[Carpal tunnel syndrome in developing pseudarthrosis following isolated fracture of os capitatum].

A rare case of well adapted and radiologically invisible capitatum fracture is reported resulting in a pseudarthrosis with pain and median nerve irritation 2 1/2 years following trauma. The difficulty in diagnosis of such well adapted rare fractures is pointed out and electromyography is demonstrated as a good method to diagnose a carpal-tunnel syndrome in cases of pseudarthrosis.


[Functional stenosis: pathogenetic principle of ganglia (author's transl)].

Ganglia originate from communicating recessus of joints. Frequently knee, wrist and ganglia at foot are seen. The theory of valve-mechanism will be expand with the principle of "functional stenosis". In the main the cause will be seen in the high viscosity of synovia in ganglia.


[Biomechanical problems in acromio plasty of the shoulder joint (author's transl)].

In the confines of the spatium subacromiale, the anatomic structures are subjected to increased wear by friction and convolution. In case of compression syndromes relief is obtained by a partial resection of the acromion, by an acromio plasty, or by a complete removal of the acromion, i.e., the acromionectomy. By analysing the anatomic preparation we have found that a resection of the ligamentum coraco-acromiale and the anterior third of the acromion can give a sufficient decompression without any substantial disturbance of the mechanism of the shoulder girdle, while the acromionectomy implies an unnecessarily extensive procedure which involves considerable disadvantages for the biomechanics of the shoulder girdle.


[A new technique for socket implantation into dysplastic acetabula (author's transl)].

This report presents a new technique of socket implantation into the dysplastic acetabulum. A major part of the resected femoral head serves as the so-called intra acetabular femoral head graft and is screwed into the lateral part of the original acetabulum. By this technique it is possible to achieve a complete bony reconstruction of the dysplastic acetabulum into which a square shaped small ceramic socket can be easily implanted using few especially designed instruments. After the description of the operative technique this report presents the clinical results of this procedure based on 11 personal cases. Finally the technical, biomechanical and biological advantages of this implantation technique are discussed in view of other similar methods described recently.


[Differential diagnosis of knee joint fluid. Enzymatic analysis (author's transl)].

Between 1971 and 1976 clinical and chemical examinations of 165 joint fluids in 149 patients were made. The comparison of clinical findings and results of laboratory tests lead us to distinguish between inflammatory and non-inflammatory joint fluids. The reported tests are simple and results can be given quickly. We do not know of any literature on the incidence of CHE in joint fluids. It seems to be of considerable importance that there is a correlation between the degree of CHE-activity and the degree of cartilagenous lesions. As far as we know no comparative tests on Caeruloplasmin in the serum and in the synovial fluid have been made. The quantitative measurement of uric acid in the synovial fluid is more efficient than other methods. The reported methods simplify the differential diagnosis of synovial fluid effusions.


Femoral neck fractures in osteoarthritis of the hip.

Femoral neck fractures seldom occur in hips with a significant degree of osteoarthritis. Experience with three hitherto undescribed fractures around hip nails suggests that the ridge of new bone which forms on the inferior part of the femoral neck in osteoarthritis increases the strength of the neck, thus decreasing the tendency to neck fractures.


Intermittent catheterization: long-term follow-up.

Intermittent catheterization for bladder retraining after spinal cord injury has been in common use in the United States for only the last 10 years. It was initiated at the Ohio State University Hospitals in 1972. In a retrospective evaluation of the effectiveness of intermittent catheterization in 100 spinal cord injured patients, 94 were found to have been discharged catheter-free, ie, without a catheter and off intermittent catheterization. After 3 to 4 years of follow-up, 63 patients were found to have remained catheter-free, 15 were no longer catheter-free and 16 had been lost to follow-up. From an analysis of the data from the study (reasons for failure, complications, etc) and comparison with those from other studies, it is suggested that a prospective study be devised to evaluate intermittent catheterization more effectively.


Sexual adjustment after lower extremity amputation.

Sexual adjustment following lower extremity amputation was evaluated by interviewing 60 adults with recent amputations, 39 men and 21 women, after they had become independent in ambulation with a prosthesis. Among the men, 77% reported a substantial decrease in the frequency of sexual intercourse following amputation, while only 38% of the women reported a decrease. The decreased frequency was greater for nonmarried men than for married men, greater for men with above-knee amputations than for men with below-knee amputations, and greater for male amputee patients in whom phantom phenomena persisted compared to those in whom phantom phenomena no longer existed. The decreased frequency of intercourse for male amputee patients was unrelated to the patient's age, education, and etiology of amputation. There was no significant change in other aspects of sexual activity, including oral-genital relations, masturbation, homosexuality, and extramarital relations.


Proper fitting of the cervical orthosis.

This study evaluated the effect of fitting of the sternal occipital mandibular immobilization (SOMI) orthosis in restricting sagittal cervical spine motion. Cervical orthotic devices are usually fitted according to the subjective tolerance of the patient. Pressures were recorded after fitting in the usual manner by a certified orthotis and when fitted more loosely. Eight able-bodied adults were fitted in this manner and were then immobilized in a straight-backed chair while lateral cervical spine films were taken in neutral, flexion, and extension. Previously standardized methods were used to measure flexion-extension of the cervical spine. The resting pressures, when fitted in the usual manner, were approximately 105 mmHg at the chin and occiput. When pressure sensors were used to fit the brace loosely, the average pressure was approximately 25 mmHg. The data indicate that regardless of fit, there was no significant difference in the mobility of the entire cervical spine or at any intervertebral level. Recommendations for fitting of the SOMI are discussed.


Fatigue curves of isokinetic contractions.

Sixteen men and 16 women volunteer college students were tested for elbow flexion fatigue curve production on the Orthotron, an isokinetic testing device. Maximal voluntary contractions were obtained repeatedly, each contraction taking approximately 2 seconds to complete, with minimal (500 msec) intertrial rest, to exhaustion. To provide a basis for plotting the isokinetic fatigue curve, the degradation of torque values from initial to final contractions, was expressed as percentage of contraction time. Results obtained following statistical analysis of the data indicated that the isokinetic fatigue curve is: (1) curvilinear; (2) independent of sex differences; and (3) a function of initial strength. Moreover, it was found that the use of 50% of initial strength to denote fatigue in repetitive rhythmic isokinetic work has merit as a conservative estimate.


Hepatic porphyria: its rehabilitative evaluation and treatment.

In the rehabilitation of patients with hepatic porphyria, participation of a multidisciplinary team, concerned with diagnosis, evaluation, and treatment, is important. An approach which includes preventive medicine, specific and supportive medical treatment, and vigorous rehabilitative treatment of the neuropathic processes can lead to control of the disease and recovery of self-care and mobility, as illustrated by a case report.


[Behavior of trace elements copper, zinc and manganese in bovine rumen. 2. Passage rate and course of trace element concentration in rumen as influenced by copper sulfate administration].

In experiments on the distribution of copper, zinc and manganese in fractions of rumen liquor (Wetzel and Menke, 1977 (1)) changes in PEG and in trace element concentration during day-time (between 8 a.m. and 6 p.m.) were measured at 2-hour intervals. Rumen volume was 61 l and passage rate 108 and 128 l/day in the two animals used. Average daily passage was calculated to be 40 mg copper, 98 mg zinc and 371 mg manganese. These values were found to be 11% higher than copper intake and 44 and 68% lower than zinc and manganese intakes, respectively. The differences indicate a remarkable endogenous secretion of copper, and a high absorption of zinc and manganese within the rumen. Concentration (mcg/g dry matter) of copper, zinc and manganese in the bacterial fraction and in total rumen liquor decreased slowly in the first hours after feeding, reaching a minimum after 4 to 6 hours and showed a slight increase thereafter. After addition of copper sulphate the curves mostly became linear. The conclusion is drawn that 1.8 ppm copper in inorganic binding does not lead to changes in trace element concentration to an extent that might be harmful to bacterial fermentation.


[Performance-related lysine requirement of fattening pigs. 4. Effect of energy and amino acid intakes on composition of gain and derivation of lysine requirement].

The results show that protein and fat syntheses proceed simultaneously in growing swine. Reducing the energy intake during the 2nd half of the fattening period in pigs of modern genetic construction (hybrid pig) is likely to cut both fat and protein deposition per day. This will result in lower daily gains, a longer fattening period and higher energy expenditure. Therfore, it is not advisable to restrict energy supply during the 2nd half of fattening of pigs derived from modern breeding lines. The optimum amino acid : energy ratio allows to make full use of the protein deposition capacity in pigs with prefixed slaughter weight. For the limiting amino acid lysine it was found to be 10.5 g/1,000 energetic feed equivalents (swine) or 3 g/Mcal NEF (swine), and, taking into account amino acid digestibility, 9.5 g dig. lysin/1,000 energetic feed equivalents (swine) and 2.7 g dig. lysin/Mcal NEF (swine), respectively.


[Dynamics of 15N-isobutylidene diurea (IBDU) in sheep. 1. IBDU conversion in the digestive tract].

Four Merino Landrace wethers averaging 47.6 kg body weight were adapted to a semi-synthetic diet containing as the only N-source 60 g of IBDU per day. After the adaptation phase, on the 1 st experimental day the IBDU of the morning feed was given in 15N-labelled form (701 mg 15N-excess). After 2 1/2, 7 1/4, 12 and 24 hours the experimental animals were killed without having been fed again. The comparison of the IBDU-concentrations in the content of the rumen bottom with the residual rumen content did not allow to draw conclusions regarding IBDU-sedimentation at the bottom of the rumen. For the 15N-decline in the rumen content, a relationship was established following y = 76.3 - 2.62 (r = 0.96) (see fig. 2). In the order of killing times the following 15N-IBDU amounts were retrieved (% of intake): I = 15.6%, II = 24.1%, III = 3.3% and IV = 3.6%. 7 1/4 hours after starting the experiment, 40% of the 15N-labelled material were found in the rumen in the form IBDU; after 12 hours it came to 10%. Except for sheep I, 15N-urea was not found but in small amounts. Only sheep I and III revealed IBDU-traces in the abomasum, but in the small intestine of all sheep 2 to 6% of the amount taken in. This fact is explained with the endogenous influx of IBDU from the blood. An additional experimental sheep provided with a ligature at the abomasum entry, revealed that IBDU is absorbed from the rumen and allowed to enter the individual segments of the intestine in small amounts.


Expression of vaccinia virion surface tubule protein as a virus specific cell surface antigen.

The sequential expression of a vaccinia virus specific antigen on the surface of infected cells has been followed by 125I-labelled anti-vaccinia IgG. After an initial drop (during the first 30 minutes of infection) the amount of viral antigen at the cell surface increased steadily for the 12 hours tested. The expression of the antigen was found to depend on protein and RNA synthesis from the start, but dependent on DNA synthesis only after 4 hours. The senitivity of the phenomenon to ultraviolet light irradiation of the virus suggests that the genetic information needed for the expression of the antigen resides in the viral genome. The antigen has been identified as the virion surface tubule, a tubule-like structure on the surface of the intact virion. It is known that vaccinia virus infection of cells starts with the fusion of the virion envelope with the host plasma membrane. It is here proposed that initially tubule from input virus is detected as viral antigen on the cell surface. Subsequently, virus tubule protein synthesised de novo migrates and is detectable as the virus specific cell surface antigen.


[Structure of the follicular epithelium of the gonads Neva lamprey Lampetra fluviatilis (classification of the follicular epithelia of vertebrates)].

During its fluvial life, the ovarian follicular epithelium of Lampetra fluviatilis undergoes polarity formation--in the animal hemisphere the follicular epithelium is flattened, in the vegetative--it undergoes a secretory specialization: as the secrete is accumulating, the follicular cells increase in volume and height, acquiring a cubical form. In connection with monotonous or differentiating morpho-physiological alterations of follicular epithelium within a single follicle during the period of rapid oocytic growth, it is suggested to widen the idea on kinds of its secondary transformation in the vertebrates. It is suggested to differ: 1) common secondary transformation, when secondary flattening (for example, in birds) or secondary specialization (for example, in fish demersal roe) embraces the whole epithelium of the follicle. In case of common secretory specialization, it can be a) isomorphous--at identical convertion of the epithelium in the whole follicle (for example, in sheatfish), b) dimorphous--when there is a certain difference in the character and degree of secretory specialization of the follicular epithelium in various follicular areas (for example in Myxine glutinosa); 2) unipolar secondary transformation when the follicular epithelium undergoes secondary flattening or secretory specialization only in the area adjacent to one of the oocyte poles (for example, in Lametra fluviatilis and Blennis follis). Follicular epithelium of the lamprey testis, unlike in other Anamnia, does not form spermatocysts; implantation of spermatids and spermia into cytoplasm of the follicular cells is not observed. Thus, histological structure of Cyclostomata testis is at a lower stage of development than in other Anamnia.


[Experimental inflammation of placenta in sheep].

The sheep desmochorionic placenta has been studied at aseptic inflammation produced by suturing it together with the uterus. The inflammatory process is evident from a decreased intensity of leucocytic phase and trophoblast participation in macrophagal phase; cytotrophoblastic elements and diplocariocytes transform into symplastic structures which seem to accomplish dendritic lysis and resorption. The foreign body incapsulation is performed with the maternal and fetal connective tissue participation.


[Embryogenesis of the venous portion of the pulmonary circulation].

The wall of the cardiac venous sinus and splanchnic plexus participate in the formation of the venous part of the pulmonary circulation. At the 4th week of the intrauterine life, splanchnic and sinusal parts come into contact. At this stage, the venous part of the pulmonary circulation is represented only by the primary pulmonary vein. Further, from the splanchnic plexus, common and lobar pulmonary veins are formed. At the 6th week of the intrauterine development, assimilation of the primary and common pulmonary veins by the heart is started--the process resulting in the formation of the primary interatrial septa at the expense of the sinusal wall and the walls of the assimilated vessels. By the end of the 8th week of the intrauterine development, the common atrium is separated into the right and left halves up to the auricular canal, the primary and common pulmonary veins are completely assimilated and the venous part of the pulmonary circulation is represented by four pulmonary veins discharging into the left atrium.


[Relationship between the dynamics of urachal microvessels and its reduction].

Three stages of the urachus morphogenesis, its stabilization, functional activity and involution find their reflection in all morphological characteristics mentioned. The stage of ductal stabilization is characterized by its tubular structure with lack of any definite anatomical criteria to subdivide it into certain parts. In the urachus wall, five constituents of the microcirculatory bed are differentiated, two microvessel network develop for its nutrition, external and internal. The stage of functional activity coincides with the 5th month of the intrauterine life. At that stage, the tubular structure of the urachus is fully developed and the main elements of its wall are formed. The vessel of the microcirculatory bed are definitely differentiated and the vascular diameter reaches its maximum. The stage of the urachus involution from the view-point of its anatomy is characterized by a possibility to distinguish three dynamically changing parts: obliterated, obliterating and open. During this period, a gradual but quite definite change of its histostructure occurs and gradually the differentiated functioning duct becomes a rudimentary connective tissue cord. Under such reduction in the wall layers of the duct, the diameter and number of microvessels reduce downward. This process is proved morphometrically.


[Ultrastructural characteristics of renal cancer cells].

The ultrastructural study of the renal carcinoma revealed some differences in the fine structure of the two main cell types: clear and dark cells. The cytoplasm of clear cells is rich in fat and glycogen. Fat is found in lipid vacuoles and glycogen in diffuse granularity in the cell cytoplasm. Dark cells contain small amounts of fat and glycogen but have well developed mitochondria and other organelles. Comparison of these cell types shows some features of their similarity such as microvilli on the cell surface, desmosomes and interdigitations in the region of cell contacts and channels between tumour cells. These features suggest that clear and dark cells have a common origin from the epithelium of the proximal tubules.


[Histological evaluation of aimed gastrobiopsy in the diagnosis of early stomach cancer].

Among 81 patients with malignancies of the stomach, the early (cancer of the gastric mucosa) was diagnosed in 10 (12.3%) patients. Timely diagnosis of the early gastric cancer requires multiple endoscopic examinations and aimed gastric biopsies of the edges and bottom of the ulcer. The analysis of the serial histological preparations permits to detect single or small aggregations of large mucous cells looking like cricoid cells (considered by the author as a sign of malignization) and less frequently like tumour tissue, in necrotic mass or in the proper layer of the mucous membrane. It is recommended that any gastric ulcer be considered as a possible ulcerative form of cancer and that the patients be mandatorily examined in specialized institutions where the differential diagnosis between peptic ulcer and ulcerative form of cancer may be performed by the method of gastroscopy with aimed biopsy.


[Pathological anatomy of defects of the prosencephalic group].

Morphological changes in the brain were studied in 14 observations of defects of the prosencephalic group. These defects were shown to be of three kinds: prosencephaly, alobar prosencephaly, and holoprosencephaly, and to be due to partial or complete undivision of the cranial part of the medullary tube. The nature of these defects is heterogeneous. Most frequently prosencephalitic defects are due to autosomal aberrrations (trisomy 13 and 18, 13-q-, 18p-, triploidy), and may also be due to gene mutations. In cases of trisomy 13 these defects are regularly accompanied by structural disorders of cytoarchitectonics of the cortex of the end brain, cerebellum, and medulla, which may be important for differential diagnosis of prosencephaly of various etiologies.


[Histogenesis and pathoanatomical diagnosis of human tumors in the light of electron microscopic data].

On the basis of the analysis of the materials obtained in the Pathological Anatomy Department of the Oncological Research Center of the USSR AMS and data from literature the author demonstrates the importance of the electron microscopic examinations for the investigation of histo- and morphogenesis of tumours and for their practical diagnosis.


[Electron-autoradiographic study of hepatic cell nuclei in chronic pathological process].

The development of a chronic pathological process in the liver induces changes in the structure and function of hepatocyte nuclei. Some of these changes, particularly an increase in the number of nucleoli, rearrangement of nucleous-associated chromatin, the appearance of ring-shaped nucleoli and intranuclear vacuoles are similar to senile changes in the nuclei. A quantitative analysis of these changes established a considerable increase in the number of ring-shaped nucleoli and intranuclear vacuoles in sick animals as compared with normal animals of any age. The number of nucleoli and nucleous-associated chromocenters also increases with the progress of the pathological process, however, it reaches the maximum level in the early stages of the process which is equal or insignificantly higher than the level in old animals, and subsequently does not change. These observations reveal a problem important for pathology, that of the existence of a limit in the development of reparative and dystrophic changes which may be attained sooner or later depending on the severity of a pathological process.


[Malignant mesothelioma of the pericardium].

An observation of malignant mesothelioma of the pericardium is described. The tumour grew as papillomatous formations and thick plaques lining the inner surface of the pericardium with transition to the epicardium and the development of massive hemorrhagic pericarditis. The mixed nodular-plate form is histological close to adenocarcinoma. There were metastases in the pleura, paratracheal and posterior mediastinal lymph nodes. Clinically the disease ran a course with angina pectoris and simulated idiopathic myocarditis not confirmed histologically.


[Characteristics of bone regeneration in distraction of bone fragments].

One of the features of distraction osteosynthesis consists in the presence of a zone of permanent growth between bone breaks. Electron microscopic examinations of the permanent growth zone at 14 and 21 days of distraction showed the presence in the zone of cells of three kinds: fibroblast-like cells, osteoblastic elements and little-differentiated preosteoblasts. Fibroblast-like cells and osteoblastic elements were found to have ultrastructural changes of the endoplasmic reticulum indicating the occurrence of intensive synthesizing processes. Specific conditions of blood supply cause hypoxic metabolism with typical enhanced formation of fibrillae which leads to the preservation of the fibrillar tissue throughout the entire period of distraction. The fibrillar tissue in diastace may be considered to be a special growth plate.


[Diagnosis of alcoholic liver cirrhosis during autopsy].

Approaches for the diagnosis of alcoholic cirrhosis of the liver at the dissecting table are given on the basis of the analysis of autopsy materials and liver biopsies. The etiological verification of cirrhosis is based on the peculiar morphological lesions of the liver and other organs. The liver is enlarged, its surface micronodal; histologically, an attack of acute alcoholic hepatitis is found not infrequently: necroses of hepatocytes, predominantly neutrophilic infiltration of the stroma and necrotic zones, alcoholic hyalin. Due to autolysis, alcoholic hyalin in the autopsy material changes somehow and is hardly detectable. A combination of alcoholic cirrhosis of the liver with alcoholic cardiopathy and/or chronic calcifying pancreatitis is typical.


[Method of obtaining semi-thin sections from paraffin blocks in the ultratome].

A method for attaching paraffine blocks with tissue in ultratomes LKB for the preparation of semi-thin sections is proposed. Paraffin is placed into gelatin capsules and to it a paraffin block with tissue is attached. This permits to place into the block-holder not the block but a capsule with paraffin. The advantage of this method is that the block with tissue is not deformed and is firmly held by the blockholder.


[Fibroblasts and the development of the connective tissue: ultrastructural aspects of biosynthesis, fibrillogenesis and catabolism of collagen].

According to the ultrastructural features, 6 types of fibroblasts are distinguished of which two are immature forms (little-differentiated and young fibroblasts) and the rest (collagenoblasts, myofibroblasts, fibroclasts, fibrocytes) are mature cells with the functional specificity participating in the collagen production, contraction, remodeling, involution and stabilization of the connective tissue, respectively. In their turn, collagenoblasts differ from each other in the intensity of biosynthesis and the mode of collagen secretion from the cell. Fibroblasts also produce proteoglycans which are important in fibrillogenesis. Proteoglycans form cuffing around each collagen fibril as well as are components of microfibrils and floccular material which form the ultrastructural basis of the cementating substance. A system of collagen-lytic enzymes and ultrastructural signs of collagen resorption have been found in fibroblasts attesting to their role in the maintenance of equilibrium between biosynthesis and catabolism of collagen under normal and pathological conditions.


An electron microscope study of fibril: matrix arrangements in high- and low-crimp wool fibres.

The crimped structure of wool fibres is generally associated with a bilateral arrangement of ortho- and paracortical cells. The most obvious difference between these cell types is in the arrangement and relative proportions of microfibril and matrix proteins that constitute the fibre cortex. In the low-crimp fibres examined there is a poorer expression of bilateral cortical asymmetry compared with the high-crimp wools together with a higher proportion of intermediate-staining mesocortical cells. These mesocortical cells exhibit much more regular arrays of microfibrils than paracortical cells. It is suggested that the packing arrangement of microfibrils in all three cell types is basically hexagonal and the variation observed in mature cells is a function of the fibril:matrix ratio.


Dexamethasone concentrations in ovine plasma during its intravenous infusion, its relation to the production of some endogenous hormones, and some of the effects on wool growth.

Plasma concentrations of dexamethasone have been measured in sheep during an 8-day infusion of dexamethasone-21 phosphate. The dexamethasone concentration profiles generally revealed a reproducible pattern with three phases--a peak during the first 48 h infusion which was followed by falling concentrations during the next 5 days, and a small increase in dexamethasone concentration during the final 24 h infusion was not uncommon. The pattern of dexamethasone concentrations was retained when dosage was arranged in such a way as to infuse increasing quantities of hormonal analogue as infusion progressed. Aspects of the metabolism of the analogue are discussed. Endogenous thyroxine and cortisol were significantly depressed during infusion. In these experiments wool was completely shed in three out of four animals dosed at a rate of 8.5 mg dexamethasone/kg0.75. The recovery of wool growth to its pretreatment values occurred by about one month after infusion. The consumption of food and body weight increases were satisfactory during the post-infusion period.


Testicular and epididymal sperm numbers and related parameters in the developing awassi ram.

Testicular parameters, parenchymal spermatid concentration, and epididymal sperm reserves were measured in tracts obtained from 16 Awassi rams classified into three age groups (1, 1.5 and 2.5 years old). Between 1 and 2.5 years of age the paired testis weight increased from 164.9 to 315.2 g, the testicular spermatid number (X 10(9)) increased from 15.9 to 55.8, and the total epididymal sperm reserves (X 10(9)) increased from 11.1 to 98.8. Significant correlations were found between the paired testis parenchyma weight and all testicular dimensions and size. Testicular spermatid numbers and concentration were correlated with paired testis weight (r = 0.91 and 0.63), paired testis volume (r = 0.92 and 0.64) and all testicular measurements, particularly paired length X depth (r = 0.92 and 0.66). Regression coefficients between all parameters were essentially linear and highly significant, allowing accurate predictions of testis weight, volume and spermatid numbers to be made. The total epididymal sperm reserves and the sperm content of each of the caput, corpus and cauda of the epididymis were correlated with the paired weights of the epididymis (r = 0.73, 0.75, 0.73 and 0.70), the testis (r = 0.76, 0.83, 0.91 and 0.72), the total spermatid numbers in testicular homogenates (r = 0.70, 0.85, 0.91 and 0.64), and spermatid concentration (r = 0.56, 0.75, 0.72 and 0.49). The storage capacity of the cauda epididymides appeared to develop more slowly than the rate of sperm production because at 2.5 years of age it was about 1.5 and 2.8 times that in rams aged 1.5 and 1 year respectively.


Gonadotrophin levels and ovarian development in the neonatal ewe lamb.

Ovarian weight has been shown to increase markedly in the newborn ewe lamb, and factors which might contribute to this growth were examined. Follicle development was studied in the ovaries of 28 lambs aged 0, 2, 4, 6, 8 and 10 weeks. Plasma samples, pituitaries and one ovary were assayed for gonadotrophin or steroid hormone content. No significant differences between age groups were found in pituitary LH concentration or mean plasma LH. Pituitary FSH concentration increased with age, except for a small decrease at 8 weeks, but no significant changes were seen in plasma FSH. Ovarian progesterone and oestradiol concentrations did not appear to be associated with follicular development. Growing follicles were most numerous at 2 weeks whilst total vesicular follicles reached a peak at 4 weeks. Most vesicular follicles were less than 1.4 mm in diameter. Advanced atresia in vesicular follicles became apparent by 4 weeks although early signs were present in younger lambs. Fluctuations in gonadotrophin levels do not appear to be responsible for variations in number and size of vesicular and growing follicles in the lamb ovary. Other possible explanations are discussed.


The effect of cycloheximide on cholecystokinin-evoked pancreatic juice of the anaesthetized rat.

Cholecystokinin (CCK) is a major stimulant for enzyme secretion by pancreatic acinar cells. In most animals this enzyme secretion is accompanied by a small volume of chloride-rich "carrier" fluid in which the enzymes are dissolved. In order to determine whether this carrier fluid is secreted independently of the pancreatic digestive enzymes, and how its composition is altered by interaction with them, we have studied CCK-evoked juice collected from anaesthetized rats before and after administration of cycloheximide, a powerful inhibitor of protein synthesis. Following treatment with cycloheximide, fluid secretion continued unchanged for 30--40 min but protein excretion declined without appreciable delay towards zero. As protein content declined, the juice bicarbonate content rose from 30 to more than 60 mmole 1(-1) so that the juice resembled that evoked in the rat by secretin. It is concluded that secretion of CCK-evoked fluid is independent of the secretion of digestive enzymes and that the carrier fluid is actually alkaline, like secretin-evoked fluid, and only becomes neutral and chloride-rich by interaction with the acidic contents of zymogen granules.


The sarcoplasmic reticulum and T-system of rat extensor digitorum longus muscles exposed to hypertonic solutions.

Excised rat extensor digitorum longus muscles were soaked in a Krebs solution made hypertonic with extra NaCl and fixed using a combination of acrolein and glutaraldehyde dissolved in the same hypertonic Krebs. Electron microscopic examination showed that the sarcoplasmic reticulum (S.R.) of these muscles occupied the same fraction of fibre volume as it did in control muscles soaked and fixed in normal Krebs solution, contradicting previous studies on frog muscle. It appears unlikely that the S.R. is the rapidly exchanging Na+-compartment of rat muscle which enlarges in hypertonic solutions, or that it is an extracellular compartment. The T-system of hypertonic muscles was swollen, but its volume is insufficient to form the rapidly exchanging Na+-compartment.


Effects of a hypercaloric high carbohydrate diet on adipose tissue metabolism in man.

The effects of a hypercaloric high carbohydrate diet on lipolysis and esterification were studied in biopsy samples of adipose tissue obtained from 5 healthy subjects. Overfeeding with carbohydrate enhanced the rate of esterification of free fatty acids (FFA) without any alteration in the rate of lipolysis. The release of FFA from adipose tissue was inhibited. These observations indicate that the utilization of glucose and FFA by adipose tissue for the synthesis of triglycerides was facilitated during weight gain on a high carbohydrate diet.


Glycoproteins of the brush border membrane of mouse duodenum.

Qualitative and quantitative chemical analyses have been made of the carbohydrate and protein moieties of the glycoproteins in purified mouse duodenal brush border membranes. Solubilized membrane protein was electrophoresed on S.D.S.--polyacrylamide gels and glycoproteins were subsequently identified by periodate-Schiff staining. A parallel experiment showed that these glycoprotein bands also incorporated 14C counts from intraluminally-administered 14C-glucosamine. Positively-staining bands were collected from several identically run gels and subjected to digestion with pronase. The sugar and amino acid compositions of the digested glycoproteins were determined. Five of the membrane proteins appeared to be complexed with sugars, and further analyses showed that there were differences between the glycoproteins with respect to sialic acid, uronic acid, amino acids, amino sugar and monosaccharide contents.


The isolation and identification of lactoferrin from bovine tears.

A two-step procedure for the isolation of pure lactoferrin from bovine tears is outlined. The amino acid composition of this lactoferrin resembles closely the amino acid composition of lactoferrin isolated from bovine milk.


The effects of methysergide on the constrictor response to serotonin on the isolated, perfused rabbit ear artery.

On the isolated, perfused rabbit ear artery methysergide 10(-9) mol.1(-1) only potentiated the constrictor response to serotonin while at a concentration of 2.5 x 10(-8) mol.1(-1) it only blocked the response. At an intermediate concentration (2.5 x 10(-9) mol.1(-1)) it produced initial potentiation followed by subsequent blockade. In contrast, at all three concentrations it only potentiated the constrictor response to noradrenaline. The results thus show that methysergide can exert a dual effect on the constrictor response to serotonin with the actual effect produced being dependent not only on the concentration of methysergide used but also on the period of time the artery is exposed to methysergide.


Habitat selection and evolutionary strategies in Drosophila: an invited address.

There is an association among resource utilization divergence, habitat selection, and taxonomic divergence in the genus Drosophila. Given permissive conditions of temperature, humidity, and light intensity, an enormous vairety of resources is used in a diversity of habitats. These resources are considered in the cosmopolitan and endemic Australian fauna, providing evidence for habitat selection in the laboratory and field. Lek behavior in picture-winged species of subgenus Hirtodrosophila, a case of parallel evolution with lek behavior in subgenus Drosophila in Hawaii, is discussed in detail. Other examples of habitat selection discussed concern behavioral reactions of larvae to alcohol and other metabolites and the avoidance by adults of extreme physical environments. Evolutionary strategies involved in habitat selection are considered at various taxonomic levels in Drosophila. These considerations show that it is essential to relate results from laboratory studies to natural environments in order to explore the genetics of habitat selection.


Behavioral development in two genetically heterogeneous mouse stocks: Swiss-Webster and Binghamton-HET.

Two genetically heterogeneous mouse stocks, Swiss-Webster and Binghamton-HET, were tested. Several measures assessing behavioral development revealed differences in rates of development between the stocks. Thus neither stock can be exclusively used to generalize about "typical" mouse behavior.


The impact of daily work load during pregnancy and/or postnatal life on the heart microstructure of rat male offspring.

The microstructure of the cardiac muscle of four groups of male rats was investigated: control (EC) and exercised (EE) offspring of mothers exercised during pregnancy, and control (CC) and exercised (CE) offspring of inactive control mothers. The differences between groups were subjected to variance analysis and multiple range testing according to Duncan. Total body weight did not differ during the entire experiment. With the exception of the subgroup CE (lower values) the absolute and relative (mg/100 g total B.W.) weight of the cardiac muscle did not differ either. The number of heart fibres and capillaries per mm2 was highest in subgroup EE, and significantly lowest in subgroup CC. The capillary: fibre ratio was significantly highest in EE; diffusion distance (D/2) was shortest in EE. Other groups were intermediate. A greater impact of prenatal than postnatal work load on the microstructure of the cardiac muscle of the offspring was demonstrated.


Pulmonary circulation in the reindeer.

Right heart catheterizations were performed in 56 sessions on 25 reindeer with determinations of blood pressure and oxygen saturation. Cardiac output was calculated from cineangiocardiograms. When the animals were placed in prone position, heart rate was about 50/min, mixed venous blood oxygen saturation 68 to 72%, right ventricular systolic pressure 27 mmHg, pulmonary arterial pressure 25/16/11 mmHg, pulmonary arterial wedge pressure 6--8 mmHg and pulmonary transfer time 4 to 5 cardiac cycles. When the animals were placed on their side, the heart rate rose to 90 b/min, mixed venous blood oxygen saturation decreased by about 4% and the right ventricular and pulmonary arterial pressures increased by about 10 mmHg; pulmonary arterial wedge pressure did not change. Hypoxemia led to an increase in heart rate, cardiac output and pulmonary arterial pressure; the latter two measures tended to increase whenever the heart rate increased. Under the conditions stated here, an increased pulmonary vascular resistance was calculated when pulmonary arterial pressure was increased.


The hypoxic, low-flow perfused rat heart: characterization as a model of global ischaemia.

In the study of the mechanism of mitochondrial damage in myocardial ischaemia, production of a small animal model of global ischaemia proved to be necessary. Thus, the effects of coronary flow rate, oxygen availability and substrate availability were studied on the functional capacity of mitochondria isolated from perfused rat hearts. The hypoxic, low-flow perfused heart eventually chosen was evaluated as a model of myocardial ischaemia by measuring tissue contents of high energy phosphate compounds and glycolytic intermediates and by assessing the effects of exogenous substrates on the above parameters.


[Evaluation of elasticity by means of length-tension relationships in a model of isolated ventricular myocardium from rat and cat papillary muscle under conditions of contracture (author's transl)].

Increase of Ca2+ concentration to 7.5 mM in the presence of 10 mM or 20 mM caffeine shifted length-tension relationships of 5 rat trabecular muscles and 5 cat papillary muscles to higher tensions. At 1max diastolic tension was enhanced from (formula: see text), for all measured points of stress-strain relationships after the increase of diastolic tension by caffeine and Ca2+ as well as under control conditions. The function E = f (sigma) = b (sigma - c) was computed by linear regression analysis (0.99 greater than r2 greater than 0.96). The average value of constant b was 13.89 +/- 2.01 in control curves of rat trabecular muscles and 13.42 +/- 1.98 in curves with 10 mM caffeine and 7.5 mM Ca2+. Likewise, in cat papillary muscles 20 mM caffeine and 7.5 mM Ca2+ did not alter the stiffness constant b in a statistically significant manner (control: 16.63 +/- 3.03, caffeine: 16.43 +/- 3.02). The results demonstrate that the stiffness constant b cannot indicate acute variations of the length-tension relationships due to caffeine and Ca2+ and may result in overestimating of myocardial distensibility. However, if the tangent modulus (E) is related to strain (epsilon), the alterations of diastolic elasticity are detectable. Mathematical considerations of these experimental results imply a new base for evaluation of diastolic elasticity of the heart by means of the tangent modulus (E). These results are of substantial importance in clinical evaluation of distensibility of myocardial tissue, as the tangent modulus related to wall stress apparently is not appropriate to realize alterations in "passive" myocardial properties due to contracture.


Thermal dilution measurement of cardiac output in dogs using an analog computer.

Thermal dilution cardiac output determinations in dogs were compared to simultaneously performed Fick oxygen measurements. The purpose of this study was to validate in dog experiments a method for thermal dilution measurement which employs a double-thermistor catheter combined with an automatic computer as described by Olsson et al. Dilution and injectate temperature are entered directly into the calculation. The method does not employ logarithmic extrapolation, integration of the dilution signal being terminated when a preset cut-off level is reached. Errors due to recirculation, thermal capacitance of the right heart and heat exchange with the catheter's dead space require the use of an empirically derived correction factor, which in dogs was found to be significantly different from the factor used for human thermal dilution curves. With the appropriate cut-off level and correction factor a good agreement was found between the results of the thermal dilution and the Fick method. The regression equation for 47 experiments was found to be COtd = 0.95 COFick + 0.08; the correlation coefficient was 0.94.


[Studies on the effects of atrazine and simazine on the N-binding bacteria Azotobacter and Beijerinckia in the ferralitic soils of Cuba].

In two Cuban locations (Santiago de las Vegas and Jovellanos) the effect was investigated of increasing atrazin and simazin applications (0, 4, 8, 16 kg/ha) to the number of Azotobacter and Beijerinckia colonies in weakly ferrallitic soils 7 and 15 days after their application during the rainy and dry seasons. The results were as follows: 1. The number of the Beijerinckia colonies is higher in all variants than that of Azotobacter. 2. Beijerinckia and Azotobacter are stimulated up to 7 days, partly also up to 15 days, after application of the triazins. Atrazin exhibits a stronger and more lasting effect than simazin, and Azotobacter are influenced more strongly than Beijerinckia. 3. This stimulation can be adduced with a high degree of probability to the inhibition of organisms which have an antagonistic effect on N-binding bacteria.


Comparative study on the effect of some chlorinated hydrocarbon insecticides on the formation of ectotrophic mycorrhizae in seedlings of Picea abies (L.) Karst.

Pot experiments were conducted to determine the effect of lindane and toxaphene on the ectotrophic mycorrhizae in Picea abies (L.) Karst. seedlings. The concentrations investigated gave variable results. Quantitative reductions were found in mycorrhizal short roots and intensity. In general the initiated lateral roots were mainly infected by mycorrhizae, even in the presence of insecticidal injuries in the root system.


Heterosis and phenotypic correlations for shank length, body weight and egg production traits in the Alexandria strains and their crosses with Fayoumi chickens.

The performance features of one paternal (PL) and one maternal line (ML) each, their reciprocal crosses and their crosses with a non-selected Fayoumi population are presented. A clear heterosis was found for hatchability only. Shank length, body weight, fertility, sexual maturity and egg weight were intermediate. Heterosis was observed for the number of eggs in the first 90 days of laying for the ML X PL cross. The performance of the PL and ML and of their reciprocal crosses was generally higher than that of the crosses with the non-selected Fayoumis.


Methemoglobin reductase in three species of Bovidae.

Isoelectric focusing of red cell hemolysates revealed several isozymes that stain for NADH-methemoglobin reductase. Evidence for two different genetic loci controlling the banding patterns was obtained. One locus controlled a single band present in all animals tested. The second locus controlled ten different banding patterns that could be accounted for by four codominant alleles. Band B occurred in Bison bison. Bands A and C occurred in Bos indicus and band D occurred in both Bos indicus and Bos taurus. Bands A, C, and D were not observed in Bison bison and bands A, B, and C were not observed in Bos taurus.


Heterozygosity in Yellowstone Park elk, Cervus canadensis.

Protein products of 24 loci from the genomes of Yellowstone Park elk were analyzed by electrophoresis. Heterozygosity was detected in only one system, making elk much less polymorphic than eastern whitetailed deer. Data for several other large mammals are compared with those for elk and reveal similarly low levels of isozymic variation. The data are consistent with the fine-grained niche theory but difficult to reconcile with bottlenecks and genetic drift.


Simplified typing of mouse hemoglobin (Hbb) phenotypes using cystamine.

Cellulose acetate electrophoresis of mouse hemoglobins modified with the disulfide reagent cystamine permits rapid, unequivocal discrimination of all combinations of the codominant mouse hemoglobin "single" (Hbbs) and "diffuse" (Hbbd and Hbbp) alleles. The single, diffuse major, diffuse d-minor, and diffuse p-minor adult hemoglobins are all resolved by this method, which depends on the presence of a cysteine in the beta chains of "diffuse" mice which is not found in the beta chain of "single" mice.


Fluorescence spectrophotometric analysis of melanins in the house mouse.

We extracted the yellow melanin (phaeomelanin), black melanin (eumelanin), and mixed type of melanin from dorsal hair of dominant yellow (Ay/a), non-agouti (a/a), and agouti (A/A) mice, respectively. Spectrophotometric and fluorescence spectrophotometric analysis demonstrated that the yellow melanin was qualitatively distinct from the black melanin and that the agouti hair contained both types of pigment.


A technique for detection and relative quantitative analysis of glucosephosphate isomerase isozymes from nanogram tissue samples.

An improved method for detecting and measuring the enzyme glucosephosphate isomerase after starch gel electrophoresis is described. Nitrocellulose filters are used in a gel overlay procedure which increases the sensitivity of the staining reaction and provides a simple means for accurate quantitation of the isozyme pattern. This staining technique may have wider application with other gel media and also with other enzymes.


Association in Long-Evans hooded rats of red cell 2,3-diphosphoglycerate levels with hemoglobin types.

Two sublines of commercially available Long-Evans hooded rats have been developed by genetic selection. These sublines have widely differing levels of erythrocyte 2,3-diphosphoglycerate (DPG) due to different alleles at a single genetic locus. In the present work, it is shown that rats from the commercial population are also polymorphic at a hemoglobin locus, probably involving two alleles of the IIIbeta-globin chain locus. Particular hemoglobin types have been found to be strongly associated with certain DPG types, not only in the high-DPG and low-DPG lines but also in the commercial population. Two explanations for this association are considered. One is a single-locus hypothesis, with hemoglobin allelic variation causing DPG variation, and the other is a two-locus hypothesis, with marked linkage disequilibrium.


An examination of the age-related patterns of decay of acid phosphatase (ACP1) in human red cells from individuals of different phenotypes.

A study has been made of the decay of acid phosphatase (ACP1) in the human red cell using red cell fractions of different mean ages prepared by density gradient centrifugation. Red cells from acid phosphatase type A and type B individuals were used in the study. Acid phosphatase activity of the red cell fractions was determined by two different assay methods. The results obtained were comparable and have been combined. Acid phosphatase type A and type B showed a biphasic decay pattern with a rapid early loss of activity, followed by a more gradual rate of decline. Type A appeared to decay more rapidly than type B in both decay phases. It is proposed that differences in stability between type A and type B in vivo may explain the observed differences in activity between the enzyme types. There was no evidence for the generation of secondary isozymes by acid phosphatase type A or type B during red cell aging.


On the two electrophoretic forms of the human superoxide dismutase A in the homozygote SOD A1.

Human superoxide dismutase A of the common phenotype SOD A1 is present in two electrophoretic forms in erythrocytes. These forms are charge isomers, easily permuting one into the other. Oxidation and reduction of the copper, partial or complete saturation of the dimers with copper, and presence of thiol groups on the enzyme do not seem to be involved.


Aconitase (E.C. 4.2.1.3) mitochondrial locus mapped to human chromosome 22: studies with Chinese hamster--human somatic cell hybrids.

Three separate somatic cell fusions were made between Chinese hamster lines and human lymphocytes containing (1) a 3/4 translocation, (2) an X/9 translocation, and (3) a 17/9 translocation. Eleven independently derived hybrids showed that only human chromosome 22 was consistently present when human ACONM was expressed and absent when human ACONM was not expressed. These studies assign a gene for human ACONM to chromosome 22, and are consistent with prior gene-mapping results.


Pigment synthesis in maize aleurone from precursors fed to anthocyanin mutants.

Aleurone tissue of c2 Pr, C2 pr, or c2 pr genotypes can utilize either of two flavanones (naringenin, homoeriodictyol) or a flavanonol (dihydroquercetin) to synthesize anthocyanin. The anthocyanins formed have substitution patterns corresponding to those of the precursors, but c2 Pr tissue can hydroxylate the 4'-OH precursor at the 3' position. The results presented suggest that C2 acts before the flavanone step and that the hydroxylation gene (Pr) can act after C2.


Gene diversity of bovid hemoglobins.

Forty-five adult hemoglobin molecular forms which include 22 electrophoretically silent forms were structurally characterized from animal species of nine genera belonging to family Bovidae. Of the 12 different bovid species studied, 11 showed either alpha or beta chain heterogeneity in their hemoglobins while eight species showed heterogeneity for both polypeptide chains. Wherever possible, the genetic basis for hemoglobin phenotypes has been suggested. By construction of a phylogenetic tree for 14 ungulate alpha-globin sequences, the evolutionary origins of duplicated alpha chain genes present in some ungulate species have been located, and the phyletic relationship of Bovids based on the alpha globin data is discussed.


Subunit size of enzymes and genetic heterozygosity in vertebrates.

There is a small but significant positive correlation between individual locus estimates of heterozygosity and subunit molecular weight in vertebrate dimeric enzymes. This correlation is smaller than that previously shown to exist for Drosophila dimers, and some possible reasons for this are explored. Data for vertebrate tetrameric enzymes are less extensive but appear to give similar trends to those shown by dimers. It is concluded that enzyme heterozygosity is influenced by both subunit size and quaternary structure.


Genetic and molecular analysis of nonrandom dimer assembly of the creatine kinase isozymes of fishes.

Species within many families of actinopterygian bony fishes (class Osteichthyes) have a two-banded allelic isozyme phenotype in individuals heterozygous at the creatine kinase A locus. This two-banded pattern is formed by the presence of the two homodimeric isozymes and the absence of the expected heterodimer. Sharks and amphibians have retained the ability to form all three allelic isozymes in individuals which are heterozygous. Reversible denaturation procedures were able to assemble the different allelic CK-A subunits within a species to form CK-A2 heterodimers. Furthermore, heterodimers were formed from different CK-A subunits from highly divergent species after this in vitro molecular hybridization process. It is concluded from these studies that the polypeptide-binding sites of creatine kinase are structurally conservative in most fishes and that the absence of a heterodimer in heterozygous individuals is not due to a structural incompatibility between the different A subunit types or to an instability of the heterodimer during electrophoresis. A temporal and/or spatial isolation of allelic CK-A subunit synthesis and assembly, within differentiated skeletal muscle, appears to have evolved in the actinopterygian bony fishes.


Genetic aspects of wheat gliadin proteins.

Inheritance of gliadin components unique to three different varieties of common wheat (Triticum aestivum L.) was studied in F1 and F2 seeds of intervarietal crosses using protein patterns obtained by polyacrylamide gel electrophoresis in aluminum lactate buffer (pH 3.2). The patterns of F1 seeds of the crosses Cheyenne X Justin and INIA 66R X Justin evidenced all the bands present in the patterns of the parents; band intensities reflected gene dosage levels dependent on whether the contributing parent was maternal or paternal in accordance with the triploid nature of endosperm tissue. Most of the gliadin components examined segregated in accordance with control by a single dominant gene, but in two instances single bands in the one-dimensional electrophoretic patterns segregated in the F2 as expected if controlled by two genes. A method of two-dimensional electrophoresis was developed that resolved these apparently single bands into two components each, which could segregate independently. Linkage analysis provided evidence of codominant alleles and closely linked genes coding for gliadin protein components in both coupling and repulsion situations. The gliadin protein components seem to be coded for by clusters of genes located on chromosomes of homoeologous groups 1 and 6 in hexaploid wheats.


Product inhibition of o-aminophenol glucuronidation by p-nitrophenyl glucuronide in detergent-treated microsomal fractions.

The glucuronidation of o-aminophenol is unaffected by p-nitrophenyl gluronide when native microsomal fractions are the source of UDP-glucuronyltransferase. When microsomal fractions treated with Lubrol detergent are the source of the enzyme, however, p-nitrophenyl glucuronide exhibits competitive inhibition of o-aminophenol glucuronidation. In addition, the apparent K1 for p-nitrophenyl glucuronide is the same whether o-aminophenol or p-nitrophenol is the acceptor substrate. The data suggest that UDP-glucuronyltransferase has one binding site for the two phenols and that the absence of inhibition observed in native microsomal fractions is dependent on an intact microsomal membrane.


Submitochondrial location of ruthenium red-sensitive calcium-ion transport and evidence for its enrichment in a specific population of rat liver mitochondria.

1. Seven fractions sedimenting at between 3000 and 120000g-min were prepared from a rat liver homogenate by differential centrifugation in buffered iso-osmotic sucrose. The following measurements were carried out on each of these fractions: Ruthenium Red-sensitive Ca(2+) transport in the absence and in the presence of P(i) as well as in the presence of N-ethylmaleimide to prevent P(i) cycling, succinate-supported respiration in the absence and in the presence of ADP, the DeltaE and -59 DeltapH components of the protonmotive force, cytochrome oxidase, uncoupler-stimulated adenosine triphosphatase, alpha-glycerophosphate dehydrogenase, P(i) content and the effect on the ;resting' rate of respiration of repeated additions of a fixed Ca(2+) concentration. 2. Ca(2+) transport either in the presence or in the absence of added P(i) and in the presence of N-ethylmaleimide exhibits significantly higher rates in the fraction sedimenting at 8000g-min. By contrast, respiration in the presence or in the absence of added ADP and the values for DeltaE and -59 DeltapH were similar in those fractions sedimenting between 4000 and 20000g-min, indicating that the driving force for Ca(2+) transport was similar in each of these fractions. 3. Experiments designed to determine the capacity of the individual fractions for Ca(2+), as measured by the effect of repeated additions of Ca(2+) on the resting rate of respiration, showed that fraction 2, i.e. that sedimenting at 8000g-min, also exhibited the greatest tolerance towards the uncoupling action of the ion. 4. Of the three enzyme activity profiles, only that of alpha-glycerophosphate dehydrogenase was similar to that of Ca(2+) transport. Because previous workers have assigned this enzyme to loci in the inner peripheral membrane [Werner & Neupert (1972) Eur. J. Biochem.25, 379-396], it is concluded that the Ruthenium Red-sensitive Ca(2+)- transport system also is located in this domain of the inner membrane. The relation of these findings to the mechanisms of mitochondrial Ca(2+) transport and the biogenesis of mitochondria is discussed.


Alkylation of deoxyribonucleic acid in vivo in various organs of C57BL mice by the carcinogens N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea and ethyl methanesulphonate in relation to induction of thymic lymphoma. Some applications of high-pressure liquid chromatography.

1. Methods were developed  for analysis of alkylpurines, O2-alkylcytosines, and representative phosphotriesters [alkyl derivatives of thymidylyl(3'-5')thymidine], in DNA alkylated in vivo, using high-pressure liquid chromatography. 2. The patterns of alkylation products in DNA in vivo at short times were closely similar to those found for reactions in vitro. Alkylation by the nitrosoureas was complete in vivo within 1 h, but with ethyl methanesulphonate was maximal at 2--4h. 3. The time course of persistence of alkylation products in vivo was determined for several tissues. In addition to the rapid loss of 3- and 7-alkyladenines reported previously for all tissues, a relatively rapid loss of O6-alkylguanines from DNA of liver was found which was more rapid at lower doses. In brain, lung and kidney, excision of O6-alkylguanine was much less marked, but was not entirely excluded by the data. In thymus, bone marrow and small bowel, all alkylated bases were lost with half-lives of 12--24h, at non-cytotoxic doses of alkylation. 4. No evidence for any marked excision of other minor products from alkylated DNA in vivo was found; thus 1-methyladenine, O2-ethylcytosine (found in appreciable amount only with N-ethyl-N-nitrosourea), 3-methylguanine, and dTp(Alk)dT persisted in alkylated DNA, including DNA of liver. 5. The induction of thymic lymphoma was determined over the range of single doses by intraperitoneal injection up to about 60% of the LD50 values, and related to the extent of alkylation of target tissues thymus and bone marrow. With N-methyl-N-nitrosourea over 90% tumour yield was attained at 60 mg/kg, and with N-ethyl-N-nitrosourea up to 52% at 240 mg/kg, but with ethyl methanesulphonate at up to 400 mg/kg only a few per cent of tumours were obtained. 6. The carcinogenic effectiveness of the agents was positively correlated with the extents of alkylation of guanine in DNA of target tissues at the O-6 atom. On the basis that at doses giving equal carcinogenic response these extents of alkylation would be equal, the chemical analyses showed that the ratio of equipotent doses to that for N-methyl-N-nitrosourea would be, for N-ethyl-N-nitrosourea, 5.3 for ethyl methanesulphonate about 21, and for methyl methanesulphonate [Frei & Lawley (1976) Chem.-Biol. Interact. 13, 215--222] about 144. These predictions were in reasonably good agreement with the observed dose-response data for these agents.


Control of sterol metabolism in rat adrenal mitochondria.

Steroidogenesis by adrenal mitochondria from endogenous precursors is stimulated by corticotropin (ACTH) and is sensitive to the protein-synthesis inhibitor cycloheximide. In the present investigation the effect of cycloheximide treatment on the metabolism of a number of analogues of the normal steroidogenic substrate, i.e. cholesterol, by rat adrenal mitochondria was studied. It was observed that the metabolism of analogues such as desmosterol, 26-norcholest-5-en-3beta-ol and 5-cholen-3beta-ol (that is with non-polar alkyl side chains like cholesterol), was sensitive to cycloheximide treatment. By contrast, the metabolism of those analogues with polar groupings on the side chain, i.e., 20alpha-, 24-, 25- and 26-hydroxycholesterols was insensitive to pretreatment with cycloheximide. The binding of added sterol to the cytochrome P-450 component of the mitochondrial sterol desmolase was studied. Similar studies on the equilibration time on addition of exogenous sterols to achieve maximum rates of pregnenolone production were also made. Both studies show that cholesterol, a non-polar sterol, penetrated slowly through the mitochondrial milieu to reach the cytochrome P-450 reaction centre whereas 24- and 26-hydroxycholesterols rapidly attained the enzymic environment. The cycloheximide-sensitive process in sterol metabolism appeared related to the transfer of non-polar sterols such as cholesterol within the mitochondria to a region in close proximity to the enzyme. The importance, and possible mechanism of action, of the cycloheximide-sensitive factor in the control of adrenal steroidogenesis is discussed.


Effect of imposed serum deprivation on growth of the mouse 3T3 cell. Dissociation from changes in potassium ion transport as measured from [86Rb)rubidium ion uptake.

Decreased serum concentrations that substantially alter the growth of normal 3T3 cells alter neither the active and non-active components of unidirectional (86)Rb(+) influx nor the intracellular K(+) content when compared with cells in exponential growth. Thus the changes in K(+) transport (measured with (86)Rb(+) as an analogue for K(+) movements) that occur on density-dependent growth inhibition of the mouse 3T3 cell are not mimicked by serum deprivation of the cells before density inhibition.


Inability of tributyltin-induced chloride/hydroxyl exchange to stimulate calcium transport in mitochondria isolated from flight muscle of the sheep blowfly Lucilia cuprina.

Tributyltin in the concentration range 1-4mum failed to stimulate Ca(2+) transport by Lucilia flight-muscle mitochondria in a medium containing KCl and respiratory substrate but devoid of P(i), despite its promotion of a rapid Cl(-)/OH(-) exchange. When 2mm-P(i) was present, concentrations of tributyltin greater than 1mum inhibited the initial rate of Ca(2+) transport and induced efflux of the ion from the mitochondria in Cl(-)- or NO(3) (-)-containing media. Lower concentrations had little effect. Oligomycin added at up to 10mug/mg of mitochondrial protein had no effect on Ca(2+) transport. By contrast, approx. 0.3mum-tributyltin completely inhibited respiration supported by alpha-glycerophosphate in either the presence or absence of added ADP. The data suggest that tributyltin can inhibit Ca(2+) transport in Lucilia flight-muscle mitochondria other than by facilitating a Cl(-)/OH(-) exchange or producing an oligomycin-like effect.


Interference by branched-chain amino acids in the assay of alanine with alanine dehydrogenase.

Commercial preparations of alanine dehydrogenase from Bacillus subtilis are contaminated to varying extents with activity towards branched-chain amino acids. The Km values for these amino acids are of the same order as for L-alanine (about 10(-3)M). The branched-chain amino acid dehydrogenase activity is lost on dialysis for 2--4h against water or 2mM-EDTA.


Sodium-dependent alanine transport in plasma-membrane vesicles from rat liver.

L-Alanine transport was studied in plasma-membrane vesicles from rat liver. A gradient of NaSCN, but not of KSCN, stimulated alanine uptake. Monensin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone abolished the observed overshoot in uptake. After equilibration of alanine, NaSCN induced uphill transport.


The biosynthesis of brain gangliosides. Ganglioside-glycosylating activity in rat brain neuronal perikarya fraction.

Rat brain homogenate and the synaptosmal and neuronal perikarya fractions from 17-day-old rats were compared for their activities in sialosylating endogenous gangliosides and transferring N-acetylneuraminic acid and galactose to several glycolipids in vitro. The sialosylation of endogenous gangliosides and the activities of sialosyltransferases acting either on lactosylceramide or haematoside as acceptors, as well as galactosyltransferase acting on Tay-Sachs ganglioside as acceptor, were between 3-and 12-fold higher in the neuronal perikarya fraction than in whole homgenate on a protein or ganglioside basis. The activities found in the synaptosomal fraction were negligible. No evidence was found to indicate that the low activities in this fraction were due to the presence of inhibitors of the transfer activities or to inacessibility of the substrates to their respective enzymes. These findings, and the time course of labelling of gangliosides of the neuronal perikarya and synaptosomes from rats that received an injection of N-[3H]acetylmannosamine, indicate that the main cellular site of glycosylation of neuronal gangliosides is in the neuronal perikarya.


Oxidative degradation of haemoglobin by nitrosobenzene in the erythrocyte.

Substitutions on the benzene ring of nitrosobenzene did not have the same effect on oxidative haemolysis as substitutions on phenylhydrazine. We previously found that the haemolytic effect of arylhydrazines paralleled their oxidative conversion into ligands of ferrihaemoglobin. In contrast, although most substituted nitrosobenzenes that are ligands of ferrohaemoglobin caused haemolysis and most that are not ligands failed to cause nitrosoarenes appeared to be related more closely to the ease of their reduction to arylhydroxylamines than to their properties as ligands. We propose a mechanism of oxidative degradation whereby the cyclic formation of phenylhydroxylamine from nitrosobenzene within an erythrocyte leads to the accumulation of H2O2, which then reacts with ferrohaemoglobin to initiate the oxidative cleavage of haem. The posulated active intermediate in this reaction is the same as that previously proposed in the oxidative degradation of haemoglobin by phenylhydrzine and in the coupled oxidation of ascorbic acid and haemoglobin.


Polyamine metabolism in liver of young rats.

Rat liver undergoes a phase of rapid growth during weaning. We followed the changes in polyamine metabolism occurring during this period of natural growth, and compared them with changes in DNA and RNA accumulation. There was a 2.5-fold increase in the number of cells per liver between suckling (18--19 days old) and weaning (30--32 days old) rats. Ornithine decarboxylase activity increased from the low value in 18-day-old rat pups and remained significantly higher (approx. 5--10-fold) than that in adult rats from day 21 to day 34. Putrescine-dependent S-adenosylmethionine decarboxylase activity was slightly but significantly increased during most of this period. Spermidine and RNA concentrations fluctuated in concert, whereas spermine content per cell doubled during the period from day 23 to day 30.


Asynchrony in the synthesis of secretory proteins in the venom gland of the snake Vipera palaestinae.

1. Venom of Vipera palastinae was subjected to isoelectrofocusing on polyacrylamide gel. The protein separation profiles were similar for different venom samples; more than 25 protein bands with a wide range of pI values could be demonstrated by this technique. 2. Labelled venom was obtained 8h after an intracardial injection of [3H]leucine. The relative radioactivities of four out of 12 main protein bands were significantly different in the venom synthesized during the 2nd day of the venom regeneration cycle as compared with the venom of the 4th day. The comparison was made in venom samples obtained from the two glands of the same snake at two different secretory stages. 3. It is concluded that the asynchronous synthesis of exportable proteins after the initiation of a new venom regeneration cycle is responsible for the non-parallel secretion of some venom proteins by the venom gland of Vipera palaestinae during the first few days after milking.


Activity and androgenic control of enzymes associated with the tricarboxylic acid cycle, lipid oxidation and mitochondrial shuttles in the epididymis and epididymal spermatozoa of the rat.

1. Enzyme activities (units/g wet wt.) were determined in the caput and cauda epididymidis and in epididymal spermatozoa of the rat. 2. The activity of most enzymes in the cauda was between 50 and 100% of that in the caput, except that ATP citrate lyase was barely detectable in the cauda. 3. Spermatozoa, unlike epididymal tissue, contained sorbitol dehydrogenase but lacked ATP citrate lyase. NADP+-malate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, succinate dehydrogenase, carnitine acetyltransferase and citrate synthase were 5 to 400 times as active in spermatozoa as in epididymal tissue. 4. 2-Oxoglutarate dehydrogenase was the least active member of the tricarboxylic acid cycle in all tissues and most closely matched the measured flux through the cycle. 5. The concentrations of hydroxyacyl-CoA dehydrogenase and carnitine palmitoyltransferase were equivalent to the more active enzymes of the tricarboxylic acid cycle, indicating the capacity for extensive lipid oxidation, and the presence of 3-hydroxybutyrate dehydrogenase suggests that these tissues can also oxidize ketone bodies. 6. Transfer of reducing equivalents from cytoplasm to mitochondrion is unlikely to occur by means of the glycerol phosphate cycle because mitochondrial glycerol 3-phosphate dehydrogenase is relatively inactive in epididymal tissue, whereas the cytoplasmic enzyme has little activity in spermatozoa, but transfer may be accomplished by the malate-aspartate shuttle. 7. Transfer of acetyl units from mitochondrion to cytoplasm could be effected by the pyruvate-malate cycle in the caput of androgen-maintained rats, but not in the other tissues because of the low activity of ATP citrate lyase. Acetyl unit transfer could take place via acetylcarnitine, mediated by carnitine acetyltransferase. 8. Castration resulted in a decrease in the concentration of nearly all enzymes, although subsequent administration of testosterone restored concentrations to values similar to those in animals maintained by endogenous androgen. The extent to which enzyme concentration was changed by an alteration in androgen status was highly variable, but was most marked in the case of pyruvate carboxylase.


The time course of the effect of hypophysectomy and oestrogen treatment on the hepatic metabolism of androst-4-ene-3,17-dione in male and female rats.

Hypophysectomy of male animals has little effect on the hepatic androst-4-ene,3,17-dione (androstenedione) metabolism, except for possible changes in the kinetics of the 16alpha-and 7alpha-hydroxylases. On the other hand, hypophysectomy of female animals leads to a "masculinization" of hepatic androstenedione metabolism, following the changes seen in Vmax. of the enzymes involved, probably due to the removal of the source of "feminizing factor" thought to maintain the "female" type of metabolism in the liver. There seems to be a temporal dissociation of the effects on the various enzymes, indicating different cellular control mechanisms for these enzymes. Oestrogen treatment of male rats causes "feminization" of the hepatic androstenedione metabolism. The time study shows an initial increase in 17-hydroxy steroid oxidoreductase and 6beta- and 16alpha-hydroxylase activities, followed by a decrease to the values in females. This biphasic effect is possibly due to an initial direct effect via the hypothalamo-pituitary system.


Measurement and kinetic study of the formation of adrenaline sulphate in vitro.

The sulpho-conjugation of [14C]adrenaline form inorganic sulphate and ATP or preformed adenosine 3'-phosphate 5'-sulphatophosphate was demonstrated in the high-speed supernatant prepared from the liver and small intestine of various animals. Hydrolysis with sulphatase indicated the sulphate nature of the conjugate. The overall sulphation reaction has a pH optimum of 9.0. Maximal activity was obtained with a ratio of ATP/Mg2+ of 1 at 4--6mM. Above their optimal concentrations, ATP and Mg2+, separately or in combination, were inhibitory. Dithiothreitol at 3 mM stimulated the reaction by about 30%. The Km for adrenaline, determined by the sulphotransferase reaction and by the three-step (sulphate-activating and sulphotransferase) reactions was 125 micrometer. The rate of synthesis of [14C]-adrenaline sulphate, expressed in pmol/min per mg of protein for the livers of dog, monkey, rat, guinea pig and rabbit were, respectively, 144, 77, 47, 11 and 6. The corresponding values for the small intestines of dog and monkey were 60 and 62. Brain and heart tissues showed no measurable activity.


The membrane potential of mouse ascites-tumour cells studied with the fluorescent probe 3,3'-dipropyloxadicarbocyanine. Amplitude of the depolarization caused by amino acids.

1. The magnitude of the K+ gradient across the plasma membrane, which was in equilibrium with the membrane potential (E) of the tumour cells, was determined by the "null point" procedure of Hoffman & Laris (1974) [J. Physiol. (London) 239, 519--552] in which the fluorescence of the dye serves as an indicator of changes in the magnitude of E. 2. A mixture of oligomycin, 2,4-dinitrophenol and antimycin was used to stop the mitochondria from interfering with the fluorescence signal. Transport functions at the plasmalemma were maintained under these conditions in the presence of glucose. 3. Physiological circumstances were found in which incubation with glycine or with glucose changed the "null point" value of E within the range--20mV to--100mV. The fluorescence intensity at the "null point" was an approximately linear function of E over that range. The procedure enabled E to be inferred form the fluorescence intensity in circumstances where titration to the "null point" was not feasible. 4. The rapid depolarization caused by l-methionine or glycine was shown in this way to have a maximum amplitude of about 60mV. A mathematical model of this process was devised. 5. The electrogenic Na+ pump hyperpolarized the cells up to about --80mV when the cellular and extracellular concentrations of K+ were roughly equal. 6. The observations show that the factors generating the membrane potential represent a major source of energy available for the transport of amino acids in this system.


Equilibrium and steady-state models of the coupling between the amino acid gradient and the sodium electrochemical gradient in mouse ascites- tumour cells.

1. The tumour cells were incubated at 37 degrees C in Ringer solutions containing glucose, 1 mM-methionine, various concentrations of Na+ and K+ and, in some instances, ouabain or valinomycin to lower the membrane potential generated by the Na+ pump. After about 30 min, when the system had reached a steady state, the ratio [extracellular Na+]/[cellular Na+] varied from about 0.6 to 3.2 with the ionic conditions. The membrane potential, determined by means of the fluorescent probe 3,3'-dipropyloxadicarbocyanine, also varied systemically from about zero to--55mV.2. the ratio [cellular methionine]/[extra-cellular methionine] varied from about 1 to 35 in these circumstances. The Na+ electrochemical gradient, measured in the same units, varied from about 1 to 30. Its magnitude in 46 assays was almost directly proportional to, though slightly smaller than, the methionine gradient. 3. A mathematical model was used to define the relation between these two gradients, which were not in equilibrium, owing to the presence of a leak pathway for the amino acid. On the assumption that the values of [cellular Na+] were correct, the methionine gradient was about 1.8 times larger than this version of the gradient hypothesis predicted.


Influence of mouse age and erythrocyte age on glutathione metabolism.

In order to determine whether the biological age of a mouse influences erythrocyte metabolism and erythrocyte aging in vivo, blood samples were collected from male C57/BL6J mice of different biological ages ranging from mature (10 months) to "very old" (37 months). In the very old mouse, compared with the mature mouse, the erythrocyte survival time was decreased, erythrocyte densities were increased, the concentrations of total free thiol and reduced glutathione, and glutathione reductase activity were decreased. Erythrocytes were separated into different density (age) groups by phthalate ester two-phase centrifugation or by albumin density-gradient centrifugation. The density-age relationship of erythrocytes was established by pulse-labelling with 59Fe in vivo and by subsequent determinations of specific radioactivity of erythrocyte fractions of different densities prepared during a chase period of 60 days. The age of erythrocytes in mice of all ages was directly related to density. Also, in older erythrocytes compared with younger erythrocytes, decreased concentrations of total free thiol and reduced glutathione, and decreased glutathione reductase activity were observed. These were the lowest in the old erythrocytes of very old mice. These results in aging erythrocytes from aging mice suggest that the glutathione status the erythrocyte may be an index of aging, not only of the cell but also of the organism.


Uptake and degradation of iodine-labelled chylomicron remnant particles by monolayers of rat hepatocytes.

1. Rat chylomicrons were labelled with 125I with 69--72% of the iodine in the protein moiety. Less than 1 nmol of iodine was incorporated per nmol of protein. Of the peptide radioactivity 44--56% was in apolipoprotein A-1, 30--40% in the C peptides and 11--15% in apolipoprotine B. The arginine-rich peptide, which accounted for about 14% of the chylomicron protein mass as determined by scanning of sodium dodecyl sulphate-polyacrylamide gels, contained very little radioactivity. 2. Chylomicron remnants generated with postheparin plasma from iodine-labelled chylomicrons showed a relative increase in the percentage of the arginine-rich peptide (76--90% of the apolipoprotein mass according to gel scanning). The major portion of the peptide iodine label was present in apolipoprotein A-1 (43--57%), B (22--32%) and C peptides (17--35%). 3. When iodine-labelled chylomicron remnants were added to rat hepatocytes in primary culture, labelled peptides were taken up and degraded by the hepatocytes by a saturable process. The Vmax. for the uptake was calculated to the 300ng of protein/h per mg of cell protein and the apparent Km as 7.7 microgram of protein/mg of cell protein. A larger proportion of the 125I-labelled lipids of the remnants (mainly polar lipids) was taken up. This suggest that these may also enter the cells by a mechanism that does not involve particulate uptake, such as phospholipid exchange. 4. The degradation of labelled peptides was inhibited by colchicine, concanavalin A, chloroquine and NH4Cl, which also inhibit degradation of the cholesteryl ester portion. All these drugs exerted their inhibition mainly after the uptake of labelled peptide. No degradation occurred at 4 degrees C, and also the uptake was markedly decreased. 5. The uptake of labelled chylomicron remnant peptide was 77 times as effective as that of labelled sucrose, which is likely to be taken up randomly by pinocytosis.


Some high-performance liquid-chromatographic studies of the metabolism of aflatoxins by rat liver microsomal preparations.

The metabolism of aflatoxin B1 in vitro was examined in rat liver microsomal preparations. 2. H.p.l.c. (high-performance liquid-chromatographic) systems were used. A silica column was used to separate non-polar metabolites. A system utilizing a reversed-phase column which separates both poar and non-polar metabolites was also developed. 3. The principal metabolites of aflatoxin B1 found were aflatoxin M1, aflatoxin Q1 and a compound which co-chromatographed with a degradation product of aflatoxin B1 2,3-dihydrodiol. 4. The time course of metabolism of aflatoxin B1 by microsomal preparations isolated from control and phenobarbitone-pretreated rats was examined. The rate and extent of metabolism was greater with microsomal preparations from the latter. The formation of aflatoxin Q1 was enhanced 4--5-fold by phenobarbitone pretreatment, whereas the production of aflatoxin M1 was only increased 1--2-fold. The formation of the degradation product of aflatoxin B1 2,3-dihydrodiol was increased 4--5-fold by the pretreatment with phenobarbitone. 5. The microsomal metabolism of aflatoxins M1, P1 and Q1 was examined. Aflatoxin M1 apparently underwent very limited microsomal metabolism to more polar compounds. Aflatoxin P1 was not metabolized. The situation with aflatoxin Q1 was complicated in that it was metabolized in the absence of NADPH to an unidentified metabolite. Aflatoxin B1 appeared as a metabolite of aflatoxin Q1 only when NADPH was present, and the formation of more polar metabolites was also then observed.


Metabolic activation of acetylenic substituents to derivatives in the rat causing the loss of hepatic cytochrome P-450 and haem.

1. A number of acetylenic-substituted steroidal and non-steroidal compounds, including 2,2-dipropargylacetamide, pregna-2,4-dien-20-yno[2,3-d]isoxazol-17-ol (Danazol) and acetylene gas, when administered to rats in vivo brought about a decrease in the concentrations of hepatic microsomal cytochrome P-450 and haem. Abnormal haem-breakdown products, ;green pigments', and porphyrins accumulated in the livers of these animals. 2. For loss of microsomal cytochrome P-450 to occur in vitro, metabolic activation of the acetylenic substituent was necessary. The enzyme system responsible required NADPH and air, and was induced by pretreatment of rats with phenobarbitone; these are characteristics typical of the microsomal mixed-function oxidases. 3. When rats were dosed with 17alpha-ethynyl-17beta-hydroxyandrost-4-en-3-one (ethynyltestosterone, 1mmol/kg) the pattern of green pigments extracted from the liver 4h after dosing and separated by t.l.c. was quite different from that in rats given 17beta-hydroxy-17alpha-vinylandrost-4-en-3-one (vinyltestosterone), suggesting that reduction of the unsaturated triple bond to a double bond is not normally part of the metabolic activation pathway of the acetylenic substituent. 4. The green pigments extracted from the livers of rats 4h after the administration of the acetylenic-substituted compounds (1mmol/kg) when separated by silica-gel t.l.c. had variable R(F) values. The number and distribution of green pigments was characteristic for each compound examined. There was little correlation between the total loss of hepatic microsomal haem and the apparent intensity of the green pigments seen on the thin-layer chromatograms. 5. After incubation of [(14)C]acetylene in vitro with microsomal preparations from phenobarbitone-pretreated rats and a NADPH-generating system, no significant covalent binding to microsomal protein was detected over a 30min incubation period, although under similar conditions there was a significant loss of cytochrome P-450.


The submicrosomal localization of acyl-coenzyme A-cholesterol acyltransferase and its substrate, and of cholesteryl esters in rat liver.

To determine the submicrosomal distribution of acyl-CoA-cholesterol acyltransferase and of cholesteryl esters, the microsomal fraction and the digitonin-treated microsomal preparation of rat liver were subjected to analytical centrifugation on sucrose density gradients. With untreated microsomal fractions the distribution profile and the median density of acyl-CoA-cholesterol acyltransferase were very similar to those of RNA. This is in contrast with hydroxymethylglutaryl-CoA reductase and cholesterol 7alpha-hydroxylase, which are confined to endoplasmic reticulum membranes with low ribosomal coating. In digitonin-treated microsomal preparations activity of acyl-CoA-cholesterol acyltransferase was not detectable. The labelling of untreated microsomal fractions with trace amounts of [(14)C]cholesterol followed by subfractionation of the labelled microsomal fraction showed that the specific radioactivity of cholesteryl esters obtained in vitro by the various subfractions was similar with all subfractions but different from the specific radioactivity of the 7alpha-hydroxycholesterol obtained in vitro by the same subfraction. These results demonstrate the existence of two pools of cholesterol confined to membranes from the endoplasmic reticulum, one acting as substrate for cholesterol 7alpha-hydroxylase and the other acting as substrate for acyl-CoA-cholesterol acyltransferase. The major part of cholesteryl esters present in both untreated and digitonin-treated microsomal fractions was distributed at densities similar to those of membranes from the smooth endoplasmic reticulum and at densities lower than those of smooth membranes from Golgi apparatus. The ratio of the concentrations of non-esterified to esterified cholesterol in the subfractions from both untreated and digitonin-treated microsomal fractions was highest at the maximum distribution of plasma membranes.


Biosynthesis of plasma-membrane proteins during myogenesis of skeletal muscle in vitro.

1. Surface labelling of plasma-membrane proteins with 125I, catalysed by lactoperoxidase, and radioactive l-fucose incorporation into glycoprotein were used as plasma-membrane markers for skeletal-muscle cells in culture. 2. Plasma membranes were prepared at various stages of myogenesis in vitro and rates of synthesis and accumulation of proteins in the membranes were compared. 3. Increased synthesis and accumulation of a protein of apparent mol.wt. 70000 occurred in the plasma-membrane fraction concomitant with the onset of myoblast fusion. 4. In cultures in which fusion of myoblasts was inhibited by 5'-bromo-2-deoxyuridine, synthesis and accumulation of the protein of apparent mol.wt. 70000 was selectively inhibited. 5. It is suggested the protein of apparent mol.wt. 70000 may be involved in the process of myoblast fusion.


Effect of zinc status of rats on the synthesis and degradation of copper-induced metallothioneins.

Injection of Zn2+-adequate and Zn2+-deficient rats with Cu2+ stimulated the incorporation of l-[35S]cysteine into a low-molecular-weight Cu2+-binding protein in both liver and kidney. No significant incorporation of l-[4,5-3H]leucine into this protein occurred, confirming the previous claim that it was metallothionein and not some other leucine-rich protein. The half-life of the protein was found to be 16.9 +/- 1.0 (S.E.)h in the liver of Zn2+-adequate rats but only 12.3 +/- 0.5h in Zn2+-deficient animals. The degradation rate of the metallothionein was similar to the rate of disappearance of Cu2+ and Zn2+ from the protein, indicating that the release of mental from the protein and its catabolism occurred simultaneously. There was no significant difference in the half-lives of the hepatic or renal copper-thioneins in Zn2+-adequate rats.


Changes in the form of Arrhenius plots of the activity of glucagon-stimulated adenylate cyclase and other hamster liver plasma-membrane enzymes occurring on hibernation.

1. Arrhenius plots of the glucagon-stimulated adenylate cyclase, 5'-nucleotidase, (Na+ + K+)-stimulated adenosine triphosphatase and Mg2+-dependent adenosine triphosphatase activities of control hamster liver plasma membranes exhibited two break points at around 25 and 13 degrees C, whereas Arrhenius plots of their activities in hibernating hamster liver plasma membranes exhibited two break points at around 25 and 4 degrees C. 2. A single break occurring between 25 and 26 degrees C was observed in Arrhenius plots of the activities of fluoride-stimulated adenylate cyclase, basal adenylate cyclase and cyclic AMP phosphodiesterase of liver plasma membranes from both control and hibernating animals. 3. Arrhenius plots of phosphodiesterase I activity showed a single break at 13 degrees C for membranes from control animals, and a single break at around 4 degrees C for liver plasma membranes from hibernating animals. 4. The temperature at which break points occurred in Arrhenius plots of glucagon- and fluoride-stimulated adenylate cyclase activity were decreased by about 7--8 degrees C by addition of 40 mm-benzyl alcohol to the assays. 5. Discontinuities in the Arrhenius plots of 4-anilinonaphthalene-1-sulphonic acid fluorescence occurred at around 24 and 13 degrees C for liver plasma membranes from control animals, and at around 25 and 4 degrees C for membranes from hibernating animals. 6. We suggest that in hamster liver plasma membranes from control animals a lipid phase separation occurs at around 25 degrees C in the inner half of the bilayer and at around 13 degrees C in the outer half of the bilayer. On hibernation a change in bilayer asymmetry occurs, which is expressed by a decrease in the temperature at which the lipid phase separation occurs in the outer half of the bilayer to around 4 degrees C. The assumption made is that enzymes expressing both lipid phase separations penetrate both halves of the bilayer, whereas those experiencing a single break penetrate one half of the bilayer only.


Zinc-binding protein in the livers of neonatal, normal and partially hepatectomized rats.

In the livers of rats after partial hepatectomy the zinc concentration began to increase soon after the operation, reached a maximum value at 14h, and decreased to the original value by 25h after the operation. In contrast, the plasma zinc concentration continued to decrease during the first 10h after the operation and remained depressed for at least 28h. The plasma and hepatic zinc concentrations were relatively unaffected by sham-operation. Synchronous with the increase in the hepatic zinc concentration after the partial hepatectomy, there was an appearance of zinc-binding protein (Zn-binding protein) in the liver cytosol. Studies with small doses of actinomycin D and cycloheximide suggest that both RNA and protein syntheses are necessary for the induction of Zn-binding protein after partial hepatectomy. A high content of the Zn-binding protein was found in neonatal rat liver. The Zn-binding protein, however, was undetectable 40 days after birth. The Zn-binding protein was also found in the adult rat liver when stimulated to proliferate after the administration of isoprenaline followed by glucagon. These findings indicate a close linkage between the appearance of Zn-binding protein in the liver cytosol and the regulation of DNA synthesis.


The biosynthetic pathway of new polyamines in Caldariella acidophila.

1. Spermidine and sym-nor-spermine (1,11-diamino-4,8-diazaundecane) were identified as the major components of the polyamine pool in Caldariella acidophila, an extreme thermoacidophilic bacterium. A minor component, a new polyamine, sym-nor-spermidine (1,7-diamino-4-azaheptane) was isolated and characterized. 2. To elucidate the biosynthetic pathway, labelled methionine, putrescine, spermidine and spermine were fed to Caldariella acidophila. Incubation of the bacterium in the presence of putrescine or spermidine labelled in the tetramethylene moiety gave unlabelled sym-nor-spermidine and sym-nor-spermine, whereas the radioactivity of propylamine-labelled methionine or spermidine was incorporated into these molecules. No radioactivity was recovered in the polyamines pool when spermine was fed to Caldariella acidophila. 3. S-Adenosylmethionine and S-(5'-adenosyl)-3-methylthiopropylamine were identified as intermediates of the biosynthetic pathway; the cellular contents of the two sulphonium compounds, measured with a new isotope-dilution technique, are 60 and 15nmol/g wet wt. of cells respectively. 4. The above results are indicative of a new pathway characterized by three propylamine-transfer reactions, decarboxylated S-adenosylmethionine being the common donor of the propylamine moiety. The reactions yielding sym-nor-spermidine and sym-nor-spermine are reported for the first time. 5. The probable intermediates related to the catabolism of the tetramethylene moiety of spermidine, gamma-aminobutyraldehyde, gamma-aminobutyric acid or Delta(1)-pyrroline were not detectable. Experiments with [3-aminopropyl-3(n)-(3)H]spermidine trihydrochloride plus [tetramethylene-1,4-(14)C]spermidine trihydrochloride gave rise to an amount of labelled CO(2) equivalent to the spermidine catabolized.


Plasma clearance of glycoproteins with terminal mannose and N-acetylglucosamine by liver non-parenchymal cells. Studies with beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, ribonuclease B and agalacto-orosomucoid.

Glycoproteins having mannose and/or N-acetylglucosamine in the terminal non-reducing position [Stockert, Morell & Scheinberg (1976) Biochem. Biophys. Res. Commun. 68, 988--993], and various lysosomal enzymes [Stahl, Schlesinger, Rodman & Doebber (1976) Nature (London) 264, 86--8] are rapidly cleared from plasma by the liver after intravenous administration. A liver cell-separation technique was used to determine the cellular localization of 125I-labelled beta-glucuronidase, ribonuclease B, agalacto-orosomucoid and asialo-orosomucoid. On a specific readioactivity basis, all ligands except 125I-labelled asialo-orosomucoid were enriched in the non-parenchymal cell fraction. Isolated cells, fixed and stained for beta-glucuronidase or N-acetyl-beta-D-glucosaminidase activity after intravenous injection of the enzymes, showed enrichment in the non-parenchymal cell fraction (probably Kupffer cells). After uptake by the non-parenchymal cells, liver lysosomal beta-glucuronidase and N-acetyl-beta-D-glucosaminidase showed degradation half-times of 2.2 and 0.4 days respectively.


Synthesis of 1 alpha-hydroxy[7-3H]cholecalciferol and its metabolism in the chick.

1. 1 alpha-Hydroxy[7-3H]cholecalciferol (specific radioactivity of 2-Ci/mmol) was synthesized, and its metabolism in chicks studied. 2. 1 alpha-Hydroxy[7-3H]cholecalciferol was metabolized very rapidly in the chick to 1 alpha,25-dihydroxy[7-3H]cholecalciferol and to a metabolite less polar than 1 alpha-hydroxycholecalciferol. Intestine exhibited highest accumulation of 1 alpha-25-dihydroxy[7-3H]cholecalciferol, and liver exhibited highest accumulation of the non-polar metabolite. 3. Tissue uptake of 1 alpha-hydroxy[7-3H]cholecalciferol and its metabolites in chicks that were dosed continuously for 16 days with 1 alpha-hydroxy[7-3H]cholecalciferol did not exceed by very much that observed in tissues obtained from chicks that were dosed with a single injection of 1 alpha-hydroxy[7-3H]cholecalciferol 24 h before killing, except for liver and kidney. 4. Lowest accumulation of metabolites was noted in muscle and bone, and for the latter, highest uptake of 1 alpha,25-dihydroxy[7-3H]cholecalciferol was noted in the epiphysial periosteum and the metaphysis. 5. Formation of 1 alpha,24,25-trihydroxy[7-3H]cholecalciferol was not observed in the chicks that were dosed continuously with 1 alpha-hydroxy[7-3H]cholecalciferol, despite the fact that plasma calcium and phosphorus were normal and despite the presence of renal 24-hydroxylase activity. 6. The vitamin D status of the chicks did not appear to affect the metabolic profile of the administered 1 alpha-hydroxy[7-3H]cholecalciferol.