxy-1,4-benzoquinones decrease vegetative growth and inhibit spore germination of 12 species of fungi belonging to six diverse genera. The nature of are substituents at the 3 and 6 positions of the quinone ring also affected their growth-inhibitory properties; generally those substituents of lower polarity inhibited growth at lower concentrations. As in the case of cochiliodinol, chemical modification of the quinone group, or the hydroxyl groups of the quinone ring, in compounds of the polyporic acid series, also led to loss of biological activity.


Effect of sodium chloride on growth and methane production of methanogens.

The effect of up to 263.7 mM sodium chloride on the growth and methane production by pure cultures of Methanospirillum hungatii GP1, Methanobacterium MOH, Methanobacterium thermoautotrophicum, and an unidentified methanogen was studied. Growth and methane production by M. hungatii GP1 were not affected up to 97.3 mM NaCl but there was some inhibition of growth at higher concentrations. Growth of Methanobacterium MOH was independent of sodium chloride concentration within the range investigated. For the unidentified methanogen, optimum growth and methane production occurred at 15.2 mM NaCl, while growth of M. thermoautotrophicum was not affected by sodium chloride concentrations up to 15.2 mM. Concentrations over 15.2 mM were inhibitory to these two organisms. The 15 mM sodium chloride concentration used by some investigators appears suitable for isolation and cultivation of methanogens since all the organisms tested in this study exhibited good growth and methane production at this salt concentration.


Effect of temperature on growth and activity of a methanogenic culture utilising acetate.

Studies with a methanogenic culture enriched for use of acetic acid showed that this culture had an optimum growth temperature of 35 degrees C, with only small differences for other temperatures between 30 and 40 degrees C. The optimum temperature was the same when determined on the basis of biomass production rate during the exponential (log) phase of growth (0.08-0.09 day-1, at 35 degrees C), amount of biomass present at the end of the log phase (100 mg/l), activity of the biomass (rate of conversion in millimoles per day per milligram (dry wt.) biomass present, 0.08 at end of log phase), or biomass yield (mg (dry wt.) biomass produced per millimole acetic acid converted, 1.0-1.1). Temperatures outside the range 30 to 40 degrees C caused marked reductions in the above parameters. The maximum temperature for growth was 42-44 degrees C; the minimum, below 15 degrees C, the lowest temperature studied. Acetic acid conversion to methane was 0.8-1.0 mol/mol, and was independent of temperature.


[Effect of aflatoxin B1 on the enzymatic activities of Bacillus thuringiensis (Berliner)].

At 20 degrees C, aflatoxin B1, at a sublethal dose, decreases the activity of alkaline phosphatase (EC 3.1.3.1), alpha-glucosidase (EC 3.2.1.20), esterase (EC 3.1.1.1), chymotrypsin (EC 3.4.21.1), leucine aminopeptidase (EC 3.4.11.1), and phosphoamidase (EC 3.9.1.1) biosynthesis in Bacillus thuringiensis (Berliner). In contrast, at 41 degrees C no significant decrease was observed. At this temperature, the mycotoxin is not destroyed or metabolized and bacterial cells are resistant to the toxin.


Back to the drawing board--the need for more realistic model systems for immunotherapy.

In experimental animals the growth of tumors which display strong immunogenicity can be slowed by immunological maneuvers that increase the magnitude of the host response to the specific tumor antigens. Such immunogeneic tumors do not, in general, cause distant metastases and may, therefore, not be relevant to the treatment of disseminated disease in man. This may explain why the current experience with immunotherapy based on such animal models has, with very few exceptions, been disappointing. Animal tumors which are not immunogenic by standard transplantation tests frequently disseminate and it seems likely that clinically useful immunotherapy has to be based on procedures which are effective against such tumors. The lack of immunogenicity detectable by transplantation may be due to the absence of tumor-specific transplantation antigens (TSTAs), in which case if there is to be any immunotherapy it will have to be direct at boosting some innate type of host resistance. Alternatively, the lack of immunogenicity may be attributable to the intervention of "escape mechanisms" which pervert the immunologically specific host response to TSTAs. In this case, the immunological maneuvre should be directed at overcoming the escape problem and not at boosting the magnitude of the specific host reaction to the TSTAs.


Acute and late effects of multimodal therapy on normal tissues.

The increasing use of combined radiation, chemotherapy, and surgery had led to an increased incidence of acute and late complications. The complications are, in general, similar to those seen with each modality alone, but occur with increased incidence. Enhanced effects of combined radiation and surgery are modest in number and consist primarily of problems with wound healing and fibrosis, as well as late gastrointestinal damage. Combinations of radiotherapy and chemotherapy have shown a greater degree of enhanced acute and late reactions. Drugs, such as actinomycin-D and Adriamycin, are particularly dangerous if the marked enhancement of radiation effects caused by the drugs in almost all organs is not appreciated and the radiation dose not adjusted accordingly. Proper selection of drugs can lead to enhanced local control by radiotherapy and/or surgery, as well as eradication of microscopic distant metastases, without increased normal tissue injury. Late induction of malignancy can occur with either radiation or chemotherapy alone and, in some cases, this appears to be enhanced when they are combined.


Some considerations in the design of new antineoplastic agents.

The design of many antineoplastic agents has been based on atom or group replacement in normal metabolites. New dimensions are being added to this approach by the synthesis of transition state, bisubstrate and "suicide" inhibitors. These provide greater potency and in some cases selectivity. Leads for differential sensitivity of viral infected cells are appearing (5'-amino-5-iodo-3',5'-dideoxy-uridine, AIU). Equally important has been the development of repository and precursor forms of active drugs along with structures having altered physical and metabolic properties. In the future combination therapy may include drugs whose intrinsic antitumor activity is relatively low but which in combination provide the means of potentiating the antitumor activity of other agents (THU, deoxycoformycin, pyrazofurin). Finally, the potential for developing macromolecular "drugs" such as therapeutic enzymes and peptide hormone analogs must be viewed as a whole new area for drug design.


Induction of aryl hydrocarbon hydroxylase and forestomach tumors ben benzo(a)pyrene.

While papillomatous tumors developed in the forestomach of female Ha/ICR mice after a 12-week chronic feeding period of benzo(a)pyrene (BP), no tumors developed in the glandular portion of stomach or in the lung or liver. Among all tissues examined, the forestomach showed the greatest increase of aryl hydrocarbon hydroxylase (AHH) activity following acute or chronic administration of BP. Single acute doses of BP induced AHH activity in forestomach, glandular stomach, lung, and small intestine, but not in the kidney and liver of these animals. Similarly, after chronic administration of BP, AHH activity was inducible in the forestomach, glandular stomach, and lung, but again not in the liver. Although the formation of tumors is associated with greater inducibility of AHH activity in the forestomach after BP administration, the relationship between tissue inducibility of AHH activity and susceptibility to BP carcinogenesis is still not clear. Further studies regarding the formation of specific carcinogenic epoxides of BP in itssues both susceptible (e.g., forestomach) and resistant to BP carcinogenesis would more clearly define the relationship between AHH inducibility and BP carcinogenesis.


Phytosterols and cholesterol in malignant and benign breast tumors.

Tissue phytosterol and cholesterol levels in 10 benign and 8 malignant breast tumors were quantitated to reexamine the hypothesis that malignant tumors had distinctive phytosterol content. Phytosterols were present in 9 of 10 benign and 7 of 8 malignant breast tumors. Mean (+/- S.E.) cholesterol, campesterol, stigmasterol, and beta-sitosterol in malignant and benign tumors (microgram/g wet weight) did not significantly differ (p greater than 0.1): (formula: see text) In the malignant tumors, tissue cholesterol correlated with campesterol (r = 0.97) and beta-sitosterol (r = 0.97) (p less than 0.01), but not stigmasterol (r = -0.06). In benign tumors, tissue cholesterol correlated with campesterol (r = 0.43), stigmasterol (r = 0.64), and beta-sitosterol (r = 0.94), with p less than 0.01 for the latter two. Phytosterols were present in four samples of normal breast tissue with mean (+/- S.E.) campesterol, stigmasterol, and beta-sitosterol (2 +/- 0.8, 15 +/- 9, 7 +/- 5 microgram/g wet weight) slightly but not significantly lower than in benign and malignant breast tumors, p greater than 0.1. The comparability of tissue phytosterols in benign and malignant breast tumors and in normal breast tissue appears to render unlikely and putative etiological relationship between phytosterols and breast carcinoma.


Fluidity difference of membrane lipids in human normal and leukemic lymphocytes as controlled by serum components.

Lymphocytes isolated from the peripheral blood of patients with chronic lymphatic leukemia and from normal healthy donors were analyzed for fluidity of membrane lipids. The degree of lipid fluidity in normal and leukemic lymphocytes was quantitatively monitored by a method based on fluorescence polarization analysis of a fluorescent probe that is embedded in lipid regions of cellular membrances. The present studies were performed on lymphocytes isolated from 26 blood samples from 16 patients with chronic lymphatic leukemia and 36 blood samples from 36 normal health donors. A signifcant increase in the degree of fluidity of membrane lipids was found in lymphocytes isolated from leukemic patients as compared to that found for lymphocytes isolated from healthy donors. In vitro incubation of leukemic lymphocytes in normal serum resulted in a decrease in the fluidity of cellular membranes, whereas incubation of normal lymphocytes in leukemic serum resulted in an increase in the fluidity of membrane lipids. These observations suggest that normal and leukemic lymphocytes can be quantitatively characterized by monitoring degree of fluidity of cellular membrane lipids and that the fluidity difference between normal and leukemic lymphocytes is controlled by components in the blood serum.


Immunochemotherapy of transplanted KMT-17 tumor in WKA rats by combination of cyclophosphamide and immunostimulatory protein-bound polysaccharide isolated from basidiomycetes.

Protein-bound polysaccharide Kureha (PS-K) isolated from Basidiomycetes was used in combination with cyclophosphamide (CY) for the treatment of a 3-methylcholanthrene- induced KMT-17 fibrosarcoma in WKA/Mk rats. A single administration of PS-K exhibited no inhibitory effect on the growth of s.c.-inoculated KMT-17 tumor at any timing and dose. However, PS-K exhibited a marked antitumor effect when it was combined with CY. The effect of PS-K dependend on the combination timing of PS-K and CY; a marked antitumor effect was observed when PS-K was administered before CY but not if it was given after CY or before tumor inoculation. When PS-K was administered on Day 1 followed by CY on Day 3, the highest survival rate of 78.5% (11 of 14) was obtained. Delayed hypersensitivity response of rats to KMT-17 was investigat ed by radioisotopic footpad assay. On Day 12, the hypersensitivity response in rats treated with PS-K on Day 1 and CY on Day 3 was significantly higher than that in nontreated rats, indicating an enhanced specific immunity to KMT-17 possibly resulting in a marked antitumor effect.


Disposition and tissue levels of [3H]vindesine in rats.

The excretion, blood levels, and tissue distribution of [3H]vindesine have been studied in the rat. After an i.v. administration of 500 microgram/kg. [3H]vindesine was found to be distributed very rapidly to tissues. After the distribution phase, blood levels  declined with a half-life near 10 hr. Excretion was mainly via the bile, and [3H]vindesine and its metabolites in bile were poorly reabsorbed in the gastrointestinal tract. Levels were relatively high in most tissues studied but appeared to be efficiently cleared from all tissues except thymus and testes. Uptake into peripheral nerves was considerably higher than into the central nervous system.


Selective inhibition of pyrimidine biosynthesis and effect on proliferative growth of colonic cancer cells.

A highly selective inhibition of de novo pyrimidine synthesis in the intact cell has been demonstrated by the action of N-(phosphonacetyl)-L-aspartate (PALA), a transition-state analog inhibitor of the reaction catalyzed by asparate transcarbamylase. The effect of pyrimidine deprivation induced by this agent on the viability and survival of human normal (WI-38) and colonic cancer cells (HT-29) was examined. The PALA-treated, pyrimidine-deprived cells failed to grow but demonstrated a normal rate of glucose utilization with impaired glycogen synthesis. Pyrimidine deprivation and lack of cell growth were maintained long after PALA removal. Growth inhibition of HT-29 cells by PALA was found to reflect an apparent steady state between newly formed and dying cells induced by limited pyrimidine availability. The highly selective nature of PALA action was confirmed by the ability of an exogenous source of pyrimidine to restore the normal growth pattern of the cell. Significant antitumor activity of PALA was found against a transplantable colonic tumor (line 26) carried in mice.


Effect of corynebacterium liquefaciens on a C3Hf mouse squamous cell carcinoma.

The antitumor effect of anaerobic Corynebacterium liquefaciens was compared with that of specific immunization. Experimental tumors were fourth or fifth generation isotransplants of a NR-Sl squamous cell carcinoma that arose spontaneously in a C3Hf/He female mouse. Specific immunization failed to exhibit an antitumor effect, whereas a single administration of the bacterium markedly inhibited the growth of the tumor. This growth inhibition was most effective when C. liquefaciens was administered 2 to 4 days before transplantation of tumor cells, but marked inhibition was also observed when this agent was administered after transplantation. The inhibitory effect was independent of dose within a range of 0.1 to 2.0 mg/mouse; a single dose of less than 0.05 mg/mouse did not exhibit antitumor effect. Multiple administrations of large doses, if given with short treatment intervals, were no more effective than one small dose. Multiple doses given at 14-day intervals resulted in marked growth retardation. The dose of cells that produced 50% tumor takes in C. liquefaciens-treated animals was not significantly different from that in nontreated animals, indicating that this bacterium exhibited no lethal effect on the tumor cells studied.


Covalent interaction of dehydroretronecine, a carcinogenic metabolite of the pyrrolizidine alkaloid monocrotaline, with cysteine and glutathione.

The covalent interaction of dehydroretronecine, a carcinogenic metabolite of the pyrrolizidine alkaloid monocrotaline, with cysteine and glutathione, has been investigated. Dehydroretronecine was allowed to react with cysteine and glutathione in an in vitro system of phosphate buffer solutions. The reaction products were identified structurally by chromatographic, nuclear magnetic resonance, infrared, ultraviolet, and mass-spectral analysis. These data indicate that the reaction products are the sulfhydryl-linked 7-thiocysteine-dehydroretronecine and 7-thioglutathione-dehydroretronecine. Active alkylation of sulfhydryl groups is a possible mechanism by which these alkaloids interact with cellular components.


Inhibition of the synthesis of polyamines and DNA in activated lymphocytes by a combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone).

The cancer chemotherapeutic drug, methylglyoxal bis(guanylhydrazone), inhibits the synthesis of spermidine and spermine, but allows continued putrescine production in small lymphocytes stimulated by concanavalin A. DNA replication in these cells is inhibited 50% while the synthesis of protein and RNA continues normally. When excess putrescine accumulation in the presence of methylglyoxal bis(guanylhydrazone) was inhibited with alpha-methylornithine, a competitive inhibitor of ornithine decarboxylase, the inhibition of DNA replication was accentuated, with still no effect on protein or RNA synthesis. No inhibition of DNA synthesis by the combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone) was observed when the inhibitors were added after accumulation of cellular polyamines. In addition, inhibition was reversed by exogenous putrescine, spermidine, or spermine. We conclude that putrescine can fulfill in part the role normally played by spermidine and spermine in DNA replication, and that blocking putrescine synthesis in the presence of methylglyoxal bis(guanylhydrazone) amplifies the polyamine requirement. The implications of this with regard to polyamine synthesis as a site of chemotherapy are discussed.


Hormone dependency of a serially transplantable human breast cancer (Br-10) in nude mice.

The human breast cancer (Br-10) serially transplanted to nude mice (BALB/c-nu/nu) grew well in female mice but very slowly or not at all in untreated male mice and female mice treated with 1 mg of testosterone i.m. twice a week. The growth in female mice was arrested by ovariectomy, and that in male mice was accelerated by 0.1 mg of estradiol i.m. once a week. Tumors in female and estrogenized male mice retained the original histology of duct carcinoma. Tumors in ovariectomized female, androgenized female, and male mice consisted of cells with smaller and more uniform nuclei, forming markedly dilated lumina in the first group and arranged in lobular patterns in the latter two groups. High-affinity 8 S and 4 S estrogen receptors were present in tumors transplanted to female nude mice, but no progesterone receptors were detected. These results provide experimental evidence for the hormone dependency of a human breast cancer in vivo and strongly suggest the important role of estrogen and androgen in the growth regulation of some estrogen receptor-positive human breast cancers.


Induction and persistence of pyrimidine dimers in the epidermal DNA of two strains of hairless mice.

The ultraviolet-light induction of DNA damage has been measured in the epidermis of hairless mice with the use of damage-specific endonucleases from Micrococcus luteus. The rates of induction of endonuclease-sensitive sites in HRS/J/Anl and Skh:hairless-1 mice were 6.1 +/- 0.5 X 10(-11) and 6.5 +/- 0.8 X 10(-11)/dalton/J/sq m from a FS40 fluorescent sun lamp (280 to 400 nm), respectively. Enzymatic photoreactivation with yeast photoreactivating enzyme showed that approximately 80% of the endonuclease-sensitive sites were cycloburyl pyrimidine dimers. In both strains of mice the pyrimidine dimers remained in high-molecular-weight DNA for 24 hr after irradiation. These data show that mouse epithelial cells in vivo have little or no capacity for the excision repair of pyrimidine dimers.


Effects of 2'-deoxycoformycin, 9-beta-D-arabinofuranosyladenine 5'-phosphate, and 1-beta-D-arabinofuranosylcytosine triple combination therapy on intracerebral leukemia 1210.

The triple combination of 2'-deoxycoformycin (2'-dCF), 9-beta-D-arabinofuranosyladenine 5'-phosphate, and 9-beta-D-arabinofuranosylcytosine was found to be very effective in the therapy of C57BL X DBA/2 F1 mice with intracerebral L1210. At the dosages and dosage scheduling used, the double combination of 2'-dCF and 9-beta-D-arabinofuranosyladenine 5'-phosphate gave minimal but significant increases in life-span. When 9-beta-D-arabinofuranosylcytosine was given at suboptimal dosage to mice with intracerebral L1210, the host toxicity caused by 2'-dCF and 9-beta-D-arabinofuranosyladenine 5'-phosphate in combination was decreased by a factor of 2, allowing a more prolonged therapy. "Cures" were obtained with the triple combination at dosages of 9-beta-D-arabinofuranosylcytosine that did not "cure". The supernatant adenosine deaminase from C57BL X DBA/2 F1 mouse brains was purified and the Ki for 2'-dCF using 9-beta-D-arabinofuranosyladenine as substrate was determined to be not more than 2 X 10(-11) M.


Distribution of technetium-99m sulfur colloid in mice bearing melanomas or mammary carcinomas.

Three groups of 24 C57BL/6J black mice were studied. One group was implanted with B16 malignant melanoma, another was implanted with mammary adenocarcinoma, and the third was not given tumor implants. After 14 to 17 days, the mice were given injections i.v. of technetium-99m sulfur colloid and killed 30 min later. Organs were weighed, and radioactivity was counted. The ratios of specific radioactivities of the spleens to those of the liver were higher only in the group of mice bearing malignant melanomas. This finding suggests that the "hot spleen" phenomenon observed in humans with malignant melanomas may be due to increased specific activity rather than increased splenic volume.


An improved assay for nuclear estrogen receptor in experimental and human breast cancer.

We have validated a hydroxylapatite assay for measuring estrogen receptor in extracts from breast tumor nuclei. By adsorption of receptor of hydroxylapatite prior to addition of radioactive ligand and warming, receptor degradation can be avoided. Total binding sites are measured at 30 degrees by exchange, and receptor sites unoccupied by steroid are measured at 4 degrees. A single saturating dose of 5 nM tritiated estradiol (with or without a 100-fold excess of nonradioactive diethylstilbestrol to estimate nonspecific binding) yields results similar to a six-dose Scatchard plot. Following in vivo injection of estradiol into rats bearing mammary tumors, receptor translocation in the tumors can be accurately quantitated with this assay. Applying the assay to human breast cancer, we find that tumor biopsies may contain cytoplasmic receptor alone or may also have appreciable nuclear receptor. The latter may be bound to estradiol or may be found in "free" form. The finding of free receptor in the nuclei in certain cases raises the possibility that unoccupied receptor might be able to stimulate cell replication in these cases, even in the absence of estrogen.


Association of macrophage activation with antitumor activity by synthetic and biological agents.

Treatment of normal BALB/c mice i.p. with a number of adjuvants, including pyran copolymer, the copolymer of polyinosinic and polycytidylic acids, Bacillus Calmette-Guérin, glucan, and dextran sulfate, rendered macrophages nonspecifically cytostatic for syngeneic tumor cells. Macrophage activation was highly dose dependent. The validity of the inhibition of DNA synthesis assay for measuring macrophage-induced cytostasis of target cells was proven by demonstrating a concurrent decrease in RNA synthesis and a reduction in viable tumor cell number. Moreover, conditioned supernatants from pyran-activated macrophages did not significantly decrease [3H]thymidine incorporation by freshly added leukemia cells. Biological or synthetic agents that activated macrophages were generally effective systemic antitumor agents against the M109 lung carcinoma. Drugs that did not activate macrophages, such as typhoid vaccine, tilorone, levamisole, WY-13876, and thymosin, were ineffective in prolonging the life of tumor-bearing mice. Pyran treatment i.p. was the most effective antitumor adjuvant in two separate tumor models, and suppression of tumor growth appeared to be related not only to an increase in macrophage tumoricidal function, but also to a larger influx of macrophages responding at the tumor site.


Carcinogenicity of benzo-ring derivatives of benzo(a)pyrene on mouse skin.

Benzo(a)pyrene (BP) and several benzo-ring derivatives of BP were tested for carcinogenic activity in mice by topical application of each compound once every 2 weeks for 60 weeks. Chronic treatment of C57BL/6J mice with (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (0.025 to 0.10 micronmole/application) indicated that the dihydrodiol was slightly more active as a complete carcinogen than the parent hydrocarbon BP. 7,8-Dihydroxy-7,8,9,10-tetrahydro-benzo(a)pyrene, a compound related to (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene but which lacks the double bond at position 9,10, was inactive as a carcinogen on mouse skin. These results indicate the importance of the double bond at position 9,10 for the carcinogenic activity of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene. Chronic treatment of mice with -.4 micronmole of the highly mutagenic (+/-)-7,beta,8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, (+/-)-7beta,8alpha-dihydroxy-9beta, 10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, or 9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene every 2 weeks for 60 weeks resulted in tumor incidences of 0, 8, and 4%, respectively, whereas BP at this dose caused a 100% tumor incidence. The high reactivity of the three epoxides may account for their inactivity or their weak carcinogenic activity on mouse skin.


High turnover rate of transfer RNA in tumor tissue.

Cancer patients and tumor-bearing animals excrete high levels of modified purines and pyrimidines some of which, e.g., N2,N2-dimethylguanosine, can originate only from transfer RNA (tRNA). Until recently, it could not be ascertained whether the high level of excretion of such compounds is due to cell death or specific tRNA turnover. However, an approach to this problem became feasible, with beta-aminoisobutyric acid as a probe. This compound is a terminal degradation product of thymine which is present in both DNA and tRNA. Since the pathway of synthesis of thymine is different in the two macromolecules, it and its end product, beta-aminoisobutyric acid can be differentially labeled with [14C]formate and [3H3]methylmethionine as precursors. Therefore the ratio of the two labels in the excreted beta-aminoisobutyric acid is a measure of the macromolecular origin of the degradation product. We have found from such analysis that tRNA's are not homogeneous in their turnover rate. There is a subpopulation that turns over much faster than the rest. The turnover rate of a subpopulation of tRNA's in tumor tissue exceeds the turnover rate of tRNA's in normal tissue. Such rapid degradation of tRNA's must be the source of the massive excretion of modified nucleosides by cancer patients which can be 10-fold higher than in normal subjects.


Repair following combined X-ray and heat at 41 degrees in plateau-phase mammalian cells.

Plateau-phase V79 Chinese hamster cells maintained at 37 degrees following X-irradiation demonstrated both sublethal damage repair and potentially lethal damage repair. When cells were kept at 41 degrees for 1 hr following irradiation, no sublethal damage repair was seen. In contrast, irradiated cells maintained at 41 degrees for 1 hr and then returned to 37 degrees were capable of repairing both sublethal and potentially lethal radiation damage; hyperthermic (41 degrees) inhibition of radiation damage repair was reversible. Repair was similar for cells heated before or after irradiation. The extent of sublethal damage repair (as measured by recovery ratios and quasi-threshold doses) and potentially lethal damage repair (as measured by recovery ratios and dose-modifying factors) was similar following combined heat and X-ray treatment and following X-ray treatment alone.


Detection of soluble tumor-associated antigens in serum of tumor-bearing rats and their immunological role in vivo.

Circulating soluble tumor antigens were detected in the serum of tumor-bearing rats. Sublethally irradiated W/Fu rats inoculated with syngeneic C58(NT)D Gross virus-induced lymphoma served as the source of tumor antigens. Soluble antigens were assessed by specific inhibition of the complement-mediated cytotoxicity of isogenic W/Fu anti-C58(NT)D antibodies against 51Cr tumor target cells. With a s.c. inoculum of 5 X 10(7) tumor cells, circulating tumor antigens were first detected at Day 8, and a maximum concentration was reached by Day 13 to 14, which coincided with the peak of tumor growth and was followed by the sudden death of the animals. Pooled serum from tumor-bearing rats was fractioned on Sephadex G-150 and resulted in one peak that contained all of the antigenic activity. The molecular weight of this fraction was estimated to be 50,000 to 60,000 daltons. Presensitization of normal rats with soluble tumor antigens resulted in a specific acceleration of tumor growth and delay in tumor rejection. Specificity was shown by lack of C58(NT)D tumor enhancement in rats presensitized with serum containing tumor antigens from a syngeneic but antigenically unrelated WR-6 lymphoma. The biological significance of circulating soluble tumor antigen mediating specific immunosuppression against an immunogenic tumor is discussed.


Inactivation of glucocorticoid receptors in cell-free preparations of rat liver.

We have examined the rates of inactivation of glucocorticoid receptors in cell-free preparations from several rat tissues. The t1/2 of inactivation of the glucocorticoid-binding ability of thymus, heart, and kidney cytosols (37,000 X g supernatants) ranges from 2 to 4 hr at 0 degrees, whereas that of liver is much slower (15 to 25 hr). The rate of inactivation of the glucocorticoid-binding capacity of soluble preparations from liver varies roughly according to the g force at which they have been centrifuged. The 100,000 X g supernatant. The ability of the particulate enzyme to inactivate glucocorticoid receptors at 0 degrees is not affected by protease inhibitors but is inhibited by fluoride and molybdate. The rapid inactivation of unbound glucocorticoid receptors that occurs in a high-speed (100,000 X g) supernatant preparation from rat liver at 25 degrees can be completely inhibited by molybdate. These observations suggest that the inactivation of glucocorticoid receptors observed in cell-free liver preparations in vitro is due to a nonproteolytic enzymatic function.


Lack of cross-resistance between certain platinum coordination compounds in mouse leukemia.

Two congeners of cis-platinum diamminodichloride, 1,2-diamminocyclohexylplatinum malonate (NSC 224964) and 1,2-cyclohexyldiamminoplatinum sulfate (NSC 250427), show approximately equal inhibitory activity in vitro against leukemia L1210 and a line of L1210 (L1210/PDD) that has developed resistance to cis-platinum diamminodichloride. These compounds are also active against L1210/PDD in vivo. These observations suggest that they be tried clinically in patients whose disease has become resistant to cis-platinum diamminodichloride.


Physicochemical considerations and pharmacokinetic behavior in delivery of drugs to the central nervous system.

The blood-brain barrier is not as rigid or as formidable as once believed. When experimental tumors of the central nervous system (CNS) and tumors of non-CNS origin are implanted intracerebrally (ic), they will grow and kill the host. It was once thought that only a few select agents, such as the nitrosoureas, were capable of crossing the blood-brain barrier and inhibiting tumor growth. There are now considerable data available which indicate that a variety of agents such as cytosine arabinoside, methotrexate, 6-mercaptopurine, 5-fluorouracil, procarbazine, melphalan, and cyclophosphamide, as well as the nitrosoureas, may significantly influence the growth rate of experimentally implanted ic tumors. A number of physicochemical factors may influence the penetration of the blood-brain barrier. These include pKa values, log-P values, and molecular size. Brain-level measurements indicate that most drugs have some accessibility to the CNS. Maximal effects against sensitive, ic implanted tumors are attained through maximal scheduling. Effective drug combinations should be sought to further enhance antitumor effects in the CNS.


Ligand-binding radioassay for the antifolate compounds: application in patients receiving methotrexate.

The general principles of ligand-binding radioassays are reviewed and a specific noncompetitive system is described for the measurement of methotrexate (MTX) using dihydrofolate reductase as the binding determinant. This type of radioassay can also be exploited to measure any antifolate compound which binds to this enzyme even if [3H]MTX is used as the tracer. The radioassay has now been used to measure MTX in all types of body fluids, tissue extracts, and rbc lysates from patients receiving this drug.


Increased cell proliferation with persistence of circadian rhythms in hamster cheek pouch neoplasms.

Squamous cell neoplasms induced by repeated topical application of 7,12-dimethyl-benz(a)anthracene in Syrian hamster cheek pouch exhibited circadian rhythms of DNA synthesis and mitotic activity. Fluctuations in the fractions of cells in mitosis and DNA synthesis observed in the tumors were approximately in phase with the circadian rhythms from normal precursor epithelium, indicating that some degree of host physiologic modulation persists during neoplastic growth. The labeling (thymidine-3H) and mitotic indices of neoplasms were considerably higher than normal throughout the 24 hr period. The duration of the neoplastic S phase--measured from the PLM curve--was 30% shorter than normal; G2 did not show detectable variation. The data demonstrated that chemically induced squamous cell neoplasms had markedly increased rates of cell production. It is postulated that applications of a carcinogen upon a cell-renewing population generate a multicompartmental cytokinetic imbalance in which: (1) a higher proportion of G0 cells is stimulated to enter the cycle; (2) the duration of the cell cycle is shortened; (3) the regulatory mechanisms fail to stimulate an accelerated rate of differentiation to compensate for the overproduction of cells; and (4) the state of proliferative hyperactivity becomes stable. An oncogenic cytokinetic mechanism based solely on a persistent decrease in cell loss (differentiation) is rule out by the present investigation, at least for squamous cell neoplasms.


Cell cycle time determinations based on liquid scintillation counting of 3H-TdR labeled mitotic cells.

Following a 10 min pulse labeling with 3H-TdR, flasks of asynchronous monolayer cultures of Chinese hamster ovary cells were subjected to mitotic selection at 2 hr intervals. The mitotic index of the selected populations was always greater than 90%. Counts per min per cell obtained by liquid scintillation counting were plotted versus time after the pulse label. Comparisons were made between cycle times obtained by the mitotic-scintillation counting method and by the standard per cent labeled mitosis technique. The resulting curves were used for calculations of the cell cycle times and the lengths of G1, S, G2 and M phases of the cell cycle. There was less than 2% difference in the cell cycle times obtained using the scintillation method as compared to times calculated from autoradiographic data obtained from individual petri dishes. The mitotic-scintillation counting technique is simple, accurate and rapid and allows the calculation of the cell kinetics parameters within 1 hr of the end of the experiment.


The effect of E type prostaglandins on the proliferation of haemopoietic stem cells in vivo.

The ability of protaglandins E1 and E2 to stimulate the proliferation of haemopoietic stem cells (CFUS) was studied in vivo. PGE2, in a dose range of 10(-4) to 10(-1) microgram/g body weight and PGE1 in a dose range of 10(-5) to 10(-1) microgram/g body weight, produced a rapid cycling wave of CFUS. The increase in the number of CFUS in S phase was not followed by a rise in the femoral CFUS content, and except for a transient increase in femoral CFUC level, no increase in differentiation was found either. Therefore, it is proposed that haemopoiesis after PG-induced CFU stimulation is ineffective. PGE2 did not stimulate regeneration of CFUS in a perturbed state (after sublethal irradiation). All these findings support  the idea that PGEs might represent potent stimulators of the haemopoietic stem cells acting in physiological doses. However, if acting concurrently with physiological control systems PGs lead to ineffective haemopoiesis (under normal conditions) or do not exert any measurable effect (after sublethal irradiation).


The effect of transposition to jejunum on epithelial cell kinetics in an ileal segment.

Epithelial cell kinetics were studied in an ileal segment after transposition to proximal jejunum. The number of cells per villus column in the transposed ileum increased after 4--7 days to reach values normal for jejunum after 14--30 days. This increase was accompanied by a simultaneous increase in the number of cells per crypt column up to 130% of values in jejunum and ileum in situ. The percentage of labelled crypt cells, after labelling with 3H-thymidine, and the relative size of the proliferative cell compartment in the crypt in the transposed ileum did not differ from values in the ileum in situ at any time interval after surgery. The total proliferative activity per crypt, which was determined by scintillation counting of isolated crypts after 3H-thymidine labelling, increased two-fold from 7 days after surgery. Cell migration studies showed that the increase in the number of villus cells was probably not caused by a change in the life span of the epithelial cells. It seems that the increase in the number of villus cells in ileal epithelium after transposition to proximal jejunum is brought about by an enlargement of the crypt, while the relative size of the proliferative cell compartment in the crypt remains unchanged.


Ultrastructure of cardiac muscle of Trochosa terricola Thor., Pardosa amentata Clerck, P. pullata Clerck, and Pisaura mirabilis Clerck (Araneae: Lycosidae, Pisauridae).

The ultrastructure of the cardiac muscle in some araneae has been investigated. The sarcolemma invaginates at the Z band and may extend into the cells through several myofibrils. Numerous T-tubules are given off both from sarcolemmal invaginations and also directly from the cell surface. In passing through the Z band, the luminal diameter of T-tubules greatly increases. Dyadic and a few triadic couplings are found mainly at the A-I level. Peripheral couplings were not seen.


Some ultrastructural effects of testosterone and insulin on the ventral prostate of rats in organ culture.

The fine structure of the secretory epithelial cells of rat's ventral prostate has been studied following organ culture. Culturing with either testosterone or insulin alone, and with the two hormones combined, were carried out to investigate how insulin modifies the action of testosterone on the maintenance of cellular integrity. After 4 days in hormone-free culture, the secretory epithelial cells showed signs of cellular atrophy and regression, involving loss of the apical microvilli, absence of the apical secretory vacuoles, atrophy of the Golgi apparatus, decrease in rough endoplasmic reticulum and the appearance of autophagic vacuoles. The presence in the medium of either testosterone or insulin alone, or combined, prevented cellular atrophy and regression. The best maintenance of cellular integrity was obtained in a culture containing both hormones. The effects of insulin was approximately equivalent to those of testosterone in the maintenance of cellular integrity.


The membrane systems of the cardiac muscle cell of Meganychtiphanes norvegica (m. sars), euphauciacea, crustacea.

The membrane systems of cardiac muscle cells of the euphausiacean Meganychtiphanes norvegica are described. Transverse tubules are found both at the Z-band level (TZ-tubules) and at the H-band level (Th-tubules). Within the sarcomere narrow longitudinal tubules branch off from the TZ-tubules. At the H-band level these tubules expand forming flattened cisternae in dyadic and triadic couplings with the sarcoplasmic reticulum (SR). Adjacent myofibrils are separated by a well developed SR. Modifications of the SR are seen at the H-band level where junctional cisternae are formed.


Ultrastructure of epidermis of Salamandra salamandra followed throughout ontogenesis.

Ventral epidermal ultrastructure of the amphibian urodele Salamandra salamandra is described and followed throughout its life cycle. Tadpoles were divided into five categories on the basis of the organization of their epidermis and the ultrastructure of its cells. In newly hatched tadpoles the epidermis is arranged in two layers and four types of cells were recognized. The number of epidermal layers increases in the metamorphosing tadpole. At this stage the layers become organized in four strata. Metamorphosis involves the disappearance of some cell types and the appearance of others, typical of the adult epidermis. The significance of these ontogenetic changes in epidermal ultrastructure is discussed in respect to aquatic and terrestrial life habits.


Irregular patterns of actin and myosin filaments in human skeletal muscle cells.

The ultrastructural study of cross sections of normal skeletal muscle cells showed the existence of irregular patterns of actin filaments in connection with the hexagonal pattern of the myosin filaments. The actin filaments surrounding each myosin filament vary in number from 6 to 11. The most frequent relationship is 9 to 1, followed by 10 to 1 and 8 to 1. The hexagonal pattern of actin filaments was observed only in the 6 to 1 arrays; as the actin filaments increase in number, they tend to form different polygons or circles around the myosin filaments. All described patterns may occur in each sarcomere. The actin to myosin filament ratio varies from 3 to 4 within each individual myofibril. The described variability of the actin filaments arrays leads to several difficulties in an explanation of the mechanism of muscular contraction.


The fine structure of distal receptors on the labium of the aphid, Brevicoryne brassicae L. (homoptera). Implications for current theories of sensory transduction.

Short peg receptors located at the distal tip of the aphid labium have the structure of mechanoreceptors. Each peg is innervated by a single sensory nerve which is anchored eccentrically to a basal cuticular tube and terminates in electron-dense material in the base of the peg. The arrangement and eccentric insertion of the eight pegs in the labial wall on one side of the stylet groove, with the eccentric insertions of their innervating neurones, provide a mirror image of the receptors on the opposite side. On the basis of a comparison of the structure of these receptors with that of tactile receptors for which electrophysiological data on sensitivity are available, it is possible to predict that the receptors detect both surface contact (pressure) and surface profile; and that the bilateral symmetry in the receptor arrangement facilitates the detection of vein contours which are preferred settling sites on the leaf. The structure of the dendritic terminal and its insertion is that of a well reinforced cytoskeleton designed to transmit tension to the cell membrane, in agreement with the concept that transduction is a membrane related phenomenon. The distal microtubules, fifty per-cent of which originate as well as terminate in the "tubular body", are packed in electron-dense material which binds to the cell membrane. The membrane in turn is attached to cuticular components of the receptor. Abrupt changes in dimension of the dendritic outer segment may be designed to modulate the conduction of a membrane potential. On the other hand, lack of continuity in the microtubules makes these organelles poor candidates for the transduction of excitation from a distal site of stimulation to a proximal region.


Myocardial cell lesions caused by an anabolic hormone.

Early changes in the composition of heart muscle cells of the rat caused by an anabolic hormone were investigated by electron microscopy. Mitochondria and myofibrils showed changes similar to those observed in early heart failure: The mitochondria were swollen and elongated. Their matrix was sparse and the cristae were few in number. The myofibrils showed either disintegration and widened and twisted Z-bands or a complete dissolution of the sarcomeric units.


In vivo and in vitro formation of the junctional complex in choroid epithelium. A freeze-etching study.

The junctional complex of choroid epithelial cells was studied during in vivo formation, disaggregation after trypsin treatment, and in vitro reaggregation. The in vivo formation begins with the occurrence of amorphous patches of particles followed by the formation of small particulate rows and polygonal-ordered particle assemblies. Further arrangement of the zonula occludens continues with the confluence of particles and smooth contoured ridges. At the 9th day stage a fully developed zonula occludens has developed. In a subsequent step nexus become integrated within the tight junction formation. Disaggregation after trypsination results in fragmentation of the zonulae occludentes. Parts of the disassembling aggregates become incorporated in vacuoles indicating an endocytotic mode of "digestion". The in vitro reconstruction of the zonula occludens proceeds from remnants of the former zonula occludens. On the 3rd to 4th day of cultivation mature tight junctions are visible. In vitro integrations of nexus were observed during a later phase. On the 7th day, cultivated choroid epithelial cells reveal well differentiated junctional complexes consisting of continuous zonulae occludentes and integrated gap junctions.


General morphology and axonal ultrastructure of the olfactory nerve of the pike, Esox lucius.

The olfactory nerve of the European pike (Esox lucius) contains 5.1 X 10(6) axons with an average diameter of 0.20+/-0,04 micron and a length of 5.5 cm in 1 meter long pike. Each axon contains an average of 4 microtubules as well as neurofilaments, smooth endoplasmic reticulum and about 500 mitochondria per centimeter. The number of neurofilaments ranges from zero in 15% of the cross sections to over 10 in 6%. Neurofilaments generally occur in clusters located opposite to microtubule regions. Smooth ER can not be identified in 14% of the cross sections suggesting that this structure may not be continuous. Microtubules often display annular regions (halos) of low electron density ranging in size from 800 to 1300 A. Halos from adjacent tubules usually merge into regional halos. The ratio of axoplasm to glial cytoplasm is 4.4:1, while the ratio of axonal plasma membrane to glial plasma membrane exceeds 7:1. A 4 cm nerve contains 1280 cm2 of axolemma. This nerve represents an extreme in high density axonal packing and is therefore exceptionally well suited for biochemical, biophysical and physiological investigations.


Some enzyme histochemical characteristics of the human hippocampus.

The histochemical distribution of acetylcholinesterase (AChE) and alpha-glycerophosphate dehydrogenase (alpha-GPDH) was studied in the area dentata and hippocampus proper of the human brain. Although differences did exist, there were many features in common with the distribution of these two enzymes in the rat and guinea pig. The laminar chemoarchitectonic picture was not as distinct in the human brain as in the rat and guinea pig. Most of the AChE reaction products were confined to the neuropil, with the strongest staining intensity in supra- and infrapyramidal zones. The layer of mossy fibres (stratum lucidum), was characteristically pale. On the other hand, AChE-positive cell bodies were observed in the hilus of the area dentata and a few scattered cells in the hippocampus proper. The AChE reaction products were sparse in the pyramidal cells, conforming similar observations in the rat and guinea pig. Based on our previous description of the AChE-positive cell bodies and fibres in the human septum and a considerable body of experimental material obtained in the rat, it is suggested that most of the AChE in the dentate area and the hippocampus proper is confined to terminals of cholinergic septal efferents in both man and other species. alpha-GPDH was particularly reactive in the cell layers, hilus fasciae dentatae and the layer of mossy fibers. These observations are similar to those described for the rat and guinea pig. They indicate, furthermore, a particular metabolic property common to the archicortex of man and other species.


'Disk shedding' in the cone outer segments of the teleost, Poecilia reticulata P.

Electron microscopical observations show that the cones in the retina of the diurnal Poecilia reticulata shed their membranous outer segment disks. This occurs at the side of the disk which is open to the extracellular space. Shedding is observed in single and twin cones and occurs at any level of the outer segment. The disks are not discarded in packages or as single disks, but are shed in small vesicular portions. This mode of 'disk shedding' may explain why in cone outer segments radioactively labelled replacement protein is diffusely distributed.


Structure and function of the amphibian follicular epithelium during ovulation.

Low concentrations of cytochalasin B (CCB) are known to inhibit ovulation in the frog, Hyla regilla. Examination of amphibian thecal cell ultrastructure reveals filaments (average diameter 71 A) arranged in bundles parallel to the surface of the oocyte. These filaments are often associated with hemidesmosome-like plaques on the basal plasmalemma, While individual filaments appear unaltered morphologically by CCB (1-5 microgram/ml), their organization into bundles, apparent relationship to the hemidesmosomes, and the highly contorted configuration of the thecal cells after oocyte expulsion, suggest that a nonmuscular contractile system residing within the follicle plays a fundamental role in ovulation. Our data suggest that the flattened epitheloid thecal cells shorten all axes that run parallel to the oocyte surface via filament bundle contractions, while they remain tightly bound together by macular attachment plaques. These cells thus increase in height to become cuboidal-low columnar in shape; the area covered by the base of each is greatly reduced. As this "thecal sac" decreases in size, the compression generated by the contractile mechanism forces the oocyte through the enzymatically weakened apex of the follicle and ovulation results.


Secretion in the rat coagulating gland (anterior prostate) after copulation.

The effect of copulation on the rat coagulating gland (anterior prostate) was studied. At 4 to 6 h after the beginning of copulation the coagulating glands of rats that had produced copulatory plugs were nearly empty of secretion. Ultrastructurally, the coagulating gland has large cisternae of rough endoplasmic reticulum (RER) and few condensing vacuoles or secretion granules. After copulation the number of secretion granules and the frequency of their expulsion into the lumen increased. Also in the lumen were "fragmentation" vesicles (50-100 nm diameter) that were bounded by a unit membrane and appeared to arise from microvilli. At 4, 6, and 7h after the beginning of copulation there was an increase in apical blebbing. Blebbing was found in both perfusion and immersion-fixed tissue. Also, after copulation there was an increase in "light cells" that were characterized by reduced RER cisternae, an electron lucent cytoplasm, and atrophic Golgi apparatus. The luminal ground substance, secretion granules, and some Golgi elements, contained polysaccharides as seen with the periodic acid-thiocarbohydrazide-silver proteinate method.


Spicule formation in the calcareous sponge Sycon ciliatum.

The spicule primordium is formed in an intercellular cavity within a group of sclerocytes. This cavity contains organic material which ensheaths the growing spicule but does not appear to determine the nature of the mineral morph (magnesian calcite) or the crystallographic orientation of the spicule. The tip of each growing spicule ray is seated in a 'dense cup' in the cytoplasm of the sclerocyte concerned. Both ends of monaxons are initially inserted each into a dense cup. As rays elongate the sclerocyte membrane around the tip becomes invaginated and forms a system of 'converging spaces' that possibly indicate high secretory activity in that region. Spicule growth involves the displacement and expansion of the organic sheath by the enlarging spicule. Fully formed spicules which are exposed to the mesohyl become surrounded by collagen fibrils. However, these fibrils are in no way concerned with the process of mineral deposition and are never found within the spicule calcite.


The rat epithalamus. I. Correlative scanning-transmission electron microscopy of supraependymal nerves.

Transmission and scanning electron microscopy of the rat epithalamus shows a regional variation in the distribution of supraependymal nerves (SN) which correlates well with supraependymal yellow fluorescence reported by Richards et al. (1974). The medial habenular nucleus, the intercommissural and suprahabenular recesses, the habenular commissure and the fibrae periventriculares thalami have the greatest density of SN/100micron of ependymal surface. The floor of the suprahabenular and intercommisural recesses is covered by non-ciliated ependyma. The significance of these findings is discussed with respect to (1) a direct functional relationship of SN with ependyma, and (2) a possible participation of the non-ciliated ependyma of the suprahabenular and intercommissural recesses in secretory activity whereby the CSF serves as a vehicle for neuroendocrine communication.


The transition of the thick ascending limb of Henle's loop into the distal convoluted tubule in the nephron of the rat kidney.

Examination of serial semithin sections of rat kidney cortex and a subsequent electron microscopic study of selected areas revealed that the characteristic epithelium of the cortical part of the thick ascending limb of Henle extends for a varying distance beyond the macula densa. The transition from the relatively thin epithelium of the thick ascending limb at this site to the three--or even four--fold thicker epithelium of the convoluted part of the distal tubule is sharply defined and occurs without the interposition of an intermediate cell type. The position of the macula densa at the end but still clearly within the ascending limb of Henle's loop is functionally interpreted to guarantee the separation of the sensor point macula densa from disturbing influences which might arise from the secretory activity of the subsequent tubular portion.


Relationship between collagen fibril diameters and body size. Study of fish derma.

Suspensions of collagen fibrils obtained from derma of Elasmobranchia and Actinopterygia of different body sizes and developmental stages were examined by transmission electron microscopy. Fibril diameters were measured and classified into groups comprising a 20 nm diameter interval. Diagrams showing fibril populations of each fish were made. The measurements were averaged and their confidence intervals and standard errors determined. For each species other diagrams were plotted in which the mean diameters were correlated to the body length of each sample. The results show that: 1) a correlation exists between an increase in diameter of collagen fibrils and somatic growth until sexual maturity is reached; 2) fibril populations are subsequently spread over a wider range due to the presence in the derma of classes of newly formed and therefore thinner fibrils. The deposition of new fibrils is possibly influenced by individual factors; 3) no relationship exists between mean fibril diameter and body size; 4) no relationship exists between phylogenetic position and pattern of diameter distribution.


Photoreceptor-like outer segments in the pineal organ of the lovebird, Uroloncha domestica (Aves: passeriformes). A scanning electron microscopic study.

In the pineal organ of the lovebird, Uroloncha domestica, bulbous, cup-shaped and elongated outer segments of photoreceptor-like pinealocytes are demonstrated by scanning electron microscopy. These scarce outer segments, 4-11 micron in length, extend into the pineal lumen. The present structural observations speak in favor of photosensitive pinealocytes in the pineal organ of Uroloncha domestica. The relation of the photoreceptor-like pinealocytes to acetylcholinesterase-positive nerve cells and a nervous connection between the pineal and the brain indicate that the pineal organ of this passeriform species may be the site of neuroendocrine and photoreceptive functions.


The connections of the sensory organ of Bellonci with the brain in Isopoda (crustacea).

The peduncle linking the organ of Bellonci with the brain was examined in Sphaeroma serratum and Anilocra frontalis. This peduncle, in its extension to the brain, becomes a nerve-like tract with bundles of pedicles originating from the sensory cell bodies located in the organ of Bellonci. It ends at the level of the medulla interna in an alveolar region resulting from the swelling of the sensory pedicle terminations. At this level three types of connections have been observed. The first is characterized by afferent synapses to the brain with, in the sensory pedicle endings, structures similar to the presynaptic ribbons noted by some authors in photoreceptors of arthropods. The two other types include nerve fibres originating from the brain, one with small electron lucent vesicles, a second displaying larger vesicles with a core of medium density. These fibres form efferent synapses to the organ of Bellonci. The sensory differentiation of the organ of Bellonci in Isopoda is confirmed but its true role is not specified.


Electron microscopic observations on the elimination of the oocyte through the peritoneal epithelium in the neonatal mouse ovary.

Numerous ovarian oocytes of primordial follicles are lost postnatally by their elimination into the peritoneal cavity during days 1-6 in young mice. It is rarely found in animals over 2 weeks old. This phenomenon has been investigated by light and EM techniques. Oocytes that become extruded from the ovary this way appear to pass between the cells of the covering epithelium. The ultrastructural changes that take place during this process are described.


Fine structure of the eyes of orb-weavers, Argiope amoena L. Koch (araneae: argiopidae). 1. The anteromedial eyes.

The anteromedial eye of the web-building spider, Argiope amoena has been examined with light and electron microscopy. The eye consists of a cornea, a lens, a vitreous body and a retina. The retina contains 400 to 500 bipolar photoreceptor cells and pigment ce-ls which envelop the receptor cells. Microvilli extend laterally from the distal process of each receptor cell beneath the vitreous body. The microvilli interdigitate with those from neighboring processes to form rhabdoms. Multivesicular bodies occur in the rhabdomeric portion, and in an intermediate segment, between the rhabdomeric portion and the receptor cell body, are found mixed lamellar vesicular bodies and lamellar bodies. A single axon extends from the receptor cell. The site of origin of the axon from the cell varies depending on the location of the cell in the retina. The axon originates on the intermediate segment, or on the lateral surface or proximal end of the cell body. The axons join together in the postero-dorsal region of the eye and then extend to the first optic glomerulus as an ocellar nerve of about 3 mm in length. The pigment cells, whose cell bodies lie among those of the receptor cells or the axons, possess cytoplasmic prolongations which reach up to the vitreous body.


Secondary sensory cells in the gravity receptor system of the statocyst of Octopus vulgaris.

The presence of secondary sensory cells in the Octopus gravity receptor system has been demonstrated. In serial thin sections of the receptor cells (hair cells) no axons were found leaving the cells. Instead, synapses were observed with synaptic vesicles lying inside the receptor cells. Both data clearly indicate that the receptor hair cells represent secondary sensory cells. In addition, efferent contacts to the receptor cells could be confirmed.


Morphologic evidence for differentiation of pinealocytes from photoreceptor cells in the adult noctule bat (Nyctalus noctula, Schreber).

An electron microscopical investigation of the pineal gland of the adult noctule bat revealed the presence of some peculiar ciliary derivatives, similar to the club-shaped outer segment of rudimentary photoreceptor cells in the pineal organ of nonmammalian vertebrates. The pinealocytes of population I can be classified in several morphological types, one of them displaying morphological features resembling those of rudimentary photoreceptor cells. These results reconfirm the concept of the sensory cell line in the vertebrate pineal organ. The question whether the pinealocytes of population II belong to the same sensory cell line is discussed.


Sequence from the assembly nucleation region of TMV RNA.

In an effort to isolate RNA sequences containing the assembly nucleation region, uniformly 32P-labeled tobacco mosaic virus RNA was partially digested with pancreatic ribonuclease, and the mixture of fragments was incubated with limited amounts of tobacco mosaic virus protein disks in conditions favorable for reconstitution. The RNA fragments which became encapsidated were purified and sequenced by conventional techniques. The sequence of the first 139 nucleotides of P1, the principal encapsidated fragment, is AGGUUUGAGAGAGAAGAUUACAAGCGUGAGAGACGGAGGGCCCAUGGAACUUACAGAAGAAGUUGUUGAUGAGUUCAUGGAAGAUGUCCCUAUGUCAAUCAGACUUGCAAAGUUUCGAUCUCGAACCGGAAAAAAGAGU. Residues 1--110 of P1 overlap the assembly origin isolated and characterized in the accompanying papers by Zimmern (1977) and Zimmern and Butler (1977). Our results, taken in conjunction with the two accompanying papers, define the sequence of much of the nucleation region as well as sequences flanking it on both sides. The features of the P1 sequence which may have role in the nucleation reaction are discussed in detail in the text.


One predominant 5'-undecanucleotide in adenovirus 2 late messenger RNAs.

Oligonucleotides containing the 5' termini of adenovirus 2 mRNA are selectively retained on columns of dihydroxyboryl cellulose. When total late adenovirus 2 mRNA was treated with RNAase T1, a single 5' terminal oligonucleotide was isolated, although in several states of methylation. This oligonucleotide has the general structure m7G5'ppp5' AmCmU(C4,U3)G. Since at least twelve individual species of mRNA must be present late after infection, this finding was unexpected and its significance is discussed.


The nucleotide sequence of two silk gland alanine tRNAs: implications for fibroin synthesis and for initiator tRNA structure.

The nucleotide sequences of two major alanine tRNAs from the Bombyx mori posterior silk gland have been determined. One of these tRNAs appears to be specific to the silk gland, where its accumulation is associated with the rapid production of fibroin. Both sequences are identical, with the exception of a single nucleotide in the anticodon stem. A striking feature of both alanine tRNAs is that loop IV contains sequences previously believed to be restricted to initiator tRNA.


Mapping of mitochondrial DNA of individual sheep and goats: rapid evolution in the D loop region.

Mitochondrial DNA (mtDNA) from sheep and goat was compared by restriction endonuclease analysis and heteroduplex mapping in the electron microscope. The fragment patterns produced by endonuclease Hae III from three individual sheep and two goat mtDNAs all differed from each other. The three sheep mtDNAs had identical Eco RI and Hind III fragments, but the two goat mtDNA patterns differed from each other as well as from sheep mtDNA. We estimate that each sheep mtDNA differs from each other by 0.5-1% of its nucleotide sequences, the two goat mtDNAs by 1-2%, and there is a 6-11% sequence difference between sheep and goat mtDNAs. We have mapped the Eco RI and Hind III sites of goat and sheep mtDNA and determined the positions of the D loop, which marks the replication origin, relative to the restriction map. The D loops are at homologous positions on the mtDNAs from both species, but the goat D loop is only 75% as long as the sheep D loop. Regions with a high degree of sequence divergence occur at both ends of the D loop. We suggest that a duplication of about 150 base pairs has occurred in the region where the sheep and goat D loops differ in length. We discuss mtDNA evolution in terms of divergence of isolated "mitochondrial DNA clones."


Globin RNA precursor molecules: biosynthesis and process in erythroid cells.

Hybridization of labeled RNA with excess amounts of DNA complementary to globin mRNA, in conjunction with a pulse-chase technique, were used to investigate the biosynthetic pathway of globin mRNA in erythroid cells. Three species of molecules sharing common sequences with globin mRNA were detected in the nuclei of these cells, two of which are larger than the cytoplasmic globin mRNA. One species was approximately 7 times larger than globin mRNA ("27S"), and the other ("15S") was only about twice the size of cytoplasmic globin mRNA. The largest species lacked poly(A) sequences, while the others contained poly(A), After chase, the large RNA species gradually disappeared ( 1/2 = 5 min), while the cytoplasmic 10S species accumulated. From these results a model is proposed describing the biosynthetic pathway of globin RNA transcription: an early transcription product is the large molecule "27S" (approximately 5000 nucleotides long) which is then cleaved into a smaller species "15S" (approximately 1500 nucleotides). This intermediate precursor is then clipped, presumably at the 5' end, and finally converted to the exported "10S" molecule (approximately 750 nucleotides) which accumulates in the cytoplasm.


Presence of histone mRNA sequences in high molecular weight RNA of HeLa cells.

Pulse-labeled HeLa cell RNA centrifuged under denaturing conditions was hybridized with DNA of recombinant phages containing sea urchin histone genes. This cross-hybridization showed the presence of histone mRNA sequences in high molecular weight RNA molecules. Treatment of the cells with actinomycin to stop RNA synthesis resulted in the rapid decay of this high molecular weight RNA followed by an increase of 9S histone mRNA in the cytoplasm. The results are consistent with the presence in HeLa cells of a high molecular weight precursor to histone messengers.


Macrophage plasminogen activator: induction by concanavalin A and phorbol myristate acetate.

The synthesis and secretion of plasminogen activator by cultured macrophages can be induced and stimulated by concanavalin A and by phorbol myristate acetate, and inhibited by such agents as glucocorticoids, mitotic inhibitors and compounds affecting cAMP metabolism. By the manipulation of stimulatory and inhibitory influences, enzyme production can be modulated continuously over a 200 fold range. In the same way, the proportion of cells that secrete detectable levels of enzyme can be varied from 1-90%. No comparable modulation of lysozyme or acid hydrolase production is observed under the same conditions. These results suggest that the physiological control of macrophage plasminogen activator production is achieved by the interacting effects of mutually antagonistic stimuli; this emphasizes the utility of this enzyme for the study of regulatory phenomena, including those relating to inflammation.


Changes in mouse mammary epithelial cell size during mammary gland development.

The average volume and size distribution of mouse mammary epithelial cells (MMEC) at different stages of functional activity of the mammary gland have been estimated. During early pregnancy, the mean MMEC volume begins to enlarge, resulting in a 21-fold cell volume increase by late lactation (Day 18). Progressive increases in mean MMEC volume during late pregnancy and throughout lactation result from the appearance and progressive hypertrophy of secretory alveolar cells. Involution of the mammary tissue brings about a precipitous decrease both in the average MMED volume and in the distribution of cell sizes established during the later phases of lactation. The progressive hypertrophy of cells particularly throughout the lactational period appear to correlate with previously reported changes in the metabolic activities of the mammary gland.


Incorporation of specific amino acids as markers of cell differentiation.

Incorporation of different amino acids into the proteins of wheat embryo has been found to be not uniform during the various stages of seed germination. It is suggested that incorporation maxima of appropriate amino acids can be used as markers of switching of specific protein synthesis during embryo development. Such markers have been found suitable for defining the functional activity of preformed messenger RNA.


Relationships between the chemical structure and the biological properties of some eremophilane compounds related to PR toxin.

The problem of the chemical structure--biological effects relationships has been studied for various eremophilane compounds related to PR toxin (PRT), a mycotoxin synthesized by Penicillium roqueforti. The biological tests were based on in vivo toxicity for male Swiss mice and inhibition of in vitro transcription and tranlation. The results showed a good correlation between the responses obtained for the three tests by the different compounds; thus, the toxic potency and the capacity of inhibiting transcription and translation should be directed by a common chemical structure. The data also indicated that the biological properties are related to the existence of an aldehyde group in position 12. In addition, evidence has been obtained demonstrating the hydrolysis of PRT imine with formation of a PRT-like compound.