Color perimetry.

Color perimetry. Seven subjects were studied to determine the reproducibility of color isopters utilizing a Tubingen perimeter with targets equated for radiant energy and separate for heterochromatic flicker luminance. Achromatic threshold recognition of targets for equal luminance gave smaller isopters with longer wavelengths (red). Color recognition thresholds, on the other hand, showed large blue, midzone red and green, and small yellow isopters. The target recognition and color recognition thresholds for equal energy targets gave smaller red isopters. The data support Traquair's contention that all color isopters would be equivalent if hue, saturation, and intensity were equated. Clinically, the detection of subtle peripheral and central field defects might reside in the use of appropriately selected equally bright-colored targets.


Subretinal tracks in ophthalmomyiasis.

Subretinal tracks in ophthalmomyiasis. Three cases of presumed infestation of the human globe by the maggot of a true fly (order of Diptera) are presented. In each case, migration of the maggot through the subretinal space produced widespread ophthalmoscopic and fluorescein angiographic changes that are believed to be pathognomonic of subretinal ophthalmomyiasis. Despite extensive  changes in the retinal pigment epithelium, each of the three patients expressed few symptoms and manifested minimal visual deficit.


Sebaceous gland carcinoma of the eyelid. A 37-year old woman had received multiple radiation treatments between the ages of 6 months and 15 years for an extensive cavernous hemangioma of the left side of the face. At the age of 27 years, a biopsy specimen from a lesion of the left buccal commissure disclosed  squamous cell carcinoma arising in chronic radiodermatitis. Multiple cutaneous lesions were excised surgically, and extensive skin grafting of the left side of the face was performed. At the age of 35, she developed a tumor of the left upper and lower eyelids that extended into the orbit, which proved to be a sebaceous gland carcinoma. Following left orbital exenteration, the tumor promptly recurred in the socket. The patient died with widespread metastatic lesions nine months after exenteration. This case, which, to our knowledge, represents the fourth example of postradiation sebaceous gland carcinoma of the eyelids, differs from the previously reported cases in that the radiation therapy had been given for a benign cutaneous condition.


Further experience with the pinch technique for repair of eyelid deformities. The pinch technique has been found to be useful in repairing cosmetic eyelid deformities. However, the local anesthetic containing hyaluronidase must be injected only in small amounts and only into the subcutaneous space. Scar tissue and skin that is firmly adherent to underlying muscle do not yield a satisfactory ridge, and therefore, the pinch technique should not be used. Ectropion can be predicted by the observation of eversion of the lid margin when even only minimal skin is pinched, and impending ectropion can be discovered by our "lean forward and look up" maneuver. An ectropion repair can then be combined with the blepharoplasty surgical operation. The pinch technique has also been found useful when upper and lower blepharoplasties are joined laterally to elevate the lateral canthus and eliminate "crow's feet." One component of a repair of trichiasis also involves the use of the pinch technique.


Vitreous membranes. A light and electron microscopical study. Patients with pigmented vitreous membranes were subjected to partial vitrectomy, and the membranes were studied by light and electron microscopy. Two yellow ochre membranes from eyes removed at autopsy were also examined. The membranes were  composed of degenerating red cells and a delicate matrix of collagen. Phagocytic cells of uncertain origin and migrating retinal pigment epithelial cells were present in some of the membranes. Vascular channels were observed in only two cases, in which the membranes had clinical features different from those in the remaining cases. The development of new techniques of vitrectomy and the relatively normal clinical appearance of the posterior pole of the retina in these cases both suggest the potential clinical value of vitreous membrane excision. Additional histopathologic studies of vitrectomy specimens will enhance our understanding of the indications for this procedure.


Aqueous ascorbate concentration in hereditary buphthalmic rabbits. Some  aqueous humor constituents, including ascorbate, protein, and sodium, potassium, and calcium ions, were analyzed in three groups of adult buphthalmic (bu/bu) rabbits from strain AXBU/J and in control rabbits of the closely related strain AX/J. Aqueous humor ascorbate concentration of the normal strain was 22.5/"2.3mg/100ml. The buphthalmic rabbits that had no meaningful clinical signs of glaucoma at the time of sample collection had lower values (11.7/"1.7mg/100ml). Much lower values were observed in the bu/bu mutant rabbits that showed mild to severe glaucoma: 8.9/"3.8mg/100 ml and 1.9"/0.4mg/100ml, respectively. It was concluded that reduction of aqueous ascorbate is a biochemical  change associated with the pathological development of buphthalmia in rabbits of the AXBU/J strain. However, the exact mechanism is still unclear.


Electron microscopical study of the Fleisher ring. The  Fleischer ring of keratoconus was studied with the transmission electron microscope in four corneal buttons. The ring was characterized by accumulations of ferritin particles in the widened intercellular spaces and/or in the cytoplasmic vacuoles of the corneal epithelium. Both changes were prominent in basal layers in three cases; in one case, ferritin-containing vacuoles were noted in wing cell layers. Ferritin particles were also scattered over the corneal epithelium in all cases. For comparison, normal human corneas and conjunctivas were studied. Ferritin particles were scattered over the corneal epithelium and throughout the basal cells of the conjunctiva. They were not found in corneal stroma or endothelium. In conjunctival stroma, numerous ferritin particles were observed in the cytoplasm of some macrophages. Possible origin of these particles and the cause of their deposition are discussed.


Intraocular penetration of the soluble antiviral, Ara AMP. Earlier studies on intraocular penetration of the antiviral drugs idoxuridine and vidarabine (formerly Ara A) reveal that either inactive metabolites from the former or very low levels of less active metabolites from the latter actually enter the aqueous. Additionally, to achieve therapeutic levels, systemically administered vidarabine must be given in large fluid volumes because of poor solubility. The present time-curve study on the intraocular penetration of the highly soluble nucleotide form of vidarabine, Ara AMP, reveals that, in rabbits, very high aqueous levels of the antiviral metabolite, Ara Hx, are achieved after intravenous does in small fluid volume. Moderately low levels are attained after subconjunctival injection, extremely low levels are attained after subconjunctival injection, and extremely low levels are present after topical administration. This indicates that intravenous  and possibly subconjunctival injection may be useful in treatment of deep ocular herpetic disease.


Visolett magnifier. Evaluation and optics. The Visolett is a glass, nonspectacle magnifier that rests on the paper, producing 1.8X to 2.5X magnification. It is one of the most useful nonspectacle magnifiers, because it is simple to use and diffusely concentrates illumination on its own field, enhancing the magnification. The optics differ from usual hand magnifiers in that the magnification does not increase with the dioptric power.


Fundus photography without a fundus camera. A camera for fundus photography can be assembled from a standard indirect ophthalmoscope, a single-lens reflex camera, and a modest amount of easily available additional equipment. This camera can be used to record moderate detail of the posterior portion of the fundus and optic nerve head and can be used in an office, a hospital room, or an operating room.


Biomicroscopical choroidoscopy (uveoscopy) and transillumination gonioscopy. A fiber optic sleeve that fits over a Goldmann three-mirror lens, as well as an independent cable used in conjunction with the Goldmann lens, are described for transillumination biomicroscopical examination of the anterior chamber angle and the uvea. High-intensity retroillumination is provided to the anterior and posterior segments of the globe without production of heat to the eye. Present clinical uses as well as possible future research with the instruments are discussed.


[Experimental examinations in plate-prebending--a contribution to the biomechanics of compression osteosynthesis (author's transl)]. The rigid fixation of bone fragments by compression osteosynthesis with plate produces a high degree of resistance against bending forces, and over the surface friction against rotatory movements. In this way, a uniform distribution of pressure extending over the whole bone section is effected. But by virtue of the asymmetric positioning of the plate on bone-shaft, a torque effect is caused when strain is applied bringing about a distraction of the cortex on the side opposite the plate. Human femoral and tibial specimens would be osteomatized and then stabilized with AO plate adapted to conform to the surface of the bones, under a tension of 70 and 140 kp. With narrow plates having a inferior rigidity there is a greater distraction than is the cause with the wide plates. To compensate against the torque effect, the plates would be prebent. Two measuring methods would be investigate by which the correlation between prestraining and prebending could be determined. To achieve a constant distribution of pressure the narrow plates would have to be prebent to a greater degree than the wide ones, and the DC plates more than the standard plates.


[Release of antibiotics from bone cement and penetration to infected cortical bone (author's transl)]. Addition of antibiotics to bone cement is used more and more in orthopedics and traumatology. Indications are discussed: 1. infection prophylaxis in replacement arthroplasty, 2. therapy and recidive prophylaxis in infected arthroplasty, 3. therapy of chronic osteomyelitis. Our own experiments show release of antibiotics from bone cement Palacos and penetration into cortical bone after experimental osteomyelitis. Carbenicillin and Cephalotin show high level of concentration for 2 weeks, after 3 weeks there is no concentration evident. Lincomycin and Gentamycin in the first days show high tissue levels, after decrease and increase again after second week. Gentamycin was examined during 10 months showing high levels after this time.


Indomethacin-induced delayed fracture healing. A patient with an ankle joint fracture-dislocation was preoperatively treated with indomethacin. As in previous animal experiments, indomethacin was found to have an inhibitory effect on bone formation. It is suggested that indomethacin should be used with caution if periosteal fracture healing is anticipated.


[The dependence of the removal torque of a leg screw a screw surface and implantation time (author's transl)]. The experiment was performed on 17 sheep tibiae in vivo. 4.5 mm ASIF leg screws with a constant insertion torque (68.6 Ncm) but different surfaces (smooth or rough) were removed after 3 s, 7, 28, 56 and 112 days after insertion. The screws with a rough surface which were implanted for 28 days and longer exhibited a significantly greater removal torque. For instance rough screws which were implanted for 112 days displayed a removal torque eight times greater than after 3 s.


[Structure and solidity of new bone in a cortical gliding hole by implanted leg screw (author's transl)]. Leg screws were implanted in 30 tibia diaphysis. 112 days later in the gliding hole there was new lamellar bone round of the screw head, neck of screw and into the both first screw threads. After screw explantation we measured in 8 cases the solidity of the new bone. Under conditions of tension measurement, we use 341 N per millimeter cortical thread surfaces to put out all new bone from the gliding hole. The results of these tension measurements were cylindric fragments. The remodelling into the gliding hole seems to be the results of static stress and dynamic compensation for reaching a defect in living cortical bone.


[Hyperextending ruptures of vessels in course of arthroplasties of the hip and knee (author's transl)]. The authors referred specific complications in the course of arthroplasties of the hip and of the knee: hyper-extending-ruptures of vessels in sequence of indirect lesions. Causes of these so far not described complications are extreme passive articulations of hip respectively knee during operation. Clinical symptoms are enumerated, the pathogenetic mechanisms explicated and directions for treatment given. Important are the knowledge of these complications and the measure for their prophylaxis.


[Causes and treatment of delayed callusformation and pseudarthrosis following fractures of the shaft of the humerus (author's transl)]. In the OHH, during the years 1969 to 1974, 28 patients were operatively treated because of delayed callusformation or pseud-arthrosis, following a fracture of the shaft of the humerus. It was possible to inquire about and to re-examine 20 patients. The operative treatment had, when possible been achieved by osteosynthesis through plates (25 cases). Each of the so treated cases lead to healing of the bone, additional nerve lesion did not in one case occur because of the operation. Objectively seen, there existed in 7 patients a slight, final restriction of movements, for the abduction and outside rotation in the shoulder joint. 6 patients complained of being sensitive to weather changes. The different treatment method of the fracture of the humerus and pseudarthrosis was discussed.


[Chronic recurrent dislocation of the elbow joint (author's transl)]. This paper discusses 3 recurrent dislocations of the elbow joint. As possible cause for the etiology of the dislocation the author offers the post-traumatic flattening of the semilunar notch of the ulna. In 2 cases, a successful repair using the Kapel procedure was performed.


[Technique for allogeneic transplantation of knee joint (author's transl)]. Ever since Lexer's first attempts to remedy destroyed knee-joints by transplanting homioplastic material, the cup joint transplantation as developed by Pap and Krompecher has proved to be expedient and suitable thanks to the recent work performed by Wolkow. In order to assure that the process is safe from the mechanical standpoint, i.e. guarantee an accurate fit of the transplant to the recipient joint portion, in particular in the femur condyle area, the author has developed a method whereby the joint cups are cut rut of the donor material in accordance with pre-produced patterns and the oecipient joint portion is shaped to the correct form by means of similar patterns produced on the basis of X-rays.


[Clinical experiences with a new total wrist prosthesis (author's transl)]. For many individuals the preservation of wrist motion is essential for augmentation of fine motor control of the hand and fingers. When significant functional disability exists at the wrist, currently available surgical treatment includes soft tissue arthroplasty, carpectomy, or arthrodesis. A total wrist joint has recently been developed which offers the patient the potential of a pain-free, stable, and mobile wrist. Based upon the principles of total joint replacement as applied to the hip and knee, the prosthesis is secured to the carpus and radius with methylmethacrylate cement following removal of the navicular, lunate, and head of the capitate. The configuration of the prosthesis interface provides for the two planes of motion normally seen at the wrist; 90 degrees of flexion and extension and 50 degrees of radial ulnar deviation are permitted by the design. Candidates for total wrist replacement are patients who exhibit far advanced disease at the wrist and who might be considered for arthrodesis, but in whom the permanent loss of motion would represent a significant handicap. To date, 20 prostheses have been inserted in 17 patients. The longest follow-up is 14 months. All but 2 patients have gained a useful range of motion. All patients have noted dramatic relief of pain and deformity, permitting them to perform tasks which were preoperatively impossible.


[The importance, indication and operative technique of dorsal synovectomy of the knee joint in rheumatic diseases (author's transl)]. From a logical basis, it is reasonable to postulate that a synovectomy must be maximally as complete as possible. The indications and operative technique of radical synovectomy of the dorsal part of the knee joint are presented. The statistically significant improvement of the results of knee synovectomy by an additional dorsal approach is referred to.


[Vascular complications following alloplastic hip replacement (author's transl)]. Two patients with complications of the pelvic vessels have been seen at our institution after total hip replacement. One had a false aneurysmn of the deep femoral artery, the other presented an arterio-venous fistula. Causes, symptomes and diagnosis of vascular complications during hip replacement are reviewed.


[Differentialdiagnostic of the kneearthrography: bursa--ganglion--capsule rupture (author's transl)]. Based on 600 arthrogramms physiologic recessus is compared with ganglion for differential-diagnostic purposes. The great variance between small and big recessus of the regio genu posterior will be demonstrated. Flexion of the knee joint favour confusion with ganglion. The radiologic and differential diagnostic valuable criteria of the physiologic recessus and ganglion is shown. Finally the characteristics of Baker's cyst as well as posterior capsule rupture are discussed.


[Statistical analysis of congenital hip dysplasia is not possible in the first months of life! (author's transl)]. The question whether congenital hip dysplasia can be defined statistically has been raised again by Kristen, Dorda and Zweymüller. These authors believe that a definition is possible by statistical methods. Compiling a normal material for statistical evaluation, however, needs a clinical separation of normal and dysplastic joints before. And this--in our opinion--is not possible during the first half year of life. The only possible way is a long-term follow-up. In our own investigation children with higher acetabular indices have been followed up to 5 years and more. Only by this clinical evaluation is it possible to say which joints have developed normally and which have not. This is a clinical diagnosis of hip dysplasia, not a statistical.


[Scoring system for evaluation of severity and progress of chronic posttraumatic osteomyelitis (author's transl)]. From the anamnestic data of 50 patients suffering from chronic posttraumatic osteomyelitis a severity scoring system was developed, based on the duration of the disease, the time of hospitalization and the number of surgical interventions. The clinical course of 18 patients, undergoing an autovaccination treatment, was registered by another scoring system ("Munich-Murnau-Scale") regarding wound morphology, bone radiology and erythrocyte sedimentation rate.


Experimental evaluation of a new implant material in frontal sinus obliteration: a preliminary report. Frontal osteoplasty with exogenous material has been uniformly unsuccessful both experimentally and clinically. Our experiment was designed to test the long-term behavior of a new poly (tetrafluoroethylene)-carbon fiber implant material (Proplast) in a canine frontal sinus model. Varying conditions such as removal of the sinus mucoperiosteum, closure of the nasofrontal ducts, exposure of dura mater, and cerebrospinal fluid leaks were studied at intervals up to one year. Proplast was 100% successful in obliteration of the canine frontal sinus. The ultraporous nature allowed rapid vascularization, collagen ingrowth, and new bone formation, and led to stabilization rather than sequestration. Frontal obliteration with Proplast may be clinically superior to osteoplasty with any other presently available exogenous material or with osteoneogenesis alone, and may even obviate the few complications encountered with adipose implants. Only longer range experimental and clinical evaluation will provide the answer.


Dermoid cysts of the head and neck in children. The sporadic occurrence of several dermoid cysts of the head and neck in children led us to investigate the relative incidence in a series of 594 epidermal cysts in which 33 such lesions were found. We review the classification of dermoid cysts according to their embryogenesis and location to help avert diagnostic or therapeutic pitfalls. This study emphasizes the importance of adequate general anesthesia and excision of these interesting lesions.


When the weeping stopped: an otologist views otorrhea and gentamicin. Gentamicin sulfate ophthalmic solution, 0.3%, was used ototopically in the treatment of 300 patients with otorrhea associated with acute and chronic ear conditions and to obtain a preoperative "dry state" in 93 cases in which surgical operation was indicated. Bacterial cultures, antibiograms, and audiometric tests were conducted before, during, and after ototopical therapy. Gentamicin-susceptible pathogens were isolated in all cases. A totally "dry state", after three weeks of therapy, was considered evidence of clinical success. Administration of gentamicin was by means of a wick or at the rate of 0.3 to 0.4 ml twice daily. Clinical success was observed in 271 cases. There were no signs of ototoxicity and only one reported instance of skin reaction. This simple, inexpensive treatment is particularly adaptable to situations in which specialized bacteriological testing facilities are unavailable and a broad-spectrum antibiotic is indicated.


Investigation of the mesotympanum's reaction to silastic and gelatin film. The section of the cat's mesotympanum, denuded of mucosa, to Silastic and gelatin film was studied and compared with the contralateral control ear, which was also denuded of mucosa. The ears were studied by horizontal pathological sections taken one, two, four, and six months post-lympanotomy and insertion of either gelatin film or Silastic. The Silastic, gelatin film and control ears were compared for inflammatory reaction, amount of fibrosis, and the quality and quantity of mucosal regeneration. The inflammatory reaction was increased in the ears with Silastic compared with their corresponding control ears or to the ears with gelatin film. The amount of fibrosis and the quality and quantity of mucosal regeneration was essentially the same in the Silastic, gelatin film, and control ears. This study shows that both substances are well tolerated in the middle ear and that neither substance stimulated or inhibited the regrowth of mucosa or fibrous tissue when compared with the control.


Mycobacterium fortuitum mastoiditis.

Mycobacterium fortuitum mastoiditis. Improved techniques of bacteriologic identification have led to increasing recognition of the clinical significance of the atypical or anomymous mycobacteria. Mycobacterium fortuitum, included in group IV of Runyon's classification because of its characteristic rapid growth, is widespread in nature as a saprophyte. Its facultative pathogenicity has received increasing attention in the literature recently with reports of a number of isolated infections, epidemics, and deaths. We report a case of mastoiditis due to M fortuitum and discuss the pathogenesis and treatment. This is the first known report of the organism causing otolaryngologic disease.


Radiation-induced salivary gland tumors: report of a case. I discuss radiation-induced salivary gland tumors, with special emphasis on those tumors thought to be secondary to childhood head and neck irradiation for benign diseases. I report such a case and review the literature. Statistically, 77.6% of irradiation-induced tumors occur in the parotid gland and 22.4% in the submaxillary and minor salivary glands. A greater proportion of malignant tumors are noted in the submaxillary and minor salivary glands. At present, there is no demonstrable relationship between tumor occurrence and the amount of radiation recieved. Young children are more susceptible to irradiation-induced salivary tumors than older individuals.


Porencephalic cyst of the mastoid.

Porencephalic cyst of the mastoid. A 10-year-old boy developed a porencephalic cyst of the mastoid in conjunction with otitic meningitis and cerebrospinal otorrhea. A defect of the tegmen tympani allowed extrusion of the cyst into the antrum and the mesotympanum. The otorrhea was controlled by closure of the defect. Our review of 251 cases in the literature showed no prior incidence of mastoid involvement by a porencephalic cyst.


Metastatic adenocarcinoma of the temporal bone. Metastatic carcinoma involving the temporal bone is an unusual occurrence. A review of the world literature reveals a total of 102 reported cases. Secondary cancers of the temporal bone arise most frequently from mammary, renal, and bronchogenic carcinomas, all of which show a tendency to metastasize to bone. The pathogenesis of spread to the temporal bone is most commonly by the hematogenous route, but extension from intracranial involvement has also been noted. The symptoms of facial nerve paralysis, otalgia, and aural discharge are consistently associated with patients found to have aural cancers. These symptoms are, however, most frequently indicative of mastoid infection; it is therefore, imperative to consider the possibility of a malignant neoplasm in patients with these symptoms.


Perichondritis of the ear as a complication of acupuncture. A 54-year-old woman developed pain, tenderness, and swelling at the site where a short acupuncture needle had been placed in the helix of the pinna. Staphylococcus aureus was isolated from this LESION. Which was treated with removal of the needle and intravenously administered nafcillin. The patient recovered completely and had no scarring.


Factors influencing the choice of fixed-level ear in the ABLB test. The use of the worse ear as the fixed-intensity one in the ABLB test has been recommended. This recommendation has been criticised on three grounds. The present work studies these arguments, both theoretically and in light of recent experimental and clinical data, in terms of accuracy of results and of various operational and empirical factors. It is concluded that for achieving each loudness balance it is markedly better to follow the recommendation to keep the worse ear at a fixed intensity and to vary the intensities delivered to the better ear.


High-sulphur proteins from alpha-keratins. I. Heterogeneity of the proteins from mouse hair. The heterogeneity of the reduced and S-carboxymethylated high-sulphur protein fraction from mouse hair has been examined by chromatography and polyacrylamide gel electrophoresis at pH values above and below the isoelectric region. Considerable heterogeneity is observed both in size (molecular weight range 12000-45000) and in charge. Amino acid analysis of a number of column chromatographic fractions shows the high-sulphur proteins to be largely composed of proteins with a carboxymethylcysteine content above 25 residues and a pronounced heterogeneity in arginine content. Their chromatographic behaviour is similar to that observed for the ultra-high-sulphur proteins from wool.


Masking level difference: another tool for the evaluation of peripheral and cortical defects. Masking level difference (MLD) due to binaural unmasking was measured for speech signals (5-word meaningful sentences) masked by broad-band noise. Tests were carried out in a group of patients with unilateral cerebral lesions of vascular origin and apparently normal pure-tone audiograms (CNS patients), in a control group (normally hearing young adults) and in 5 other groups of patients (conductive symmetrical hearing loss, conductive asymmetrical hearing loss, bilateral presbyacusis, unilateral sudden deafness, Menière's disease). Testing pattern implied three or more S/N ratios in the listening conditions, of SmNm, Sdelta tNo and SmNu (noise correlated), and the speech signal intensity was 70 dB SPL re 20 muPa for the control group, whilst for the pathological cases, speech level intensities were established by means of alternate binaural loudness balance and simultaneous balancing median-plane localization procedures to assess subjective suprathreshold sound image localization at the midline. The results obtained in the control group and in the patients are discussed. With specific reference to the CNS patients, a statistically significant tendency was evident for the binaural condition to produce more MLD when the ear leading in time was ipsilateral to the normal hemisphere. The importance of this and of sensitized speech testing methods in CNS disorders is discussed on the basis of the results obtained in the CNS patients.


Solid-phase assay for luteinizing hormone in mouse plasma. A solid-phase tube assay for measuring LH levels in mouse plasma is described. The assay utilizes an antiserum to ovine LH and ovine LH standards and it measures LH levels in 20 mul of plasma with a sensitivity of less than 0.6 ng/ml. Various parameters affecting the sensitivity and specificity of the assay were investigated. Serial dilutions of plasma from pregnant mice, a pituitary homogenate from mice and plasma from hypophysectomized mice, injected subcutaneously with ovine LH, ran parallel with ovine LH standards in plasma from hypophysectomized mice and plasma with low LH levels from intact mice. Ovine TSH showed about 12% cross reaction in the assay system, whilst rat FSH and prolactin and also ovine FSH, prolactin and GH showed practically no cross reaction. Measurements of plasma LH levels have been made in hypophysectomzied mice after injection with different vehicles containing 10 or 50 mug LH or 50 mug FSH per animal. Daily measurements of LH levels throughout pregnancy in the mouse show a rise in LH level prior to implantationand a further rise around mid-pregnancy which drops off sharply to levels which remain fairly constant until parturition when there is another rise.


High-sulphur proteins from alpha-keratins. II. Isolation and partial characterization of purified components from mouse hair. The present paper continues the study of the reduced and S-carboxymethylated high-sulphur proteins from mouse hair. Fractions have been obtained in a substantially purified form by fractional precipitation with ammonium sulphate at pH 6, followed by ion exchange chromatography on cellulose phosphate at pH 2.6. Approximately 80% by weight of the high-sulphur proteins fall into the ultra-high-sulphur category (carboxymethylcysteine content greater than 26 residues per 100 residues), and they cover a molecular weight range of 17000-28000. The components show a remarkable diversity in amino acid composition; for example the contents of arginine and glycine each vary by about 3:1. The remainder of the proteins contain 17-20 residues per 100 residues of carboxymethylcysteine, are smaller in size (molecular weight 11500), and also show great diversity in overall amino acid composition. Molecular weights were determined by chromatography on controlled-pore glass and confirmed by gel filtration on Sephadex G-100 and Sepharose 6B. A comparison of the results suggests that the values obtained should be reliable to within 10%.


In vitro culture, storage and transfer of goat embryos. Goat embryos collected 5 and 7 days after mating, were cultured in vitro at 37 degrees C for 2 days, or stored at 5 degrees C for 1 or 2 days and then cultured for 2 days, or stored in liquid nitrogen (-196 degrees C) for 2-4 weeks and then cultured for 1 day. After culture some of the embryos were transferred to recipient does. Culture and storage was carried out in Dulbecco phosphate buffer enriched with 25% goat serum. 1M glycerol or 2M dimethylsulphoxide (DMSO) was added to the media used for frozen storage. Thirteen of 15 embryos cultured without prior storage showed apparently normal development in culture. Ten of the 13 were transferred and five kids were born. Twenty of 38 embryos stored at 5 degrees C developed in culture and six kids were born following the transfer of 17 embryos. Duration of storage at 5 degrees C had no marked effect upon subsequent development. Six of 48 frozen stored embryos developed in culture. All six were transferred and three kids were born.


The acoustic reflex at low sensation levels. The acoustic stapedial reflex, which normally occurs at a hearing level of about 80 dB, can be detected at lower levels by means of preactivation. Two techniques are presented, which allow the detection of reflex activity at intensities from 30 to 50 dB HL. It was the authors' intention to find a way to determine the hearing threshold from the stapedius reflex threshold even in patients, in whom the hearing threshold was not horizontal.


Repression, derepression and activation of nitrogenase in Azotobacter vinelandii. Ammonium ion repressed nitrogenase in cells fixing N2 gas. Immunological tests and electrophoresis in various gels show that components I (Fe-Mo-S protein) was completely repressed by ammonium, whereas component II (Fe-S protein) apoprotein was not markedly affected. Component II from ammonium-grown cells, however, was inactive since it did not cross react with component I to reduce C2H2 to C2H4. The inactive component II apoprotein is immunologically identical to its active counterpart from cells fixing N2. Identical protein patterns were also observed in various gel-electrophoresis systems. Oxygen-inactivated component II may be reactivated with FeSo4. This salt is preferable to ferrous ammonium sulphate which inactivated component I. Immunodiffusion under aerobic conditions shows that purified component I is composed of aggregated and non-aggregated forms which are antigenically distinct. The aggregate was dissociated by treatment with sodium dodecyl sulphate (SDS) into a single antigenic species which was further resolved into two subunits on SDS disc polyacrylamide gel electrophoresis.


Tracking of complex binaural images.

Tracking of complex binaural images. Subjects were presented with an initially stationary binaural image formed by the fusion of two identical pulses, without interaural time difference (ITD), at the two ears. The image was then made to traverse the subject's auditory perceptual space by introducing ITD, varying linearly with time, under computer control. The direction of movement, i.e. towards the right or left ear, could be reversed by the listener, by pressing a button. Subjects were requested to keep the image central, by pressing the button when they judged that deviation from subjective centre had occurred. Experiments of this type can be considered as analogous to Békésy audiometry, where the subject automatically traces his threshold of hearing, in that here the listener traces out his auditory perceptual centre as it varies with time. Hence, equivalent analyses to those employed for Békésy audiometry are possible. Subsequent to the initial part of the experiment, an additional pulse was added to one channel, preceding the original pulse, to form a pulse pair. The monaural masking of the original pulse by this additional pulse thus acts to shift the pre-existing binaural image. The effect of varying the amplitude and onset time of the masking pulse, relative to the original pulse, on the Békésy-type trace was examined.


[Early potentials evoked by acoustic stimuli: further results (author's transl)]. The research work carried out during the last years explains only partially the true nature of the short-latency (less than 50 ms) auditory evoked responses (PEP) in man. Widespread support is given to the hypothesis suggesting a double origin: myogenic and cortical with varying predominance between the two according to the position of the active electrodes and the stimulation parameters. PEP appears to have different characteristics according to the electrode coupling, i.e. inion-lobe, vertex-parietal, and vertex-mastoid. Experiments carried out on normally hearing subjects, on subjects with normal hearing but with marked vestibular signs, and on subjects with severe hearing loss and normal vestibular findings show that the presence of auditory function is one of the essential conditions for the generation of the PEP. Conversely, no influence on PEP is apparent following simultaneous and bilateral inhibition of the steady action potential of the ampullary nerve. Whichever the theoretical origin of the PEP is, it may be concluded that, since the results obtained during the clinical work are still incomplete and controversial, it is not possible to consider the PEP as a true expression of the vestibular function.


Production of endogenous acetate by the liver in lactating ewes. The production of endogenous acetate by the liver has been investigated in lactating ewes using animals with indwelling arterial, and portal and hepatic venous cannulae. The capacity of the liver to produce acetate from acetyl-CoA in vitro has also been examined using homogenates prepared from liver biopsy samples. Mean arterial, portal and hepatic venous blood acetate concentrations in four ewes at 4 weeks lactation were 0.40, 1.00 and 1.46 mM respectively. The mean exogenous and endogenous acetate production rates were 56 and 54 mmol/h respectively, giving a total of 110 MMOL/h. The mean portal-hepatic venous difference in free fatty acid concentration was 81 muM. Converting this uptake of free fatty acids by the liver (based on palmitate as a standard) to 2-carbon equivalents, the acetate produced accounted for 70% of the fatty acids taken up. The correlation coefficient (r2) between uptake of free fatty acids and production of acetate by the liver was 0.83 (P less than 0.01). Calculation of the net acetate production in vivo gave a mean value for the production of acetate of 0.75 nmol/min. Calculation of the in vitro enzymic capacity of the liver to produce acetate from acetyl-CoA gave a mean of 0.94 mmol/min. These results indicate that enzymic production of acetate from acetyl-CoA, via carnitine acetyltransferase and acetylcarnitine hydrolase (see Costa and Snoswell 1975a), can adequately account for the substantial production of acetate by the liver in lactating ewes.


Intensity and frequency discrimination monitored by evoked response audiometry: clinical application. Delta 1 and delta f repetitive stimuli superimposed on continuous 0.5-, 1- and 4-kHz carrier tones were administered to 20 normally hearing persons, 8 adults with sensorineural hearing loss, 10 adults with conductive hearing loss and 22 children subjected to evoked response audiometry (ERA) for assessment of hearing acuity. The intensity modulation depth ranged from + 1 to 10 dB. In the subjects with normal hearing and in the adult patients, three carrier tone levels were used whenever possible: 20, 40, and 60 dB SL. The frequency modulation depth ranged from -1 to -10% of the carrier tone frequency. The same levels for the carrier tone were used: 20, 40, and 60 dB SL. With a carrier tone level of 20 dB SL, a clear-cut vertex response was noted in 80% of the tests carried out in the normal adults with 5-dB delta i stimuli and with delta f stimuli of -3 to -5% in magnitude; the same figures are valid for the patients with sensorineural and conductive hearing loss. The patients with sensorineural hearing loss showed a tendency to give vertex responses with lower delta i stimuli than subjects with normal hearing. However, this difference was not statistically significant. According to our results, the children tested can be roughly divided into two groups, the first including subjects with responses to delta i stimuli of +5 and +10 dB and delta f stimuli of -5% and -10%; the second group including subjects with no clear-cut responses to these stimuli.


Studies on monotreme proteins. VII. Amino acid sequence of myoglobin from the platypus, Ornithoryhynchus anatinus. Myoglobin isolated from skeletal muscle of the platypus contains 153 amino acid residues. The complete amino acid sequence has been determined following cleavage with cyanogen bromide and further digestion of the four fragments with trypsin, chymotrypsin, pepsin and thermolysin. Sequences of the purified peptides were determined by the dansyl-Edman procedure. The amino acid sequence showed 25 differences from human myoglobin and 24 from kangaroo myoglobin. Amino acid sequences in myoglobins are more conserved than sequences in the alpha- and beta-globin chains, and platypus myoglobin shows a similar number of variations in sequence to kangaroo myoglobin when compared with myoglobin of other species. The date of divergence of the platypus from other mammals was estimated at 102 +/- 31 million years, based on the number of amino acid differences between species and allowing for mutations during the evolutionary period. This estimate differs widely from the estimate given by similar treatment of the alpha- and beta-chain sequences and a constant rate of mutation of globin chains is not supported.


Influence of tonic reflex activities on the slow auditory evoked potential. Possible influences of tonic reflex activities on the slow auditory evoked potential were investigated. For this purpose, the slow auditory evoked potential was picked up in 18 volunteers with normal hearing in relaxed position and in postures corresponding to reflex-induced positions and in 15 patients with cerebral palsy (CP) in tonic reflex patterns. No influence on latency for N120 and P215 due to postures could be observed. Latency within CP patients was age-dependent. The effect of the reflex-inhibiting pattern (RIP) - the pattern inhibiting tonic reflex activity in CP patients as far as possible - on potential amplitude was different in the two groups. The effect of RIP in CP patients was comparable to the relaxed position in volunteers. This difference points toward an influence of tonic reflex activity on the slow auditory evoked potential.


Haemoglobins of the shark, Heterodontus portusjacksoni II. Amino acid sequence of the alpha-chain. The amino acid sequence of the alpha-chain of the principal haemoglobin from the shark, H. portusjacksoni has been determined. The chain has 148 residues and is acetylated at the amino terminal. The soluble peptides obtained by tryptic and chymotryptic digestion of the protein or its cyanogen bromide fragments were isolated by gel filtration, paper ionophoresis and paper chromatography. The amino acid sequences were determined by the dansyl-Edman procedure. The insoluble "core" peptide from the tryptic digestion contained 34 residues and required cleavage by several prosteases before the sequence was established. Compared with human alpha-chain there are 88 amino acid differences including the additional seven residues which appear on the amino terminal of the shark chain. There is also one deletion and one insertion. The chain contains no tryptophan but has four cysteinyl residues which is the highest number of such residues recorded for a vertebrate globin.  In the alpha1beta1 contact sites there are four changes in the oxyhaemoglobin form and six deoxy form. Nine of the 16, alpha1beta1 contact sites show variation while three of the haem contact sites have changed in comparison to the residues known to be involved in these interactions in horse haemoglobin alpha-chain. Use of the sequence data to estimate a time of divergence of the shark from the main vertebrate line yielded the value of 410 +/- 46 million years. The data, in general, support the palaeontological view that bony fishes arose before the elasmobranchs.


Insect-control chemicals from plants. III. Toxic lignans from Libocedrus bidwillii. Feeding tests showed that the powdered dried leaves and leaf extracts of L. bidwilli are toxic to the larvae of the housefly (Musca domestica), and the codling moth (Laspeyresia pomonella). The powdered material was not toxic to the light-brown apple moth (Epiphyas postvittana). The most active toxin is the lignan beta-peltatin-A methyl ether (II) and at a concentration of 100 ppm in a chemically defined diet it gave 98% mortality of housefly larvae.


Pulmonary diseases of cattle in the Kimberley District of Western Australia. A survey of pulmonary disease in cattle slaughtered in the Kimberley district of Western Australia, incidental to the monitoring of lungs for contagious bovine pleuropneumonia (CBPP), was initiated in 1969. The results of the first 3 years are reported and indicate the complete absence of CBPP, the occurrence of actinobacillosis as the most common pulmonary lesion, the existence of phycomycosis in a few lungs each year, the frequent presence of subacute and chronic bronchopneumonias and interstitial pneumonias possibly as sequelae of actinobacillosis, tuberculosis and the pneumomycoses, and the relatively low incidence of fibrinous and necrotic pneumonias of other bacterial origin.


Vaccination of lambs against infection with Taenia ovis. A trial was designed to test the efficacy of vaccination of lambs against field challenge with T. ovis eggs using 3 different levels of pasture contamination. Three concentric paddocks (1.62 hectares each) were used and the inner paddock was heavily contaminated with T. ovis eggs by tethering 2 dogs, experimentally infected with T. ovis, at 5 different points in the paddock over a period of 1 month. Four groups of lambs were used. Sixty lambs, from a property known to be free of T. ovis for 2 years previously, were divided into 2 groups of 30 each; one group was vaccinated with T. ovis culture antigen (vaccinated group=V) while the other was vaccinated with culture medium without antigen (sham-vaccinated group=SV). Thirty mature sheep were obtained from a property with a recent record of T. ovis infection (previous natural exposure to infection group=PNE) and 15 lambs were artificially reared from birth under cestode-egg-free conditions (artificially reared groups= AR). Ten animals from groups V, SV, and PNE, and 5 AR lambs were grazed on each of the 3 concentric paddocks for 6 weeks, and then all animals were slaughtered and their total muscle mass (including heart, diaphragm, tongue and masseters) examined, by slicing, for T. ovis cysticerci. The mean total numbers of T. ovis cysticerci recovered were as follows: Inner paddock, PNE 45.5, AR 47.6, SV 107.8, V 6.5; Middle paddock, PNE 5.2, AR 9.0, SV 8.2, V 0.3; Outer paddock, PNE 2.9, AR 2.8, SV 2.3, V 0.2. There were 4 major conclusions from this study. Firstly, vaccination was more effective than previous natural exposure to infection in preventing establishment of new cysticerci. Secondly, although vaccination effectively reduced the numbers of cysticerci, those that became established were mostly viable at the time of slaughter. In contrast, PNE sheep had greater numbers of cysticerci but the majority were degenerating. Vaccination, therefore, did not seem to stimulate a complete immunological response. Thirdly, the presence of T. hydatigena in lambs did not prevent subsequent infection with T. ovis. Fourthly, T. ovis eggs were dispersed from the inner paddock to the outer paddock.


Genetic resistance to Marek's disease.

Genetic resistance to Marek's disease. A programme to control MD by genetic selection was tested by selecting breeding sires and dams whose progeny were found to have above average resistance to MD following inoculation at day old with infectious material. Under conditions of natural exposure, Australorp pullets from parents selected for resistance had a lower incidence of MD lesions than an unselected Australorp group. In experimentally challenged Australorp chickens the incidence of MD was significantly lower in progeny derived from mating resistant Australorp sires with unselected dams than in progeny from an unselected Australorp line. Under conditions of experimental and natural exposure to MD, crossbred chickens derived from resistant White Leghorn sires and Australorp dams had a lower incidence of MD lesions than the chickens derived from susceptible White Leghorn series and unselected Australorp dams.


Gastro-intestinal parasites of feral cats in New South Wales. A total of 146 feral cats from several localities in New South Wales were examined for the presence of gastro-intestinal parasites. The cestodes recorded, and their prevalence, were Taenia taeniaeformis (21.9%), Spirometra erinacei (61.0%) and Dipylidium caninum (11.6%). One animal contained 31 T. serialis scolices considered to be the contents of a recently ingested Coenurus serialis. The common nematodes encountered and their prevalence were Toxocara cati (21.9%) and Ancylostoma spp (23.3%). Uncinaria stenocephala were found in 2 cats and a stomach wall tumour containing Cyathospirura dasyuridis was found in 1 cat, An acanthocephalan resembling Oncicola canis was the most prevalent parasite among feral cats in this survey. High burdens of S. erinacei, D. caninum, Ancylostoma spp and Oncicola sp were found. Samples from 4 localities in New South Wales were compared and there were major differences in the prevalence of T. taeniaeformis, D. caninum and T. cati. Ancylostoma spp occurred only in the Sydney (53.2%) and the Warren (4.8%) areas. Oncicola sp was not present in the Sydney area, but occurred in the Young area in 23.5% of the feral cats examined and in the Warren area (33.3%) and was most prevalent in the Menindee area (79.2%).


The comparative incidence of foot rot in Bos taurus and Bos indicus cattle. Data on incidence of foot rot were collected over 2 years in a mixed herd of both Bos taurus and Bos indicus breed types. The level of infection was significantly higher in the Bos taurus breeds than in the Bos indicus breeds and raises the possibility of using resistant breeds where foot rot constitutes a major economic problem.


Dermal ulceration of mullet (Mugil cephalus). Phycomycotic granulomas are described in the skin of 7 mullet (Mugil cephalus) from extensive outbreaks of dermal ulceration in fish in south-east Queensland. The fungus involved was probably a member of the Family Saprolegniaceae, but was not cultured from the lesions. A myxosporidian infection was present in some fish. The primary cause of the disease is uncertain.


Effect of furazolidone in feed on immune response of turkeys vaccinated with live Pasteurella multocida in drinking water. In 2 trials, turkey poults were fed diets containing 0.011% furazolidone: in one trial until 2, 3, 4, 5, 6, or 7 days before vaccination with live Pasteurella multocida (CU) in the drinking water; and in a second trial with the furazolidone added to the diet 4 days before the vaccination or 1, 3 or 5 days after. This 0.011% level of medication did not appear to reduce the immune response.


Isolation of adenoviruses and reoviruses from avian species other than domestic fowl. Adenoviruses and reoviruses were isolated from pigeons and mallard ducks. In addition, adenoviruses were isolated from budgerigars and a bantam and a reovirus was isolated from a turkey. Primary identification of these viruses was by electron-microscope examination. It was further possible to assign the 4 adenoviruses to recognized fowl serotypes, and the reoviruses shared a common antigen with fowl reoviruses. These viruses were isolated from a variety of clinical conditions.


Further observations on the use of a bivalent bacterin against Haemophilus gallinarum. Chickens vaccinated with two doses of a bivalent Haemophilus gallinarum bacterin were protected against seven strains of the organism. Vaccinated and unvaccinated birds with antibody to Mycoplasma gallisepticum had increased HI titers when challenged with H. gallinarum. Birds positive for antibody to Mycoplasma gallisepticum which were not challenged showed no increase in HI titer.


Congenital anomalies of domestic fowl: seven cases. In 13,483 necropsies of domestic fowl, six anomalies were recorded: bifurcation of the metasternum, duplication of the ileum, pancreas divisum (partial), vesica fellea occulta, aplasia of the left cecum, and aplasia of the right kidney (2 cases).


Isolation of Mycobacterium avium serotype 3 from a white-headed tree duck (Dendrocygna viduata). Tuberculous lesions were observed at necropsy in the liver of 1 of 10 White-headed Tree ducks (Dendrocygna viduata) imported from Nigeria. Microscopic examination revealed granulomas with acid-fast bacilli; Mycobacterium avium serotype 3 was isolated. Two chickens inoculated intraperitoneally with the isolant had gross and microscopic granulomas in the liver at necropsy 62 days after inoculation. Isolants from chickens were serologically similar to the strain isolated from the duck.


Air sac and renal mucormycosis in an African gray parrot (Psittacus erithacus). A case of mucormycosis in an African gray parrot (Psittacus erithacus) involving air sac and kidney is described. The causal fungus, Absidia corymbifera, had produced sporangia in the air-sac lesion.


An outbreak of trematodiasis in domestic geese. Losses greater than 3% were incurred in a large flock of domestic geese on summer pasture. Necropsy of the sick birds revealed no significant bacterial or viral isolates. The trematodes Echinostoma revolutum and Notocotylus attenuatus were present in considerable numbers, respectively in the small and large intestines.


Enhancement of hemagglutinating activity of Haemophilus gallinarum by trypsin. It is shown that hemagglutination (HA) activity of Haemophilus gallinarum is enhanced by trypsin. HA activation was more effective at 37 C than at room temperature.


Neuropsychological servosystems, consciousness, and the problem of embodiment. Recent work in neurophysiology has begun to close the paradigmatic gap between neurophysiology and the psychology of embodied behavior and movement. The present paper surveys these developments, traces the origins of the split between neurophysiology and neuropsychology and suggests that recent work by Pribram may provide a link between the psychology of consciousness, the neurophysiology of movement, and the growing body of work on nonverbal communication.


[Influence of autolysis on the quantitative cytoarchitecture of rat hepatocytes. (An ultrastructural morphometric study) (author's transl)]. Autolysis is very often a reason for cell damage and is also super-imposed on many other cell damages. Already one hour of autolysis causes serious changes in cell metabolism, which can be demonstrated morphometrically. Experiments were made with 10 male adult Wistar rats. 5 animals were for control purposes and 5 animals had to undergo a 1-hour's autolysis. The morphometric analysis of the liver parenchymal cells was based on the information of Weibel et al. (1968) with the help of a computer program. The consequence of the drop in energy caused by autolysis is an enlargement of the liver cell which is possibly due to a breakdown of the energy-dependent ionic pumps. One of the earliest observable cell changes is a so-called "Kernwandhyperchromatosis" and chromatin condensation within the nuclei, whereby the Kernwandhyperchromatosis is seen to be a direct consequence of the increase in lactate and decrease of pH. ATP-sufficiency causes a disturbed function of mitochondrial membranes. The microchondria are swollen, the number of mitochondrial grana is clearly reduced. An enlargement of the mitochondrial outer membrane takes place by folding while the surface of mitochondrial cristae remains unchanged. As a consequence of the altered membrane activities also the peroxisomes swell at reduced numerial density. At unchanged total volume of RER the surface of the granulated membranes of the RER decrease by 50%. This decrease caused by ribosome detachment of the granulated membranes corresponds to the enlargement of the degranulated membrane parts of the nedoplasmic reticulum. The vesiculation is caused by an unspecific damage of cytoplasm. While the density of its volume and the membrane surface remain unaltered, the SER also shows a tendency to small vesiculation caused by an unspecific damage of cytoplasm. The increase in number and volume of the lysosomes and vacuoles of unknown origin speaks for a lysosomal activity. The cell compartment responsible for protein synthesis shows the most impressive morphometric and morphologic changes, which eventually can be explained by a decrease of protein synthesis which is needed to obtain enough energy for a well operating physiological equilibrium.


Paneth cell tumor. An additional case of duodenal adenoma with malignant change. A papillary adenoma with malignant change consisting of only Paneth cells and their precursors is described. Neoplastic proliferation of Paneth cells is discussed from a point of view of cell biology.


Variations in rat liver chromatin composition during growth and the heterotic response. The manifestation of hybrid vigor with regard to the inheritance of body weight was established as a postweaning phenomenon in rats. Liver weight gain generally corresponded to body weight gain and also reflected hybrid vigor. The protein/DNA ratios of the chromatin from livers of inbred and heterotic hybrid male rats were found to increase with increasing age in growing rats. Hybrid chromatin had higher protein/DNA ratios throughout the age range studies. The difference between hybrids and inbreds were greatest in the early postweaning stages of growth and maturation. The RNA/DNA ratios of chromatin from livers of inbred and hybrid male rats were also found to exhibit trends within certain developmental periods.


Gene action in fish of tetraploid origin. IV. Ribosomal DNA amount in clupeoid and salmonoid fish. Phylogenetically tetraploid species of the fish order Isospondyli generally have twice the mean ribosomal gene content as closely related species on the phylogenetically diploid level. Considerable intraspecific variation of rDNA amount was observed. These findings are discussed in view of the hypothesis that selective loss of ribosomal genes may account for diminishing genic activity in phylogenetically tetraploid organisms.


Allelic polymorphism in arabian camel ribonuclease and the amino acid sequence of bactrian camel ribonuclease. Pancreatic ribonucleases from several species (whitetail deer, roe deer, guinea pig, and arabian camel) exhibit more than one amino acid at particular positions in their amino acid sequences. Since these enzymes were isolated from pooled pancreas, the origin of this heterogeneity is not clear. The pancreatic ribonucleases from 11 individual arabian camels (Camelus dromedarius) have been investigated with respect to the lysine-glutamine heterogeneity at position 103 (Welling et al., 1975). Six ribonucleases showed only one basic band and five showed two bands after polyacrylamide gel electrophoresis, suggesting a gene frequency of about 0.75 for the Lys gene and about 0.25 for the Gln gene. The amino acid sequence of bactrian camel (Camelus bactrianus) ribonuclease isolated from individual pancreatic tissue was determined and compared with that of arabian camel ribonuclease. The only difference was observed at position 103. In the ribonucleases from two unrelated bactrian camels, only glutamine was observed at that position.


Strain distribution and linkage relationship of a mouse embryonic hemoglobin variant. Search for structural variants of three globin chains (x, y, z), synthesized only during mouse embryonic hematopoiesis, was carried out by electrophoretic analysis of blood from 12-day embryos, all with C57BL/6 mothers, and fathers from 115 inbred stocks selected for their diverse genetic origins. Structure of the beta-chains of adult hemoglobins differed among the tested strains, with 57 carrying the Hbbs allele, 56 the Hbbd allele, and two the Hbbp allele. The search revealed no x- or z-chain variants but confirmed and extended knowledge of a previously described y-chain variant. Blood of all embryos sired by males from the 57 Hbbs strains contained only y'-chains, while blood of au embryos sired by Hbbd or Hbbp males contained y2-chains as well as the y1-chains inherited from their C57BL/6 mother. The locus controlling structure of the y-chain of mouse embryonic hemoglobins is thus extremely closely linked to the locus controlling structure of adult hemoglobin beta-chain, with maximum possible recombination frequency less than 0.019.


Genetic and developmental variation of hemoglobin in the deermouse, Peromyscus maniculatus. A genetic investigation of electrophoretic hemoglobin variants of the deermouse, Peromyscus maniculatus, shows three alleles, Hblf, Hblr, and Hblo, at a duplicated site controlling the six adult phenotypes. The Hblf allele has not been described previously. The hemoglobin locus is not closely linked to the albino locus. Fetal hemoglobin is distinct from any of the adult components and has a slower electrophoretic mobility. The fetal phenotype changes to the adult type between the days 15 and 18 of prenatal life.


Isolation, determination of structure and synthesis of the acid-labile conjugate of aldosterone. 1. After administration of 600mg of 3H-labelled aldosterone to human volunteers, 57 mg of homogeneous acid-labile conjugate was isolated from the urine and identified as aldosterone 18 beta-D-glucosiduronic acid. 2. Esterification and acetylation of the conjugate gave a tetra-acetate methyl ester, which, by measurement of the optical rotation and nuclear-magnetic-resonance spectrum, was shown to be a beta-glucosiduronate. This tetra-acetate methyl ester was synthesized in approx. 10% yield by the Koenigs-Knorr procedure. 3. Removal of the acetyl and methyl ester groups from the tetra-acetate methyl ester with alkali was accompanied by almost complete isomerization at C-17 to give 17-isoaldosterone 18 beta-D-glucosiduronic acid. 4. To prevent inversion at C-17 during removal of the acetate and ester groups of beta-glucosiduronate (a) the 3,20-disemicarbazone was prepared, (b) the acetate and ester groups were removed from the disemicarbazone by treatment with alkali, and (c) the semicarbazone groups were removed from the product at pH 2.0, and aldosterone 18 beta-D-glucosiduronic acid was obtained in 47% overall yield. 5. In the presence of components used to synthesize beta-glucosiduronate by the Koenigs-Knorr reaction this substance is converted slowly into the alpha-glucosiduronate; this conversion is responsible, in part, for the low yield of beta-glucosiduronate. 6. Two additional conjugates were obtained in the Koenigs-Knorr reaction; a provisional structure was assigned to one substrate. The other substance is a C-18 alpha-glucosiduronate. Removal of the acetyl and ester groups from C-18 alpha-glucosiduronate gave the alpha-glucosiduronic acid in 84% yield and the 17-isoaldosterone alpha-glucosiduronic acid in 12% yield. 7. The rate at which several types of beta-glucuronidase hydrolyse the foregoing steroidal alpha- and beta-glucosiduronic acids is given.


The role of tetrahydrofolate dehydrogenase in the hepatic supply of tetrahydrobiopterin in rats. The reduction of 7,8-dihydrobiopterin to 5,6,7,8-tetrahydrobiopterin by rat liver tetrahydrofolate dehydrogenase (5,6,7,8-tetrahydrofolate-NADP+ oxidoreductase, EC 1.5.1.3) is competitively inhibited by trimethoprim lactate (apparent Ki 0.285 muM). An apparent Michaelis constant of 43 muM for dihydrobiopterin was obtained, which is 430 times higher than the reported Km for dihydrofolate with this enzyme. The reduction of dihydrobiopterin is thus more susceptible to inhibition by trimethoprim lactate than is the reduction of dihydrofolate. However, intraperitoneal administration of trimethoprim had no significant effect on the hepatic supply of tetrahydrobiopterin in rats.


Studies on the cholesterol ester hydrolase of Trogoderma (Coleoptera). The enzyme cholesterol ester hydrolase (EC 3.1.1.13) was detected in the larvae of the khapra beetle, Trogoderma granarium (Everts). The pH and temperature optima for the enzyme were 6.6 degrees and 37 degrees C respectively. The mol.wt. of the enzyme was 76000-80000. The enzyme was equally effective in hydrolysing cholesteryl acetate, stearate and oleate. Cholesterol derivatives, namely the chloride and the methyl ether, inhibited the enzyme activity almost completely. It was also inhibited completely by p-hydroxymercuribenzoate. This inhibition was reversed by the addition of dithiothreitol, reduced glutathione or cysteine. The enzyme activity was associated predominantly with the 104000 g fraction.


Dipeptidyl peptidase IV, a kidney brush-border serine peptidase. Dipeptidyl peptidase IV, an enzyme that releases dipeptides from substrates with N-terminal sequences of the forms X-Pro-Y or X-Ala-Y, was purified 300-fold from pig kidney cortex. The kidney is the main source of the enzyme, where it is one of the major microvillus-membrane proteins. Several other tissues contained demonstrable activity against the usual assay substrate glycylproline 2-naphthylamide. In the small intestine this activity was greatly enriched in the microvillus fraction. In all tissues examined, the activity was extremely sensitive to inhibition by di-isopropyl phosphorofluoridate (Dip-F), but relatively resistant to inhibition by phenylmethylsulphonyl fluoride. It is a serine proteinase which may be covalently labelled with [32P]Dip-F, and is the only enzyme of this class in the microvillus membrane. The apparent subunit mol.wt. estimated by sodium dodecyl-sulphate/polyacrylamide-gel electrophoresis and by titration with [32P]Dip-F was 130 000. Gel-filtration and sedimentation-equilibrium methods gave values in the region of 280 000, which is consistent with a dimeric structure, a conclusion supported by electron micrographs of the purified enzyme. Among other well-characterized serine proteinases, this enzyme is unique in its membrane location and its large subunit size. Investigation of the mode of attack of the peptidase on oligopeptides revealed that it could hydrolyse certain N-blocked peptides, e.g. Z-Gly-Pro-Leu-Gly-Pro. In this respect it is acting as an endopeptidase and as such may merit reclassification and renaming as microvillus-membrane serine peptidase.


The separation and characterization of the methylumbelliferyl beta-galactosidases of human liver. 1. A previously uncharacterized form of human liver acid beta-galactosidase (EC 3.2.1.23), possibly a dimer of molecular weight 160 000, was resolved by gel filtration. It has the same ability to hydrolyse GM1 ganglioside as the two other acid beta-galactosidase forms. 2. The low-molecular-weight forms of acid beta-galactosidase undergo salt-dependent aggregation. 3. The high-molecular-weight component may consist of the low-molecular-weight forms bound to membrane fragments. It can be converted completely into a mixture of these forms. 4. The neutral beta-galactosidase activity can be resolved into two forms by DEAE-cellulose chromatography. They differ in their response to Cl-ions. 5. A new nomenclature is suggested for the six beta-galactosidases so far found in human liver. 6. The enzymic constituents of the beta-galactosidase bands resolved by electrophoresis were re-examined. The A band contains three components. A two-dimensional electrophoretic procedure for resolving the A band is described. 7. The effect of neuraminidase treatment on the behaviour of beta-galactosidases in various separation systems is examined.


A steady-state kinetic study of the reaction catalysed by the secondary-amine mono-oxygenase of Pseudomonas aminovorans. 1. Secondary-amine mono-oxygenase (proposed EC group 1.14.99.-) was partially purified from trimethylamine-grown Pseudomonas aminovorans by (NH4)2SO4 fractionation, gel filtration, hydrophobic chromatography on 5-aminopentylamino-Sepharose, and affinity chromatography on Sepharose-bound NADH. 2. Some problems in the affinity-chromatography step are discussed. 3. A steady-state kinetic analysis varying substrate, oxygen and electron-donor concentrations was performed, which, over the concentration range studied, gave a series of families of approximately parallel double-reciprocal plots. From secondary and tertiary plots, Michaelis constants of 0.160 mM, 0.086 mM and 0.121 mM were obtained for dimethylamine, NADPH and oxygen respectively. 4. Product-inhibition studies supported the postulated Hexa Uni Ping Pong (triple-transfer) reaction mechanism.


Spectroscopic studies of the binding of bilirubin by ligandin and aminoazo-dye-binding protein A. Ligandin and aminoazo-dye-binding protein A both bind bilirubin at a single site. Quantitative studies of the interactions using difference spectrophotometry show that at pH 7.0, protein A binds the tetrapyrrole with an association constant (K) greater than or equal to 2 X 10(7) litre/mol, whereas binding by ligandin is slightly weaker (K = 7 X 10(6) litre/mol) at this pH. The protein-bilirubin complexes give rise to absorption and fluorescence spectra quite different from those of unbound bilirubin and also to large Cotton effects. It appears that on binding to both proteins, the ligand is forced into a rigid twisted configuration in a hydrophobic environment. Ligandin and protein A resemble serum albumin in their interactions with bilirubin.


Creatine kinase. Modification of the working enzyme. Protection against inhibition of creatine kinase by iodoacetamide is measured by the decrease in the rate constant for the inhibition reaction; A mixture of purified substrates at equilibrium protects quite strongly when all the components of the mixture are nearly saturating. The protection by substrates 'working' in the forward direction only (from creatine and MgATP) was measured by carrying out the experiment rapidly at low concentrations of the enzyme; by varying the concentration of substrate it was found that the amount of protection when the substrates of the forward reaction are saturating is about 80% (100% protection would imply a value of zero for the rate constant of the inhibition reaction). The effects of Ca2+ and Mg2+ are compared. It is already known that the complex creatine-NO3--MgADP, which is considered to be either a transition-state analogue or an analogue of an intermediate in the reaction pathway, protects fully against iodoacetamide, whereas creatine and MgADP alone, or together without NO3-, do not protect. This suggests that the degree of protection by the working enzyme represents the proportion of enzyme molecules that have a conformation complementary to a creatine-PO3-MgADP intermediate.


Presence of type III collagen in guinea-pig dermal scar. Guinea-pig dermal scar was shown to contain type III collagen, and, from densitometric analysis of gel electrophoretograms, it was shown to have a higher concentration than the surrounding dermis. This finding is consistent with the 'embryonic' nature of newly formed dermal wound tissue, reflected in increased hydroxylation of collagen lysine and the presence of dihydroxylysinonorleucine (after reduction) as the major cross-link.


Types I and II collagens in intervertebral disc. Interchanging radial distributions in annulus fibrosus. Intervertebral disc is a highly specialized cartilaginous tissue, containing two genetic types of collagen (I and II). Analysis of peptides from a CNBr digest of collagen showed that the proportions of I and II varied gradually and inversely across pig annulus fibrosus, with exclusively type I at the extreme outer edge and exclusively type II in the nucleus pulposus.


Elongation of (omega-14C)oleic acid and (omega-14C)nervonic acid. During feeding experiments with [omega-14C]oleic acid and [omega-14c]nervonic acid to adult rats, 14C-labelled C26, C28 and C30 fatty acids were recovered from the intestinal mucosa, liver, plasma, kidney and stools. The structures of these fatty acids were determined by g.l.c., radio-g.l.c. and mass spectrometry. The Schmidt and Ginger degradation methods indicated that most of the 14C found in these extra-long fatty acids remained in the omega position. These radioactive extra-long fatty acids were found mainly in the polar lipids of rats killed 3 or 15 h after being fed on labelled oleic acid or nervonic acid. Rats killed 63 h later yielded only traces of these extra-long fatty acids. When the rats were given antibiotics or received the same radioactive fatty acids by intravenous injection, the labelled extra-long fatty acids could not be detected in any of the tissues. We conclude that they were probably synthesized by elongation of oleic acid and nervonic acid by intestinal micro-organisms (probably yeasts) and then absorbed by the intestinal mucosa.


Higher-plant mitochondrial ribosomes contain a 5S ribosomal ribonucleic acid component. Ribosomes from higher-plant mitochondria contain 5S rRNA, in contrast with the mitochondrial ribosomes of animals and fungi, in which such a component has not been detected. In common with the ribosomes of prokaryotes and chloroplasts, higher-plant mitochondrial ribosomes do not appear to contain an RNA equivalent to the 5.8 S rRNA that is found in eukaryoytes hydrogen-bonded to the largest of the cytoplasmic rRNA species.


An explanation of apparent sudden change in the activation energy of membrane enzymes. If an enzyme-lipid mixture forms phases of pure lipid and enzyme-lipid solution, and enzyme activity depends on the composition of the enzyme-lipid solution, the temperature-dependence of lipid solubility in the enzyme-lipid solution leads to apparent sudden changes in enzyme activation energy without activity discontinuities at lipid phase transition temperatures.


Turkey liver xanthine dehydrogenase. Relation between nicotinamide-adenine dinucleotide oxidoreductase activity and the content of functional enzyme. The turkey liver xanthine dehydrogenase-catalysed oxidation of NADH by Methylene Blue, by ferricyanide or by O2 is not dependent on the integrity of the active-centre persulphide groups. By contrast, the NADH-dichlorophenol-indophenol oxidoreductase and NADH-trinitrobenzenesulphonate oxidoreductase activities are directly proportional to the content of functional enzyme. These findings help to identify the sites of egress of electrons to oxidizing substrates.


Subcellular localization of ornithine decarboxylase in liver of control and growth-hormone-treated rats. 1. Ornithine-2-oxo acid aminotransferase activity was inhibited by amino-oxyacetate (10(-5) M). This permitted the measurement of ornithine decarboxylase in the presence of mitochondria by using the 14CO2-trapping technique. 2. Subcellular fractionation of rat liver by differential centrifugation, followed by the assay of ornithine decarboxylase in the presence of amino oxyacetate and of marker enzymes for each fraction, demonstrated that ornithine decarboxylase was located in the cytosol. 3. The greatly increased ornithine decarboxylase activity observed after growth-hormone administration was also found to be localized in the cytosol. 4. The Km of ornithine decarboxylase from rat liver for ornithine was 28 muM. Administration of growth hormone 4 h before death did not affect the apparent affinity of ornithine decarboxylase for ornithine.


Active-site determinations on forms of mammalian brain and eel acetylcholinesterase. Three forms of brain acetylcholinesterase were purified from bovine caudate-nucleus tissue and determined by calibrated gel filtration to have mol.wts. of approx. 120 000 (C), 230 000 (B) and 330 000 (A). [3H]Di-isopropyl phosphorofluoridate (isopropyl moiety labelled) was purified from commercial preparations and its concentration estimated by an enzyme-titration procedure. Brain acetylcholinesterase preparations and enzyme from eel electric tissue were allowed to react with [3H]di-isopropyl phosphorofluridate in phosphate buffer until enzyme activity was inhibited by 98%. Excess of [3H]di-isopropyl phosphorofluoridate that had not reacted was separated from the labelled enzyme protein by gel filtration, or by vacuum filtration or by extensive dialysis. The specificity of active-site labelling was confirmed by use of the enzyme reactivator, pyridine 2-aldoxime. The forms of brain acetylcholinesterase were calculted to contain approximately two (C) four (B) and six (A) active sites per molecule respectively. Acetylcholinesterase (mol.wt. 250 000) from electric-eel tissue was estimated to contain two active sites per molecule. Gradient-gel electrophoresis was used to confirm the estimation of molecular weights of brain acetylcholinesterase forms made by gel filtration. Under the conditions of electrophoresis acetylcholinesterase form A was stable, but form B was converted into a species of approx. 120 000 mol. wt. Similarly, form C of the brain enzyme was converted into a 60 000-mol.wt. form during electrophoresis. These results are in general accord with the suggestion that the multiple forms of brain acetylcholinesterase may be related to the aggregation of a single low-molecular-weight species.


The purification and properties of ferritin from human serum. 1. Ferritin has been isolated from the serum of four patients with iron overload by using two methods. 2. In method A, the serum was adjusted to pH 4.8 and heated to 70 degrees C. After removal of denatured protein, ferritin was concentrated and further purified by ion-exchange chromatography and gel filtration. In most cases, only a partial purification was achieved. 3. In method B, ferritin was extracted from the serum with a column of immuno-adsorbent [anti-(human ferritin)] and released from the column with 3M-KSCN. Further purification was achieved by anion-exchange chromatography followed by the removal of remaining contaminating serum proteins by means of a second immunoadsorbent. Purifications of up to 31 000-fold were achieved, and the homogeneity of the final preparations was demonstrated by polyacrylamide-gel electrophoresis. 4. Serum ferritin purified by either method has the same elution volume as human spleen ferritin on gel filtration on Sephadex G-200. Serum ferritin has a relatively low iron content and iron/protein ratios of 0.023 and 0.067 (mug of Fe/mug of protein) were found in two pure preparations. On anion-exchange chromatography serum ferritin has a low affinity for the column when compared with various tissue ferritins. Isoelectric focusing has demonstrated the presence of a high proportion of isoferritins of relatively high pI. 5. Possible mechanisms for the release of ferritin into the circulation are briefly discussed.


Contemporaneous isolation of deoxyribonucleic acid-dependent ribonucleic acid polymerase and poly(A) polymerase from rat liver mitochondria. 1. Poly(A) polymerase and DNA-dependent RNA polymerase from rat liver mitochondria can be completely separated by using two different chromatographic procedures. 2. Poly(A) polymerase can only incorporate ATP into acid-insoluble material and strongly depends on the addition of an endogenous factor (probably containing a mixture of oligoribonucleotides), but it is not stimulated by DNA. 3. RNA polymerase is fully DNA-dependent and rifampicin-sensitive, but was not stimulated by the endogenous factor mentioned above. 4. The chromatographic behaviour of the two enzymes, together with the properties described, suggest that they represent two different protein molecules.


Isolation of albumin from whole human plasma and fractionation of albumin-depleted plasma. The dye Cibacron Blue F-3-GA was conjugated to Sepharose to provide an affinity column for serum albumin. Passage of whole human plasma through a column of Cibacron Blue-Sepharose results in the removal of approx. 98% of the albumin. The latter can be quantitatively recovered by desorption with NaSCN. Albumin-depleted plasma can be readily resolved into discrete fractions by a combination of conventional biochemical techniques. In particular, the resolution of plasma proteins with properties similar to those of native human plasma albumin can readily be accomplished by ion-exchange chromatography of the Sepharose-dye-treated plasma on DEAE-cellulose.


Studies on sequencing of peptides from the carboxyl terminus by using the thiocyanate method. We report on our experience in applying the thiocyanate method developed by Stark (1968) (Biochemistry 7, 1796-1807) to the sequencing of short peptides from the carboxyl end in free solution. Yields fell to very low levels after three cycles of degradation. The method was time-consuming because of the filtration and freeze-drying stages involved. To overcome these problems, peptides were attached to modified polystyrene polymers for sequential degradation in the solid phase, and a maximum of six amino acids was determined. Also, ribonuclease was attached to active-ester glass beads and sequential degradation was carried out to determine six amino acids at the C-terminal end of this protein.


The amino acid sequence of pike-whale (lesser-rorqual) pancreatic ribonuclease. Pancreatic RNAase (ribonuclease) from the pike whale (lesser rorqual, Balaenoptera acutorostrata) was isolated by affinity chromatography. The protein was digested with different proteolytic enzymes. Peptides were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. The amino acid sequence of peptides was determined by the dansyl-Edman method. Although we do not have an amino acid composition for the whole protein, all peptide bonds were overlapped by one or more peptides. Residues 85-96 are bridged by a peptide of unstaisfactory composition and the sequence here depends, at least in part, on homology for its confirmation. Another region in which a similar situation obtains is residues 39-40. This pancreatic RNAase differs at 24-33% of the positions from all other mammalian pancreatic RNAases sequenced to date, except for pig RNAase, from which it differs by 19%. This indicates that whale RNAase has evolved independently during the larger part of the evolution of the mammals. Lesser-rorqual pancreatic RNAase is partially glycosidated (30%) at asparagine-76 in an Asn-Ser-Thr sequence (residues 76-78). Pig RNAase also has carbohydrate attached to asparagine-76 and is identical with lesser-rorqual RNAase in residues 76-98. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50066 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms ginen in Biochem. J. (1976) 135, 5.


Enzymes of 2-oxo acid degradation and biosynthesis in cell-free extracts of mixed rumen micro-organisms. The enzymes of 2-oxo acid decarboxylation and 2-oxo acid synthesis (EC 1.2.7.1 and EC 1.2.7.2) were isolated and partially purified from cell-free extracts of rumen micro-organisms. The lyase was active with pyruvate, 3-hydroxypyruvate and 2-oxobutyrate. The synthase was active with acetate, 2-oxoglutarate or succinate. Pyruvate synthase was separated from pyruvate lyase by Sephadex G-200 gel filtration. With Sephadex filtration, approximate mol.wts. of 310000 and 210000 were determined for pyruvate lyase and pyruvate synthase respectively.


Deviation from Michaelis-Menten kinetics for fumarase. A study of the steady-state kinetics of fumarase over an extended concentration range, using novel methods of analysis, reveals an initial-rate equation of at least fourth degree for malate as substrate at pH 7.0, with no kinetically significant dead-end complex formation even up to concentrations of 100 mM. In the absence of demonstrable enzyme-aggregation phenomena, this is interpreted as indicating co-operative effects overlooked previously, although a mixture of isoenzymes, each individually of high degree and giving a complex curve, may be a contributing factor.


Comparative activity of arylsulphatases A and B on two synthetic substrates. Rat liver and human skin fibroblasts arylsulphatase A and B activities on both 4-methylumbelliferyl sulphate and 4-nitrocatechol sulphate were compared. The intracellular distribution of activity differed markedly when 4-methylumbelliferyl sulphate was used from that observed with 4-nitrocatechol sulphate. No discrimination between control and metachromatic leucodystrophy or mucopolysaccharidosis (type VI) could be achieved when 4-methylumbelliferyl sulphate was used as substrate. These results contrast sharply with those obtained with 4-nitrocatechol sulphate and cast doubt on the validity of 4-methylumbelliferyl sulphate as substrate for the determination of arylsulphatase A and B activities.


Biotin-binding protein from chicken egg yolk. Assay and relationship to egg-white avidin. 1. Biotin in chicken egg yolk is non-covalently bound to a specific protein that comprises 0.03% of the total yolk protein (0.8 mg/yolk). This biotin-binding protein is not detectable by the normal avidin assay owing to the biotin being tightly bound. Exchange of [14C]biotin for bound biotin at 65 degrees C is the basis of an assay for this protein. 2. Biotin-binding protein from egg yolk is distinguishable from egg-white avidin on Sephadex G-100 gel filtration, although the sizes of the two proteins appear quite similar. 3. Biotin-binding protein is denatured at a lower temperature and freely exchanges biotin at lower temperatures than does avidin. 4. The biotin-binding protein in egg yolk is postulated to be responsible for the deposition of biotin in egg yolk. D-[carboxyl-14C]Biotin injected into laying hens rapidly appears in the egg bound to yolk biotin-binding protein and avidin. Over 60% of the radioactivity is eventually deposited in eggs. The kinetics of biotin deposition in the egg suggests a 25 day half-life for an intracellular biotinyl-coenzyme pool in the laying hen.


The interactions of haem with ligandin and aminoazo-dye-binding protein A. 1. The interactions of ferriprotoporphyrin IX with ligandin and aminoazo-dye-binding protein A result in absorption spectra in the Soret region characteristic of the ligand in its monomeric state. 2. Both proteins are able to bind ferrous as well as ferric haem. 3. Ferriprotoporphyrin IX is bound at a single site on both proteins. At pH7.0, I 0.16M, difference-spectrophotometric measurements gave association constants of 10(7) and 4 X 10(6) LITRE/MOL FOR LIGANDIN AND PROTEin A respectively. Under the same conditions fluorescence-quenching experiments gave an association constant of 2 X 10(7) litre/mol for ligandin. 4. Bilirubin, bromosulphophthalein and oesterone sulphate each compete with haem for binding by the two proteins. 5. Ferriprotoporphyrin IX bound to both ligandin and protein A is able to form co-ordination complexes with CN-, but not, to any measurable extent, with either N3- or F-. From these results it is suggested that binding by the two proteins may not involve the haem iron atom. 6. Both haem-protein complexes give rise to measurable extrinsic Cotton effects in the Soret region. 7. The formation and properties of the ligandin- and protein A-haem complexes are compared with those of haem-albumin, haemoglobin, myoglobin and other haemoproteins.


The identification of 2,8-dihydroxyadenine, a new component of urinary stones. Stones passed by a child homozygous for a deficiency of the enzyme adenine phosphoribosyltransferase have been identified by u.v., i.r. and mass spectrometry as 2,8-dihydroxyadenine.


Methods for fitting equations with two or more non-linear parameters. 1. Descriptions are given of two ways for fitting non-linear equations by least-squares criteria to experimental data. One depends on solving a set of non-linear simultaneous equations, and the other on Taylor's theorem. 2. It is shown that better parameter estimates result when an equation with two or more non-linear parameters is fitted to all the sets of data simultaneously than when it is fitted to each set in turn.


Two methods for fitting a rectangular hyperbola to data from several animals. Two methods are described for fitting the Michaelis-Menten equation to sets of data with a common michaelis constant but different maximum velocities. One of them uses the method of least squares, and the other is based on the direct plot of Eisenthal & Cornish-Bowden [Biochem. J. (1974) 139, 715-720].


Some properties of a soluble methane mono-oxygenase from Methylococcus capsulatus strain Bath. Soluble extracts of Methylococcus capsulatus (Bath), obtained by centrifugation of crude extracts at 160000g for 1h, catalyse the NAD(P)H- and O2-dependent disappearance of bromomethane, and also the formation of methanol from methane. Soluble methane mono-oxygenase is not inhibited by chelating agents or by most electron-transport inhibitors, and is a multicomponent enzyme.


Interaction of pepstatin with pig pepsinogen. In contrast with pepsin, pepsinogen does not bind pepstatin at pH values between 5.3 and 2.5. Pepsinogen is not retarded by pepstatin immobilized on to aminohexyl-Sepharose at pH5.3 or 4.1, whereas at pH3.0 activation takes place during the chromatography, with retardation of the resultant pepsin.


Instability of rat liver chromatin and other nuclear non-histone proteins in alkaline solution. Non-histone proteins from rat liver nuclei and chromatin were shown to be hydrolysed in 0.1M or-1M-NaOH solutions both at 4 degrees and 18 degrees C; 24h in 1M-NaOH at 18 degrees C is sufficient to break down approx. 77% of these proteins to low-molecular-weight peptides. Loss of protein material banding in the region of pH5.5-8.0 has been demonstrated by isoelectric focusing in polyacrylamide gels, and fine high-molecular-weight bands are no longer visible on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The results indicate that care must be taken when analysing non-histone-protein fractions to avoid exposure to alkaline pH conditions.


The 3H-labelling of bilirubin.

The 3H-labelling of bilirubin. A simple method has been developed for the preparation of 3H-labelled bilirubin IX-alpha from bilirubin dimethyl ester. The label is located in the propionic acid side chains, and there is no isomerization of the bilirubin during the preparation.


Isolation of (copper, zinc)-thioneins from the livers of copper-injected rats. The mixed copper- and zinc-binding proteins, with mol.wt. of approx. 12000, induced in rat liver after injection of copper were isolated and characterized as metallothioneins. Three separate forms were obtained, with 7-11% total metal in the protein, equivalent to 2.6-1.6 cysteine residues/metal atom.


Radioimmunological identification of tropoelastin. Antiserum was prepared in sheep against insoluble elastin isolated from embryonic-chick aortae. In an indirect immunoprecipitation test, the antiserum reacted quantitatively with small amounts of radioactively labelled purified tropoelastin prepared from embryonic-chick aortae. The antiserum did not cross-react with chick procollagen, and the antiserum uas used to identify radioactively labelled tropoelastin secreted by chick aorta cells in suspension culture.


Regulation of phosphoenolpyruvate carboxykinase (GTP) in adipose tissue in vivo by glucocorticoids and insulin. 1. The regulation of the synthesis of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in epididymal adipose tissue, liver and kidney in vivo was studied immunochemically. 2. Phosphoenolpyruvate carboxykinase (GTP) synthesis in adipose tissue is increased by starvation, diabetes and noradrenaline, and decreased by re-feeding and insulin. These changes were also seen in adrenalectomized rats and are qualitatively similar to those observed for the liver enzyme. This indicates the involvement of cyclic AMP as an inducer and insulin as a de-inducer in the regulation of phosphoenolpyruvate carboxykinase (GTP) in both tissues. (Induction and de-induction are defined as selective increase and decrease respectively in the rate of enzyme synthesis, regardless of the mechanism involved.)3. Adrenalectomy had little effect on phosphoenolpyruvate carboxykinase (GTP) synthesis in liver and kidney, but increased the synthesis rate of the adipose-tissue enzyme. Starvation and adrenalectomy had additive effects in increasing the synthesis rate of adipose-tissue phosphoenolpyruvate carboxykinase (GTP). In adrenalectomized diabetic rats glucocorticoids increased phosphoenolpyruvate carboxykinase (GTP) synthesis in liver and kidney while decreasing enzyme synthesis in adipose tissue. De-induction of adipose tissue phosphoenolpyruvate carboxykinase (GTP) is therefore regulated independently by glucocorticoids and insulin. 4. Although liver, kidney and adipose-tissue phosphoenolpyruvate carboxykinases (GTP) are seemingly identical, there is an apparent tissue-specific differentiation in regulatory systems for the enzyme.


The effect of cobalt on the uptake of plasma iron by the liver. 1. After an intraperitoneal injection of 59Fe the recovery of radioactivity in the liver, but not in other tissues, was increased in cobalt-pretreated rats. There was no proportional increase in the radioactivity recovered from liver haem. 2. Rats were injected intravenously with serum containing protein-bound 59Fe and 125I-labelled albumin as a marker. At various times after injection the specific radioactivities of iron in plasma and of non-haem iron in liver were determined; corrections were applied for the content of plasma in samples of liver. In cobalt-pretreated rats there was a more rapid loss of 59Fe radioactivity from the plasma and a corresponding increase in the uptake of 59Fe into liver non-haem iron. 3. The results are discussed in relation to the possible sites of action of cobalt, and the possibility is considered that only a fraction of the liver non-haem iron may be involved.


Induction of benzo[a]pyrene Mono-oxygenase in liver cell culture by the photochemical generation of active oxygen species. Evidence for the involvement of singlet oxygen and the formation of a stable inducing intermediate. 1. The photochemical generation of excited states of oxygen in liver cell culture by the mild ilumination of culture medium containing riboflavin, results in stimulation of benzo[a]pyrene 3-mono-oxygenase, a cytochrome P-450-linked mono-oxygenase. 2. The same large increase in mono-oxygenase activity was found when medium containing riboflavin was illuminated in the absence of cells and then stored in the dark for 24h before contact with the cells. From this it may be inferred that stimulation is due to the formation of a stable inducer in the culture medium. Further experiments indicate that the stable inducer is due to the photo-oxidation of an amino acid. 3. Evidence that singlet oxygen is responsible for initiating the stimulation of the mono-oxygenase is based on the use of molecules that scavenge particular active oxygen species. Of all the scavengers tested, only those that scavenge single oxygen inhibited the stimulation. 4. A hypothesis is developed to relate the stimulation of the mono-oxygenase by singlet oxygen in cultured cells to the regulation of the cytochrome P-450 enzyme system in vivo. It is suggested that single oxygen generation within cells may be a common factor linking the many structurally diverse inducers of the enzyme system.


Anti-ketogenic effect of glucose in the lactating cow deprived of food. 1. The aim of this study was to determine the effect of a constant infusion of glucose on the ketosis that is observed when dairy cows are deprived of food in early lactation. 2. Cows in early lactation were first deprived of food for 4 days (96h) to induce a 'fasting ketosis'. Glucose was then infused intravenously at a constant rate of 0.75 g/min for 48h while deprivation of food was maintained. At the end of this 48 h period, blood and liver ketone-body concentrations had decreased to values well below those found in healthy fed cows. 3. On the assumption that the anti-ketogenic effect of glucose was mainly due to suppression of hepatic ketogenesis, it was concluded that two anti-ketogenic mechanisms had been identified. These were (a) a decrease in the availability of free fatty acids for hepatic oxidation, and (b) anti-ketogenic changes within the liver itself. 4. These latter anti-ketogenic changes were twofold. The first was a major increase in the hepatic concentrations of citrate and 2-oxoglutarate. The second was an increase in the degree of oxidation of the hepatic cytosol. It was proposed that both these intrahepatic changes might indicate an augmentation of the quantity of oxaloacetate available for condensation with acetyl-CoA derived from fat oxidation. 5. Hepatic glycerol 1-phosphate concentration fell substantially after glucose infusion. 6. Glucose infusion into fed cows produced qualitatively similar effects to those observed in the unfed cows. However, blood and liver ketone-body concentrations were not decreased to the same extent in the fed cows as in the unfed cows.


The metabolism and binding of catecholamines by the hepatic microsomal mixed-function oxidase of the rat. Noradrenaline and adrenaline were metabolized by an NADPH- and oxygen-dependent process located within the hepatic microsomal fraction of the rat. Metabolism was inhibited by CO and compound SKF 525A, but not by pargyline, an inhibitor of monoamine oxidase, or by 3,4-dimethoxy-5-hydroxybenzoic acid, an inhibitor of catechol O-methyltransferase. It is concluded that the enzyme system responsible for the metabolism of the catecholamines was the microsomal mixed-function oxidase. The Km for noradrenaline was 2.4 mM and for adrenaline 1.0 mM, and V 15.6 and 3.6 nmol/min per mg of microsomal protein respectively. Both catecholamines bound to the microsomal fraction, producing a type II spectral change, with a Ks for noradrenaline of 0.9 mM and for adrenaline of 1.0 mM, and showed other characteristics of type II compounds by inhibited the reduction of cytochrome P-450 by NADPH and exhibiting an enhanced metabolism in the presence of acetone. The major product of catecholamine metabolism was an as yet unidentified alkali-labile compound, which did not correspond to any of the recognized catecholamine metabolites.


Serum glycoprotein synthesis after partial hepatectomy in the rat. The incorporation in vivo of D-[1-14C]glucosamine into serum glycoproteins and proteins of liver microsomal fractions shows a decrease in the early stages (24h) after partial hepatectomy compared with sham-operated animals; 72h after partial hepatectomy the specific radioactivity of hexosamines bound to liver microsomal fractions reaches the same value as for sham-operated animals.