Man was for woman made and woman made for man.

An acute bed shortage for geriatric patients two years ago resulted in a change of admitting procedure. As a result we ran a mixed sex ward as a rehabilitation unit. The nursing staff felt the atmosphere, rate of recovery and personal relationships improved from male/female company.


Immunocytoma, cancer and other associations of monoclonal gammopathy: a review of 224 cases.

The clinical, haematological and biochemical correlations of 224 consecutive unselected examples of monoclonal gammopathy have been studied. The paraprotein frequency detedted was IgG 62 percent, IgA  15.2 percent, IgM 10.3 percent, Bence Jones protein 8.9 percent and in 3.6 percent the paraproteins were not identified. In half the monoclonal gammopathy was associated with an immunocytoma (myeloma in 82 and lymphoma in 30). In three cases the associated clinical disease was amyloidosis. In 36 cases (16.1 percent) the associated clincial disease was a nonlymphoproliferative malignant tumour. Monoclonal gammopathy may be a significant marker of malignancy in such cases. In 73 cases (32.6 percent) the associated clinical conditions were unrelated to the gammopathy although only 55 of these cases were sufficiently investigated to warrant classification as examples of benign monoclonal gammopathy. There was a strong correlation between Bence Jones proteinuria and malignancy. Sixty-five patients demonstrated Bence Jones proteinuria and in 59 of these a malignancy was detected. The association was strong between hypercalcaemia and malignancy as this was present in all 27 of the cases who had hypercalcaemia. The relationship between Bence Jones proteinuria and hypercalcaemia was also strong and Bence Jones proteinuria was detected in 73 percent of the hypercalcaemic patients as opposed to 36.7 percent in the whole series. Hypercalcaemia and Bence Jones proteinuria, when found in a patient with monoclonal gammopathy have a grave clinical connotation.


Progeny, pregnancy and phenylketonuria.

Two sisters were diagnosed as having phenylketonuria at the age of 13 years and eight years and having Wechsler IQs of 48-58 and 71-81 respectively. Neither girl was treated with diet. At the age of 21 years the older girl became pregnant. Her blood phenylalanine level was 23mg/100 ml. A low phenylalanine diet, instituted from the 10th week of gestation, maintained her blood phenylalanine levels below 6mg/100ml for the rest of the pregnancy. A female baby, of birth weight 3216g and normal skull size, was delivered at term. The baby died at 14 days of congenital heart disease. Pregnancy in a phenylketonuric woman carries high risks to the fetus. A generation of treated phenylketonuric girls is approaching reproductive life, and doctors and the girls themselves need to be alerted to these risks and the need for strict dietary control during pregnancy. There are probably unrecognised women in the community with phenylketonuria or with hyperphenylalaninaemia whose babies face similar risks. Identification of these women could be achieved by antenatal Guthrie testing.


The impact of sports injuries in winter on medical services.

2529 injured sportsmen were treated at the Hutt and Wellington accident and emergency departments from 1 April to 31 August 1972. Almost two-thirds (62 percent) of these injuries were sustained during Saturday games, but only one-third (36.7 percent) of those injured presented on Saturdays. In view of the necessity to x-ray 75 percent, refer 18 percent to other hospital departments and admit 7.6 percent to hospital, a sports medicine clinic is suggested within the hospital complex. Such a clinic would ease the A and E work load and, at the same time, provide improved treatment for injured sportsmen.


Retroperitoneal mesonephric cyst: case report.

A retroperitoneal cyst of mesonephric origin is presented. There may be difficulty in identifying the cause of such a cyst.


Xerocephalography.

Xeroradiography used with standard cephalometric analysis has proved clinically to be extremely beneficial in the orthodontic and orthognathic evaluation of patients. We have used the negative mode of xeroradiogrpahy routinely and have termed this application xerocephalography. We have described the methods and the equipment and have presented examples of the technique. Xerocephalography provides an improved method which may be universally used in orthodontic and orthognathic treatment.


Chemical protection against postirradiation dental caries.

Experience with dental caries in twenty-four patients receiving irradiation for malignant lesions of the head and neck demonstrates the critical importance of cooperation by the patient, a program of strict oral hygiene, and daily self-treatment with 0.4 per cent stannous fluoride gel. The ravaging form of dental caries associated with the postirradiation period can be essentially eliminated in cooperating patients. In nine patients who were uncooperative, fifty-seven crowns were amputated and an additional seventy-five carious surfaces were found 3.75 years after irradiation. In six patients who cooperated and used the gel on a daily basis, no crowns were amputated and only one carious area was found over the same time period.


Oral considerations of the myofascial pain dysfunction syndrome.

The oral health of twenth-two patients with a diagnosis of myofascial pain dysfunction syndrome was evaluated. Radiographs, photographs, study casts, visual and digital clinical examinations, hematologic data, blood pressure, and periodontal examinations were used with each patient. A review of the literature indicated a variety of causes for facial pain, including Costen's syndrome, muscle fatique and spasm, occlusion, and psychogenic factors. This study revealed the following trends: (1) The periodontal health of patients with the myofascial pain dysfunction syndrome appears to be better than anticipated. (2) Bruxism accounts for a healthy dental apparatus when other diseases are not present. (3) Groups of muscles, other than the masticatory group, may contribute to the myofascial pain dysfunction syndrome. (4) Patients presented with various ranges of malocclusions and normal occlusions, deep overbites and overjets, complete dentitions, and missing teeth (either equally missing right and left or unequally missing right and left). This article also discusses clinical considerations in the diagnosis of the myofascial pain dysfunction syndrome and offers a practical, physiologic approach to treatment. We conclude that how one uses his mandible is more of a causative factor than the relationships of the teeth.


Dental anesthetic management of epidermolysis bullosa: a new approach.

Epidermolysis bullosa is a rare blister-producing skin disorder that has been of some concern to dentists in the past. Dental treatment has been attempted on an outpatient basis with local anesthesia. In some of these patients, local anesthesia has caused large intraoral bullae. Other methods of treatment have been sought, including the use of general anesthesia and intramuscular sedation. This is a report of a case in which intravenous ketamine hydrochloride was used with good results. We believe this method has much to offer in the treatment of these patients.


Dental fluorosis associated with hereditary diabetes insipidus.

Among the characteristics of hereditary pituitary diabetes insipidus are polydipsia and polyuria from early infancy. Drinking of large amounts of water, even with lower than accepted fluoride content, can produce fluorosis of the teeth. A mother and her four children affected by this disorder presented different degrees of fluorosis directly related to the stage at which hormonal therapy was introduced.


Dental management of the patient with biliary atresia.

Biliary atresia is a disease of unknown origin characterized by partial or total absence of the biliary tract. While this condition is rare, the medical and surgical management makes the ramifications for dental treatment increasingly complex. This article reviews the disease and its complications and documents dental treatment of two patients.


The calcifying odontogenic cyst. Eight new cases and a review of the literature.

Eight new cases of the calcifying odontogenic cyst have been reported. In addition, a review of the literature produced an additional sixty-five cases. This lesion can occur in any age group, but it shows a peak incidence in the second decade. It is evenly distributed between the maxilla and the manidible and occurs with equal frequency in males and females. There does not appear to be any race predilection. The calcifying odontogenic cyst may occur in association with other pathologic entities. It is postulated that it does not in every case represent a separate pathologic entity but that it may represent a secondary phenomenon which occurs within preexisting lesions.


The ultrastructure of geographic tongue.

Two typical cases of "geographic tongue" were investigated by means of the electron microscope. Biopsy specimens of both including the white elevated and the smooth erythematous areas of the lesions were, after routine processing, studied in semithin and ultrathin sections with a JEM 7A electron microscope. Results revealed, in the white elevated areas of the lesions, subepithelial polymorphonuclear infiltrates and microabsesses, leukocyte invasion into the epithelial layer, interepithelial edema, rupture of cell juctions, rich glycogen deposits in epithelial cells, lack of differentiation into keratinized cells, and cell necrosis with exfoliation of necrotic epithelial cells and leukocytes in the surface layer. The erythematous, smooth areas revealed pathomorphologic features of healing : some mononuclear infiltrate cells in sub- and interepithelial positions, two types of basal cell, occurring in groups, with features of either typical epidermal basal cells of immature basal cells, filament and filament bundle formation in the spinous layer, the lack of a stratum granulosum, and incomplete keratinization or parakeratinization in the surface layer. Differentiation into filiform  papillae  was lacking in this area. The findings have been discussed in relation to the disturbances of the keratinization process.


Familial polyostotic fibrous dysplasia.

A case of polyostotic fibrous dysplasia of the craniofacial type is presented, together with substantial evidence that this condition had a genetic basis in this patient. A review of the literature indicates that there is absolutely no previous evidence of a genetic basis to this condition. The possibility that the propositus suffered from polyostotic fibrous dysplasia of the Jaffe or the Albright type was excluded.


Jaw and pulpal metastasis of an adrenal neuroblastoma.

Hematogenous spread of malignant tumors to the dental pulp is very rare. A case of adrenal neuroblastoma in a 71/2-year-old boy which metastasized to the mandible and dental pulp is described. Tumor cells were found within the pulpal blood vessels of a deciduous molar tooth.


Dimensional stability of temporary restoratives.

The effects of thermal cycling on the dimensional stability of temporary restoratives  were studied. Stability appeared to be affected significantly by water content. On thermal cycling, Cavit showed less linear dimensional change than two cements containing zinc oxide-eugenol and gutta-percha temporary stopping.


The effects of thermal injury on pulpal hydrolases.

The effects of thermal injury on pulpal protein levels and the activities of selected pulpal hydrolases were determined 48 hours following injury. At this time protein levels were significantly greater while enzyme activities were significantly lower in thermally insulted teeth. The implications of these studies are discussed.


Transferred drug-resistance in Eimeria maxima.

A series of experiments is described in which two drug-resistant strains of Eimeria maxima were passaged together in untreated chicks. The resultant oocysts were then inoculated into chicks treated with both drugs. When strains resistant to methyl benzoquate and sulphaquinoxaline or clopidol and sulphaquinoxaline were used the resultant infections were not controlled by the double treatment, indicating the acquisition of resistance factors by one strain from the other. When strains resistant to clopidol and methyl benzoquate were used the phenomenon was not observed.


Dural sinus occlusion in leukemia.

The case of a 7-year-old child with acute lymphocytic leukemia who, while in relapse, developed a superior sagittal sinus thrombosis is documented by angiography and post-mortem examination. Review of the literature suggests that cerebral spinal fluid (CSF) pleocytosis which usually accompanies the arachnoid infiltration of central nervous system (CNS) leukemia may be absent in some patients as in this case. In the absence of CSF pleocytosis, cerebral venous sinus thrombosis may mimic the other signs and symptoms of CNS leukemia and consequently may be a more frequent complication of acute leukemia than is presently appreciated. Therapy directed to the CSF axis generally would not under these circumstances be appropriate.


Effect of spinal deafferentation on temperature regulation and spinal thermosensitivity in pigeons.

1. To study the effect of spinal deafferentation on temperature regulation and spinal thermosensitivity in acute experiments, the spinal cord of pigeons was transected at Th4 and the dorsal roots cut carefully on both sides from Th4 to C6 or C4 (13 or 15 segments); only afferent signals from the upper neck and the head could reach the CNS. Selective changes of the spinal cord temperature in the deafferented region were performed by a thermode in the vertebral canal. 2. At thermoneutral ambient conditions (Ta 23-30 degrees C) the deafferented pigeons were able to maintain a normal body temperature (about 41 degrees C). During ambient cooling (Ta 1-10 degrees C) the core temperature was regulated at a lower level of about 38 degrees C, strong shivering occurred, and heat production was increased. 3. If the decreased spinal cord temperature at low Ta was adjusted experimentally to normal values (about 41 degrees C) then shivering stopped and oxygen consumption decreased. This decrease in heat production was followed by a continuous fall in rectal temperature to values as low as 33-34 degrees C without any initiation of shivering or extra heat production. This means that shivering in the deafferented pigeons must be elicited by cold sensors in the spinal cord alone and that there are no important cold sensors in the non-deafferented region including the brain. 4. Selective spinal cooling of the deafferented region at thermoneutral Ta was followed by an immediate onset of shivering and an increase in heat production. Spinal heating resulted in an increase in wing temperature which served as an indication of vasodilatation, i.e., an activation of a heat loss mechanism. This is a confirmation of the assumption that the spinal temperature sensors are indeed located in the spinal cord and that the responses to experimental changes of spinal canal temperature are not mediated by extraspinal thermoreceptors. The results show clearly that the regulation of body temperature in pigeons at moderate thermal loads can be mediated by these spinal sensors alone. 5. Continued spinal cooling resulted in an increase in body temperature by about 2 degrees C and a subsequent regulation at this high level. This means that there must exist warm sensors in the non-deafferented cranial region.


[Microretention of plastic filling materials on acid etched enamel].

In order to study the strength of the physical bond between composite materials and acid etched enamel, various commonly used composites were applied to the conditioned surface of extracted young permanent bicuspids. The specimens were stored in saline at 37 degrees C for one week and nine weeks and then subjected to tensile strength test. The following composites were tested: Restodent, Nuva Seal/Nuva Fil, Cosmic, Smile, Adaptic and Concise Enamel Bond. The latter was used in 4 different ways. The results of the study indicate that all the tested materials have a fairly good retention on the conditioned enamel. However, when using Concise Enamel Bond, care must be taken not to let the pure, liquid resin polymerize on the enamel before applying the composite, because this will give a poor bonding between the enamel and the filling. In addition to the tensile strength test, the materials were submitted to scanning electron microscop, to examine their penetration ability into conditioned enamel. The results of this study seem to indicate that the resin part of viscous composites (Cosmic and Concise pastes) was able to penetrate into the crevices of the etched enamel as well as the more fluid types (Restodent, Nuva Seal and diluted Concise).


[Electrostatic air filters for dental practice].

The filtrering effect of an electrostatic filtrattion equipment (Vortronic mod. 75) for air filtration was examined by sampling the air in a general dental practice by means of a Casella Slit Sampler for Airborne Bacteria (C. F. Casella & Co. Ltd., London). A reception, a sterilization, two treatment chairs and a resting cabinet were seperated by 180 cm high walls in a rectangular room of 33 m2 and with an air volume of approximately 80 m3. The staff including one dentist and one or two assistents worked from 8 a.m. till 4 p.m. with a 30 min. lunch break. A mean of 17 patients visited the practice every day. The electrostatic filter was placed in front of one of the treatment chairs and the slit sampler was, for one period, placed at a distance of 5 m from the filter. In the next period both filter and slit sampler were placed within 140 cm from the treatment chair. The air was sampled at noon and at 4 p.m. Micro-organisms from the air samples were cultured on blood agar by incubation at 37 degrees C for 48 hours. After incubation all colonies visible to the eye were counted. A total of 36 air samples were examined. Without filtration of the air the bacterial contamination amounted to 14 cultivable organisms per ft3 air at noon and 54 organisms per ft3 at 4 p.m. The filtration equipment proved to reduce the air contamination to 6 cultivable organisms per ft3.


[Systems for financing and delivery of dental care].

This paper gives a survey of the most common systems for financing delivery of dental services in the USA and Europe. Emphasis is placed on the systems prevailing in the Scandinavian countries. The background and development of the Norwegian dental health policy are discussed, and the current programs and plans are reviewed. The need for and attitudes towards the introduction of new programs are appraised. It is concluded that new dental health programs directed towards disadvantaged individuals, adolescents and small children should be developed in Norway. The "mixed" system, i.e. public dental services together with dental health insurance, seemingly have certain advantages.


[Encephalic pseudotumoral toxoplasmosis in the course of blood diseases].

The authors report two cases, the first pathological and the second clinical of pseudo-tumoural cerebral toxoplasmosis, occurring during the course of blood diseases. They refer to the conditions of appearance of this rare complication, with only twenty cases recorded in the literature, during malignant blood diseases. They stress the primordial role of immunological deficiency related either to the underlying disease (especially Hodgkin's disease) or to immuno-suppressive therapy. The clinical picture, with little in the way of typical features, is thus that of a diffuse méningo-encéphalitis. Pseudo-tumoural forms are rare. The authors also stress the minimum conditions required for the validity of the dye test and the value of the diagnosis of cerebral toxoplasmosis. An association of sulphadiazine-purimethamine, active and effective, cured the second patient.


Mobility of cholera toxin receptors on rat lymphocyte membranes.

Fluorescein-labeled cholera toxin binds detectably to 40-60% of rat mesenteric lymph node cells and induces a temperature-dependent redistribution (patch and cap formation) of cell surface toxin receptors. The redistribution is inhibited by several "metabolic," "microtubule," and "microfilament" inhibitors, by concanavalin A, and by anticholera toxin IgG. Various studies indicate that cholera toxin is at least bivalent, and that this property may be related to both the induction of receptor redistribution and to the activation of adenylate cyclase. Membrane components which are probably identical to the sialo-glycolipid, GM1 ganglioside, appear to be mobile in the plane of the membrane. The possible role of toxin multivalency and receptor mobility in the mechanism of toxin action is considered.


Theoretical studies of metal-phosphate interactions: interaction of Li+, Na+, K+, Be++, Mg++, and Ca++ with H2PO4- and (CH3O)2PO2-: implications for nucleic acid solvation.

Model phosphate-metal solvation complexes have been studied by ab-initio self-consistent-field techniques. The complexes studied include (RO)2PO2-(R = H or CH3) with Li+, Na+, K+, Be++, Mg++, Ca++, H2O, and Cl-. The geometries of the complexes were chosen to approximate reasonable model solvation complexes for phosphate groups in a nucleic acid environment. Calculated energies of formation vary as Be++ greater than Mg++ greater than Ca++ greater than Li+ greater than Na+ greater than K+ for all isostructural complexes, consistent with experimental binding trends. These results suggest that site binding of this type can successfully account for the relative specificities of ion binding in polynucleotides and other phosphate-containing molecules.


Model of protein folding: inclusion of short-, medium-, and long-range interactions.

A hypothesis for protein folding is proposed, in which the native structure is formed by a three-step mechanism: (A) formation of ordered backbone structures by short-range interactions, (B) formation of small contact regions by medium-range interactions, and (C) association of the small contact regions into the native structure by long-range interactions. Empirical interaction parameters (free energy of formation of a contact) between amino-acid residues were evaluated from the frequency of contacts in the x-ray structures of native proteins. On the basis of this mechanism, a Monte Carlo simulation of protein folding (with an accompanying decrease in the total contact free energy) was carried out for bovine pancreatic trypsin inhibitor. The predicted three-dimensional structure is in fairly good agreement with the experimental one.


Matrix method for fluctuations and noise in kinetic systems.

In a series of papers we were concerned with the question of how to calculate the concentration noise power spectra of an ensemble of multi-state linear kinetic systems when the rate constants of the systems are assumed to be known. We have used a standard eigenvalue-eigenfunction method to solve the differential equations which govern the regression of the means and derived the noise power spectrum as a function of the eigenvalues and eigenfunctions of the relaxation matrix of the system. In this paper, we have obtained an equation which relates the noise spectrum matrix of the fluctuations directly to the relaxation matrix of the means. As a result, the noise power spectrum can be calculated through matrix operations without the necessity of an eigenvalue-eigenfunction calculation. The present formalism is particularly useful in the evaluation of kinetic rate constants when the noise spectrum data of concentration fluctuations are given. Possible applications to biochemical systems are briefly discussed.


Translation of thyroglobulin 33S messenger RNA as a means of determining thyroglobulin quaternary structure.

Thyroglobulin is a 19S protein of approximately 660,000 daltons and unknown quaternary structure. We have previously shown that a 33S mRNA purified from mammalian thyroids promoted synthesis in the Xenopus oocyte of a peptide immunologically related to thyroglobulin. Chemical identity to the native protein is now presented by means of a tryptic peptide analysis. Moreover, the 33S mRNA is shown to contain all the information required for the synthesis of a complete 19S thyroglobulin molecule. Gel filtration in Sepharose under denaturing conditions indicates that the reduced polypeptide encoded by the 33S mRNA is larger than 210,000 daltons. A model of a dimeric thyroglobulin with about 300,000 dalton subunits is presented.


Imbalanced globin chain synthesis in heterozygous beta-thalassemic bone marrow.

Globin synthesis was studied in the bone marrow of seven heterozygous beta-thalassemic subjects. We found evidence of significant imbalance of alpha- and beta-chain production particularly at short times of incubation. There was a progressive decrease in alpha/beta-chain production ratio with increasing incubation time which was due to a decreased rate of net alpha-chain production, indicating that a large proportion of the newly synthesized alpha chains are degraded, particularly in bone marrow, within a few minutes of synthesis, leading to relatively low alpha/beta ratios if these are measured solely at incubation times greater than 10 min. The significant degradation of excess alpha chains explains why inclusion body formation and ineffective erythropoiesis, notable in beta-thalassemia homozygotes where there is gross chain imbalance, are not observed to any marked degree in heterozygotes.


Very short-lived and stable mRNAs from resting human lymphocytes.

The kinetics of degradation of newly synthesized cytoplasmic poly(A)-bearing RNA have been examined in resting human lymphocytes. Two classes were identified, a very labile component with a half-life of less than 17 min and a stable component which remains apparently undiminished during 24 hr of observation. Both classes have molecular weights between 2.5 and 3.5 x 10(5) but the stable material has a narrower size distribution and a slightly lower average molecular weight than the short-lived component. The fate of stable RNA synthesized in the resting cell was also examined after growth stimulation with phytohemagglutinin after 2 and 6 hr of treatment. No transfer of stable material into the labile pool could be discerned; the amount of stable material remained constant. The existence of two species of mRNAs with different lifetimes in animal cells provides a potential means for regulation of protein synthesis by controlling the supply of specific messages. Furthermore, such a short-lived mRNA species may explain the observed disparity between the amount of poly(A)-bearing heterogeneous RNA produced in the nucleus and the amount of mature message found in the cytoplasm.


Relationship between in vivo degradative rates and isoelectric points of proteins.

Previous studies have shown that in mammalian cells proteins of large molecular weight are degraded more rapidly than small ones. Evidence is presented here that half-lives of proteins are also related to their isoelectric points. A double-isotope method was used to compare degradative rates of soluble proteins separated by isoelectric focusing. In rat liver, skeletal muscle, kidney, and brain, more rapid rates of catabolism were found for acidic protein fractions than for neutral or basic ones. Acidic proteins also tended to be degraded faster in several mouse tissues. A literature survey confirmed this trend. For 22 proteins from rat liver, a highly significant correlation was found between rates of degradation and isoelectric points (r = 0.824; P less than 0.01). This relationship between isoelectric point and half-life appears to be distinct from that between protein size and half-life.


Cell-free synthesis of human interferon.

With mRNA prepared from induced human fibroblasts biologically active human interferon was synthesized de novo in a cell-free extract from mouse cells. The identity of the antiviral activity as human interferon was demonstrated by its species and antigenic specificity.


Major nonhistone proteins of rat liver chromatin: preliminary identification of myosin, actin, tubulin, and tropomyosin.

Two major nonhistone polypeptides from rat liver chromatin have been identified as myosin and actin. Preliminary observations indicate that three other chromatin polypeptides of molecular weights 50,000, 34,000, and 32,000 are tubulin and heavy and light tropomyosin, respectively. A sixth component of molecular weight 65,000 which has been purified and electrophoreses as a single band on sodium dodecyl sulfate-polyacrylamide gels may be composed in part of protease-digested myosin. These six polypeptides together account for as much as 38% of the nonhistone protein mass of chromatin in this tissue.


Position-specific oxygenation of benzo(a)pyrene by different forms of purified cytochrome P-450 from rabbit liver.

High-pressure liquid chromatography was used to detect oxygenated products of benzo[a]pyrene formed in a reconstituted microsomal mixed-function oxidase system containing cytochrome P-450 (P-450LM), phospholipid, and NADPH-cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4). Three cytochrome fractions purified from a single source, hepatic microsomes from phenobarbital-treated rabbits, were studied; the various forms of the cytochrome are designated by their relative electrophoretic mobilities. The total benzo[a]pyrene oxygenation rate was greatest for P-450LM1,7, intermediate for P-450LM2, and least for P-450LM4. The phenolic products were eluted in two peaks, A and B, that contained primarily 9-hydroxy- and 3-hydroxybenzo[a]pyrene, respectively. The ratio of peak A to peak B phenols was 0.11 for P-450LM2 and 0.45 for P-450LM4. Thus, the relative amounts of the various phenols formed by these two cytochrome fractions differ markedly. The positional specificity of the hydroxylation is also indicated by large differences in the fluorescence spectra of the phenolic products formed by the two cytochromes. P-450LM2 and P-450LM4 did not form benzo[a]pyrene dihydrodiols, thereby showing that benzo[a]pyrene oxide hydratase activity was absent from these purified preparations. Ninety percent of the phenols formed by P-450LM1,7 were eluted in peak B; the metabolites produced by this preparation also included dihydrodiols, thus indicating the presence of hydratase activity. The positional specificities of different forms of cytochrome P-450 may channel polycyclic aromatic hydrocarbon metabolism into the various activation and detoxification pathways and thereby help determine the cytotoxic and carcinogenic activity of these compounds.


Direct identification of the calcium-binding amino acid, gamma-carboxyglutamate, in mineralized tissue.

A direct approach has been developed for quantitative identification of the calcium-binding amino acid, gamma-carboxyglutamate, in proteins. This should be advantageous for the study of numerous systems where specific roles for the binding of calcium or other divalent cations are suspected. Investigation of mineralized tissue, where calcium-binding proteins are implicated in the mineralization process, revealed that gamma-carboxyglutamate was present in proteins solubilized from chicken bone with neutral aqueous ethylenediamine tetraacetic acid. This was established by direct isolation of the amino acid from alkaline hydrolysates and its quantitative conversion to glutamic acid by decarboxylation in 0.05 M HCl at 100 degrees. The kinetics of decarboxylation and chromatographic behavior are identical to those of gamma-carboxyglutamate from human prothrombin. After resolution of the soluble bone proteins by phosphate gradient elution from hydroxyapatite, gamma-carboxyglutamate was found to be concentrated primarily in one BaSO4-adsorbable anionic protein species; bone collagen was devoid of the amino acid. In view of the recently discovered requirement of vitamin K for generation of calcium binding sites (gamma-carboxyglutamate) by gamma-carboxylation of specific glutamic acid residues in prothrombin, our findings may implicate vitamin K metabolism in normal bone development and suggest a role for the gamma-carboxyglutamate-rich protein in regulation of calcium salt deposition in mineralized tissues.


Translation of hormone-induced messenger RNA in amphibian oocytes: I. Induction by estrogen of messenger RNA encoded for vitellogenic protein in the liver of the male African clawed toad (Xenopus laevis).

Induction of the synthesis of the vitellogenic proteins, lipovitellin and phosvitin, in the liver of the male African clawed toad (Xenopus laevis) was investigated as a function of time after treatment with estradiol-17beta [1,3,5(10)-estratriene-3,17beta-diol]. The appearance of mRNAs encoded for lipovitellin and phosvitin in the cytoplasmic fraction of the liver was assayed by microinjections of hepatic mRNA preparation [either polyribosomes or poly(A)-rich RNA] into oocytes obtained from mature female toads. Oocytes were then incubated in the presence of radioactive amino acid(s) at 19 degrees for periods of time varying from 4 to 18 hr after microinjection. The results show that at 2 hr after hormone treatment more mRNA was present in the cytoplasm, and that from 2 to 72 hr after treatment the level of induced mRNA increased almost linearly to 110% above the control values. Experiments employing specific lipovitellin antiserum indicated no radioactive lipovitellin among the proteins synthesized in oocytes microinjected with hepatic mRNAs isolated from 3 to 9 hr after hormone treatment. However, a marked synthesis of immunoprecipitable, radioactive lipovitellin and an enhanced incorporation of [3H]serine occurred in the oocytes microinjected with hepatic mRNA preparations obtained from toads treated with hormone for 12 or more hr. The identities of the proteins encoded by the mRNAs induced early in estrogen action (2-9 hr) in the male amphibian liver are unknown. It is surmised that some of these proteins may function in the regulation of the subsequent synthesis of the vitellogenic proteins.


De novo synthesis of a polymer of deoxyadenylate and deoxythymidylate by calf thymus DNA polymerase alpha.

In a reaction mixture containing calf thymus DNA polymerase alpha (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7), calf thymus DNA unwinding protein, DNA, deoxyadenosine 5'-triphosphate and deoxythymidine 5'-triphosphate, a copolymer of deoxyadenylate and deoxythymidylate is synthesized after a lag period of 1-2 hr. In the presence of the four deoxyribonucleoside triphosphates only deoxyadenylate and deoxythymidylate are incorporated into the polymer and the rate of synthesis is decreased. The reaction variably occurs in the absence of DNA or DNA unwinding protein but with a greatly entended lag period. The optimal Mg2+ concentration for synthesis of the polymer of deoxyadenylate and deoxythymidylate is 1 mM, in contrast to an optimal Mg2+ concentration of 8 mM for DNA synthesis with activated DNA as template. Characterization of the product of de novo synthesis indicates that it is the alternating copolymer, poly(dA-dT).


Regulation of synthesis of hepatic fatty acid synthetase: binding of fatty acid synthetase antibodies to polysomes.

Mammalian fatty acid synthetase was shown to be composed of two peptides, molecular weight 240,000, after dissociation with sodium dodecyl sulfate. Rat liver polysomes that synthesize fatty acid synthetase were identified by sucrose gradient analysis of polysomes that had been reacted with 125I-labeled antibody against fatty acid synthetase. The binding of 125I-labeled antibody to polysomes was found to correlate with the rate of hepatic fatty acid synthesis in various nutritional conditions.


Analysis of DNA of isolated chromatin subunits.

Partial digestion of rat liver nuclei with staphylococcal nuclease results in the liberation of nucleo-protein complexes consisting of one or more upsilon bodies. By velocity centrifugation we have isolated the monomeric subunit in relatively pure form. We find that this subunit contains 185 base pairs of DNA and 240,000 daltons of protein, resulting in a protein to DNA ratio identical to that of unperturbed chromatin. The isolated monomeric particle is further susceptible to internal nuclease attack resulting in the solubilization of 46% of the monomeric DNA. Analysis of the resistant DNA reveals a complex but highly reproducible pattern of DNA fragments ranging from 160 to 60 base pairs in length. Analysis of the reassociation kinetics of the isolated subunit DNA reveals that most, if not all genomic sequences, are involved in this basic subunit structure. No special frequency class of DNA is absent from upsilon bodies. Furthermore, virtually all liver sequences transcribed into mRNA are present in upsilon body DNA. These results indicate that upsilon body formation may be random with respect to DNA sequence and suggest that the mere presence of upsilon bodies over a specific region of DNA is not sufficient to restrict its transcription.


The turning wheel: a study in steady states.

Subject to the limitation of one-step transitions, a system composed of a polyfunctional macromolecule (say a polyfunctional enzyme), present in fixed amount together with its ligands (substrates) in a fixed volume, can be represented by an r-dimensional cube of which each of the 2r corners corresponds to one of the microscopically different forms. In the steady state we may predict circulation of the macromolecule around the edges of the cube. This would provide a molecular mechanism whereby one enzymatic reaction could drive another, the enzyme itself acting as a transducer. Such a system is asymptotically stable and subject to a set of linkage relations derivable by the cofactor principle from the steady state equations.


A theory of the chain melting phase transition of aqueous phospholipid dispersions.

A model for the chain melting phase transition in dilute aqueous phospholipid bilayer dispersions is presented. This model includes interactions between head groups, between hydrocarbon chains, and within the chains. The head groups are modeled as hard disks which are constrained to lie on a two-dimensional surface separating the aqueous and hydrocarbon regions. The chain statistics problem is treated in an approximate manner using an approach motivated by scaled particle theory to describe the inter-chain steric repulsions in a mathematically tractable way. In this approach the whole system interacts with any given chain through an average lateral pressure which is proportional to the hard disk pressure. Following Nagle, we assume that the steric repulsions between chains and between head groups and the trans-gauche rotation energies are the dominant interactions in determining the transition and we describe the effect of the other interactions with a mean field approximation. Using the known transition temperature of a series of 1,2-diacyl phosphatidyl cholines to adjust two parameters in the theory, the model gives enthalpy and area changes that are in quite reasonable agreement with experiment. Moreover, the curvature observed in the plot of the transition temperature against acyl chain length is reproduced.


Magnitude of subunit inequivalence for oxygen release from hemoglobin: reinvestigation of the oxygen-pulse experiment.

Two hypotheses have been presented to explain the grossly biphasic oxygen release kinetics observed when hemoglobins are studied with the oxygen pulse technique [Gibson (1973) Proc. Nat. Acad. Sci. USA 70, 1-4]. Hypothesis I suggests that the two phases result from cooperativity, with the fast phase being oxygen release from the low affinity (T) state and the slow phase being oxygen release from molecules that have switched to the high affinity (R) state. Hypothesis II suggests that the biphasic curves are due to a large (factor of 20-30) difference in oxygen release from the two types of subunits within deoxyhemoglobin. In order to experimentally discriminate between these two hypotheses, we reinvestigated the oxygen pulse reaction for hemoglobin Kansas (alpha2 beta2 102 Asn leads to Thr) in the absence and presence of inositol hexaphosphate, since recent high resolution nuclear magnetic resonance studies have shown that this allosteric cofactor stabilizes hemoglobin Kansas in T even when fully liganded [Ogawa, Mayer, and Shulman (1972) Biochem. Biophys. Res. Commun. 49, 1485-1491]. The results of these studies clearly favor hypothesis I over hypothesis II as being the correct interpretation for the oxygen pulse results. However, we have found evidence that suggests that oxygen release and binding in T are surprisingly faster than previously observed. Furthermore, within T, there is some spectral and kinetic heterogeneity for oxygen release from adult hemoglobin and hemoglobin Kansas. The magnitude of this kinetic heterogeneity in T appears to be about the same as that seen in the high affinity, R, state. The exchange of hypothesis II for hypothesis I more strongly favors views of cooperative oxygen binding involving both types of subunits, as required if the allosteric model of Monod, Wyman, and Changeux [(1965) J. Mol. Biol. 12, 88-118] is considered operative.


Determination of the iron-sulfur distances in rubredoxin by x-ray absorption spectroscopy.

The high intensity x-ray flux from the synchrotron radiation at the Stanford Synchroton Radiation Project has been used to study the extended x-ray absorption fine structure (EXAFS) of the iron-sulfur protein Peptococcus aerogenes rubredoxin. Absorption measurements were made from 7080 eV, which is below the K-edge of iron, to about 650 eV above the edge and structure was obtained over the entire region. By means of a model iron-sulfur compound for evaluating the phase shifts, the variation of the absorption above the edge of lyophilized, oxidized rubredoxin was converted to iron-sulfur distances. The data were fitted with a least squares program to a model in which three distances R3 were kept equal and the fourth R1 was allowed to differ. The mean square error was constant over a region of this parameter space, becoming twice as large at R3 = 2.217, R1 = 2.389 and R3 = 2.268, R1 = 2.108 A. These values, which are the extreme differences allowed by the present data, are definitely closer to being equal than those found by the determination of the x-ray diffraction crystal structure of the similar protein from Clostridium pasteurianum. However, the average distance from our experiment is in excellent agreement with the average distance from the crystal structure determination. Preliminary EXAFS measurements were also made on the oxidized rubredoxin in solution at pH 7.0. The spectra were unchanged, indicating that the average iron-sulfur distance change is less than 0.02 A. Upon reduction the average iron-sulfur bond length increased by about 0.05 A. Since the EXAFS measurements can give accurate determinations of distances in proteins both in crystals and solution, the technique should be widely applicable.


Infection of preimplantation mouse embryos and of newborn mice with leukemia virus: tissue distribution of viral DNA and RNA and leukemogenesis in the adult animal.

Explanted mouse embryos derived from low leukemia incidence strains were infected with Moloney murine leukemia virus (M-MuLV) at the 4-8 cell stage of development. After cultivation in vitro to the blastocyst stage, the embryos were surgically transferred to the uteri of pseudo-pregnant surrogate mothers. Of 15 animals born, one developed a leukemia at 8 weeks of age. When autopsied, this leukemia was found to be of the lymphatic type, as is typical for the M-MuLV-induced disease. In addition, infectious M-MuLV virus was isolated from the serum. Molecular hybridization tests for the presence of M-MuLV-specific sequences were conducted on DNA and RNA extracted from eight different organs. The DNA-DNA reannealing experiments revealed the presence of two classes of M-MuLV-specific sequences in equal concentrations in all tissues tested. The less abundant class of M-MuLV-specific sequences was not detected in tissues from uninfected animals or in non-target tissues of leukemic animals infected at birth. The results are consistent with the working hypothesis that the virus was integrated in all cells of the animal, possibly including the germ line. Fifty to 100 times more M-MuLV-specific RNA was detected in tumor tissues than was found in non-target organs such as liver, brain, and testes. Since all organs contained the same amount of virus-specific DNA, these results indicate that the M-MuLV-specific DNA can be differentially expressed in different tissues.


Evidence for the translocation of 5'-nucleotidase across hepatic membranes in vivo.

Hepatic 5'-nucleotidase (EC 3.1.3.5; 5'-ribonucleotide phosphohydrolase) activity has been studied in cisternal elements of the Golgi complex and in secretion vacuoles, both isolated after ethanol administration to rats in vivo. The enzyme in secretion vacuoles was latent, so that a 5-fold increase in activity was observed when incubations were carried out in the presence of detergent; evidence is presented that the latency is caused by the impermeability of the membrane to substrate. Essentially no latency was observed in Golgi cisternae. Confirming the results of Farquhar et al. [(1974) J. Cell Biol. 60, 8-25], reaction product from 5'-nucleotidase was localized by cytochemical procedures on the inside of secretion vacuoles and on the cytoplasmic side of Golgi cisternae. After solubilization in detergent, the enzyme from both fractions reacted almost identically with both antibody to the purified enzyme and concanavalin A. In contrast, when intact fractions were incubated with an excess of antibody or concanavalin A, only 22-23% of the enzyme was inhibited in secretion vacuoles whereas 51-84% was inhibited in Golgi cisternae. Sonication of secretion vacuoles in the presence of antibody or concanavalin A increased the inhibition 2- to 3-fold. It is suggested that during the formation of secretion vacuoles from the Golgi cisternae, 5'-nucleotidase is translocated from the cytoplasmic side of the membrane to the inside.


Assembly of microtubules onto kinetochores of isolated mitotic chromosomes of HeLa cells.

The kinetochores of isolated HeLa cell chromosomes attached to an electron microscope specimen grid, fixed in formaldehyde, and stained with alcoholic phosphotungstic acid are visible as dark, preferentially stained structures distinct from the chromatin with which they are associated. When unfixed chromosomes are immobilized by attachment to grids and incubated with chick brain tubulin, microtubules are observed to assemble onto the kinetochores. This demonstrates the competence of kinetochores in isolated chromosomes to act in vitro as microtubule assembly sites and suggests that they also possess this capacity in vivo. In addition, the results provide a possible means for isolating and characterizing kinetochores.


Synthesis of acetylcholine receptor during differentiation of cultured embryonic muscle cells.

Acetylcholine receptor, a component of the specialized muscle membrane, appears during the differentiation of embryonic myogenic cells in tissue culture. Demonstration of incorporation of the radioactive precursor L-[35S]methionine into purified receptor polypeptides is presented as evidence for its synthesis de novo. The identity of the purified radioactive species is established by cosedimentation of the [35S]receptor with [3H]alpha-toxin binding activity on sucrose gradients and by crossreaction with antiserum to purified acetylcholine receptor of Electrophorus electricus.


Increased cellular levels of spermidine or spermine are required for optimal DNA synthesis in lymphocytes activated by concanavalin A.

There are large increases in cellular levels of the polyamines spermidine and spermine in lymphocytes induced to transform by concanavalin A. The anti-leukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) blocks synthesis of these polyamines by inhibiting S-adenosylmethionine decarboxylase. Previous results showed that when cells are activated in the presence of MGBG the synthesis and processing of RNA, as well as protein synthesis, proceed as in the absence of the drug. In contrast, the incorporation of [methyl-3H]thymidine into DNA and the rate of entry of the cells into mitosis are inhibited by 60% in the presence of MGBG. Several experiments suggest that MGBG inhibits cell proliferation by directly blocking polyamine synthesis and not by an unrelated pharmacological effect: (1) the inhibitory action of MGBG is reversed by exogenously added spermidine or spermine; (2) inhibition of DNA synthesis by MGBG shows the same dose-response curve as does inhibition of spermidine and spermine synthesis; and (3) if MGBG is added to cells which have been allowed to accumulate their maximum complement of polyamines, there is no inhibition of thymidine incorporation. MGBG-treated and control cultures initiate DNA synthesis at the same time and show the same percentage of labeled cells by autoradiography. Therefore, it appears that in the absence of increased cellular levels of polyamines, lymphocytes progress normally from G0 through G1 and into S-phase. Furthermore, these experiments suggest that the increased levels of spermidine and spermine generally seen in rapidly proliferating eukaryotic systems are necessary for enhanced rates of DNA replication.


Normal and mutant thermotaxis in the nematode Caenorhabditis elegans.

When grown at a temperature from 16 degrees to 25 degrees and placed on a thermal gradient, the nematode Caenorhabditis elegans migrates to its growth temperature and then moves isothermally. Behavioral adaptation to a new temperature takes several hours. Starved animals, in contrast, disperse from the growth temperature. Several mutants selected for chemotaxis defects have thermotaxis defects as well; these behaviors depend on some common gene products. New mutants selected directly for thermotaxis defects have unusual phenotypes which suggest mechanisms for thermotaxis.


Induction by alkylating agents of sister chromatid exchanges and chromatid breaks in Fanconi's anemia.

Sister chromatid exchanges, which may reflect chromosome repair in response to certain types of DNA damage, provide a means of investigating the increased chromosome fragility characteristic of Fanconi's anemia. By a recently developed technique using 33258 Hoechst and 5-bromodeoxyuridine, it was observed that the baseline frequency of sister chromatid exchanges in phytohemagglutinin-stimulated lymphocytes from four males with Fanconi's anemia differed little from that of normal lymphocytes. However, addition of the bifunctional alkylating agent mitomycin C (0.01 or 0.03 mug/ml) to the Fanconi's anemia cells during culture induces less than half of the increase in exchanges found in identically treated normal lymphocytes. This reduced increment in exchanges in accompanied by a partial suppression of mitosis and a marked increase in chromatid breaks and rearrangements. Many of these events occur at sites of incomplete chromatid interchange. The increase in sister chromatid exchanges induced in Fanconi's anemia lymphocytes by the monofunctional alkylating agent ethylmethane sulfonate (0.25 mg/ml) was slightly less than that in normal cells. Lymphocytes from two sets of parents of the patients with Fanconi's anemia exhibited a normal response to alkylating agents, while dermal fibroblasts from two different patients with Fanconi's anemia reacted to mitomycin C with an increase in chromatid breaks, but a nearly normal increment of sister chromatid exchanges. The results suggest that chromosomal breaks and rearrangements in Fanconi's anemia lymphocytes may result from a defect in a form of repair of DNA damage.


Meiosis in Phycomyces.

A four-factor cross between two strains of Phycomyces involving two auxotrophic, one color, and the mating type marker is described. Samples of 40 germspores from 84 individual fertile germsporangia were characterized. The results show: (i) The germspores of a germsporangium are derived from one meiosis in approximately 78% of the cases. (ii) The four markers are on separate chromosomes. They are nonselective. (iii) Analysis of a large sample of germspores from 106 pooled germsporangia confirms that the four markers are unlinked. (iv) From the ditype/tetratype ratios it is inferred that each marker is located about 15 map units from its centromere.


Inheritability of plasmids and population dynamics of cultured cells.

The compositions of growing bacterial cultures containing F' plasmids are developed in theoretical terms that will be helpful in designating experiments to determine the genetic and physiological parameters involved. The genetic parameter is the inheritability of the plasmid defined as the probability, h, that a daughter bacterium will inherit the plasmid and thus be F' rather than F-. The value of h determines the chance that the plasmid will survive in a clone initiated by a single F' bacterium. If 0 less than or equal to h less than 0.5, the probability of plasmid survival is zero, whereas if 0.5 less than h less than 1 the survival is (2h - 1)/h2. While clone sizes are demonstrated to be erratic, the proportion of F' bacteria does converge to an equilibrium value if log2 h greater than alpha - 1, where the physiological factor, alpha, is the ratio between the division times of F' and F- bacteria. A general expression of this equilibrium is derived. The two cases of alpha = 0, implying that only the F' bacteria multiply on a selective medium, and alpha = 1, implying a completely nonselective medium, are analyzed in detail. It is shown that the above considerations apply generally to growing cultures of cells in which irreversible mutations occur.


Linkage disequilibrium over space and time in natural populations of Drosophila montana.

The previously described allelic frequencies and linkage disequilibrium among the active and null alleles of four tightly linked loci coding for the alpha-esterases were found to be maintained by one population for 5 years, and were found to be present in two other populations which were shown to be genetically distinct from the first. It appears that enzyme variants coded by these highly polymorphic loci are being maintained in the populations by selective forces.


Purification and synthesis of eosinophilotactic tetrapeptides of human lung tissue: identification as eosinophil chemotactic factor of anaphylaxis.

Preferential eosinophil chemotactic activity exhibiting a molecular weight comparable to that released from sensitized human lung fragments challenged with specific antigen and designated eosinophil chemotactic factor of anaphylaxis has been isolated from extracts of human lung fragments by sequential purification on Sephadex G-25, Dowex-1, Sephadex G-10, and paper chromatography. Two eosinophilotactic tetrapeptides of amino acid sequence Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu were recovered from the extracts in 4-12% overall yield of the low molecular weight peak from Sephadex G-25. Purified eosinophil chemotactic factor of anaphylaxis and the synthetic tetrapeptides were maximally active in amounts from 0.1 to 1.0 nmol per chemotactic chamber, and the activity was dependent on both the NH2-terminal and the COOH-terminal residues. Both natural and synthetic peptides were preferentially chemotactic for eosinophils and rendered them unresponsive to a subsequent stimulus.


Hypoxanthine phosphoribosyltransferase deficiency: association of reduced catalytic activity with reduced levels of immunologically detectable enzyme protein.

In the present study hemolysates from fourteen patients with a genetically determined deficiency of hypoxanthine phosphoribosyltransferase (EC 2.4.2.8; IMP:pyrophosphate phosphoribosyltransferase) activity were examined immunologically for the presence of material that crossreacts with the normal enzyme. A quantitative assay for crossreacting material in enzyme-deficient hemolysates was based on the inhibition of the immunoprecipitation of the normal enzyme. As little as 3% of normal crossreacting material could be detected. One patient in this series was found to have a normal amount of crossreacting material, whereas the remainder had no detectable crossreacting protein. The lack of detectable crossreacting material in these patients raises the possibility that a defect in synthesis or degradation of enzyme protein may be present in many patients deficient in hypoxanthine phosphoribosyltransferase.


Radioimmune determination of hypoxanthine phosphoribosyltransferase crossreacting material in erythrocytes of Lesch-Nyhan patients.

We have developed a sensitive radioimmunoassay capable of detecting and quantitating 20 ng of hypoxanthine phosphoribosyltransferase (EC 2.4.2.8; IMP:pyrophosphate phosphoribosyltransferase) protein. For this assay, hypoxanthine phosphoribosyltransferase from human erythrocytes was iodinated with 125I under mild conditions using hydrogen peroxide and lactoperoxidase attached to Sepharose-4B. Antisera prepared against homogeneous human hypoxanthine phosphoribosyltransferase precipitates the iodinated enzyme as effectively as the unlabeled enzyme. The radioimmunoassay has been used to look for hypoxanthine phosphoribosyltransferase crossreacting material in hemolysates from sixteen different patients with a marked genetic deficiency of this enzyme characteristic of the Lesch-Nyhan syndrome. Fifteen hemolysates contained no detectable (less than 1% of normal) crossreacting material. One hemolysate contained a normal amount of crossreacting material. Hypoxanthine phosphoribosyltransferase from this patient (E.S.) has been shown to be a Km mutant enzyme.


Bis-(N-maleimidomethyl) ether: an antisickling reagent.

The interaction of bis-(N-maleimidomethyl) ether with oxyhemoglobin results in covalent linkages of both maleimide groups, converting them to succinyl derivatives of beta93 Cys and beta97 His at their sulfhydryl and imidazolyl side chains, respectively. The resultant hemoglobin is stable, and reveals a left-shifted oxyhemoglobin equilibrium curve in which cooperativity is abolished. This reagent readily traverses the red cell membrane and prevents the sickling reaction upon deoxygenation. It appears to affect none of the activities of the red cell enzymes adversely, nor does it appear to affect the red cell membrane. Since there are several defined effects on the stereochemical status of the molecule conferred by interaction with bis-(N-maleimidomethyl) ether, the precise mechanism of the anitsickling effect remains to be elucidated. A more subtle perturberant will be required to specify a precise antisickling effect. By use of bis-(N-maleimidomethyl) ether a precise locus on the beta chain of human hemoglobin S can be perturbed to produce the desired effect.


Body weight and food intake at early estrus of rats on a high-fat diet.

Body weight, food intake, and age at vaginal opening and estrus were studied for two groups of weanling rats (age 21 days), fed on high-fat (24.6% by weight) and low-fat (5.0%) diets. Fat was substituted isocalorically for carbohydrate in the high-fat diet. The high-fat rats had estrus at 33.3 +/- 0.8 days, significantly earlier (P less than 0.001) than the age at estrus, 37.4 +/- 0.7 days, of the low-fat rats. Estrus was simultaneous with vaginal opening in 81% of the high-fat rats, in comparison to 48% of the low-fat rats. The caloric intake per 100 g of body weight of the high-fat and low-fat rats did not differ at vaginal opening or at estrus, whereas the two groups differed significantly at both events in age, body weight, absolute food intake (g/day), and relative food intake (g/100 g of body weight per day) and absolute caloric intake (calories/day). Caloric intake/100 g of body weight as a function of chronological age first increased and then decreased steadily before estrus for both high-fat and low-fat rats. The findings support Kennedy's hypothesis that a food intake signal, now further defined as caloric intake/100 g of body weight, is a signal for puberty, and are in accord with the hypothesis that a critical body composition of fatness is essential for estrus in the rat, as in the human female.


Neurohypophyseal hormones, analogs, and fragments: their effect on puromycin-induced amnesia.

Neurohypophyseal hormones and several of their analogs, as well as N-terminal and C-terminal fragments, have been studied for their ability to attenuate puromycin-induced amnesia in mice. [8-Lysine]vasopressin, [8-arginine]vasopressin, and the analogs des-9-glycinamide-[8-lysine]vasopressin, [1-beta-mercaptopropionic acid, 8-lysine]vasopressin, [1,6-aminosuberic acid, 8-lysine]vasopressin, [4-leucine, 8-lysine]vasopressin, glycyl-glycyl-glycyl-[8-lysine]vasopressin, [1-beta-mercaptopropionic acid, 8-D-arginine]vasopressin, and [1,6-aminosuberic acid, 8-arginine]vasopressin are active. [8-Arginine]oxytocin as well as oxytocin and all of its other analogs tested are inactive with the striking exception of glycyl-glycyl-glycyl-oxytocin. The structural aspects of the neurohypophyseal hormones which appear to be important for significant activity in memory consolidation include the combination of a cyclic moiety containing the Tyr and Phe residues along with a basic residue in position 8. Another series of active compounds comprises C-terminal neurohypophyseal peptides and analogs thereof, including the naturally occurring Pro-Leu-Gly-NH2 and, most surprisingly, Leu-Gly-NH2, as well as its derivatives D-Leu-Gly-NH2 and the diketopiperazine, cyclo(-Leu-Gly-).


Chemoreceptors and feeding in calanoid copepods (Arthropoda: Crustacea).

Ultrastructural studies of the mouthparts of the calanoid copepod Diaptomus pallidus have revealed the presence of numerous chemoreceptors, and the apparent absence of mechanoreceptors. The setae contain no muscles, and the setules are noncellular extensions of their chitin wall. This allows a new insight into the selective feeding of zooplankters.


Raman resonance of electron donor/acceptor complex.

The resonance Raman excitation profiles of a number of charge transfer transitions in electron donor/acceptor complexes with tetracyanoethylene as acceptor in solution at room temperature are reported and compared with absorption and fluorescence spectra of these complexes. All complexes show distinct anomalies which cannot be accounted for by existing theories unless they are extended. In particular, the excitation profiles peak at the low energy side of the absorption profiles by amounts of the order of 1000-2000 cm-1 and also, in the cases where two charge transfer bands are present, resonance occurs independently for the two bands. The latter observation suggests that the two bands are due to distinct species of the complex with differing geometrical configurations. The former observations is interpreted, in connection with the known asymmetry of the absorption profile and the large Stokes gap between absorption and fluorescence peaks, as arising from the relatively stronger contributions of the pure electronic and vibronic levels in the Stokes gap to the Raman scattering cross-section of the complex, and a frequency dependent damping of the vibronic transitions contributing to resonance. This provides important physical insights into the nature of charge transfer transitions of electron donor/acceptor complexes in general. In our discussion we also refer to similar anomalies in the work of others on the resonance Raman effect of the iodine visible absorption band in solution.


The extraordinary dielectric properties of biological materials and the action of enzymes.

From very general theoretical considerations it is concluded that many biomolecules (i) should have metastable excited states with very high dipole moment, and (ii) should be capable of strongly excited giant dipole vibrations with frequencies near 10(11) Hz. Experimental evidence available so far seems to support these postulates. It is suggested that the two postulates should be of importance for the action of enzymes, and relevant experiments are proposed.


Resonance Raman spectroscopic studies of the interactions between trypsin and a competitive inhibitor.

Raman spectroscopy was used to study the interactions between bovine trypsin and a competitive inhibitor. For this purpose, a chromophoric substrate analogue, 4-amidino-4'-dimethylamine azobenzene, was synthesized. This compound competitively inhibits the enzyme with a 1:1 stoichiometry and an inhibition constant Ki of 2.3 muM at pH 6.08 and 15 degrees. Resonance Raman spectra in aqueous solution of free or enzyme-bound inhibitor were analyzed. The main spectral changes observed upon enzyme-inhibitor complex formation were changes in the relative intensities of four bands (1171, 1206, 1315, 1608 cm-1) while no large frequency shifts occurred. The binding of the inhibitor molecule to the enzyme did not induce a twisting of the phenyl groups around the N=N bond. Some modifications of the band widths are interpreted in terms of a restriction of rotational motions in the inhibitor molecule. The possible involvement of specific interactions between trypsin and the benzamidinium ion part of the inhibitor molecule is discussed.


Allosteric mechanism for codon-dependent tRNA selection on ribosomes.

We suggest that the interaction between a codon and its cognate tRNA induces conformational changes in the tRNA. We further suggest that sites on the ribosome preferentially bind tRNA in those conformations which require proper matching of codon and anticodon. According to this model, the codon functions as an allosteric effector which influences the conformation at various sites in the tRNA. This is made possible by the ribosome, which we suggest traps tRNA molecules in those conformation states that maximize the energy difference between cognate and noncognate codon-anticodon interactions. Studies of the interactions between tRNA molecules and their cognate codons in the absence of the ribosome have suggested that triplet-triplet interaction between codon and anticodon is far too weak to account for the specificity of the tRNA selection mechanism during protein synthesis. In contrast, we suggest that such affinity measurements do not adequately describe the interaction between a codon and its cognate tRNA. Thus, such experiments can not detect conformational changes in the tRNA, and, in particular, those stabilized by the ribosome.


Nucleotide sequence of microvariant RNA: another small replicating molecule.

Microvariant RNA, a small self-replicating molecule (114 nucleotides long), has been isolated from Qbeta replicase reactions incubated in the absence of exogenous template. Its complete nucleotide sequence has been determined. Comparison with MDV-1 RNA, a somewhat larger endogenous Qbeta replicase product (220 nucleotides long) that had previously been characterized, revealed no significant sequence similarity. Since Qbeta replicase can mediate the synthesis of both of these disparate RNA molecules, primary sequence cannot be the sole determining factor in the processes of enzyme recognition and replication. This implies that the key is to be found in the secondary or tertiary structures. The availability of two different replicating molecules of defined sequence should aid in identifying these critical structural features.


Temporal changes in tectal cell surface specificity induced by nerve growth factor.

The change in cell surface adhesive specificity previously shown to occur between day 7 and 8 of development in the chick optic tectum ]Gottlieb et al. (1974), Proc. Nat. Acad. Sci. USA 71, 1800-1802] can be induced in rotating cultures of tectal cells by the addition of 10(-7) M mouse submaxillary gland nerve growth factor. Insulin, proinsulin, dexamethasone, and performic-acid-oxidized nerve growth factor are individually inactive in this system, but nerve growth factor in which the three tryptophan residues/subunit have been oxidized with N-bromosuccinimide is active. Thus, the specificity of this system for nerve growth factor is different than that observed with embryonic dorsal root or sympathetic ganglia, where the oxidized tryptophan derivative is inactive in stimulating neurite production. It is possible that in this system nerve growth factor serves as an analog of another specific trophic factor, presumably structurally related to nerve growth factor, may be active at much lower concentrations.


Action of nicking-closing enzyme on supercoiled and nonsupercoiled closed circular DNA: formation of a Boltzmann distribution of topological isomers.

Highly purified nicking-closing enzyme from mouse cells in 20-fold enzyme/substrate excess converts closed circular native PM2, ColE1, and Minicol DNA into limit product sets of DNAs. Each set has a mean degree of supercoiling of approximately zero. The individual species in the sets differ by deltatau = +/-1, +/-2, etc., and the relative masses fit a Boltzmann distribution. It was also demonstrated that "nonsupercoiled" closed circular duplex molecules serve as substrates for the nicking-closing enzyme, and that a distribution of topological isomers is generated. Polynucleotide ligase, acting on nicked circular DNA, forms under the same conditions, the same set of closed DNAs. The latter enzyme freezes the population into sets of molecules otherwise in configurational equilibrium in solution.


Functional level of rat liver tryptophan 2,3-dixoygenase messenger RNA during superinduction of enzyme with actinomycin D.

Tryptophan 2,3-dioxygenase [EC 1.13.11.11; L-tryptophan:oxygen 2,3-oxidoreductase (decyclizing)] activity is induced by glucocorticoid hormones and superinduced by actinomycin D. Previous experiments had shown that hormonal induction of the enzyme activity is accompanied by parallel increases in tryptophan 2,3-dioxygenase mRNA level. In this study, we measured the tryptophan 2,3-dioxygenase mRNA levels during superinduction as well as hormonal induction, to determine whether superinduction of the enzyme activity is also mediated through changes in mRNA concentration. Tryptophan 2,3-dioxygenase mRNA was measured in a Krebs ascites cell-free protein synthesizing system supplemented with rabbit reticulocyte initiation factors. We found that during superinduction of the enzyme activity by actinomycin D, the mRNA level is identical to that of the actinomycin D-free controls. Our results do not, therefore, support the hypothesis that hormonal induction and/or superinduction of tryptophan 2,3-dioxygenase mRNA are regulated by a rapidly turning over repressor.


Characterization of DNA condensates induced by poly(ethylene oxide) and polylysine.

High-molecular-weight DNA is known to collapse into very compact particles in a salt solution containing polymers like poly(ethylene oxide) [(EO)n] or polyacrylate. The biological relevance of this phenomenon is suggested by our recent finding that high concentrations of the highly acidic internal peptides found in the mature T4 bacteriophage head, as well as poly(glutamic acid) and poly(aspartic acid), can collapse DNA in a similar manner. The structure of DNAs collapsed by various methods has been studied with electron microscope. We find (EO)n collapses T4 or T7 bacteriophage DNA into compact particles only slightly larger than the size of the T4 and T7 head, respectively. In contrast, polylysine collapses DNA into different types of structures. Double-stranded DNA collapsed with (EO)n is cut by the single-strand specific Neurospora crassa endonuclease (EC 3.1.4.21) into small fragments. Extensive digestion only occurs above the critical concentration of polymer required for DNA collapse, demonstrating the (EO)n-collapsed DNA contains enzyme-vulnerable regions (probably at each fold), which are preferentially attacked. The size of the DNA fragments produced by limit-digestion with the nuclease ranges between 200 and 400 base pairs when DNA is collapsed by (EO)n. Only fragments of DNA which are larger than 600 base pairs are cut by the endonuclease in (EO)n-containing solution.


Kinetics of binding of carbon monoxide to lumbricus erythrocruorin: a possible model.

This paper represents a kinetic study of the binding of carbon monoxide by Lumbricus erythrocruorin. Observations on the quantum and the relaxation of the system both to equilibrium and to the steady state realized in the presence of constant illumination under various conditions are reported. The results, besides indicating the existence of at least two types of binding sites, give indications as to the behavior of a complex polyfunctional molecule, such as an enzyme, working under steady-state conditions.


Genome-sized RNA from reovirus particles.

Spider-like forms of purified reovirus RNA have been characterized by electron microscopy. These structures contained 11.3 +/- 0.8 mum of double-stranded RNA and up to 20 arms, each of distinct length. Length measurements of 64 "spiders" suggest that in these structures the viral genome is folded fairly specifically.


Pituitary growth hormones: further evidence for evolutionary conservatism based on immunochemical studies.

Immunochemical relatedness of preparations of purified somatotropins (growth hormones) of somatotropins in pituitary extracts from various vertebrate species was investigated by applying an antiserum to a purified somatotropin from a submammalian species, the snapping turtle. With the exception of monkey somatotropin, all mammalian, reptilian, and avian preparations tested showed reactions of identity or near identity by immunodiffusion studies in agar gel. Radioimmunoassay employing labeled rat somatotropin as a tracer and for standards, revealed that these same pituitary preparations gave steep inhibition slopes that were parallel or nearly parallel to each other. Purified somatotropins or somatotropins in pituitary extracts of subreptilian species, including an amphibian and existing primitive fishes, showed partial yet substantial relatedness to mammalian (ray) or reptilian (turtle) somatotropins by both immunodiffusion and radioimmunoassay. Our evidence indicates that the immunochemical relatedness of somatotropins from various vertebrate species appears to be even closer than has been suggested previously, and that a high degree of conservation of structure occurs during evolution.


General model for nutritional responses of higher organisms.

A general saturation equation is derived which is shown to describe a wide variety of nutrient-response relationships in higher organisms. Iterative multiple linear regression analysis is used to obtain least squares estimates of the constants defining theoretical nutrient-response curves. Curves thus generated accurately predict experimentally observed responses. From this treatment, response parameters are developed which are analogous to Vmax and Km of enzyme kinetics. It is proposed that this model be applied in evaluating nutritional requirements and in assessing the relative biological efficiency of nutrient sources.


Regulation of synthesis of hepatic fatty acid synthetase: polysomal translation in a cell-free system.

Polysomes were isolated from livers of rats fed various diets and were translated in a protein-synthesizing system derived from cultured Chang liver cells. One of the labeled products was identified as complete subunit(s) of fatty acid synthetase by indirect immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized immunoprecipitate. The relative amounts of fatty acid synthetase synthesized by polysomes from livers of rats fed a normal diet, starved rats, and rats starved and refed a fat-free diet for 16 hr were 1, 0.1, and 10, respectively. Induction of synthesis of fatty acid synthetase after fat-free refeeding of starved rats began by 2 hr (3-fold increase over starved animals), was increasing rapidly by 5 hr (19-fold over starved animals), and reached a high level by 16 hr (95-fold over starved and 10-fold over normally fed).


Acetylcholine-receptor-mediated ion flux in electroplax membrane preparations.

The kinetics of acetylcholine-receptor-mediated sodium efflux from electroplax microsacs of electrophorus electricus has been analyzed. This led to the discovery that only a small fraction of the observed efflux is affected by chemical effectors such as carbamylcholine. Experimental conditions were chosen so that the receptor-mediated flux could be analyzed without the measurements' being obscured by efflux from the nonexcitable microsacs. Near equilibrium the efflux follows a single exponential decay. The apparent first order rate constant for sodium-22 efflux was determined as a function of effector concentration and is considerably higher than previously estimated. The process does not show cooperativity under the experimental conditions, in agreement with the binding isotherms of effectors and the same membrane preparation. The presence of potassium ions inhibits the receptor-mediated sodium flux. It is suggested that interaction of inorganic ions with the receptor may play an important role in the cooperative effects observed in electrophysiological experiments.


Brain receptors for antipsychotic drugs and dopamine: direct binding assays.

In order to test the suggestion that antipsychotic drugs act by blocking dopamine receptors in the brain, the direct effects of such neuroleptic drugs were tested on the stereospecific binding of [3H]dopamine and of [3H]haloperidol to rat brain striata and their subfractions. The stereospecific component of binding was defined as that amount of [3h]dopamine or [3H]haloperidol bound in the presence of (-)-butaclamol (an inactive drug) minus that bound in the presence of (+)-butaclamol (a potent neuroleptic drug); 100 nM butaclamol was used for the [3H]haloperidol assay, while 1 muM butaclamol was used for the [3H]dopamine assay. Various antipsychotic drugs inhibited this stereospecific component in both the dopamine and haloperidol assays. These inhibitory potencies correlated with the clinical doses used for controlling schizophrenia.


Role of carnitine in hepatic ketogenesis.

The enhancement of long-chain fatty acid oxidation and ketogenesis in the perfused rat liver, whether induced acutely by treatment of fed animals with anti-insulin serum or glucagon, or over the longer term by starvation or the induction of alloxan diabetes, was found to ba accompanied by a proportional elevation in the tissue carnitine content. Moreover, when added to the medium perfusing livers from fed rats, carnitine stimulated ketogenesis from oleic acid. The findings suggest that the increased fatty acid flux through the carnitine acyltransferase (carnitine palmitoyl-transferase; palmitoyl-CoA:L-carnitine O-palmitoyltransferase; EC 2.3.1.21) reaction brought about by glucagon excess, with or without insulin deficiency, is mediated, at least in part, by elevation in the liver carnitine concentration.


Initiation of in vitro mRNA synthesis from the wild-type lac promoter.

An in vitro transcription system, dependent on catabolite gene activator protein (CAP), utilizing a 200 base-pair restriction fragment, has been used to show that the initiation site of the wild-type Escherichia coli lac mRNA, and that of two mutants, 8d and ps, are identical to that previously reported for the CAP-independent promoter mutant UV5. Order of addition experiments are used to show that the binding of lac repressor to the operator is competitive with that of the RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) to the promoter, thus demonstrating functional overlap of the operator and promoter sites.


Influence of insertions on packaging of host sequences covalently linked to bacteriophage Mu DNA.

Insertions in bacteriophage Mu DNA have been identified. These insertions are responsible for at least seven X mutations, all of which eliminate essential Mu functions. The insertions are about 800 base pairs long and are located to the left of the cleavage site of restriction endonuclease EcoRI, near the immunity end of Mu DNA. We have found that such insertions cause a reduction in the length of nonhomologous terminal sequences which are seen as split ends in denatured and renatured Mu DNA molecules. These heterogeneous sequences apparently arise from packaging of host DNA from maturation precursors in which Mu and host DNA are covalently linked. We infer that a single Mu genome length is too short to be cut during morphogenesis, and thus some host DNA is packaged into mature virions. Since the insertions increase the length of Mu DNA, they decrease the amount of host DNA needed for packaging.


Structure of transcriptionally active chromatin.

Rat-liver chromatin has bee fractionated into transcriptionally active and inactive regions [Gottesfeld et al. (1974) Proc. Nat. Acad. Sci. USA 71, 2193-2197] and the distribution of nuclease-resistant complexes in these fractions has been investigated. About half of the DNA of both fractions is resistant to attack by tne endonuclease DNase II. The nuclease-resistant structures of inactive chromatin are DNA-histone complexes (v-bodies) which sediment at 11-13 S. Template-active chromatin yields two peaks of nuclease-resistant nucleoprotein. These complexes sediment at 14 and 19 S, and contain DNA, RNA, histone, and nonhistone chromosomal proteins. Polyacrylamide gel electrophoresis reveals a complex pattern of chromatin proteins, suggesting that the complexes are heterogeneous in composition.


Fusion of dipalmitoylphosphatidylcholine vesicle membranes induced by concanavalin A.

The temperature dependence of fatty acid spin label resonance spectra and freeze fracture micrographs of sonicated dipalmitoylphosphatidylcholine vesicles in the absence and presence of concanavalin A demonstrate a strong interaction of concanavalin A with these lipid membranes, which results in fusion of the vesicles. The rate of this reaction as followed with use of magnetic resonance exhibits a pronounced maximum at 36 degrees, the midpoint of the phase transition range of dipalmitoylphosphatidylcholine vesicles. This maximum is discussed in terms of structural fluctuations, which are maximal in the phase transition range of the membranes.


Resonance Raman effect in mu-oxo-bis[iron(III) tetraphenylporphyrin].

Resonance Raman spectra of mu-oxo-bis[iron(III) tetraphenylporphyrin] have been observed and compared to Raman spectra of the monomers iron(III) tetraphenylporphyrin chloride and bis(piperidine)iron(II) tetraphenylporphyrin. Selection rules for the Raman effect under conditions of resonance of the incident photons with electronic states are presented for the exact symmetry group (C2) and the pseudo-symmetry group (D4d): emphasis is placed on the physical processes involved in scattering via vibronic states of the dimer. These two models are experimentally distinguishable in the behavior of the depolarization ratios of the dimer vibrational doublets. Experimental data favor the assignment of D4d as the symmetry group relevant to a description of the various states of the dimer. The usefulness of resonance Raman spectra of the mu-oco-dimer of Fe(III) tetraphenylporphyrin has been discussed with respect to several biochemical systems where there is strong evidence of two or more closely interacting chromophores.


Chemical induction of sperm abnormalities in mice.

The sperm of (C57BL X C3H)F1 mice were examined 1, 4, and 10 weeks after a subacute treatment with one of 25 chemicals at two or more dose levels. The fraction of sperm that were abnormal in shape was elevated above control values of 1.2-3.4% for methyl methanesulfonate, ethyl methanesulfonate, griseofulvin, benzo[a]pyrene, METEPA [tris(2-methyl-l-aziridinyl)phosphine oxide], THIO-TEPA [tris(l-aziridinyl)phosphine sulfide], mitomycin C, myleran, vinblastine sulphate, hydroxyurea, 3-methylcholanthrene, colchicine, actinomycin D, imuran, cyclophosphamide, 5-iododeoxyuridine, dichlorvos, aminopterin, and trimethylphosphate. Dimethylnitrosamine, urethane, DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane], 1,1-dimethylhydrazine, caffeine, and calcium cyclamate did not induce elevated levels of sperm abnormalities. The results suggest that sperm abnormalities might provide a rapid inexpensive mammalian screen for agents that lead to errors in the differentiation of spermatogenic stem cells in vivo and thus indicate agents which might prove to be mutagenic, teratogenic, or carcinogenic.


Local anesthetics affect transmembrane cytoskeletal control of mobility and distribution of cell surface receptors.

Tertiary amine local anesthetics facilitated concanavalin A-induced redistribution of lectin receptors on murine BALB/3T3 cells and enhanced the susceptibility of these cells to agglutination by concanavalin A. In contrast, these drugs at similar concentrations inhibited ligand-induced capping of immunoglobulin receptors on mouse lymphocytes. We propose that these differing effects of local anesthetics on membrane receptor mobility in fibroblasts and lymphocytes result from the action of anesthetics on membrane-associated microtubules and microfilaments involved in the transmembrane control of receptor mobility. We present electron microscopic evidence of structural alterations in microtubule and microfilament organization in anesthetic-treated cells, together with data on changes in the responsiveness of anesthetic-treated cells to drugs that act on microtubules and/or microfilaments. This evidence supports the proposal that anesthetics affect the organization of cytoskeletal components or their plasma membrane attachment points. The effects of local anesthetics on ligand-induced redistribution of membrane receptors in both 3T3 cells and lymphocytes can be duplicated by treating cells with colchicine (or Vinca alkaloids) together with cytochalasin B. We propose that the participation of membrane-associated microtubules and microfilaments in the transmembrane control of receptor mobility is such that microtubules and microfilaments play opposing roles in regulating the mobility and topography of cell surface receptors.


Adenovirus-2 mRNA is transcribed as part of a high-molecular-weight precursor RNA.

The order of transcription and the length of nascent RNA transcripts from adenovirus-2 (Ad-2) DNA in the nucleus of infected cells has been deduced by labeling the growing RNA chains in vivo for a very brief period, separating the RNA on the basis of size, and hybridizine to the ordered EcoRI restriction endonuclease fragments derived from Ad-2 DNA. The majority of the virus-specific RNA molecules are synthesized as very high-molecular-weight units beginning at a common point at least 25-30,000 base pairs from one end of the Ad-2 DNA. These molecules can be reduced in size without further RNA synthesis. The experiments indicate the obligatory origin of Ad-2 mRNA from a high-molecular-weight precursor molecule.


Selective effects of inhibitors of protein synthesis on metabolism of nuclear anc cytoplasmic proteins: evidence for coordinate synthesis of non-histone chromosomal proteins.

We have compared the effects of inhibitors of protein synthesis on the metabolism of nuclear and cytoplasmic proteins of HeLa S-3 cells. L-1-tosylamido-2-phenylethyl chloromethyl ketone, a potent inhibitor of polypeptide chain initiation, was shown to preferentially inhibit the synthesis of cytoplasmic proteins and of histones at concentrations that permit continued amino acid incorporation into nuclear non-histone proteins. Comparisons of the molecular weight distributions of newly synthesized proteins in the presence and absence of L-1-tosylamido-2-phenylethyl chloromethyl ketone have revealed striking differences between nuclear anc cytoplasmic protein fractions. Differential effects on the synthesis of cytoplasmic proteins, acid-soluble nuclear proteins, and residual nuclear proteins have also been obtained with the antibiotic, pactamycin, another inhibitor of polypeptide chain initiation. The incorporation of radioactive amino acids into nuclear non-histone proteins shows resistance to inhibition by pactamycin, but is strongly inhibited by agents such as puromycin and cycloheximide which block chain elongation. The possibility that proliferating cells have developed specialized mechanisms for the coordinate synthesis of chromosomal proteins, possibly involving polycistronic messenger RNAs, is tested and discussed.


Ectopic neurons and aberrant connections during neural development.

During the normal development of the nucleus of origin of centrifugal fibers to the chick retina (the so-called isthmo-optic nucleus) a significant number of neurons are misrouted during their migration from the neural epithelium in which they are generated. Like the cells in the isthmo-optic nucleus, these ectopic neurons can be identified by the retrograde transport of horseradish peroxidase (donor: hydrogen peroxide oxidoreductase, EC 1.11.1.7) injected into the eye. Between 500 and 600 such cells have been identified on one side of the brain of 12 to 15-day-old embryos, but only about 10% of this number is seen beyond hatching. In addition, during development some of the cells in the nucleus send their axons to the ipsilateral retina, whereas in post-hatched animals they all project to the contralateral retina. The number of such neurons with aberrant axons is greatly increased (in some cases by as much as two orders of magnitude) in chicks from which one eye was removed early in development. Although normally about 60% of the neurons in the isthmo-optic nucleus degenerate between the 13th and 17th days of incubation, they can almost all be labeled by intraocular injections of horseradish peroxidase on the 12th day, indicating that the axons of virtually all the cells, including those neurone that subsequently die, reach the retina.


Turning on of activities in unfertilized sea urchin eggs: correlation with changes of the surface.

Unfertilized sea urchin eggs exposed to low concentrations of ammonia enter into a number of activities which normally appear after fertilization. It is shown that the effects are attributable to ammonia, rather than to NH4+ ions of elevated pH. The same effects are obtained by exposure to isotonic urea and to glycerol at very low ionic strengths. All treatments which produce these changes (such as the turning on of chromosome replication and condensation in unfertilized eggs) also bring about changes of the outer cell surface which are visible in the scanning electron microscope. The most striking indicator is the elongation of the microvilli which cover the surface of the unfertilized egg. The changes of the surface are interpreted as the dissociation of a component from the outer surface layer. This component is not the "vitelline" sheet as defined morphologically or by the ability of the egg to form a fertilization membrane upon insemination. It is proposed further that this component is a peripheral component of the plasma membrane, whose removal modifies the membrane functionally and leads to the derepression of various processes within the egg.


Relationship between release of surface proteins and metabolic activation of sea urchin eggs at fertilization.

Macromolecular components are released from sea urchin eggs when their metabolism is activated at fertilization or by incubation in ammonia. When the released material is dialyzed, concentrated, and added back to partially activated eggs the rate of protein synthesis is suppressed to the level of the unactivated egg. The surface proteins of the unfertilized eggs can be labeled with 125I by a lactoperoxidase procedure. When fertilized or activated with various parthenogenetic agents, 15-25% of the total labeled protein is released; most of the label is associated with a 150,000-dalton glycoprotein. The extent of metabolic activation, as assessed by measuring increased protein synthesis, is correlated with the amount of surface label released. Several other proteins are released during activation but are not labeled by the lactoperoxidase procedure in the intact cell. We have not yet identified which of these components is responsible for suppressing protein synthesis, nor do we know if any of the other metabolic changes of fertilization such as K+ conductance and DNA synthesis are also suppressed. We suggest that these released components are surface molecules involved in maintaining the low metabolic state occurring at the end of oogenesis and that removal of these components during fertilization results in the release of the suppression of the egg.


Serum rapidly stimulates ouabain-sensitive 86-RB+ influx in quiescent 3T3 cells.

Serum causes a 4-fold increase in 86Rb+ (a K+ tracer) influx in quiescent 3T3 mouse fibroblast cells. It is one of the earliest changes caused by serum, being seen in 2 min and reaching a maximum in 10 min. Removal of serum causes rapid reversal of this effect. Serum acts mainly by increasing the maximum velocity, Vmax, of entry. Ouabain inhibits entry of 86Rb+ (82-90%) both in the presence and absence of serum, but does not alter exit. The rapid increase in cation influx is unaffected by cycloheximide and by changes in cyclic AMP and GMP. Low concentrations of insulin, epidermal growth factor, and prostaglandins (E1 and F2alpha) produced a smaller (80%) activation of 86Rb+ entry. Ouabain, at a level that inhibits cation influx, also prevents the onset of DNA synthesis following serum addition; this is reversible effect dependent on the concentration of K+ in the medium. This suggests that cation pumping activity may be required for initiation of DNA synthesis.


Specific cleavage analysis of mammalian mitochondrial DNA.

Mitochondrial DNA from several mammalian species has been digested with a site-specific restriction endonuclease (HaeIII) from Haemophilus aegyptius. A quantitative analysis of the resulting specific fragments indicates that the mtDNA of any individual mammal is predominantly a single molecular clone. Gel analysis of specific cleavage products has proven quite sensitive in detecting differences in mtDNA: mtDNAs from the more distantly related mammals studied (e.g., donkey and dog) are found to have few bands in common and very closely related mammals (e.g., donkey and horse) share only about 50% of their bands. This procedure has detected several intraspecies mtDNA differences. Six distinct human patterns have been found, with one pattern usually differing from another in two or three bands. mtDNAs from different organs of single individuals have also been analyzed, and no differences have been found.


Use of gene 32 protein staining of single-strand polynucleotides for gene mapping by electron microscopy: application to the phi80d3ilvsu+7 system.

A method for visualizing RNA-DNA duplex regions along a single strand of DNA in the electron microscope is described. A preparation of RNA molecules is hybridized to a long DNA strand containing the coding sequences (genes) for some of the RNAs. T4 gene 32 protein, which binds selectively and cooperatively only to the single-strand regions, is added, followed by glutaraldehyde. The resulting nucleic acid-gene 32 complex is adsorbed to the surface of an electron microscope grid in the presence of ethidium bromide. The single-strand regions are relatively thick (8.5 nm) compared to the duplex (RNA-DNA hybrid) regions (3.5 nm), so that the two kinds of regions are readily recognized by electron microscopy. In favorable cases, tRNA-DNA hybrids of length about 80 nucleotide pairs can be recognized (although with difficulty). The positions of a number of interesting genetic sequences on the DNA of the transducing phage phi80d3ilvsu+7 have been mapped. The r strand contains 16S, 23S, and 5S rRNA coding sequences in that order. The spacer between 16S and 23S genes has a length of 500 nucleotides and contains the coding sequence for a tRNA2Glu gene in agreement with previous biochemical observations. The spacer between the 23S and 5S genes has a length of 180 nucleotides. The su+7 tRNATrp coding sequence has been mapped on the l strand at a position just to the left of the ilv genes. Secondary structure loops due to short inverted repeat sequences flanking the 16S, 23S, tRNATrp, and F sequences in the DNA have been observed.


Analysis of changes in collagen biosynthesis that occur when chick chondrocytes are grown in 5-bromo-2'-deoxyuridine.

Chick embryo chondrocytes, in primary culture, initially synthesize only Type ii collagen (chain composition, [alpha1(II)13), as judged by two criteria: (i) carbosymethyl-cellulose chromatography of the denatured collagen, and (ii) carbosymethyl-cellulose chromatography of the cyanogen bromide peptides derived from the isolated chains. After a period of growth in 5-bromo-2'-deoxyuridine, however, synthesis of two different types of collagen could be detected after differential salt precipitation of the newly synthesized native collagens from neutral salt solutions at 2.2 M NaCl and subsequently at 0.01 M Na2hPO4. By criteria indicated above, the collagen precipitating at 2.2 M NaCl was identified as Type I collagen (chain composition, [alpha(I)]2alpha2), whereas the collagen subsequently precipitated at 0.01 M Na2HPO4 was found to be comprised entirely of alpha1(I) chains, indicating a chain composition, [alpha](I)]3. We propose to designate the latter type of molecule as the Type I trimer.


Colchicine permeation is required for inhibition of concanavalin A capping in Chinese hamster ovary cells.

Several antimitotic, tubulin-binding agents, such as colchicine, colcemid, and podophyllotoxin, inhibit the capping of fluorescent-labeled concanavalin A in Chinese hamster ovary cells. By comparing the effects of these agents on parental cell lines on several independently selected colchicine-resistant mutants with decreased drug permeability, we have demonstrated that permeation of these drugs in required for inhibition of capping. These data support the hypothesis that these antimitotic agents interact with an intracellular component, probably microtubules, to prevent the directional movement of concanavalin A receptors on the surface membranes of Chinese hamster ovary cells.