A microsomal butyrylesterase appearing in rat livers during development, regeneration, and carcinogenesis, and after phenobarbital treatment. A microsomal butyrylesterase (L-I) was purified from the livers of male W rats treated with phenobarbital, and an antiserum against this purified L-I was raised in a rabbit. By the Ouchteriony double-diffusion test, a precipitin line was observed between the anti-L-I antiserum and each Triton X-100 extract of livers during development, regeneration after partial hepatectomy, and carcinogenesis and of hyperplastic nodules and hepatomas, all of which revealed L-I in their esterase isoenzyme patterns. These precipitin lines exhibited esterase activity. The fusion of the lines of these tissue extracts and that of the purified L-I indicated the presence of an antigen site common to their esterases. The extracts of adult and fetal livers and also of hepatomas resembling fetal liver in the esterase isoenzyme pattern did not produce a precipitin line with anti-L-I antiserum.


Carcinogenicity test of six nitrosamides and a nitrosocyanamide administered orally to rats. Six nitrosamides [ethylnitrosourea (ENU), 2-hydroxyethylnitrosourea (HENU), carboxymethylnitrosourea, 1-nitroso-5,6-dihydrouracil (NDHU), 1-nitrosohydantoin, and N-methyl-N-nitrosobenzamide (MNB)] and ethylnitrosocyanamide (ENC) were administered chronically in sodium citrate-buffered drinking water to MRC Wistar rats. ENU induced tumors of the reticuloendothelial system (RES) (50% incidence), mammary glands, and large intestine. NDHU in drinking water produced hepatocellular carcinomas (96% incidence), but NDHU injected ip caused mostly tumors at the injection sites (54% incidence). HENU produced bone tumors (38% incidence) and RES tumors (28% incidence). ENC produced nasal cavity tumors (36% incidence). Papillomas and/or carcinomas of the forestomach, tongue, and pharynx were induced by most of the compounds, with the highest incidence in the forestomach (47% for MNB); these tumors were attributed to local action when the compounds were ingested. Carcinogenicity was not quantitatively correlated with direct mutagenicity for Salmonella typhimurium TA1535.


Complement-dependent cytotoxic antitumor antibody. II. Quantitative determination of cell-bound immunoglobulin M1. A method for the quantitative determination of rabbit IgM bound to cell surfaces has been developed. This method was based on the ability of goat IgG specific for rabbit IgM heavy chains to bind 125I-labeled protein A when bound to the antigen. With the use of this technique the production of specific IgM antitumor antibodies in New Zealand White rabbits after immunization with guinea pig hepatoma cells line-1 and line-10 was followed. Differences in the production of IgM were observed between the different bleedings from rabbits immunized with line-1. No significant IgM antibody was produced following immunization of rabbits with line-10 tumor cells. This indirect method for determining IgM on the cell surfaces was objective, easy to perform, and detected complement-fixing and noncomplement-fixing antibodies. In addition, this technique could be applied to quantify other components on the cell surface for which a suitable specific antibody was available.


Lung tumorigenesis in mice after chronic exposure in early life to a low dose of dimethylnitrosamine. Strain A female mice were exposed to 10 ppb dimethylnitrosamine (DMN) in their drinking water for 4 weeks before mating. Treatment was continued through pregnancy and lactation and after weaning until the progeny were 22 weeks old. The incidence of primary lung tumors among the treated progeny (23%) was significantly higher (P less than 0.021) than that among controls (8%). The effect of the DMN was greatest among the males: 32% had lung tumors, compared with 4% of the control males (P less than 0.016). The DMN-exposed females also had a higher lung tumor incidence than did the controls, but the difference was not of statistical significance. These results demonstrate carcinogenicity of DMN at a dose approaching amounts possibly encountered by the human population as a result of environmental exposure.


Increased levels of lipid-bound sialic acid in thymic lymphocytes and plasma from leukemic AKR/J mice. Lipid-bound sialic acid levels for thymic lymphocytes and plasma were elevated twofold in leukemic AKR/J mice as compared to the levels observed in young, nonleukemic mice. In contrast, lipid-bound phosphorus levels were similar for thymic lymphocytes from both groups of mice.


Remissions of mammary adenocarcinoma in hypothyroid mice given 5-fluorouracil and chloroquine phosphate. In two separate experiments, treatment of C3H/He mice bearing transplantable mammary adenocarcinomas (C3HBA) with a regimen of 6-propylthiouracil (PTUra) and 5-fluorouracil (FUra) plus chloroquine phosphate (CP) resulted in complete remissions of 77 and 65%, respectively. Treatment with PTUra alone resulted in 41% remissions in experiment 1 and 35% remissions in experiment 2. None of the nontreated control mice in either experiment had spontaneous remissions, and all controls died in each experiment. The principal effect was apparently due to the treatment with PTUra, inasmuch as most of the tumors disappeared during the 21-day treatment period. This observation indicated that the proper timing of the thyroid treatment with PTUra was crucial to achieve the best results. The combined FUra+CP regimen augmented the effects of the thyroid treatment and resulted in an increase in remissions.


Transplantable tumor cell line derived from a spontaneous fibrosarcoma in a gerbil. A cultured tumor cell line was derived from a spontaneous fibrosarcoma in a gerbil (Meriones unguiculatus). The cell line consistently produced large tumors with a high incidence of metastases in gerbils of all ages and in both sexes.


[Reversible visual impairment caused by leukemic fundus changes (author's transl)]. Monocular impaired visual acuity was the initial symptom of chronic myeloic leukemia in a 41 year old man. A subretinal infiltration had caused foveal retinal thinning. 3 1/2 months after the onset of therapy retinal thickness had normalized and the vision was fully restored.


Recombination in phage T4 gene-43 (DNA polymerase) mutants. The effect of phage T4 gene 43 (DNA polymerase) mutations on recombination between adjacent base pairs was measured in rII amber and opal mutants. The mutator allele tsL56 did not promote recombination frequencies at the two sites in which its effect was studied. The antimutator allele tsCB87 caused slight or no reduction in recombination frequencies at five sites.


Proposed uniform nomenclature for mammalian ribosomal proteins. The numbering systems for mammalian ribosomal proteins used in several laboratories have been correlated and a proposal for a standard system is presented.


Construction and some properties of packageable plasmid F. A derivative of plasmid F which is packageable in lambda phage coat was constructed using techniques of in vitro recombination. This plasmid is composed of three DNA fragments generated by restriction enzyme EcoRI: a miniF fragment (fragment f5 of F'lac) which is able to replicate autonomously, a DNA fragment from Staphylococcus plasmid that carries the beta-lactamase gene, and a portion of guaA  (B) transducing lambda phage DNA carrying lambda cohesive ends (cos site) along with almost all the late genes but devoid of all those genes and sites that are needed for replication, regulation, and recombination. The hybrid plasmid has a molecular weight of 2.7 x 10(7) daltons, about 84% size of lambda phage genome, and can be packaged in lambda coat when helper phage replicates in the plasmid-carrier cell. The packaged plasmid and the helper lambda phage particles are separated by CsCl density gradient centrifugation. The replication characteristics of the recombinant plasmid are all those of F including the copy number, incompatibility, and curing with acidine orange. The packaged plasmid is injected into an F- cell and establishes a plasmid state with normal efficiency. In F+ or Hfr cells, the resident F factor hinders this process.


Construction in vitro of "phage-plasmid" chimerae: a new tool to analyse the mechanism of plasmid maintenance. In this paper, we report the construction in vitro of chimerae between lambdoid replacement vectors (Murray et al., 1977) and the miniF Apr plasmid: pSC138 (Timmis et al., 1975). lambdaF recombinants were shown to be chimerae between the lambda and the F replicons. By genetical tests, we have demonstrated that both lambda and F replication mechanisms are functional: the lambdaF recombinant behaves as a non defective plaque forming phage on lambda sensitive bacteria and establishes itself as a stable plasmid on recA F- homoimmune bacteria. In the extra-chromosomal state, the lambdaF recombinant apparently retains the controlled autonomous replication and the FI incompatibility characteristics of the F plasmid. The potential experimental uses of these phages are discussed.


In vitro constructed plasmids containing both ends of bacteriophage Mu DNA express phage functions. The construction of a plasmid carrying the right end PstI . B fragment of bacteriophage Mu DNA and of plasmids containing in addition the left end EcoRI.C fragment of Mu DNA into the vector pBR322 is described. Inversion of the G segment still occurs in all these plasmids. By marker rescue and complementation experiments the right PstI cleavage site was located to the left of gene Q. The composite plasmids inheriting also the left end EcoRI fragment of Mu DNA express both the immunity and killing functions of Mu and direct the in vitro synthesis of presumably Mu-specific polypeptides. These results demonstrates that Mu-specific functions can be analyzed from cloned fragments.


Transposon-facilitated recombination in Vibrio cholerae. Improved Vibrio cholerae donors were constructed by introducing the ampicillin transposon, Tn1, into both the conjugative plasmid, P, and the bacterial chromosome to provide "portable regions of homology." The resulting Tfr (Transposon-facilitated recombination) donors transferred genes at high frequency from origins specified by the chromosomally inserted Tn1 copies. Tn1 was transposed into the chromosome from a deleted P::Tn1 vector, which was eliminated from the cells by superinfection with a thermosensitive P::Tn9 (chloramphenicol) mutant plasmid. After eliminating the thermosensitive plasmid, the chromosomally resistant isolates were converted into donors with a P::Tn1 conjugative plasmid. Tfr donors were also obtained by isolating Tn1 insertion mutations in a gene for thymine biosynthesis. Chromosomal sites of Tn1 relative to bacterial genes were determined by measuring gene transfer frequencies and genetic linkage. In one case, linkage of the amp gene to the chromosomal genes that defined its location was demonstrated. Chromosomal transfer by Tfr donors was reversed by isolating P::Tn1 plasmids that contained Tn1 inserted in the opposite orientation.


A method which facilitates the ordering of DNA restriction fragments. The Southern transfer technique has been used to provide a generally applicable method for ordering DNA restriction fragments. It involves electrophoresis of partially digested DNA, transfer to nitrocellulose filter paper and annealing to a 32P-labelled fragment. Only those partials containing that particular fragment will reanneal to the probe and produce bands on autoradiography. The size of each partial in the labelled set is the sum of the sizes of the fragment used as probe and of one or more adjacent fragments. Thus the size of the adjacent fragments can be determined from the size increments of this set of partials. The method is illustrated by the mapping of certain BamHI sites on coliphage 186 DNA.


Bacteriophage T4 rIIB protein synthesis with a temperature-sensitive mutation in the rIIB initiation codon. In protein synthesis, the incorporation of an N-terminal formylmethionine residue is directed by an initiation codon. The most frequently used codon is AUG, although initiation at GUG and UUG codons has also been observed. The HD263 mutation is an AUG to AUA change in the rIIB initiation codon. Evidence is presented here that wild type and HD263 rIIB proteins, whether synthesized in vivo or in vitro, have identical fmet peptides. It is concluded that translation began at the AUA mutant initiation codon in vitro and in phage T4 infected cells. In the in vitro translation system used in these studies, the rIIB protein synthesized at 25 degrees no longer contains the N-terminal formyl group whereas a large proportion of the formyl group is retained at 37 degrees.


Analysis of lethal events induced by ultraviolet in a heterokaryon of Neurospora. The relative frequencies of heterokaryons and the two kinds of homokaryons have been scored among colonies from conidia harvested from a heterokaryon and treated with UV, in order to determine which kinds of lethal mutations were induced. Recessive lethal mutations were scored directly. The pattern of surviving types indicated that recessive lethals and mitotic lethals (events destroying whole nuclei) occurred with similar frequencies. But the absolute frequency of these mutations was not sufficient to account for the observed kill, suggesting that dominant lethals and/or cytoplasmic lethals were also induced at a similar rate.


Restriction cleavage maps of coliphages 186 and P2. The restriction enzymes BamHI, Bg/II, EcoRI, HindIII, PstI, XbaI and XhoI have been used to cleave DNA isolated from the related coliphages P2 and 186 for analysis on 1% agarose gels. Three approaches were used to map the sites of cleavage: a) analysis dependent upon the existence of cohesive termini and availability of viable P2-186 hybrids; b) analysis of double digests and redigests of isolated fragments with a second enzyme and c) analysis of partial digests by transfer to nitrocellulose and hybridization with a single fragment. This last approach and the results obtained from it are detailed in a separate paper (Saint and Egan, 1979). The number of sites of each enzyme are as follows: a) 186, BamHI-7, Bg/II-1, EcoRI-3, HindIII-2, PstI-22, XbaI-0 and Xho-I-1; b) P2, BamHI-3, Bg/II-2, EcoRI-3, HindIII-0, PstI-O, XbaI-1 and XhoI-O. All of these sites have been mapped with the exception of PstI for 186, where only the five sites in the right 35% (the control region) have been mapped.


A restriction endonuclease cleavage map of bacteriophage P2. A restriction endonuclease cleavage map of phage P2 was constructed. The enzymes used and, within parenthesis, the number of their cleavage sites on the P2 lg cc DNA molecule were: AvaI(3), BalI(1), BAMI(3), BglII(3), HaeIII (more than 40; only three were mapped), HindIII(0), HpaI(10), KpnI(3), PstI(3), SalI(2) and SmaI(2). The EcoRI cleavage sites (3), as determined earlier, were used as reference points for this study. The DNAs of a variety of P2 mutants carrying chromosomal aberrations (dell, del2, del3, del6, vir22, vir37(2), vir79 and vir94) were also similarly examined.


Spot position of rat liver ribosomal proteins by four different two-dimensional electrophoreses in polyacrylamide gel. Separation of the proteins from rat liver 40S and 60S ribosomal subunits and polysomes was done in four different two-dimensional polyacrylamide gel electrophoresis systems. The first dimension was run at acidic or basic pH, the second dimension either with sodium dodecyl sulphate or at acidic pH in 18% acrylamide. The position of each individual protein of both subunits and polysomes was determined in each system. This identification resulted from a new method avoiding any pervious purification of individual proteins. The new "proposed uniform nomenclature for mammalian ribosomal proteins" (McConkey et al. in press) was used for numbering the proteins in the four systems.


The prognostic significance of radiological and symptomatic bone involvement in childhood acute lymphoblastic leukaemia. Full skeletal survey was performed on 193 consecutive children presenting over a 6-year period with acute lymphoblastic leukaemia (ALL). A record was made of the degree of bone pain in these patients and 60 others presenting immediately prior to the availability of skeletal surveys. Bone pain was present in 76 out of 253 patients (30%) and radiological bone changes characteristic of leukaemia in 125 out of 193 (65%). A significant correlation was found between the severity of bone pain and the number of bones involved on X ray (r = 0.25, P = less than 0.001), but not between initial white cell counts and bone score (r = -0.14, P = 0.08). There was no significant correlation between the degree of bone pain and survival or length of first remission (P = 0.29 and 0.86). Similarly, the extent of radiological bone disease was of no prognostic significance with relation to survival (P = 0.41) or length of first remission (P = 0.21).


Keeping abreast of the times: the Tauranga Infant Feeding Survey. An infant feeding survey revealed that 77% of 187 mothers, whose babies were born over a three month period in the Tauranga maternity annexe, were breast feeding on discharge. Fifty-seven percent of the total breast fed for at least three months. The feeding pattern did not vary with ethnic group nor with previous breast feeding experience. Short-term breast feeders were younger and had fewer children than either the bottle feeders or long-term breast feeders. Bottle feeding tended to occur with extremes of maternal age. Breast feeders sought more advice during pregnancy and confinement. Overall, the most common reason for changing to bottle feeding was "insufficient lactation", although this reason rarely stood alone. Many mothers experienced difficulties with artifical feeds and made at least one milk change, nearly half receiving no advice. Solid food was most commonly introduced at three months.


Mode of delivery and developmental characteristics in a thousand Dunedin three year olds: a report from the Dunedin Multidisciplinary Child Development Study. A study of 1037 representative Dunedin children delivered by a variety of modes was described. The results showed that there were no significant differences in maternal general mental ability, training in child development, or background of child experiences among the five delivery groups studied. Also, there were no significant differences between children who were spontaneous deliveries or any of the other groups in age at which the developmental milestones were achieved, nor in any of the developmental characteristics assessed.


Hypothyroidism in elderly hospital patients. Three hundred and ninety-six patients aged 65 years and over who were admitted to Waitakere Hospital in 1967-77 were screened for hypothyroidism using an adjusted serum thyroxine, serum thyrotrophin, or both. Nine new cases were found. Eleven other patients were already under treatment with thyroxine, though one had defaulted; in six of these the validity of the diagnosis was confirmed. A further five patients had marginally raised levels of serum thyrotrophin, of doubtful significance. There were therefore 15 proven cases of hypothyroidism, an incidence of 3.8%.


In-vitro activity of cefoxitin against recent clinical bacterial isolates. The susceptibility of 635 clinical isolates of bacteria to cefoxitin was determined. Isolates from the urinary tract showed 97% sensitivity, isolates from infected wounds showed 93% sensitivity and 82% of blood culture isolates were sensitive. Streptococcus faecalis isolates showed only 50% sensitivity and pseudomonas organisms were predominantly resistant. Bacteria isolated from unprepared heart valves submitted for use as homografts showed only 71% sensitivity due to the large proportion of Streptococcus and Pseudomonas species isolated.


Fulminant pneumococcal septicaemia in an adult following splenectomy: case report. A case is reported of fulminant pneumococcal septicaemia occurring in a 22-year-old male six years after surgical removal of the spleen.


Piretanide (HOE 118): a new potent diuretic: preliminary communication. A preliminary study of piretanide (HOE 118) on healthy subjects indicates it was as effective as frusemide for diuresis.


Rising cervical cancer mortality in young New Zealand women. New Zealand age-standardised mortality rates for cervical cancer in women 20-34, 35-64 and 65 years and older show that mortality has been falling steadily for women 35 years and older ever since 1941, 14 years before cytology screening was introduced. Mortality has risen significantly since about 1959 in women 20-34 years old. These findings cast doubt on the value of screening. Possible causes of the increased mortality in young women include a cohort effect, increased promiscuity, and the effect of steroidal compounds on cervical epithelium.


Mandibular odontogenic infection with serious complications. Orbital cellulitis usually begins as an infection of the paranasal sinuses. While a small percentage of cases are of dental origin, these usually involve the maxillary teeth. In the case reported here orbital cellulitis originated from an infection in the mandible and spread through the pananasal sinuses, deep facial circulation, and orbital tissues, resulting in unilateral blindness. Principles of management and possible pathways for the spread of the infection are discussed.


Pedunculated pharyngeal lesions.

Pedunculated pharyngeal lesions. Dentists enjoy a unique position in the health care field because they examine and treat more intraoral disease than any other specialty. It is, therefore, the responsibility of the dentist to examine the entire oral cavity and not just the teeth. Thorough intraoral examination may reveal undiagnosed pathological lesions which arise outside the oral cavity but are pedunculated and present in the mouth or oropharynx. The article presents three cases of such lesions which are potentially life threatening but may be cured if detected and treated early.


Peridontal manifestations and treatment in a case of Sturge-Weber syndrome. A case of Sturge-Weber syndrome, uncommon in its intraoral extent, is presented. The pathologic process involved both the soft and osseous tissues of both the maxilla and mandible of the affected side. The first known histopathologic description of alveolar bone involvement is presented. The patients was treated by means of extractions and periodontal flap surgery on both an outpatient and an inpatient basis, with good results and no untoward sequelae. common clinical findings in Sturge-Weber syndrome and specific signs and symptoms manifested by this patient are discussed.


Sensory deprivation.

Sensory deprivation. The loss of vision, hearing, and speech, even on a temporary basis, may be responsible for strange, unpredictable, or bizarre behavior. The placement of obtundent surgical dressings may be responsible for the symptoms of sensory deprivation.


An unusual case of trismus.

An unusual case of trismus. Described is a case in which a patient suffered multiple facial injuries resulting in significant trismuss. Many extracapsular factors have been reported to results in trismus. Most common are those involving the coronoid process and fibrous tissue adhesions of associated structures. This case involves a situation in which multiple foreign-body fragments led to this patient's being unable to open his mouth adequately.


Retained tooth vitality after 15 years' displacement into floor of mouth. Report of a case. A case in which a tooth traumatically displaced into the floor of the mouth retained its vitality for 15 years is reported. Although the presence of the tooth and soft-tissue cuff which appeared to represent the original attached gingiva presented a source for complications, no significant pathologic changes were noted. The requirement for a thorough radiographic examination before definitive treatment of maxillofacial injuries is emphasized.


Postinjection trismus due to formation of fibrous band. A persistent form of trismus which sometimes follows inferior alveolar nerve block is described. Clinical data from sixteen cases indicate that in most cases the condition responds to conservative therapy.


Tuberculosis of the tongue. A case report with immunologic study. A case of tuberculosis of the tongue in a 59-year-old woman with active pulmonary tuberculosis is described. The lingual tuberculosis was considered to be a secondary infection from the pulmonary disease, but the oral lesions were, in fact, noticed prior to recognition of the pulmonary lesion. This case was marked by a multiplicity or oral lesions arising on the bilateral surfaces of the tongue. Immunologic investigation revealed that cell-mediated immune responses in the patient were within the normal range in terms of the PPD skin test, DNCB skin test, lymphocyte transformation test, and subpopulation of the peripheral blood lymphocytes. Rosette-forming assay on the frozen sections disclosed that T-lymphocytes and macrophages were predominant in the lymphoid cells infiltrating the tuberculous lesion.


Unusual gingival epithelial proliferation: primary pseudoepitheliomatous hyperplasia. A primary pseudoepitheliomatous hyperplasia arising in the gingiva of a 9-year-old-boy with histologic features of carcinoma was surgically excised and has not recurred after 2 years. It is suggested that the histogenesis is atypical proliferation of an aborted gingival cyst arising from dental lamina.


Benign nerve sheath myxoma: light and electron microscopic features of two cases. Two new cases of the rare benign nerve sheath myxoma (NSM) are presented. The light microscopic findings are compared with previously reported cases. In addition, the ultrastructural features of three constitutent cell types are described. The lesion is confirmed as a neural sheath neoplasm, and its similarities to other myxomas are discussed.


Submucous fibrosis in Taiwan.

Submucous fibrosis in Taiwan. Thirty-five cases of submucous fibrosis have been collected in Taiwan. The fibrotic changes occurred most often in the buccal and palatal mucosa, and between the ages of 30 and 50 years. The occurrence of the disease correlated highly with the habit of betel nut chewing. Tobacco, hot spicy foods, and liquor were not important etiologic factors.


Ultrastructural features of monomorphic adenoma of the parotid gland. A monomorphic adenoma of the parotid gland (basaloid type) was studied with the electron microscope. In general, the tumor consisted of a single cell type, except in areas of squamous metaplasia. The cells were polygonal with prominent nuclei and scant cytoplasmic organelles. Intercellular junctions and plasmalemmal projections (filopodia) were conspicuous. Myoepithelial cells and secretory granules were absent. On the basis of these ultrastructural features, it is proposed that the interlobular duct, rather than the intercalated duct, is the probable site of origin of this tumor.


Endodontic perforations which resulted in alveolar bone loss. Report of five cases. Five cases in which perforations of mandibular molars into the furca occurred during endodontic instrumentation of the canal are presented. This resulted in alveolar bone loss. All were relatively asymptomatic, which could present a problem in diagnosis of the periodontal defect. The prognosis is questionable. The only tooth retained was treated surgically by an apically positioned flap and opening of the furca area to make it accessible to cleansing. The best treatment is the avoidance of the perforation. This is accomplished by considering the configuration of the canals and the size of the reamer than can follow it. Overinstrumentation with an endodontic instrument that is too large in diameter should be avoided.


Antimicrobial effectiveness in endodontic therapy using formocresol and two new alcoformal agents. Mechanical preparation during the second visit. Antimicrobial effectiveness of alcoformol two agents (AF 8.85 and AF 3.5) was studied in a clinical trial by means of bacteriologic examination of the root canals. After the initial culture was taken, one of the disinfectants was sealed in the pulp chamber for one week. At the second visit, the root canal was reamed and a second culture was taken. It was found that AF containing 3.5 percent formaldehyde is a satisfactory disinfectant for root canals of vital teeth. A concentration of 8.75 percent was needed for disinfection of teeth with necrotic pulps.


Antimicrobial effectiveness in endodontic therapy using formocresol and two new alcoformol agents. Mechanical preparation during the first visit. Antimicrobial effectiveness of two alcoformol agents (AF 8.75 and AF 3.5) was studied in a clinical trial by means of bacteriologic examination of the root canals. After the initial culture was taken, the root canal was reamed and a second culture was taken. One of the disinfectants was sealed in the pulp chamber for one week. At the second visit, a third culture was taken. No statistically significant differences were found between the effectiveness of the disinfectants; the cumulative effect of mechanical preparation and disinfection was such that initially positive teeth did not differ significantly from initially negative teeth when treated by this procedure.


Stress relaxation of interim restoratives. Stress relaxation of four temporary restoratives was studied. In the vicinity of ambient mouth temperature, the relaxation characteristics of an unmodified zinc oxide-eugenol cement were more favorable than those of IRM and Cavit. The plastic behavior of gutta-percha temporary stopping precluded assessment of its relaxation at temperatures in excess of 22 degrees C.


Xeroradiographic detection of tooth and bone pathology. Xeoradiography has gained limited use in oral surgery and orthodontics. Its wide exposure latitude, detailed image, and low operating costs recommend its use in general dentistry. In studies on real and simulted hard-tissue lesions, it was found to be slightly superior to conventional radiography in the detection of small lesions. When intraoral plates are marketed and low-KVP techniques are perfected, xeroradiography could assume an important role in dental radiology.


Radiopacity of impression materials.

Radiopacity of impression materials. The radiographic densitites of twenty-six impression materials were measured and the values expressed as an equivalent thickness of aluminum. Under simulated clinical conditions, only ten of the materials tested could be consistently distinguished from the bone structure in periapical radiographs. It was far more dificult to detect objects with beveled margins than objects of uniform thickness. The minimum radiopacity of a material which could be detected in the periodontal tissue on a periapical radiograph was estimated.


Squamous-cell carcinoma arising in a lateral periodontal cyst. A rare case of well-differentiated squamous-cell carcinoma arising in the epithelial lining of a lateral periodontal cyst is reported. Submission of surgically removed tissues for histopathologic evaluation is emphasized. The surgical site has been reconstructed and functions well. Because of early diagnosis and treatment, the patient has no evidence of clinical disease 2 1/2 years postoperatively.


Congenital granular-cell epulis. Report of a case. The congenital granular-cell epulis is an uncommon pedunculated tumor of the alveolar mucosa found only in newborn infants. This lesion can present an alarming appearance and interfere with efforts at nursing. The generally accepted treatment is excision through the pedicle, and recurrence is not to be anticipated. An example of a tumor fulfilling the clinical and histopathologic criteria of this lesion is reported.


Talon cusps: a dental anomaly in the Rubinstein-Taybi syndrome. Dental casts of fourteen cases of Rubinstein-Taybi syndrome (RTS) were studied. Two exhibited talon cusps, a dental anomaly which is rare in the normal population. Three others exhibited prominent cingula, which were interpreted as representing a tendency toward talon cusp formation. While additional cases should be examined for this anomaly to determine its frequency in RTS, the presence of talon cusps in a suspected case supports that diagnosis. Teeth exhibiting talon cusps are especially prone to caries and often require early treatment if they are to be retained.


Neurofibroma and schwannoma of the oral cavity. A clinical and ultrastructural study. The clinical parameters of fifty-five cases accessioned as neurofibroma and schwannoma of the oral cavity were reviewed. An electron microscope study was performed on several cases. The component cells of neurofibroma were spindle shaped, with bundles of collagen fibrils between cells. Junctional complexes were observed between adjacent cells. Component cells of the schwannoma showed numerous convoluted cytoplasmic processes. Reduplication of basal lamina was frequently observed and formed the material in the hyalinized area of Verocay bodies.


Acquired toxoplasma lymphadenitis.

Acquired toxoplasma lymphadenitis. Toxoplasmosis is a common parasitic infection that is widely distributed throughout the world, including the United States. Its occurrence in the oral cavity, however, is uncommon. This report concerns a case of toxoplasmosis that manifested as an intraoral lymphadenitis in a 14-year-old white girl. The etiology, clinical, microscopic, and laboratory findings, and treatment are reviewed. The importance of recognizing lymphadenopathy as a clinical sign of this disorder is discussed.


Angiolymphoid hyperplasia with eosinophils in oral mucosa. Angiolymphoid hyperplasia with eosinophils is a rare, benign angioproliferative lesion with a predilection for the facial skin. A unique instance of the lesion in oral mucosa is described, and the classification, differential diagnosis, and pathogenesis of the lesion are reviewed.


The solvent action of sodium hypochlorite on fixed and unfixed necrotic tissue. This study was carried out to answer the questions concerning the percentage of sodium hypochlorite needed to dissolve necrotic tissue and parachlorophenol- or formaldehyde-fixed necrotic tissue and the effect of the combined use of sodium hypochlorite and hydrogen peroxide. The results of this investigation indicate that a 3 percent concentration of sodium hypochlorite is optimum for the production of an adequate solvent action on necrotic tissue. A combination of sodium hypochlorite with 3 percent hydrogen peroxide did not result in an increased solvent action; therefore, their simultaneous use is not recommended.


Use of an inclined footrest for panoramic dental radiography. An inclined footrest was evaluated as a positioning aid for dental panoramic radiography. Experimental and control groups of students were examined by a conventional panoramic technique, with and without the addition of an inclined footrest. All radiographs obtained were evaluated for six parameters in a single-blind fashion by three dentists. Results indicated that the inclined footrest reduced spinal-shadow artifacts and improved over-all image quality.


[A variant of the Eggens filmholder for use in the posterior molar region]. Dr. Eggen's filmholder for long-cone-paralleling-technique is today available in two new, supplementary devices. These devices have an angulation in the metal rod, see fig. 1, and are meant to facilitate the film-positioning in the 3rd-molar area. A test was carried out to evaluate the new film-holders. Pairs of radiographs were taken in all 3rd-molar-areas, one radiograph with the straight film-holder, one with the angled device. Difference in horizontal film-position was measured. The results showed that in 93% the most posterior film-position was obtained with the angled film-holder.


[Disseminated intravascular coagulation (D.I.C.) and fibrinolysis in patients with acute leukemia (author's transl)]. Biological symptoms of D.I.C. were investigated in 43 patients with acute leukemia. Ten of them were found to be positive either at the onset or at the relapse of the disease and in some cases D.I.C. was triggered by chemotherapy. Among the ten positive cases 3 patients had an acute promyelocytic leukemia, 4 had an acute lymphoblastic leukemia, 2 a myeloblastic and 1 a monoblastic leukemia. D.I.C. was found either in patients with an hypercellular form of the disease or in patients with a normal or low white cell count. Symptoms of D.I.C. in acute leukemia must be systematicaly sought at the onset and during treatment by chemotherapy and treated with heparin and platelet transfusions as it is now admitted for acute promyelocytic leukemia.


Approximate steady-state properties of lattices of interacting three-state enzyme molecules: a novel phase transition. Previous work on the cooperative behavior of lattices of interacting two-state enzyme molecules at steady state is extended here to interacting three-state enzyme molecules with a one-way cycle. The Bragg-Williams (mean field) approximation is used. A phase-transition example with a bifurcation point is discussed. Compared to conventional phase transitions (with a van der Waals loop),  several new and complicated features appear. A second paper on this subject will contain a number of other examples of three-state systems.


Interferon induction: a conformational hypothesis. The ability of polynucleotides or polynucleotide duplexes such as poly(I).poly(C) to induce interferon production is proposed to depend on the existence of certain stable glycosidic orientations. It appears that a slight increase in instability of 1--3 kcal/mole (1 cal = 4.184 J) in the conformational regions near 20 degrees, 80 degrees, and 160 degrees leads to a loss of potency with respect to interferon induction. Thus, it is proposed that, for a polynucleotide to exist in the overall conformation necessary for interferon induction, stability of glycosidic orientations near 20 degrees, 80 degrees, and 160 degrees may be necessary to confer flexibility and activity on polynucleotide structures. This proposed conformational triad of stable conformational regions essential to interferon induction is based on the results of conformational energy calculations of the glycoside rotational profiles of adenosine, 7-deazaadenosine, inosine, and 7-deazainosine, as well as the conformational properties of other purine nucleoside analogs, and on inferences derived from calculations about the conformational effect in polynucleotides of removing the 2'-OH group.


Semihistone protein A24 replaces H2A as an integral component of the nucleosome histone core. The semihistone protein, A24, was shown to be a stable minor component of purified salt-washed nucleosome core particles. A24 was also shown to become integrated into nucleohistone during reconstitution in a manner characteristic of the core histones. Purified A24 in solution was shown to exhibit the same specificity of interaction with histone H2B as is exhibited by histone H2A. We conclude that A24 in chromatin replaces H2A as a stable integral component of certain nucleosome histone cores.


Effect of estrogen on gene expression in chicken oviduct: evidence for transcriptional control of ovalbumin gene. The transcription of structural and intervening sequences of the chicken ovalbumin gene was studied in nuclei isolated from the oviduct, liver, and spleen of chickens in different states of estrogen simulation. The concentration of transcripts of structural and intervening DNA sequences was determined by hybridizing the newly synthesized [(3)H]RNA to filters containing cloned ovalbumin cDNA (pOV230) or fragments of the natural ovalbumin gene (pOV2.4 and pOV1.8). Of the RNA synthesized by oviduct nuclei from chickens chronically stimulated with diethylstilbestrol, 0.23% corresponded to ovalbumin mRNA and 0.17% were transcripts of intervening sequences. No detectable ovalbumin mRNA sequences were synthesized by nuclei from spleen and liver. After 60 hr of hormone withdrawal, synthesis of ovalbumin mRNA by oviduct nuclei could not be detected. After readministration of estrogen, a gradual increase in ovalbumin mRNA synthesis was observed which began at 1 hr and reached a plateau by 8 hr. For the intervening sequences, similar kinetics were observed for the initial 4 hr. Previously we had identified multiple species of putative precursors of ovalbumin mRNA in oviduct nuclei from chickens chronically stimulated with diethylstilbestrol. We demonstrate here that withdrawal of diethylstilbestrol resulted in a depletion of high-molecular-weight ovalbumin RNA and of mature ovalbumin mRNA and that readministration of the estrogen induced the nuclear accumulation of both forms of ovalbumin RNA. These findings indicate that: (i) a method exists to assay synthesis of hormone-inducible specific eukaryotic [(3)H]mRNA in vitro; (ii) the estrogen-mediated preferential expression of the ovalbumin gene is maintained in isolated oviduct nuclei; (iii) after hormone withdrawal, a single injection of diethylstilbestrol induces transcription of ovalbumin structural and intervening sequences, with nuclear accumulation of high-molecular-weight ovalbumin RNA and mature ovalbumin mRNA; and (iv) these results are consistent with regulation of ovalbumin mRNA at the level of ovalbumin gene transcription.


Aliphatic groups of sperm whale myoglobin: 13C NMR study. The aliphatic region of the 13C NMR spectrum of sperm whale cyanoferrimyoglobin has been examined at 67.9 MHz. Fifty partially resolved or well-resolved resonances, representing at least half of the aliphatic carbons in the molecule, are observed in the spectral region from 9 to 29 ppm downfield of tetramethylsilane. Analyses of the spin lattice relaxation times (T1) and nuclear Overhauser enhancements for these resonances reveal considerable motion freedom of the aliphatic side chains. In the spectral region from 9 to 15 ppm, eight single carbon resonances are observed and tentatively assigned to Cdelta 1 of eight of the nine isoleucine residues. In at least five cases the reorientational motion of the isoleucine side chains could not be characterized solely by rotation of the Cdelta 1 methyl groups. The simplest model consistent with the data is a restricted diffusion model with two degrees of internal rotation [Wittenbort, R. J. & Szabo, A. (1978) J. Chem. Phys. 69, 1722--1736]. In light of the packing densities within the myoglobin molecule these results are taken to imply concerted motions of the buried aliphatic residues.


Regulation of protein synthesis in rabbit reticulocyte lysates: additional initiation factor required for formation of ternary complex (eIF-2.GTP.Met-tRNAf) and demonstration of inhibitory effect of heme-regulated protein kinase. Heme deficiency in rabbit reticulocytes and their lysates leads to the activation of a heme-regulated translational inhibitor (HRI) which causes the cessation of polypeptide initiation. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 binds Met-tRNA(f) and GTP in ternary complex. As a continuation of the studies on the molecular basis of the inhibition of the formation of 40S ribosomal subunit-Met-tRNA(f) complexes by HRI [Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., Roy, R. & Gupta, N. K. (1978) Proc. Natl. Acad. Sci. USA 75, 745-749], we describe here the isolation and some characteristics of a factor that is required for the HRI-catalyzed inhibition of eIF-2-promoted ternary complex formation. In the presence of 1 mM Mg(2+), ternary complex formation by eIF-2 is dependent on the presence of this stabilization factor (SF). Under these conditions, SF increases the rate and the extent of ternary complex formation. This finding suggests that the interaction of SF with eIF-2 causes a conformational change that stabilizes eIF-2 and promotes efficient ternary complex formation by increasing the affinity of eIF-2 for GTP and Met-tRNA(f). In the absence of Mg(2+), however, eIF-2 efficiently forms the ternary complex and SF has little effect on its ternary complex formation capacity-hence, the name eIF-2 stabilization factor (SF). In the presence of SF, HRI markedly inhibits (70-80%) the ternary complex formation capacity of eIF-2. The inhibitory effect requires both HRI and ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of eIF-2. Both the rate and the extent of the SF-dependent ternary complex formation are inhibited. These findings are consistent with the idea that phosphorylation causes a conformational change in eIF-2 such that its interactions with other initiation factors in the formation and the binding of ternary complex to 40S ribosomal subunits are inhibited.


Structure of the A2m(1) allotype of human IgA--a recombinant molecule. The complete amino-acid sequence of the constant (C) region of the alpha2 heavy chain of a human IgA2 protein of the A2m(1) allotype has been determined. Excluding the hinge region and the carbohydrate content, this alpha2 allotype differs from the alpha1 chain in only 14 amino-acid positions; all of these are identical to the A2m(2) allotype of the alpha2 chain and confer subclass (or isotypic) character on the alpha2 chains. However, the A2m(2) allotype differs in six positions where A2m(1) and alpha1 are identical; the first two are just before the hinge and the other four are in the last (C(H)3) domain. The A2m allotypic character of alpha2 chains is attributed to several conformational factors in the sequence at positions 211-221, just before the hinge. The isoallotypic determinant shared by alpha1 chains and the A2m(1) allotype of alpha2 resides in the identity of their C(H)3 domains. Thus, the A2m(1) allotype appears to be a hybrid chain that is identical with alpha1 in the C(H)3 domain and identical with the A2m(2) alpha2 chain in the C(H)1 and C(H)2 domains and in the hinge, except for the allotypic determinants arising from four structural differences from residues 211-221. The genetic origin of isotypes, allotypes, and isoallotypes of the alpha chain has involved several events of homologous crossing over and neutral point mutations accumulated late in the evolutionary development of IgA immunoglobulins. Since the crossing over appears to occur between C(H)2 and C(H)3, heavy chain domains may be coded for by independent units in embryonic DNA that are analogous to the variable (V) and C segments of light-chain genes.


Rapid helix--coil transitions in the S-2 region of myosin. Temperature-jump studies on the long S-2 fragment (100,000 daltons) isolated from myosin show that this structure can undergo alpha-helix--random coil transitions in a time range approximating the cycle time of a crossbridge. Two relaxation times are observed after temperature jumps of 5 degrees C over the range 35--55 degrees C, one in the submillisecond (tau f) and the other in the millisecond (tau s) time ranges. Both processes exhibit maxima near the midpoint of the helix--coil transition (tm = 45 +/- 2 degrees C) as determined by optical rotation melt experiments. Similar results were observed for the low temperature transition (tm = 45 degrees C) of the myosin rod. Viscosity studies reveal that the S-2 particles has significant flexibility at physiological temperature. Results are considered in terms of the Huxley--Simmons and helix--coil transition models for force generation in muscle.


Asymmetric lateral distribution of unshielded phosphate groups in nucleosomal DNA and its role in DNA bending. We suggest that an asymmetric charge neutralization of DNA phosphate groups produces part of the driving force for nucleosome folding. In nucleosome core particle DNA, many of the phosphate groups are neutralized by histones, and a lateral alignment of these histones along the core DNA has been demonstrated [Mirzabekov A. D., Shick, V. V., Belyavsky, A. V. & Bavykin, S. G. (1978) Proc. Natl. Acad. Sci. USA 75, 4184--4189]. Histones appear to shield DNA phosphatases asymmetrically at one side of the surface of the DNA double helix along all its length inside the core. The external side of the DNA helix remains unneutralized. The electrostatic repulsion between negatively charged unneutralized phosphates may fold the nucleosomal DNA towards the side occupied by histones.


Interaction of papain with derivatives of phenylalanylglycinal: fluorescence studies. Fluorescence studies have been performed on the interaction of papain with active-site-directed inhibitors of the type mansyl-(Gly)n-Phe-glycinal, where n = 0, 1, 2. It has been found that whereas the mansyl [6-(N-methylantilino)-2-naphthalene sulfonyl] fluorescence of mansyl-Phe-glycinal is greatly enhanced, that of the two longer mansyl compounds is not, although all three are equally effective as inhibitors of papain action. Measurements of fluorescence polarization and rotational relaxation time support the conclusion that the fluorescent probe group of the two longer mansyl compounds protrudes into the solvent to a greater degree than that of mansyl-Phe-glycinal. Considerable energy transfer from papain tryptophan to the mansyl group is evident for all three inhibitors, however, although it is most marked with mansyl-Phe-glycinal. Stopped-flow fluorescence measurements have shown that, after initial rapid interaction, the first-order conformational changes in the active-site region of papain in the complex with mansyl-Phe-glycinal are approximately 1/10(4) those observed with comparable mansyl oligopeptide substrates, and approximately 1/10(2) those with acetyl-Phe-glycinal.


Cross-sectional views of hemoglobin S fibers by electron microscopy and computer modeling. Fibers of deoxyHb S have been investigated by thin-section electron microscopy, utilizing a tannic acid embedding procedure. On the basis of numerous measurements of cross-sectional center-to-center distances for adjacent fibers in pairs or arrays, fiber diameters (mean +/- SD) of 205 +/- 5 A in embedded cells and 212 +/- 8 A in embedded hemolysates were obtained. This is an agreement with values obtained by conventional embedding procedures [Crepeau, R. H., Dykes, G., Garrell, R. L. & Edelstein, S. J. (1978) Nature (London) 274, 616--617]. The use of tannic acid has resulted in improved resolution of fiber cross sections, revealing individual strands of Hb S molecules. Because the section thickness corresponds to approximately one-fifth of the fiber helical repeat distance, the strands in projection superimpose to form characteristic image patterns. Additional superposition patterns arise in sections taken at small deviations from perpendicularity to the longitudinal fiber axis. These patterns are consistent with the 14-strand structure for hemoglobin S fibers [Dykes, G., Crepeau, R. H. & Edelstein, S. J. (1978) Nature (London) 272, 506--510], as indicated by computer models of cross-sectional patterns for various thicknesses and angular deviations of sections.


Prostaglandin synthesis in isolated rat kidney glomeruli. Isolated rat kidney glomeruli converted octatritiated arachidonic acid to several prostaglandins whose production was inhibited by meclofenamate. These were, in order of decreasing abundance, prostaglandin F2 alpha, prostaglandin E2, 6-oxo-prostaglandin F1 alpha, thromboxane B2, and prostaglandin D2. These products were identified by thin-layer chromatography, before and after treatment with potassium hydroxide or sodium borohydride. Prostaglandins F2 alpha and E2 were also determined by radioimmunoassay. The major product made by glomeruli was an unidentified substance(s), whose appearance was partially inhibited by meclofenamate, and was likely to be a hydroxylated fatty acid(s). The specific activity of glomerular fatty acid cyclo-oxygenase (EC 1.14.99.1), based on radioimmunoassay for prostaglandins E2 and F2 alpha, was 10- to 40-fold higher than that of cortical tubular enzyme. These data demonstrate that glomeruli have the capability of synthesizing an array of end-products from arachidonic acid. These prostaglandins may exert important physiologic effects, because renin secretion and arteriolar resistance are regulated by the glomerulus and the afferent and efferent arterioles.


Identification and isolation of a collagen-binding fragment of the adhesive glycoprotein fibronectin. We have identified and purified a polypeptide region containing the collagen-binding site of the adhesive glycoprotein fibronectin. Chicken cellular fibronectin isolated from cultured embryonic fibroblasts was permitted to bind to gelatin coupled to agarose beads and was then digested extensively with chymotrypsin. A prominent 40,000-dalton fragment of fibronectin consisting of a single polypeptide chain was detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of material remaining bound to the gelatin-agarose. This fragment appeared within 10 min after the digestion was initiated and persisted for more than 20 hr. This proteolytic fragment was isolated in electrophoretically pure form and retained its affinity for collagen. Plasma fibronectins from chicken and human blood also contained collagen-binding proteolytic fragments of similar size. This finding suggest that the collagen-binding sites of cellular and plasma fibronectins are homologous.


Alkaline phosphatase of mouse teratoma stem cells: immunochemical and structural evidence for its identity as a somatic gene product. The immunochemical and structural characteristics of the alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] from mouse teratoma stem cells derived from the OTT-6050 teratoma (ascitic and solid tumors and the F9 and PCC4 cell lines) have been compared to those of the alkaline phosphatases expressed in normal mouse placenta and several adult organs. Crossreactivity of the stem cell alkaline phosphatase with antisera reacting with placental, kidney, liver, and brain alkaline phosphatases indicated that the stem cell enzyme had common antigenic determinants. Structural studies utilizing two-dimensional electrophoresis of the (32)P-labeled alkaline phosphatase subunits showed that the stem cell, placental, and kidney alkaline phosphatases differed only in their sialic acid content and comigrated after removal of terminal sialic acid by neuraminidase digestion. Furthermore, one-dimensional peptide mapping of partial proteolysis fragments from (32)P-labeled enzymes demonstrated identical fragmentation patterns for the stem cell and somatic enzymes. These immunochemical and structural data indicate that the stem cell alkaline phosphatase is the same core enzyme as that produced in the mouse placenta and kidney, with different amounts of terminal sialic acid. The one mouse alkaline phosphatase examined that differed from the other enzymes was the intestinal alkaline phosphatase. This isoenzyme was not immunochemically crossreactive with the other alkaline phosphatases, did not comigrate in two-dimensional electrophoresis after neuraminidase digestion, and did not give identical peptide maps after partial proteolysis.


Molecules in mammalian brain that interact with the colchicine site on tubulin. Colchicine, a plant alkaloid, is a potent inhibitor of mitosis and other physiological processes that involve microtubules. These effects are mediated by the specific binding of colchicine to a high-affinity receptor site on tubulin, the major protein of microtubules. It seemed possible that the colchicine site on tubulin might also be the receptor for endogenous cellular molecules. We now report that mammalian brain does, in fact, contain a class of molecules that interact with the colchicine site on tubulin. Tubulin-agarose affinity chromatography has been used to isolate factors from soluble extracts of bovine brain that interact with tubulin. Certain of these factors inhibit the binding of [3H]colchicine to tubulin. Using the inhibition of colchicine binding as an assay, I have partially resolved at least two species. One is a protein and another may be a heat-stable peptide. In parallel to the action of colchicine, the smaller species inhibits the assembly of isolated microtubules. This class of molecules may include the endogenous ligands for which the colchicine site on tubulin is the receptor. They may have a significant role in the regulation of cellular microtubule function and assembly.


Models of fibrin.

Models of fibrin. High-symmetry models of the fibrin fiber are proposed that reproduce the experimentally observed high specific volume of the fiber. The models meet the following criteria: fibrin monomers have the three-domain Hall and Slayter structure; the monomers are arranged lengthwise into strands (protofibrils) in which successive monomers half overlap; the monomers' alignment is nearly parallel to the fiber axis; and the monomers make adequate longitudinal and lateral contacts, as required by observed fiber properties and the high affinity of monomers for one another. All the models contain helical protofibrils related to each other by rotation axes parallel to the fiber axis; as a consequence the protofibrils are in register in the fiber direction. The protofibrils may contain two, three, or four monomers per helical turn and can be packed in four different symmetries (space groups). A large specific volume is achieved only if the D-domain (which are presumed to contain the lateral polymerization sites) are somewhat displaced from the helical axes of the protofibrils. This displacement may involve either a lateral shift of the monomers away from the helix axis or a tilt of the monomers, which swings the D-domains away from the helix axis. It is shown that the fiber containing tilted monomers is more highly interconnected; the two D-domains of each tilted monomer form lateral contacts with different adjacent protofibrils, whereas the two D-domains of each nontilted monomer contact the same adjacent protofibril(s).


Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea. This communication demonstrates that all de novo fatty acid biosynthesis in spinach leaf cells requires acyl carrier protein (ACP) and occurs specifically in the chloroplasts. Antibodies raised to purified spinach ACP inhibited at least 98% of malonyl CoA-dependent fatty acid synthesis by spinach leaf homogenates. Therefore, the presence of ACP in a compartment of the spinach leaf cell would serve as a marker for de novo fatty acid biosynthesis. A radioimmunoassay capable of detecting 10(15) mol (10(-11) g) of spinach ACP was developed to measure the levels of ACP in leaf cell components isolated by sucrose gradient centrifugation of a gentle lysate of spinach leaf protoplasts. All of the ACP of the leaf cell could be attributed to the chloroplast. Less than 1% of the ACP associated with chloroplasts resulted from binding of free ACP to chloroplasts. Of interest, ACP from Escherichia coli, soybean, and sunflower showed only partial crossreactivity with spinach ACP by the radioimmunoassay. These results strongly suggest that, in the leaf cell, chloroplasts are the sole site for the de novo synthesis of C16 and C18 fatty acids. These fatty acids are then transported into the cytoplasm for further modification and are either inserted into extrachloroplastic membrane lipids or returned to the chloroplast for insertion into lamellar membrane lipids.


Glycosylation of human chorionic gonadotropin in mRNA-dependent cell-free extracts: post-translational processing of an asparagine-linked mannose-rich oligosaccharide. Placental RNA has previously been shown to direct the synthesis of an asparagine-linked mannose-rich glycosylated form of the alpha subunit of human chorionic gonadotropin (hCG-alpha) in lysates derived from mouse ascites tumor cells. Glycosylation was dependent on the presence of homologous microsomal membranes, and the glycosylated protein was sequestered into the microsomal vesicles. Here we show that when Triton X-100 is added after 60 min of translation and the incubation is continued, there is a shift of this glycosylated form to new discrete lower molecular weight proteins. The formation of these new proteins was not the apparent result of proteolysis because (i) treatment of the fully glycosylated protein or the proteins formed in the presence of Triton with endoglycosidase H resulted in the formation of a single protein migrating at the same rate on sodium dodecyl sulfate gels; (ii) the migration of nonglycosylated hCG-alpha synthesized in the presence of membranes isolated from tunicamycin-pretreated ascites tumor cells was not changed upon Triton addition; and (iii) the Triton-induced change was inhibited by mannonolactone, yeast mannan, and purified mannose oligosaccharides. It was also shown that little processing of the mannose-rich glycoprotein occurred in the presence of microsomal membranes alone. However, addition of the ribosome-free supernatant fraction to the glycoprotein resulted in processing. These data suggest that processing of the oligosaccharide core is a compartmentalized process in which removal of sugar, presumably mannose, requires a transfer of the glycoprotein from the endoplasmic reticulum to another component of the secretory cascade.


Correlation between structure and function of heparin. We have fractionated crude porcine heparin to obtain highly active as well as relatively inactive species of molecular weight approximately 7000 with specific anticoagulant activities of 360 and 12 units/mg, respectively. Nitrous acid degradation of both of these polymers yielded a tetrasaccharide fraction, 1beta, that contained equimolar amounts of iduronic and glucuronic acids, possessed an internal N-acetylated glucosamine, and carried anhydromannitol at the reducing end position. The 1beta tetrasaccharide derived from the highly active heparin, 1betaa, was recovered in a yield of 1.1 mol/7000 daltons. Our analyses indicate that at least 95% of the 1betaa is a single structure that consists of the following unique monosaccharide sequence: L-iduronic acid --> N-acetylated D-glucosamine-6-sulfate --> D-glucuronic acid --> N-sulfate D-glucosamine-6-sulfate. The 1beta tetrasaccharide fraction from relatively inactive mucopolysaccharide, 1betai, was recovered in a yield of 0.3 mol/7000 daltons and was a mixture of several components. Only 8.5% of the 1betai tetrasaccharide fraction exhibited the same uronic acid placement and sulfate group position found in 1betaa. Thus, 2.6% of relatively inactive mucopolysaccharide molecules contain the unique tetrasaccharide sequence found within each molecule of highly active heparin. Given the correlation between abundance of this unique 1betaa tetrasaccharide sequence and biologic potency, we suggest that this structure represents the critical site responsible for anticoagulant activity.


Nerve growth factor-induced transformation of immature chromaffin cells in vivo into sympathetic neurons: effect of antiserum to nerve growth factor. Pre- and postnatal injections of nerve growth factor, initiated with one dose on day 17 of gestation and continued after birth with daily subcutaneous administration until day 10 of life, produce massive transformation of chromaffin in sympathetic nerve cells in the rat adrenal medulla. Large sympathetic ganglia, absent in controls, adhere to the medial external surface of the gland. Nerve fibers produced by the transformed chromaffin cells invade the inner and outer cortical zones of the organ, producing cell depletion and substantial alteration of the structure of the cortical layers. When the growth factor treatment is initiated after birth, only a partial replacement of chromaffin with nerve cells takes place. The treatment is ineffective after the second postnatal week. Injections of a specific antiserum to nerve growth factor in 17-day-old rat fetuses, which were continued after birth, produce progressive and massive destruction of chromaffin cell precursors and of immature chromaffin cells in the adrenal medullary gland.


Nerve growth factor in the nucleus: interaction with receptors on the nuclear membrane. Cells of a continuous line of rat pheochromocytoma (PC12) were incubated with 125I-labeled beta nerve growth factor (beta NGF), and at given time intervals the cell nuclei were isolated by a procedure that used the detergent Triton X-100. NGF was detectable in the nucleus after 20 min and continued to accumulate in a linear fashion for several hours after the total binding to the cell had reached steady state. After 17 hr at 37 degrees C, about 60% of the NGF bound to the cell was in the nucleus, NGF was not translocated to the nucleus at 4 degrees C. When nuclei were purified from PC12 cells and incubated with 125I-labeled beta NGF, specific binding sites were found. Binding was saturable and consistent with two sites: a high-affinity site with a Kd of 0.08 nM (+/- nM) and a lower-affinity site with a Kd of 9.0 nM (+/- 2.0 nM). The receptors in the nucleus were shown to be localized to the nuclear membrane. Membrane-free chromatin did not bind NGF specifically. The translocation of NGF to the nucleus was accompanied by a commensurate decrease in the cell-surface binding capacity. In the nucleus, however, the receptor capacities of both sites were increased when PC12 cells were grown in the presence of NGF.


Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Fibroblasts can condense a hydrated collagen lattice to a tissue-like structure 1/28th the area of the starting gel in 24 hr. The rate of the process can be regulated by varying the protein content of the lattice, the cell number, or the concentration of an inhibitor such as Colcemid. Fibroblasts of high population doubling level propagated in vitro, which have left the cell cycle, can carry out the contraction at least as efficiently as cycling cells. The potential uses of the system as an immunologically tolerated "tissue" for wound healing and as a model for studying fibroblast function are discussed.


Induction of terminal differentiation in human promyelocytic leukemia cells by tumor-promoting agents. Human promyelocytic leukemia cells (HL-60) were induced to differentiate into mature cells by the tumor-promoting agent phorbol-12-myristate-13-acetate and other related phorbol diesters. Differentiation was determined by an increase in the percent of myelocytes, metamyelocytes, and other mature myeloid cells as well as by an increase in the percent of phagocytizing cells. Induction of differentiation could be determined after 2 days of treatment with phorbol-12-myristate-13-acetate at a dose as low as 6 X 10(11) M. A correlation was found between reported tumor-promoting activity of a series of phorbol esters and their ability to induce myeloid differentiation and to inhibit cell growth. It is suggested that tumor-promoting agents like chemicals that induce terminal differentiation in these cells, at extremely low concentrations, may be used as a tool in the study of the control of cell growth, cell differentiation, and malignancy in human leukemic cells.


Changes in the synthesis of actin and other cell proteins after stimulation of serum-arrested cells. The synthesis of both cytoplasmic and nuclear proteins has been studied as quiescent, serum-deprived Swiss mouse 3T3 cells are stimulated to transit the cell cycle. In serum-arrested cells a 200,000 dalton cytoplasmic protein and a 51,000 dalton nuclear protein were found to be preferentially synthesized. In serum-stimulated cells the first major protein whose synthesis was seen to increase had a molecular mass of 42,000 daltons. This protein also showed the greatest change in synthesis during the transit from G0 to S phase. Its synthesis rose to a maximum 4--6 hr after stimulation and then declined as cells entered S phase. The protein was present in both nuclear and cytoplasmic extracts. It was identified as actin on the basis of its mobility on sodium dodecyl sulfate and isoelectric focusing polyacrylamide gels. Other proteins synthesized preferentially by stimulated cells had molecular masses of 57,000 daltons (cytoplasmic), 33,000 daltons (cytoplasmic and nuclear), and 15,000 daltons (nuclear). The synthesis of the 57,000 and 33,000 dalton proteins increased gradually after stimulation and remained high during S phase. The 15,000 dalton proteins began to be synthesized as cells entered S phase. The preferential synthesis of these proteins provides biochemical markers for the transition from quiescence to proliferation.


Separate roles for calcium and magnesium in their synergistic effect on uridine uptake by cultured cells: significance for growth control. The uptake of uridine by BALB/c3T3 cells is markedly inhibited by reducing the concentration of Mg(2+) in medium containing only traces of Ca(2+). When physiological [Ca(2+)] is present in the medium, omission of Mg(2+) has no effect on uridine uptake, and when Mg(2+) is present, omission of Ca(2+) has only a slight inhibitory effect. When both Ca(2+) and Mg(2+) are omitted, the concentration of Ca(2+) in the cells is not reduced, but that of Mg(2+) is reduced to about one-half in 3 hr. The concentration of K(+) is also reduced, and that of Na(+) is increased, suggesting increased membrane permeability to cations. The rate of diffusion of the nontransported hexose, L-glucose, into the cells is greatly increased. Changes in intracellular Na(+) and K(+) concentrations do not in themselves affect uridine uptake. When Ca(2+) alone is restored to the medium of cells that had been deprived of both Ca(2+) and Mg(2+), there is no increase in the greatly depressed rate of uridine uptake, but when Mg(2+) alone is restored, the rate of uridine uptake returns to control values. We conclude that the omission of Ca(2+) from the medium has no direct effect on uridine uptake, but acts by increasing the exchange of Mg(2+) between cells and medium and by otherwise altering the availability of Mg(2+) for this reaction. A similar conclusion is reached in considering the role of these ions in the regulation of other reactions of the coordinate response, including the initiation of DNA synthesis and the control growth.


Intact microtubules are required for rapid turnover of carboxyl-terminal tyrosine of alpha-tubulin in cell cultures. In cultured muscle cells the carboxyl-terminal tyrosine of alpha-tubulin was shown to exchange rapidly with free tyrosine. The rapid turnover of this residue was dependent upon the presence of intact microtubules. Half-life determinations were made by two methods: (i) the cells were pulse-labeled in hypertonic medium, in which the major tyrosine incorporation was post-translational, and then chased with isotonic medium; and (ii) the cells were pulsed and chased in isotonic medium, and the post-translational component of the radioactivity of purified alpha-tubulin was calculated. Both methods yielded a half-life of 37 min or less for the terminal tyrosine residue, whereas the half-life of tubulin itself was shown to be greater than 48 hr.


Analysis of the constancy of DNA sequences during development and evolution of the nematode Caenorhabditis elegans. In order to test for the occurrence of rearrangements in DNA during development and to assess the rate of DNA divergence during evolution, we have compared restriction fragments derived from DNA from four sources: sperm cells and somatic tissues of one strain of the nematode Caenorhabditis elegans, somatic tissues of a second strain of the same species, and whole animals of a closely related species. Restriction fragments were detected by hybridizing radioactive cloned fragments to restriction digests that had been fractionated by size on agarose gels and transferred to nitrocellulose sheets. In this way, approximately 50 BamHI restriction fragments were visualized and compared. Fragments from sperm and somatic DNAs were found to be identical; 15% differed in size between the two strains. Little cross homology was found between the two species. We conclude that, if rearrangements occur in C. elegans DNA during development, they must affect fewer than a few percent of the restriction fragments or restriction sites. The difference found between the two strains and the two species is surprisingly great.


Gene localization by chromosome fractionation: globin genes are on at least two chromosomes and three estrogen-inducible genes are on three chromosomes. Chicken metaphase chromosomes were partially purified by rate zonal centrifugation, and DNA was prepared from each of the fractions of the sucrose gradient. The DNA was digested with various restriction enzymes and subjected to electrophoresis in agarose gels. The DNA was transferred to nitrocellulose filters (as described by Southern), and the filters were hybridized with cDNA probes. Four globin genes alpha A, alpha D, beta, and rho or epsilon are located on at least two chromosomes, and three of the estrogen-inducible genes of the hen oviduct--ovalbumin, ovomucoid, and transferrin--are on three different chromosomes. These experiments also confirm our earlier assignment of the endogenous viral sequence related to Rous-associated virus-0 to a separate (and larger) chromosome than the cellular sequence related to the transforming gene of avian sarcoma virus (cellular sarc), although it now appears that cellular sarc is on a small macrochromosome, rather than on a microchromosome.


Mutations in nine chloroplast loci of Chlamydomonas affecting different photosynthetic functions. Chloroplast components known to be coded by chloroplast DNA include chloroplast rRNAs, tRNAs, and the large subunit of ribulose-bisphosphate carboxylase. Because these components comprise less than 3% of the estimated coding capacity of the chloroplast genome, most chloroplast gene functions have yet to be identified. One approach to this problem is the isolation and characterization of mutations in the chloroplast genome affecting specific photosynthetic functions. Recently we have found that such mutations can be preferentially recovered by using arsenate selection on cells previously grown in 5-fluorodeoxyuridine. Sixteen mutants thus isolated have been localized into nine chloroplast loci, based on their ability to recombine and produce photosynthetically competent progeny. Mutants at two loci show the characteristic syndrome of photosynthetic defects that results from a deficiency in chloroplast protein synthesis. These have been found to lack chloroplast ribosome monomers. Mutants at three loci are missing chlorophyll-protein complex I in their thylakoid membranes. Mutants at three other loci are deficient in membrane polypeptides known to be associated with the chloroplast coupling factor.


Measurement of mutagenesis in mammalian cells. A method using mammalian cells in vitro for detection and quantitation of mutagenic actions that appears to be useful for screening for carcinogenesis and genetic damage by environmental agents is presented. The method involves use of stable human--Chinese hamster ovary hybrid cells that have retained a single human chromosome not necessary for cell reproduction. Forward mutations are detected in genes necessary for production of specific human cell surface antigens. Such mutants form colonies in the presence of specific antisera and complement that destroy the unmutagenized cells. Use of the method is illustrated for the action of x-irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, and caffeine. The method appears to be unique in that it permits assessment of lesions that cause loss of all or most of the chromosome as well as various localized gene mutations. The former action is particularly important because of the major involvement of chromosomal lesions in an extremely important class of human genetic disease.


DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. In this report, we demonstrate the feasibility of transforming mouse cells deficient in adenine phosphoribosyltransferase (aprt; AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) to the aprt+ phenotype by means of DNA-mediated gene transfer. Transformation was effected by using unfractionated high molecular weight genomic DNA from Chinese hamster, human, and mouse cells and restriction endonuclease-digested DNA from rabbit liver. The transformation frequency observed was between 1 and 10 colonies per 10(6) cells per 20 microgram of donor DNA. Transformants displayed enzymatic activity that was donor derived as demonstrated by isoelectric focusing of cytoplasmic extracts. These transformants fall into two classes: those that are phenotypically stable when grown in the absence of selective pressure and those that are phenotypically unstable under the same conditions.


Measurement and purification of human chromosomes by flow cytometry and sorting. The 24 human chromosome types of normal diploid fibroblast cell strain were classified into 15 groups by high-resolution flow cytometry on the basis of 33258 Hoechst fluorescence. Chromosomes associated with each group were flow sorted onto microscope slides and identified by quinacrine banding analysis. DNA cytophotometry of metaphase chromosomes from the same cell strain supported and extended this identification. Four of the groups purified were due to chromosomes of a single type--namely, chromosomes 5, 6, 13, and 17. Eight additional groups were also separated and found to contain the following chromosomes: 1 and 2; 3 and 4; 7, 8, and X; 9--12; 14 and 15; 16 and 18; 20 and Y; and 19, 21, and 22. The average purity for the 12 sorted fractions was 78%.


DNA fragments of the Mus musculus beta globin haplotypes Hbbs and Hbbd. Two alternative haplotypes at the complex locus controlling hemoglobin beta chain synthesis in Mus musculus were compared at the DNA level. As expected, Hbbd homozygotes--which as adults synthesize two species of beta chain--have two genes for beta globin. Adult mice homozygous for the Hbbs haplotype make only a single type of beta polypeptide, yet they also have two beta globin genes. Apparently the two Hbbs genes encode identical proteins, or one of the two genes is not detectably expressed. The Hbbs and Hbbd haplotypes are thus more similar at the DNA level than studies of their polypeptide products have indicated.


Role of methylation in the modification and restriction of chloroplast DNA in Chlamydomonas. The different metabolic paths followed by homologous chloroplast DNAs of maternal and paternal origins in zygotes of Chlamydomonas were examined by prelabeling parental cells, before mating them, with [3H]adenine, [3H]thymidine, and [3H]deoxycytidine. Within 6 hr after mating, maternal chloroplast DNA was extensively methylated to 5-methylcytosine and its bouyant density decreased. Paternal chloroplast DNA was largely degraded. Some radioactivity from deoxycytidine of maternal origin reappeared in thymine, and residual paternal DNA contained radioactivity in a base tentatively identified as uracil. These results confirm and extend our previous findings and support our hypothesis that modification (methylation) and restriction enzymes determine maternal inheritance of chloroplast DNA and that the two parental DNAs have different metabolic fates within the zygote.


Fc-receptor variants of a mouse macrophage cell line. Variants of the J774 mouse macrophage cell line that lack immunologically important membrane receptors were isolated. After mutagenesis, variants were selected in a metrizamide gradient that separated cells heavily rosetted with sheep erythrocytes (E) coated with rabbit anti-E IgG (EIgG) from poorly rosetted cells. Stable variants that exhibited altered binding were found with a frequency of <10(-7), and five clones were studied in detail. The variants failed to bind E opsonized with a monoclonal mouse IgG2b anti-E antibody but bound monomeric IgG2a normally when compared to the parental J774 line (K(a) 4 degrees C= approximately 1x10(8) M(-1); approximately 2x10(5) sites per cell). This demonstrates the independence of the receptor for mouse IgG2b complexes (FcRII) from the trypsin-sensitive receptor for mouse IgG2a monomer (FcRI). The variants bound an average of 10-15 EIgG per cell, compared to >20 per cell for J774. After trypsinization, three variants bound only three to five EIgG per cell; the J774 line was not affected by this treatment. Monomeric IgG2a could inhibit the binding of soluble rabbit IgG-antigen complexes to the variants but not to the parent line. Finally, E coated with IgM and complement (EIgMC) were bound poorly by all the variants, relative to the J774 parent. These results show that rabbit IgG complexes are bound by both FcRI and FcRII on mouse macrophages. The impairment of EIgMC rosetting in the variants suggests that the C3b receptor and FcRII are related.


Biochemical characterization of alien H-2 antigens expressed on a methylcholanthrene-induced tumor. A methylcholanthrene-induced tumor of BALB/c (H-2d) origin had been shown to express H-2 antigens normally associated with the H-2k haplotype. The molecules expressing the alien antigens have been partially purified and shown to be distinct from molecules expressing normal H-2d antigens. Like normal H-2 antigens, the alien antigens have a molecular weight of 48,000, are glycoproteins, and are noncovalently bound to beta 2-microglobulin. The alien antigens differ from normal H-2 antigens in their susceptibility to papain digestion, behavior during gel filtration, and overall stability. Possible mechanisms accounting for the expression of the alien antigen are discussed.


Detection of specific hybridoma clones by replica immunoadsorption of their secreted antibodies. A sensitive and rapid method for the detection of monoclonal antibodies secreted by hybridomas is described. Mouse myeloma cells are fused with spleen cells from immunized mice and directly cloned in soft agarose containing selective medium; hybrid clones can be seen after a week. Nitro-cellulose filters that have been coated with a specific protein antigen, with antigen-coupled erythrocyte ghosts, or with other cells used as antigens are then placed on the agarose surface. After incubation to allow immunoadsorption of any secreted antibodies specific for the filter-bound antigen, the filter is removed and overlaid with a suspension of antigen-coupled erythrocytes that react with the adsorbed antibodies; after unbound erythrocytes are allowed to fall off the filter, red spots delineate the sites at which antibody-forming clones are present in the agarose. Alternatively, the filter may be treated with radiolabeled antigen followed by autoradiography. The reliability and sensitivity of the method are demonstrated with alpha-(1 leads to 3)-specific antidextran myeloma J558, and the method's applicability is established by detecting hybridomas with specificities for sheep erythrocytes and for alpha-(1 leads to 3) dextran.


Immunological tolerance to allergenic protein determinants: a therapeutic approach for selective inhibition of IgE antibody production. Administration of stable conjugates prepared by coupling protein antigens such as ovalbumin or antigen E of ragweed extract to the synthetic random copolymer of D-glutamic acid and D-lysine (D-GL) is effective in inducing a state of long-lasting, antigen-specific immunological tolerance in experimental animals. A striking aspect of the tolerance induced by protein-D-GL conjugates is the remarkable selectivity of the tolerance for antibody responses of the IgE class. Protein-D-GL conjugates of either type were capable of inducing such tolerance both in unsensitized and in previously sensitized animals when administered in appropriate doses. Comparable doses of unconjugated proteins were likewise capable of suppressing IgE antibody production, although the duration of suppression in these cases was significantly less than that observed with protein-D-GL conjugates. If such conjugates act in man as they do in experimental animals, they could be of great value as therapeutic agents in selectively diminishing IgE antibody production while sparing antibody production in the IgG class.


Influence of major histocompatibility haplotype on autoimmune disease varies in different inbred families of chickens. Three partially inbred substrains of Obese strain chickens were studied for the spontaneous development of autoimmune thyroiditis. The influence of the major histocompatibility complex (B haplotype) was marked in one, less marked but still significant in a second, and barely detectable and transient in a third substrain. These differences in the effect of B haplotype may be due to the overriding action of genes other than those within the B haplotype.


Colorectal carcinoma-specific antigen: detection by means of monoclonal antibodies. Fusion of P3 X 63 Ag8 mouse myeloma cells with splenocytes obtained from mice immunized with cells derived from human colorectal carcinomas resulted in the production of antibody-secreting hybridomas. Two hybridomas (1083-17 and 1116-56) and their clones secreted antibodies binding specifically to human colorectal carcinoma cells either grown in culture or obtained from patients, but did not bind to normal colonic mucosa or other normal and malignant human cells. The binding specificity was consistent in three assays: radioimmunoassay, mixed hemadsorption, and immunofluorescence. Adsorption of these antibodies to colorectal carcinoma cell lines totally eliminated their specific binding.


Neurotoxicity of human eosinophils.

Neurotoxicity of human eosinophils. Eosinophils contain a substance that is neurotoxic when injected intracerebrally or intrathecally into laboratory animals-an effect known as the "Gordon phenomenon." We found neurotoxic activity in eosinophils from three patients with eosinophilic syndromes by injecting cell preparations into rabbits and guinea pigs. These animals developed a syndrome of muscular rigidity and ataxia, progressing to severe paralysis. No neurotoxic activity was found in preparations of polymorphonuclear or mononuclear leukocytes from normal donors. Examination of the brains of affected animals confirmed widespread loss of Purkinje cells, as described by earlier investigators. A new finding was severe spongy change occurring in the white matter of the cerebellum, brainstem, and spinal cord. Electron microscopic examination showed that vacuoles formed within the myelin sheaths of axons by separation of lamellae. Associated axonal degeneration was common and was also seen occasionally in peripheral nerves. Gray matter in the cerebral hemispheres and spinal cord was normal. This eosinophil-derived neurotoxin was partially purified by ultracentrifugation of sonicated eosinophils and fractionation of the supernate by gel filtration. Fractions with neurotoxic activity eluted at a position consistent with a molecular weight of approximately 15,000. The neurotoxic activity of this material withstood lyophilization and dialysis but was destroyed by heating to 90 degrees C. Injection of eosinophil-derived neurotoxin into laboratory animals may provide a useful short-term experimental model for study of mechanisms of damage to myelinated nerve fibers. The clinical significance of the Gordon phenomenon has yet to be established.


Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting. Fetal cells, potentially usable for prenatal diagnosis, were sorted from maternal blood samples taken as early as 15 weeks of gestation. Immunogenetic and cytogenic criteria established the fetal origin of the observed cells: Y-chromatin-containing (male) cells were detected in the sorted sample if and only if the newborn proved to be male and carried cell-surface antigens detected by the fluorescent-labeled antibody used for sorting with the fluorescence-activated cell sorter.


Amidolytic properties of single-chain activated Hageman factor. Activation of Hageman factor (Factor XII) upon exposure to negatively charged agents has been attributed to proteolytic cleavage of this molecule. To examine this question, purified Hageman factor was exposed to Sephadex gels to which ellagic acid had been adsorbed. Such Hageman factor, separated from the gels and studied in the fluid phase, was amidolytic. Nonetheless, no cleavage of Hageman factor treated in this way could be demonstrated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Thus, activation of Hageman factor by negatively charged agents was not necessarily accompanied by molecular scission.


Evidence for internal homology in bovine preproparathyroid hormone. By means of a statistical method for detecting distant evolutionary relationships between proteins, we found a high probability that internal homologies exist in preproparathyroid hormone, the biological precursor of proparathyroid hormone and parathyroid hormone. The regions so defined were -27 to +22 of preproparathyroid hormone vs. +26 to +74 parathyroid hormone and -23 to +29 of preproparathyroid hormone vs. +30 to +81 of parathyroid hormone. Examination of these regions for possible similarities of internal secondary structure (beta turns, alpha helix, and beta sheet) by a second type of predictive formulation indicated that most of the regions calculated to be related on an evolutionary basis were likewise homologous in terms of structure. These results and other considerations suggest that a primitive form of parathyroid hormone, possibly of pituitary origin, existed (or still exists). Parathyroid hormone of higher species likely was formed by gene doubling and fusion of the primitive gene.


Hemoglobin synthesis in cultures of hepatic erythroid cells from the human fetus. A recent theory of the control of human fetal hemoglobin synthesis, based on studies in cultured adult marrow, proposes that the phenotypic expression of fetal hemoglobin is largely dependent on the level of differentiation of the parental stem cells; that is, the earlier the progenitor, the greater the ability of its progeny to express fetal hemoglobin [Papayannopoulou, Th., Brice, M. & Stamatoyannopoulos, G. (1977) Proc. Natl. Acad. Sci. USA 74, 2923-2927]. To test this relationship with fetal tissue, we have studied hemoglobin synthesis in cultured human fetal liver, comparing gamma chain synthesis in the descendants of the early progenitors ("bursts") with that in the descendants of the later progenitors ("colonies"). Cells from the livers of midtrimester fetuses were cultured in methylcellulose with erythropoietin. The beta/(beta + gamma) globin synthetic ratio on days 5 to 7, when colonies predominated, was 0.09-0.11, a value characteristic of fetal reticulocytes, and on days 11 and 12, when bursts predominated, was 0.15-0.17. Thus, in fetal liver, the descendants of the earlier progenitor, the burst-forming unit, may be making more beta chains rather than more gamma chains, compared to descendants of the later progenitor, the colonyforming unit. Our data on fetal liver, taken together with the data on adult marrow by others, suggest that the erythroid colonies express the gene characteristic of the age of the organism to a greater degree than bursts, which express beta and gamma genes less specifically. Thus, the capacity for highly selective gene expression characteristic of differentiated cells appears to be less well developed in the burst-forming unit than in the colony-forming unit.


Sparing of metabolic stress in Purkinje cells after maximal electroshock. Experimental seizures were induced in mice by application of 50 mA for 0.2 sec via corneal electrodes. The reproducible conclusive behavior was characterized by a sequence of 2 sec of tonic flexion, 13 sec of tonic extension, and 8 sec of clonus followed by a postictal depressive stage. The animals were frozen and tissues were prepared for analysis according to Lowry and Passonneau [Lowry, O. H. & Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis (Academic, New York)]. Freeze-dried samples (1-10 ng) of pyramidal cell bodies and adjacent neuropil from the parietal cortex and of Purkinje cell bodies and adjacent neuropil from the cerebellum were analyzed for glucose, ATP, and P-creatine (0.01-0.05 pmol). There were marked decreases in these energy stores after the maximal electroshock in three of the areas examined. In the Purkinje cell bodies, however, the metabolic stress was dampened; glucose concentrations decreased, but the levels of ATP were maintained and, to a lesser extent, those of P-creatine. The results indicate that the output from the Purkinje cells is less than in the other regions examined in the excitable stages of the convulsion. The lesser energy debt probably reflects lower energy demand as well as a lower discharge intensity. The fact that Purkinje cells are spared from the metabolic stress imposed on other regions may be a partial explanation of the seizure activity. A diminished output from the Purkinje cells could be a situation that permits cortical convulsive activity.


Retrograde transport of nerve growth factor in chicken embryo. We have demonstrated retrograde transport of nerve growth factor to the dorsal root ganglia in the chicken embryo. Polyacrylamide gel pellets impregnated with 125I-labeled nerve growth factor were implanted subcutaneously in the leg of stage 36 (10-day) chicken embryos. The embryos were killed 8 hr after implantation and were processed for routine autoradiography. Examination revealed significant labeling of all lumbar dorsal root ganglia on the side of the implanted pellet. The lateral motor columns and ventral roots as well as the dorsal roots on both sides were unlabeled. These observations show that in the chicken embryo, during the period of sensory cell sensitivity to nerve growth factor, there is a selective uptake and retrograde transport of nerve growth factor from the target area to the dorsal root ganglia. These observations supplement reports from other laboratories in which selective retrograde transport of nerve growth factor in neonatal and adult rodents has been demonstrated.


Inactivation of Ca conductance dependent on entry of Ca ions in molluscan neurons. Inactivation of the Ca channel of Aplysia neurons was studied in the absence of potassium current in cells that were cesium-loaded with the aid of the ionophore nystatin. Inactivation was substantially decreased by methods that limited Ca entry. Depolarizations commensurate with the equilibrium potential of Ca resulted in minimal inactivation. Replacement of extracellular Ca by Ba also decreased inactivation. It is concluded that inactivation of the Ca channel is a function of the extent of Ca entry rather than membrane potential, thus differing fundamentally from the purely voltage-dependent mechanism for sodium inactivation.


Inosine, an endogenous ligand of the brain benzodiazepine receptor, antagonizes pentylenetetrazole-evoked seizures. Partially purified extracts of bovine brain were previously found to inhibit competitively the binding of [3H]-diazepam to rat brain synaptosomal membranes. The purines inosine and hypoxanthine were subsequently identified as the compounds responsible for this inhibitory activity. Intracerebroventricular administration of inosine to mice of the C3H/HEN and NIH general purpose strains caused a dose- and time-dependent increase in the latency to clonicotonic seizures produced by intraperitoneal administration of the convulsant pentylenetetrazole. Intracerebroventricular administration of equimolar doses of 2'-deoxyinosine, which is more potent than inosine in inhibiting the binding of [3H]diazepam in vitro, significantly increased pentylenetetrazole-evoked seizure latency. In contrast, both 7-methylinosine and thymidine were ineffective in inhibiting the in vitro binding of [3H]diazepam and increasing the latency to pentylenetetrazole-induced seizures in vivo. These results suggest that endogenously occurring purines such as inosine exhibit diazepam like effects when administered intracerebroventricularly, and these effects may be related to the interaction of inosine and related compounds with benzodiazepine receptors in the central nervous system.


Evidence for selection by male mating success in natural populations of Drosophila pseudoobscura. Gene arrangement frequencies were determined at two stages in the life history of Drosophila pseudoobscura taken from nature. Three populations in the central highlands of Mexico were each sampled twice during 1976. Gene arrangement frequencies were measured in adult males and in larvae that were the offspring of females collected at the same time. The adult males were in all likelihood a representative sample of those who fathered the larvae produced by the wild females. Differences in gene arrangement frequency between these two life stages should indicate the operation of natural selection. One-third of our comparisons of common gene arrangement frequencies in males and in larvae from the next generation were statistically significant, as were one-third of our comparisons of total frequency arrays in the two life stages. We consider the components of selection that could produce such frequency changes and reason that male mating success must be the major one. Gene arrangement frequencies in the Mexican populations fluctuate within wide bounds. Selection must act to retain the polymorphism in the face of this flux in gene arrangement frequencies, and we suggest that male mating success plays an important role.


Extrahepatic tissue distribution, covalent binding, and toxicity of halothane in control and phenobarbital-pretreated rats. Single anesthetic (11.5 mmole/kg) and nonanesthetic (4.6 mmole/kg) doses of 1-14C-halothane were administered separately to control and phenobarbital-pretreated rats by the intraperitoneal route. Tissue distribution and covalent binding of the anesthetic agent in the liver, lung, and kidney were determined at 4 and 24 hours after administration. Histopathological examination of the tissues revealed that halothane produced necrosis only in the livers of phenobarbital-pretreated rats and only after a period of 24 hours following the anesthetic dose. The lung and kidney appeared normal in all test animals. The liver was the major organ for the uptake, metabolism, and covalent binding of halothane metabolites to tissue proteins. Although similar metabolic activity was observed in the kidney and lung, toxic levels of the halothane metabolites were apparently not attained in these extrahepatic tissues in either control or phenobarbital-pretreated rats.