beta-Galactosidase from Aspergillus niger. Separation and characterization of three multiple forms.

The enzyme beta-galactosidase (EC 3.2.1.23) from Aspergillus niger was purified and resolved into three multiple forms, using molecular sieving, ion-exchange, an hydrophobic chromatography. The isolated enzyme forms accounted for 83%, 8%, and 9% of the total beta-galactosidase activity, respectively. They were glycoproteins with estimated molecular weights of 124,000, 150,000 and 173,000, isoelectric points of about 4.6, and pH optima between 2.5 and 4.0. Amino acid and carbohydrate analyses showed that multiplicity was mainly due to dissimilar carbohydrate contents (about 12.5%, 20.5% and 29% neutral carbohydrates, respectively). The multiple form pattern might depend on the culture conditions. The beta-galactosidase forms were heat-stable up to about 60 degrees C. The Km values for lactose ranged from 85 mM to 125 mM, whereas those for the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside were equal to about 2.4 mM. The V values obtained at 30 degrees C for lactose and o-nitrophenyl-beta-D-galactopyranoside were 104 units/mg enzyme protein and 121 units/mg enzyme protein, respectively (weighted averages for the three enzyme forms). The slight reactional dissimilarities between the three enzyme forms are unlikely to be physiologically relevant. The biological significance of A. niger beta-galactosidase multiplicity might be related to the observed differences in carbohydrate content, as suggested by recent reports on other microbial glycoprotein enzymes.


Characterization and comparison of chloramphenicol acetyltransferase variants.

1. Variants of chloramphenicol acetyltransferase from a variety of bacterial species have been isolated and purified to homogeneity. They constitute a heterogeneous group of proteins as judged by analytical affinity and hydrophobic ('detergent') chromatography, native and sodium dodecyl sulfate electrophoresis, sensitivity to sulfhydryl specific reagents, steady state kinetic analysis, and reaction with antisera. 2. The most striking observation is that three variants of chloramphenicol acetyltransferase (R factor type III, Streptomyces acrimycini, and Agrobacterium tumefaciens) possess an apparent subunit molecular weight (24,500) which is significantly greater than that of all other variants examined (22,500). The three atypical variants are not identical since they show marked differences in a number of important parameters. 3. Although the fundamental mechanism of catalysis may prove to be identical for all chloramphenicol acetyltransferase variants, there is a wide range of sensitivity to thiol-directed inhibitors among the enzymes studied. 4. Amino acid sequence analysis of the N-termini of selected variants suggests that the qualitative differences among chloramphenicol acetyltransferase variants is a reflection of structural heterogeneity which is most marked in comparisons between variants from Gram-positive and Gram-negative species.


Structure and properties of the putrescine carbamoyltransferase of Streptococcus faecalis.

Ornithine and putrescine carbamoyltransferases from Streptococcus faecalis ATCC11700 have been purified and their structural properties compared. The molecular weight of native ornithine carbamoyltransferase, measured by molecular sieving, is 250 000. It is composed of six apparently identical subunits with a molecular weight of 39 000, as determined by cross-linking with the bifunctional reagent glutaraldehyde followed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Using the same method, putrescine carbamoyltransferase is a trimer of 140 000 consisting of three identical subunits with a molecular weight of 40 000. Ornithine carbamoyltransferase displays a narrow specificity towards its substrate, ornithine. In contrast, putrescine carbamoyltransferase carbamoylates ornithine and several diamines (diaminopropane, diaminohexane, spermine, spermidine, cadaverine) in addition to its preferred substrate, putrescine, but with a considerable lower efficiency than for putrescine. The kinetic mechanism of putrescine carbamoyltransferase has been investigated. Initial velocity studies yield intersecting plots using either putrescine or ornithine as substrate, indicating a sequential mechanism. The patterns of protection of the enzyme by the reactants during heat inactivation as well as the results of product and dead-end inhibition studies provide evidence for a random addition of the substrates. The putrescine inhibition that is induced by phosphate does, however, suggest that a preferred pathway exists in which carbamoylphosphate is the leading substrate. The different kinetic constants have been established. The properties of putrescine carbamoyltransferase are compared to the known properties of other carbamoyltransferases. The evolutionary implications of this comparison are discussed.


A study of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Improved purification, relative molecular mass, and amino acid composition.

The purification procedure for p-hydroxybenzoate hydroxylase has been modified by replacement of the DEAE-cellulose (DE-32) column in the original procedure by a Sephadex--Cibacron-blue affinity column. In this way the yield of enzyme could be improved from 16% to about 40--50%. Preparative gel chromatography indicated that the enzyme does not exist as a monomeric species as earlier believed but mainly as a dimer. Sodium dodecyl sulfate gel electrophoresis of purified enzyme revealed a minimum relative molecular mass (Mr) of 43000--45000. Analytical gel chromatography, sedimentation equilibrium and sedimentation velocity experiments showed that the enzyme exists in solution mainly as a dimer but also in higher-order quaternary structures (presumably tetramer and hexamer). Temperature dependence of the distribution of the oligomers suggests that the association is of hydrophobic nature. The amino acid composition of the enzyme is also presented. The enzyme contains no disulfide but five sulfhydryl groups. In the native state of the enzyme only one sulfhydryl group is accessible to N-ethylmaleimide or 5,5'-dithiobis(2-nitrobenzoic acid). The iso-electric point of the enzyme was found to be 5.8.


Nucleotide sequences adjacent to the proteins covalently linked to the cowpea mosaic virus genome. Sequence determination after labelling in vitro using RNA ligase.

The sequences of the first 17 nucleotides of cowpea mosaic virus middle and bottom RNAs adjacent to the covalently-linked proteins have been determined. Sequences of the oligonucleotides, produced by complete T1 RNase digestion, were established after labelling of the 3' termini in vitro using RNA ligase. Both sequences are A/U-rich, the first nine nucleotides being identical.


The influence of vagotomy on bile kinetics in the rhesus monkey.

An increased incidence of gallstones has been reported following truncal vagotomy and gastric drainage but never conclusively proven. In the Rhesus monkey, bile composition and flow is similar to man. A model of biliary drainage was established which permits continuous monitoring of bile kinetics. Following truncal vagotomy and pyloroplasty, a significant fall in the bile acid concentration was observed, accompanied by a rise in cholesterol concentration. This resulted in an overall rise in cholesterol saturation. Similar changes in hepatic bile composition were, however, also seen following pyloroplasty alone, and suggests that the vagotomy itself may not be the important factor in producing bile changes.


Clonidine effect in chronic angina pectoris. Double-blind, crossover trial on 60 patients.

Increased adrenergic activity, often manifested in chronic angina, is likely to influence adversely the course of the disease. In view of the inhibitory effect of clonidine (CL) upon the adrenergic nervous system, the effectiveness of small doses of CL in chronic angina was evaluated in a double-blind crossover study on 60 patients suffering at least 5 coronary pains per week in spite of routine medication. CL was given orally in a dose of 2 x 75 microgram/day for a 2 wk. Reduction in frequency of coronary pains by at least 50% was observed in 53.7% of patients, total nitroglycerin consumption decreased from 322 to 174 tablets/week, and ergometric performance increased from 168 to 283 W x min/patient. Urinary excretion of adrenaline and noradrenaline diminished. Blood pressure and heart rate were not considerably changed. Mild and transient side effects occurred in 10 patients, 9 of them completed the trial. It is concluded that CL in low doses is effective and safe in patients with chronic angina, presumably by alleviating adrenergic strain.


Effect of intracerebroventricular administration of thyrotropin-releasing hormone upon the electroenteromyogram of rat duodenum.

Electroenteromyographic activity (EMG) of the duodenum was recorded in pentobarbital-anesthetized rats. TRH intraventricularly administered to rats produced changes in EMG such as increased amplitude, decreased frequency of slow waves and the association of bursts of spike potentials with nearly every cycle of the basal electric rhythm (BER). The effect was selectively prompt and marked in the EMG of proximal duodenum. The response was abolished by vagotomy or atropine injection and no response was elicited in the neonatally 6-OHDA-treated rat. Hypophysectomy, cord-transection or acute i.v. injection of 6-OHDA did not block the response. In the brain, TRH seems to stimulate the neuronal system controlling the vagus efferents involved in the regulation of the duodenal enteric nervous system which in turn modulates the myogenic excitability of the duodenum.


Motilin excites neurons in the cerebral cortex and spinal cord.

The 22 amino acid polypeptide motilin was tested by iontophoretic application onto neurons in the rat cerebral cortex and by perfusion over the isolated hemisected toad spinal cord. Motilin (25--150 nA) excited identified cortico-spinal neurons and other deep spontaneously firing cortical cells. Excitation developed relatively rapidly and lasted for up to 60 sec after the application. Motilin was a potent excitant (threshold concentration 2.5 x 10(-9) M) of neurons in the amphibian spinal cord, eliciting a depolarization of dorsal root terminals and motoneurons. Its effects were substantially reduced after tetrodotoxin, suggesting a primary site of action on spinal cord interneurons.


Immunofluorescent and immunoelectron microscopic demonstration of primate heart muscle antigens cross-reactive with Streptococcus pyogenes.

The presence of antigens in heart muscles cross-reactive with antibodies against Streptococcus pyogenes were examined in heart samples of seven primate species (chimpanzee, rhesus macaque, stump-tailed macaque, hamadryas baboon, capuchin monkey, green monkey and patas monkey). Cross-reactive antigens were localized by means of the indirect immunofluorescent technique in nearly all samples tested. The fluorescence pattern was the same as found in the heart muscle of other mammals. Using the indirect immunoferritin technique the substructural distribution of the antigens was investigated in heart muscle of rhesus macaque and hamadryas baboon. Beside the location on the cell surface the antigens were also demonstrated within the cell. Above all there was a strong labelling along the intercalated discs and in the sarcoplasmatic reticulum.


Developmental G6PD polymorphism in Drosophila melanogaster: evidence for non-structural variants.

G6PD isozyme variation in Drosophila melanogaster is investigated in the larval stage through electrophoretic and genetic analyses. As current structural models for this gene-enzyme system fail to apply in these laboratory populations, the authors suggest a regulatory hypothesis to explain their observations.


Effects of estrogens on the characteristics of [3H]spiroperidol and [3H]RU24213 binding in rat anterior pituitary gland and brain.

Since estrogens have been found to exert a marked inhibitory effect on dopaminergic action at the anterior pituitary and striatal levels in the rat, the effect of estrogen treatment has been studied on the binding characteristics of the dopamine (DA) antagonist [3H]spiroperidol and of the new DA agonist [3H]N-n-propyl-N-phenylethyl-beta-(3-hydroxyphenyl)ethylamine hydrochloride ([3H]RU24213) in rat striatum, nucleus accumbens + olfactory tubercle, frontal cortex and anterior pituitary gland. Specificity of binding was carefully examined in order to investigate possible changes of the agonist and antagonist states of the DA receptor. Estrogen treatment led to a small increase (approx. 20%) of [3H]spiroperidol and [3H]RU24213 binding in rat striatum, nucleus accumbens + olfactory tubercle and frontal cortex while no significant effect was found in the anterior pituitary gland. That the increased binding is due to a corresponding increased number of binding sites and not to higher affinity is indicated by the absence of effect of estrogen treatment on the IC50 values for displacement of the two labeled ligands by a variety of unlabeled compounds. Specificity of binding of DA agonists and antagonists remained unchanged after estrogen treatment. The present data suggest that the potent desensitizing effect of estrogen on DA action at the striatal and pituitary levels is exerted at a step subsequent to binding of DA to its receptor.


Effects of early undernutrition on motivation and discrimination learning in rats.

Offspring were either adequately nourished or undernourished during the suckling period. They had free access to food from weaning, and were given 2 behavioral tests between 13 and 19 weeks of age. Motivation for water was tested on a variable interval (VI) schedule at 3 levels of deprivation and a test of light-dark discrimination made at 23.5 hr of water deprivation. The results indicated (1) no clear difference in VI response rate, but (2) significant differences in discrimination performance between the 2 groups. A learning ability or certain relevant abilities rather than motivation for water appeared responsible for the difference in discrimination learning.


Another Kasahara-variant alkaline phosphatase in renal cell carcinomas.

Another Kasahara-variant alkaline phosphatase isoenzyme was found in 2 out of 25 human renal cell carcinoma tissues. This enzyme electrophoresed in a single diffuse band which is cathodal to but continuous with the liver alkaline phosphatase. After neuraminidase treatment, this enzyme electrophoresed in the same position as that of neuraminidase-treated Kasahara isoenzyme. The enzymic properties of another neuraminidase-treated Kasahara-variant enzyme such as inhibitions by L-phenylalanine, L-homoarginine, L-tryptophan, and L-leucine, effects of inorganic phosphate, urea, and sodium dodecyl sulfate, heat stability, and the reactivity with concanavalin-A are consistent with those of Kasahara isoenzyme. On Ouchterlony's double diffusion, the precipitin lines of Kasahara and the new variant enzyme produced by antibody to Kasahara isoenzyme fused completely. These facts may indicate the occurrence of another Kasahara-variant isoenzyme.


[Spontaneous transformation characteristics of Bacillus subtilis bacteria. I. The relatively low transforming activity of spontaneously released DNA for markers located close to the origin and termination points of chromosome replication].

Relative efficiencies of spontaneous Bacillus subtilis transformation for markers placed in different areas of the cell chromosome were studied. As donor of genetic material, an untransformable strain BD224 trpC2 thr5 rec4 was used during its early log-phase. It was found that for markers placed near points of origin and termination of the chromosome replication the relative transformation efficiencies are significantly lower than those in the case of transformation with DNA extracted from the same donor cells. If a contact of spontaneously released DNA with the recipient cells was delayed for about 60 minutes ("separate" experiment) this difference proved to be less pronounced for ade16 placed near the origin, but remained practically unchanged for metB placed near the termination point. The results obtained can be explained by permanent attachment of chromosome regions, carrying "origin" and "termination" points, to a cytoplasmic membrane. During spontaneously release of cellular genetic material, "origin" and "terminal" DNA fragments carrying ade16 and metB respectively, can retain the contact with components of cell membrane. Hence, their penetration in the recipient cell and (or) participation in recombination can be violated. The first of two fragments becomes free from structurating substances more easy.


[Mutations in the purine nucleoside phosphorylase (pup) gene of Escherichia coli K-12 characterized by leaky damage to enzymatic activity and a pleiotropic effect].

The phenotype of earlier obtained mutants AIR38 and AIR6 is caused by leaky mutations of the structural gene for purine nucleoside phosphorylase (pup). These mutants are unable to grow on the medium with inosine as the only carbon source in the presence of thymidine. In contrast to ordinary leaky mutations, AIR38 and AIR6 are dominant in heterozygotes. When the strain F' with mutations AIR38 or AIR6 on episomes was used for conjugational matings with F- recA (pup+), recombinants with unexpected phenotype were observed: about 20% of recombinants F' became pup+ and thus the convertion of AIR38 and AIR6 alleles to pup+ took place. AIR38 mutation, unlike AIR6, is mapped in the proximal region of the pup gene and is characterized by pleyotropic effect on deo-genes: the inosine-induction of purine nucleoside phosphorylase in AIR38 mutant is absent and the induction of thymidine phosphorylase by exogenous thymidine is decreased. A speculation was made that the mutation AIR38 altered the structure of purine nucleoside phosphorylase in the site responsible for its interaction with membrane.


[Riboflavin auxotrophs of Escherichia coli].

Escherichia coli riboflavin auxotrophs having a different level of riboflavin requirement were isolated. This auxotrophic mutations are located near cysB93 and trpA62 markers. The complementary effect of Bacillus subtilis riboflavin operon linked with pPR1 hybrid plasmid with rib8-1 and rib1-1 mutations was obtained.


[Problems in the genetics of peptic ulcer. II. An analysis of the associations of the disease with certain simply heritable traits].

The research of distribution of blood group ABO, Rhesus, Lewis, Secretor, C5+-component of choline esterase and the ability to taste PTC among Moscow population patients suffering from duodenal ulcer is carried out in comparison with the control. Statistically authentic association of the disease with 0(I) blood group, unsecretor and the association of joint signs (coefficients of association are 1.32, 2.17 and 2.62 respectively) is found. Authenticity of relation with disease is not proved during the investigation of other signs. The values of risk to fall ill for the patients possessing and not possessing the signs of duodenal ulcer are obtained (concerning separate factors and joint factors). It is established that the combination of 0(I) blood group and unsecretor increases the risk of the diseases in 2.4 times as compared with the patients possessing A, B, AB blood groups and secretors.


Synthesis of proteins coded by plasmid vectors of pCV series (Apr, Tcr) and their recombinant derivatives (pDm) in E. coli minicells.

Polypeptide synthesis directed by vector plasmids of pCV series conferring ampicillin and tetracycline resistance (Apr, Tcr) and by recombinant plasmids (pDm) have been analyzed using the minicell system. It has been found that a polypeptide of 34 000 daltons is responsible for the Tcr phenotype and regulated from the promoter near the HindIII site. Cloning of DNA fragments into HindIII site allowed to conclude that DNA from Drosophila melanogaster contains nucleotide sequences which may act as promoters for a 34 000 dalton polypeptide gene. beta-Lactamase is expressed as five proteins of 24 000, 26 5000, 27 000, 28 500 and 29 500 daltons. Insertion of DNA fragments into PstI site prevents the synthesis of all five polypeptides. Recombinant clones Dm39 and Dm187 produce additional proteins of 19 000, 23 000, 24 000 and 27 000 daltons.


Modification of a DNA cloning vehicle to give a high strand-separation melting temperature.

We have constructed a recombinant DNA cloning vector with a high strand-separation melting temperature. This feature is the result of an insertion of a short sequence of dG:dC base pairs. The new vector is useful for cloning DNA segments in anticipation of forming R-loops for the purpose of either transcriptional mapping or gene isolation.


Age-related defense against infection with intracellular pathogens.

Young adult (6--12 weeks old) and aged (20--24 months old) NMRI mice were infected with various intracellular parasites. The following results were obtained: (1) After a sublethal infection with Listeria monocytogenes, aged mice were found to show a resistance similar to that of young adults. A challenge infection with this pathogen was followed by specific immunity of long duration in both age-groups. (2) On the other hand, young animals were significantly more resistant to Salmonella typhimurium than aged mice. It was concluded that this was due to the LD50 which was 14 times greater for 2-month-old than for 20-month-old mice. Furthermore, during 7 weeks after infection there were more S. typhimurium in the spleens of senescent mice than in those of young adult controls. (3) Aged mice showed highly increased susceptibility to the weakly virulent DX strain of Toxoplasma gondii. Almost all aged animals died whereas the control mice survived. When death of the aged mice was prevented by treatment with sulfadiazine after infection with the DX strain, the aged mice were found to be as well protected against subsequent infection with the strongly virulent BK strains as the young adult mice. These results suggest that the susceptibility of the aged animal to infectious agents may considerably vary from one pathogen to another.


Craniofacial growth in olive baboons (Papio cynocephalus anubis): browridge formation.

The examination of a developmental series of skulls of olive baboons has led to the generation of a model for browridge formation. Evidence in support of the model comes from surface configuration of browridge bone as studied by scanning electron microscopy, histology of the bone by the analysis of thin sections and by measurements that reflect both depositional cycles in browridge bone and the biomechanics of mastication. The model relates sudden changes in the biomechanical system caused by the tooth eruption cycle to cyclical changes in the formation of browridge bone. We suggest that browridge bone is added during periods of peak stress caused by the positive response of the masticatory muscles to relatively sudden reduction of their biomechanical efficiency.


Control of glycogen metabolism in the developing turkey poult.

An experiment was conducted with young turkey poults to evaluate factors controlling glycogen metabolism in the period following hatching. Glucose and sucrose solutions were given along with a standard starter diet. Liver and carcass glycogen were measured on days 1, 4 and 6. Liver glycogen synthetase (EC 2.4.1.21) and phosphorylase (EC 2.4.1.1) were also assayed at these times. The characteristics of active and inactive glycogen synthetase at these times were determined and sensitivity of the active and inactive forms were related to physiological concentrations of glucose-6-phosphate. Supplemental glucose or sucrose increased carcass glycogen in comparison to controls; however, but sucrose was more effective than glucose in promoting liver glycogen synthesis in 4- and 6-day-old poults. There was an age dependent increase in carcass glycogen between days 1 and 6, but a decrease in liver glycogen between days 4 and 6. The activation of liver glycogen synthetase is incomplete in 1 day old poults but activity increases during the 1st week of life. Activation of glycogen synthetase decreased the apparent Ka for glucose-6-phosphate. Phosphorylase inactivation in vitro was not affected by age. Liver glucose-6-phosphate increases rapidly after hatching and the concentration is related to the in vitro Ka derived for both active and inactive synthetases. Both glucose and sucrose increased liver glucose-6-phosphate at days 4 and 6 as well as glycogen synthetase activity. The increase in enzyme activity may be caused indirectly by an allosteric effect of glucose-6-phosphate. Phosphorylase, while not affected by supplemental carbohydrates, did decrease in activity between days 4 and 6. The decrease in activity could affect the phosphorylase a/ synthetase a ratio and change glycogen metabolism.


Aeration sensitizes Streptococcus faecalis to hydroxyurea.

Hydroxyurea in up to 60 mM concentration did not inhibit growth or DNA synthesis in nonaerated cultures of Streptococcus faecalis ATCC 8043. In contrast, in cultures aerated by shaking already 1 mM hydroxyurea decreased the rate of net DNA synthesis and in higher concentrations of the drug the growth of the total cell mass also slowed down and the number of cells per chain increased from 1-2 to 10. The differential rate of DNA synthesis, but not the growth of the total cell mass, could be restored almost to the control level by adding thymidine to the medium. Thus there are at least two targets for hydroxyurea in the cells of S. faecalis grown in aerated cultures.


Gestation and inter-birth intervals in the owl monkey (Aotus trivirgatus griseimembra).

A single, timed mating indicated a gestation period of 133 days for Aotus trivirgatus griseimembra. This figure is confirmed by approximate correspondence with minimum inter-birth intervals, results from radioimmunoassay of urinary oestrogens during pregnancy, and general agreement with a logarithmic plot of gestation period against body weight for simian primates. Birth data indicate that owl monkey females in present colonies in captivity breed throughout the year and will, on average, produce 3 infants every 2 years. There is no evidence of a characteristic post-partum oestrus.


An analysis of tooth and body size relationship in five primate taxa.

The strength and the nature of the covariance between tooth and body size was investigated in Homo, Gorilla, Pan, Papio and Colobus. When sexes are combined in each taxon, the correlations are strong enough to compare the allometry coefficients of taxa, and the non-human taxa show a sufficiently strong linear relationship to compute 'interspecific' allometry coefficients. Allometry coefficients for each variable were not uniform among the taxa, and coefficients also differed from one variable to another. Computed 'intra' and 'inter' specific allometry coefficients from these data suggest that canine size will usually scale at a higher level than molar crown area, which is at most isometric, and not positively allometric with respect to body size. The consequence is that larger representatives of a taxon would be expected to combine relatively larger canines with a proportional, or relatively smaller, molar crown area. It is pointed out that these differences do not correspond to those found between 'gracile' and 'robust' australopithecines.


Female aggression and male membership in troops of Japanese macaques and olive baboons.

A detailed comparison of Macaca fuscata and Papio anubis reveals that female aggression to males has an effect on male association with females in both species, but only in M. fuscata do females appear to be able to limit the number of males in their troop. It is suggested that this is the reasons why 'solitary' males are commonly reported in M. fuscata, but rare in P. anubis, and may be related ultimately to the different degrees of sexual dimorphism in the two species.


The primates in Darfur, Republic of Sudan.

The paper describes the distribution, group size and relative abundance of Erythrocebus patas and Papio doguera in Darfur Province, Sudan. Less detailed information is given on Cercopithecus aethiops and Galago senegalensis. Detailed observations were carried out in the field and by low-level aerial survey during 1972--1978 and have been supplemented by references to the literature where appropriate.


Fetal sex ratio in the rhesus (Macaca mulatta).

Fetal sex ratio was determined in several hundred timed Macaca mulatta pregnancies terminated by hysterotomy between 75 and 179 days' gestation. From 75 to 149 days' gestation a greater number of females occurred in utero, while after 150 days a greater number of males was observed. However, this difference was not statistically significant.


[Hunger and intellectual development].

Severe hunger in infancy and early childhood is associated with reduced head size, reduction in cell number and impaired chemical composition of the brain. Intellectual deficits correlating with malnutrition persist into later life. Because malnutrition and poor socio-economic conditions often exist together the additional unfavorable influence or lack of environmental stimuli on intellectual development has to be taken in account.


[Thyroxine-binding globulin (TBG). Clinical studies on the regulation of TBG concentration in serum and the value of TBG for the evaluation of thyroid function].

1. The radioimmunoassay (RIA) and the competitive ligand binding assay (CLBA) are convenient routine methods for the precise and reproducible measurement of TBG in large numbers of serum samples. 2. There is an age dependent variation of the TBG-concentration in serum. There is a steady decrease of TBG with increasing age with a minimum between the 20th and 50th year. In higher age TBG increases again significantly. 3. There are significantly negative correlation between TBG-serum levels on the one hand and free T4- and T3- fractions on the other. The low TBG-level in hyperthyroid patients increases gradually to normal during treatment with thyroid blocking drugs, the elevated TBG-concentrations in hypothyroid patients decrease to normal during treatment with thyroid hormones. 4. Estrogen stimulates TBG-synthesis in the liver. During enhanced endogenous estrogen production (pregnancy) as well as during exogenous estrogen application a rise occurs in TBG-concentration in serum, which seems to be dose related. 5. Androgens induces a decrease of the TBG-concentration in serum. 6. During viral hepatitis and in compensated cirrhosis of the liver TBG-concentration is significantly elevated. In cirrhosis of the liver with poor hepatic function the TBG-concentration is decreased. 7. The T4/TBG-quotient is a good parameter to estimate free T4-concentration in serum.


Plant plasma membrane. Correlation between glucan synthase II activity and a mannosyl transferase activity.

In cotyledons of germinating cucumber seeds (Cucumis sativus), plasma membranes were investigated biochemically and partially characterized. Glucan synthease II was utilized as a marker to locate plasma membrane vesicles within fractions obtained by differential centrifugation or within sucrose gradients used either in zonal centrifugations or in sedimentations to equilibrium density. Thorough homogenization led to a homogeneous population of plasma membrane vesicles which could be clearly separated from mitochondria by centrifugation at 150000 x g for 4 h in a zonal rotor. The profiles of glucan synthase II activity and naphthylphthalamic acid binding coincided with that of a mannosyl transferase activity, monitored by direct transfer of mannose from GDPmannose to endogeneous acceptors.


Hemagglutinin activity of human plasma fibronectin.

Purified human plasma fibronectin at concentrations of about 30 microgram/ml was found to agglutinate trypsin-treated erythrocytes from certain species. The hemagglutination reaction was inhibited by specific antibodies to fibronectin, by relatively low concentrations of polyamines and by higher concentrations of basic amino acids and nonacetylated amino sugars. The divalent cations Ca2+ and Mg2+ and the chelating agent ethylenediaminetetraacetate did not affect the reaction. None of the neutral amino acids, neutral sugars or polyanions tested was inhibitory. The results imply that plasma fibronectin is capable of interacting with cell surfaces and support the idea of a similarity between cellular and plasma fibronectins.


Polysaccharide meningococcal vaccines--current status.

Two separate periods of intensive research, with a 25-year hiatus after the advent of the sulfonamides, have resulted in two good, but imperfect, antimeningococcal vaccines--against serogroups A and C. Neither is recommended for routine use, but both have been effective in halting epidemics. Lack of an effective group B immunogen remains the major obstacle to the control of meningococcal disease.


Peptic ulcer: antacids or cimetidine?

Controlled studies indicate that both high-dose antacids and cimetidine can be highly effective. Thus, the choice must rest on other considerations--acceptability, compliance, side effects, and cost. The problem of recurrence is addressed.


Assignment of structural beta-galactosidase loci to human chromosomes 3 and 22.

Hybrid cell lines isolated after fusions between Chinese hamster E36 cells and normal human white blood cells were analyzed for human beta-galactosidase isoenzymes and for human chromosomes, especially 3, 12, and 22, the candidates for bearing a beta-galactosidase locus. Results of neuraminidase treatment of the cell lysates and immunological studies showed that in man two structural beta-galactosidase loci are present and can be assigned to chromosomes 3 and 22. No correlation was found between the expression of human beta-galactosidase and the presence of human chromosome 12.


Human erythrocyte carbonic anhydrase polymorphism in Kenya.

In this study, 1736 Western Kenyans were examined for red cell carbonic anhydrase (CA) variants. No CA I variants were detected, but the CA II2 isozyme was found with a calculated gene frequency of 0.054.


Pokeweed mitogen induced differentiation of human B cells: evaluation by a protein A haemolytic plaque assay.

Using the protein A plaque assay, the number of human cells secreting immunoglobulin of various classes after pokeweed mitogen stimulation was determined. At optimal response (on day 5-7) a mean of 58,354 IgM PFC/10(6), 34,207 IgG PFC/10(6) and 10,525 IgA PFC/10(6) cells was found when using peripheral blood lymphocytes. In spleen cells, peak values which were slightly higher than in blood, were obtained at day 4-6. The proportions of cells secreting light chains of either type were found to be comparable to those of unstimulated cells thus supporting the notion of the polyclonality of the response. Pokeweed mitogen stimulation of peripheral blood lymphocytes was found to be totally T-cell dependent whereas the response of spleen cells was not. When assayed for antigen-specific precursor cells in cultures stimulated by mitogen, the frequency of SRBC-specific IgM producing cells was found to be 1.3/1000 cells. This frequency was regularly found to be independent of medium supplement.


Biochemistry and genetics of gangliosidoses.

The gangliosidoses comprise an-ever increasing number of biochemically and phenotypically variant diseases. In most of them an autosomal recessive inherited deficiency of a lysosomal hydrolase results in the fatal accumulation of glucolipids (predominantly in the nervous tissue) and of oligosaccharides. The structure, substrate specificity, immunological properties of and genetic studies on the relevant glycosidases, ganglioside GM1 beta-galactosidase and beta-hexosaminidase isoenzymes, are reviewed in this paper. Contrary to general expectation, only a poor correlation is observed between the severity of the disease and residual activity of the defective enzyme when measured with synthetic or natural substrates in the presence of detergents. For the understanding of variant diseases and for their pre- and postnatal diagnosis, the necessity of studying the substrate specificity of normal and mutated enzymes under conditions similar to the in vivo situation, e.g., with natural substrates in the presence of appropriate activator proteins, is stressed. The possibility that detergents may have adverse affects on the substrate specificity of the enzymes is discussed for the beta-hexosaminidases. The significance of activator proteins for the proper interaction of lipid substrates and water-soluble hydrolases is illustrated by the fatal glycolipid storage resulting from an activator protein deficiency in the AB variant of GM2-gangliosidosis. Recent somatic complementation studies have revealed the existence of a presumably post-translational modification factor necessary for the expression of ganglioside GM1 beta-galactosidase activity. This factor is deficient in a group of variants of GM1-glangliosidosis. Among the possible reasons for the variability of enzyme activity levels in heterozygotes and patients, allelic mutations, formation of hybrid enzymes, and the existence of patients as compound heterozygotes are discussed. All these may result in the production of mutant enzymes with an altered specificity for a variety of natural substrates.


The neonatally thymectomized rat: a model for compensatory IgM antibody formation in exocrine secretions.

The effect of T-cell deprivation on IgM antibodies in saliva was studied in rats. IgM was never detected in salivas obtained from normal or sham-thymectomized rats, but was detected in saliva samples from 8/15 (53.3%) rats that had undergone neonatal thymectomy. All (6/6) neonatally thymectomized rats exhibited an IgM antibody response to DNP in salivary secretions after local immunization with a T-dependent antigen (DNPBGG), while no IgA anti-DNP activity was detected in saliva from these antimals. IgM antibodies were detected in saliva from 5/7 thymectomized rats following local injection with a T-independent antigen (DNP-Lys-Gicoll). This was accompanied by detectable but substantially reduced levels of secretory IgA antibody in saliva from 7/7 rats. The results suggested that absent or decreased salivary IgA responses accompanying T-cell deprivation in the rats are compensated for by secretion of IgM antibodies into the saliva. The neonatally thymectomized rat may thus provide a model for the study of synthesis, secretion and protective potential of exocrine IgM antibodies.


Inhibition of human eosinophil chemotaxis by IgA paraproteins.

Sera from patients with IgA myeloma inhibit normal human eosinophil chemotaxis. No correlation was noted between inhibition and the absolute concentration of IgA or lambda-K light-chain type. Eosinophil chemotactic inhibitory activity was associated with isolated IgA paraproteins and was found to be cell directed and stable at 56 degrees C. Pepsin digestion of IgA paraproteins resulted in loss of both IgA Fc fragment and eosinophil chemotactic inhibitory activity. Polymeric IgA accounted for most of the inhibitory activity as evidenced by sucrose density gradient centrifugation studies and a loss of inhibitory activity following dithiothrietol reduction and iodoacetamide alkylation which converted polymeric IgA to monomeric IgA. Comparative studies with neutrophils showed that both neutrophil and eosinophil chemotaxis and chemokinesis were effectively inhibited by IgA paraproteins. The mechanisms of suppression of eosinophil and neutrophil chemotaxis by IgA paraproteins appear to be similar and possibly may involve a membrane receptor for IgA.


Hemodynamic effects of nitroglycerin.

The authors have studied the hemodynamic effects of sublingually administered nitroglycerin (NG). Obtained data indicate that the response to NG is time-dependent. A first phase, lasting about 2 minutes, is characterized by a reduction of total systemic resistance (TSR) and an increase of cardiac output (CO), without significant variations of left ventricular filling pressure (FP) and blood pressure (BP). A modest increase of heart rate (HR) is observed. The second phase is characterized by a reduced FP and later, after 5 minutes, of BP. Subsequently HR increases to a greater degree, CO decreases and TRS presents a variation toward control values.


Rhesus monkey aqueous humor composition and a primate ocular perfusate.

The composition of rhesus monkey aqueous humor has been studied in large-volume, pooled samples. Replicate determinations of the concentrations of a number of constituents have been carried out for both aqueous humor and serum from large veins by means of automatic analyzing equipment. Since aqueous humor  has been obtained by anterior chamber paracentesis, it is a mixture of anterior and posterior chamber aqueous. When compared to serum, the pooled aqueous contains an excess of chloride, bicarbonate, ascorbate, lactate, uric acid, and several neutral amino acids. Rhesus monkey aqueous humor is deficient in calcium, urea nitrogen, phosphates, glucose, protein, creatinine, iron, bilirubin, cholesterol, triglycerides, a number of serum enzymes, acidic and basic amino acids, and several neutral amino acids. Sodium, potassium, magnesium, and two neutral amino acids (cysteine and valine) are of equal concentration in aqueous humor and serum. Glutathione concentration is very low in both aqueous humor and serum. Pooled rhesus monkey aqueous humor and serum are isosmolar, with measured osmolality being about 303 mOsm. Based upon the chemical analysis, a new solution has been formulated to substitute for primate aqueous humor during anterior ocular perfusion. This new solution causes very little change in the physiologic integrity of the outflow pathways during prolonged, repeated perfusion. In this respect, its effects are very similar to those of pooled rhesus monkey aqueous humor during perfusion of rhesus monkey eyes. In contrast, perfusion of rhesus monkey eyes with glutathione-bicarbonate-Ringer's solution has been shown to cause progressive increase of the total facility. To minimize physiologic alterations during operative procedures, a solution similar to this new one could be formulated for irrigation of the inside of the human eye.


Ocular fluorophotometry in streptozotocin diabetes mellitus in the rat: effect of pancreatic islet isografts.

Fluorophotometry was used to evaluate the integrity of the blood-ocular barriers to fluorescein in experimental diabetes mellitus in rats. This technique allowed quantitation of ocular fluorescein concentrations following intravenous injection. Streptozotacin-induced diabetes resulted in an increased fluorescein accumulation in the anterior chamber (1.52 +/- 0.17 microgram/ml, mean +/- S.E.M.) and vitreous (0.82 +/- 0.11) over baseline nondiabetic levels (0.68 +/- 0.80 and 0.40 +/- 0.03, respectively). Fluorophotometry was repeated at 5, 13, and 20 days following portal vein pancreatic islet transplantation. At 5 days anterior chamber (1.40 +/- 0.17) and vitreous (0.61 +/- 0.08) fluorescein concentrations remained elevated. However, at 13 and 20 days following islet transplantation, ocular fluorescein concentrations were identical to levels observed prior to the induction of diabetes. Intravenous glucose (0.5 gm/kg) tolerance testing was performed 5 and 13 days following transplantation. The glucose responses to the tolerance test were normal and similar at both times. However, at 5 days the insulin response was abnormal with a decreased initial peak and an absent second peak. At 13 days there was a normal biphasic insulin response. In experimental diabetes mellitus ocular vascular permeability was more closely correlated with insulin than blood glucose abnormalities.


Increase in axial length of the macaque monkey eye after corneal opacification.

The cornea of one eye was opacified in two young macaque monkeys by multiple stromal injections of a suspension of polystyrene particles (latex). Ultrasound measurements showed that the eye with opaque cornea grew at a faster rate, so that after 1 year it was more than 1 mm longer than the normal eye. This difference in axial length was due to elongation of the posterior segment, since lens thickness, depth of anterior chamber, and corneal curvature were identical in both eyes. At histological examination, no pathological changes were observed in the anterior segment of the latex-injected eye except for a scant vascularization of the corneal opacity. The result of this experiment demonstrates that opacification of the corneal has effects on axial length similar to, although less marked than, those on lid fusion and therefore supports our previous conclusion that the myopia caused by lid fusion is triggered by the abnormal visual impact and involves central visual pathways.


Retinal detachment from hyperosmotic intravitreal injection.

Hyperosmotic solutions were injected into the rabbit vitreous to study their effects upon the retina. Injection of 0.05 ml of a 1000 mOsm solution caused rapid whitening of the posterior retina followed by the development of a large detachment and permanent retinal degeneration. The weakest solutions which produced ophthalmoscopically visible changes in the retina (after an injection of 0.05 ml) were near 500 mOsm. Sodium chloride, sodium aspartate, EDTA, mannitol, sucrose, and penicillin were effective at similar osmolarities. An osmotic load in the vitreous caused immediate loss of the c-wave of the electroretinogram (ERG), and a slower decline of the a- and b-waves. The reported intravitreal toxicity of some drugs may relate to osmotic rather than pharmacologic effects. Osmolarity should be accounted for in planning the amount and location of any vitreous injection.


Programming, budgeting, and control in health care organization: the state of the art.

The planning, budgeting, and controlling processes (PBCP) largely subsume all of the planning and controlling activities of an organization. This paper discusses these activities within the context of a single management control system, focusing on three topics. First, a brief historical perspective of management concerns which relate to PBCP is presented and several important external pressures currently imposed on the health care industry are discussed. Second, normative models of the processes--programming, budgeting, and controlling--are presented. The discussion focuses on the elements and relationships of these processes, and numerous references to the literature are provided. Third, several issues related to the gap between the state of the art in PBCP for hospitals and the current state of practice are discussed.


Sharing arrangements in the nonprofit hospital industry.

The major task of this paper is to develop hypotheses about voluntary sharing arrangements (SAs) from a plausible economic analysis of the hospital industry. The second task of the paper is to review some emerging evidence about SAs. Our research suggests that some SAs could or actually do reduce hospital costs to the community. However, there are reasons which indicate that cost reduction is neither a necessary nor a sufficient result for the success of many SAs.


The effects of various fixatives on the relative thickness of cellular membranes in the ventral lobe of the rat prostate.

A desitometric method was utilized in the measurement of the relative thickness of the cellular membranes in the ventral lobe of the rat prostate. Potassium permanganate, glutaraldehyde, osmium tetroxide, and ruthenium tetroxide solutions were used as fixatives. During preparation for electron microscopy, the tissues were given standardized treatments to reduce methodological errors; latex particles were applied to the thin sections to serve as reference particles of a known size. The most remarkable observation of the study was that the densitometric method yielded reproducible results and that the different fixatives gave significantly different values for the relative thickness of cellular membranes. Glutaraldehyde, or glutaraldehyde followed by ruthenium tetroxide post-fixation, gave the highest values for membrane thickness, while osmium tetroxide and potassium permanganate gave the lowest values. Glutaraldehyde treatment, prior to osmium tetroxide or potassium permanganate post-fixations, rendered the membranes thicker than after osmium tetroxide and potassium permanganate treatments alone. Ruthenium tetroxide appeared to be very suitable for fixation of cellular membranes.


The relative thickness of intracellular membranes in epithelial cells of the ventral lobe of the rat prostate.

The relative thickness of intracellular membranes of epithelial cells in the ventral lobe of the rat prostrate was measured by a densitometric method. Glutaraldehyde perfusion followed by ruthenium tetroxide immersion fixation appeared to be the most suitable method for membrane thickness measurements. By thickness, the membranes could be roughly subdivided into three groups. The inner and outer membranes of the mitochondrion made up the thinnest membranes of the cell. The second group of membranes consisted of the membranes of the rough-surfaced endoplasmic reticulum and the Golgi apparatus, the different faces of the latter organelle, and the Golgi vesicles. The thickest group of membranes included those of the cell membrane, secretory granules, condensing vacuoles, lysosomes, autophagic vacuoles and multivesicular bodies. The differences in thickness of the membranes are probably due to the varying protein/lipid ratio, and the qualities and proportions of the different lipids in the membranes.


The separation of basal and differentiating cells from human epidermis for DNA cytofluorometry.

A method for the selective separation of human epidermal cells, in which the Feulgen-stainable material suffers minimal damage, was investigated. The principal stage involves alpha-chymotrypsin treatment of skin specimens as well as shaking in an isotonic solution. With respect to DNA distribution pattern, there was good agreement between that of epidermal cells separated with the microdissection-ultrasonic irradiation method, reported previously by us, and those separated by the present method.


Carbonic anhydrase activity of the cat hind leg.

The accessibility of tissue carbonic anhydrase to plasma was studied in five surgically isolated cat hind legs. After the leg was skinned and the paw circulation occluded with a tourniquet, it was perfused with a solution that contained neither red cells nor carbonic anhydrase. Solutions containing either H14CO3- or 14CO2 were injected with 125I-albumin, 22Na+, and 3H2O into the femoral artery and the concentrations of each were measured in the femoral venous outflow. Under control circumstances the outflow patterns of H14CO3- and 14CO2 were very similar. However, after carbonic anhydrase inhibition with 20 mg/l acetazolamide in the perfusion solution, the initial exchange of H14CO3- ("extraction") was greatly decreased, whereas the extraction of 14CO2 was slightly increased. Because there was insignificant carbonic anhydrase activity in the venous outflow, these data suggest the presence of carbonic anhydrase at a readily accessible site, possibly bound to the endothelial surface. In this location it would promote CO2 exchange and minimize disequilibrium between plasma HCO3- and CO2.


Herbicidins C and E, two new necleoside antibiotics.

Streptomyces saganonensis No. 4075 used for production of herbicidins A and B has also produced other two antibiotics, herbicidins C and E in submerged culture. Isolation of the antibiotics was performed by adsorption on resinous adsorbent followed by elution with aqueous MeOH and completed by silica-gel chromatography. Physico-chemical characteriation revealed that herbicidins C and E are new nucleoside antibiotics having an adenine moiety in their structures. Herbicidins C and E indicated inhibitory activity against germination of chinese cabbage at 12.5 micrograms/ml and 25 micrograms/ml, respectively, and Anacystis nidulans, a blue green algae, at 100 approximately 200 micrograms/ml.


Teaching mending skills to mentally retarded adolescents.

This experiment presents a model for analyzing community living skills and teaching them to mentally retarded adolescents. A task analysis of three mending skills was developed and validated, aided by consultation with persons having expertise in home economics and mental retardation. The task analysis was modified to compensate for the constraints imposed by the trainees' disabilities. Five moderately retarded youths received training on sewing hems, buttons, and seams. Sewing skills were acquired rapidly and maintained. The behavior generalized from trained to untrained tasks on their common components for all subjects. A multiple baseline across participants combined with a multiple baseline across responses demonstrated the combined effectiveness of an objectively validated, detailed task analysis; graduated sequence of prompts; and response consequences in training and maintaining community living skills with mentally retarded adolescents.


High mobility group nonhistone chromosomal proteins also exist in Tetrahymena.

High mobility group (HMG) nonhistone chromosomal proteins have been shown to exist also in the ciliated protozoan Tetrahymena pyriformis. One or two histone-like components were extracted with 0.25 M HCl from the chromatin, in addition to five histone species. These proteins were also extracted selectively with 0.5 M HClO4, 0.35 M NaCl, or 4 mM spermidine, together with H1 histone, and were characterized as HMG proteins on the basis of the following criteria: high mobilities on polyacrylamide gel electrophoresis, relatively low molecular weights, amino acid compositions rich in lysine and glutamic acid, and relative contents in chromatin. This extends the distribution of the HMG proteins to all four eukaryotic kingdoms, and suggests the possibility that they have some universal role in chromatin structure and function.


Studies on Ca2+-Mg2+ binding sites of frog skeletal muscle myosin.

From skeletal muscle myosin light chains readily dissociate from the myosin oligomer in the absence of divalent cations, and unlike rabbit skeletal muscle myosin light chains, the released light chains of frog skeletal muscle myosin have a high Ca2+ binding affinity. Whereas each Ca2+ binding light chain of frog skeletal muscle myosin, when in association with the heavy chains bound 1 mol of Ca2+, when in the dissociated state bound 0.5 mol of Ca2+; the latter were readily displaced with low Mg2+ concentrations. Whereas 10(-5) M Mg2+ displaced all of the Ca2+ binding sites on the released light chains at Ca2+ concentration ranges of 10(-7) to 10(-4) M, there was negligible displacement of the Ca2+ binding sites with native frog skeletal muscle myosin under these same conditions.


Porous titanium endosseous dental implants in Rhesus monkeys: microradiography and histological evaluation.

Artificial tooth roots with porous surface coatings were fabricated by sintering spherical powder of titanium alloy to solid cylindrical cores. The tooth roots were implanted subgingivally in healed mandibular premolar extraction sites of fifteen Rhesus monkeys. Supracrestal abutments were screwed into pretapped holes in the superior aspect of the primary subgingival stage four to eight weeks after implantation of the root. Clinical evaluations were performed monthly. Ten animals were sacrificed for histological evaluation of the functioning free standing implants. Of twenty-nine implants placed, three were lost and four were rated failures on the basis of histological evaluation. Postmortem evaluations revealed bone growth into the porous surface coating of the primary stage of all the implants. The most characteristic features which could be used to describe differences in the implant histology were the buccal and lingual crestal bone heights measured in relation to the root porosity. Twelve of sixteen implants had crestal bone heights within one millimeter of the superior aspect of the root. Four other implants displayed excessive bone recession, revealing as much as one half of the root porosity supracrestally. The four implant failures could be related to unfavorable features of recipient bone sites. The results demonstrate that the bone growth into the porous surface coatings of artificial tooth roots is an efficacious method of dental implant fixation.


Immobilized enzyme electrodes for the potentiometric measurement of glucose concentration: immobilization techniques and materials.

Glucose oxidase, catalase, and bovine serum albumin were co-immobilized with glutaraldehyde around a platinum screen or around a single platinum-iridium wire. The potential difference between this dual enzyme electrode and a Ag/AgCL reference electrode was proportional to the logarithm of the glucose concentration over the range from 10 to about 150 mg glucose per 100 ml in buffered solution at pH 7.4 and 37 degrees C. The enzyme electrode responded in serum only if coated with a semipermeable film, such as cellulose acetate, to exclude serum macromolecules. The potentiometric results were similar to those obtained with the two enzymes co-immobilized in polyacrylamide gel around a platinum screen or with only one of the enzymes, glucose oxidase, covalently coupled to a platinum screen. The results so far suggest that glucose for development of a continuous in vivo glucose sensor.


Spectrin rearrangement early in erythrocyte ghost endocytosis.

The endocytic vacuoles induced in white ghosts were found to be depleted of spectrin and therefore it was proposed that they arose from spectrin-free areas in the erythrocyte membrane. To follow changes in spectrin distribution during endocytosis, affinity-purified rabbit antispectrin antibodies were produced. Quantitative techniques were developed for the use of a highly specific 125I-F(ab')2 antispectrin, and these showed that before the appearance of vacuoles, as assessed by phase microscopy, there was a reproducible decrease in immunoreactive spectrin. To determine whether this spectrin decrease represented a local or diffuse spectrin loss or a spectrin rearrangement, morphologic studies were undertaken using transmission electron microscopy on samples treated with rabbit antispectrin and ferritin-conjugated goat anti-rabbit immunoglobulin. These studies showed that endocytosis was preceded by the creation of extensive spectrin-free areas separated by discrete spectrin-containing zones. Pretreatment of ghosts with alkaline phosphatase blocked all forms of endocytosis and prevented the creation of spectrin-free areas. Therefore, it is proposed that under the impetus of endocytosis inducers, phosphorylated spectrin is redistributed so that spectrin-free zones are created, and that endocytic vacuoles form and fuse in spectrin-free areas.


The innominate steal syndrome.

Two patients with isolated innominate artery occlusion presented with symptoms of significant right-sided cerebrovascular insufficiency. One of these also noted progressive pain and weakness of the right upper extremity. Both were treated with dacron bypass grafts from the ascending aorta to the innominate bifurcation with complete relief of all symptoms. Unlike the subclavian steal, innominate artery occlusion induces distinct and much more significant hemodynamic alterations in extracranial arterial flow and is rarely asymptomatic. Three distinct patterns of blood flow have been described secondary to this lesion. Correction is best achieved by either innominate artery endarterectomy or dacron bypass grafting which the authors favor. Extra-anatomic bypasses represent a less satisfactory solution except in the poor risk patients. Long term relief of symptoms and patency of the reconstruction have been generally achieved by either of the recommended techniques.


Reversible inhibition of Chlamydomonas flagellar surface motility.

Chlamydomonas exhibits force transduction in association with its flagellar surface; this can be visualized by the saltatory movements of attached polystyrene microspheres. This flagellar surface motility has been quantitated by determining the percentage of attached microspheres in motion at the time of observation (60% in the case of control cells at 25 degrees C). A number of experimental treatments reversibly inhibit flagellar surface motility. These include an increase in sodium or potassium chloride concentration, a decrease in temperature, or a decrease in the free calcium concentration in the medium. Many of the conditions that result in inhibition of flagellar surface motility also result in an induction of flagellar resorption. Although both flagellar stability and flagellar surface motility are dependent on the availability of calcium, the two processes are separable; under appropriate conditions, flagellar surface motility can occur at normal levels on flagella that are resorbing. Inhibition of protein synthesis results in a gradual loss of both the binding of microspheres to the flagellum and the flagellar surface motility. After resumption of protein synthesis, both binding and movement return to control levels. The effect of the inhibition of protein synthesis is interpreted in terms of selective turnover of certain components within the intact flagellum, one or more of these components being necessary for the binding of the microspheres and their subsequent movement. If this turnover is inhibited by keeping the cells below 5 degrees C, the absence of protein synthesis no longer has an effect on microsphere attachment and motility, when measured immediately after warming the cells to 25 degrees C.


Late results after extra-anatomic bypass routes.

The long-term results of 37 carotido-subclavian grafts was 100% till the end of the third year. This method is now our preferred method for correction of subclavian lesions. The operative mortality was 0%. The long-term results of the femoro-femoro bypasses was 79.7% from the third through the fifth year. 5 out of 7 axillo-femoral grafts remained patent. In our opinion the axillo-femoral bypass should only implanted in patients with an infected area in the aortoiliac segment but not routinally.


Lactose synthetase activity in mouse mammary glands is controlled by thyroid hormones.

Epithelial cells in explants from the mammary glands of euthyroid mature virgin mice are proliferatively dormant. They must undergo DNA synthesis and traverse the cell cycle in vitro before they are able to differentiate fully in response to insulin, hydrocortisone, and prolactin, and synthesize enzymatically active alpha-lactalbumin (measured as lactose synthetase activity). In contrast, glands from hyperthyroid mature virgin mice do not require DNA synthesis in vitro to differentiate. Explants from the euthyroid virgin tissue overcome their dependence on DNA synthesis when 10(-9) M 3,5,3'-triiodo-L-thyronine is added directly to the cultures in addition to the other three hormones. Explants from involuted mammary glands from euthyroid primiparous mice do not require DNA synthesis in vitro to make the milk protein even though they, like explants from mature euthyroid virgin tissue, are proliferatively dormant and do not contain detectable lactose synthetase activity in vivo. Glands from primiparous animals made mildly hypothyroid by ingestion of 0.1% thiouracil in drinking water during 7 wk of involution remain morphologically indistinguishable from glands of their euthyroid counterparts. However, explants from the glands of these hypothyroid animals revert to a state of dependence on DNA synthesis to differentiate functionally. These observations suggest that the dependence on DNA synthesis and cell cycle traversal for hormonal induction of lactose synthetase activity in the mouse mammary gland is controlled by thyroid hormones.


Contributions of lipids and proteins to the surface charge of membranes. An electron microscopy study with cationized and anionized ferritin.

The surface charge of cultured neurons was investigated with the electron microscope markers anionized ferritin (AF) and cationized ferritin (CF). To determine which membrane components could react with the markers, model reactions were used. Both protein-coated Sepharose beads and lipid vesicles were reacted at physiological pH. Results with these model reactions indicate that the following groups may contribute to the surface charge: acidic groups--the sialic acid of both glycoproteins and gangliosides, the carboxyl group of proteins, and the phosphates of phospholipids; basic groups--the amines of proteins. The effect of chemical fixation on the surface charge was investigated. Glutaraldehyde fixation was shown to increase the charge of neutral proteins but not by a mechanism involving unbound aldehydes. Glutaraldehyde fixation of phospholipid vesicles in the presence of CF showed that amine-containing phospholipids were cross-linked to CF. This cross-linkage was seen with the electron microscope as the clumping of CF and the burying of CF in the membrane. Paraformaldehyde fixation had a lesser effect on the charge of proteins but did react with phospholipids as did glutaraldehyde. It is concluded that at physiological pH: (a) most of the charged proteins and lipids on cell surface can contribute to the membrane surface charge, and (b) the membrane surface charge of cells can be greatly changed by chemical fixation.


Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin.

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.


Dissociation of prolactin response to thyrotropin-releasing hormone and metoclopramide in chronic renal failure.

PRL secretion was evaluated in 14 males with chronic renal failure on long term hemodialysis. Twelve had basal hyperprolactinemia. Ten subjects were challenged with TRH in doses ranging from 200--1000 micrograms. None of them responded to 200 or 500 micrograms TRH, although 2 of 4 subjects tested did respond to 1000 micrograms TRH. In contrast, all 4 subjects challenged with metoclopramide did have PRL responses which were indistinguishable from those of the controls. These results indicate that there is a dissociation in responsiveness to metoclopramide and TRH in chronic renal failure.


Normalization of insulin and glucagon secretion in ketosis-resistant diabetes mellitus with prolonged diet therapy.

Glucose tolerance and insulin and glucagon secretion were examined sequentially during 6 months of calorie and carbohydrate restriction in an obese, recent-onset, ketosis-resistant diabetic adult. The subject was then followed for 9 additional months, during which some weight was regained. Fasting plasma glucose levels returned to normal after 6 week of calorie restriction and remained normal during periods of carbohydrate refeeding. Normalization of 2-h plasma glucose concentrations after a standard oral carbohydrate load required 5 months, and glucose disposal after an iv glucose load did not return to normal until the end of the study. Insulin secretion in response to oral glucose reached maximal levels during the first months of weight reduction and then decreased as glucose tolerance continued to improve. Acute phase insulin release in response to iv glucose gradually increased throughout the study. Glucagon stimulation by iv arginine and suppression by iv glucose also returned to normal levels slowly over several months. Abnormalities in glucose tolerance and glucoregulatory hormone secretion of ketosis-resistant diabetes are totally reversible with prolonged dietary therapy. Reduction in tissue resistance to the action of insulin also appeared to be of major importance in the recovery of normal glucose tolerance in this subject.


Teichoic acid antibody determination by agar-gel diffusion: effect of using dilute antigen preparations.

Because the reported frequency of teichoic acid precipitins in controls and various patient groups has varied considerably among laboratories, we studied the effect of using various concentrations of staphylococcal extracts in agar-gel diffusion tests for teichoic acid antibodies. Of 25 normal sera, only 1 was positive against an undiluted extract, but 4 were positive against a 16-fold-diluted extract. Of nine sera from patients with staphylococcal bacteremia, two were positive at a higher titer against the diluted extract. A false-positive serum against the undiluted extract had a twofold titer increase against the diluted extract. Because human immune serum globulin is generally used as a positive teichoic acid antibody control, the variability of five different lots was studied. Three lots ahd teichoic acid antibody titers of 1:4, whereas one each had titers of 1:2 and 1:8. Based on this study, we feel that staphylococcal extracts should not be diluted. If immune serum globulins are used to determine the adequacy of ultrasonic extracts, newly acquired globulin lots should be standarized against an ultrasonic extract of proven sensitivity and specificity.


Enzyme immunoassay for detecting Brucella antibodies in cow's milk.

An enzyme immunoassay (EIA) was developed for detecting Brucella antibodies in milk of cows infected with Brucella abortus. The enzyme immunoassay using heat-killed cells of B. abortus strain 19 was of comparable sensitivity to the Brucella ring test in detecting antibodies in milk of a reference positive control cow experimentally infected with B. abortus strain 2308 and in milk of 16 naturally infected cows from which B. abortus was isolated. No detectable enzyme immunoassay reactions were present in milk of 11 noninfected controls. The enzyme immunoassay is a procedure which can be readily automated so that screening tests for brucellosis could be conducted at a reference laboratory where uniform conditions can be maintained.


Bactericidal antibody response to Pseudomonas aeruginosa by adults with urinary tract infections.

In this investigation we found that adults with upper urinary tract infections caused by Pseudomonas aeruginosa produced serum antibodies with bactericidal activity against the bacterium. Seventeen of 20 infected adults showed bactericidal activity with a titer range of 1:10 to 1:10,000.


Action and interaction of penicillin and gentamicin on enterococci.

The action and interaction of benzylpenicillin and gentamicin on Streptococcus faecalis was studied using mainly turbidimetric methods. The minimum antibacterial concentration (MAC) of each antibiotic lay considerably below the conventionally determined minimum inhibitory concentration, and levels of the two agents exceeding the MAC were necessary in order to obtain a synergic interaction. Evidence was obtained that gentamicin interfered with bacterial lysis induced by penicillin, and this suggests that the aminoglycoside is responsible for the bactericidal activity of the combination, the role of the penicillin being solely to facilitate access of the aminoglycoside to its target site. Our findings do not, however, fully support the generally held view that the increased permeability of enterococci to aminoglycosides is due to penicillin-induced cell wall damage. 'Persisters'--cells surviving prolonged exposure to the optimum lethal concentration of penicillin--were not killed by subsequent exposure to gentamicin if the penicillin was removed but were killed if the penicillin remained present.


Immunochemical demonstration of J chain: a marker of B-cell malignancy.

Many B-cell lymphomas can be shown to contain cytoplasmic immunoglobulin which is characteristically monotypic with respect to light chains. In Hodgkin's disease, however, the Reed-Sternberg cells have been shown to contain both immunoglobulin light chains. This finding, which is also present in some other lymphomas, has been used as evidence both for and against a B-cell derivation of these cells. J chain is present in normal immunoblasts irrespective of the class of immunoglobulin being synthesised and, thus, should be present in tumour cells that synthesise cytoplasmic immunoglobulin. In a series of lymphomas, in which the cells could be shown to contain immunoglobulin, J chain was present only in those tumours exhibiting a monotypic light chain staining pattern. J chain was not present in Reed-Sternberg cells and other cells staining polytypically for light chains. Demonstration of J chain is thus a useful marker for B-cell lymphomas; its absence in Reed-Sternberg cells indicates that the immunoglobulin in these cells is not synthesised by them and cannot be used as evidence for their derivation from B-cells.


Combined melanocytoma-mastocytoma in a case of nodular mastocytosis.

A patient with long-standing nodular mastocytosis developed a slate-blue lesion on the scalp which produced symptoms of histamine release on mechanical and thermal stimulation. Light and electron microscopic examination of the lesion showed zones of mast cells and zones of melanized cells. A transition zone showing cells with dual (melanosomal and mast) granulation, as well as granules representing intergrades between classical mast granules and melanosomes, was present. Features of this lesion add to the accumulated evidence that there is a histogenetic relationship between mast cells and melanocytes.


Adenylate energy charge: a method for the determination of viable cell mass in dental plaque samples.

The biochemical function, adenylate energy charge (AEC), correlates with the viable count of S. mutans. AEC may be used to estimate the percent viable fraction of bacteria in dental plaque samples. An interactive computer program designed to process the AEC data is described.


Cellular mechanics of dentinal bridge repair using 3H-thymidine.

Cellular reorganization in the pulp following mechanical pulp exposure involves three steps: First, lysis and macrophage resolution of the clot form; second, there is an invasion of the clot area by fibroblasts and endothelial cells, i.e., formation of granulation tissue; third, an organization and differentiation of these cells into functional odontoblasts occurs as early as 9 days after exposure. Autoradiographic results showed an increased DNA synthesis in the fibroblast and endothelial cell populations which coincided with a histologically-observed increase in those populations. A relative increase in fibroblastic activity, as compared to endothelial cell activity, suggested that fibroblasts may be the cells that replace odontoblasts.


Clinical studies of plaque control agents: an overview.

Dental plaque is massed packed bacterial cells which accumulate on the supra- and subgingival surfaces of the teeth as well as on the oral mucosa. The microorganisms of plaque have been shown to be associated with both dental caries and periodontal disease. This overview of clinical studies of plaque control agents reviews the properties and effects of chemical compounds which have demonstrated a potential for the control of plaque microorganisms. The search for clinically effective antiplaque agents has been stimulated by findings in laboratory and animal studies of plaque dynamics. Based upon these in vitro and in vivo experiments, chemotherapeutic agents such as antibiotics, antiseptics, enzymes, detergents, bacteriosides, antimetabolites, and oxidizing agents have been evaluated against human plaque microorganisms using the ultimate biological model -- man. Continued study of chemotherapeutic agents should be encouraged because many of these drugs have been shown to be safe for human use and may require only the development of a delivery system to potentiate their concentration in a specific local site. Use of these chemotherapeutic agents, which can be self-administered, becomes an attractive way of providing the public with a cost-effective method of preventing caries and periodontal disease.


Auditory lateralization in monkeys: an examination of two cues serving directional hearing.

In assigning binaural ongoing time differences (phase) as the cue for localization of low frequencies, and binaural intensity differences as the cue for localization of high frequencies, the duplex theory has successfully accounted for human directional hearing of tones. Sensitivity of monkeys to these cues was examined in two experiments. The dependencies on frequency of interaural intensity difference thresholds (lateralization experiment I) and time difference thresholds (lateralization experiment II) were determined behaviorally on three monkeys (M. nemestrina). The range of frequencies was from 125 Hz to 8 kHz in experiment I and from 250 Hz to 2 kHz in experiment II. The results indicate that the duplex theory is applicable to monkeys. However, monkeys are less sensitive than man to both binaural cues. The shortest time disparity monkeys discriminate is 42 microseconds at 1.5 kHz and the smallest intensity difference is 3.5 dB at 500 Hz. Good agreement between the present findings and localization measurements [C. H. Brown et al., J. Acoust. Soc. Am. 63, 1484-1492 (1978)] suggests: (a) that monkeys utilize time disparity cues through higher frequencies than man; and (b) that inaccurate localization by monkeys at high frequencies reflects decreasing sensitivity to interaural intensity cues.


Dietitians' effectiveness and patient compliance with dietary regimens. A pilot study.

Demonstration of dietitians' effectiveness is a necessary prerequisite in documenting the cost-effectiveness of untritional care. In this report, a framework for the study of dietitians' effectiveness and patient compliance with dietary regimens is developed, and the results of a pilot study are presented. Personal, attitudinal, and situational variables that affect adherence to diets were investigated. Findings suggest that (a) disclosure of patient non-compliance varies, depending on the type of question asked and the sequence of questions in an interview; and (b) dietitians with higher "Orientations to Social Influence" use more influence strategies, involve patients in counseling sessions more, and tend to have patients with more appropriate health attitudes and behaviors.


Productivity improvement and job satisfaction among public health nutritionists.

A workshop for public health nutritionists which scrutinized ways to improve productivity and job satisfaction is reported. Participants believed that productivity could be improved most by improving the execution of tasks, followed by better planning of programs, office management, and skills in group education, and by delegation of non-professional activities to lesser-trained personnel. Job satisfaction of public health nutritionists could be increased by reducing stress and discomfort and promoting feelings of personal effectiveness and efficiency through role clarification and by management training. There is a large potential for greater productivity in the profession. Realization of this potential will contribute measurably to the cost-effectiveness of nutritional services.


Circulating prolactin and its response to TRH following administration of testosterone undecanoate in normal men.

To investigate the effect of orally administered testosterone undercanoate (TU) on circulating prolactin (PRL) and PRL response to TRH stimulation, 8 eugonadal male volunteers, aged 19--30, presenting with normal plasma levels of FSH, LH, testosterone (T), estradiol (E2) and PRL, were given 120 mg/day of TU for 6 days. Plasma PRL levels were measured daily during the pre-treatment phase (3 days), treatment phase (6 days) and post-treatment phase (3 days) by radioimmunoassay. The TRH Test (200 micrograms iv) was done on the 3rd day of the pretreatment phase and on the treatment phase. No significant changes in circulating PRL levels or in PRL response to TRH stimulation were observed. Plasma T and E2 levels showed a slight, but not significant tendency to increase, while gonadotropin levels remained unchanged.


Effects of tourniquet ischemia and postischemic edema on muscle metabolism.

In vivo interstitial muscle pressures measured by wick catheter, tissue gas tensions measured by mass spectrometer, and glucose and high-energy phosphate metabolism measured fluorometrically were studied in the anterior tibial (AT) and vastus lateralis (VL) muscles of primate limbs during and following tourniquet ischemia (2.5 hours; 400 mm Hg) to elucidate postischemic edema and its metabolic consequences. During ischemia, interstitial pressure in the VL rose, while in the AT it decreased, but 24 hours later pressures in both experimental muscles were significantly greater than those in the controls. In both experimental muscles PO2 decreased significantly within 15 minutes of ischemia. PCO2 increased significantly in the AT at 30 minutes and at 75 minutes in the VL muscle. Twenty-four hours later only PO2 in the experimental AT was significantly different than its matched control. During ischemia glucose and phosphocreatine (CrP) decreased significantly, and G-6-P and lactate increased significantly in both muscles, but at 24 hours glucose levels were 25% lower and G-6-P 16.2% higher in the experimental AT and CrP 34% lower in the experimental VL. This study shows that there are significant acute and delayed alterations in primate muscle metabolism following tourniquet ischemia and suggests that these changes may be related to the anatomic location of the muscle studied and the type of trauma it has sustained.


An unlabeled antibody macromolecule technique using hemocyanin for the identification of type B and type C retrovirus envelope and cell surface antigens by correlative fluorescence, transmission electron, and scanning electron microscopy.

The present resolution (75-100 A) of the conventional scanning electron microscope (SEM) and its ability to image the surfaces of large numbers of whole cells in situ permit the approach of problems such as viral and cell surface antigen localization by immunological labeling with visual markers. Identification of virus and cell surface antigens in situ has been accomplished in indirect reactions by unconjugated markers. Hemocyanin (Hcy) from whelk, Busycon canniculatum, has been developed as an immunospecific marker for virion and cell surface labeling in the electron microscope. Its size (30 x 50 nm) and distinct cylindrical shape permit easy visualization in the SEM and the transmission electron microscope (TEM). The Hcy method involves the preparation of antisera to Hcy in appropriate hosts for use in an unlabeled antibody macromolecule procedure based exclusively on antigen-antibody affinity to couple the macromolecule to the antigen site. Further correlative data from fluorescence microscopy can be obtained from similarly labeled samples by binding fluorescein to the bridging antibodies used in the Hcy technique. The usefulness of the Hcy marker system was demonstrated by employing highly specific antisera to the major envelope and cell surface glycoprotein (gp70) of Rauscher murine leukemia virus (R-MuLV), a type C retrovirus. The antiserum was shown to bind to the virion and cell surfaces of virus-infected cells in the homologous virus-infected cell system. It also demonstrated the expression of R-MuLV gp70-related antigens on a murine cell line Mm5mt/c1 which produces mouse mammary tumor virus (MuMTV), a type B retrovirus. Furthermore, when used in the Hcy marker system the anti-gp70 serum was able to distinguish type B from type C budding virus on the same cell. Methods for the preparation of immunoreagents and labeling of cells are discussed.


Antibody binding by native and denatured myosin and paramyosin.

By the techniques of immunodiffusion and fluorescent immunohistochemistry we show that antibodies to both the native and the SDS-denatured forms of the proteins, paramyosin and myosin, react with the native, SDS-denatured and glutaraldehyde-fixed forms of their respective antigens. Anti-denatured myosin also binds to both native and denatured forms of the proteolytic subfragments of myosin: globular subfragment-1 and alpha-helical LMM. Anti-native myosin, on the other hand, while able to bind to both native and denatured LMM or rod and to native and glutaraldehyde-fixed S-1, does not bind to SDS-denatured S-1.


Microbial contamination of topical medicaments used in the treatment and prevention of pressure sores.

Topical medicaments used in the treatment and prevention of pressure sores in patients in three hospitals were examined for Pseudomonas aeruginosa and Staphylococcus aureus contamination. Contamination rates were found to vary between hospitals and were affected by differences in the packaging of the product and in the method of application used by the nursing staff.


Comparison of serological methods for the detection of B. abortus antibodies in sera from vaccinated and non-vaccinated cattle.

A total of 4551 sera from 863 Strain 19 vaccinated and non-vaccinated adult cattle, independent of disease status, were tested by five serological methods to detect the presence of antibodies to B. abortus. Results from Standard Agglutination Tube (SAT), Buffered Brucella Antigen or card (CT), Complement Fixation (CF), Enzyme Linked Immunosorbent Assay (ELISA) and Rivanol (Riv) methods were compared. There was a 95% probability for agreement among CT negative sera, between serological methods, for all groups of vaccinated and non-vaccinated cattle. The agreement between tests with Riv Positive sera, excluding the calfhood and adult vaccinated group tested by the CF method, was 91-100%. The probability of a serum which was serologically negative by other methods being Riv negative was 98%. The usefulness of serological results from Riv (greater than or equal to 1/50) tests for classifying the reactor status of cattle are of doubtful supplemental value to confirm card test positive results. Vaccination history is an important consideration when evaluating serological data on cattle sera particularly from SAT and CF methods.


Serological differentiation between infected and vaccinated cattle by visual and quantitative immunofluorescence using Brucella abortus antigen coupled sepharose beads.

The ability to differentiate cattle infected with brucellosis from animals vaccinated against the disease is of considerable economic advantage in countries adopting eradication schemes. Current serological tests such as bacterial tube agglutination do not readily distinguish cattle in this way. An immunofluorescent procedure was investigated in an attempt to overcome this problem. Antigenic extract of brucella abortus strain 544/W were coupled to sodium metaperiodate oxidised Sepharose CL4B beads. Following coupling, the beads were chemically reduced or blocked with rhodamine labelled BSA prior to reduction. Characterised bovine serum samples were tested by indirect immunofluorescence on both types of bead and by standard bacterial tube agglutination. Serological discrimination between infected and vaccinated cattle was achieved by fluorescence quantitation using unblocked beads and by visual determination of colour using rhodamine-albumin blocked beads. Bacterial tube agglutination failed to distinguish between the two groups of sera.


Use of 2-mercaptoethanol to facilitate detection and classification of IgM abnormalities by immunoelectrophoresis.

IgM immunoglobulins in elevated serum concentrations have a tendency to polymerize and form aggregates. When subjected to immunoelectrophoreses these proteins may deposit at the point of origin. This can result in failure to detect an IgM abnormality or the masking of other serum protein abnormalities migrating near the area of application. This paper demonstrates the importance of using reducing agents such as 2-mercaptoethanol not only to detect and/or to confirm the monoclonal IgM gammopathies but also to unmask other protein abnormalities.


Comparison of radioimmunoprecipitation assays for the detection of human anti-tumor virus antibodies.

The demonstration of human antibodies reactive in radioimmunoprecipitation assays (RIAs) with primate tumor virus (oncornavirus) antigen has implications for a possible previously published negative findings and led to considerable scientific controversy. We feel much of the discrepancy may be of methodological origin. An attempt is therefore made in this communication to resolve these apparent discrepancies by comparing various published parameters of the RIAs used in the search for human antibodies reactive with oncornavirus antigens.


Diffusion in gel-enzyme linked immunosorbent assay (DIG-ELISA): a simple method for quantitation of class-specific antibodies.

A new method for quantifying class-specific antibodies is presented. The method has been named Diffusion-In-Gel-Enzyme-Linked-ImmunoSorbentAssay (DIG-ELISA), and is briefly as follows. Antiserum ia allowed to diffuse from wells in a gel layered over an antigen-coated plastic surface. The gel is then removed and the preparation is incubated with enzyme-conjugated anti-immunoglobulin. The enzyme is then visualised in situ by a colour reaction produced by pouring a substrate-containing gel over the plastic surface. Bovine serum albumin and rabbit-anti-BSA were used as a model system, and horseradish peroxidase or alkaline phosphatase as enzymes for visualization.


Improved detection of immune complexes in human and mouse serum using a microassay adaptation of the C1Q binding test.

A microassay adaptation of the [125I]C1q binding test for the detection of circulating immune complexes is described. This technique is more rapid to perform, requires smaller volumes of serum and reagents, and surprisingly, increases the sensitivity of the assay when compared to the previously reported C1q binding method.


A modified cytoplasmic antigen of Candida albicans for serodiagnosis of systemic candidiasis.

A modified cytoplasmic antigen, prepared by Natamycin degradation of Candida albicans cells is described. The reactivity of this antigen in detecting precipitins to Candida albicans proved tobe very similar qualitatively and quantitatively to a standardised reference antigen, prepared by X-press disruption when both were compared by immunological techniques; the majority of antigenic components in each proving to be identical. When tested against 127 human sera of unknown antibody content the two antigens showed 100% correlation by counterimmunoelectrophoresis and 87.5% correlation by double diffusion. The modified antigen proved to be reproducible and reliable in use and is easily prepared in the routine laboratory.


Detection of 3-hydroxy fatty acids at picogram levels in biologic specimens. A chemical method for the detection of Neisseria gonorrhoeae?

A method for the detection of 3-hydroxy dodecanoic acid at low picogram levels is described. The procedure involves preparation of a heptafluorobutryl derivative of the butyl ester of the fatty acid and its detection by gas-liquid chromatography using an electron capture detector. The method was adapted for use with biological specimens. Potential of the method for screening for gonococcal infection is discussed. Limitations of the method are that about 105 Neisseria gonorrhoeae cells are required for detection and that interfering substances are a major problem working at maximum sensitivity of the electron capture detector necessitating complex purification procedures. The method eliminates the need to maintain the viability of cells in specimens, thus facilitating collection and transport of specimens.


The effects of UVB and 7, 12, dimethylbenz(alpha)anthracene (DMBA) on epidermal melanocytes of the tail in C57BL mice.

The morphological and numerical changes in the epidermal melanocyte system of the tail of C57BL mice were studied after exposure to 7, 12 dimethylbenz(alpha)anthracene (DMBA) followed by UVB irradiation. The dorsal aspect of the tail was exposed for 5 days per week, for a total duration of 10 weeks, to a daily dosage of 0.1 J/cm2 UVB (peak 310 nm). Once a week, 0.2 ml of 0.15% DMBA in acetone was locally applied to the irradiated areas. Biopsies were studied by the combined skin-splitting DOPA and electron microscopic techniques. After 10 weeks of DMBA treatment the following changes were observed: DMBA treatment the following changes were observed: the original brick-like arrangement of melanocytes became confluent, melanocytes were irregularly shaped, dendrites shortened and clumped together, and the outer root sheaths of the hair follicles became covered with melanocytes. There was a significant increase in the number of DOPA positive melanocytes at the end of the first week in DMBA and DMBA + UVB treated skin. Ultrastructurally, an increase in melanosome formation in melanocytes and transfer into keratinocytes was found, as well as redistribution of melanosomes from singlets and doublets into larger groups. Damage of melanocytes by the DMBA treatment was seen, but no inflammation or tumor formation was observed.


Heterogenity of serotypes of Neisseria meningitidis that cause endemic disease.

Three collections of strains of Neisseria meningitidis that caused meningococcal disease during nonepidemic periods were serotyped to determine whether serotypes that cause endemic disease are more heterogeneous than those responsible for epidemic disease. Thirty-four strains isolated from pediatric patients in Houston, Texas, from February 1977 to March 1978 were of three separate serogroups and 11 serotypes; 27 contemporary (1977-1978) strains from predominantly military populations, obtained nationwide, were of six serogroups and six serotypes, while 11 strains isolated at military posts in the southwest United States from 1970 through 1976 were of four serogroups and five serotypes. Between 9% and 20% of the strains were nontypable, while type II strains which were responsible for the epidemics in the Northern and Western Hemispheres earlier in the 1970's, accounted for only 20%-44% of the strains. In contast to epidemics, which appear to be caused by a single serotype, endemic meningococcal disease appears to be caused by a broad, heterogeneous distribution of serotypes. Thus, development of a serotype-specific vaccine may have limited application to the prevention of endemic meningococcal disease.


In vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody.

Five pairs of juvenile, dengue virus-susceptible rhesus monkeys were given normal or dengue-immune human cord-blood serum injected intravenously to a final dilution of 1:300. The pool of immune human cord-blood serum had a titer of antibody to dengue type 2 virus (D2V) of 1:140 in the plaque-reduction neutralization test and a titer of human monocyte infection enhancement of greater than 1:2,000,000. Fifteen minutes after inoculation of serum, animals were infected with D2V (strain no. 16681). Daily titers of viremia were always higher in the animals that had received antiserum to D2V than in animals that had received normal cord-blood serum. Ratios of infection enhancement ranged from 2.7 to 51.4. The demonstration of antibody dependence of dengue virus infection in subhuman primates--a complex, outbred experimental host--supports the hypothesis that the severity of dengue in humans is regulated by antibody.


Effect of bacille Calmette-Guérin on the immune response of BALB/c mice to a tumor allograft.

The effect of dosage and route of inoculation of bacille Calmette-Guérin (BCG) on immune response to allogeneic tumor cells was investigated. BALB/c mice were tested 14 and 21 days after injection of EL-4 lymphoma for spleen-cell cytotoxicity against EL-4 cells in vitro and for complement-dependent, antibody-mediated lysis of tumor cells. BCG treatment had no measurable effect on the antibody-mediated lysis of tumor cells, but spleen-cell cytotoxicity was significantly increased in mice treated with 10(4) or 10(8) BCG by the intraperitoneal route; no such increase occurred when BCG was given by the oral or subcutaneous routes. The cytotoxic effector cells were primarily thymus-derived, since treatment of spleens with rabbit antiserum to mouse brain serum decreased cytotoxicity titers by approximately 90%. Within the framework of these experiments, the intraperitoneal route of BCG inoculation resulted in a more effective immune stimulation than the oral or subcutaneous routes.