Tissue damage from electrosurgical power output variations in hamster tongues.

An investigation was designed to determine the differences in the amount of tissue damage produced by variations in the amount of cutting power delivered to the hamster tongue with electrosurgery. By dividing the power of an electrosurgical instrument into 5 W intervals, from the lowest setting which would produce a cut up to the 50 W setting, histometric comparisons were made in the hamster tongue. Histologic analysis showed the following: 1. The 30 W power interval produced less tissue damage than any other power interval. 2. The 25 and 35 W intervals produced significantly greater damage than the intervals from 40 thorugh 50 W. 3. Histologically the experimental defect showed an area of coagulation necrosis approximately 50 mu wide along the margin of the incision. Tissue damage decreased laterally and inferiorly from the surface of the defect except at the 30 W setting, where the damage in the epithelium and the damage in the connective tissue were essentially equal. 4. There is some validity to the sparking-dragging test for setting the power interval of an electrosurgical instrument. 5. Higher power values which produce excessive sparking should be avoided by the clinician during electrosurgery due to the imprecise nature of the clinical incision.


Geriatric nutrition II: Dehydration in the elderly.

Water balance in the elderly is critical in preventing tissue dehydration. Negative water balance, occurring when more water is lost than is retained, results in a severe decrease in all secretions. This "drying out" of tissues and organs, including muscles and joints, may lead to premature aging. Xerostomia is a major cause of denture discomfort and failure. The prosthodontist is advised to inspect and, if possible, correct dehydration of the oral tissues prior to constructing the denture. Denture failures may often result more from tissue deficiencies in the aged than from technical inadequacies. Water balance is efficiently achieved in the elderly through ingestion of soups containing many of the solubilized nutrients essential to tissue health. This is more beneficial than merely increasing the intake of plain water or beverages.


The effect of anteroposterior inclination of the occlusal plane on biting force.

With the experimental design of this study the following conclusions were reached. 1. Biting force during maximum clenching was the greatest when the occlusal plane was made parallel to the ala-tragus line. It decreased when the occlusal plane was inclined about 5 degrees anteriorly or about 5 degrees posteriorly. 2. The efficiency of biting force exertion during maximum clenching showed the best value when the occlusal plane was made parallel to the ala-tragus line. 3. Muscle activity during clenching at various given forces was least when the occlusal plane was made parallel to the ala-tragus line. The anteroposterior inclination of the occlusal plane tends to affect the biting force, and the method with the ala-tragus line seems to be the most reasonable for occlusal plane orientation.


A spectrofluorometric protein assay of plaque on dentures and of denture cleaning efficacy.

A spectrofluorometric method of protein analysis using fluorescamine was shown to be a meaningful method of evaluating denture cleanser efficacy. This assay was useful in the presence of most currently used denture cleanser ingredients, materials which cause other protein assay methods to fail. The determination of dental plaque protein was shown to be an adequate measure of dental plaque dry weight.


A quantitative and subjective characterization of tarnishing in low-gold alloys.

1. The quantitative objective characterization of tarnish gave results which are identical with those obtained from a blind subjective analysis. 2. The low-gold alloys fall into three classes. The alloy Ney Cast III is superior in performance to the other low golds and approximately equivalent to the high-gold alloy Firmilay. The intermediate alloys, including Midacast, Stabilor G, Mowrey No. 46, Tiffany, and Midas are similar in performance to the high-gold alloy Rx O.R.Y. The low-gold alloys Dent Cast 44, Minigold, and Progold tarnish in a manner which, in our opinion, makes them unacceptable.


Myofacial pain dysfunction: a manifestation of the short-face syndrome.

The practitioner who treats patients with MPD should be aware that certain anatomic variations may predispose a patient to developing the syndrome. We feel that the newly described entity, the short-face syndrome, is such an example. Patients exhibiting short-face syndrome are overclosed. Their condition appears to be improved by establishing an increased vertical dimension of occlusion. This is first accomplished with the use of a processed acrylic resin occlusal splint. If a patient has been asymptomatic at the new occlusal relationship, a maxillary osteotomy is recommended to achieve a more permanent functional and cosmetic result.


Immediate dental root implants from synthetic dense calcium hydroxylapatite.

In this article, root implants implanted directly after extraction of the natural teeth were described. The root implants were made of densely sintered or hot-pressed calcium hydroxylapatite (CHA). The significance of using such implants as a method of preserving the bulk of the alveolar ridge and as possible anchoring sites for a post and core for fixed partial dentures was discussed.


A method to measure operating variables in electrosurgery.

Variables affecting tissue response to electrosurgical wounding have been identified and discussed. An electronic measuring device was designed and built to record actual power generated at the active electrode during surgical use of an electrosurgery instrument. In addition, this electronic device measures the amount of time the active electrode is in contact with tissue. Coupled with an electrosurgery unit producing a documented waveform and an electrosurgery electrode having a controlled-depth gauge, variables affecting tissue response to electrosurgery wounding may be measured.


Stabilized record bases for complete dentures.

A step-by-step technique has been described to construct stabilized record bases which allows the dentist to take advantage of undercuts through the use of a resilient denture liner material in the undercut regions. The resilient denture liner material protects the rugae and prevents damage to the casts in the undercut regions during repeated placing and removal from the cast. Shellac denture base material at the posterior limit of the record base permits an easy method of checking the posterior palatal seal. The incorporation of the posterior palatal seal in the record base allows for adequate retention during the recording of the maxillomandibular jaw relation records and try-in procedures. The record bases made by the sprinkle-on technique are preferred to those made using the dough method. The bases are rigid, stable, not easily broken or warped, and fit accurately.


Occlusal splint fabrication.

There are many methods of fabricating an occlusal splint. The advantages of the technique described are as follows: (1) little intraoral adjustment is required, (2) there is ease in construction, and (3) only one set of diagnostic casts can serve for both occlusal analysis and splint construction. The disadvantage is that an occlusion with group function cannot be produced by this method.


Undesirable effects of gold salts.

A review of recent studies of undesirable effects of gold salts is presented. Marrow aplasia is potentially the most serious side effect with a poor prognosis. Its prevalence is undetermined but may have been overestimated. Gold nephropathy and thrombocytopenia although more common usually have a good prognosis. Both can be satisfactorily treated without prolonged morbidity or mortality. Mucocutaneous reactions although more common are rarely of serious importance and need not necessarily prevent completion of a therapeutic course of gold salts. When indicated the side effects of gold salts can be adequately treated by cessation of therapy and use of corticosteroids. The value of chelating agents is desirable.


The technique of electron probe x-ray analysis and the atomic composition of aurosomes.

Electron probe x-ray analysis is an exciting new method of elemental analysis at the ultrastructural level. This is a relatively non-destructive form of analysis by which elements from Na onwards (i.e. above Z number 10) can be simultaneously detected and displayed as a spectrum. The best spatial resolution that can be achieved is of the order of 20 nm. The sensitivity of detection varies with the elements but about 10-18 g of an element can be detected. With the available computer programs elemental ratios are readily determined without the need of standards. The special appeal of this technique stems from the fact that it is the only way of analyzing minute inclusions in situ within the cell and so correlate morphology with atomic composition. With this method, gold has been demonstrated in aurosomes produced in various sites after the administration of soluble gold salts to experimental animals and man. A more detailed analysis of aurosomes found in the synovial membrane 3 days and 18 months after injection of gold sodium thiomalate into the rabbit knee joint revealed that the aurosome contains gold, phosphorus, and sulphur, and that the atomic ratios of these elements do not alter over the above mentioned time interval.


Trace element determination in serum by proton-induced x-ray emission.

We report the use of proton-induced x-ray emission (PIXE) as an analytical method in clinical research. The experimental set-up and target preparation procedures are briefly discussed together with the methods of automatic data acquisition and analysis. Results from a clinical project involving rheumatoid patients receiving chrysotherapy are presented.


X;15 translocation in a retarded girl: X inactivation pattern and attempt to localise the hexosaminidase A and other loci.

Cytogenetic studies on a retarded girl showed a complex S;15 translocation, karyotype 45,X,-15,+t(X15). The translocation X chromosome was non-randomly partially inactivated, the inactivation being mainly confined to the X segment and in some cells only to the X long arm. Gene marker studies failed to show anomalous segregation of the hexosaminidase A gene or any other gene markers tested.


Isolation of a recombination deficient Agrobacterium tumefaciens mutant.

The isolation of a recombination deficient (Rec-) strain of Agrobacterium tumefaciens is described. Strain LBA 4011 was mutagenized with nitrosoguanidine and after segregation 18,000 colonies were replica plated and UV irradiated. Twentytwo UV sensitive strains were isolated and tested for methylmethanesulphonate (MMS) sensitivity. Six of these strains were more MMS-sensitive than LBA 4011. A Ti plasmid that was genetically marked with Tn 1 (CbR) was introduced in these strains and the rescue of the CbR marker during superinfection with an incompatible cointegrate plasmid Ti::R 702 was determined. One strain exhibited a large reduction in rescue frequency. It is concluded that the latter strain was recombination deficient. This property did not influence the induction of plant tumours.


Rough and fine linkage mapping of the Rhizobium meliloti chromosome.

A circular linkage map of the Rhizobium meliloti chromosome, obtained from R68.45-mediated crosses, has been revised by cotransductional analysis, after general transduction by DF2 phage. Three short chromosomal regions have been mapped by cotransduction. Comparison between conjugal and cotransductional data suggests that R68.45-mediated linkage measures are indeed rough. Cotransduction seems to be a useful tool for improving the linkage map of R. meliloti.


Kinetics of hybridization to human DNA of heterogeneous nuclear RNA isolated from normal human lymphoblasts and acute leukemia blast cells.

Heterogeneous nuclear RNA was extracted from normal PHA-stimulated human lymphocytes and acute myeloid leukemia blast cells. Experiments were performed to determine the hybridization kinetics of these RNA's to human DNA. The best least squares solutions indicate in the hybridization reaction of both normal and leukemic RNA two main components. For leukemic cell RNA the rate constants of both components were significantly different from that of normal cell RNA. In particular, the difference between the rate constants of the second lower component suggests that the slowly hybridizing sequences in leukemic cell RNA have a degree of repetition higher than of the corresponding sequences of normal cell RNA.


[A contribution to hereditary sensory neuropathy. Electron microscopical studies (author's transl)].

A female infant nine months of age, hospitalized several times because of feeding and thriving problems as well as statomotoric retardation, at the occasion of a combustion showed no sign of perceiving pain. Based on clinical symptoms, histological and electron microscopical examinations the disease could be classified as hereditary sensory neuropathy type III.


Primary hyperparathyroidism in a New Zealand community: a review of 40 cases.

Forty cases of primary hyperparathyroidism presenting over a 15 year period, have been reviewed. The disease was equally prevalent in both sexes with highest incidence in the sixth decade. Most patients presented with renal colic or calculi (73 percent) and skeletal disease was found in only 13 percent. A single parathyroid adenoma was found in 32 cases and hyperplasia was diagnosed in only two cases. Follow-up questionnaires were sent to 32 patients, and in 27 of these a full clinical and laboratory assessment was undertaken. Of 26 patients with renal colic preoperatively only six continued to experience colic one year after parathyroid surgery. Thirty percent of all patients were hypertensive preoperatively, and in only two patients did blood pressure normalise after surgery. Fourteen of 27 patients followed-up were found to be hypertensive. A highly significant fall was noted in serum calcium, chloride, alkaline phosphatase and urine calcium excretion postoperatively. Recurrence of the disease was low and less than 8 percent in this series. The low incidence (1 per 10 000 population per year) suggests that primary hyperparathyroidism is under diagnosed in the Christchurch community.


Blood selenium values in an adult Auckland population group.

New Zealand top soils are low in selenium with some regional variation. Grain and cereal products are the main source of dietary selenium intake but in New Zealand their content is low due to widespread top soil deficiency. Whole blood levels reported from Oago, 68 +/- 13ng/ml, are a half to one-third of those in North America and also lower than in Auckland residents, 79 +/- 15.7ng/ml. The variation between New Zealanders is due to regional differences in top soil levels and grass content affecting secondary selenium sources such as sheep meat and beef. Regular consumption of selenium rich chicken and fish is associated with a significant increase above average values, but age, sex, smoking, alcohol and the use of oral contraceptives, are unimportant factors.


Age, adiposity, blood pressure and blood lipids in a rural New Zealand population.

Multiple regression analysis of data on age, blood pressure, adiposity and blood lipids from a rural New Zealand population of over 1200 adults has been undertaken. The results show that rises in blood lipids over time in the population are independent of age and correlate significantly with adiposity. Thus plasma cholesterol and triglycerides correlate with adiposity (expressed as Quetelet's index or skinfold thickness) in men whereas significant correlation in women was only between adiposity and plasma triglycerides. Systolic and diastolic blood pressure in men was strongly correlated with their plasma triglycerides, but not cholesterol, when the effect of age and adiposity was removed. In women however only a weak correlation was observed between plasma triglycerides and systolic blood pressure. The significance of the findings is discussed.


Tokelau Islands Children's Study: scabies infestation in children.

Tokelau children have been examined for scabies infestation in three groups--those living in their home atolls--those living in New Zealand in 1972-73, and those living in New Zealand in 1975. There was less infestation in Tokelau than in New Zealand. In 1975 the rate of infestation in New Zealand had increased since 1972. The manifestation of the mite in children in New Zealand varied from the classical descriptions, in that the mite itself, and its burrows, were seldom found, and the skin lesions were more often seen on the body than on the wrists and hands. A treatment regime which was found satisfactory is described. It was found essential to treat the entire family at the same time.


Eye injuries: a twelve-month survey.

Patients who were admitted to Auckland Hospital with an eye injury during 1975 have been considered. The cause of injury, type of injury and visual outcome have been recorded. At least 60 percent of the eye injuries occurred during leisure activities. In this study more patients were admitted with an hyphaema following sports injuries and fewer admitted after motor mower injuries than in an earlier study. Some preventative measures in eye injury are discussed.


Fetal respiratory movements observed by ultrasound during abnormal pregnancies.

Observations of fetal respiratory movements were carried out during 35 pregnancies with complications likely to indicate increased fetal risk. Fetal breathing was demonstrated in 25 of 35 patients studied, and in 29 of 47 separate examinations. Of 17 patients delivering after 36 weeks gestation in whom observations were made with two weeks of delivery, three did not demonstrate fetal breathing. Infants born from these pregnancies were severely growth retarded in two instances and light for dates in the other. Although absent fetal breathing is likely to detect increased fetal hazard near term, the time and expense needed for accurate recording of fetal breathing will restrict application of the technique in the meantime.


Sequential complement changes after childhood acute nephritis.

A group of 29 children with acute nephritis have been studied in relation to complement changes during and after their illness. Eighty-two percent had evidence of a streptococcal aetiology. C3 levels were initially depressed in 96.5 percent, but by 12 months all children were normal clinically and had normal complement status. The role of complement is discussed in relation to acute nephritis.


Photochemotherapy of psoriasis (PUVA).

Photochemotherapy for psoriasis using ordinary fluorescent blacklight combined with orally administered methoxalen was used in the treatment of 20 patients suffering from long standing psoriasis. Ten of the patients had complete clearing of their lesions, two had 75 percent or more clearance, one patient had 50 percent improvement and there was only slight improvement in two further cases. Side effects were minimal.


Contraceptive practice among New Zealand women.

In May 1976 the contraceptive practices of New Zealand women were surveyed among a national random probability sample of 1200 women aged 15 years and over. The survey found 93 percent of women aged 15-44 years considered themselves at risk of pregnancy. Of all women surveyed, almost half (two-thirds of those at risk) currently practised contraception. The highest incidence was among women aged 25-44 years. Single women at risk contracepted less frequently than did married women. The most frequently used method was the contraceptive pill, favoured particularly by younger women. It was followed by sterilisation of either partner, which was most frequent among women aged 25-44 years.


The investigation of colorectal disorders.

The investigation of colorectal disorders follows from a thorough clinical examination, examination of stools, and proctosigmoidoscopy. In many patients, barium enema examination will also be required, and the value of good air contrast examinations is stressed. Colonoscopy has revolutionised the diagnostic and therapeutic capabilities in colonic disease, but this examination is not readily available to all, its indications limited, and the risks and discomfort associated with it by no means small. In New Zealand, colorectal cancer is as common as anywhere else in the world, and the major hope of any improvement in this area is with preclinical diagnosis. This could be achieved by more widespread faecal occult blood testing especially in high risk groups (Table 2) and thorough investigation of those with positive results. Serological tests for colorectal cancer (carcino-embryonic antigen) have proved disappointing, and much less specific than originally hoped.


Racial and environmental factors in acute haematogenous osteomyelitis in New Zealand.

Data on the hospital incidence of acute haematogenous osteomyelitis in New Zealand children between 1965 and 1973 are presented. The incidence in Maori children is four times that in European children. The condition is more common in the North Island than the South Island, but this difference is mainly due to the distribution of the Maori people. Admissions to hospital are common in late summer and autumn. The relationship of osteomyelitis to skin and ear sepsis and some possible reasons for ethnic difference, are discussed.


Coconut water as a rehydration fluid.

The recent epidemic of cholera on the Pacific Ocean atoll of Tarawa, Gilbert Islands renewed interest in the use of coconut water as a rehydration fluid. Fifty-one samples of coconut water from Tarawa were analysed for a variety of constituents to assess its potential usefulness in the oral and parenteral rehydration of patients with cholera and other severe forms of gastroenteritis. Compared to oral rehydration fluids known to be effective in cholera, coconut water was found to have adequate potassium and glucose content, however was relatively deficient in sodium, chloride and bicarbonate. The addition of table salt to the coconut water is suggested to compensate for the sodium and chloride deficiency. In areas of the world where coconuts are plentiful, the advantages of sterility, availability and acceptability make coconut water theoretically feasible for the oral rehydration of patients with severe gastroenteritis when conventional fluids are unavailable.


A screening programme for congenital hypothyroidism: preliminary results.

Highly sensitive and precise radioimmunoassays for thyroxine (T4) and thyrotropin (TSH) in dried blood spots on filter paper cards have been developed and are used to screen newborn infants for congenital hypothyroidism. Blood spot TSH levels are measured in samples for which blood spot T4 levels fall in the lower 10 to 15 percent. There was a low recall rate of approximately one infant in every 550 screened. During a 17-month period 5225 infants have been screened for congenital hypothyroidism and two cretins have been detected. Due to very early diagnosis, both infants were commenced on T4 replacement therapy before the age of three weeks.


Susceptibility of New Zealand isolates to anaerobic bacteria to tinidazole.

Minimum inhibitory and minimum bactericidal concentrations (MIC, MBC) of tinidazole for 80 New Zealand isolates of anaerobic bacteria were determined. Growth of 95 percent of the isolates was inhibited by 4 micrograms/ml or less of tinidazole. MBC values were the same as, or one dilution higher than, the MIC.


The effect of mandibular osteotomy in three patients with hypersomnia sleep apnea.

Hypersomnia sleep apnea (HSA) is characterized by apneic episodes during sleep and daytime hypersomnolence. Patients afflicted as a result of upper airway obstruction have been treated traditionally with permanent tracheostomy. Three patients with HSA and mandibular retrognathism are presented. Each patient had a retrognathic mandible that stemmed from a different cause. Surgical advancement of their underdeveloped mandibles corrected the symptoms of HSA rapidly. The literature concerning HSA is reviewed and the advantages of mandibular surgery in selected cases are discussed.


Comparative evaluation of the 30-gauge dental needle.

The results of these tests demonstrated that aspiration through 30-gauge needles is possible, but since the flow rate is so diminished its clinical application is questionable. The evaluation of the physical properties of these needles proved them to be tough, durable, and suprisingly resistant to breakage, even under the most extreme manipulations and stresses. The insertion of needles into the plastic material clearly showed that the needles are always deflected toward the side without the bevel (the side that contains the point). Finally, the deposition patterns of the injected fluid in tissue have no relationship to the bevel.


Apicoectomy and retrograde amalgam in mandibular molar teeth.

The apicoectomy with retrograde amalgam is an excellent and accepted surgical technique for salvaging teeth. Anatomic considerations in molar teeth are not a contraindication to this technique.


Facial cellulitis caused by unrecognized foreign body.

Reported is a case of acute infection thought to be of odontogenic origin. The cause of the infection turned out to be a foreign body which had remained asymptomatic for more than 25 years.


A bizarre penetrating head wound with intraoral communication. Report of a case.

A case of a transcervical-intraoral penetrating foreign body in a 49-year-old man is presented. This is of interest because of the unusual mechanism of this potentially fatal injury, including the oral cavity, and because of the absence of serious neurologic sequelae.


Anterior lingual mandibular bone concavity. Report of a case.

A lingual mandibular bone concavity in the region of the left canine is described. The radiographic appearance of the concavity in this anterior location is an uncommon finding. Seven similar bone cavities with soft-tissue contents beneath the periosteum have been reported in the literature. Surgical exploration revealed this cavity to have no contents subperiosteally.


Adenosine in the treatment of recurrent herpes labialis.

The effectiveness of adenosine 5'-monophosphate was tested in thirty-six patients with recurrent herpes labialis. The compound displayed an immediate effect upon the healing of the recurrent lesions. Furthermore, twenty-three patients were free of recurrences for varying intervals extending over 2 years. In the remainder of the treated patients, a single recurrent episode was observed.


Hereditary angioedema controlled with danazol. Report of a case.

Hereditary angioedema is a condition which should be approached with caution and concern for the patient. The fact that the disease can be fatal cannot be overemphasized. The routine medical history should include questions aimed at identifying these patients. If the angioedema patient is to be treated, provisions must be made for maintenance of an adequate airway. This should include intubation if deemed necessary, and the practitioner should be prepared for a tracheostomy. Close observation following extubation is indicated. A case report in which the patient was controlled with danazol, a synthetic androgen, has been presented. The primary advantage of this drug is a decrease in the various side effects which have been associated with other therapeutic agents used previously in the treatment of hereditary angioedema.


Nodular fasciitis of the orofacial region.

The clinical and histologic findings from a study of forty-one cases of nodular fasciitis occurring in the orofacial region are presented. These findings reveal no essential differences in the biologic conduct of nodular fasciitis, whether it occurs in the orofacial area or in its preferred sites, i.e., the extremities and trunk. Various histologic findings are illustrated, none of which, including central necrosis, are indicative of any adverse biologic behavior. Accumulation of a large series of nodular fasciitis of the orofacial region indicates that this anatomic site is perhaps uncommonly but certainly not rarely affected. Pathologists, therefore, need not be so reluctant, as was found in this study, about assigning the diagnosis of nodular fasciitis to orofacial lesions.


Prostatic carcinoma presenting as a primary parotid tumor.

A patient who presented with a clinically solitary intraparotid metastasis from a prostatic carcinoma is reported. The entity of metastatic disease in the parotid is reviewed. The importance of clinical and histologic distinction between primary and metastatic neoplasms of the parotid is stressed.


Pulpal responses to operative procedures in rats receiving systemic methotrexate.

The systemic administration of methotrexate in nonlethal doses impairs the ability of the laboratory rat to localize an inflammatory response to operative dental procedures that result in exposures within the pulpal tissue. The pulpal healing response within these animals is thus greatly compromised.


Intraoral radiographic errors.

The purpose of this investigation was to investigate intraoral radiography in regards to the frequency of errors, the types of error necessitating retakes, and the relationship of error frequency to the teeth area examined and type x-ray cone used. The present study used 283 complete mouth radiographic surveys made, and 890 radiographs were found to be clinically unacceptable for one or more errors in technique. Thirteen and one-tenth errors per one hundred radiographs were found in this study. The three major radiographic errors occurring in this study were incorrect film placement (49.9 percent), cone-cutting (20.8 percent), and incorrect vertical angulation (12.5 percent).


General legal aspects of diagnostic dental radiography.

Recent concern about the effect of ionizing radiation on man has focused attention on the frequent use of diagnostic radiographs in dentistry. The clinician is expected to conduct his actions according to guidelines which reflect new information and changing technology in diagnostic dental radiography. Failure to do so may have severe legal consequences. Protection of the patient must be a matter of normal course, for each time the federal or state governments legislate toward safety in the healing arts, health professionals lose credibility and the confidence of the public. Implications of the federal radiation protection guidelines are discussed.


[Autotransplantation of teeth: indication, technic and prognosis].

An account is given of the most important indications for tooth transplantation. The developmental stage of the tooth most suitable for transplantation and the special demands to the recipient site, are considered. Each main group of indication is illustrated with case reports. Finally, factors influencing the prognosis of the transplant are discussed.


[Parotidomegaly in psychiatric dysorexia. 10 cases (author's transl)].

Had markedly anarchic dietary behaviour with alternating anorexia and bulimia, or even self-induced vomiting. These disturbances in dietary behaviour fall within the context of an unusual neurotic syndrome, predominantly hysterical. There were also menstrual problems, most often amenorrhoea. The physiopathological relations between these symptoms and signs may be envisaged as follows: the initial psychological problems explain the dysorexia; the menstrual difficulties are related at one and the same time to the psychological problems and the dysorexia; the latter is responsible for the parotidomegaly and for disturbances in carbohydrate and lipid metabolism.


[Quantitation of myelofibrosis in blood diseases by electronic image analysis (author's transl)].

Normal and pathologic reticulin networks colored black by silver nitrate can be automatically quantitated by electronic image analysis. By using this technique, different parameters can be obtained, such as the average density, the surface of network meshes, the thickness of the fibers, the complexity of the reticulum, and the heterogeneity of the myelofibrosis distribution. All of these parameters were obtained in 83 osteomedullar biopsies of blood diseases (primary splenomegaly, chronic myeloid leukemia, polycythermia vera, acute leukemia, and aplastic anemia). We have shown that there is no relation between the different parameters obtained and the medullary richness, hematopoietic center, or patient survival. On the other hand, the histomorphometric parameters can be used to distinguish acute leukemia and chronic myeloid leukemia myelofibrosis, while the parameters in primary splenomegaly are shown to be very heterogeneous.


Some specific neurological complications of acute lymphocyte leukaemia of childhood.

Aggressive chemotherapy and prophylactic central nervous system irradiation have increased the survival time of children with acute lymphatic leukaemia. As a result of treatment complex neurological problems are appearing. Computerized axial tomography allows more definite elucidation of these problems. 7 of 11 treated patients studied by this technique showed the presence of intracranial calcification and patchy cerebral atrophy, raising the questions of the quality of survival and the relationship of the findings to the cytotoxic drugs used.


L-glucosylceramide: synthesis, properties, and resistance to catabolism by glucocerebrosidase in vitro.

Procedures for the synthesis and radioactive labeling of L-glucosylceramide are described. This compound is a stereoisomeric analogue of D-glucosylceramide which occurs in nature and accumulates in pathological quantity in the organs and tissues of patients with Gaucher disease. The properties of L-glucosylceramide that have been examined so far have been found to be indistinguishable from those of the naturally occurring glycolipid. However, L-glucosylceramide is completely refractory to enzymatic hydrolysis by purified placental glucocerebrosidase and enzyme(s) present in whole tissue extracts. It is anticipated that L-glucosylceramide will be a uniquely useful substance for exploring pathogenetic processes in animal analogues of Gaucher disease.


Polyadenylylation of proteins in reovirions.

The reovirus oligoadenylates exist in two states within the virion: free and bound to viral proteins. The latter class of oligonucleotides, after digestion with Penicillium (P1) nuclease, yields adenylic acid and an adenosine-containing compound that is positively charged at pH 1.7, 3.5, or 6.5. In a mixture of [35S]methionine- and [3H]adenosine-labeled reovirus disrupted by sodium dodecyl sulfate/urea, approximately 4% of the radioactivity in [35S]methionine-labeled proteins coelutes with [3H]adenosine-labeled material at a net charge of -1.5 when analyzed by ion-exchange chromatography on DEAE-cellulose. This material migrates in sodium dodecyl sulfate/polyacrylamide gels with mu polypeptides and with a small protein, viii. Radioactivity is not released when the complex is boiled in buffer containing sodium dodecyl sulfate and urea or boiled in 80% dimethyl sulfoxide or when viral RNA is extracted with phenol. Digestion with Pronase converts the [3H]adenosine-labeled compound to oligomers of net charge -8 to -12 which contain nuclease P1- and alkaline phosphatase-sensitive adenylic acid residues as well as adenosine in a P1- and phosphatase-resistant linkage. These data indicate that reovirus contains structural proteins that are covalently bound to an oligoadenylate moiety.


Sequence relationships among defective interfering influenza viral RNAs.

Each clone of ts-52 and ts+ WSN influenza virus, when serially passaged at high multiplicity, gives rise to defective interfering (DI) virus with a unique set of new RNA species. The new RNAs (DI RNA) from several DI viruses were examined by the technique of RNase T1 oligonucleotide two-dimensional electrophoresis. It was found that each DI RNA arises from a specific segment of standard viral RNA. All DI RNA studied arose from the viral polymerase genes (P1, P2, and P3). DI RNAs originating from the same polymerase gene were interrelated. Certain of these DI RNAs appeared to contain completely overlapping nucleotide sequences. Others contained both overlapping and nonoverlapping nucleotide sequences. The latter DI RNAs may be formed from the progenitor viral RNA segment by a mechanism other than a common initiation (or termination) point and a simple deletion from one end.


Resolution of the ATP-dependent proteolytic system from reticulocytes: a component that interacts with ATP.

The ATP-dependent proteolytic cell-free system from reticulocytes has been resolved into three components, each of which is absolutely required for acid solubilization of 125I-labeled bovine serum albumin radioactivity. In addition to the previously reported heat-stable polypeptide [Ciechanover, A., Hod, Y. & Hershko, A. (1978) Biochem. Biophys. Res Commun. 81, 1100-1105], we now describe a protein of high molecular weight (approximately 450,000) that is labile at 42 degrees C. The extremely heat-labile factors is remarkably stabilized by ATP. GTP and CTP, which do not stimulate protolysis, do not stabilize this factor. Adenylate nucleotides such as ADP or the nonhydrolyzable beta,gamma imido or methylene analogues of ATP cause stabilization although they do not activate proteolysis. A third protein component of the protease system, stable at 42 degrees C, has been separated from the heat-labile species by salt precipitation. All three components are required with ATP for proteolytic activity, but thus far only the heat-labile factor has been shown to interact directly with ATP.


Binding of ribosomes to 5'-terminal leader sequences of eukaryotic messenger RNAs.

Tobacco mosaic virus and turnip yellow mosaic virus genome RNAs, which have long (greater than or equal to 69 nucleotides) 5'-terminal leader sequences preceding the AUG initiation codon, can form disome initiation complexes when incubated with a wheat germ extract and sparsomycin. In tobacco mosaic virus RNA, whereas one ribosome in such complexes presumably occupies the AUG-containing initiation site, the other is located within the RNA leader sequence. The results favor a model of eukaryotic initiation in which ribosomes first bind to the 5' end of the mRNA independently of AUG, then advance to the AUG initiation codon, where protein synthesis ensues.


Structural studies of human urinary kallikrein (urokallikrein).

Human urinary kallikrein (urokallikrein) has been purified by affinity chromatography with aprotinin coupled to CH-Sepharose and by gel filtration. The isolation procedure, which was performed under mild conditions, was completed in a 36-hr period and yielded an overall recovery of more than 75% and a purification of 1727-fold. Homogeneity of the urokallikrein was demonstrated by three criteria: the coincidence of the stained protein band and functional urokallikrein in duplicate gels after alkaline disc gel electrophoresis; the appearance of a single stained band of molecular weight 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis of reduced and unreduced enzyme; and the finding of a single amino-terminal residue, namely alanine after dansylation and acid hydrolysis of purified enzyme. The Km of urokallikrein on N alpha-p-tosyl-L-arginine methyl ester was 400 microM, and the Vmax was 194 mumol/min per mg of protein, which is higher than that observed with any previous preparations. The molecular weight of 48,700 determined on gel filtration and the molecular weight of 48,000 observed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis are in good agreement with the molecular weight of 48,213 calculated from the amino acid composition. The finding of a molecular weight higher than those previously reported, namely 27,000-43,500, the increased functional activity on tosylarginine methyl ester, and the detection of a single amino-terminal residue are consistent with the isolation of a more native protein by the procedure described in this paper.


Vitamin K-dependent gamma-carbon-hydrogen bond cleavage and nonmandatory concurrent carboxylation of peptide-bound glutamic acid residues.

The pentapeptide Phe-Leu-Glu-Glu-Leu, tritiated at the gamma carbon of each Glu residue, has been synthesized. In a system using microsomal preparations derived from rat liver, vitamin K-dependent tritium release from the L-Glu residues of this substrate can occur without the concurrent gamma-carboxylation of Glu. This tritium release reaction, which indicates cleavage of the gamma C-H bond, although easily uncoupled from CO2-dependent gamma C carboxylation, does require the reduced (hydroquinone) form of vitamin K and oxygen. The data argue against a concerted mechanism for the cleavage of the gamma C-H bond and carboxylation and against a mechanism in which the vitamin functions solely to transfer or activate CO2. Although the tritium release is related clearly to the oxidation of vitamin KH2, it is not yet established how the subsequent carboxylation proceeds. However, two carboxylation mechanisms compatible with the results are discussed.


Purification and distribution of a major protein in rat prostate that binds estramustine, a nitrogen mustard derivative of estradiol-17 beta.

A protein in rat ventral prostate cytosol that binds estramustine [estradiol 3-bis(2-chloroethyl)carbamate) was purified to homogeneity by using chromatography on DEAE-cellulose, Sephadex G-100 (superfine), octyl-Sepharose (CL-4B, and polyacrylamide gel electrophoresis. The estramustine-binding protein was found to have a Mr of 46,000 as estimated by gel filtration. After analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the protein was found to consist of two subunits with Mr of about 20,000 and 18,000. After reduction of disulfide bridges, the protein was decomposed into three components with Mr of about 12,000, 11,000, and 8000. Amino acid analysis indicated that the protein is a glycoprotein. Antibodies against the protein were raised in rabbits and a radioimmunoassay was developed for it. The estramustine-binding protein constituted about 18% of the total protein in rat ventral prostate cytosol, was present in the dorsal and lateral lobes of the prostate, and was also detected in the pituitary gland, cerebral cortex, submaxillary gland, thyroid gland, adrenal gland, seminal vesicle, coagulating gland, epididymis, and preputial gland of the male rat. In female rats the protein was detected in cerebral cortex. Because the estramustine-binding protein is predominantly found in the accessory sexual glands of the male rat, it may be of importance for maintaining male fertility.


Glucagon-induced autophagy and proteolysis in rat liver: mediation by selective deprivation of intracellular amino acids.

Amino acid deprivation and glucagon are both potent inducers of autography and proteolysis in liver. Because glucagon enhanced the metabolic utilization of some amino acids, the catabolic response to both of these stimuli could be achieved by a lowering of intracellular amino acid pools. Alternatively, glucagon could act independently of amino acids. To clarify the mode of hormonal action and also the relationship between the two cellular responses, livers from fed rats were perfused, with and without glucagon, with plasma amino acids over a concentration range of 0 to 10 times normal. Individual amino acids constancy at each level was ensured by perfusion in the single-pass mode. Amino acids alone strongly regulated autophagy and proteolysis in a coordinated fashion; maximal suppression was achieved at twice normal concentration; both effects increased rapidly to maximum at less than normal concentration. Corresponding effects of glucagon, however, could be elicited only at intermediate amino acid levels. None was noted at 4 and 10 times normal; at 0, hormonal stimulation was minimal. The amino acid inhibition was selective because it did not block cyclic AMP production or glycogenolysis. Intracellular pool measurements and systematic alteration of perfusate amino acid composition indicated that the autophagic and proteolytic effects of glucagon are mediated by a hormonally induced depletion of glycine, alanine, glutamate, and glutamine; of these, glutamine alone is the most effective. We conclude that the stimulation of intracellular protein degradation in liver is a manifestation of deprivation-induced autophagy which results from a decrease in certain intracellular glucogenic amino acids, notably glutamine.


Nucleoside triphosphate regeneration decreases the frequency of translation errors.

The addition of naturally occurring polyamines and inorganic ions to an in vitro protein-synthesizing system improved the extent and fidelity of translation. In such an optimized system, regeneration of the nucleoside triphosphates with phosphoenolpyruvate and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) reduced further the missense error frequency to the in vivo level as well as enhanced the extent of translation. The effect of nucleoside triphosphate regeneration was shown to be due primarily to the increase in the ratio of adenosine and guanosine triphosphates to their hydrolysis products and only marginally due to the increase in the absolute concentrations of the nucleoside triphosphates.


Arachidonic acid metabolism in polymorphonuclear leukocytes: unstable intermediate in formation of dihydroxy acids.

An unstable intermediate was detected in the transformation of arachidonic acid into 5,6-dihydroxyicosatetraenoic acids (two isomers) and 5,12-dihydroxyicosatetraenoic acids (three isomers) in rabbit peritoneal (glycogen-induced) polymorphonuclear leukocytes. Addition of 10 vol of methanol, ethanol, or ethylene glycol to short-term incubations (30-45 sec) led to the formation of the corresponding 12-O-alkyl derivatives of the 5,12-dihydroxy acids. The time for 50% disappearance of the intermediate (37 degrees C), as measured by formation of 5-hydroxy-12-O-methylicosatetraenoic acids (two isomers) upon trapping with methanol, was about 1 min in live cell preparations (pH 7.4) and about 4 min in water/acetone (1:1), pH 7.4. At pH 6.0 or below, the hydrolysis of the intermediate was too rapid to be measured by the method employed. Data supporting both enzymatic and nonenzymatic hydrolysis of the intermediate into dihydroxy acids are presented. Incubation of the cells with arachidonic acid under an atmosphere of 18O2 led to incorporation of 18O into the 5,6-dihydroxy acids and 5,12-dihydroxy acids only at C-5. The 5-hydroxyicosatetraenoic acid was also labeled at C-5. Considering the chemical reactivity of the intermediate and the structures of the derivatives obtained, it is proposed that the intermediate is 5(6)-oxido-7,9,11,14-icosatetraenoic acid.


Developmental and mutational changes of glycoproteins in the mouse neuronal retina: studies with bovine galactosyltransferase.

Bovine galactosyltransferase (lactose synthase; EC 2.4.1.22) which catalyzes the transfer of galactose from UDPgalactose to glycoproteins with N-acetylglucosamine as the terminal residue of their oligosaccharide side chains was used to label glycoproteins of mouse retina with [14C]galactose. The glycoproteins were separated by isoelectric focusing in the first dimension and by sodium dodecyl sulfate gel electrophoresis in the second dimension. Their position on the gel was determined by autofluorography. With this method, quantitative as well as qualitative changes in the glycoprotein composition of the neuronal mouse retina during postnatal development were observed. Furthermore, it was found that the photoreceptor loss in mice with retinal degeneration was paralleled by the disappearance of certain glycoprotein bands.


Inactivation of phage T7 by near-ultraviolet radiation plus hydrogen peroxide: DNA-protein crosslinks prevent DNA injection.

A nonlethal concentration of H2O2 (0.05%) greatly enhances near-ultraviolet (NUV) inactivation of phage T7. Simultaneous treatment with H2O2 and NUV reduces the amount of DNA injected into the bacterial host, but not the number of phage adsorbed. Not only were recombination and gene expression of late markers reduced upon treatment of phage T7 with NUV plus H2O2, but also a gradient of recombination resulted, with markers injected first reduced to a lesser extent than those injected last. Double-strand DNA breaks were not detected; however, DNA-protein crosslinks were observed upon NUV plus H2O2 treatment of double-labeled T7. Previous studies demonstrated that single-strand DNA breaks did not account for phage death by NUV plus H2O2. It is concluded that the DNA-protein crosslinks prevent normal injection of T7 phage DNA; such crosslinks may be important lesions in NUV cellular damage.


Diglyceride lipase: a pathway for arachidonate release from human platelets.

We provide evidence that the mechanism for arachidonate release from stimulated human platelets involves two enzymes: a phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) and a diglyceride lipase. After incubation of platelets with thrombin for 15 seconds, 1.2 nmol of 1-stearoyl-2-arachidonoyl diglyceride per 10(9) platelets, was isolated. Arachidonate was released from this substrate by the action of diglyceride lipase located in the particulate fraction of platelets. The enzyme has a pH optimum of 7.0, is stimulated by calcium ions and reduced glutathione, and liberates 31 nmol of fatty acid per min per mg of platelet particulate protein. The diglyceride lipase has sufficient activity to account for the 5-10 nmol of arachidonate released per 10(9) platelets upon thrombin stimulation. That only arachidonate is released upon thrombin stimulation may be explained by the fact that the diglyceride substrate in platelets contains only arachidonate in the 2 position. The lipase activity found in platelet membranes can also hydrolyze the 1-position fatty acid. Stearate is not released when intact platelets are stimulated with thrombin, and the fate of this fatty acid remains to be elucidated.


Antineoplastic activity of poly(L-lysine) with some ascites tumor cells.

We have found that poly(L-lysine) can be a very effective agent in preventing the growth of Ehrlich ascites tumors in mice. When given optimal doses of poly(L-lysine) (Mr 60 x 10(3)) intraperitoneally for 5 consecutive days, beginning on day 1 after inoculation with Ehrlich ascites cells. White Swiss mice show nearly a 100% remission from subsequent tumor growth. Rechallenge of "cured" animals with tumor cells, however shows no long-term immunological protection. In tissue culture, poly(L-lysine) shows a related potent cytotoxicity with HeLa cells; interestingly, the D isomer. In addition, there is a strong molecular weight dependence in that the small polylysine (Mr 3 x 10(3)) possesses less than 1/20th the cytotoxicity of large polymers (Mr 70 x 10(3)) on a weight basis in both cell culture and animal studies. At the same time, none of these lysine polymers gives any significant increase in life span to BDF1 mice infected with L1210 murine leukemia cells. We have also further explored the mechanism by which the polylysines express their cytotoxicity. These data indicate that lysine polymers show cell specificity in their action and in some cases they may be beneficial as potent antineoplastic agents, particularly when molecular weight is taken into consideration.


A factor in serum and amniotic fluid is a substrate for the tRNA-modifying enzyme tRNA-guanine transferase.

Q factor, a substance found in animal serum that enables cultured mammalian cells (L-M) to produce tRNA containing queuine (the base of "nucleoside Q", queuosine), has been purified to homogeneity from bovine amniotic fluid. Q factor causes the appearance of Q-containing tRNAAsp in the L-M cells cultivated in serum-free medium, and this was used as an assay to monitor the purification of Q factor. Q factor is a competitive inhibitor of guanine for rabbit reticulocyte tRNA-guanine trnsferase, with a K1 of 4.5 x 10(-8) M. Q factor is inactivated in both the L-M cell and tRNA-guanine transferase assays by treatment with periodate or cyanogen bromide, both of which react with queuine. In L-M cells, nearly complete conversion of Q-free to Q-containing tRNAAsp is observed within 24 hr after addition of pure Q factor to the medium; actinomycin D, cycloheximide, and cycloleucine, inhibitors of RNA synthesis, protein synthesis, and nucleic acid methylation, respectively, do not inhibit this conversion. The product of the reaction, catalyzed by pure rabbit reticulocyte tRNA-guanine transferase, between Q factor and rabbit reticulocyte Q-free tRNAHis is chromatographyically indistinguishable from Q-containing tRNAHis.


In vitro core particle and nucleosome assembly at physiological ionic strength.

Nucleosome core particles have been efficiently assembled in vitro by direct interaction of histones and DNA at physiological ionic strength, as assayed by digestion with DNases, supercoiling of relaxed circular DNA, and electron microscopy. Reconstitution was achieved either by the simultaneous addition of all core histones, or by the sequential binding of H3 . H4 tetramer and H2A . H2B dimer to DNA. Micrococcal nuclease digestion and electron microscopy studies indicated that there is heterogeneity in the spacings at which core particles are assembly on the DNA. Length measurements of oligomeric DNA produced during the course of the digestion suggest that the core histone octamer can organize 167 (+/- 4) rather than 145 base pairs of DNA, the extra 20 base pairs being quickly digested. Binding of histone H1 to core particles resulted in the protection of about 165 base pairs of DNA from nuclease attack. Because the core histone octamer is fully dissociated into H3 . H4 tetramer and H2A . H2B dimer at physiological ionic strength, our results would suggest that in vivo core particle assembly may also occur by interaction of these two complexes on the nascent DNA.


Moloney murine sarcoma virions synthesize full-genome-length double-stranded DNA in vitro.

Moloney murine sarcoma virus (MSV) virions incubated under optimal conditions were shown to support extensive synthesis of double-stranded DNA. The major product, a 5950-base-pair (6-kilobase-pair DNA) double-stranded DNA, was characterized by cleavage with restriction endonucleases and shown to contain a 600-nucleotide-long direct repeat at both ends of the MSV genome. Linear DNA molecules made in vivo shortly after infection were compared to the linear double-stranded DNA synthesized in vitro. The restriction maps of both viral DNA products were indistinguishable. The 600-base-pair repeat results in a progeny DNA molecule that is longer than the parental MSV genomic RNA. The generation of this repeat must involve a mechanism that allows the viral reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) to copy 5'- and 3'-terminal genomic (+) strand sequences twice.


Biofunctional evaluation of a hydrogen bond linking the ring and tail beta-turns of oxytocin.

Deamino-[8-N-methylleucine]oxytocin and deamino-[8-alpha-hydroxyisocaproic acid]oxytocin were synthesized to study the importance of hydrogen bonding between the carboxamide carbonyl of asparagine and the peptide N-H of leucine in stabilizing the biologically active conformation of oxytocin. The analogs were synthesized by coupling deaminotocinoic acid with Pro-Leu(Me)-Gly-NH2 and Pro-HyIc-Gly-NH2, respectively. (HyIc is alpha-hydroxyisocaproic acid). Deamino-[8-N-methylleucine]oxytocin was found to possess 48 +/- 7 units of uterotonic activity, 33 +/- 5 units of avian vasodepressor activity, and 3.15 +/- 1.5 units of antidiuretic activity per mg; deamino-[8-alpha-hydroxyisocaproic acid]oxytocin possessed 134 +/- 12 units of uterotonic activity, 31 +/- 3 units of avian vasodepressor activity, 9.6 +/- 3.0 units of antidiuretic activity, and 0.26 +/- 0.02 unit of pressor activity per mg. Neither of the analogs possesses the peptide N-H at residue 8 required for the formation of a hydrogen bond with the asparagine carboxamide; however, both can assume the conformation needed to evoke the characteristic biological activities of oxytocin although in lower potency. It is concluded that such a hydrogen bond does not constitute a conformational constraint that is essential for hormone action.


Membrane damage caused by irradiation of fluorescent concanavalin A.

Visible light irradiation of fluoresceinated concanavalin A (f-Con A) bound to the outside of resealed erythrocyte membranes caused crosslinking of as much as 50% of the membrane proteins. Crosslinking was absent in controls in which equivalent amounts of f-Con A were added to the membranes but prevented from binding by the presence of 10 mM alpha-methylmannoside. The photodamage was not accompanied by a change in the membrane permeability barrier or membrane shape. Although fluorescein bleaching accompanies the formation of protein aggregates, the amount of aggregated protein is not simply a function of the number of fluoresceins bleached. The percentage of aggregated protein decreases when the same dose of light is given in a shorter time. Although certain antioxidants and free-radical scavengers had no detected effect on the crosslinking, reducing agents such as cysteamine and reduced glutathione either blocked or reversed the protein crosslinking. The mechanism of photoinduced oxidation and the implications of these results for fluorescence studies of cell membranes are discussed.


Acoustic microscopy: resolution of subcellular detail.

Recent advances now permit the use of scanning acoustic microscopy for the analysis of subcellular components. By sequential viewing of identified fixed cells with acoustic, light, and electron microscopy, we have established that the acoustic microscope can readily detect such features as nuclei and nucleoli, mitochondria, and actin cables. Under optimal conditions, images can even be obtained of filopodia, slender projections of the cell surface that are approximately 0.1-0.2 micron in diameter. Small objects separated by as little as 0.5-0.7 micron can successfully be resolved. Three aspects of the acoustic micrographs prepared in this preliminary survey seem especially prominent. These are, first, the extraordinary level of acoustic contrast that can differentiate the various cytoplasmic organelles, even in regions of very thin cytoplasm; second, the reversals in acoustic contrast that occur when altering the plane of focus; and third, the sensitivity of the acoustic response to overall cytoplasmic thickness. The acoustic microscope uses a novel source of contrast that is based on local mechanical properties. In addition, it can provide a degree of resolution that is comparable to that of the light microscope.


Growth of Madin-Darby canine kidney epithelial cell (MDCK) line in hormone-supplemented, serum-free medium.

Madin-Darby canine kidney (MDCK) cells can grow in synthetic medium supplemented with five factors--insulin, transferrin, prostaglandin E1, hydrocortisone, and triiodothyronine--as a serum substitute. These five factors permit growth for 1 month in the absence of serum and a growth rate equivalent to that observed in serum-supplemented medium. Dibutyryl adenosine 3',5'-cyclic monophosphate substitutes for prostaglandin E1 in the medium. Potential applications of the serum-free medium are discussed. The medium permits a defined analysis of the mechanisms regulating hemicyst formation by hormones and permits the growth of primary kidney epithelial cell cultures in the absence of fibroblast overgrowth.


Role of carbohydrate in biological function of the adhesive glycoprotein fibronectin.

We have investigated the role of the carbohydrate moiety in the biological activity of fibronectin in vitro by using tunicamycin to inhibit the glycosylation of this glycoprotein. Tunicamycin is a glucosamine-containing antibiotic that specifically inhibits glycosylation of protein asparaginyl residues mediated by dolichol pyrophosphate. Fibronectin synthesized in the presence of 0.5 microgram of tunicamycin per ml was not glycosylated, as determined by amino sugar analysis, lack of incorporation of [14C]glucosamine and [3H]mannose, and concanavalin A binding studies. Nonglycosylated fibronectin that was isolated from chicken embryo fibroblasts and added to transformed cells in vitro was as effective as the glycosylated protein in promoting a more normal fibroblastic phenotype, including cell flattening, elongation of cell processes, and parallel alignment of cells. The nonglycosylated protein was also as effective as the glycosylated species in mediating cell attachment to collagen and spreading on plastic, as well as in agglutination of formalin-fixed sheep erythrocytes. The nonglycosylated protein was twice as sensitive as the glycosylated protein to proteolytic hydrolysis in vitro as had been suggested by previous studies with intact cells [Olden, K., Pratt, R.M. & Yamada, K.M. (1978) Cell 13, 461-473]. We conclude that the carbohydrate moiety of fibronectin is not required for the mediation of a number of biological activities characteristic of this glycoprotein.


Water permeability changes studied by 17O nuclear magnetic resonance during differentiation of Friend leukemia cells.

Water permeability of Friend leukemia cells was studied by 17O nuclear magnetic resonance during differentiation induced by dimethyl sulfoxide (Me2SO). While in noninduced cells water permeability was essentially constant during the growth period, in the Me2SO-induced cells there were two distinct periods at which the water permeability was increased by at least an order of magnitude. These periods correspond to approximately one doubling time and 6 days of growth. This change in water permeability is not due to direct interaction of Me2SO with the membrane but must be ascribed to structural changes in the membrane during the course of differentiation.


Ganglioside inhibition of fibronectin-mediated cell adhesion to collagen.

Fibronectin mediates the adhesion of cells to collagen by first binding to the collagen substrate, followed by attachment of the cells to the fibronectin-collagen complex. Bovine brain gangliosides were found to block fibronectin-mediated cell adhesion to collagen in a concentration-dependent manner. The gangliosides did not block the binding of fibronectin to collagen but did prevent the attachment of the cells to the fibronectin-collagen complex. Of the individual gangliosides tested, GT1 and GD1a were the most effective inhibitors followed by GD1b greater than GM1 greater than GM2; GM3 was not an inhibitor. The inhibition of cell adhesion also was observed with the oligosaccharide portion of the gangliosides, but not with ceramides or with a variety of free sugars or glycosaminoglycans. Mild periodate oxidation of mixed gangliosides or of GD1a modified their sialic acid residues and the oxidized gangliosides were no longer inhibitory; subsequent reduction with NaBH4 did not restore the inhibitory activity of the modified gangliosides. These results suggest that specific gangliosides or related sialic acid-containing glycoconjugates on the cell surface may act as the receptors for fibronectin.


Bimane fluorescent labels: labeling of normal human red cells under physiological conditions.

The bimane fluorescent labels, monobromobimane, dibromobimane, and monobromotrimethylammoniobimane, are derivatives of syn-9,10-dioxabimane:1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione. They efficiently label hemoglobin (reactive thiol groups), membrane proteins, and glutathione of normal human red cells under physiological conditions. Monobromobimane and dibromobimane are effective on intact cells while red cell membranes may be impermeable to the positively charged monobromotrimethylammoniobimane, the latter being effective only on lysed cells. These bimane labels provide a class of labeling agents that may have wide applicability in biological materials.


Junctional modulation in mouse embryonal carcinoma cells by Fab fragments of rabbit anti-embryonal carcinoma cell serum.

Mouse embryonal carcinoma PCC4 cells are connected by extensive gap and tight junctions. When the cells are incubated in a medium containing Fab fragments against embryonal carcinoma F9 cells, they round up and a process of junctional removal is initiated. In particular, gap junctions are internalized and after 30 hr of incubation with the anti-F9 Fab fragments both tight and gap junctions are no longer present at the cell surface; however, the cells are still in contact by means of small attachment sites.


Transport of tricarboxylic acid cycle intermediates by membrane vesicles from renal brush border.

The uptake of citrate and alpha-ketoglutarate by membrane vesicles from rabbit renal brush border was studied by a rapid filtration technique. Both compounds exhibited transport characteristics similar to those seen for the sodium-dependent cotransport systems previously described for sugars and amino acids in brush border membranes. The estimated sodium-dependent Vmax and Km were 17 nmol per mg of protein per min and 0.18 mM for citrate and 17 nmol per mg of protein per min and 1.0 mM for alpha-ketoglutarate. The initial rate of citrate transport was 5 times that of sugars and amino acids under comparable conditions. Uptake rates of 0.1 mM citrate and alpha-ketoglutarate were inhibited by greater than 90% by 10 mM succinate, malate, fumarate, or oxaloacetate, indicating the presence in the brush border membrane of a transport system highly specialized for the renal conservation of intermediates of the tricarboxylic acid cycle.


Sustained growth and three-dimensional organization of primary mammary tumor epithelial cells embedded in collagen gels.

We have developed a method for embedding cells within a collagen matrix which allows sustained growth of mouse mammary tumor epithelial cells in primary culture. A characteristic and reproducible pattern of organization and growth occurs: the cells rearrange themselves and produce duct-like structures extending into the matrix, resulting in a three-dimensional outgrowth. Autoradiography showed continuous [3H]thymidine incorporation during 8 weeks in culture. An increase in DNA content of the cultured cells as a function of time was observed. Mouse mammary tumor cells cultured in the conventional monolayer system failed to show any significant increase in cell number during a culture period of 6 weeks. In addition, in such monolayer systems, cells progressively became detached from the dishes in long-term culture. The mammary epithelial cell origin of the collagen gel cell outgrowths was shown by electron microscopic demonstration of polarized cells containing tight junctions and budding mammary tumor virus particles. In addition, in vivo transplantation of collagen gel outgrowths resulted in the development of mammary adenocarcinoma histologically similar to the donor tumor. Cellular outgrowth patterns resembling those from tumor cells were also seen in similar collagen gel cultures of normal mammary cells from mouse and human and of hyperplastic alveolar nodule cells from mouse. The significance and usefulness of this system in comparison to the conventional monolayer system are discussed.


Human T gamma globin chain is a variant of A gamma chain (A gamma Sardinia).

Isoelectric focusing, cellulose acetate electrophoresis, and carboxymethylcellulose chromatography in the presence of Nonidet P-40 allow the separation of pure gamma chains into two fractions. Amino acid analysis of their cyanogen bromide fragment 3 (gamma CB3) identifies these fractions as the separated G gamma (Gly-136) and A gamma (Ala-136) globin chains. Fingerprint and amino acid analyses of the gamma Tp9 tryptic peptide from the purified A gamma and G gamma fractions from two different patients demonstrate that the commonly occurring gamma Sardinia variant (gamma 75 isoleucine leads to threonine), also known as T gamma chain, has alanine in position 136. From this analysis we suggest that the T gamma gene is an allele of the A gamma locus (A gamma Sardinia) rather than a third gamma locus.


Attempt to immunize guinea pigs against L2C leukemia with leukemia cells inactivated by gamma irradiation.

In previous studies, intradermal inoculation of small doses of L2C leukemic cell suspensions into strain 2 guinea pigs induced immunity against challenging reinoculation with leukemic cells; however, 50% of the guinea pigs developed leukemia in the course of immunization. We have now attempted to induce immunity by inoculation of L2C leukemic cells inactivated by in vitro gamma irradiation ranging from 750 to 8000 rads (1 rad = 0.01 gray). In preliminary experiments, irradiation with 750 to 2750 rads had no significant effect on leukemogenic potency of leukemic cells; however, doses exceeding 3000 rads inactivated the leukemogenic potency of L2C cells. Eighty-nine guinea pigs that survived intradermal, subcutaneous, or intraperitoneal inoculations with irradiated (1000-8000 rads) L2C cells were subsequently challenged by reinoculation with nonirradiated leukemic cells, and 83 of them (93%) developed leukemia. L2C leukemic cells contain spherical particles, about 103 nm in diameter; it is reasonable to assume that these particles represent the causative virus responsible for the development of L2C leukemia. Inactivation of leukemogenic potency of L2C leukemic cells by gamma irradiation in vitro does not necessarily imply that the virus particles consistently present in these cells were also inactivated. In previous experiments carried out on mouse leukemia, doses exceeding 1,000,000 rads were needed in order to inactivate the mouse leukemia virus in vitro.


Light-induced damage to ocular lens cation pump: prevention by vitamin C.

The cation pump activity of the ocular lens was damaged by exposure to light in the presence of riboflavin phosphate. The intensity of light was similar to that used for reading purposes. The observed light-induced damage was due to superoxide or its derivatives, the superoxide being produced photochemically. Such damage was attenuated by vitamin C in amounts comparable to that in the aqueous humor. Thus, a new role for the high ascorbate level present in the anterior chamber fluid and the lens has been suggested. Ascorbate in other tissues also might have this novel physiological function of protecting against damage due to superoxide and its derivatives produced during normal cellular oxidation.


Tyrosine administration reduces blood pressure and enhances brain norepinephrine release in spontaneously hypertensive rats.

Administration of L-tyrosine to normotensive or spontaneously hypertensive rats reduces blood pressure. The effect is maximal within 2 hr of injection. In spontaneously hypertensive rats, a dose of 50 mg/kg, intraperitoneally, reduces blood pressure by about 12 mm Hg (1 mm Hg = 1.33 x 10(2) pascals); a dose of 200 mg/kg produces the maximal effect, a reduction of about 40 mm Hg. Tryptophan injection (225 mg/kg) also lowers blood pressure in spontaneously hypertensive rats, but only by about half as much as an equivalent dose of tyrosine. Other amino acids tested (leucine, isoleucine, valine, alanine, arginine, and aspartate) do not affect blood pressure. Tyrosine injection appears to reduce blood pressure via an action within the central nervous system, since the effect can be blocked by co-administering other large neutral amino acids that reduce tyrosine's uptake into the brain. That tyrosine's antihypertensive action is mediated by an acceleration in norepinephrine or epinephrine release within the central nervous system is suggested by the concurrent increase that its injection produces in brain levels of methoxyhydroxyphenylethylglycol sulfate.


Detection of glucocorticoid receptors on Friend erythroleukemia cells.

The inhibition of dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells by steroids led us to examine these cells for the presence of glucocorticoid receptors. Direct assessement of dexamethasone binding revealed high-affinity dexamethasone receptors on the untreated cells. The specific binding of [3H]dexamethasone was dose dependent. At a concentration of 10(-8) M, almost all binding sites were occupied. The mean number of binding sites per cell in two separate experiments was 8045 and 7191, respectively, and the Kd varied between 3.38 and 3.49 x 10(-9) M. Dimethyl sulfoxide treatment led to a decrease in the number of dexamethasone binding sites on the cells induced to differentiate. After 5 days of treatment, the mean number of sites per cell was reduced to 1216 and 896 in two experiments, with a Kd of 5 x 10(-9) M. Dexamethasone treatment resulted in a moderate decrease in the efficiency of colony formation within 72 hr after the cells were plated in methylcellulose. The mechanism of this inhibitory effect is unknown. However, it was also dose dependent and could be abrogated by appropriate concentrations of progesterone or 11-deoxycortisone. These results suggest that the steroid effects on growth and differentiation of the erythroleukemia cells may be mediated via glucocorticoid receptors.


Role of pain in placebo analgesia.

The hypothesis that perceived pain intensity can influence placebo analgesia was tested. One hundred and seven subjects rated their pain from from 0 to 10 on a visual analog scale after a standard wisdom tooth extraction. The expected course of such postoperative pain in the absence of therapy or placebo is a steady increase; this was confirmed by blind administration of the placebo. When placebos were given intravenously in view of the patients, some (placebo nonresponders) reported that their pain increased, whereas others (placebo responders) reported that their pain either decreased or remained the same over the next 60 min. A placebo response was more likely to occur if the pain rating 5 min prior to placebo administration (initial pain) was greater than 2.6. Furthermore, placebo responders with initial pain above this 2.6 level reported significantly greater mean analgesia than those with lower initial pain. Indeed, responders with initial pain less than 2.6 reported no change in pain during the 60 min after administration of a placebo. When their initial pain level was greater than 2.6, they reported a steady decline in pain over this period. However, above the 2.6 level there was no obvious relationship between the magnitude of the placebo analgesia and the initial pain.


Isolation and culture of motoneurons from embryonic chicken spinal cords.

A method is described for isolating cholinergic alpha motoneurons from the spinal cord of chicken embryos at stage 17-18 (Hamburger and Hamilton numbering), at the time when it has been shown that motoneurons withdraw from the mitotic cycle but neurons of other types and glia are still mitotic. Fragments of the ventral half of the spinal cord are incubated for 24 hr in the presence of 10 microM 1-beta-D-arabinofuranosylcytosine in order to eliminate dividing cells and are subsequently dissociated into a suspension of single cells. The following evidence has been obtained and suggests that these cells are neuronal and appear to be alpha motoneurons: (i) they are resistant to the lethal effect of arabinofuranosylcytosine, and thus are postmitotic at stage 17-18; (ii) when grown in vitro, they exhibit morphological characteristics similar to those of ventral spinal neurons, which include the ability to be stained with silver, Nissl, methylene blue vital stain at pH 6.5-7.0, and choline acetyltransferase histochemistry; (iii) they have high choline acetyltransferase activity; (iv) they are capable of forming functional synapses with muscle.


Localization of horseradish peroxidase-alpha-bungarotoxin binding in crustacean axonal membrane vesicles and intact axons.

A conjugate of alpha-bungarotoxin with horseradish peroxidase was used to visualize alpha-bungarotoxin binding sites at the fine structural level in isolated axonal membrane vesicles from lobster walking leg nerve. These plasma membrane vesicles have previously been shown to exhibit saturable binding of [3H]nicotine and [3H]acetylcholine. Binding of the toxin was identified in the axon plasma membrane and could be blocked by pretreatment with excess free alpha-bungaratoxin or d-tubocurarine. Binding sites for alpha-bungarotoxin were identified by the same technique in sections of intact nerve fibers from both lobster and spider crab and were found to be localized primarily in the axolemma rather than in the Schwann cell membrane.


Degradation of junctional and extrajunctional acetylcholine receptors by developing rat skeletal muscle.

We have examined the rate of degradation of the total acetylcholine receptor content of diaphragm muscles of young rats and have found that even in muscles from 1-day-old rats some receptors are metabolically more stable than adult extrajunctional receptors. Further experiments have shown that acetylcholine receptors at junctional regions from young rats are degraded slowly, whereas those in extrajunctional regions are degraded rapidly. The results demonstrate that junctional acetylcholine receptors in rat diaphragm are degraded at a slow rate characteristic of adult junctional receptors at all ages after birth.


Immunocytochemical localization of rat peripheral nervous system myelin proteins: P2 protein is not a component of all peripheral nervous system myelin sheaths.

Specific antibodies have been developed against P1, P2, and P0 myelin proteins and were used to study the localization of these proteins in the rat peripheral nervous system. Both peripheral and central nervous system myelin sheaths contain P1 protein. P0 and P2 proteins are found exclusively in peripheral nervous system myelin sheaths. Antisera to P1 and P0 proteins stain all peripheral nervous system myelin sheaths uniformly. P2 protein is not a component of all peripheral nervous system myelin sheaths. In sheaths that do contain P2 protein, it is concentrated in the area of the Schmidt-Lanterman incisures.


Proton magnetic resonance studies of cholinergic ligand binding to the acetylcholine receptor in its membrane environment.

Proton magnetic resonance has been used to monitor binding of choline, a known partial agonist, to acetylcholine receptor-enriched membrane preparations from Torpedo californica electroplax. The interaction between choline and receptor led to a broadening of the resonance of the choline methyl groups and this effect was reversed by alpha-bungarotoxin, a quasi-irreversible antagonist of the acetylcholine receptor. From the concentration dependence of line broadening the equilibrium dissociation constant for choline was obtained (Kd = 190 +/- 65 microM). The temperature dependence of the parameters observed in the choline titrations gave an enthalpy of binding delta H less than 1.5 kcal/mol and allowed estimates for the dissociation rate constant of the receptor-choline complex (kdiss greater than 1.6 x 10(3) S(1) and the respective activation energy, Ea (Kdiss) approximately 5.5 kcal/mol. The association of other ligands with the membrane-bound receptor could also be studied by observing effects of varying concentrations of such ligands on the choline methyl group linewidth at a constant choline concentration.


Dynamics of activated processes in globular proteins.

A procedure for the dynamical simulation of activated processes, such as ligand binding and enzymatic reactions, in a globular protein is outlined. Preliminary calculations of the transition state geometry and barrier crossing trajectories are presented for a model reaction, the rotation of an aromatic ring in the bovine pancreatic trypsin inhibitor. The results show that repulsive nonbonded interactions between the ring atoms and the atoms in the surrounding protein matrix determine the dynamical character of the reorientation process; the nonbonded interactions are the source of the rotational barrier and of the impulses that speed up or slow down the ring motion during the barrier crossings.


Photosensitized electron transport across lipid vesicle walls: quantum yield dependence on sensitizer concentration.

An amphiphilic tris(2,2'-bipyridine)ruthenium(2+) derivative that is incorporated into the walls of phosphatidylcholine vesicles photosensitizes the irreversible oxidation of ethylenediaminetetraacetate(3-) dissolved in the inner aqueous compartments of the vesicle suspension and the one-electron reduction of heptylviologen(2+) dissolved in the continuous aqueous phase. The quantum yield of viologen radical production depends on the phospholipid-to-ruthenium complex mole ratios. A kinetic model is used to derive an order-of-magnitude estimate for the rate constant of electron transport across the vesicle walls. The results are inconsistent with a diffusional mechanism for electron transport and are interpreted in terms of electron exchange.


Nick-free formation of reciprocal heteroduplexes: a simple solution to the topological problem.

Because the individual strands of DNA are intertwined, formation of heteroduplex structures between duplexes--as in presumed recombination intermediates--presents a topological puzzle, known as the winding problem. Previous approaches to this problem have assumed that single-strand breaks are required to permit formation of fully coiled heteroduplexes. This paper describes a simple, nick-free solution to the winding problem that satisfies all topological constraints. Homologous duplexes associated by their minor-groove surfaces can switch strand pairing to form reciprocal heteroduplexes that coil together into a compact, four-stranded helix throughout the region of pairing. Model building shows that this fused heteroduplex structure is plausible, being composed entirely of right-handed primary helices with Watson-Crick base pairing throughout. Its simplicity of formation, structural symmetry, and high degree of specificity are suggestive of a natural mechanism for alignment by base pairing between intact homologous duplexes. Implications for genetic recombination are discussed.


Subunit interaction during catalysis: alternating site cooperativity in photophosphorylation shown by substrate modulation of [18O]ATP species formation.

Pronounced substrate modulation of incorporation of water oxygen into ATP formed by photophosphorylation is observed, as measured by 31P NMR analysis of products formed from ADP and highly 18O-labeled Pi. A marked increase occurs in oxygen exchange per ATP formed as ADP or Pi concentration is decreased. This is explainable by the binding-change mechanism for ATP synthesis, in which the energy-linked release of ATP from one site requires the binding of ADP and Pi at an alternate site. Analysis of the distribution of 18O-labeled species arising from the ATP formed eliminates explanations for substrate modulation based on preexisting or induced enzyme heterogeneity. Furthermore, the results, together with other related findings, make participation of control sites unlikely. The occurrence of alternating site catalysis cooperativity in ATP synthesis by chloroplasts thus appears to be reasonably well established.


An adenovirus type 5 early gene function regulates expression of other early viral genes.

We have identified an adenovirus type 5(Ad5) early gene function located in early region 1 which is required for the production of early cytoplasmic mRNAs corresponding to early regions 2, 3, and 4. Mutant dl312 (lacks the segment between 1.5 and 4.5 map units) grows as well as wild-type  virus in 293 cells (Ad5-transformed human embryonic kidney cells), but its growth is severely restricted in HeLa cells. We detect no viral RNAs in the cytoplasm of dl312-infected HeLa cells. Viral RNA sequences are present, however, in dl312-infected HeLa cell nuclei.


Adenine aminohydrolase: occurrence and possible significance in trypanosomid flagellates.

Adenine aminohydrolase (EC 3.5.4.2) from four species of Leishmania and from Crithidia fasciculata was examined for specific activities, affinity for substrate (adenine), and stability to heat. All were found to be strongly and non-competitively inhibited by both coformycin and deoxycoformycin, two tight-binding inhibitors of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4). Deoxycoformycin is the more potent inhibitor of the two. Neither inhibitor was active against the purine phosphoribosyltransferases. When deoxycoformycin was added to the defined growth medium containing hypoxanthine as the purine source, the growth of C. fasciculata was unaffected, but when adenine was the purine source for the organism, severe inhibition resulted. This implies that hypoxanthine is the obligatory base for nucleotide synthesis and that the adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) is, in some manner,idenied access to exogenous substrate.


Proton nuclear magnetic resonance investigation of structural changes associated with cooperative oxygenation of human adult hemoglobin.

The structural changes associated with cooperative oxygenation of human adult hemoglobin as a function of oxygen saturation in aqueous media at neutral pH and at 25-27 degrees C have been investigated by high-resolution proton nuclear magnetic resonance spectroscopy at 250 and 360 MHz. By monitoring the intensities of two hyperfine shifted proton resonances (at about -12 and -18 ppm from H(2)O) and two exchangeable proton resonances (at about -6.4 and -9.4 ppm from H(2)O) as a function of oxygenation, the amount of oxygen bound to the alpha and beta chains of a hemoglobin molecule can be determined and the relationship between tertiary and quaternary structural changes under a given set of experimental conditions can be investigated. These results suggest that: (i) in the absence of organic phosphates, there is no preferential O(2) binding to the alpha or beta chains; (ii) in the presence of organic phosphates, the alpha hemes have a higher affinity for O(2) as compared to the beta hemes; (iii) the ligand-induced structural changes in the hemoglobin molecule are not concerted; and (iv) some cooperativity must be present within the deoxy quaternary state during the oxygenation process. The variations of the exchangeable proton resonances as a function of oxygenation strongly suggest that the breaking of one or more inter- or intrasubunit linkages of a ligated subunit can affect similar linkages in unligated subunits within a tetrameric hemoglobin molecule. Thus, the present results show that two-state allosteric models are not adequate to describe the cooperative oxygenation of hemoglobin. In addition, the present results provide direct correlation to the ligand-induced structural changes (such as in the heme pockets and subunit interfaces) observed to occur in the crystals of deoxy- and oxy-like hemoglobin molecules and in the solution state.


Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate.

We describe a technique for transferring electrophoretically separated bands of double-stranded DNA from agarose gels to diazobenzyloxymethyl-paper. Controlled cleavage of the DNA in situ by sequential treatment with dilute acid, which causes partial depurination, and dilute alkali, which causes cleavage and separation of the strands, allows the DNA to leave the gel rapidly and completely, with an efficiency independent of its size. Covalent attachment of DNA to paper prevents losses during subsequent hybridization and washing steps and allows a single paper to be reused many times. Ten percent dextran sulfate, originally found to accelerate DNA hybridization in solution by about 10-fold [J.G. Wetmur (1975) Biopolymers 14, 2517-2524], accelerates the rate of hybridization of randomly cleaved double-stranded DNA probes to immobilized nucleic acids by as much as 100-fold, without increasing the background significantly.


Titin: major myofibrillar components of striated muscle.

Electrophoretic analyses of protein components of striated muscle myofibril purified from various vertebrate and invertebrate species revealed that proteins much larger than myosin heavy chain are present in significant amounts. To define possible roles of these heretofore unidentified proteins, we purified a combination of two uncommonly large proteins, designated as titin, from chicken breast myofibrils. Chemical and immunological studies indicated that titin is distinct from myosin, actin, and filamin. Specific titin anti body crossreacts with similar protein in both skeletal and cardiac myofibrils of many vertebrate and invertebrate species. Immunofluorescent staining of glycerinated chicken breast myofibrils indicated that titin is present in M lines, Z lines, the junctions of A and I bands, and perhaps throughout the entire A bands. Similar staining studies of myofibrils from other species suggest that titinlike proteins may be organized in all myofibrils according to a common architectural plan. We conclude that titin is a structurally conserved myofibrillar component of vertebrate and invertebrate striated muscles.


Production and characterization of a monoclonal antibody to chicken type I collagen.

We have shown that lymphocyte-myeloma cell hybridization can be used to produce large amounts of extremely high-titer specific antibodies against type I collagen, a macromolecule normally of low immunogenicity. In a passive hemagglutination assay the antibody had a high titer against chicken type I collagen but showed no activity against chicken type II or rat type I collagen. By using a two-step fluorescence histochemical procedure on sections of embryonic chicken tibia, strong fluorescence was observed in the perichondrium and surrounding connective tissue (known to contain type I collagen) but not over the cartilage (characterized by type II collagen). When used in conjunction with Staphylococcus aureus as a solid phase immunoadsorbant, the antibody was shown to bind to labeled collagen synthesized in vitro by embryonic chicken calvaria.


Human leukocyte interferon: relationship between molecular structure and species specificity.

Human leukocyte interferon can be separated into two classes of subspecies by polynucleotide-agarose affinity chromatography; 30-40% of the molecular species have the polynucleotide-binding property and 60-70% lack affinity for the polynucleotide ligand. When analyzed on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the former class of interferon has a slower mobility corresponding to the migration of a polypeptide of 21,000 daltons, while the latter class has a faster mobility corresponding to a polypeptide of 13,500-15,000 daltons. By analogy to the behavior of other interferons and a class of nucleotidyl transferases on the polynucleotide-agarose chromatography, we suggest that the human leukocyte interferon having the polynucleotide-binding site is in a possibly "native" conformation and the loss of affinity for polynucleotide results from a degradative alteration of the native molecules. Moreover, the alteration of interferon is accompanied by an increase in heterospecific activity on bovine cells. It is suggested that the polypeptide domain responsible for species specificity may be closely related to the polynucleotide binding area. The modified interferon molecule, however, still conserves its antiviral activity. The simplicity and the high capacity of polynucleotide-agarose chromatography make this a powerful technique for the purification of interferon. The easy separation of these two classes of human leukocyte interferon makes the purification procedures more rational and will facilitate the preparation of both subspecies to a high degree of molecular homogeneity.


Platelet-derived growth factor: purification and partial characterization.

A cationic protein that stimulates DNA synthesis in human cultured cells was isolated from human platelets by ion exchange chromatography, hydrophobic chromatography, gel chromatography, and gel electrophoresis in sodium dodecyl sulfate. The electrophoretic behavior of biologically active or radioiodinated and reduced growth factor indicated that the native protein (approximately 30,000 daltons) was composed of two different polypeptides (approximately 13,000-14,000 and 16,000-17,000 daltons, respectively) linked via reduction-susceptible bonds. The stimulatory activity on human glial cells of the purified product at a concentration of approximately 4 ng/ml (0.13 nM) was equal to that of 1% human serum.


Covalently bound non-coenzyme phosphorus residues in flavoproteins: 31P nuclear magnetic resonance studies of Azotobacter flavodoxin.

In addition to the 5'-phosphate ester on its flavin mononucleotide (FMN) moiety, flavodoxin from Azotobacter vinelandii contains 2 moles of tightly bound phosphate. One non-coenzyme phosphate group is covalently bound to the protein, as it remains with the protein on acid precipitation, whereas the other phosphate is released. The invariance of the (31)P nuclear magnetic resonance chemical shift of the covalently bound phosphate (-0.8 ppm relative to 85% phosphoric acid) with pH, even in the presence of protein denaturants, implies it is in a diester linkage to the protein. Because no evidence could be found for the presence of covalently bound sugars, nucleotides, or phospholipids, it is suggested that the phosphate residue forms a diester linkage with two hydroxyl amino acids in the protein. The only other suggestion of a phosphodiester linkage in proteins is from previous studies on pepsin and pepsinogen [Perlmann, G. E. (1955) Adv. Prot. Chem. 10, 1-30]. The observed changes in (31)P chemical shift with pH show that the covalent phosphorus in pepsinogen has ionization properties of a monoester rather than a diester. The (31)P resonance of the FMN phosphate occurs at -5.6 ppm in native Azotobacter flavodoxin. No ionization of the protein-bound FMN phosphate is observed since the chemical shift does not change appreciably in the pH range of 5.5-9.5. The chemical shift data suggest, but do not prove, that the coenzyme phosphate in its protein-bound form is dianionic. Chemical analysis of several other flavoenzymes from a variety of sources shows the presence of covalently bound phosphorus in quantities stoichiometric with the flavin content in most of the enzymes tested. Thus, the presence of covalent phosphorus in flavoenzymes may be a general phenomenon with currently unknown catalytic significance.


The smallest genome RNA segment of influenza virus contains two genes that may overlap.

The genome of influenza virus consists of eight segments of single-stranded RNA, each of which encodes a different polypeptide. In addition to the eight recognized gene products, the virus specifies a distinct smaller nonstructural polypeptide (NS2), which is translated from a separate species of virus-specific mRNA. The location on the virus genome of the gene encoding this polypeptide was investigated by hybridization of the NS2 mRNA with isolated subgenomic RNA species, and by correlation of the inheritance of a strain-specific NS2 with inheritance of particular genome RNA segments during recombination between two different virus strains. The genetic information for NS2 was found to reside in the smallest genome RNA segment of the virion, which also encodes the NS1 polypeptide. Considering the sizes of the molecules involved, it is likely that the coding sequences for the two polypeptides overlap.


Immunological and chemical identification of a neurophysin-containing protein coded by messenger RNA from bovine hypothalamus.

The biosynthetic origin of the 10,000 molecular weight neurophysins, carriers of the peptide hormones oxytocin and vasopressin, has been studied by cell-free synthesis, Poly(A)-RNA was isolated from bovine hypothalamus and translated in a wheat germ system containing (35)S- or (3)H-labeled amino acids. A number of unique [(35)S]cysteine- but few [(35)S]-methionine-labeled proteins were coded by hypothalamic mRNA. A single, major, isotopically labeled protein (molecular weight 23,000-25,000) was immunoprecipitated from these translation mixtures by addition of purified antibodies against bovine neurophysin II and subsequent addition of Cowan I strain of Staphylococcus aureus. Specificity of the immunoprecipitation was demonstrated by competition with unlabeled authentic neurophysins and the absence of competition with structurally unrelated ovalbumin. Furthermore, neither nonimmune serum nor purified antibodies against ribonuclease immunoprecipitated the protein. The [(35)S]cysteine-labeled protein that was specifically immunoprecipitated was oxidized with performic acid and digested with trypsin in the presence of unlabeled, authentic bovine neurophysin II. Peptide mapping revealed that most of the major [(35)S]cysteine-labeled peptides (of the translation product) were identical to major cysteine-containing peptides of authentic neurophysin. The data show that hypothalamic mRNA directs the translation of several unique cysteine-rich proteins in an in vitro cell-free system. Furthermore, one of these proteins, which has a higher molecular weight than authentic neurophysin, is recognized by purified antibodies to bovine neurophysin II and has cysteine-containing tryptic peptides in common with those of authentic neurophysin. The data suggest that this protein is the primary translation product, pre-pro-neurophysin.


Identification of separate mRNAs coding for the alpha and beta subunits of thyrotropin.

A 9S mRNA, purified from mouse thyrotropic pituitary tumors by sucrose density gradient centrifugation of poly(A)-enriched mRNA, directed the synthesis of only alpha and beta subunits of thyrotropin in the reticulocyte lysate translation system. Analysis of radioiodinated 9S mRNA, repurified by oligo(dT)-cellulose chromatography and sucrose gradient centrifugation, yielded two species of RNA on urea/polyacrylamide gel electrophoresis. The major RNA species contained 620 nucleotides, and the minor RNA species contained 560 nucleotides. Unlabeled 9S mRNA was further purified by urea/polyacrylamide gel electrophoresis; the mRNAs were separately eluted from slices of the gel containing material migrating with an apparent length of 620 and 560 nucleotides. Translation of these mRNAs in the reticulocyte lysate showed that the longer mRNA coded for the alpha subunit and the shorter mRNA coded for the beta subunit of mouse thyrotropin. Because more alpha than beta subunit of thyrotropin was consistently synthesized, unbalanced amounts of thyrotropin subunits appear to be synthesized by translation of unbalanced amounts of individual mRNAs. We have demonstrated that the synthesis of thyrotropin is directed by two separate mRNA molecules, each coding for a different subunit of the hormone.