Alborixin, a new antibiotic ionophore: isolation, structure, physical and chemical properties. Alborixin, a polycyclic polyether ionophorous antibiotic, active against Gram-positive bacteria and fungi, was isolated from cultures of a strain of Streptomyces albus. The isolation, structure and physiochemical properties of this antibiotic are reported. Some derivatives have been prepared and their structures and properties are also described in this paper.


U-43,120; a new antitumor antibiotic. I. Production, biological activity, microbiological assay and taxonomy of the producing microorganism. A new antitumor antibiotic, U-43,120 was discovered. It is produced by fermentation of a new species of Streptomyces, designated Streptomyces paulus DIETZ sp. n. Its antimicrobial activity is limited to bacteria. A microbiological assay with Bacillus subtilis was developed that can be detect concentrations of 1 approximately 2 mug/ml of the drug in fermentation liquors. U-43,120 was active in vivo against P-388 leukemia in mice.


Samarosporin, a new peptide antibiotic. I. Fermentation, isolation ahd characterization. A new antibiotic, samarosporin, was isolated in a crystalline state from a mycelial extract of a strain of Samarospora species (an ascomycete). Samarosporin is a neutral cyclopeptide and has a molecular formula of C72H111N15O19. Samarosporin shows a broad antimicrobial activity against various test microorganisms.


Structure of a new isoflavone from fungi and Streptomyces inhibiting dopa decarboxylase. A new dopa decarboxylase inhibiting isoflavone has been isolated from culture filtrates of fungi and streptomyces and shown to be 3',4',5,7-tetrahydroxy-8-methoxyisoflavone.


Biosynthesis of formycin. Incorporation and distribution of labeled compounds into formycin. 1. Two carbons, carbon-2 and one of carbons-3 to -5, of lysine seemed likely to be incorporated into one of carbon of the chromophore moiety of formycin. 2. From the results of radioisotopic studies with glutamate, gamma-amino-n-butyrate or organic acids related to tricarboxylic acid cycle, the important role of glutamate in the biosynthesis of formycin was strongly suggested. 3. The incorporation of nitrogen(s) of lysine into three nitrogens, including two nitrogens of pyrazole ring, of formycin was suggested by mass spectroscopy. 4. Ribose was estimated as a direct precursor moiety of formycin, whereas the biosynthesis of ribose was shown to occur via the pathway other than hexose monophosphate shunt or glucuronate pathway. 5. In replacement culture, the salvage synthesis of formycin from its chromophore moiety was not observed and it was also evident that the chromophore moiety or pyrazofurin (pyrazomycin) inhibited the biosynthesis of formycin.


Formation of 3-methylthioacrylic acid from methionine by Streptomyces lincolnensis. Isolation of a peroxidase. Cultures of Streptomyces lincolnensis accumulated 3-methylthioacrylic acid in amounts directly related to the concentration of methionine in the medium. The metabolite was labeled by L-(methyl-14C) but not by DL-(carboxyl-14C) methionine, indicating biosynthesis from the amino acid with loss of the carboxyl group. S. lincolnensis mycelium contained sufficient peroxidase activity to catalyse oxidative decarboxylation of L-methionine to 3-methylthiopropionamide as the initial step of a biosynthetic sequence. The enzyme, partially purified by ammonium sulfate precipitation, chromatography on a DEAE-cellulose column ang gel filtration, had a molecular weight of approximately 350,000, a pH optimum of 6.0, with o-dianisidine as electron donor and a Km value of 7.5 x 10(-4) M with respect to hydrogen peroxide. Cultures of S. lincolnensis supplemented with 3-(methyl-14C) methylthiopropionic acid gave labeled 3-methylthioacrylic acid. However, 3-(methyl-14C) methylthiopropionamide did not label the metabolite, suggesting that the first intermediate in the pathway may be the keto acid, which is then oxidatively decarboxylated to 3-methylthiopropionic acid.


CGP 9000: a new orally active, broad-spectrum cephalosporin. CGP 9000, (7-[D-2-amino-2-(1,4-cyclohexadienyl)-acetamido]-3-methoxy-3-cephem-4-carboxylic acid), is a new broad-spectrum cephalosporin antibiotic. Its antibacterial activity in vitro (MIC) is similar, but its bactericidal efficacy superior to that of cephalexin and cephradine. Upon oral administration to mice infected with various bacteria, CGP 9000 is, in general, 2 to 7 times more effective than either cephalexin or cephradine.


In vitro anti-mycoplasmal activity of amphotericin B methyl ester. The polyene macrolide antibiotic amphotericin B (AB) and its chemically modified derivative amphotericin B methyl ester (AME) were tested for in vitro activity against Acholeplasma laidlawii, Spiroplasma citri and Mycoplasma gallisepticum. Both polyene macrolide preparations demonstrated anti-mycoplasmal activity. However, AME was mycoplasmacidal toward all three strains of mycoplasma at levels which previous studies have indicated would be permissible for most cell culture systems, whereas the levels of AB required for similar activity would be physiologically intolerable for tissue culture cells. In addition, AME was 100 fold more active than AB toward A. laidlawii, 10 fold more active than AB toward S. citri and demonstrated equivalent activity as AB toward M gallisepticum. The in vitro anti-mycoplasmal activity of AME and AB was directly correlated with polyene macrolide antibiotic levels and the number of treated mycoplasma.


Studies on luciferase from Photobacterium phosphoreum. VIII. FMN-H2O2 initiated bioluminescence and the thermodynamics of the elementary steps of the luciferase reaction. Bacterial luciferase from Photobacterium phosohoreum was found to produce bioluminescence on reaction with FMN and H2O2 in the presence of aldehyde. This luminescence is presumably produced by the same X1 intermediate as that found in FMNH2-O2 initiated luminescence. From the ratio of the light intensities of the FMN-H2O2 initiated reactions, we calculated the association constant of the reaction, luciferase+FMN+H2O2in equilibriumX1, and estimated its temperature dependence. From these results we calculated the thermodynamic parameters of the reaction, luciferase+FMNH2+O2in equilibriumX1. We found that the free energy level of X1 is only 3.2 kcal below that of fr-e FMN and H2O2. We also estimated the thermodynamic parameters of other steps of the luminescent reaction. The values obtained showed that the formation of X1 from luciferase, FMNH2 and O2 involves a positive entropy change, but the intermediate is in a state stabilized against decomposition. Results also suggest a considerable degree of electronic rearrangement on formation of the excited-state molecule from the X1-aldehyde complex.


Thermodynamic characteristics of the adsorption of sulfanilamide, phenol, and n-butanol on bio-gel beads. The thermodynamic properties of the adsorption of sulfanilamide, phenol and n-butanol on Bio-Gel beads have been studied. Bio-Gel was chosen as the adsorbent as it possesses both hydrophobic and hydrophilic sites on its surface. Adsorption of the former two adsorbates was found to be exothermic, and the relevant thermodynamic parameters at 20 degrees are in the ranges: deltaH degrees = -2.7 to -5.4 kcal/mole; deltaF degrees = -6.0 to -7.6 kcal/mole; deltaS degrees = +7.7 to +11.6 e.u. In the presence of urea, adsorption of sulfanilamide and phenol was partially disrupted. This, together with the large entropy gain of the process, indicates that both hydrogen bonding and hydrophobic bonding contribute cooperatively to the adsorption. On the contrary, adsorption of n-butanol, which was not susceptible to urea, was an endothermic process with the parameters, deltaH degrees = +5.8kcal/mole, deltaF degrees = -1.8 kcal/mole, and deltaS = +26.1 E.U. at 20 degrees. These data conform to the thermodynamic properties of hydrophobic bond formation. Finally, possible implications of these data in the structural assembly of lipoprotein molecules are discussed.


Snake venom proteinase inhibitors. III. Isolation of five polypeptide inhibitors from the venoms of Hemachatus haemachatus (Ringhal's corbra) and Naja nivea (Cape cobra) and the complete amino acid sequences of two of them. Five proteinase inhibitors which all inhibit the activity of bovine trypsin [EC 3.4.21.4] were isolated from African Elapid venoms of Hemachatus haemachatus (HHV, Ringhal's cobra) and Naja nivea (NNV, Cape cobra). All the inhibitors were essentially homogeneous by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecylsulfate. Amino acid analysis and terminal analysis also supported their chemical homogeneities, except for one of the two inhibitors from Hemachatus haemachatus venom. The isolated inhibitors had a molecular weight of about 6,500, consisting of 52 to 57 amino acid residues, and they were all devoid of tryptophan. However, their amino acid compositions differed from each other. One of the three inhibitors isolated from Naja nivea venom, designated NNV inhibitor Ia, was unique, in that 4 half-cystinyl residues per mole fof the polypeptide were present, whereas all the others contained six residues. Of the isolated proteinase inhibitors, the complete amino acid sequences of two major inhibitors were established by manual and automatic Edman degradations and standard enzymatic techniques. Each of the inhibitors, designated HHV inhibitor II and NNV inhibitor II, consisted of 57 amino acid with arginine and glycine at the NH2- and COOH-termini, respectively. Both contained six half-cystines in disulfide linkages, and their overall amino acid sequences were similar, showing 91% homology. The two inhibitors differed in sequence by only five amino acid replacements, Asp-3 to Arg; Tyr-17 to Arg; Leu-25 to Arg; Gln-32 to Glu; and Arg-52 to His, in the 57 residue peptide chain. Comparing the amino acid sequences of these two cobra venom inhibitors with those of Russell's viper venom inhibitor II and bovine pancreatic trypsin inhibitor (BPTI), about 50% homology was found in their sequences. The 6 half-cystinyl residues of these inhibitors were in the same linear positions. Moreover, the regions which are structurally and functionally important in the well-known BPTI molecule were found with extremely high sequence homology in the cobra venom inhibitors. These findings strongly suggest that the cobra venom inhibitors as well as Russell's viper inhibitor II have very similar conformations to that established for BPTI.


Existence of glucosaminyl lactosyl ceramide (amino CTH-I) in human erythrocyte membranes as a possible precursor of blood group-active glycolipids. A novel glycolipid containing N-acetylglucosamine (lacto-N-triose II ceramide, Amino CTH-I) was isolated as a minor component from the ceramide trihexoside fraction of human erythrocyte lipids. This glycolipid was purified by column chromatography and preparative high-speed liquid chromatography on Iatrobeads. Its structure was found to be N-acetylglucosaminyl(beta 1-3)galactosyl(beta 1-4)glucosyl-(beta 1-1)ceramide, so it is suggested that it may be a precursor of paragloboside and blood group-active glycolipids.


Isolation and characterization of acid invertase inhibitor from sweet potato. An inhibitor of sweet potato acid invertase [EC 3.2.1.26] was found in fresh sweet potato root tissue, and partially purified. The inhibitor is possibly a thermostable protein with a molecular weight of about 19,500. The inhibitory activity is highly specific to acid invertase. The inhibitor binds to the enzyme reversibly in a non-competitive way, inhibiting the enzyme activity.


Studies on cyclodextrin glycosyltransferase. IV. Enzymatic synthesis of 3-O-alpha-D-glucopyranosyl-L-sorbose and 4-O-alpha-D-glucopyranosyl-D-xylose using cyclodextrin glycosyltransferase. The acceptor specificity of the transglycosylation reaction of cyclodextrin glycosyltransferase[EC 2.4.1.19] was investigated using various sugars and sugar alcohols. L-Sorbose, D-xylose, and D-galactose, which contain configurational or structural changes relative to the D-glucopyranose unit at positions other than position 1, were also shown to be efficient acceptors in the transglycosylation reaction of this enzyme. It was shown by chemical and enzymatic methods that this enzyme could transfer glycosyl residues only to the C3-hydroxyl group of L-sorbose and C4-hydroxyl group of D-xylose, producing oligosaccharides terminated by 3-O-alpha-D-glucopyranosyl-L-sorbose and 4-O-alpha-D-glucopyranosyl-D-xylose at the reducing ends, respectively.


Quantitative microanalysis of capsaicin, dihydrocapsaicin and nordihyrocapsaicin using mass fragmentography. A mass fragmentographic method for the quantitative microanalysis of capsaicin, dihydrocapsaicin and nordihydrocapsaicin in the fruits of Capsicum annuum has been developed. The molecular ions at m/e 377, 379 and 365 in the mass spectra were used for monitoring the trimethylsilyl derivatives of capsaicin, dihydrocapsaicin and nordihydrocapsaicin, respectively. The ratios of the height of each molecular ion to that of an internal standard (cholestane) were linear over the range 5-60 ng. The purification of individual capsaicinoids by high-performance liquid, thin-layer and gas-liquid chromatography is also described.


A highly efficient carboxylic acid analyzer and its application. A new highly efficient carboxylic acid analyzer, employed with liquid chromatography and a specific detection method, has been designed for the quantitative analysis of carboxylic acids in foods and biological samples. This apparatus is particularly advanced in terms of its sensitivity and resolution as a result of the extensive modifications to our previous system. The applications of this analyzer to the analyses of juices, beer, human serum and urine are presented.


Interaction between asymmetric solutes and solvents. Diamides derived from L-valine as stationary phases in gas-liquid partition chromatography. The resolution of enantiomers of (+/-)-a-amino acid derivatives on optically active diamides derived from L-valine, R1CONHCH[CH(CH3)2]CONHR2, as gas chromatographic stationary phases was studied. The effects of the structure of R1 and R2 in the stationary phases, the structure of the racemic a-amino acid derivatives, temperature, etc., on the separation factors are reported. In the light of these and related results, a more probable mechanism of resolution is presented.


Gas-liquid chromatographic determination of sulphonamides. Simultaneous qualitative analysis of fourteen sulpha drugs and their individual quantitative determinations have been performed by gas-liquid chromatography on solutions of dimethylformamide dialkylacetal derivatives of the drugs in acetone. Linear calibration curves were obtained for each sulpha drug under appropriate conditions. The structures of the above derivatives are assumed to be N1-methyl, N4-acetal sulphonamides, which can be detected with an electron capture detector with highly sensitive response. Columns used were 10% OV-101, 5% XE-60 and 5% OV-225.


Quantitative determination of propranolol, propranolol glycol and N-desisopropylpropranolol in brain tissue by electron capture gas chromatography. A method for the quantitative determination of propranolol and two of its active metabolites, 3-(alpha-naphthoxy)-1,2-propanediol (propranolol glycol) and N-des-isopropylpropranolol, in brain tissue of mice is described. Tissues are homogenized in perchloric acid-acetonitrile. Propranolol and its metabolites are isolated from the supernatant by solvent extraction and separated and detected as their trifluoroacetyl derivatives by electron capture gas chromatography. Chemical structures of the derivatives were confirmed by gas chromatography-mass spectrometry. The electron capture detector response of all three compounds is high, 0.7-2.0 X 10(-16) moles/sec. Brain levels of 10-250 ng/g can be detected of all three compounds with high specificity and good precision.


Determination of the content and purity of ergotamine preparations by means of high-pressure liquid chromatography. A reversed-phase system of high-pressure liquid chromatography with solvent gradient is described for testing the purity of ergotamine as an active substance and for checking its concentration in pharmaceutical preparations. Because of its good resolution, this system can be used not only for the selective assay of ergotamine but also for the identification and quantitative determination, in the same chromatogram, of seven known isomerization and hydrolysis breakdown products. Simultaneous detection at two different UV wavelengths also makes it possible to measure further breakdown products formed by addition at the 9,10 double bond (lumi-compounds). The advantages of the system lie in the determination of all of the products within ca. 20 min, direct injection of low-dosage injection solutions and sensitive detection of polar breakdown products. The system is reproducible with regard to retention times and quantitative determination. It is suitable as a quality-control method for the routine determination of the content and purity of ergotamine preparations.


Procedural difficulties in the gas-liquid chromatographic assay of the arylalkylamines. A gas-liquid chromatographic method for the estimation of 2-phenylethylamine in rat brain tissue is described. The procedure involves initial separation of the amine from homogenates with ion-exchange chromatography, subsequent acetylation of the eluate and extraction of the N-acetylated amine with ethyl acetate. The N-acetyl-2-phenylethylamine derivative is then converted to the pentafluoropropionyl derivative and quantitated with electron capture detection. Difficulties involved with alternative separative procedures and derivative formation are discussed.


Separation and quantitation of some urinary arylalkylamines. The arylalkylamines m- and p-tyramine, beta-phenylethylamine and tryptamine in their unconjugated forms have been identified and quantitated in urine collected from human volunteers. Their excretion levels (mean+/-standard error of the mean in mug/g creatinine) were, respectively, 67 +/- 5, 419 +/- 37, 4.6 +/- 1.2, and 82 +/- 11.


Autotransfer chromatography in the characterization of pyrroles. Chemistry of multiple-spot phenomena. A characteristic pattern of five interconverting "component" zones is obtained on autotransfer chromatography when pure kryptopyrrole (KP) is adsorbed from aqueous solution onto charcoal and then eluted with methanol. Specific oxidation products (lactams) derived from synthetic KP matched four of the five zones. Zone 1 was obtained directly from the a'-hydroxy-a-lactam. Zone 3 was produced directly by the a'-methoxy-a-lactam, while zones 2 and 5 were formed as a consequence of its acid hydrolysis during equilibration and chromatographic development. The formation of the methoxy lactam itself depended upon the presence of added methanol in the extraction process. In the absence of methanol during extraction only zone 1 (sometimes accompanied by zone 4) was formed.


Cannabis sativa L. (Marijuana). VII. The relative specificity of the RIM test. A total of 526 non-marijuana plant samples representing 427 different plant species were subjected to the RIM test (Rutgers Identification for Marijuana test). Although a few samples gave Fast Blue B reactive spots when examined by thin-layer chromatography, the spots could easily be distinguished from the marijuana cannabinoid spots. No plants were found to exhibit false positive results and hence the method is considered to be relatively specific for marijuana.


Serum 17 beta-estradiol and estrone levels in postmenopausal women with and without endometrial cancer. Serum 17 beta-estradiol (E2) and estrone (E1) levels were measured before and 6-8 weeks after ovariectomy in 16 postmenopausal women with endometrial cancer, and in 10 postmenopausal women with normal endometrium (preovariectomy only). For E2, no significant difference in the mean baseline (+/-SE) level was found between the tumor (13.5 +/- 1.1 pg/ml) and non-tumor patients (11.7 +/- 1.4 pg/ml). For the same patients, the mean baseline E1 levels of 33.8 +/- 2.4 pg/ml and 28.5 +/- 4.7 pg/ml were also not significantly different. The mean body weight of the tumor patients (141.1 +/- 7.3 lbs.) was similar to the mean weight of the non-tumor subjects (137.5 +/- 6.3 lbs.). This was of importance since both E2 and E1 levels correlated significantly with body weight and excess fat in these postmenopausal women. The circulating estrogen levels did not correlate significantly with the patients' height, age, or years of menopause. In the patients with endometrial cancer the mean E2 (14.1 +/- 1.7 pg/ml) and E1 (39.5 +/- 7.3 pg/ml) levels after ovariectomy were not significantly different from the preoperative concentrations. These data are consistent with the concept that in postmenopausal women most, if not all, circulating estrogen is produced by peripheral conversion of androgens and that this conversion is influenced by obesity. Circulating estrogen levels are not significantly different in postmenopausal patients with endometrial cancer compared with women of a similar age and weight who do not have the tumor.


Normal glucagon response to arginine infusion in "prediabetic" Pima Indians. To determine whether abnormalities in glucagon secretion might precede the onset of hyperglycemia in diabetes mellitus, 32 prediabetic Pima (American) Indians, 27 normal Pima Indians and 34 normal Caucasians received an infusion of arginine monochloride (5 mg/kg/min for 40 minutes) with measurement of glucose, insulin, and glucagon. [Prediabetes is the period between conception and the development of diabetes. In most studies the term is used to characterize patients who on genetic grounds are believed to be at high risk of developing the disease, including the normoglycemic monozygotic co-twin of a diabetic or the normoglycemic offspring of two diabetic parents. The latter definition is used in the present study recognizing that in the final analysis the true prediabetic can be identified only in retrospect after the development of diabetes.] The three groups had similar mean fasting glucagon levels. During arginine infusion, the prediabetic Indians reached a mean maximum glucagon level of 315 +/- 14 pg/ml (mean +/- 1 SEM) compared with 294 +/- 20 pg/ml in the normal Indians and 292 +/- 25 pg/ml in the normal Caucasians. The calculated mean areas above baseline under the glucagon curves were 5704 +/- 324 pg-min/ml in the prediabetics, 5189 +/- 446 pg-min/ml in the normal Indians, and 4239 +/- 613 pg/min/ml in the normal Caucasians. The differences among the groups in these variables were not statistically significant. Thus, arginine induced hyperglucagonemia could not be identified as a characteristic of the prediabetic state in Pima Indians.


Dissociation of renin and aldosterone during dehydration: studies in a case of diabetes insipidus and adipsia. The regulation of aldosterone was studied in a child with diabetes insipidus and adipsia, associated with holoprosencephaly. Plasma ADH was low and unresponsive to dehydration. Plasma renin concentration ranged from 52 to 1350 ng ml-1 h-1 at various degrees of hydration, and plasma aldosterone ranged from 4.7 to 104 ng/100 ml. Despite these wide ranges, the levels of the two hormones were not correlated. The aldosterone-renin ratio (log.) was inversely related to the plasma sodium concentration, while the plasma renin concentration (log.) was directly related to plasma sodium. Reduced values of both extracellular fluid volume (radiosulphate and sodium spaces) and total exchangeable sodium were measured when plasma sodium was elevated. Sodium depletion at the time when the patient was in a dehydrated state appeared to be caused, at least partly, by defective renal sodium conservation. Thus, in the dehydrated state, the patient showed the following unusual combination of abnormalities: hypernatraemia, sodium depletion, hyperreninaemia, and low to normal plasma aldosterone. The abnormal aldosterone-renin ratio was probably not caused by an intrinsic adrenal abnormality, since high levels of aldosterone were measured as long as a certain degree of hydration had been achieved with or without exogenous ADH, and since plasma cortisol was normal and responsive to exogenous ACTH. The results suggest that the responsiveness of the adrenal cortex to angiotensin may vary with extracellular sodium concentration. The direction of this effect, that is, suppression of aldosterone with increased sodium concentration, is not different from what is observed under experimental conditions, when extra-cellular sodium concentration is raised by infusions of hyperosmolar saline.


Effect of normal and reversed sleep-wake cycles upon nyctohemeral rhythmicity of plasma thyrotropin: evidence suggestive of an inhibitory influence in sleep. The relation of nyctohemeral variation in plasma TSH to sleep-wake cycles was examined in 10 normal young men who had their sleep polygraphically monitored and their blood sampled every 20 min for 24,36, or 48 h periods. Studies of normal sleepwake cycles in which sleep was allowed from the usual bedtime to 0630 h totalled 21 nights (night = 1840-0620 h) and their corresponding 16 days. TSH was measured by a sensitive RIA. On 17 nights, the mean nightly TSH significantly exceeded that of the day's and, on 18 nights, clear nyctohemerally maximal peaks in TSH were seen in the 2100-0100 h interval. Greater amplitude, duration and rhythmic repetition over several nights distinguished 2100-0100 h maxima from a background of persistent briefly episodic release. These nyctohemeral peaks were pre-sleep maxima, as rises uniformly began, and on 15 nights, the peaks occurred prior to the onset of sleep. The peaks clustered within the 30 min just before (12 nights) or after (3 nights) entry into sleep. TSH release then declined across sleep. Other evidence suggestive of an inhibitory influence in sleep upon TSH release was that sleep began early on the 3 nights without clear 2100-0100 h TSH maxima and that the mean 2100-0100 h TSH peak was significantly reduced when sleep began prior to the usual 2300-0000 h interval and significantly increased when the onset of sleep was delayed or postponed. After a 24 h baseline, 4 men underwent phase-reversal of their sleep-wake cycles for 48 h, in which sleep was shifted to the 1100-1830 h interval. On the first wakeful night of reversal, the 2100-0100 h peak began normally, but, in the absence of sleep, the enhanced TSH release then simply continued across this night, delaying achievement of the nyctohemeral maxima. On the second wakeful night of reversal, the maximum in mean TSH lay in the same 0400-0600 h interval as that of first reversal night, and the mean 2100-0100 h peak was no longer evident. The TSH of the second 24 h of reversal also was significantly reduced, suggestive of a negative feedback effect of enhanced release of the first reversal day. No shift of basal pre-sleep TSH peaks to the 0900-1300 h interval or of sleep-enhanced TSH release was seen during reversal. Thus, despite the persistence of TSH's nyctohemeral rhythmicity across acute sleep-wake reversal, its pattern changed significantly in relation to shifts in sleep. We currently view these results as consistent with the origin of TSH's nyctohemeral rhythmicity in a circadian mechanism whose expression is subject to modulation by the inhibitory influences of feedback and sleep.


The effects of ovarian wedge resection on circulating gonadotropin and ovarian steroid levels in patients with polycystic ovary syndrome. Serum gonadotropin, estrogen, and androgen levels were measured daily before and up to 35 days after surgery in 8 patients with polycystic ovary syndrome (PCO) undergoing ovarian wedge resection (WR). To serve as controls, similar assessments were made in 5 women having hysterectomies for non-ovarian disease during the early follicular phases of their cycles. Preoperatively, LH but not FHS, estrone (E1) but not estradiol 17 beta (E2), and both androstenedione (delta) and testosterone (T) levels were higher in the PCO patients than in the normal women. In the PCO patients, surgery had no discernible effect on FSH levels. In the 5 PCO patients who apparently ovulated after WR, LH levels did not fluctuate significantly until the midcycle LH peaks occurred 13-25 days after surgery. In the 3 patients who did not ovulate, there was a transient fall of LH, which reached a nadir on the sixteenth postoperative day and then returned to preoperative levels. In the total PCO group there was a significant fall of E1 (P less than 0.05) and a decrease of E2, which reached nadirs on the third postoperative day. Preovulatory rises of both E1 and E2 were seen only in patients who ovulated following WR. For the androgens, significant decreases (P less than 0.05) of both T and delta were seen during the first 3 days after WR. These were followed by steady increases of both androgens back to preoperative levels. Delta levels remained elevated while T concentrations fell again and were significantly lower (P less than 0.005) at the end of the sampling period than preoperatively. This pattern was seen whether the patients did or did not ovulate. Except for a small transitory fall of androgens, surgery had no discernible effect on the circulating levels of any of the hormones in the control subjects. These results show that in PCO patients, ovarian WR is followed by a profound, temporary reduction of ovarian delta secretion and a persistent reduction of T secretion. Smaller, but significant, decreases of estrogen production were also observed. The mechanism responsible for ovulation following WR appears to be local (intra-ovarian) rather than central since the alterations in ovarian hormone secretion had no discernible effect on circulating gonadotropin levels prior to the onset of midcycle surges.


Inhibitory effect of an ergoline derivative, methergoline, on growth hormone and prolactin levels in acromegalic patients. Plasma levels of growth hormone (GH) and prolactin (PRL) were measured in twelve acromegalic patients after acute administration of an ergoline derivative (methergoline) which has been proposed as a specific serotoninergic blocking agent. The administration of methergoline (4 mg p.o.) was followed by a significant decrease in plasma GH and PRL concentrations. The administration to the same subjects of CB 154 (2.5 mg p.o.), a known stimulator of dopaminergic receptors, led to results almost superimposable to those obtained with methergoline although the suppressive effect of CB 154 on GH and PRL levels was more sustained. Also on the ground of results obtained in these patients with the use of cyproheptadine, phentolamine, or pimozide, we have concluded that methergoline inhibition of GH and PRL release is, in acromegalic patients, most probably due to a dopaminergic mechanism of action.


Evidence for higher proportion of "little" growth hormone with increased radioreceptor activity in acromegalic plasma. Plasma growth hormone (GH) from both normal and acromegalic patients comprises multiple immunoreactive components that can be separated by G-100 Sephadex gel filtration and measured by radioimmunoassay (RIA) and radioreceptor assay (RRA). The higher molecular weight immunoreactive components, from both normal and acromegalic subjects, have a lower RRA/RIA than does the lower molecular weight "little" GH component. The "little" GH component comprises a higher proportion of the total immunoreactive GH in acromegalic plasma than in normal plasma whether the data are expressed only in terms of the "big" and "little" components (89 vs. 71%), or as a function of total immunoreactive GH (76 vs. 55%), or whether the plasma is obtained in the basal or stimulated state for the acromegalic patients. When the RRA/RIA x 100 is determined for the isolated "little" component, the acromegalic has a significantly greater ratio than the normal (110 vs. 75%). We conclude that acromegalic plasma contains a higher proportion of the more radioreceptor active "little" GH component than does the normal and, in addition, that the "little" component from the acromegalic is more radioreceptor-active than the normal.


The fate of 17 beta-estradiol in the plasma of premenopausal and postmenopausal patients with cancer. The patterns of radioactive estrogen metabolities in the plasma of patients with a variety of malignancies were determined after the injection of physiological amounts of 17 beta-estradiol-6,7-3H or of estrone-6,7-3H. The metabolites were separated and isolated from the plasma by the following procedures: a) alumina chromatography, b) modified Brown procedure, and c) gas liquid chromatography. This study showed that the conjugated state of the estrogens varied with the subjects's age. There was a dramatic decrease in the relative quantity of the plasma free estrogens which are converted to sulfates as the age of the patient increased. This change was noted just prior to menopause, suggesting that there are alterations in the conjugation of plasma estrogens which are detectable before clinical menopause.


Unconjugated cortisol in human amniotic fluid: relationship to lecithin/sphingomyelin ratio. A specific and thoroughly validated competitive protein binding radioassay has been developed for the measurement of unconjugated cortisol in human amniotic fluid. Cortisol levels rose throughout normal gestation from 8.6 +/- 0.8 ng/ml at less than 20 weeks to 19.8 +/- 1.5 ng/ml at 30-40 weeks. Levels were significantly higher in amniotic fluid samples having mature lecithin/sphingomyelin (L/S) ratios (greater than 2), and good correlation with individual cortisol values was obtained (r = 0.79; P less than 0.001). These results support the hypothesis that cortisol plays a role in fetal lung maturation.


Free and protein-bound plasma estradiol-17 beta during the menstrual cycle. Methods are described for the measurement of the estradiol-binding capacity of TeBG and of the free, TeBG-bound, and non-specifically protein-bound fractions of plasma estradiol. Each determination used undiluted plasma at 37 C, and a total volume of less than 2.0 ml of plasma was required to complete all the assays. The measurement of the per cent of free estradiol was affected by changes in plasma dilution. The measurement of the other fractions of estradiol was not influenced by changes in either the dilution or the volume of plasma. The distribution of plasma estradiol was determined daily throughout 5 individual menstrual cycles. The per cent of free, the per cent of TeBG-bound, and the TeBG binding capacity of estradiol remained constant throughout the cycle with mean values of 2.21 +/- 0.04% (SE), 38.4 +/- 0.7%, and 16.6 +/- 0.43 ng/ml, respectively. The mean association constant of TeBG for estradiol was 6.58 +/- 0.25 x 10(7)M-1. The concentration of the free and non-specifically protein-bound fractions of estradiol paralleled the total plasma concentration of estradiol. The results show that biologic events related to normal cyclic changes of plasma estradiol may be attributed to fluctuations in the free estradiol and to estradiol which is bound with low affinity to non-specific plasma proteins.


Preoperative diagnosis of occult parathyroid hyperplasia by calcium infusion in patients with multiple endocrine neoplasia, type 2a. Hereditary multiple endocrine neoplasia, type 2 (MEN 2) comprises medullary thyroid carcinoma (MTC) and bilateral pheochromocytoma; the syndrome includes two major variants, MEN 2a (normal appearance, parathyroid disease common) and MEN 2b (mucosal neuromas, Marfanoid habitus, parathyroid disease rate). Patients with MEN 2a may be normocalcemic, with normal basal serum immunoreactive parathyroid hormone (iPTH) levels, yet have parathyroid hyperplasia discovered during thyroid surgery. In an attempt to predict the presence of this occult parathyroid hyperplasia, we performed calcium infusion (15 mg Ca++/kg/4 hours) in six patients with MEN 2a, seven patients with MEN 2b, and eight normal subjects. iPTH was measured in samples taken at one hour intervals during the infusion with a sensitive radioimmunoassay (antiserum GP 1M [Arnaud]). The iPTH values (expressed as per cent of the basal value) in the normal, MEN 2a, and MEN 2b groups at the fourth hour were 38.0 +/- 3.8% (mean +/- SE), 79.2 +/- 7.2%, and 47.8 +/- 5.2%. These iPTH values for patients with MEN 2a were significantly different from normal (P less than 0.001) and from those of the patients with MEN 2b (P less than 0.001). All MEN 2a patients had parathyroid hyperplasia at cervical exploration; parathyroid histology in the MEN 2b patients was normal. Failure of iPTH suppressibility in the MEN 2a patients was not due either to the plasma calcitonin or to achieved serum calcium concentrations. The normal iPTH suppressibility in MEN 2b is consistent with the concept that the parathyroid disease in MEN 2a is genetically determined, and not secondary to MTC and high plasma calcitonin concentration. Calcium infusion combined with the measurement of serum iPTH levels may detect occult parathyroid hyperplasia in patients with MEN 2a, normocalcemia, and normal basal iPTH values. The identification and follow-up of similar patients should establish the natural history of this unusual parathyroid disease.


Dissociation of plasma renin activity and aldosterone in essential hypertension. Plasma renin activity (PRA) and plasma aldosterone (PA) were measured in 401 and 98 patients, respectively, with mild essential hypertension after overnight recumbency and on a 135 mmol/day sodium diet. The mean PRA values declined with each increasing decade of age, whereas the mean PA concentrations did not, and those PRA and PA values determined simultaneously were not correlated, suggesting that PA concentrations are inappropriately elevated relative to the PRA in patients with essential hypertension in the older decades of age.


Circadian rhythm of insulin-induced hypoglycemia in man. Four healthy men were given an insulin tolerance test (0.1 IU/kg BW) six times in different days at the following clock hours: 0400 h, 0800h, 1200 h, 1600 h, 2000 h, and 2400 h, in random order. Four days separated two consecutive tests. The glucose disappearance rate (K value) was calculated for each test between 5 min and 25 min after iv injection of insulin. The statistical evaluation by Cosinor test showed a statistically significant circadian rhythm for the hypoglycemic action of exogenous insulin with a peak at 1030 h (95% condifence limits: 0830 to 1430 h).


Bromocriptine increases plasma estradiol-17 beta concentration in amenorrhea patients with normal serum prolactin. In a series of 23 patients bromocriptine increased the plasma estradiol-17 beta level from 44.0 +/- 9.5 (mean +/- SE) to 144.1 +/- 31.8 pg/ml after 3 - 5 weeks' treatment (p less than 0.01). In normoprolactinemic patients (N = 12) the level increased from 59.6 +/- 16.3 to 186.5 +/- 50.5 pg/ml (p less than 0.05), and in hyperprolactinemic patients (N = 11) the corresponding values were 27.0 +/- 6.2 and 97.8 +/- 34.6 pg/ml (p less than 0.05). Bromocriptine treatment did not significantly alter the FSH and LH levels. The results suggest that bromocriptine treatment induces endocrine recovery also in patients whose clinical findings give no indication of prolactin suppression.


Significance of urinary isolates of coagulase-negative Micrococcaceae. Of 16,347 urine cultures submitted to the hospital laboratory, 68 (0.4%) specimens from 50 patients yielded greater than 10(4) coagulase-negative staphylococci/ml in pure culture. A total of 62 of 63 organisms available for study were staphylococci: 45 Staphylococcus epidermidis (predominantly subgroup 1), 15 Staphylococcus saprophyticus (subgroup 3), and 2 Staphylococcus aureus. Twenty-one patients had "probable" urine infections. Eight patients had two or more positive urine cultures, and all isolates from the same patients were identical (by morphology, antibiotic susceptibility, and hemolytic pattern). Nine (75%) of the 12 isolates of S. saprophyticus, which were novobiocin resistant and nonhemolytic on the synergistic hemolysis test, were from patients with probable urinary infection. Eight were young women with acute symptoms and pyuria. Differences in the glucose and mannitol fermentation tests with different media may lead to difficulties in identification. Novobiocin resistance cannot be relied upon to differentiate isolates of S. saprophyticus from S. epidermidis.


Differential quantitation of surface and subsurface bacteria of normal skin by the combined use of the cotton swab and the scrub methods. By testing adjacent sites on the hypothenar eminence of the palm, enriched with bacteria by massaging the forehead, we found that the numbers of bacteria recovered from the skin surface by a wet cotton swab in 30 s were not significantly different from the numbers obtained by a brisk scrubbing with a blunted Teflon policeman for 120 s. This was true of aerobes (gram-positive cocci) and anaerobes (propionibacteria). If the same site on the palm was swabbed two times for 15 s each time, 67 to 94% of the total recovered bacteria were obtained on the first swab. Differential localization of bacteria into surface and subsurface populations was accomplished by first swabbing a test skin site to assay the surface flora and then scrubbing the same site to test for subsurface organisms. On the palm the swab yielded more aerobes and anaerobes than did the subsequent scrub. On the forehead the scrub yielded three to eight times as many anaerobes as the preceding swab. In some tests gram-positive cocci were distributed on the forehead like propionibacteria (large excess in scrub specimen); in other tests their numbers were similar in the swab and scrub specimens or there was a large excess in the swab specimen. These results indicate that there was no substantial subsurface flora on the palm. On the forehead propionibacteria were predominantly in deeper locations in all tests; gram-positive cocci were variable: in some test sites they were largely at the surface, whereas at other sites a predominance of cocci was in subsurface locations.


Serogrouping of Bacteroides fragilis subsp. fragilis by the agglutination test. The agglutination technique was used to establish a serological classification scheme for 98 strains of Bacteroides fragilis subsp. fragilis isolated from clinical specimens and normal human feces. Absorbed antisera were prepared to seven strains of B. fragilis subsp. fragilis. These seven absorbed antisera were species as well as subspecies specific and provided the basis of the serological classification scheme. This scheme was composed of 21 serogroups; seven of these serogroups contained only one group component. There was a total of 45 serological patterns. This serological scheme may be used for the serological classification of strains of B. fragilis subsp. fragilis and to study the epidemiology of this organism.


Differential agar medium (A7) for identification of Ureaplasma urealyticum (human T mycoplasmas) in primary cultures of clinical material. A differential agar medium for the identification of Ureaplasma urealyticum in primary cultures of clinical specimens is described. The differential medium (no. A7) is specific for the identification of U. urealyticum and other members of the genus Ureaplasma. Large-colony, classical Mycoplasma and Acholeplasma species and Proteus L colonies are unreactive on this differential medium. The medium incorporates the biochemical principle of the direct spot test for urease in colonies of Ureaplasma and contains added urea and a sensitive indicator of ammonia, manganous sulfate. Ureaplasma colonies on this medium are identified as dark golden-brown or rich deep-brown colonies, in sharp contrast to the light background of the medium, when viewed by direct transmitted illumination under the low power of the microscope.


Large-scale purification of hepatitis B surface antigen. Hepatitis B surface antigen was concentrated and purified from plasma by two simple steps of purification. In the first step the antigen was purified 24-fold by polyethylene glycol precipitation. An additional 10-fold purification was achieved by batchwise adsorption to hydroxylapatite and subsequent elution with 0.02 M sodium phosphate buffer.


Long-chain fatty acids of Sporothrix (Sporotrichum) schenckii. A number of strains purporting to belong to the species Sporothrix schenckii were examined for their fatty acid content. The majority of the strains were isolated from cases of sporotrichosis. Two strains were reputedly saprophytic. In all cases except the two saprophytic ones the major fatty acid was a C18 diene. Considerable amounts of palmitic acid and C18 monoene were found in all strains.


The organization and postnatal development of the commissural projection of the rat somatic sensory cortex. Anterograde and retrograde tracing experiments have been used to demonstrate the origin and terminal distribution of commissural fibers in the first somatosensory cortex (SI) of the rat. The commissural fibers originate from pyramidal cells of all layers, but predominantly from layers III and V. The fibers terminate in a series of approximately vertical bands. In each of these there are concentrations of terminals extending from the inner portion of the molecular layer to the deep portion of layer III as well as in the superficial part of layer V, and in layer VI. Discrete vertical bands of cortex are reciprocally connected across the midline to give both the origin and terminal regions of the projection a patchy or "columnar" appearance. The commissural fibers arise from and terminate in areas of the cortex that lie between and alongside the aggregations of granule cells that distinguish SI of the rat. No commissural fibers terminate within the aggregations of layer IV cells themselves but the more superficial terminal ramifications may come to overlie these aggregations. A heterotopic projection to the contralateral second somatosensory cortex has been observed and is similar in form to the homotopic projection to SI. Many commissural fibers have crossed the midline in the corpus callosum by the day of birth but lie in the underlying white matter and do not enter the cortical plate until at least the third postnatal day. During the first postnatal week these fibers grow somewhat diffusely into the maturing cortex and their topographic and laminar pattern of distribution attains its adult characteristics by the end of the first week. Commissural axons, thus, arise from immature cells but the maturation of cell form seems to precede the ingrowth of these axons and the acquisition of commissural synapses.


The effect of age on the reversibility of cellular atrophy in the LGN of the cat following monocular deprivation: a test of two hypotheses about cell growth. Monocular closure soon after birth is known to reduce the growth of the deprived cells in the LGN provided that there are competing cells with normal input. When the closed eye was opened and the open eye closed after three of six weeks of monocular closure, the originally deprived cells in the LGN were able to recover their normal size. However, it was found that after 14 weeks of monocular closure there was no recovery. The ability of the initially closed eye to excite cells in the visual cortex is known to depend on the age when eye closure is reversed in a similar manner. Thus the growth of cells in the LGN is correlated with the possession of effective synapses upon cortical cells. This result is compatible with the possession of effective synapses upon cortical cells. This result is compatible with the hypothesis that competition occurs at the cortical level. It is argued that the alternative hypothesis of competition within the LGN predicts reversibility at any age. In some kittens, the closed eye was opened and the opposite optic nerve crushed. Some evidence was then found of structural recovery even after 14 weeks of deprivation. The hypothesis of cortical competition predicts functional recovery in such kittens, but this remains to be tested.


Light and electron microscopy of the ground squirrel retina: functional considerations. Light and electron microscopy of Golgi-impregnated ground squirrel retinas have revealed a range of morphological subtypes of bipolar, amacrine, and ganglion cells. There are at least seven subtypes of bipolar cells. Those subtypes in which the somata were high (sclerad) in the inner nuclear layer (3 subtypes) had axon terminals low (vitread) in the inner plexiform layer, and those with somata low in the inner nuclear layer (4 subtypes) had axon terminals high in the inner plexiform layer. The bipolar subtypes with high axon terminals made flat contacts with receptor cells, whereas all but one of the bipolar subtypes with low axon terminals made ribbon-related contacts with receptor cells. There are at least five subtypes of amacrine cells. The two subtypes which the Golgi method revealed most frequently were a broad-field, unistratified neuron with a dendritic spread in excess of 1,000 mum and a narrow-field, diffuse neuron with a dendritic spread of about 30 mum. The broad-field, unistratified cell had the lowest proportion of amacrine vs. bipolar cell synaptic input of the amacrine subtypes (43%), whereas the narrow-field, diffuse cell had one of the greatest proportions of amacrine cell input (96%). There are at least 15 subtypes of ganglion cells. The proportion of synaptic inputs to these cells ranged from 21% to 100% amacrine cell synapses. An attempt has been made to relate this new knowledge of retinal circuitry to the physiological output of the ganglion cells.


Polarization of the avian retina. Ocular transplantation studies. The optic fiber layer of the avian retina, as seen in flat mounts, contains morphological markers of vertical and horizontal polarity. Optic vesicles were rotated in chick embryos prior to the appearance of the optic fiber layer. Later, the optic fiber pattern was examined to determin if its polarity was inverted, or upright or if its right-left asymmetry changed. Large grafts showed no tendency toward axial realignment, regardless of the stage of operation. Moderate size grafts became realigned mainly by derotation. In this process the vertical axis of the graft showed a greater tendency to derotate than the horizontal axis. Thus, left-to-right transplants, when performed prior to embryonic stage 12, developed as left retinas with normally aligned vertical axes, regardless of the degree of operative rotation. Small grafts mainly showed cross-axial regulation, as opposed to derotation; the polarity of the horizontal axis became fixed prior to the vertical axis. The equatorial zone of the optic vesicle, which lies about midway between the distal tip of the vesicle and the midline of the embryo, was included in moderate size, but not in small grafts. This zone appears critical in the transfer of morphological information to the developing retina.


Alterations in receptive field properties of superior colliculus cells produced by visual cortex ablation in infant and adult cats. To determine if functional alterations in the superior colliculus might account for recovery of visual behaviors following visual cortex removal in infant cats, the receptive field characteristics of single units in the superior colliculus of cats whose visual cortex was removed within the first week of life were compared with those of cats which sustained visual cortex lesions in adulthood and with those of normal cats. In the normal superior colliculus, 90% of all cells responded to moving stimuli irrespective of shape or orientation. Sixty-four percent of these units were directionally selective, responding well to movement in one direction but poorly or not at all to movement in the opposite direction. Ninety percent of units were binocular, the vast majority of these responding equally to stimulation of either eye or showing only slight preference for stimulation of the contralateral eye. Responses to stationary flashes of light were observed in only 33% of all visually activated cells in the normal superior colliculus. After visual cortex ablation in adult cats, only six percent of movement sensitive cells were directionally selective. Binocular preference was shifted following adult visual cortex lesions such that sixty percent of all cells responded exclusively or predominantly to stimulation of the contralateral eye. Seventy-one percent of all visually responsive units responded to stationary lights flashed on or off within their receptive field boundaries. Lesions limited primarily to area 17 had the same effect as larger lesions of visual cortex. Infant visual cortex lesions resulted in receptive field alterations similar to those observed after adult ablation. Only fifteen percent of motion sensitive units were directionally selective. Seventy-one percent responded exclusively or predominantly to stimulation of the contralateral eye. Seventy-six percent of visually responsive cells were activated by stationary light. Lesions largely confined to area 17 produced the same alterations as more extensive lesions of visual cortex. Thus, no evidence was found that the superior colliculus is involved in the functional reorganization presumed to occur following visual cortex ablation in infant cats. Recovery of visual behaviors following neonatal injury may therefore not involve alterations in the receptive fields of single cells.


Cortical cells projecting to the dorsal column nuclei of cats. An anatomical study with the horseradish peroxidase technique. The retrograde transport of horseradish peroxidase (HRP) has been used to label cortical neurons which give origin to descending projections to the dorsal column nuclei (DCN) in kittens and adult cats. HRP was injected unilaterally or bilaterally into the dorsal medulla at, or rostral to, the level of the obex, and, in most cases, retrogradely transported HRP was visualized in cortical neurons by incubation of serial 40 mum coronal or sagittal frozen sections. In the remaining cases, 1 mm coronal or sagittal slabs through the anterior part of the cerebral hemisphere were cut and incubated en bloc, and embedded in celloidin for sectioning (40 mum). HRP-positive neurons identifiable as the source of cortical projections to the DCN were layer V pyramidal cells whose largest diameter ranged from 20 to 45 mum: no "giant" cells were labelled. The cells appeared concentration in the fore-and hindlimb regions of the sensorimotor cortex and, to a lesser extent, in the second somatosensory area contralateral to the injected side. Labelled neurons were especially numerous in the upper bank of the cruciate sulcus and in the medial wall of the posterior sigmoid gyrus which respectively form parts of areas 4 and 3a (Hassler and Muhs-Clement, '64). The number of labelled neurons progressively diminished in the first somatosensory area proper (areas 3b, 1 and 2). Ipsilaterally, fewer labelled neurons were present, mainly in areas 4 and 3a. These results are in genral agreement with previous anatomcial and electrophysiological studies. It is suggested that previous results based upon lesions or electrical stimulation of the cerebral cortex in cats have failed to reveal adequately the cortical regions containing the bulk of the cells projectng to the DCN, since these regions have escaped selective experimental manipulation due to their relative inaccessibility.


Modification of the pigeon's visual startle reaction by the sensory environment. A series of seven experiments related amplitude and latency of the pigeon's startle response, elicited by an intense visual stimulus, to antecedent auditory and visual events in the sensory environment. The data indicated that (a) within broad limits the amplitude of the reflex is a positive function of the intensity of the sensory background prevailing at the time of startle elicitation, (b) a change in the sensory environment occurring 15-2,000 msec prior to the startle-eliciting stimulus inhibits the amplitude of the response, and (c) a change in the sensory environment less than 10 msec prior to the startle-eliciting stimulus reduces the latency of the response. These findings are consistent with previous research on acoustic elicited startle in the rat. The overall configuration of the results suggests that a pathway including the reticulospinal tract and the bulbopontine reticular nuclei could be the major mediator of startle. In these terms, latency-reduction effects would occur because of partial activation of this pathway, amplitude inhibition would occur because of cerebellar influence, and amplitude facilitation would reflect cerebral or striatal influences.


Ontogeny of defensive reactions in Norway rats. Young rats of several ages were presented with stimuli (a caged domestic cat, a footshock, and a suddenly moving object) known to be aversive to adults and disruptive of behavior in mature animals. Twenty-day-old rats were relatively unaffected by these events, while rats aged 30 days and older tended to reduce their locomotion and freeze upon the presentation of these cues. These data are consistent with Bolles' hypothesis that shock-elicited responses are innate defensive reactions. We also suggest that the inefficient passive avoidance learning in juvenile rats may result from their deficit in shock-induced freezing.


Influence of physostigmine upon consolidation of memory in mice. The influence of physostigmine upon the consolidation process was investigated in a series of four experiments. Male mice of a DBA/2J strain were trained to escape shock in a Y-maze. In Experiment 1, physostigmine (.4 mg/kg, ip) impaired a previously learned task 2 days after initial learning but improved performance 11 days after initial learning. In experiments 2 and 3, it was determined that the impairment of memory found on Day 3 was transitory and no longer evident once the drug was no longer active in an animal's system. The findings of Experiment 4, in which the Day 12 facilitory effect was examined, were essentially the same as those of Experiments 2 and 3. The results of the experimentation tend to support an inhibitory or motivational hypothesis rather than a consolidation hypothesis.


Effects of lesions of the gustatory necortex on taste aversion learning in the rat. Normal rats and rats with lesions of the gustatory neocortex (GN) were compared for their ability to learn aversions to taste cues paired with toxicosis. When the taste presentation was followed immediately by toxicosis, normal rats and rats with lesions of the posterior (visual) neocortex learned aversions to sucrose, sodium chloride, quinine hydrochloride, and hydrochloric acid solutions. Rats with GN lesions learned aversions to all solutions except sucrose. In preference tests, all solutions were shown to be discriminable from water by both normal rats and rats with GN lesions. Under conditions in which a 6-hr delay separated taste presentation and toxicosis, normal rats again learned specific aversions to all four solutions, but rats with GN lesions failed to learn specific aversions to sucrose, sodium chloride, and hydrochloric acid solutions. It was shown that the ability of rats with GN lesions to learn aversions to sucrose and quinine depended on stimulus concentration; and it was proposed that the data could be accounted for by postulating a change in the threshold for taste-illness associations following GN lesions.


Conditioned taste aversions and neophobia in rats with hippocampal lesions. In the first experiment extensive hippocampal lesions retarded, but did not prohibit, the conditioning of a strong taste aversion to physiological saline (the conditioned stimulus; CS) when illness (the unconditioned stimulus; UCS) was induced by injecting rats with apomorphine 15 min following ingestion of the saline. In the second experiment hippocampal lesions reduced the aversiveness of novelty in a drinking fluid for the thirsty rat. It was suggested that the mild impairment of taste aversion learning in the rats with hippocampal lesions was not the result of destruction of mnemonic mechanisms that serve to span the long CS-UCS interval but rather that the reduced intensity of the aversion resulted from a lesion-altered neophobic disposition that weakened the saliency of the novel flavor CS.


Cued DRL training: effects on the permanence of lesion-induced overresponding. The purpose of the present study was to determine the degree to which lesions in the septum and other anatomically related structures result in the presence and/or permanence of an overresponding symptom on a differential-reinforcement-of-low-rate (DRL 20 sec) schedule. Animals were given 15 days of training to determine the presence or absence of overresponding. Then, animals that overresponded were divided into two groups, with one receiving 15 days of cued DRL training and 15 days of regular DRL training while the other received 30 days of regular DRL training. Overresponding occurred following lesions in septum, hippocampus, medialis dorsalis, and ventral thalamus pars dorsalis. While in effect, cued DRL facilitated performance in controls and in operated animals but did not facilitate performance following its removal in septals. Although the hippocampals continued to overrespond with extended training on a regular DRL schedule, exposure to the cued DRL allowed hippocampals to reduce responding and increase the frequency of obtained reinforcements. Lesions in medialis dorsalis and ventral thalamus led to an overresponding that disappeared with prolonged regular DRL training. Finally, it was shown that the cued DRL training actually functioned as a time-out from DRL training. The variations in the permanence of the overresponding symptom according to lesion locus preclude the identification of the lesion-induced dysfunction based solely on the presence or absence of overresponding.


Facilitation and nonfacilitation of active avoidance behavior of rats with septal lesions in the shuttle box and running wheel. It was hypothesized that facilitation of avoidance performance of rats with septal lesions occurs only in tasks that punish responses having the same topography as the avoidance response, such as intertrial responses, or tasks that have aversive consequences for making the avoidance response, such as a brightly illuminated safe compartment. Twenty-eight male rats were trained in two shuttle box tasks, and 24 were trained in two running-wheel avoidance tasks under conditions of punishment or nonpunishment of intertrial responses. Rats with septal lesions performed better than control rats in both the shuttle box and the wheel tasks when intertrial responses were punished. When intertrial responding was not punished, experimental and control groups did not differ in avoidance performance. Avoidance performance of punished and unpunished rats with septal lesions did not differ from each other or from unpunished control rats in either wheel or shuttle box tasks. These results were discussed in the context of the species-specific defense reaction (SSDR) avoidance theory of Bolles. It was suggested that septal lesions interfere with the suppression of ineffective SSDRs.


Mechanisms for enhanced feeding in the cold in rats. The effect of environmental cold upon feeding, food-motivated behavior, and gastric clearance of food was studied in rats. Rats ate liquid diet in either a 5 degrees C or 22 degrees C ambient temperature (TA) following (a) 24-hr food deprivation at TA of either 5 or 22 degrees C, or (b) 0- or 48-hr food deprivation at TA 22 degrees C. Rats ate more at 5 degrees C than at 22 degrees C regardless of the ambient temperature during deprivation. Rats increased feeding in the cold by increasing meal frequency but not meal size. Cold (5 degrees C) TA also enhanced the urge to eat. Rats bar pressed for food more often in the cold TA on a variable interval 30-sec schedule when gastric clearance of food was prevented by a pyloric noose and when food cleared from the stomach normally. Also, quinine adulteration of food suppressed food intake only in the 22 degrees C TA. Cold (5 degrees C) TA enhanced gastric clearance of liquid food within 20 min of ingestion. These results support the view that the peripheral sensation of cold is an adequate stimulus for feeding and that cold TA can stimulate feeding directly by increasing the urge to eat and indirectly, secondary to increased gastric clearance.


Response to lactating dairy cows to abomasal infusion of amino acids. Essential amino acids most limiting for milk production and/or secretion of milk protein were determined in five trials involving infusion of amino acids into the abomasum of lactating Holstein cows. Cows were fed corn-based rations containing 10.7 to 11.5% crude protein (dry matter basis) which exceeded standard allowances for energy. All trials were Latin-square designs with 9-day periods. The abomasal infusion of methionine had no effect on secretion of milk, milk protein, or milk fat. Lysine infusion resulted in 16% of the total response in yield of milk protein that was obtained with either the 10 essential amino acids or sodium caseinate while infusion of lysine and methionine together accounted for 43% of the total response. This suggested that lysine and methionine were first and second limiting, or co-limiting, for secretion of milk protein when rations consisting primarily of corn, corn silage, and alfalfa-grass hay were fed. The primary effect was on content of milk protein rather than milk yield. In general, amino acid infusions had no effect on feed intake, milk fat, and nonprotein nitrogen content of milk. Little change occurred in concentrations in plasma of other amino acids when lysine and methionine were infused together. When concentrations were lowered, indicating increased protein synthesis, changes were too small to be statistically significant.


Nutritive value of potato-based silages for dairy bulls. Diet acceptability, nutrient digestibility, and nitrogen and energy partition were measured in growing Holstein bulls to compare corn silage with three potato-based silages which consisted of (fresh basis): 1) 50% potatoes, 20% hay; 2) 60% potatoes, 20% hay, 20% whole oats; 3) 50% potatoes, 25% hay, 25% whole oats. The 50% potato 50% hay silage dry matter was consumed at 3.0% of body weight per day which was greater than for any of the other three silages. Consequently, daily consumption of each nutrient was generally higher for this silage even though it was lower in content of many nutritional components. Except for ether extract, the digestion coefficients for the 50% potato 50% hay silage were higher than for the other three silages. The addition of whole oats to the potato-based silage tended to depress apparent digestibility of the nutritional components below those for corn silage. Both nitrogen and energy utilization were higher for the 50% potato 50% hay silage than for any of the other three experimental silages. Efficiency of use of absorbed nitrogen was higher for the oat-containing potato-based silages than for corn silage while the oat-containing silages were comparable to corn silage in energy efficiency. The 50% potato 50% hay silage was equal or superior to corn silage in dry matter intake, overall overall digestibility, nitrogen and energy balance for growing Holstein bulls.


Plasma lipids, ketone bodies, and glucose concentrations in calves fed high- and low-fat milk replacers. Twenty-four 5-day-old male calves were fed twice daily milk replacers containing either 5% (low-fat) or 25% (high-fat) lard. Plasma lipids, blood glucose, and ketone bodies were determined in jugular blood before feeding and every hour during 8 h after feeding. The high-fat diet caused in the 1st h after feeding a sharp increase of triglycerides and phospholipids followed by a sharp decrease; these two increased slowly during the following 5 h. Within the first 2 h after feeding, there was an increase of cholesterol esters, free cholesterol, and nonesterified fatty acids. With the low-fat diet, triglycerides and cholesterol esters showed a small increase during the 4 h following meal whereas phospholipids, free cholesterol, and nonesterified fatty acids were not affected significantly. With both diets, blood glucose reached a maximum of 110 mg/100ml 1 h after feeding; ketone bodies were not altered significantly. With the high-fat diet, lipid digestion would occur in two phases; firstly, part of the fat would be lipolyzed quickly by pregastric esterase before clot formation in the abomasum; secondly, the rest of the lipids, slowly released by progressive lysis of the coagulum would be digested under the action of gastric and pancreatic lipases. The first phase did not occur with the low-fat diet.


Influence of environment on passive immunity in calves. Passive immunity in neonatal calves is influenced by environment. Placing newly born Holstein calves (108 head) in three different housing environments (shade, cooled shade, hutch) during hot weather produced differences in body temperature, serum cortocosteroids, immunoglobulin IgG1 concentrations, and mortality. Experimental design permitted examination of effects due to treatments, time, differences in colostrum, and climatic environment in an analysis of variance. Calves exposed to the hotter, less desirable environment responded by having a higher mortality, higher serum corticosteroid concentration, and lower serum immunoglobulin IgG1 at 2 and 10 days after birth. All of these were correlated. Calves that died had serum immunoglobulin IgG1 falling below the mean for all experimental calves.


Synthesis of ruminal microbial protein and volatile fatty acid production in vitro. Growth of a mixed ruminal population taken from a sheep on a protein-free (all urea) purified diet was estimated by an in vitro fermentation technique including precipitation of microbial protein by trichloracetic acid. Volatile fatty acid production in vitro was determined, and the associated a denosine triphosphate was estimated as moles volatile fatty acids X 2.4. On this basis, the quantity of microbial protein synthesized per mole of adenosine triphosphate increased at higher microbial growth rates.


Changes in neurologia and myelination in the white matter of aging mice. Changes with age were investigated in the anterior and posterior limbs of the anterior commissure of the brains of 5-mo-old and 18 mo-old mice. In the 18-mo-old animals the total number of glia was reduced by 40% in both limbs. All types of glia were reduced in numbers, the astrocytes being most affected. Ultrastructural changes were also most obvious in astrocytes. There was no significant change in the number of axons, percentage myelination, axonal diameter (myelinated or unmyelinated) nor in the mean number of myelin lamellae in either limb in the 18-mo-old animals. The percentage of redundant myelin sheaths, however, increased significantly in 18-mo-old animals.


Fatty marrow in the vertebrae. A parameter for hematopoietic activity in the aged. Fatty alteration of bone marrow was analyzed in upper lumbar vertebrae obtained from 94 autopsies. Of these, 85 were from individuals over 60 years of age. Fatty marrows were classified grossly into three groups: Group I, a massive type, Group II, a moderate or less significant type; Group III, a nonfatty type. Fatty marrows of Groups I and II were found most frequently in the older subjects. They were not observed (0/9) below 60 years of age, but were present in 42% (5/12) in the 6th decade, 61% (23/38) in the 7th decade, 76% (25/33) in the 8th decade, and 100% (2/2) in the 9th decade. This may relate to a decreasing volume of a reservoir stem cell compartment for blood cell generation. Since the vertebral marrow is the last reserve site for hematopoietic activity, the volume of fatty marrow in the vertebrae may be used as a parameter for the estimation of hematopoietic activity in the entire skeletal system in the aged.


Age-related decrement in hearing for speech. Sampling and longitudinal studies. A 10-year study was conducted in which 282 adults in age decades of 20 through 80 were tested in a comprehensive battery of tasks requiring the perception of speech under a variety of conditions in which the speech signal was degraded. The original results were plotted to reveal the differences in performance of the population samples of each decade. In follow-up studies, after 3 years and again after 7 years, samples of the original subject population were retested. The results of the first, stratified sampling and of the longitudinal studies are compared. The two most apparent trends are: (1) the perception of degraded (distorted and competed) speech undergoes a noticeable decline beginning with the 5th decade of life, and (2) the decline is sharply steeper in the 7th decade.


A relationship between functional age and health status in female subjects. A multiple regression equation was used to predict age from seven clinical variables in 1080 apparently well female subjects. A multiple correlation coefficient of R = 0.77 was achieved by five of the variables: timed forced expiratory volume, systolic blood pressure, plasma urea nitrogen, cholesterol, and alkaline phosphatase. On the basis of selection by medical questionnaire responses and other objective criteria, 9% of the subjects were nonsmokers and healthier than the rest. These selected subjects showed a significant reduction in preducted age. Within this group, subjective perception of health was associated with differences in predicted age: poor health with an increase and good health with a decrease in functional age. This study of functional age was based on the healthiest segment of the population in order to minimize the effect of overt pathological processes on the aging rate. An association has been demonstrated between health impairment and predicted age as a measure of the aging rate.


Effects of age and frequency of stimulus repetitions on two-choice reaction time. Binary choice-reaction times for 123 males, 25-79 years of age, were obtained in a sequence of discrete trials in which the proportion of occasions that the same stimulus was presented twice in succession varied from .25, .50, and .75 or from .75, .50, and .25 over successive thirds of the sequence. Stimulus alternations were responded to more rapidly than stimulus repetitions by subjects in all age groups, especially in trial blocks with .75 alterations. The absolute differences in mean reaction time to stimulus alterations and repetitions increased with age. The results are consistent with the hypothesis that reaction times are affected both by pre-existing expectations for stimulus alternations as well as expectations for stimulus alternations or repetitions developed during the course of the experiment. Mean response latency as well as between- and within-subject variability in performance increased with age. Analyses of frequency distributions of the reaction times indicated that the psychomotor component of reaction time increases with age while the decision-making component becomes more variable.


Age differences in personality structure: a cluster analytic approach. A cluster analytic approach was used to determine possible age differences in the structure of personality as measured by the Cattell Sixteen Personality Factor Questionnaire. Subjects were 969 adult male volunteers, originally divided into three groups: 25 to 34, 35 to 54, and 55 to 82. Two familiar dimensions of personality, Anxiety-Adjustment and Introversion-Extraversion, were found to be cross sectionally stable. A third cluster dimension, interpreted as openness to experience, showed age-related differences. Young subjects showed openness to feelings; middle-aged subjects showed openness to ideas; and old subjects showed a balanced openness to both feelings and ideas. Repartitioning of the sample into two groups (ages 25 to 43 and 44 to 82), and three groups (25 to 39, 40 to 47, and 48 to 82) confirmed these age differences in the third cluster. The interpretation of the third cluster is discussed in terms of Jung's concepts of psychological functions.


Cautiousness in adulthood as a function of age and instructions. Forty-eight younger (average CA = 21.71, SD = 3.58) and older (average CA = 69.31, SD = 3.84) males participated in a vocabulary task involving varying degrees of risk under neutral, supportive, and challenging instructions. The results indicated that older adults were more cautious than younger adults. Moreover, they selected tasks at which they would have higher probabilities of success; relative to their younger counterparts, they were less likely to raise their level of aspiration following success. No effect of instructions on cautiousness was found for either age group. The results were interpreted as indicative of the significant age differences in the achievement motivational dispositions of the groups. The findings suggest that older adults choose relatively easy tasks as a means of protecting themselves from engaging in self-evaluation of important ability dimensions.


Age differences in humor comprehension and appreciation in old age. Ninety-six subjects in three age groups (50-59, 60-69, 70-79) who resided at home were tested on their appreciation and comprehension of 12 conservation and 12 nonconservation jokes. The conservation jokes depended on the Piagetian concepts of conservation of mass, weight, and volume for their comprehension. Cognitive ability of the subjects on the three conservation tasks was also tested. Appreciation increased with age and comprehension decreased with age for both types of jokes. The ability to conserve volume declined with age. Among conservers of volume, appreciation of conservation of volume jokes increased with age; among nonconservers, it tended to decrease. The study provided some support to the hypothesis that the cognitive perceptual theory of humor applies in old age as it does in childhood, with appreciation depending on a match between ability level and the cognitive demand of the joke.


A seven-year follow-up of intelligence test scores of foster grandparents. After 7 years, a group of originally nonemployed poverty-level older people (over 60) who had been employed as foster grandparents were retested with the WAIS. Four WAIS subtests - Vocabulary Similarities, Digit Span, and Block Design - were employed. Of the original group of 39, complete data were available for 28; 18 of these were still working on the project, and the other 10 had dropped out. Dropouts as a group tested lower originally and also showed more deterioration in functional health ratings over time. For the total group of 32 foster grandparents, three subtest scores showed stability over the 7 years. Only Digit Span showed a statistically significant drop. Neither age nor the initial level of health or WAIS scores was related to test-score changes over time.


Attitudes toward aging and towards the needs of older people. In order to explore the underlying dimensionality of beliefs about aging, two sets of opinion statements (one dealing with general attitudes toward aging and the other with programmatic issues) were factor analyzed for two age groups: persons under 60 (N = 290) and persons 60 and older (N = 181). For the first set of social-psychological belief statements, a common factor structure was found for the two age groups. For the second set of programmatic statements, there was some communality between age groups, but most of the items loading on the factors tended to be age-specific. The belief structure of the older group was generally more complex and variegated than was the case with the younger group. Except where the items were personal relevance, positive and negative items tended to load on separate, unipolar factors.


Predicting the dimensions of life satisfaction. Taking multidimensional life satisfaction as the basic premise of this study, a four-equation multiple regression model was constructed for its prediction. The set of regressors included some conventional biographical characteristics, a number of activity variables, and three indicators of retirement experience. A stepwise regression estimation method was adopted. Results indicated that the pattern of regressor influence varied greatly between equations, providing fairly specific evidence on a number of previously espoused hypotheses. In particular, the Activity perspective on aging was found to be important in the prediction of over-all affect or mood tone, but of little relevance for the other dimensions of life satisfaction, and a modicum of support for the Disengagement perspective was observed.


Expectancy theory prediction of the preference to remain employed or to retire. Vroom's (1964) expectancy theory model was used to predict older workers' choices between continued employment or immediate retirement. It was hypothesized that a person's preference for one of the two alternatives would be a function of the differences between the instrumentality of employment and the instrumentality of retirement for the attainment of outcomes, multiplied by the valence of each outcome, summed over outcomes. To test this, 290 Israeli male workers, aged 57 to 64, were interviewed. Measures included: preference for employment or retirement; valences of 35 outcomes; perceived instrumentalities of employment and retirement. The results supported the hypothesis (R = .40; p less than .01). It is suggested that further research along these lines could equip organizations with a tool for assisting older employees in the transition to retirement or for encouraging those who are still capable of making a significant contribution to remain employed.


Inpatient care or outplacement: which is better for the psychiatric medically infirm patient? Eighty-four geriatirc ward patients were randomly assigned to groups targeted for outplacement planning or inpatient care. During the following year, half of the former were placed in community settings while nearly all of the latter remained in the hospital. Neither the physical health, level of self-care, or psychiatric condition ratings of the two groups changed differentially over that time. However, the mean Morale Inventory score of the outplacement sample improved while that of the in-patient group remained static. Additional analyses were run between three groups - outplacement planning subjects who were released, those who were not released, and inpatient planning men who were not released. These results confirmed those of the first analyses; the only significant difference appeared on the Morale Inventory, the patients who left showing more improvement than the other two groups. The results argue for an increased emphasis on outplacement programs among geriatric patients.


Cytochemical demonstration of extraperoxisomal catalase. I. Sheep liver. In sheep hepatocytes catalase activity was demonstrated both within peroxisomes and within the cytosol. In the cytosol the catalase reaction product is contiguous to the plasma membrane and surrounds the nuclei, rough endoplasmic reticulum, cisternae, mitochondria and Golgi apparatus. This is the first cytochemical demonstration of guine extraperoxisomal catalase. No catalase reaction product was seen in the cytosol of nonparenchymal cells. To demonstrate catalase, both glutaraldehyde and formaldehyde fixation were used, followed by a diaminobenzidine technique modified from Novikoff and Goldfischer. Control reactions were performed to distinguish catalase reaction product from adsorption of oxidized diaminobenzidine and from precipitate due to oxidase-, peroxidase- or heat-stable peroxidatic activities. The results were evaluated in the light and electron microscopes.


Induction of resistance to Toxoplasma gondii in human macrophages by soluble lymphocyte products. Autoradiography and light microscopy were used to study the effects of lymphocyte culture supernatants, prepared under a variety of conditions, on the course of intracellular Toxoplasma gondii infection in human monocyte-derived macrophages in vitro. Supernatants prepared by incubating lymphocytes of dye test- (DT) positive subjects with T. gondii lysate antigen (TLA), lymphocytes of DT/negative subjects with streptokinase-streptodornase (SK-SD), or both populations of lymphocytes with concanavalin A (Con A) were capable of activating macrophages to inhibit or kill intracellular T. gondii. Supernatants prepared with the homologous antigen (TLA) to the target organism appeared more active in conferring resistance to infection with T. gondii on macrophages than those prepared with a heterologous antigen (SK-SD) or mitogen (Con A). The number of lymphocytes was critical in preparing active supernatants. These results suggest that soluble lymphocyte mediators can activate human macrophages in vitro to inhibit or kill T. gondii.


Purification of native properdin by reversed affinity chromatography and its activation by proteolytic enzymes. A highly purified preparation of human properdin (P) has been obtained in a simple two-step procedure utilizing reversed application of the technique of affinity chromatography. The method involved precipitation of properdin from human serum and subsequent passage through an immunoadsorbent column of Sepharose anti-RP globulin which bound the contaminating proteins. The immunochemical properties of the isolated properdin (P) was found to be different from those of activated properdin (P). P was shown to be a 6.1S, beta2 globulin with a mean subunit m.w. of 57,900. P on the other hand was a 5.1S protein with gamma2 mobility and a subunit m.w. of 53,000 daltons. Double diffusion analysis using anti-P revealed a reaction of identity between P and P. However, when the reaction was developed with anti-P, a reaction of partial identity was obtained and the precipitin line of P was seen to spur over the line developed with P. Mild treatment with plasmin or trypsin converted P to P. Unlike P, P was ineffective in triggering the activation of the Properdin System in RP unless trace amounts of zymosan were added. Under these conditions P was found to be converted to P. The results indicate that properdin is present in fresh serum in a precursor form and its activation to P involves a limited proteolytic cleavage of the molecule.


Control of Fc and C3 receptors on myeloid leukemic cells. An experimental system has been developed to study in cloned lines of cells the control of Fc and C3 receptors by different compounds. The cells used were clones of mouse myeloid leukemic cells and the compounds used were the protein MGI2 (macrophage and granulocyte inducer) in serum from mice injected with bacterial endotoxin and the steroid inducer (SI) dexamethasone. Eight clones were isolated which could be divided into three groups. One group (MGI+SI+) was induced to form EA and EAC rosettes by MGI and only EAC rosettes by SI, the second group (MGI-SI+) was not inducible by MGI but was induced by SI to form EA or EA and EAC rosettes, and the third group (MGI-SI-) was not inducible for EA or EAC by MGI or SI. There were two types of MGI+SI+ clones, one type (D+) could be induced by MGI to differentiate to mature macrophages and granulocytes, and the other type (D-) could not be induced to differentiate to mature cells. The MGI-SI+ and MGI-SI- clones were all D-. The results indicate that there are different cellular sites for MGI and SI and that induction of EA and EAC rosettes did not seem to be mediated by cyclic AMP. Experiments with specifically bound 3H-BSA-anti-BSA complexes have indicated that there was an increase in the amount of 3H-BSA-anti-BSA bound per rosette-forming cell following induction by MGI or SI, and there were differences in the amount of 3H-BSA-anti-BSA bound per rosette-forming cell in different clones. These clones also showed differences in the shape of the curve for the number of EA rosette-forming cells obtained with erythrocytes coated with decreasing concentrations of antibody. The results suggest that such curves and those obtained with EAC rosettes can be used to determine the relative abundance of EA and EAC receptors on rosette-forming cells. EA rosettes on the myeloid leukemic cells, like those on normal macrophages and granulocytes, were specifically inhibited by IgG2a and by the Fc but not the Fab fragment of IgG. The EAC rosettes were inhibited by destroying the C3 component of complement. The different clones maintained their specific properties for at least 6 months in culture. The present system should, therefore, also be useful for studies on the genetic control of the regulation of Fc and C3 receptors.


Immunoglobulin binding by mouse intestinal epithelial cell receptors. The IgG receptors involved in transporting maternal antibodies to the blood stream of the young mouse were studied by feeding internally labeled mouse myeloma proteins and enzymatically generated specific fragments of these proteins. The data obtained from competition experiments showed the following: 1) intestinal receptors have a single specificity which is directed against IgG Fc determinants; 2) IgG subclass proteins differ in their affinities for the receptors and thus have similar but not identical Fc recognition sites; 3) the order of affinities of the IgG subclass proteins for the receptors correlates with the relative concentrations of the four subclass proteins in mouse milk; and 4) several subfractions of the Fc fragment were inactive in binding to the receptors. In many of their properties the intestinal receptors resemble the Fc receptors on other cell types, but they are apparently unique in their ability to bind tightly non-aggregated IgG molecules.


Strain-, age-, and tumor-dependent distribution of terminal deoxynucleotidyl transferase in thymocytes of mice. The distribution of terminal deoxynucleotidyl transferase (TdT) peak I and peak II in thymuses and peripheral lymphoid tissues was examined in a variety of mouse strains in relation to tumor burden and age. TdT peak I was found to be present in all strains at comparable levels, which did not change with age. TdT peak II levels were also comparable for the strains examined at 1 week of age. In contrast to peak I, however, in NIH Swiss and AKR mice, peak II activity decreased rapidly at 2 weeks of age and by 6 weeks of age less than 2% of the initial activity remained. In C57BL/6 mice there was a similar loss of peak II activity with age although this change started at about 4 months of age and by 8 months of age approximately 15% of the initial activity remained. These changes did not appear to be due to the presence of an inhibitor. Leukemic C57BL/6 and AKR mice were also examined for TdT. Both strains characteristically had TdT peak I in peripheral lymphoid tissues infiltrated with transformed thymocytes. AKR mice had only TdT peak I in the thymus, whereas C57BL/6 thymuses had both peak I and II at levels comparable to age-matched controls. No aberrant distribution of TdT was observed in a spontaneous reticulum cell sarcoma or Rauscher MuLV-induced erythroblastosis.


C3 requirements for formation of alternative pathway C5 convertase. Although alternative pathway C3 and C5 convertases both have active proteolytic sites dependent on the same protein, Bb, the quantitative requirements for the expression of these activities are sufficiently different to permit their delineation in terms of B input an cell-bound C3b. That the labile component of each active site is Bb was established by their parallel decay rates, regeneration of the original specificities with B in the presence of D, and stabilization of each convertase by C3NeF. The evidence that the spatial relationships of Bb and C3b on the cell surface for C3 and C5 convertase activities are distinct is based not only upon the decay and regeneration of each original convertase but more so upon their interconversion. C3 convertase is converted to C5 convertase by interaction with additional C3 whereas C5 convertase reverts to a C3 convertase by treatment with C3 INA. The capacity of C3 INA treatment to abolish C5 convertase sites without affecting C3 convertase sites indicates the existence of two functional species of C3b, one of which is protected in the C3bBb complex whereas the other is exposed.


Altered lymphocyte functions in rats bearing syngeneic Moloney sarcoma tumors. II. Suppressor cells. Spleens from tumor-bearing rats contained a population of adherent cells that suppressed lymphocyte responses to Con A and LPS and cell-mediated cytotoxicity (CMC). By use of various fractionation procedures, Ig+ cells, T cells, and null cells were excluded as candidates for the suppressor cell activity. Procedures that removed macrophages abolished the suppressive activity whereas those procedures that enriched for or involved direct addition of macrophages resulted in augmented suppression. On the basis of these results, we conclude that the suppressor elements in spleens of tumor-bearing rats were macrophages.


In vitro activation of mouse macrophages by rat lymphocyte mediators. Macrophage-activating factors (MAF)3 were released by presensitized rat lymphocytes stimulated in vitro with the appropriate antigens. Different supernatants of presensitized rat lymphocytes specifically stimulated in vitro with several different mouse, dog, and rat tumor or normal cells were capable of rendering normal rat and mouse macrophages nonspecifically cytotoxic in vitro to their respective syngeneic tumor cells. The release of active mediators by rat lymphocytes sensitized in vivo was dependent upon immunologically specific recognition of an antigen in vitro. When rat lymphocytes were incubated in vitro with antigens unrelated to the in vivo sensitizing antigens, no release of MAF occurred. Once rat MAF was released, it activated both syngeneic (rat) and xenogeneic (mouse) macrophages to kill tumor cells in vitro. These activated marcophages destroyed all syngeneic tumor targets. Such cytotoxicity was obtained even when the cells used to elicit release of MAF were totally unrelated to the target tumor cells. The data thus demonstrated that MAF can cross strain and even species specificities and can activate macrophages to kill tumors in a nonspecific manner. The cytotoxicity mediated by in vitro activated mouse macrophages decreased with time once the macrophages were removed from MAF; and by 7 days postactivation, the macrophages were not cytotoxic. However, when incubated again with MAF, significant reactivation was observed. This suggested that activation of macrophages in vivo may be a continuous process of lymphocyte-macrophage interaction.


Radioimmunoassay for thymopoietin.

Radioimmunoassay for thymopoietin. A radioimmunoassay for thymopoietin was developed that detects thymopoietin concentrations greater than 5 ng/ml. A critical point in development of the radioimmunossay was preparation of 125I-labeled thymopoietin by the Bolton-Hunter method, since direct iodination of tyrosyl residues impaired the immunoreactivity of thymopoietin.


Mammalian epidermal protein synthesis: initiation factors. There is a deficiency of initiation in protein-synthesizing systems prepared from mammalian epidermis. These systems do not respond to inhibitors of initiation although they remain sensitive to elongation inhibitors and exogenous ribonuclease. In spite of this deficiency of initiation, active protein factors which support initiation reactions are present in the potassium chloride extract of mammalian epidermal ribosomes. A factor corresponding to the reticulocyte factor IF-MP has been isolated. An inhibitor of initiation is also present in the epidermal KCl wash.


The synthesis of specific proteins in adult mouse epidermis during phases of proliferation and differentiation induced by the tumor promoter TPA, and in basal and differentiating layers of neonatal mouse epidermis. Mouse epidermis can be induced to undergo sequential waves of proliferation and keratinization by treatment with the tumor promoter TPA (12-O-tetradecanoyl-phorbol-13-acetate). This model system offers a unique possibility to study the synthesis of differentiation-specific proteins in the epidermis. During these studies it could be shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis that several previously undetected proteins appear in the epidermis during the phase of differentiation. These have been fractionated according to solubility in 1 M phosphate buffer, pH 7.0, or 0.1 M sodium citrate buffer, pH 2.6. Of the soluble proteins, a band of approximate molecular weight 13,500 (band 1) appears early during the differentiative phase (24 hr after treatment with TPA). Another previously undetected protein of approximate molecular weight 27,000 (band 2) appears in the soluble fraction after 48 hr, at a time when many of the typical manifestations of increased keratinization can be observed. Both of these soluble proteins, although not detectable in normal adult mouse epidermis, are shown to be present in newborn mouse epidermis, which demonstrates histologically a well-developed layer of keratinizing and cornified cells. In addition, by separation of neonatal and TPA-treated adult mouse epidermis into "basal" and "cornified" layers, it could be demonstrated that these two proteins are localized exclusively in the upper layer of the epidermis.