The passive hamstring stretch test: a comparison of clinical estimates with tension gauge measurements. A comparison of the clinical estimate of the available passive stretch in the hamstring muscles is made against the changing tension in a spring gauge, as the leg is raised. It was found that the angle at which rotation was palpable in the contralateral anterior superior spine occurred after there was a sharp rise in the tension measurements. The spring gauge was incorporated in a pulley system which allowed the leg to be pulled up and the tension readings were taken at 5 degrees increments of raise. Comparison of the clinical estimates with the tension gauge measurements show the latter to be a more reliable and objective research tool.


An assessment of vulval pigmentation.

An assessment of vulval pigmentation. Thirty-five vulvectomy specimens were obtained from post-mortem material and studied to assess the distribution of melanin pigment. The information obtained was compared with the melanin distribution in melanoma-in-situ and melanosis vulvae. A lesion had to be larger than 3mm before it was recognised clinically. Melanosis vulvae is but an exaggeration of the normal distribution of melanin pigment. Melanoma-in-situ, however, cannot be reliably distinguished from the benign causes of pigmentation on clinical grounds. The correct diagnosis can only be made histologically. Other causes of vulval pigmentation are also discussed.


Reticuloendothelial neoplasia of the female genital tract. Twelve patients thought to have gynecologic tumors subsequently were found to have lymphoma or leukemia. Eleven tumors involved the genital organs; one involved the retroperitoneal pelvic lymph nodes. A lymphoma originated in the genital tract in 7 patients, and secondary involvement occurred in 2 patients. One patient with a previously treated lymphoma and 1 with leukemia in remission developed ovarian tumors as the first sign of relapse. The difficulty in diagnosis, need for appropriate treatment, and prognostic importance of the genital organs affected are discussed.


Trismus after injection of local anesthetic. Persistent pain at the site of injection is the most common complication of local anesthesia in the oral cavity. The complication of trismus after local anesthetic injection is rare and may be prevented by the use of short needles for maxillary posterior injections, and by the avoidance of multiple injections in  a short period time. Once trismus develops, its progression to chronic hypomobility and fibrous ankylosis may be prevented by the early institution of treatment consisting of heat, analgesics, muscle relaxants, and exercises.


Jawbone cavities and trigeminal and atypical facial neuralgias. The possible role of dental and oral disease in the etiology of idiopathic trigeminal and atypical facial neuralgias has been examined. Among thirty-eight patients with idiopathic trigeminal neuralgia and twenty-three patients with atypical facial neuralgia, there was in nearly all instances a close relationship between pain experienced and the existence of cavities in alveolar bone and jawbone of the patients. The cavities were at the sites of previous tooth extractions and, although at times more than 1 cm. in a given diameter, were usually not detectable by x-rays. A new method for their detection and localization was developed empirically, based on the observation that peripheral infiltration of local anesthetic into or very close to the bone cavity rapidly abolished trigger and pain perception by patients during persistence of the anesthetic action. Histopathologic examination of bone removed from cavities by curettage revealed, in both idiopathic trigeminal and atypical facial neuralgias, a similar pattern characterized by a highly vascular abnormal healing response of bone. Some lesions presented a mild chronic inflammatory (lymphocytic) infiltration. Preliminary microbiologic studies of material from the walls of the cavities showed the existence within them of a complex, mixed polymicrobial aerobic and anaerobic flora. Treatment consisted of vigorous curettage of the bone cavities, repeated if necessary, plus administration of antibiotics to induce healing and filling-in of the cavities by new bone. Responses of patients to the above treatment consisted of marked to complete pain remissions, the longest of which has been for 9 years. Complete healing leads to complete and persistent pain remissions. It was concluded that in both idiopathic trigeminal and atypical facial neuralgias, dental and oral pathoses may be major etiologic factors.


Dental management for children with chronic renal failure undergoing hemodialysis therapy. Patients with chronic renal failure who are undergoing hemodialysis therapy demonstrate problems of significant importance for dental treatment. A case is presented in which secondard hyperparathyroidism, resulting from renal failure and hemodialysis, was noted in a child. Other relationships between renal failure, hemodialysis, and dental care were also presented.


Cold agglutinin disease. Report of a case. Cold agglutinin disease is an autoimmune hemolytic anemia with varying causes. Two major types are recognized, with different clinical manifestations. Cold agglutinins are circulating antibodies capable of erythrocyte agglutination. However, the reaction is readily reversible. Treatment of the disease is variable. A case is presented to illustrate that minor oral surgical procedures are feasible, provided the proper precautions are taken.


Simultaneous presence of partial anodontia and supernumerary teeth. The literature is reviewed and three cases of simultaneous presence of partial anodontia and supernumerary teeth are reported. In all three cases the abnormality involved both the maxilla and the mandible. The role of the panoramic radiograph in the detection of rare cases of this kind is emphasized and illustrated.


Ultrastructure of a plasma-cell myeloma in the mandible. Tumor cells exhibited large amounts of rough endoplasmic reticulum arranged in a lamellar pattern, large numbers of mitochondria with a perinuclear distribution, and a prominent Golgi complex, but only a small number of immunoglobulin granules. No crystalline structures of immunoglobulins were observed. Nuclei exhibited patchy condensation of chromatin and large nucleoli. The ultrastructural features suggest that this case could be a "resecretory" myeloma.


Scanning electron microscope study of a new irrigation method in endodontic treatment. The effectiveness of a newly developed perforated endodontic irrigation needle, with its distal aperture sealed, was compared to conventional endodontic irrigation procedures. In all instances 5 percent sodium hypochlorite was used since the delivery system, rather than the irrigant, was being evaluated. A base line group in which all soft tissue was described was also included. The results indicate that the perforated endodontic irrigation needle is a very effective instrument for the cleansing of instrumented root canals.


Clinical and radiographic study of dens invaginatus. After a determination of the frequency of dens invaginatus by means of radiographic observations, various problems of its identification are discussed. The 188 teeth showing a distinct loop-shaped invagination apically on the radiograph were studied for the presence or absence of caries and periapical lesions. It was demonstrated that periapical lesions caused by caries are mostly due to interproximal caries and that the frequency of periapical lesions caused by caries in the dens invaginatus area was less than previously reported. Comments are made on the dens invaginatus cases having periapical lesions without caries and the parameters for future studies.


Cephalometric studies of patients with ankylosis of the temporomandibular joint following surgical treatment. Cephalometric studies of eighteen cases of ankylosis of the temporomandibular joint, treated surgically, were undertaken. Arthroplasty tended to correct the characteristic facial deviation of patients with ankylosis and approximate the facial features of these patients to those of a normal person. These changes were found to occur in a larger percentage of the patients and to a greater magnitude 6 months postoperatively than 1 week following surgery. These findings support the concept that the mandible grows in response to functional stimulation and emphasize the importance of early surgical treatment of ankylosis of the temporomandibular joint.


Fibrositic myofascial pain in intermittent claudication. Effect of anesthetic block of trigger points on exercise tolerance. The blocking of trigger points in the calf by the local injection of an anesthetic agent was performed in 15 patients with intermittent claudication. Reactive and exercise hyperemia, work load and duration of exercise were recorded before and after infiltration of the trigger points. Reactive hyperemia does not change, but the exercise tolerance of the leg significantly improves after local blocking of the trigger areas and the exercise hyperemia increases because of the higher work load. The pain pattern of intermittent claudication can be activated by the summation of abnormal input from muscles, due to contraction in the presence of anoxia, with activity from trigger points. Local infiltration of trigger areas blocks theirir activity. The vicious cycle of pain is interrupted and the exercise tolerance of the leg is increased, without improving blood circulation.


Osteosarcomas in NZBR/BI mice.

Osteosarcomas in NZBR/BI mice. Osteosarcomas occurring in untreated and paraffin-treated NZBR/BI mice are described. Tumour tissue from one paraffin-treated animal was propagated by subcutaneous transplantation into young syngeneic male mice and the growth pattern and histological appearance of successive transplants were studied. The ability to form bone was retained and a propensity for metastasis to the lungs was observed. The latent period between the time of transplantation and clinical evidence of tumour formation diminished as passaging proceeded but there was no enhancement of the transplantability of osteosarcomas. The incidence of osteosarcomas in NZBR/BI mice is compared with that observed in other strains of mice.


[Cerebro-meningeal involvement in acute myeloblastic leukaemia and myeloproliferative syndromes in acute transformation. Cytological, histological and clinical study of 62 cases (author's transl)]. Clinical and histopathologic study of central nervous system (CNS) was performed in 46 acute myeloid leukemia (AML) and 16 chronic granulocytic leukemia in the blastic phase (CGL). Involvement of the CNS developed in 28 cases. Eighteen patients out of these 28 had neurological symptoms. The frequency of meningeal leukemia depends on the number of lumbar punctures and on the survival time. Post-mortem examination was performed on 45 patients. Eighteen had evidence of CNS leukemic infiltration (18/45 arachnoid, 10/31 dura, 5/45 brain). Hemorrhages are frequent even without CNS involvement (19/27). CNS leukemic infiltration is common enough in AML and CGL to justify agressive diagnostic, therapeutic, and prophylactic measures.


Photoisomerization, energy storage, and charge separation: a model for light energy transduction in visual pigments and bacteriorhodopsin. A simple model for the early events in visual pigments and bacteriorhodopsin is proposed. The model makes use of the likelihood that a negatively charged amino acid forms a salt bridge with the positively charged nitrogen of the retinylic chromophore. The photochemical event is a cis-trans isomerization in visual pigments and a trans-cis isomerization in bacteriorhodopsin, which in each case cleaves the salt bridge and thus separates charge in the interior of the protein. We propose that this is how the energy of a photon is transduced into chemical free energy of the primary photoproduct. The use of photoisomerization of a flexible chromophore to achieve charge separation provides a general mechanism which may be applicable to other systems. Our model explains many of the fundamental properties of visual pigments and their photoproducts. First, the extraordinarily low rate of thermally populating the ground state of the primary photoproduct, as determined from psychophysical and electrophysiological measurements, is seen as resulting from the large barrier to thermal isomerization about a double bond, perhaps enhanced by electrostatic attraction in the salt bridge. Second, the increase in energy and the spectral red shift that characterize the primary photochemical events are natural consequences of the separation of charge. Proton-dependent processes detected with picosecond techniques are proposed to be ground-state relaxation processes following the primary photochemical event. Finally, the charged groups of the salt bridge, repositioned by photoisomerization, provide a simple mechanism for vectorial proton translocation in bacteriorhodopsin.


"Alternating B-DNA" conformation for the oligo(dG-dC) duplex in high-salt solution. The high resolution 1H and 31P NMR spectra of the (dG-dC)8 duplex have been recorded in low- and high-salt solutions in order to evaluate the structural aspects of the salt-induced transition of oligo(dG-dC) in solution  [Pohl, F. M. & Jovin, T. M. (1972) J. Mol. Biol. 67, 375-396]. The NMR data require that the (dG-dC)8 duplex in 4 M NaCl adopt an "alternating B-DNA" conformation for which the symmetry unit repeats every two base pairs. By contrast, the oligomer duplex in low-salt solution is of the regular B-DNA type in solution. The chemical shift parameters for oligo(dG-dC) in high-salt solution demonstrate that every other glycosidic torsion angle and phosphodiester linkage adopts a different conformation from that observed in regular B-DNA. We demonstrate further that the generation of the "alternating B-DNA" structure is facilitated by introduction of halogen atoms at the 5 position of pyrimidine and that this probably reflects the greater overlap of this position with adjacent base pairs in high salt solution. An "alternating B-DNA" model has recently been proposed for alternating deoxy purine-deoxy pyrimidine polynucleotides based on the x-ray structure of pdA-dT-dA-dT [Klug, A., Jack, A., Viswamitra, M.A., Kennard, O., Shakked, Z. & Steitz, T.A. (1979) J. Mol. Biol., in press].


Cyclized dipeptide model for a beta-bend. A cyclic dipeptide in which L-Ala-Gly was cyclized with epsilon-aminocaproic acid has been synthesized as a model for a beta-bend. Its conformational properties have been examined by means of conformational energy calculations and nuclear magnetic resonance, infrared, Raman, and circular dichroism spectroscopy in various solvents. These calculations and experiments suggest that a type II beta-bend exists in the Ala-Glymoiety, with an NH...O = C hydrogen bond in the epsilon-aminocaproic acid portion of the molecule, and that the molecule adopts a unique conformation in solution. In contrast, an open-chain analog of this compound exists in solution as an ensemble of conformations but with a significant amount of a type II beta-bend structure in the ensemble.


Paracatalytic modification of aldolase: a side reaction of the catalytic cycle resulting in irreversible blocking of two active-site lysyl residues. Paracatalytic enzyme modifications result from the oxidation of enzyme-substrate carbanions by extrinsic oxidants. During the oxidation of enzyme-activated substrates, transiently reactive intermediates are generated which, without being released from the enzyme, modify groups at the active site. For enzymes producing carbanion intermediates, the combination of the normal substrate with a suitable electron acceptor has thus been proposed as a highly specific binary system for their active site-directed modification. In this study, the structural features of paracatalytically modified fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from rabbit muscle have been elucidated. This enzyme is completely inactivated within 60 min in the presence of fructose 1,6-bisphosphate in saturating concentration and 0.5 mM hexacyanoferrate(III) (pH 7.6, 25 degrees C). The inactivation is caused by covalent incorporation of one triosephosphate derivative per subunit. Peptide analysis showed that the triosephosphate derivative forms an intrachain crosslink between lysine-146 and lysine-227. According to previous independent experimental evidence, both lysyl residues are located at the active site: the epsilon-amino group of lysine-227 forms a Schiff base intermediate with the carbonyl group of the substrate [Lai, C. Y., Nakai, N. & Chang, D. (1974) Science 183, 1204-1206] and alkylation of lysine-146 by the affinity labeling reagent N-bromoacetylethanolamine phosphate inactivates the enzyme [Hartman, F. C. & Brown, J. P. (1976) J. Biol. Chem. 251, 3057-3062]. The present data thus establish paracatalytic modification as a mode of active site-directed enzyme modification.


Polyamine-mediated phosphorylation of a nucleolar protein from Physarum polycephalum that stimulates rRNA synthesis. An acidic nucleolar phosphoprotein with a subunit M(r) of 70,000 was purified as an apparent dimer of 139,000 from isolated nuclei of the slime mold Physarum polycephalum. The protein was purified without the aid of strong dissociating agents after its selective phosphorylation in isolated nuclei by a polyamine-mediated reaction. Its amino acid composition resembled that of a nucleolar phosphoprotein from Novikoff hepatoma ascites cells. The phosphoprotein stimulated rRNA synthesis 5-fold by RNA polymerase I within a nucleolar, ribosomal deoxyribonucleoprotein complex isolated from nucleoli of P. polycephalum. It was also identified as a component of the complex. It bound with high affinity and specificity to the palindromic ribosomal DNA of 38 x 10(6)M(r) from P. polycephalum, which contained two coding sequences for 5.8S, 19S, and 26S rRNA. It also bound to three fragments of ribosomal DNA of M(r) 21.2 x 10(6), 17.1 x 10(6), and 8.1 x 10(6), prepared by cleavage with restriction endonucleases HindIII, PstI, and BamHI, respectively. All of these fragments included the symmetry axis of the palindromic ribosomal DNA. The phosphoprotein that had been treated with alkaline phosphataseagarose to hydrolyze the phosphate groups did not stimulate transcription and did not bind to ribosomal DNA or to the restriction fragments indicated. We have thus isolated a specific phosphoprotein with the capacity to stimulate transcription of a specific set of genes in a eukaryote. These findings suggest that this phosphoprotein may specifically regulate functions of ribosomal DNA in a manner dependent on its degree of phosphorylation.


Asymmetric and globular forms of acetylcholinesterase in mammals and birds. We have identified six molecular forms of acetylcholinesterase (AcChoE: acetylcholine hydrolase, EC 3.1.1.7) in extracts from bovine superior cervical ganglia. We show that three of them resemble the collagen-tailed forms of Electrophorus AcChoE in their hydrodynamic parameters, low-salt aggregation properties, and collagenase sensitivity. The six molecular forms of bovine AcChoE appear structurally homologous to the six forms of electric fish AcChoE that have previously been characterized. They include globular molecules (monomers, dimers, and tetramers) and asymmetric aggregating molecules that possess a collagen-like tail associated with one, two, and three tetramers. We propose to call the globular forms G1, G2, and G4 and the asymmetric forms A4, A8, and A12, the subscripts indicating  the number of catalytic subunits. In spite of quantitative differences in their molecular parameters, the AcChoE forms from rat and chicken are clearly homologous to those of bovine AcChoE. Thus the nomenclature we introduce is very probably valid for the main AcChoE molecular forms, at least in vertebrates, and should help to clarify structural relationships and homologies among them. This model, however, does not claim to represent entirely the complex polymorphism of AcChoE, because more or less hydrophobic variants of the G forms have been observed, and because other molecular associations cannot be excluded. We discuss the significance of the globular and collagen-tailed structure for the molecular localization of AcChoE.


Staphylococcal nuclease: proposed mechanism of action based on structure of enzyme-thymidine 3',5'-bisphosphate-calcium ion complex at 1.5-A resolution. The structure of the staphylococcal nuclease (EC 3.1.4.7)-thymidine 3',5'-bisphosphate-Ca(2+) (enzyme-inhibitor) complex has been extended to 1.5-A resolution by using much additional data and a phase refinement scheme based on an electron-density map modification procedure. By correlating this structure with the known properties of the enzyme, a mechanism of action is proposed that involves nucleophilic attack on phosphorus by a water molecule, which is bound to Glu-43, in line with the 5'-CH(2)O(H) leaving group. The carboxylate of Glu-43 promotes this attack by acting as a general base for the abstraction of a proton from the attacking water molecule. Nucleophilic attack is further facilitated by polarization of the phosphodiester by an ionic interaction between a Ca(2+) ion and a phosphate oxygen atom and by four hydrogen bonds to phosphate oxygen atoms from guanidinium ions of Arg-35 and Arg-87. These interactions may also catalyze the reaction by lowering the energy of a trigonal bipyramidal transition state. The hydrolysis of nucleic acid substrate proceeds by cleavage of the 5'-P-O bond to yield a free 5'-hydroxyl group and a terminal, 3'-phosphate monoester group. In the inhibitor complex the only general acid group found in a position to donate a proton to the leaving 5'-oxygen is the guanidinium ion of Arg-87. Alternative proton donors, presently lacking direct structural support, could be the phenolic hydroxyl group of Tyr-113 or a water molecule. The precision and rigidity of the location of the reactants at the active site and the probable dual binding and catalytic roles of the guanidinium ions of Arg-35 and Arg-87 are especially noteworthy.


Synthesis and biological properties of pinane-thromboxane A2, a selective inhibitor of coronary artery constriction, platelet aggregation, and thromboxane formation. Pinane-thromboxane A2 (PTA2, [1alpha,2 beta(Z),-3 alpha (1E,3R*),5 alpha]-7-(3-(3-hydroxy-1-octenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-yl)-5-heptenoic acid) has been synthesized and tested for biological activity in systems responsive to thromboxane A2, stable prostaglandin endoperoxide (PGH2) analogs, and prostatacyclin (PGI2). At low concentrations, PTA2 inhibited cat coronary artery constriction induced by stable prostaglandin endoperoxide analogs, and it stabilized liver lysosomes. At slightly higher concentrations, it inhibited platelet aggregation. At still higher concentrations, PTA2 inhibited thromboxane synthetase, but it had no effect on prostacyclin synthetase. The analog also had no effect on the inhibition of platelet aggregation by PGI2 or prostaglandin D2. It is suggested that PTA2 has a suitable biochemical profile for use as an antithrombotic agent.


Specific binding of perhydrohistrionicotoxin to Torpedo acetylcholine receptor. Torpedo californica postsynaptic membrane fragments were treated with base, which resulted in membranes that were depleted of many nonacetylcholine receptor polypeptides and contained acetylcholine receptor subunits of Mr 40,000, 50,000, 60,000, and 65,000 (Raftery, M.A., Vandlen, R.L., Reed, K.L. & Lee T. (1975) Cold Spring Harbor Symp. Quant. Biol. 40, 193-202). A 43,000-Mr polypeptide and some other components were quantitatively extracted. Base-treated membranes retained the capacity to bind [3H]perhydrohistrionicotoxin and the local anesthetics dibucaine and tetracaine. The regulation of this binding by carbamylcholine, as well as the kinetic mechanism of perhydrohistrionicotoxin binding, was unchanged. [3H]Perhydrohistrionicotoxin binding activity was largely reconstituted from 2% sodium cholate extracts of base-treated membranes. Therefore, the perhydrostrionicotoxin binding site appears to be located on one or more of the acetylcholine receptor polypeptides, and the reconstitution of that binding site from detergent extracts does not require the presence of a 43,000-Mr polypeptide.


Mechanism of monensin-induced hyperpolarization of neuroblastoma-glioma hybrid NG108-15. Addition of the ionophore monensin to mouse neuroblastoma-rat glioma hybrid NG108-15 cells leads to a 20 to 30-mV increase in the electrical potential across the plasma membrane as shown by direct intracellular recording techniques and by distribution studies with the lipophilic cation [3H]-tetraphenylphosphonium+ (TPP+) [Lichtshtein, D., Kaback, H.R. & Blume, A.J. (1979) Proc. Natl. Acad. Sci. USA 76, 650-654]. The effect is not observed with cells suspended in high K+ medium, is dependent upon the presence of Na+ externally, and the concentration of monensin that induces half-maximal stimulation of TPP+ accumulation is approximately 1 microM. The ionophore also causes rapid influx of Na+, a transient increase in intracellular pH, and a decrease in extracellular pH, all of which are consistent with the known ability of monensin to catalyze the transmembrane exchange of H+ for Na+. Although ouabain has no immediate effect on the membrane potential, the cardiac glycoside completely blocks the increase in TPP+ accumulation observed in the presence of monensin. Thus, the hyperpolarizing effect of monensin is mediated apparently by an increase in intracellular Na+ that acts to stimulate the electrogenic activity of the Na+,K+-ATPase. Because monensin stimulates TPP+ accumulation in a number of other cultured cell lines in addition to NG108-15, the techniques described may be of general use for studying the Na+,K+ pump and its regulation in situ.


Delimitation of a promoter for RNA polymerase III by means of a functional test. A Xenopus laevis DNA segment containing the structural gene for tRNA1Met and 22 base pairs at the 5' side of the gene is active in tRNA production. The DNA segment was ligated to sea urchin histone DNA and was also inserted into plasmid pCR1. Both recombinant DNAs were shown to produce mature tRNA1Met at a high rate when injected into centrifuged Xenopus oocytes.


Intermolecular crosslinking of fatty acyl chains in phospholipids: use of photoactivable carbene precursors. Phospholipids containing photolysable carbene precursors (beta-trifluoro-alpha-diazopropionoxy and m-diazirinophenoxy groups) in omega-positions of sn-2 fatty acyl chains were prepared. Photolysis of their vesicles produced crosslinked products in 40-60% yields. Crosslinking was mostly intermolecular and occurred by carbene insertion into the C-H bonds of a second fatty acyl chain. Crosslinking products were characterized by (i) their gel permeation behavior, (ii) analysis of products formed by base-catalyzed transesterification, (iii) degradation with phospholipases A2 and C, (iv) gas chromatography/mass spectrometry, and (v) use of mixtures of phospholipids carrying the carbene precursors and a phospholipid containing radioactively labeled fatty acyl groups. Nitrenes generated from the aliphatic or aromatic azido groups in phospholipids were unsatisfactory for forming crosslinks by insertion in C-H bonds.


Serum RNase in the diagnosis of pancreatic carcinoma. Serum RNase (RNase I; ribonuclease 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) activity (mean +/- SD) with polycytidine as substrate was determined in normal individuals (24.9 +/- 3.0 units/ml) and in patients with pancreatic cancer (37.3 +/- 14.8), pancreatitis (38.5 +/- 12.6), nonpancreatic diseases (48.7 +/- 14.8), or renal failure (175.8 +/- 92.8). Patients with pancreatic cancer could not be distinguished from those with pancreatitis or with nonpancreatic disease, although the RNase activities in all of these differed from the activity in normal individuals. The serum RNase activities of four patients with resectable "curable") pancreatic carcinoma and two others with advanced pancreatic cancer without obstructive jaundice were normal. After total pancreatectomy, serum RNase activity remained in the high-normal range. The data presented here and data in the literature show that serum RNase cannot be of primarily pancreatic origin. The present study also demonstrates that measurement of its activity is not useful in early detection of pancreatic cancer.


Chemoattractants stimulate degradation of methylated phospholipids and release of arachidonic acid in rabbit leukocytes. When rabbit peritoneal leukocytes were treated with chemoattractants such as fMet-Leu-Phe, an apparent decrease of [3H]methyl incorporation into the lipid fraction from L-[methyl-3H]methionine was observed. This decrease was a result of increased degradation of methylated phospholipids, not of decreased synthesis. Chemotactic peptides did not affect the metabolism of the phospholipids in which [methyl-14C]choline was incorporated. The disappearance of the [3H]methyl group was associated with the release of [1-14C]arachidonic acid from phospholipids prelabeled with these compounds. These findings suggested the activation by chemoattractants of phospholipase A2, an enzyme that removes an unsaturated fatty acid from phospholipids. The order of potency of chemoattractants for the stimulated degradation of phospholipids was in good agreement with that for chemotaxis. Mepacrine (quinacrine) and hydrocortisone inhibited and a phorbol ester enhanced both chemotaxis and phospholipase A2 activity. These results, taken together, suggest close association of the metabolism of methylated phospholipids with chemotaxis in rabbit peritoneal leukocytes.


Fibronectin and the multiple interaction model for platelet-collagen adhesion. A rapid, sensitive, and reproducible assay to determine the adhesion of platelets to collagen has been developed. Collagen fibers and adherent platelets are retained on polycarbonate membrane filters. Chemical modification of collagen by acetylation and of platelets by treatment with chymotrypsin markedly reduces adhesion. The role of fibronectin in the collagen-platelet interaction has been examined. Treatment of platelets with purified antibody or Fab' fragments to fibronectin only slightly reduces adhesion. Preincubation of platelets with high concentrations of gelatin reduces adhesion by only 22% but fails to inhibit aggregation. Thus, fibronectin has only a limited role in the adhesion of platelets to collagen and is either not involved in the adhesion that leads to aggregation or is only one of several adhesion mechanisms, any of which alone can initiate aggregation.


Messenger RNA of opsin from bovine retina: isolation and partial sequence of the in vitro translation product. Opsin, the apoprotein of the visual pigment rhodopsin, is synthesized on membranes of the rough endoplasmic reticulum and subsequently passes through the Golgi apparatus to the rod outer segment. This pathway parallels the early stages of biosynthesis of some secretory proteins and viral membrane glycoproteins. Most of these proteins are initially synthesized as precursor molecules with a short-lived hydrophobic extra peptide segment at the NH(2) terminus. Therefore we investigated whether or not the immediate translation product of opsin mRNA contains a similar short-lived NH(2)-terminal extra peptide. The mRNA coding for opsin was isolated from bovine retina polysomes precipitated by antibodies to opsin. The mRNA directed the cell-free synthesis of a protein comparable in size to opsin that was specifically precipitated by anti-opsin antibodies. Sequence analyses of the immunoprecipitated protein labeled with six radioactive amino acids (Met, Asn, Pro, Phe, Tyr, Val) provided the following result: [Formula: see text] (X is unknown). This partial sequence of the cell-free product corresponds exactly to the published NH(2)-terminal segment of native opsin (21 residues long) and extends beyond this region. Met-1 was shown to be the initiator methionine residue, because only the initiator [(35)S]Met-tRNA(1) (Met)-not the internal [(35)S]Met-tRNA(2) (Met)-donated the NH(2)-terminal methionine. This finding essentially rules out the possibility that Met-1 was preceded by a peptide that was rapidly cleaved. Thus opsin, and not a precursor, is the immediate product of opsin mRNA translation.


Determination of enzyme mechanisms by radiationless energy transfer kinetics. Rigorous definition of the elementary steps of an enzymatic reaction requires visualization of transient enzyme-substrate (ES) complexes. Measurement of radiationless energy transfer (RET) between enzyme tryptophan residues and a fluorescent dansyl (5-dimethylaminonaphthalene-1-sulfonyl) substrate provides a sensitive means to observe ES complexes directly. Analysis of the rate of formation and breakdown of ES complexes by RET can serve as the basis of a rapid kinetic approach to enzyme mechanisms. Both pre-steady-state and steady-state kinetics can be performed in the same RET experiment. Analysis at steady state precisely determines k(cat) and K(m) values by multiple means. Analysis at pre-steady state determines the number of intermediates, the type of reaction mechanism, and all the individual binding and rate constants. Chymotrypsin was chosen as a standard of reference for RET kinetics because extensive investigations have established both the existence of transient intermediates in the course of its catalytic process and the range of values to be expected for pertinent kinetic constants. As predicted, RET kinetics readily detects the two known intermediates in the alpha-chymotrypsincatalyzed hydrolysis of specific ester substrates. The results are both qualitatively and quantitatively in accord with data derived for this enzyme from classical kinetics. Hence, this experimental study both validates and demonstrates the theoretical advantages and potential of RET kinetics. The generality of the approach has been investigated by synthesizing a family of dansyl-labeled substrates designed to meet the specificity requirements of a number of metallo- and nonmetallo- exo- and endopeptidases. In all cases, the ES complex is observed readily at micromolar or lower concentrations of enzyme under stopped-flow conditions. The success of the RET kinetic approach on proteolytic enzymes shows its broad utility.


De novo biosynthesis of an enzymatically active precursor form of bovine pancreatic RNase. The de novo biosynthesis of RNase (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) was studied in a cell-free rabbit reticulocyte translation system using a polyadenylylated fraction of mRNA isolated from bovine pancreas. Analysis of the [35S]methionine-labeled translation products of pancreas mRNA by polyacrylamide gel electrophoresis indicated the presence of several proteins, one of which corresponded to 16,500 daltons, or approximately 2800 daltons greater than native RNase A. This protein was specifically precipitated from the mixture of products by anti-RNase antibodies. Partial sequence determination of the NH2-terminal region of the anti-RNase antibody-precipitable species indicated that it is a precursor form of RNase A with 25 additional amino acids on its NH2 terminus. The precursor nature of the protein was confirmed by demonstration that a mixture of RNase A and a glycosylated form of the enzyme, RNase B, is formed when translation of the mRNA is conducted in the presence of dog pancreas membranes. Assay of the putative precursor form of RNase for catalytic activity with polycytidylic acid as substrate indicated that the protein has a specific enzymatic activity identical to that of native RNase A.


Initial glycosylation of proteins with acetylgalactosaminylserine linkages. Epithelial glycoprotein like that produced by the gastric surface consists of a polypeptide chain rich in serine and threonine; to these amino acid residues oligosaccharide chains of variable length are linked. The linking sugar is acetylgalactosamine. To find out whether the initial glycosylation takes place at the ribosomal level. I treated purified peptidyl-tRNA, derived from rat gastric membrane-bound polysomes, with alkali in the presence of boro[3H]hydride. Alkali specifically splits glycosidic bonds between serine or threonine and oligosaccharide side chains (beta-elimination reaction). The linking sugar is converted to an alditol and simultaneously labeled. GalNAc was identified as the linking sugar by paper chromatography. Furthermore, nascent peptides with lengths between 40 and 60 amino acid residues already contained this linking sugar. Gel filtration on Bio-Gel P-2 of 3H-labeled saccharides revealed that nascent chains contained mainly monosaccharides, but some di- or trisaccharides were found with GalNAc as the linkage sugar. These findings demonstrate that initial glycosylation of epithelial glycoprotein occurs at the ribosomal level rather shortly after the nascent peptide chain has reached the cisternal lumen of the endoplasmic reticulum.


Helical parameters of DNA do not change when DNA fibers are wetted: X-ray diffraction study. We have measured the helical parameters of DNA in concentrated solutions by x-ray fiber diffraction methods. Fibers of the sodium salt of DNA were swollen with water within capillaries; the capillary served to limit water uptake, slowing dissolution. Samples containing  up to 80% water gave essentially a B-form diffraction pattern and had virtually the same helical parameters [9.91 base pairs per turn (SD = 0.14); 3.34 A axial rise per residue (SD = 0.019)] as did the initial fibers [9.95 base pairs per turn (SD = 0.15); 3.33 A axial rise per residue (SD = 0.015)]. Hence, under highly solvated conditions in which the interactions between molecules should be greatly decreased, DNA maintains its classical B-form structure.


Arachidonic acid is responsible for the smooth muscle-contracting activity of G-acid. G-acid, a smooth-muscle-contracting substance isolated from human blood plasma, was previously identified as 3-octadecenoic acid. Synthetic cis- and trans-3-octadecenoic acid, however, are biologically inactive. G-acid actually consists of a mixture of fatty acids, characteristic of animal tissues. The biological activity is caused by arachidonic acid, which contracts smooth muscle upon in situ conversion to prostaglandins.


Modulation of binding and bioactivity of insulin by anti-insulin antibody: relation to possible role of receptor self-aggregation in hormone action. Incubation of physiological concentrations of 125I-labeled insulin with liver membranes in the presence of anti-insulin IgG results in a 7- to 15-fold increase in the specific binding of the hormone. The low-affinity/high-capacity binding sites are replaced by an apparently homogeneous class of high-affinity sites, and the nonlinear Scatchard plots are converted to linear plots without a change in the maximum number of binding sites. Similarly, the binding of insulin to receptors in 3T3 fibroblasts is increased substantially in the presence of anti-insulin antibody, and the biological activity of subactive concentrations of insulin is enhanced by antibody in these cells. However, the affinity of 125I-labeled epidermal growth factors (EGF) in fibroblasts is not affected by anti-EGF IgG. In adipocytes anti-insulin IgG in the same concentration range only inhibits the binding of insulin and suppresses insulin-mediated glucose oxidation. Monovalent Fab' fragments from anti-insulin IgG inhibit the binding of the hormone, indicating that the enhancement of binding in liver membranes and fibroblasts requires the bivalency of the antibody.


Tactoidal state and phase transitions in systems of linear polymers of variable length. The tactoidal state in systems containing long, rod-like molecules consists of partially aligned solute molecules in equilibrium with and at a concentration not much higher than that in the conjugate isotropic phase. Under the liquid lattice model of Flory [Proc. R. Soc. London Ser. A, (1956) 234, 73-89], as well as under other models, tactoid formation by molecules of fixed axial ratio depends on nonideality induced by excluded volumes; the process is wholly entropy driven and requires no direct interactions between rods. Many rod-like biological polymers exhibit reversible polymerization, so that axial ratio and length are not fixed. Polymerization and rod length will then not only induce nonideality, alignment, and phase separation, but will be affected by these. In this work these interrelations are treated under the model of Flory, modified to include a free energy of polymerization and to permit reversible changes in rod length. The primary conclusion is that, in contrast to the situation for fixed lengths, excluded volume-dependent nonideality alone does not suffice to induce a tactoidal phase separation. In the absence of attractions or repulsions between rods the anisotropic phase is highly concentrated. This phase only becomes tactoidal when a minimal level of repulsive interaction between rods is reached. Under this model, tactoid formation in systems such as deoxygenated hemoglobin S and tobacco mosaic virus depends on repulsive interactions or metastability or both. As a secondary result it is shown that rod length in the anisotropic phase is much greater than in the conjugate isotropic phase.


Kinetics and mechanism of tobacco mosaic virus assembly: direct measurement of relative rates of incorporation of 4S and 20S protein. The mechanism of assembly of tobacco mosaic virus has been investigated under conditions in which the rates of incorporation of the 4S and 20S proteins can each be directly measured by analytical centfrifugation. Under these conditions, pH 6.5, 6.5 degrees C, 0.10 M ionic strength potassium orthophosphate, the protein can be made to exist as a metastable 20S aggregate that is necessary for efficient reconstitution. The overall assembly process consists of an initiation (nucleation) reaction that requires two to three 20S disk aggregates per RNA molecule and is followed by an elongation (growth) reaction. In the elongation phase of assembly the 4S protein is incorporated 50 to 70 times faster than the 20S disk, calculated on the basis of a steady-state kinetic analysis. Therefore, under these conditions, in which the rate of assembly is about 0.06 of that at pH 7, 20 degrees C, 0.10 M ionic strength orthophosphate, the 4S protein preferentially participates in the elongation phase. At this slow reconstitution rate intermediate assembly states (about 70-168 S) can be observed. The kinetics of both protein incorporation and nucleoprotein formation suggest that the elongation process is composed of at least two different, possibly sequential, rate-limiting reactions.


Measured work of deformation and repulsion of lecithin bilayers. We used three complementary techniques to vary the chemical potential of water in lipid/water mixtures; we measured the work of removing water from the multilayer lattice formed in water by the zwitterionic phospholipid egg lecithin. By x-ray diffraction, we observed the structural consequences of water removal. There are no discrete classes of "bound water" in this system; the work of removal is a continuous function of water content and lattice repeat spacing. From 30 to 3 A separation between bilayers there exists an exponential "hydration force" repulsion with a 2.6 A decay length. This interaction translates into a very large force to prevent contact between vesicles and planar membranes. It may be an important feature in controlling vesicle-to-cell fusion. As water is removed, bilayers not only move closer, but thicken as the lipid polar groups on the same bilayer move closer together. It is possible to divide the applied work into that of direct bilayer repulsion and that of bilayer deformation. We thus obtained a first determination of the lateral pressure required to create large increases in bilayer thickness and concomitant decreases in bilayer area. The lateral pressure reaches 25 dynes/cm for a 25% decrease in bilayer area. Systematic measurements of the mechanical properties of bilayers suffering such large deformation will allow critical tests of theories on bilayer stability and phase transition.


Electron flow through plastoquinone and cytochromes b6 and f in chloroplasts. With dark-adapted chloroplasts in which the plastoquinone was oxidized, a partial reduction of cytochrome b6 was obtained upon illumination with a pair of short saturating flashes. The second flash of the pair was much more effective than the first, and the reduction was inhibited by the system II inhibitor diuron. When the plastoquinone pool was reduced, both the reduction and the oxidation of cytochrome b6 were accelerated. The cytochrome b6 oxidation appeared to proceed in association with the reduction of cytochrome f, although these cytochromes are not simply connected in series. From these observations it is inferred that electron flow to the secondary donors of system I alternately caused the reduction and the oxidation of cytochrome b6. An interpretation is offered that also accounts for the transmembrane proton translocation that is associated with the oxidation of plastohydroquinone.


Selective hepatic uptake of human beta-hexosaminidase A by a specific glycoprotein recognition system on sinusoidal cells. Intravenously administered (125)I-labeled human beta-hexosaminidase A (beta-N-acetylglucosaminidase; 2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) was rapidly cleared from the circulation of rats and accumulated in the liver. When hepatic cells were subsequently isolated, the label was recovered from both sinusoidal cells and, to a lesser extent, hepatocytes. Clearance was inhibited by the simultaneous infusion of mannan but not by a galactose-terminated glycoprotein. Studies in vitro, in which (125)I-beta-hexosaminidase was incubated with isolated hepatic cells, detected no uptake of the labeled ligand by hepatocytes. In contrast, uptake by sinusoidal cells was shown to be temperature dependent and approached saturability. Prior treatment of sinusoidal cells with Pronase resulted in markedly decreased uptake of (125)I-beta-hexosaminidase by these cells. Mannan and partially deglycosylated glycoproteins bearing terminal nonreducing N-acetylglucosamine or mannose residues were shown to be potent inhibitors of the cellular uptake of (125)I-beta-hexosaminidase; native orosomucoid and desialylated (galactoseterminated) orosomucoid were not inhibitory. Of six simple sugars tested, including N-acetylglucosamine, only mannose was an effective inhibitor of the cellular uptake of (125)I-beta-hexosaminidase. The kinetics of uptake of beta-hexosaminidase and mannose-terminated orosomucoid by sinusoidal cells were shown to be similar. These findings suggest that the hepatic uptake of the lysosomal glycosidase beta-hexosaminidase A is mediated by a receptor on sinusoidal cells which recognizes and binds mannose-terminated glycoproteins.


Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester. When suspension cultures of human promyelocytic leukemia cells (line HL60) were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA; 1.6-160 nM), more than 80% of the cells adhered to the plastic substrate within 24 hr. Within the same time period the immature azurophilic granulations typical of HL60 promyelocytic cells disappeared and the nuclear chromatin became more condensed, but the nucleolus was retained. The attached cells stopped dividing and synthesizing DNA. The phenomenon was irreversible and independent of the continuous presence of TPA. Approximately 60% of the untreated cells and of TPA-treated cells bore surface Fc receptors for IgG. Under the experimental conditions used, about 10% of the TPA-treated cells were also able to phagocytize IgG-coated erythrocytes and more than 80% were able to phagocytize latex beads, but untreated controls were unable to do so. Cellular levels of NADase, acid phosphatase, and non-specific esterase were markedly increased after treatment with TPA, whereas little or no increase was seen after treatment with dimethyl sulfoxide (Me2SO), a drug that induces myeloid differentiation of HL60 cells. Peroxidase activity was lower in TPA-treated and Me2SO-treated cells than in HL60 cells. More lysozyme was found in the medium of TPA-treated cells than in the medium of untreated or Me2SO-treated cells. These data indicate that, after treatment with TPA, human promyelocytic leukemia cells can differentiate into cells that have several characteristics of macrophages.


Mitotic factors from mammalian cells induce germinal vesicle breakdown and chromosome condensation in amphibian oocytes. Cytoplasmic extracts of HeLa cells synchronized in various phases of the cell cycle were injected into fully grown Xenopus laevis oocytes to monitor the presence of factors that can induce meiotic maturation: i.e., germinal vesicle breakdown and chromosome condensation. Extracts from GI and S phase cells had no activity. The maturation-inducing activity, which was found to be low during early and mid G2 phases, increased rapidly during late G2 and reached a peak in mitosis. The results of this study suggest that the factors that regulate the breakdown of nuclear membrane and chromosome condensation during mitosis, meiosis, and premature chromosome condensation appear to be very similar, if not identical, throughout the animal kingdom.


Intracellular localization of 125I-labeled insulin in hepatocytes from intact rat liver. We have shown that 125I-labeled insulin initially localizes to the plasma membrane of isolated rat hepatocytes. The ligand is subsequently internalized and preferentially localizes to lysosomal structures. Further, we have observed that labeled insulin localizes to regions of the cell rich in lysosomal and Golgi elements. In the present study in intact rat liver we found that, approximately 10 min after a pulse injection of 125I-labeled insulin, 56% of the label was internalized by the cell. When all grains are considered there is a preferential localization of grains to the biliary pole of the cell and these grains are almost all internalized and preferentially associated with lysosomes. These data, therefore, demonstrate that the lysosome-Golgi-rich area of the isolated hepatocyte corresponds to the biliary pole of the cell and there is a movement of the labeled hormone from its initial binding site on the plasma face of the cell membrane toward the biliary pole of the cell.


Regulation of dihydrofolate reductase synthesis in an overproducing 3T6 cell line during transition from resting to growing state. We have isolated a methotrexate (MTX)-resistant clone of mouse 3T6 cells, designated M50L3, which grows normally in the presence or absence of 50 muM MTX and produces a level of dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate:NADP(+) oxidoreductase, EC 1.5.1.3) that is increased about 300-fold compared to the parental 3T6 cells. The cells retain the ability to rest in the G(0) state when maintained in medium containing 0.5% calf serum and can be stimulated to reenter the cell cycle by increasing the serum concentration to 10%. The rate of accumulation of DHFR in resting M50L3 cells is about 1/25th of that in exponentially growing cells. When resting cells are stimulated to reenter the cell cycle, the rate of accumulation of DHFR starts to increase at about 8 hr and reaches a maximum (25-fold increase) at about 16 hr after stimulation. Pulse-labeling experiments show that the increase in DHFR accumulation is due to an increased rate of synthesis. This increase occurs at about the same time the cells enter S phase. However, inhibitors of DNA synthesis have no effect on the increase in DHFR accumulation after serum stimulation, indicating that there is no tight coupling of the two events. Actinomycin D inhibits the subsequent increase in DHFR accumulation if added 8 hr after stimulation but has no effect if added 16 hr after stimulation. This is consistent with the idea that the increase in DHFR gene expression depends on transcription of the gene and that DHFR mRNA synthesis begins at about the time the cell initiates DNA replication. DHFR gene expression appears to be regulated in the same manner in the overproducing cells as we found in the parental 3T6 cells [Johnson, L. F., Fuhrman, C. L. & Wiedemann, L. M. (1978) J. Cell. Phys. 97, 397-406]. Therefore, the alterations that are responsible for DHFR overproduction (presumably DHFR gene amplification) do not interfere with the ability of the cell to regulate the rate of synthesis of the enzyme after serum stimulation.


Induction by lithium ion of multiplication of mouse mammary epithelium in culture. Lithium ion at concentrations between 2 and 20 mM simulated the stimulatory effects of insulin on the uptake of alpha-aminoisobutyric acid, synthesis of RNA and DNA, and cell multiplication in mouse mammary gland explants cultured in a chemically defined synthetic medium. Other monovalent cations were virtually ineffective. In most instances the stimulatory effect of lithium ion was somewhat smaller than and additive to that of insulin. However, lithium ion was incapable of substituting for the action of insulin in augmenting milk protein synthesis in mammary explants cultured with other lactogenic hormones, prolactin, and glucocorticoid. The observed similarities of the responses of mammary cells to lithium and insulin suggest that possible importance of cation(s) in the regulation of mammary cell proliferation, which may be a common basis for the action of the two agents. On the other hand, the observed inability of lithium to mimic the lactogenic effect of insulin indicates a specific function of the hormone in the functional differentiation of mammary cells.


Human lymphoblastoid cell variants defective in cell-cell adhesion. Adhesion mutants were selected from a human lymphoblastoid cell line. Initially, cells were selected on the basis of survival in serum-free medium. Subclones that grow as single cells rather than macroscopic aggregates were selected from the serum-independent variant. The defect in cell-cell adhesion is stable over many generations and is not corrected by growth in serum or the presence of serum in the culture medium. Analysis of mixed cultures composed of adhesive cells and nonadhesive cells indicates that the two cell types do not interact to form mixed aggregations. Furthermore, those results suggest that the adhesion-deficient phenotype does not result from the production of a transferable inhibitor. In a previous study [Whipple, A.P., Dalvin, M. & Millis, A.J.T. (1978) Exp. Cell Res. 116, 457-461], we found that the growth rate in serum-containing medium is identical for the two classes of cells. This suggests that cell-cell adhesion is not a critical factor in the growth of these cells.


Fixation of a deleterious allele at one of two "duplicate" loci by mutation pressure and random drift. We consider a diploid population and assume two gene loci with two alleles each, A and a at one locus and B and b at the second locus. Mutation from wild-type alleles A and B to deleterious alleles a and b occurs with mutation rates va and vb, respectively. We assume that alleles are completely recessive and that only the double recessive genotype aabb shows a deleterious effect with relative fitness 1-epsilon. Then, it can be shown that if va greater than vb mutant a becomes fixed in the population by mutation pressure and a mutation-selection balance is ultimately attained with respect to the B/b locus alone. The main aim of this paper is to investigate the situation in which va = vb exactly. In this case a neutral equilibrium is attained and either locus can drift to fixation for the mutant allele. Diffusion models are developed to treat the stochastic process involved whereby the deleterious mutant eventually becomes fixed in one of the two duplicated loci by random sampling drift in finite populations. In particular, the equation for the average time until fixation of mutant a or b is derived, and this is solved numerically for some combinations of parameters 4Nev and 4Ne epsilon, where v is the mutation rate (va = vb = v) and Ne is the effective size of the population. Monte Carlo experiments have been performed (using a device termed "pseudo sampling variable") to supplement the numerical analysis.


Continuous variation caused by genes with graduated effects. The classical polygenic theory of inheritance postulates a large number of genes with small, and essentially similar, effects. We propose instead a model with genes of gradually decreasing effects. The resulting phenotypic distribution is not normal; if the gene effects are geometrically decreasing, it can be triangular. The joint distribution of parent and offspring genic value is calculated. The most readily testable difference between the two models is that, in the decreasing-effect model, the variance of the offspring distribution from given parents depends on the parents' genic values. The more the parents deviate from the mean, the smaller the variance of the offspring should be. In the equal-effect model the offspring variance is independent of the parents' genic values.


Familial hyperproinsulinemia: partial characterization of circulating proinsulin-like material. Familial hyperproinsulinemia is an autosomal dominant defect that is associated with strikingly elevated levels of serum proinsulin-like material. Our studies show that trypsin converts familial hyperproinsulinemia proinsulin to insulin more slowly than it converts a 131I-labeled porcine proinsulin marker. Molar yields of insulin indicated that the material may be an intermediate proinsulin. Studies with two human C-peptide antisera that differ in their relative immunoreactivity with human C-peptide and proinsulin showed that the two antisera reacted equally with familial hyperproinsulinemia proinsulin, suggesting that it is a partially cleaved proinsulin intermediate. Sulfitolysis of highly purified material to break the inter- and intra-chain disulfide bridges and subsequent adsorption on a specific B-chain antibody covalently bound to Sepharose beads showed that the C-peptide was still connected to the B-chain. These data indicate that familial hyperproinsulinemia proinsulin is normally cleaved at the C-peptide-A-chain linkage site. A structural abnormality appears to underlie familial hyperproinsulinemia proinsulin, which impairs its cleavage at the B-chain-C-peptide linkage site.


Association of HLA antigens and total serum immunoglobulin E level with allergic response and failure to respond to ragweed allergen Ra3. We have applied rigorous statistical analysis to human immune response and HLA data in allergic subjects having varying sensitivity to the minor ragweed allergen, Ra3. By using regression analysis on a series of subsets of Ra3 responders and nonresponders, we obtained data supporting the concept of discrete "responder" and "nonresponder" phenotypes. We found significant positive associations between Ra3 response and total serum IgE and the presence of HLA antigens of the A2 crossreacting group (A2 and A28), and significant negative associations with antigens of the A3 crossreacting group (A3 and A11). Our results suggest that alleles of the HLA-A locus may function either as immune response or immune suppressor genes or that such alleles are epistatic to the response or suppressor genes. Our data add further support to previous findings that HLA-associated specific IgE response is strongly influenced by genetic regulation of basal IgE level.


Phospholipid methylation in macrophages is inhibited by chemotactic factors. Chemotaxis by human monocytes has been shown to require methylation mediated by S-adenosyl-L-methionine(AdoMet), but the specific transmethylation reaction necessary for this function was not elucidated. In an attempt to define the methylation requirement for chemotaxis, we examined the effect of chemotactic agonists and antagonists on protein carboxy-O-methylation of protein and methylation of phospholipid in guinea pig macrophages. Chemotactic agents tested over a wide dose and time range produced no alteration in carboxy-O-methylation. However, these agents did produce an effect on the methylation of phosphatidylethanolamine by macrophages. AdoMet-mediated phospholipid methylation was inhibited by as much as 73% by chemotactic factors, and there was excellent correlation (r = 0.99) between their concentrations for producing half-maximal chemotactic responses and for inhibiting phospholipid methylation. The inhibition of methylation by chemotactic factors was observed at all incubation times and could not be explained by an increased turnover of membrane phospholipid. Neither the chemotaxis antagonist fPhe-Met nor the nonchemotactic tripeptide Met-Met-Met significantly depressed phospholipid methylation. Immune phagocytosis by macrophages similarly did not alter phospholipid methylation. The chemotactic factors produced no alteration in total macrophage phospholipid synthesis or in the phospholipid methylation in a nonchemotactic cell type. The formation of newly methylated derivatives of phosphatidylethanolamine in macrophages was decreased by a biologically active dose of chemotactic factor. These findings indicate that chemotactic factors are capable of altering the methylation of phosphatidylethanolamine in chemotactically responsive cells. The inhibition of phospholipid methylation by chemotactic factors may be necessary for the translation of a chemotactic signal on the surface of the cell into directional cell movement.


Cell surface antigens of human melanoma identified by monoclonal antibody. Mouse NS-1 myeloma cells were fused with spleen cells from mice that had been immunized with cells from a human melanoma, M1804. Hybrid cells were grown in selective medium and tested for production of antibody to surface antigens of M1804 cells. Three hybrids that produced antibodies that bound to the melanoma cells but not to autologous skin fibroblasts were cloned. Antibodies produced by two of the clones were cytotoxic to M1804 cells in the presence of rabbit complement. Extensive specificity tests showed that the antibodies produced by the clones bound strongly only to M1804 cells; significant, although weaker, binding occurred with 2 of 11 allogeneic melanomas. Apart from weak binding of the antibody produced by one of the clones to a breast carcinoma, binding assays of five carcinomas, one sarcoma, and fibroblasts from 17 individuals were negative, as were cytotoxic tests of 10 lymphoblastoid cell lines and peripheral blood lymphocytes from 68 normal donors and 12 chronic lymphocytic leukemia patients. This suggests that we have identified one or more determinants of a melanoma-associated antigen(s), whose expression is limited to a small proportion of melanomas.


Clonal evolution of myeloma cells leads to quantitative changes in immunoglobulin secretion and surface antigen expression. We report that a cloned population of tumor cells can rapidly produce variants that differ in their quantitative expression of surface proteins and in their rate of immunoglobulin secretion. A fresh clonal isolate of S107 myeloma cells possessing large amounts of surface IgA was continuously passaged in vitro for 2 years. During this period, fluorescence-activated cell sorter analysis indicated the development of subpopulations possessing decreased amounts of surface IgA. Cells from these variant subpopulations were isolated by first using the cell sorter to enrich for cells with decreased amounts of surface IgA and then cloning the selected population in soft agar. The 50 sublines that were isolated showed heritable differences in their levels of surface IgA and H-2 antigens and in their rates of myeloma protein secretion. Sublines having either large amounts, intermediate amounts, or absence of surface IgA also had corresponding large amounts, intermediate amounts, or absence of myeloma protein secretion. In contrast, a decrease or loss of surface Ig did not correlate with a decrease or loss of viral envelope glycoprotein gp71 and H-2 antigens. The variants did not resemble the phenotypes of less-differentiated normal lymphocyte populations of the B-cell lineage. The isolation and characterization of these variants allows us to explore the mechanisms and pathways of tumor cell differentiation as well as to study the regulation and function of cell surface proteins.


Idiotypic analysis of monoclonal antibodies to poly(Glu60Ala30Tyr10). Fifteen hybridoma anti-poly(Glu60Ala30Tyr10) (anti-GAT) antibodies were analyzed for the presence of a common set of idiotypic specificities associated with murine anti-GAT antibodies, termed CGAT idiotype, which are present on the anti-GAT antibodies of all mouse strains. Thirteen of these monoclonal anti-GAT antibodies expressed a major fraction of CGAT idiotypic specificities. However, the remaining fraction of CGAT idiotypic specificities were not detected in individual or pooled hybridoma anti-GAT antibodies. Anti-idiotypic antisera made against each of the 15 hybridoma anti-GAT antibodies preferentially bound homologous ligand and showed minimal binding activity to specifically purified serum anti-GAT antibodies. Furthermore, the diversity of the hybridoma anti-GAT antibodies was demonstrated by the presence of individual idiotypic specificities on each of the hybridoma anti-GAT antibodies. However, relatedness among some of the hybridoma antibodies was also apparent since idiotypic analysis revealed that some hybridoma anti-GAT antibodies shared cross-reactive idiotypic specificities not associated with CGAT idiotype. The genetic mechanisms which could account for the generation of such antibody diversity are discussed.


Structural analysis of human platelet membrane glycoprotein I complex. The glycoprotein I complex, consisting of two polypeptides of Mr 210,000 and 150,000, was isolated from human platelet membranes by wheat germ lectin affinity chromatography. Glycocalicin, a soluble loosely bound membrane glycoprotein of Mr 150,000 related to the glycoprotein I system, was also purified. The isolated polypeptides were radioiodinated in sodium dodecyl sulfate/polyacrylamide gels and digested with trypsin, and the labeled peptide digest was analyzed by two-dimensional high-voltage electrophoresis and thin-layer chromatography. The two polypeptides of Mr 210,000 and 150,000 in the glycoprotein I complex had essentially identical radioactive peptide maps. Glycocalicin had a completely different tryptic peptide map. These studies shed light on the molecular relationships of some of the components of the platelet membrane glycoprotein I system. The possibility is raised that the receptorlike function of the intrinsic platelet membrane glycoproteins may be related to the polymeric subunit associations of the constituent polypeptides.


Isolation and characterization of somatostatin from pigeon pancreas. Most of the somatostatin-like activity from pigeon pancreas was found to correspond to small species with an apparent molecular weight of 1500--2500. This species was isolated under conditions minimizing intermolecular interactions and protease activities. The isolated product was characterized by two somatostatin radioimmunoassays, a bioassay, endgroup determination, and amino acid analysis. The structure of the isolated compound was determined to be H-Ala-Gly-cyclo-(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys)-OH. Additionally, small amounts of des-Ala1-somatostatin, a possible degradation product of pancreatic somatostatin, and a large somatostatin-like species with an apparent molecular weight of 11,000--12,500 were detected. It is concluded that the main somatostatin-like polypeptide isolated from pigeon pancreas is identical to the mammalian hypothalamic tetradecapeptide somatostatin.


Morphine and endorphins modulate dopamine turnover in rat median eminence. The is evidence that some of the actions of both endogenous and exogenous opioids (e.g., stimulation of prolactin release) are mediated by interaction with catecholaminergic systems. Morphine (1.67, 5, and 15 mg/kg of body weight, intraperitoneally) altered dopamine turnover as measured by the alpha-methyl-p-tyrosine method in the median eminence, neostriatum, and frontal cortex of male Sprague-Dawley rats. The turnover rate of dopamine was reduced in the median eminence and frontal cortex but accelerated in the neostriatum. In the frontal cortex all doses were effective in decreasing dopamine turnover; however, in the median eminence the lowest dose of morphine did not significantly alter dopamine turnover. All three doses accelerated dopamine turnover in the neostriatum. Naloxone effectively reversed the effects of morphine at all doses in all brain areas, whereas it had no effect on turnover when given alone. In the median eminence, neostriatum, and frontal cortex, intraventricular injection of [D-Ala2,D-Leu5]-enkephalin (25 micrograms) or beta-endorphin (15 micrograms) produced the same effects on dopamine turnover as morphine. The actions of these peptides were blocked by naloxone. It is hypothesized that opiates and opioid peptides increase prolactin release by reducing the activity of the tuberoinfundibular dopaminergic system.


Iontophoresis of Li+ antagonizes noradrenergic synaptic inhibition of rat cerebellar Purkinje cells. Li salts provide effective therapy for manic-depressive psychosis, but the site and mechanism of this effect are not known. We have tested the ability of Li, applied by microiontophoresis, to modify the responsiveness of rat cerebellar Purkinje neurons to iontophoretic applications of norepinephrine and gamma-aminobutyrate and to the inhibition produced by stimulation of the noradrenergic ceruleo-cerebellar pathway. As previously reported for rat hippocampal neurons, acute exposure to Li produces selective, reversible antagonism of the effects of norepinephrine and the noradrenergic pathway but does not affect inhibitory actions of gamma-aminobutyrate. Collectively, these selective antagonisms of noradrenergic sympatic inhibitions in the cerebellum and hippocampus may indicate a general effect of Li suitable for extended observations in rats exposed to Li for the chronic periods needed to achieve therapeutic effects in man.


Immunoreactive and biologically active somatostatin-like material in rat retina. Somatostatin-like activity, as determined by radioimmunoassay and bioassay, is present in HCl extracts of rat retina. The concentrations of immunoreactive somatostatin are 612 +/- 43 (mean +/- SEM)pg per whole retina of 0.621 +/- 0.044 pg/microgram of protein in retinas from rats killed by decapitation, values which are not significantly different from those in retinas from rats killed by ether inhalation. The immunoreactive somatostatin was partially purified and concentrated by immunoaffinity chromatography. Both the crude retinal extracts and the immunoaffinity-purified immunoreactive somatostatin inhibited, in a dose-related manner, the release of rat growth hormone from dispersed rat anterior pituitary cells in culture. When the immunoaffinity-purified immunoreactive somotostatin was subjected to gel filtration chromatography, 96% of the recovered somatostatin eluted as a peak corresponding in position to that of synthetic somatostatin. Retinas from a group of rats with hereditary degeneration of the photoreceptor cells and another group of rats studied 1 year after transection of the optic nerves demonstrated an increased concentration of immunoreactive somatostatin compared to controls.


Dissociation of action potentials from contraction in single crab muscle fibers. In single crab fibers (Callinectes danae) bathed in Ca2+-free media, Ba2+ action potentials did not elicit tension. In contrast, Sr2+ spikes evoked twitches similar in amplitude to those accompanying the control Ca2+ spikes. Tension development in these fibers, therefore, depends on the ionic species carrying the inward current during membrane excitation. The Ca2" or Sr2+ influx appears insufficient to evoke the observed twitch tensions, and it seems necessary to postulate mobilization of an intracellular source of Ca1+. Procaine, which suppresses release of Ca2+ from sarcoplasmic reticulum, depressed twitch tension but did not reduce the overshoot or duration of Ca2+ or Sr2+ spikes. This finding is compatible with the suggestion that the contractions results from release of Ca2+ from the sarcoplasmic reticulum initiated by signals given by the influx of Ca2+ or Sr2+, but not Ba2+.


Middle ear muscles of the frog.

Middle ear muscles of the frog. The anuran middle ear in its complete form includes two skeletal elements, the columella and operculum, each occupying a portion of the oval window of the otic capsule and each provided with a middle ear muscle. The two elements have an interlocking arrangement of a form that makes it possible for these muscles to exercise a high degree of control of sound transmission from tympanic membrane to inner ear receptors. From the anatomical relations it is inferred that the two muscles operate as antagonists so that contraction of the opercular muscle and relaxation of the columellar muscle leave the columella free to move in and out of the oval window in response to sound vibrations, whereas a contraction of the columellar muscle and relaxation of the opercular muscle tend to immobilize the columella and reduce the transmission inward. The frog thus achieves a degree of control of sound reception that probably is unmatched among vertebrate ears. The purpose of the middle ear mechanism is no doubt the protection of the inner ear receptors (the amphibian and basilar papillae) from overstimulation by sounds, including the animal's own cries and the intense clamor produced by a group of frogs calling in chorus.


Receptoral and postreceptoral visual processes in recovery from chromatic adaptation. The time course of recovery from chromatic adaptation in human vision was tracked by determining the wavelength of light that appears uniquely yellow (neither red nor green) both before and after exposure to yellowish green and yellowish red adapting lights. Recovery is complete within 5 min after steady light exposure. After exposure to the alternating repeated sequence 10-sec light/10-sec dark, the initial magnitude of the aftereffect is reduced but recovery is retarded. The results are interpreted in terms of two processes located at different levels in the hierarchical organization of the visual system. One is a change in the balance of cone receptor sensitivities; the second is a shift in the equilibrium baseline between opposite-signed responses of the red/green channel at the opponent-process neural level. The baseline-shift mechanism is effective in the condition in which repeated input signals originating at the receptors are of sufficient strength to activate the system effectively. Hence, this process is revealed in the alternating adaptation condition when the receptors undergo partial recovery after each light exposure, but receptor adaptation during continued steady light exposure effectively protects the subsequent neural systems from continued strong activation.


[2 cases of priapism treated by cavernous body shunts]. The authors present their personal experience in connection with two cases to priapism, one of which was solved by unilateral saphenocavernous anastomosis in a leukemic patient, and the second one by bilateral caverno-spongious anastomosis in a patient with idiopathic priapism. Both interventions had a good outcome. The emergency character is stressed, of this type of intervention, that should b performed bilaterally and followed by anticoagulant treatment.


[Malformations of the spino-cervical joint and of the cervical spine associated with Binder's syndrome (author's transl)]. The authors found in 53.5% of the cases of Binder's Syndrome, where a radiological work up had been carried out on the cervical spine and the spino-cervical joint, malformations of varying importance in these related structures. This lesional association had not as yet been pointed out. After pathological groupings and an analytical study of the anomalies encountered, the question of a common inducer factor is raised, or at least a factor contemporary to the two dysplasias, maxillo-nasal and vertebral. In practical terms, it would seem vital that all individuals with Binder's syndrome have a radiological examination of the cervical spine and the spino-cervical joint. In those subjects presenting with malformations and in particular, complex corporeal dysplasias, the authors strongly insist on the necessity of regular follow up in order to make an early diagnosis of eventual neurological repercussion.


[Diagnostic and therapeutic problems posed by a congenital lateral fistula of the neck (author's transl)]. Cysts and congenital lateral cervical fistulas are related to the persistance of the normally transitory branchial pouches and their embryologico-anatomic arrangement. Due to this fact they have a preferential topography. An atypical fistula of the second branchial cleft is reported. It is a congenital supra-hyoidian lateral cervical fistula with an intra-parotid route which terminates in the tonsils. The extreme variability of the external orifice, the pathogenic nature of the internal orifice and of the fistular route, as well as the initial discovery of the facial nerve and tonsillectomy in lateral fistulas of the neck are, by the same occasion, brought to mind.


[Factors favouring necrosis after curietherapy for oral cavity epitheliomas (author's transl)]. The authors studied the factors which favour necrosis after curietherapy, in a series of 82 patients with epithelioma of the oral cavity and a previous series of 280 cases. The principal factor is the size of the tumor. The other factors (macroscopic appearance, dose, quantity of radioactive material, degree of inhomogeneity, distance between the lines, association with external irradiation), are or only moderate importance, as shown by the variations observed, though they are difficult to evaluate in an exact manner, partly for methodological reasons. The frequency of necrosis of bone tissue is directly relaxed to the number of radioactive lines in contact with the maxilla. Alcohol and tabacco abuse have a definite influence but this cannot be calculated. In conclusion, therefore, it would appear possible to reduce the frequency of necrotic lesions by reducing the tumoral size by curietherapy, by applying no more than 2 lines in contact with the mandible for bony necrotic lesions, and by stopping the abuse of alcohol and tobacco.


[Dental complications of irradiation : fluoride prophylaxis (author's transl)]. The authors review the studies describing dental changes after radiotherapy or curietherapy of the cervicofacial region published over the last 50 years. They discuss more particularly: --direct and indirect effects on the teeth,--lesions of the salivary glands,--dental lesions,--fluoride prophylaxis for dental lesions after irradiation.


[Micrographic aspect of mandibular osteoradionecrosis (author's transl)]. Fragments of the lower jaw from 42 patients with carcinoma of the oral cavity or oropharynx, and treated primarely with fraction radiotherapy, were excised for radiation osteonecrosis 6 month to 10 years after irradiations. The excised specimens are X-rayed before being coated with methylmetacrylate. They are then cut into thick sections along lines detailled on the X-ray films. The sections are trimmed to 80 microns and examined microradiographically. Two observations are sufficient to demonstrate destruction or reorganisation of the bone tissue. Reabsorption of unusual appearance is seen around the Haversian canals. The newly formed tissue resembles either embryonic bone matter, or lamellar tissue as seen in osteomyelitis.


[Recent advances in the therapy of osteoradionecrosis of the maxillomandibular bones (author's transl)]. In spite of the considerable technical progress made over the last few years, no type of irradiation used at the present time for treating malignant tumors in the maxillofacial region can protect the patient from the always dreaded appearance of a secondary osteoradionecrosis of the maxillomandibular bones. Extablished osteoradionecrosis is not a "true" necrosis of these bones but a particularly severe osteitis which evolves in bone, which is pathological because it has been irradiated, and is surrounded by tissues which are also pathological for the same reason. Very high doses of antibiotics, comparable to those used in the treatment of osteomyelitis, mandibular resection by the endobuccal route, and the possibility of treating perimaxillomandibular tissues by reconstructive surgery, have completely transformed the prognosis of this dreaded affection, which is being increasingly prevented by daily fluoride applications applied locally.


[Twenty-five cases of bone resection for mandibular osteoradionecrosis (author's transl)]. Good morphological and functional results are obtained in lateral osteoradionecrosis after bone resection by the endobuccal route, and this operation should be performed at an earlier stage. Median resections give poor results and in these cases it is reasonable to temporize in the normal manner. Long-term prognosis is only moderate as tumoral recurrences are frequent in these patients.


[Dystrophy and necrosis following radiotherapy for maxillary cancer (author's transl)]. About 10% of patients developed severe sequelae following radiotherapy, which had been associated with surgical treatment, even though they were, or appeared to be cured. Complications affected the skin and soft tissues (causing skin retraction, loss of substance, and sometimes trismus) and bone (necrosis required surgical treatment, and when there was associated cutaneous dystrophy, resulted in exposure of the bone and severe functional and esthetic problems). A critical study of the radiotherapeutic techniques used suggests that the dose given should be reduced, the fields modified, and Cobalt used in preference to electrons in most cases.


[Maxillodental problems after irradiation in children (author's transl)]. The treatment of a certain number of children with very severe dentomaxillary sequelae after radiotherapy for facial and intracranial lesions raises many problems. These include the type of reconstructive therapy, the protection of the maxillodental structures, the value of local and alimentary hygiene, and the quantity and quality of dental treatment needed. Further difficulties are encountered because of dental malformations (absence of roots), increased rate of eruption of the teeth in the arch (contrary to the accepted opinions), and objectives should be modest when attempting maxillofacial orthopedic procedures.


[Disturbances in development of the maxilla following interstitial radium therapy for a facial rhabdomyosarcoma at the age of 3 years (author's transl)]. Radium therapy at the age of 3 years for an embryonic rhabdomyosarcoma of the paranasal fold resulted in severe cutaneous lesions but also relatively limited disturbances of growth of the corresponding maxilla: inclusion of the underlying canine and version of neighbouring teeth, very incomplete development of the nearest dental roots, absence of air filling of the maxillary sinus. The relatively slight nature of these developmental disturbances is certainly related to the techniques of interstitial radium therapy developed and used at the Gustave Roussy Institute. Routine orthodontic study of the cases of children surviving after treatment of a malignant tumor of the face would undoubtedly offer even more precise information concerning therapeutic indications.


[Maxillofacial radiation-induced cancers (author's transl)]. Radiation-induced cancers are fortunately a very rare complication of radiotherapy for cancers. Sagerman, however, reported 21 cases in a series of 243 patients with retinoblastomas, examined after a period of at least 5 years, and 6 cases were observed in a similar series of 112 children seen at the Curie Institute. This report describes 10 cases of irradiation-induced cancers of the maxillofacial region seen at the Curie Institute over a period of 30 years. The exceptional nature of this risk is such that it cannot represent a contra-indication to irradiation therapy of a malignant tumor, especially now that low-energy radiation has been abandoned, and with the modern therapeutic procedures available. The risks of cancer are greater, however, after irradiation of benign bone lesions and benign affections of the cervico-facial region. Treatment of these benign affections in children or young adults by low or reduced doses of conventional low-energy radiations should be forbidden, and the indication for treatment with radiations, which must be of high voltage, should be exceptional.


In vitro susceptibility testing of anaerobic bacteria. In vitro susceptibility testing of anaerobic bacteria should be limited to isolates from persistent or recurrent infections that have been treated adequately and appropriately with antimicrobial agents and, in reference centers, to collections of isolates in order to monitor alterations in susceptibility of species to various antimicrobial agents. An agar dilution reference method is being evaluated currently; however, practicality limits sporadic testing of single isolates to disk elution or broth dilution techniques. No single disk diffusion method has yet been found to be acceptable for testing anaerobic bacteria, and the results obtained with standardized procedures for aerobic and facultatively anaerobic bacteria are not applicable to anaerobic bacteria.


Experimental Bacteroides fragilis abscess in the rat liver. Liver abscess was induced in rats by injecting a culture of B. fragilis ss. fragilis (approx. 5X10(6) cells) into devascularized liver tissue. Mixed infection with B. fragilis and aerobes occurred in all animals subjected to repeated laparotomies for bacteriological sampling (4, 7, 14 and 28 days after inoculation). In animals sampled only at 8 or at 42 days after injection, B. fragilis was isolated in pure culture from the majority of animals. The size of abscesses containing B. fragilis in pure culture decreased markedly between 8 and 42 days after inoculation. In inoculated animals and in sham-operated animals with secondary infection histological examination revealed an abscess containing necrotic liver tissue and numerous granulocytes. In sham-operated animals with no bacterial growth the histological examination microscopy revealed a liver infarct with incipient organization and no accumulation of granulocytes.


The humoral antibody response to Bacteroides fragilis infections in humans. The humoral antibody response to Bacteroides fragilis infections in humans, with particular reference to ss. fragilis, was studied using an enzyme immunosorbent assay (EIA). Phenol-water extracted polysaccharide fractions (PS) from B. fragilis ss. fragilis, ss. ovatus, ss. distasonis and ss. vulgatus were used as antigens. Antibody titer determinations were done on sera collected from 57 patients where B. fragilis had been cultured and from 50 controls. In patients with septicemia caused by B. fragilis ss. fragilis a significant titer increase (greater than or equal to doubling) against PS from strain 9343 was seen during the course of the illness. In sera from patients with appendicitis, and where B. fragilis was isolated from the appendix, a titer increase against B. fragilis ss. fragilis 9343 was observed for 6 of 17 patients. However, in sera from 9 of the 17 patients a titer increase against B. fragilis ss. ovatus was also seen. No titer increase was observed in sera from most of the patients with salpingitis where B. fragilis had been isolated. In sera from 10 patients with Crohn's disease the median antibody titer against all four B. fragilis antigens was slightly higher than in sera from the controls. The median antibody titer against B. fragilis ss. ovatus was, however, significantly higher. Our experience is that a humoral antibody response against B. fragilis ss. fragilis can be expected in patients where the organism is isolated from blood. In diseases like appendicitis and salpingitis titer increases are less common. When they occur they are not necessarily directed against ss. fragilis only but are as frequent against ss. ovatus. Titer increases against ss. vulgatus and ss. distasonis are also seen. This raises the question if the observed titer increases are a consequence of a specific pathogenic role of B. fragilis in these infections, or if they merely represent the result of an antigenic stimulus as a result of an increased permeability of the mucosal barriers caused by inflammation and/or surgical manipulation.


Anaerobic bacteria in maxillary sinusitis. The anaerobic bacterial flora of purulent maxillary sinusitis was investigated in 30 patients and the different anaerobic species identified and tested with respect to antibiotic susceptibility. Most strains were identified as anaerobic cocci. Non-sporeforming Gram-positive rods and Gram-negative rods other than Bacteroides fragilis were also isolated. The majority of the strains were susceptible to benzylpenicillin, cefoxitin, clindamycin, doxycycline, metronidazole and chloramphenicol.


Bronchial carcinoma. IV. A methodological evaluation of the vitagram index for measurement of quality of survival. The validity of the Carlens Vitagram Index was tested by using the result of therapy in 48 patients with bronchial carcinoma in advanced disease, 54 patients with localized disease and 34 deceased treated surgically. The Carlens Vitagram Index, which is additional and composed of six items, was correlated to a set of additive indexes including the same items but other item weights, which were chosen with non-controversial pre-specified restrictions. The lowest correlation achieved was an indicator of the lowest degree of consistency that could be attributed to the Carlens Index given. The definition of the index was operationalized so far that each item in the set of six items included in the index was regarded as valid, and it was also considered reasonable that only these six items should be included; that each item had a weight; that the index was obtained as a product (weight x item value) sum and that the pre-specified conditions were accepted. By using restrictive conditions the lowest correlation between the index giving the lowest correlation and the Carlens Index for the 48 patients with localized disease was 0.98, for the 54 patients with localized disease 0.91 and for the surgically treated ones 0.83. In addition to the total index, the average index per month was also studied and the corresponding correlations were 0.96, 0.86 and 0.81 respectively. The Carlens Index and its application per month was found to have good validity for inoperable patients and acceptable validity for surgically treated patients. The Carlens Index was shown to be robust and there is reason to believe that it lies close to the optimal index.


Bronchial carcinoma. V. A methodological evaluation of the vitagram index for measurements of quality of survival by means of the "MaxMin" correlation method. The validity of the Carlens Vitagram Index for measuring the quality of survival in bronchial carcinoma was further tested on the basis of the result of therapy in 48 patients with advanced disease, 54 patients with localized disease and 34 deceased patients surgically. Different approaches for index construction are discussed and the "MaxMin" correlation method, in which the highest of the lowest correlations is sought, is presented. The principal algorithms of the method are given, and the mathematical proof and the procedures are described and exemplified. By finding the "MaxMin" correlation a new index was constructed with weights of 20, 12.36, 12.36, 8.18, -6.15 and 0 points. The corresponding weights of the Carlens index are 20, 16, 12, 8, -4 and -2 points. Thus the Carlens index lies very close to the optimal index found with the "MaxMin" method. This result gives further proof that the Carlens index is valid for use in the types of bronchial carcinoma described above.


Bronchial carcinoma. VI. Some aspects of the therapy of inoperable patients. In small cell anaplastic carcinoma with localized disease the median survival for radiotherapy, single drug chemotherapy or operation was 10.1 months. The median survival for radiotherapy or single drug chemotherapy for small cell anaplastic carcinoma with extensive disease was 2.8 months and for untreated controls 0.5 months. The difference in the quality of survival was moderate, 2.0 vitagram points per month. Untreated epidermoid carcinoma patients with localized disease had a median survival of 11.4 months, which is the level reached by most non-surgical therapeutic regimes today.


Bronchial carcinoma. I. A prospective five year study on an unselected carcinoma population in a Swedish county. No prospective clinical investigations of unselected total materials of bronchial carcinoma have been reported. The clinical profile of 273 patients with histologically verified bronchial carcinoma from a Swedish county during 1971-1976 is presented. The difficulties in making valid comparisons with other literature reports makes it necessary to describe in detail the total material. Such a description is given in the present paper. In order to avoid different selection mechanisms an attempt was made to collect an almost total material. No patients were lost in the follow-up. Autopsy was performed in 95% of the deceased patients. The distribution of the histological WHO groups in the total material was: 44% group I, 18% group II, 29% group III and 9% group IV. The material was used for randomized investigations of radiotherapy, chemotherapy and placebo treatment in inoperable patients with the purpose of studying the quality of survival in addition to survival time. For the measurement of the quality of survival the Carlens vitagram index was used (Carlens et al. 1970). The influence of a general health survey with mass miniature chest X-ray was also studied, since such a survey was performed in the region of the study. The expected 5-year survival is estimated to be 7%. Only 20 patients with resectional therapy without known signs of metastases are expected to survive. Nine (45%) of these patients were discovered by mass miniature chest X-ray and thus for a limited number of patients the detection by the general health survey gives a better prognosis.


Bronchial carcinoma. II. Quantitative measurements of the quality of survival. A prospective randomized study of the result of therapy in inoperable patients with advanced disease. Forty-eight bronchial carcinoma patients in clinicoanatomical stage 4 of the disease (advanced disease) were randomly assigned to groups for radiotherapy, chemotherapy (cyclophosphamide) and placebo treatment, respectively. The results were assessed by the survival time and quality of survival. The median survival time was 4.7 months for radiotherapy, 4.7 months for cyclophosphamide and 1.7 months for placebo. The median total sum of vitagram points was 28.1 for radiotherapy, 20.7 for cyclophosphamide and 6.8 for placebo. When calculated per month, the median sum of vitagram points was 5.9 for radiotherapy, 5.7 for cyclophosphamide and 4.8 for placebo. Statistically the results give no reason to believe that placebo is better than radiotherapy, but it cannot be excluded that radiotherapy patients could have a much longer survival. As for cyclophosphamide versus radiotherapy, the differences are to uncertain for any conclusion to be drawn.


Serologic analysis of human tumor antigens. II. Reactivity of sera from eleven osetogenic sarcoma patients against autologous and allogeneic tumor cells. Sera from eleven patients bearing osteogenic sarcomas were tested for reactivity against autologous and allogeneic normal and tumor cells using a sensitive microcytotoxicity assay. Following absorption with human fetal tissue cultured lines, sera from all 11 patients lost reactivity to autologous and allogeneic normal fibroblasts in culture. However, sera from four patients retained reactivity against both autologous and allogeneic osteogenic sarcoma, sera from five patients retained reactivity to allogeneic but not autologous tumor, and sera from two patients lost all reactivity to tumor. Sera from nine normal individuals did not react with either normal or osteogenic sarcoma cells after fetal cell absorption. Thus it appears that nine of 11 patients bearing osteogenic sarcoma contained antibody to a common tumor-associated antigen. This tumor-associated antigen appears to be expressed on tumor cells and not normal cultured cells, and reactivity to it is present in tumor-bearing but not normal individuals.


Reaction of the human dental pulp to silver amalgam restorations. The effect of enclosing amalgam of initially high plasticity in intermediate depth or deep cavities. From earlier studies using amalgam of known physical and sealing properties it was concluded that the initially high mercury content was the main reason for pulpal changes and that clinical leakage had been less important. The purpose of the present investigation was to study how a possible sealing insufficiency may contribute to the pulpal changes beneath restorations of silver amalgam. The influence of bacteria at the tooth/filling interface upon pulpal changes was also studied. The material consisted of 21 contralateral pairs of premolars. Intermediate depth cavities were prepared in 13 pairs and deep cavities in 8 pairs. All cavities were filled with silver amalgam by the wet technique. The restoration in one tooth of each pair was sealed off by glued metal foil protected by a cemented orthodontic band. After 1 week the teeth were extracted and examined histologically. In the pulp of teeth with restorations exposed to the oral environment, the frequency of dilated capillaries and inflammatory cells in the odontoblast - cell-rich zone boundary, associated with the dentinal tubules involved in the cavity preparation, was insignificantly higher statistically than in those with seal-off restorations. A few scattered bacteria were found on the cavity wall in some cases. It was concluded that, in the short run, the pulp reaction was influenced by sealing insufficiency, which may develop during the experimental period as a result of external factors such as tooth deformation and/or changes in temperature. No influence from bacteria on the cavity wall could be established.