Somatostatinoma of the duodenum.

An adenocarcinoma of the second portion of the duodenum in a 26-year-old male is presented. The patient was suffering from pain in the epigastrium. Immunofluorescent studies revealed that it consisted almost exclusively of cells with a distincly positive somatostatin-like immunoreactivity. Ultrastructurally, the cytoplasm of the tumor cells had numerous large round granules (about 400 micrometers) with variable electron density. Most of these cells closely resembled the D cells normally seen in the duodenum and the islets of the pancreas, although a few argyrophil cells could be demonstrated by light microscopy. Radioimmunoassay of extracts of the tumor revealed a large amount of somatostatin (2260 pg/mg); substance P and VIP were detected also. Somatostatinoma has been known to occur in the pancreas, but this seems to be the first somatostatinoma found in the intestine.


The basal cell nevus syndrome: disasters occurring among a series of 36 patients.

We have reviewed a series of 36 patients with the basal cell nevus syndrome seen at Roswell Park Memorial Institute from 1950 to 1978. Among these patients a number of disastrous complications occurred. These include extensive uncontrolled basal cell epithelioma of the face resulting in one death from direct cerebral invasion via the orbit, one facial nerve palsy, two bilateral and two unilateral eye enucleations, and several severe facial mutilations in young patients. There were three patients who developed severe bradycardia and hypotension during induction of general anesthesia required postponement of the intended operation, and one patient with congenital hemiparesis who on investigation was found to have an absent internal carotid artery. A review of the syndrome complex is presented with a plan for recognition and management.


Oncobytic papillary cystadenomatosis of the larynx: a clinicopathologic entity.

A patient with numerous oncocytic lesions of the larynx is reported. Laryngectomy for a concurrent epidermoid carcinoma made it possible to study the entire laryngeal mucosa. A hyperplastic component to the oncocytic metaplasia of the ductal epithelium of the seromucinous glands resulted in a spectrum of lesions varying from simple oncocytic cysts to solid lesions that may be erroneously interpreted as true neoplasms (oncocytomas). Oncocytic papillary cystadenomatosis is a condition affecting the larynx in a diffuse fashion suggesting the need for follow-up on patients with biopsy proven oncocytic lesions.


Multiple disparate cranial neoplasms.

The case of a three-year-old girl with fibrosarcoma of the right orbit is presented. She was initially treated with surgery and later with radiotherapy. After an interval of thirty-three years she developed a suprasellar epidermoid cyst and two years later a right frontal meningioma. The two benign tumors fulfill the usual criteria for irradiation induced tumors and the possible role of radiotherapy in their induction is discussed.


Acute hepatic vein thrombosis occurring during therapy for Hodgkin's disease: a case report.

A case of fatal acute hepatic vein thrombosis occurred during treatment for Hodgkin's disease. At necropsy no evidence of Hodgkin's disease or obliterative venoocclusive disease of the liver was found. We speculate that Adriamycin, DTIC and/or vinblastine may have been responsible for this unusual disorder.


Transsphenoidal hypophysectomy in breast cancer: evidence for an individual role of pituitary and gonadal hormones in supporting tumor growth.

Transsphenoidal hypophysectomy was performed in 212 consecutive patients with metastatic breast cancer: 11 died within 30 days, two of surgical complications and nine of advanced metastatic disease. Two patients were unevaluable because of inadequate follow-up in one and simultaneous radiation treatment in the other. Of 199 evaluable patients 42% had an objective remission. Duration of remission averaged 18+ months with 10 out of 84 patients still in remission. Presence of estrogen receptors in the tumor significantly predicted response to hypophysectomy. Of 156 patients in whom completeness of hypophysectomy was assessed, 128 were thought to have a complete removal as shown by the fact that their growth hormone and prolactin were undetectable after stimulation with arginine or chlorpromazine, respectively. Of 26 patients in whom TRH test was performed, TSH and prolactin were undetectable in 20. Of 23 patients where autopsy was performed only six had microscopic pituitary tissue remaining. Hypophysectomy induced remission in eight of 15 patients who had previously responded and then relapsed to the antiestrogen Tamoxifen and in four of 17 who had failed. Conversely, antiestrogen therapy induced remission in six of 26 patients who had previously responded to hypophysectomy and in whom serum estrogens were present in small amount. These data indicate that both gonadal and pituitary hormones play a role in the growth of some human breast cancers.


Indications and results of omental pedicle grafts in oncology.

Sixty omental grafts were performed in our department. Sixty-two percent concerned breast cancer patients. Other grafts were undertaken for other cancers: head and neck, gynecologic urologic and intestinal, skin and soft tissue tumors. These grafts were indicated for radionecrosis or chemonecrosis in 33 cases and for cancer recurrence in 26 cases (among whom 24 were previously irradiated). Only one graft was performed for lymphoedema treatment. Overall, fifty four patients (83.5%) had successful grafts, and six (16.5%) had graft failures. According to the treated lesion we obtained 82% of successful treatment among patients treated for radio or chemonecrosis, and 92% for patients treated for recurrences.


Transplantation of azaserine-induced carcinomas of pancreas in rats.

Two pancreatic adenocarcinomas which had been induced in Wistar/Lewis rats by azaserine treatment were transplanted into rats of the same strain by subcutaneous and intraperitoneal injection of minced tumor. Subsequently, we have serially transplanted into non-radiated recipients. Transplanted tumors have maintained evidence of acinar cell differentiation including the presence of zymogen granules in tumors studied by electron microscopy, and of lipase, amylase and trypsin activity in the supernatant of tumor homogenates. Histologically, the tumors vary from poorly differentiated solid carcinomas to well differentiated variants which form acini. Transplanted tumors are locally invasive and have metastasized to lung and liver in some recipients.


Urinary metabolite of 1-(2,4,6-trichlorophenyl)-3,3-dimethyltriazene with an intact diazoamino structure.

The tumour-inhibiting substance 1-(2,4,6-trichlorophenyl)-3,3-dimethyltriazene is metabolised in rats to the corresponding substituted 1-O-(triazenyl-methyl) glucuronic acid. The urinary metabolite was purified by ion exchange chromatography and gel filtration, and isolated from the enriched fractions by freeze-drying. Cold acid cleavage into the 2,4,6-trichlorobenzene-diazonium cation and hydrolysis to glucuronic acid and formaldehyde indicated the presence of an O-glycosidic bond through an enzymically-introduced hydroxymethyl oxygen. This novel type of glucuronoside structure was established by chemical evidence, and confirmed by NMR and field-desorption mass spectrometry. It is conceivable that this metabolite represents a stabilised carrier form of the biologically-active triazene that transports the methylating agent from its site of formation to its ultimate target.


Effect of graded dietary levels of selenium on tracheal carcinomas induced by 1-methyl-1-nitrosourea.

An investigation was conducted to determine the effect of the dietary level of selenium on the induction of tracheal cancer by 1-methyl-1-nitrosourea (MNU). Male Syrian golden hamsters received intratracheal instilations of a 0.5% MNU solution once weekly for 12 weeks. Two weeks prior to the initiation of carcinogen treatment, animals were placed on a semisynthetic, 30% torula yeast diet to which either no selenium or 1 or 5 mg selenium/kg of diet as sodium selenite was added. Animals were maintained on their respective diets for the duration of the study which was terminated 195 days after the first MNU treatment. No significant differences among groups in the incidence of either benign lesions or carcinomas was observed and the distribution of tumor type was similar irrespective of selenium treatment. The results of this study indicate that selenium exerts no chemopreventive effect against MNU-induced tracheal carcinogenesis.


UV-induced DNA repair in hairy cell leukaemia patients.

We analysed by liquid scintillation counting and by autoradiography the DNA repair synthesis induced by UV irradiation in mononucleated cells from peripheral blood of 'hairy cell leukaemia' patients. In 9 out of 12 patients the repair activity values obtained in repeated assays were found to be significantly lower than those of healthy donors.


Purified human fibroblast interferon in vivo: skin reactions and effect on bone marrow precursor cells.

Human interferon from normal diploid fibroblasts, purified by sequential chromatography on concanavalin A-agarose and phenyl-sepharose, was administered parenterally in 4 subjects. Fever, marked skin hypersensitivity reactions and suppression of marrow stem cells (estimated by the count of myeloid colony-forming cells), side-effects common for less purified fibroblast and leukocyte interferons, were absent. Purified fibroblast interferon retained antiviral and immunomodulatory activity, evidenced by reduction of the blastogenic response of peripheral lymphocytes and decrease of hepatitis B virus markers in a patient with chronic hepatitis B infection treated with this substance.


Repair and de novo replication of rat lung DNA following instillation of 3-methylcholanthrene.

Structural analysis of rat lung DNA was made by stepwise elution of the nucleic acid from benzoylated--DEAE-cellulose with 1.0 M NaCl and 30% formamide solutions respectively, the DNA content of collected fractions being determined by absorbance. The proportion of the minor fraction, which was eluted with formamide and thought to contain DNA having single-stranded regions, was increased by intratracheal instillation of 3-methylcholanthrene prior to death of the animal. Maximum initial increase was observed 14 h after treatment. Analysed up to 10 days after treatment, there was a biphasic response, the early maxima being dose dependent and the second apparently independent of the amount of hydrocarbon administered. The data implicate DNA repair processes and cellular proliferation stimulated by 3-methylcholanthrene as being involved in recovery of the lung from this potentially carcinogenic insult.


Identification of S-(carboxymethyl)-L-cysteine and thiodiglycolic acid, urinary metabolites of 2,2'-bis-(chloroethyl)-ether in the rat.

S-(carboxymethyl)-L-cysteine (CMC) and thiodiglycollic acid (TGA) were identified by gas chromatographic-mass spectrometric measurements in the urine of rats after intraperitoneal injections of 2,2'-bis-(chloroethyl)-ether (BCEE). It is therefore probable that BCEE is O-dealkylated by a mixed-function oxidation. The hepatocarcinogenic effect of BCEE may be explained by the liberation of chloroacetaldehyde in vivo.


Induction of aryl hydrocarbon hydroxylase in human peripheral blood lymphocytes by chrysene.

Many of the polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene (B[a]P), benzanthracene (BA), 3-methylcholanthrene (3-MC)) are not only carcinogenic, but also induce AHH in human tissues. Recently, chrysene has been implicated as an etiologic determinant of chemical carcinogenesis. Here we describe the ability of chrysene to induce AHH in cultured human lymphocytes. Lymphocytes were obtained from 9 healthy subjects, divided into 2 sets, and cultured in duplicate, triplicate, or quadruplicate for 48 h. Chrysene (25 microM final concentration) in acetone was then added to the induced culture set and the control set received acetone alone. Lymphocytes were then cultured an additional 24 h before harvesting. AHH was quantitated by a fluorometric analysis of the phenolic metabolites produced by incubating the lymphocytes with B[a]P for 35 min. A significant increase in enzyme induction occurred in the chrysene-induced cultures compared with control (non-induced) cells (one-tailed student t-test; P less than 0.001). It was also observed that the interindividual variation in AHH inducibility seen with other PAHs is also observed with chrysene.


Selenium effects on the carcinogenicity and metabolism of 2-acetylaminofluorene.

Addition of 4 ppm Se to the drinking water of male albino rats fed diets containing 0.03% 2-acetylaminofluorene (AAF) provided protection against hepatic damage and also resulted in at least 50% reduction in liver tumor incidence. An in vitro assay system utilizing microsomes from Se supplemented or non-supplemented 3-methylcholanthrene (MC) induced rats was used to determine the effect of oral Se intake on the metabolism of AAF. Oral Se administration led to an increase in ring hydroxylation and a decrease in N-hydroxylation. Addition of Se to the microsomal assay system increased 3-OH AAF formation and decreased N-OH AAF formation, thus shifting the balance of metabolism toward detoxification pathways.


Carcinogenicity of N-nitroso-ethylenethiourea in female mice.

The nitrosation product of ethylenethiourea (ETU), N-nitroso-ETU, was tested for tumorigenicity in female ICR mice. Ten weekly oral administrations of this compound (0.66-2.64 mg/dose) caused apparently dose-dependent increases in the number of mice with pulmonary and lymphocytic neoplasms.


Induction of aryl hydrocarbon hydroxylase in primary cultures of type II alveolar lung cells and binding of metabolically activated benzo[a]pyrene to nuclear macromolecules.

Alveolar-like structures cultured on a gelatin sponge substrate are composed of cells resembling type II alveolar pneumonocytes. These cells contain aryl hydrocarbon hydroxylase (AHH) as determined by fluorescent measurements of water-soluble product from the metabolism of polycyclic aromatic hydrocarbons (PAH). Of 3 compounds tested, benzo[a]pyrene (BP) stimulates the highest level of AHH activity. Activity reaches a peak in 6 day cultures and remains relatively stable for a culture period of 12 days. Metabolites of tritium labeled BP interact with nuclear macromolecules in these cells as determined by measurements of 3H binding to DNA, histone and non-histone chromosomal proteins. A preferential binding to the non-histone protein fraction occurs.


Potentiation of antitumor effect of cyclophosphamide and hydrazine sulfate by treatment with the antidiabetic agent, 1-phenylethylbiguanide (phenformin).

It was shown that 1-phenylethylbiguanide (phenformin) administered orally to mice at a dose of 2 mg/day potentiated the antitumor effect of cyclophosphamide on transplantable squamous cell cervical carcinoma, hepatoma-22a and Lewis lung tumor, but did not alter the effect of cyclophosphamide on sarcoma-180 and L-1210. Oral administration of phenformin (5 mg/day) to rats with transplanted Walker 256 carcinoma enhanced the antitumor effect of hydrazine sulfate.


Relationship between dose, time, and tumor yield in mouse dimethylhydrazine-induced colon tumorigenesis.

Colorectal tumor yield and volume data were obtained using 355 CF1 mice serially sacrificed up to 84 weeks following various dose levels of the carcinogen 1,2-dimethylhydrazine dichloride (DMH). Several conclusions were reached: (a) With increasing doses of DMH, there was an increased tumor yield and decreased latency period. (b) With repeated doses, there was a rapidly cumulative tumor yield. (c) New tumors continued to accumulate in the colon and rectum even at long intervals after the DMH treatments. This was substantiated by a positive correlation between the number of tumors per colon and the delay after DMH. In addition, when several tumors were present in the same mouse, their sizes were graded rather than uniform. These observations are consistent with a 2- or multi-step carcinogenesis mechanism. The latter implies that DMH induces a permanent transmissible alteration within some cells which thereafter will be at risk for further alterations capable of initiating cancer growth.


Relative extents of hydrogen-deuterium exchange of nitrosamines: relevance to biological isotope effect studies.

Relative extents of base-catalyzed, hydrogen-deuterium exchange have been determined for a number of nitrosamines. Observed trends in the exchanges are discussed in terms of substitution, ring size and conformation. The relevance of the exchanges to deuterium isotope effects in carcinogenesis tests is discussed. Those compounds which give pronounced biological isotope effects undergo exchange only to a small extent. No biological isotope effect is found for compounds which undergo extensive exchange.


Alpha-hydroxylation of N-nitrosopyrrolidine and N'-nitrosonornicotine by human liver microsomes.

The environmental carcinogens N-nitrosopyrrolidine (NPYR) and N'-nitrosonornicotine (NNN) were incubated with human liver microsomes to test for the presence of enzymes catalyzing alpha-hydroxylation, which is likely to be an activation mechanism for these compounds. Both nitrosamines underwent alpha-hydroxylation; rates were higher for NPYR than for NNN, as observed in rats. The results indicate that both NPYR and NNN can be metabolically activated by human liver microsomes.


The stimulation of murine hepatic aryl hydrocarbon hydroxylase activity in vitro by caffeine.

The activity of aryl hydrocarbon hydroxylase (AHH) of murine liver is increased by caffeine. However, other analogs of caffeine, such as theophylline, theobromine xanthine, hypoxanthine and uric acid, do not significantly alter AHH activity. The maximal stimulation is observed at a caffeine concentration of approx. 3 mM. The Km of AHH for benzo[a]pyrene is decreased in the presence of caffeine. Thus, the inhibition by caffeine of the binding of polycyclic aromatic hydrocarbons (PAH) to DNA may be related to its effect on AHH.


The retrospective study of the carcinogenic hydrocarbon benz[a]pyrene in the biosphere.

The major aim of this study was to determine taking, as an example, the carcinogenic polycyclic aromatic hydrocarbon benz[a]pyrene (BaP), the present biologically active compounds in the early historical and geological epochs with the following assessment of the degree of danger of such compounds in the modern times. In the first section of work, study results of soil samples in the areas of eternal frost confirmed the presence of BaP in the frozen layers of soil aged 10 years, 100 years, 3000--4000 and 10,000 years of age. In the second part of the work, results are furnished on the BaP content in the ice of modern glaciers and their moraines, located in Kamchatka. BaP was found in 11 samples in the concentration of 0.001--0.003 microgram/l. These data represent the first results in the retrospective study of carcinogenic substances in the biosphere.


The possibilities of reproducing effects of benzo[a]pyrene in a transformation test using Syrian hamster fetal lung cells.

Various dose levels of benzo[a]pyrene (BP) were tested on short-term cultured foetal hamster lung cells for transforming effects in vitro in 6 similar experiments. The statistical evaluation of the results indicated a weak reproducibility (P = 0.09). Factors which inherently influence such reproducibility were considered.


Tumor initiating activity of 5,11-dimethylchrysene and the structural requirements favoring carcinogenicity of methylated polynuclear aromatic hydrocarbons.

The tumor initiating activities of 5,11-dimethylchrysene and 5-methylchrysene on mouse skin were compared. After initiating doses of 30 microgram or 10 microgram, with promotion by 3 times weekly applications of tetradecanoylphorbol acetate, both compounds were highly tumorigenic, inducing tumors in 70--85% of the treated animals. Since 5,12-dimethylchrysene had previously been shown to be only a weak tumor initiator, these results support the generalization that the structural requirements favoring carcinogenicity among the methylated chrysenes and other polynuclear aromatic hydrocarbons (PAH) are a bay region methyl group and a free peri position, both adjacent to an unsubstituted angular ring.


Significance of the karyorrhectic index in 1,2-dimethylhydroazine carcinogenesis.

Specimens from the liver, kidney, stomach and various intestinal segments of BD IX male rats were analyzed for mitotic index (MI) and the proportion of karyorrhectic figures (karyorrhectic index, KI) at various intervals after a s.c. injection of 1,2-dimethylhydrazine. In the intestinal specimens, a severe drop in MI (maximum after 12--14 h) and rise in KI (maximum after 6 h) were observed, with all karyorrhectic figures found in the proliferative compartment of the mucosa. Within the large intestine, there was a good correlation between the KI of each segment and its susceptibility to further tumor development.


The relationship of regional coronary blood flow to mitochondrial function during reperfusion of the ischemic myocardium.

The relationship of changes in regional coronary flow to the nature and degree of biochemical disturbances during occlusion of branches of the left anterior descending coronary artery and following reestablishment of flow was investigated in two groups of dogs: group I, moderate ischemia before reflow, and group II, severe ischemia prior to reflow. Regional coronary blood flow was determined before ligation, after 60 min of ischemia and after 15 min of reflow using labelled microspheres. Hearts made ischemic for 60 min but not reperfused served as controls. Groups I and II were distinguished by the following features. Group II showed a marked exacerbation of biochemical damage on reperfusion of the ischemic region (reduced levels of ATP, impairment of mitochondrial oxygen consumption and mitochondrial calcium binding). This was accompanied by significant subendocaridial hyperemia. Reperfusion in group I, on the otherhand, partially reversed these changes (increased level of ATP in the ischemic-reperfused region, improved mitochondrial oxygen consumption and calcium binding). Mitochondrial calcium uptake and oxidative phosphorylation (ADP/O ratio) were not affected in any group. These data illustrate that the degree of biochemical damage following reperfusion of the ischemic myocardium is determined by the degree of ischemia, and suggest that interference with ATP production by the mitochondria is not responsible for the damage.


Effect of a conditioning program in patients taking propranolol for angina pectoris.

17 patients with stable angina pectoris due to coronary occlusive disease had serial graded exercise tests performed while on no medication, after treatment with propranolol and after an 8-week exercise reconditioning regimen while taking propranolol. The patient whose angina-free exercise capacity on beta-blockade is 2.5 METs or greater is likely to benefit from an exercise program.


Excitation-contraction coupling in normal and myopathic hamster hearts I: identification of a calcium pool involved in contraction.

The results of this study indicate that two min of Ca-free perfusion did not significantly alter the exchange kinetics of mannitol or Ca in isolated heart preparations. A numerical solution for series Ca exchange between a superficial Ca pool, Ca1, and an intracellular pool, Ca2, was developed in terms of the coefficients and constants determined from a parallel analysis. The series and parallel models for Ca efflux yielded a high correlation between contractile activity and the rate of exchange and Ca content of Ca1. The kinetics of Ca influx were best described by a series model of exchange and agreed closely with the Ca contents and exchange properties measured during Ca efflux. No differences were detected between normal and myopathic hamster hearts in the kinetics of Ca exchange or the role of Ca1 in the E-C coupling process.


Excitation-contraction coupling in normal and myopathic hamster hearts II: changes in contractility and Ca pools associated with development of the cardiomyopathy.

Myocardial contractility and Ca exchange in the pool responsible for E-C coupling was studied in cardiomyopathic (B10 14.6) and control (B10.RB) hamster hearts at three stages in the development of the disease. Contractility (dP/dt) was significantly reduced in the B10 14.6 strain at 26 to 30 days of age, and at all subsequent stages of the disease. Reduced contractility was related directly to a reduction in the Ca content of a superficial pool, Ca1, representing Ca contained in the interstitial space and bound to the sarcolemma and other structures in the interstitium. Increased entry of Ca into cellular pools was associated with the reduction of superficial Ca content. The results suggest that the reduction in surface Ca binding sites may be responsible for the reduction in contractility.


Electrocardiographic changes in vitamin B6 deficient rats.

Serial electrocardiograms of vitamin B6 deficient rats show significant changes from those of rats on a complete diet. Animals fed a totally deficient diet from weaning grow slowly and die after about 12 weeks, often with a terminal weight loss. R and T amplitudes increase, then terminally decrease and are significantly greater than those of control animals. The PR interval is significantly shorter at times. Individuals have shown transient AV block, irregular sinus rhythm, wandering pacemaker, and inverted T waves. Autopsy findings include hypertrophy (89%), dilatation (61%) and atrial thrombi (44%). ECG's of adult animals remained normal for about three months after being fed the deficient diet. The two males developed premature ventricular contractions of several ectopic foci. Finally, split QRS complexes were observed. The female had a normal ECG until the final record at 234 days when some minor changes were seen. Extensive cardiomyopathic changes were found in two out of the three adults at autopsy.


Endothelial injury of arteries following catheterisation with polyethylene tubes: experimental studies on rabbit aorta using the Seldinger technique.

The effect of catheterisation on the rabbit aortic wall is investigated employing a technique identical to that used for continuous monitoring of blood pressure in humans. The catheters were introduced into the aorta through the right femoral artery of the rabbit using the Seldinger technique, except that the catheters were introduced directly into the femoral artery and not percutaneously. It was found that catheterisation for 24 h was followed by intimal and medial injuries. 3 to 60% (mean 20.4%) de-endothelialisation of the aortic surfaces was observed. The present experiments appear to explain some of the complications observed after continuous registration of blood pressure in humans. Case histories are referred to in which the brachial artery was catheterised.


Cytotoxic effects of colcemid or high specific activity tritiated thymidine on clonogenic cell survival in B6CF1 mice.

High specific activity tritiated thymidine (HSA-[3H]TdR) and colcemid were given in cytotoxic doses and regimens to B6CF1/Anl mice. The number of cells per intestinal crypt was reduced by the S-phase-specific (HSA-[3H]TdR and the metaphase blocking and cytotoxic effect of multiple injections of colcemid. In 50-day-old mice, the cytotoxic effect of multiple injections of colcemid reduced both the number of cells per crypt and the clonogenic cell survival. However, the number of surviving intestinal clonogenic or stem cells, assayed by the microcolony technique, did not change in 110--130-day old mice. These data suggest that most of the cells at risk from these cytotoxic agents are not clonogenic in adult 110--130-day old mice but are the cells in amplification division. However, since the stem cells of young mice are more susceptible to colcemid, they are apparently in a more rapid cell cycle than those of older mice. The clonogenic cell survival measured in 110--130-day old mice after a single radiation dose of 14 Gy (1400 rad) responded in a non-linear way to increasing time of continuous colcemid cytotoxicity. These data suggest that the intestinal stem cells can respond to amplification compartment cell death by a shortening of their cell cycle and thus, over time, the number of stem cells at risk to colcemid cytotoxicity increases.


Mitotic index, influx and mean transit time in the hamster cheek pouch epithelium, a partially synchronized cell system. Presentation of a mathematical model based on a non-stationary probability density function for the transit time in a compartment.

Mitotic activity was followed in the epithelium of the hamster cheek pouch for about 12 hr in two experiments under different conditions of noise and light intensity. A mathematical model based on a non-stationary probability density function for the transit time through mitosis was developed, making an analysis of this partially synchronized cell system possible. The most important result of the two experiments is the indication of a non-stationary mean transit time for cells in mitosis. In the first experiment (70 dB, 103 lux), which produced high mitotic indices (maximum 2.0%), the influx changed from 0. to 0.9%/hr (mean of 7-hourly determinations: 0.4%/hr), and the mean transit time from 1.9 to 5.5 hr. In the second experiment (70 dB, 265 lux), which had a lower level of MI (maximum 1.3%), the influx changed from 0 to 1.8%/hr (mean of 13-hourly determinations: 0.4%/hr), and the mean transit time from 0.3 to 3.3 hr. It was difficult to say precisely whether the variation in influx or in mean transit time was the main factor in the increase and decrease of MI. The data could not be simulated with a stationary mean transit time. It is suggested that changes in MI due to different external circumstances are mainly the result of variation in the mean transit time.


Limitations in the use of [3H]thymidine incorporation into DNA as an indicator of epidermal keratinocyte proliferation in vitro.

The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [14C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [3H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.


Analysis of intestinal cell proliferation after guanethidine-induced sympathectomy. II. Percentage labelled mitoses studies.

Neonatal administration of guanethidine-sulfate results in an alteration of the cell proliferative pattern of the small intestinal epithelium of the young adult rat. Sympathectomy with guanethidine has previously been shown to depress mitotic, labelling, and total cellular migration indices while increasing the generation cycle time (Tc) of small intestinal crypt cells as measured by a stathmokinetic method. The present study showed that the G1, S and G2 phases of the crypt cell cycle are altered by sympathectomy, G1 accounting for most of the increase in Tc. In addition, the percentage of [3H]-thymidine labelled crypt cells is reduced and the duration of crypt cell transit is lengthened by guanethidine-induced sympathectomy.


Variability of cell generation times in a hepatoma cell pedigree.

Time-lapse cinematographic analysis of a clone of HTC rat hepatoma cells showed variations in interdivision time within the clone. A positive correlation was found between the interdivision times of mother and daughter cells. The variability of the differences between interdivision times of cells of sister, cousin, second cousin or second-second cousin relationship was calculated. The proportion of cells with large differences in intermitotic times was found to increase with decreasing relationship. The clonal division pattern observed suggests strongly that 'inherited' factors goven the process leading to cell division but that their effects can be modified.


The cardiac ultrastructure of Chimaera monstrosa L. (Elasmobranchii: Holocephali).

The ultrastructure of the heart in Chimaera monstrosa L. is described. The endocardial and the epicardial cells are similar in the three cardiac regions. Myocardial cells show small variations. The myofibred, 4--6 microns thick, contains one or a few myofibrils. Each myosin filament is surrounded by six actin filaments. The sarcomere banding pattern includes the Z-, A-, I-, M-, N-, and H-band. End-to-end attachments between myofibres are composed of alternating desmosomes and fasciae adhaerentes. Desmosomes and nexuses occur between longitudinally oriented cell surfaces. The sarcoplasmic reticulum is poorly developed but well defined. Peripheral coupling-like structures are common, T-tubules are absent. Membrane bound dense bodies occur in all regions. Areas with ribosomes and single myosin filaments are often seen. The epicardial cells have a regular hexagonal surface and are much thicker than the endocardial cells. Numerous short and few longer cytoplasmic extensions face the pericardial cavity. The flat endocardial cells contain a large nucleus and small amounts of cytoplasm.


Fine structure of the ependyma and intercellular junctions in the area postrema of the rat.

Ependymal cells and their junctional complexes in the area postrema of the rat were studied in detail by tracer experiments using horseradish peroxidase (HRP) and colloidal lanthanum and by freeze-etch techniques, in addition to routine electron microscopy. The ependyma of the area postrema is characterized as flattened cells possessing very few cilia, a moderate amount of microvilli, a well-developed Golgi apparatus and rough endoplasmic reticulum. Numerous vesicles or tubular formations with internal dense content were found to accumulate in the basal processes of ependymal cells; the basal process makes contact with the perivascular basal lamina. It is suggested that the dense material in the tubulovesicular formations is synthesized within the ependymal cell and discharged into the perivascular space. The apical junctions between adjacent ependymal cells display very close apposition, with a gap of 2--3 nm, but no fusion of adjacent plasma membranes; they thus represent a transitional form between the zonulae adhaerentes present in the ordinary mural ependyma and the zonulae occludentes in the choroidal epithelium. A direct intercommunication between the ventricular cerebrospinal fluid (CSF) and the blood vascular system indicates that a region exists lacking a blood-ventricular CSF barrier.


Pathways and fine structure of neurons forming the nervi corporis allati II of the cockroach Periplaneta americana (L.).

By back-filling the nervus corporis allati II (NCA2) with Co2+ and precipitating the sulfide, two groups of somata (A and B) are revealed on the ipsilateral side of the subesophageal ganglion (SG). These occur anteroventrally, adjacent to the midsaggital plane. Group A consists of two cells; group B of five. Their processes form two discrete tracts issuing dorsoposteriorly into the neuropile between and slightly behind the circumesophageal connectives (CEC). After producing separate arborization fields in the dorsal neuropile, the tracts circumscribe the base of the ipsilateral CEC, unite, and their seven fibers enter NCA2 anteriorly. Prograde diffusion reveals 4--6 NCA2 axons penetrating the corpus allatum (CA) near a cap-like neurohemal organ. These axons form the transverse allatal tract (TAT), from whence they branch amongst the CA cells, and into the "cap", the postallatal nerves, and the opposite CA. Electron microscopy of transverse sections demonstrates nine neurosecretory axons entering the SG through NCA2. Proximal to the CA, NCA2 consists of a central bundle of neurosecretory axons and a peripheral zone confluent with the CA "cap". Depending upon the level of sectioning, there are 7--20 axons at the center, and seven pass into the TAT. The peripheral zone has the structure of a neurohemal organ.


Hypertrophic smooth muscle. III. Increase in number and size of gap junctions.

The smooth muscle cells of the circular musculature of the guinea pig ileum are connected by gap junctions (nexuses) which occupy about 0.21% of the cell surface. When the muscle hypertrophies in the portions of the ileum oral to an experimental stenosis, the muscle cells increase in size and number. Gap junctions become markedly larger than in control muscles and occupy 0.49% of the cell surface. While the cells double their surface area, the number of nexuses per unit surface remains unchanged (47--48 per 1000 microns2). The packing density of intramembrane particles (or pits) in the nexuses of hypertrophic muscle cells is 6700 . microns-2, which is slightly less than in control muscle cells (7200 . microns-2). A characteristic grouping of the particles (or the pits) within the nexus is often observed. Nexuses between two processes originating from the same cell are common. Nexuses do not occur in the longitudinal muscle.


Light and electron microscopical investigations on the tanycyte differentiation during the perinatal period in the rat.

The differentiation of tanycytes was studied light and electron microscopically during the perinatal period in rats, the time when functional connections between hypothalamus and hypophysis are established. The 3rd ventricle is slit-like between 16 and 18 days of the prenatal period. Its wall is formed by intensively proliferating matrix cells with apical processes, ovoid perikarya and a basal process. The ventral region of the 3rd ventricle becomes funnel-shaped on the 20th day of the prenatal period. As the cells differentiate, the apical process becomes shorter and broader. Moreover, on day 20 of prenatal life cells without apical processes appear. Their number increases during the postnatal period. The concentration of endoplasmic reticulum, mitochondria, polysomes, lipid droplets, dense bodies (lysosomes), lamellated and multivesicular bodies increases. Initially the cells are similar but from the 3rd day of postnatal life differentiation occurs in different regions of the infundibular recess. After the 5th day, there are no marked changes in the structure and distribution of these cells.


Ultrastructural heterogeneity of the mesocoxal muscles of Periplaneta americana.

Ultrastructural studies have been performed upon the posterior coxal depressor muscle (136) and a coxal branch of the main depressor group (135d') from the mesocoxa of the cockroach, Periplaneta americana. The quantitative stereometric analyses performed have shown the latter muscle to consist of a dorsal band of fibers having 25.% mitochondria and 13.6% sarcoplasmic reticulum (SR) and T-tubules (TTS), and a ventral group of fibers with only 4.4% mitochondria and 26.6% SR/TTS. The volume fractions characteristic of the ventral fibers of muscle 135d' are also typical of muscle 136.


Met enkephalin-like immunoreactivity in Merkel cells.

The Merkel cells from sinus hair follicles of rats were investigated by immunohistochemistry using different antisera against neuropeptides and gastroenteropancreatic (GEP)-hormones. For the first time it has been demonstrated that Merkel cells exhibit an immunoreactivity towards metenkephalin (methionine-enkephalin). The met-enkephalin immunoreactivity was restricted to Merkel cells and was not found in associated nerve axons or terminals. Denervation of Merkel cells did not affect the met-enkephalin immunoreactivity. Antisera leu-enkephalin (leucine-enkephalin) and other polypeptides did not produce an immunoreaction. The demonstration of met-enkephalin-like immunoreactivity supports the concept that the Merkel cell is a member of the paraneuronal system and a potential neuroreceptor cell.


Immunocytochemical localization of somatostatin-containing neurons in the rat hypothalamus.

The rat hypothalamus was studied at the light microscopic level with the use of single and double immunocytochemical staining methods. It was shown that the rat supraoptic and paraventricular hypothalamic nuclei, and their accessory neurosecretory nuclei, do not contain magnocellular somatostatin neurons. The distribution of the hypothalamic parvocellular somatostatin cells is described. The parvocellular component of the rat hypothalamic paraventricular nucleus is, at least partly, composed of somatostatin cells: they form a fairly well circumscribed periventricular cell mass. The rat suprachiasmatic nuclei contain separate somatostatin neurons and vasopressin neurons. Scattered somatostatin cells are present in the entire arcuate nucleus. In addition to the periventricular somatostatin cells located in the preopticanterior hypothalamic area in the arcuate nucleus, the rat hypothalamus also contains numerous scattered somatostatin cells located distant from the third ventricle.


A fine structural survey of the development of the adult fat body of Leptinotarsa decemlineata.

The fat body of a female Colorado potato beetle, Leptinotarsa decemlineata, at adult ecdysis, contains a large number of protein granules which are composed of light and dark zones. Part of the light zone in some of these granules is believed to be urate. During the first two days after adult ecdysis, fat body development is not essentially different in females reared either under long- or short-day conditions. Protein granules and large  vacuoles disappear and the first cell organelles are regenerated. The effect of the photoperiod on the histological structure of the fat is expressed after these events. In females reared under long-day conditions, the fat body becomes specialized for vitellogenin synthesis. Under short-day conditions, the fat body stores massive amounts of lipid until day 6 after adult ecdysis. Then the first electron-dense protein granules develop near the nucleus, and on day 10 the first autophagic vacuoles are seen. These structure changes are discussed in connection with the known biochemical properties of the adult faty body of Leptinotarsa.


Ultrastructural changes in the glial cells at neuromuscular synapses of Locusta migratoria occurring after nerve stimulation and subsequent rest: a morphometric analysis.

The glial processes ensheathing the motor nerve terminals on the retractor unguis muscle of Locusta migratoria are described. Ultrastructural changes observed after electrical nerve stimulation (20 Hz, 7 min) without or with subsequent rest (2 min, 1 h) are analysed morphometrically. Immediately after stimulation both the average terminal circumference (+ 23%) and its proportion covered by glial processes (+ 16%) are significantly increased. The mean number of Schwann cell processes per micron of terminal circumference (without stimulation: 0.86 +/- 0.04) is also affected: Immediately after stimulation it is increased by about 15% and after 2 min of rest even by 36%. The periaxonal cleft (without stimulation: 16.5 nm +/- 0.36) becomes wider immediately after stimulation by about 19%, an effect which is almost reversed after 1 h of rest. It is suggested that these changes are a consequence of the enlargement of the nerve terminal's surface upon massive exocytotic activity and that they are possibly mediated by mechanical attachment between glial and terminal plasma membranes.


A study of TSH-synthesis of spontaneous hypertensive rats by electron microscopic morphometry and autoradiography.

In order to compare the functional state of the anterior pituitary of spontaneously hypertensive rats (SHR) with that of nonmotensive Wistar Kyoto rats (WKR), the anterior pituitary was examined by morphometry and autoradiography at the level of electron microscopy. The relative number and the relative volume of thyrotrophs in the anterior pituitary were significantly greater in SHR compared with age-matched WKR at 0, 7, 30-33 days and 10 months of age, while the relative number of somatotrophs in SHR was significantly smaller at 1 and 10 months of age. Electron microscope autoradiographic analysis of uptake of 3H-lysine by thyrotrophs of both strains at the age of approximately one month showed that 3H-lysine was incorporated into protein and transported finally to secretory granules which migrated to near the cell membrane to be discharged. Silver grains were significantly more numerous over the thyrotrophs of SHR than over those of WKR at 30 min, 1 h, and 4 h after the injection of 3H-lysine. The present study has ascertained morphologically than a congenital hypersynthesis of TSH by the anterior pituitary occurs in SHR.


Investigations on the lectin-producing cells in the sponge Axinella polypoides (Schmidt).

The localization of two carohydrate binding proteins, so-called lectins, was studied in the sponge tissue of Axinella polypoides by light and immunofluorescence microscopy. They do not occur at the cellular surface of any cell type, but they are stored in vesicles of the "spherulous cells". After short formaldehyde fixation spherulous cells can be isolated and they release the active lectins upon lysis in distilled water. Electron microscopical studies of spherulous cells show that they contain almost nothing else but a small nucleus and vesicles of different size and number. Small vesicles are full of an electron dense material, whereas the content of large vesicles has a fluffy and fibrillar structure. Spherulous cells are large and tightly packed in the outer layer of the ectosome and in the meshwork of the spongin fibres of the central axis. The are small and scattered in the inner layer of the ectosome, and they are found throughout the choanosome. The function of the lectins is not clearly defined, and different alternatives such as participation in glycoprotein synthesis, immunological defense, or carbohydrate transport are possible.


"Intracisternal microtubules" in neurons of dog ganglia.

Ultrastructure, age-incidence, and topographic distribution of so-called intracisternal microtubules (ICMT) were studied in neurons of autonomic and ganglia of dogs. Microtubules packed within dilated cisterns of rough ER are about 24 nm in diameter; the thickness of the walls is about 10 nm. These facts suggest that intracisternal microtubules are identical with cytoplasmic microtubules. The ICMT show 12, 13, and 14 protofilaments. ICMT wer more frequently observed in autonomic than in somatic ganglia. In 23 dogs over 7 years old, ICMT was positive in 20 dogs. These structures therefore, seem related to ageing. The mechanism of production has not yet been established, but the cause may be a disturbance of protein synthesis and/or of the protein tubulin transport system.


On the presence of conspicuous electron dense bodies in the pinealocytes of the pig.

The pinealocytes of the pig contain a large number of aldehydefuchsin positive granules. In order to determine their nature an ultrastructural study was carried out. Numerous bodies having a maximal diameter of about 1,600 nm were found. These elements showed a great variety of internal structure, ranging from a "homogeneous" content and amorphous dense aggregates to lamellate bodies. Although only a few of them displayed a positive reaction for acid phosphatase, their morphological appearance strongly suggests that they belong to the lysosomal system.


Intercellular junctions in the hypodermis, salivary gland and Gené's organ of the cattle tick, Boophilus microplus.

The intercellular junctions that occur in the hypodermis, Gené's organ, and the salivary glands of the tick, B. microplus, are described. The epithelial cells of the hypodermis are connected by spot desmosomes and septate junctions and the secretory cells of Gené's organ by septate and gap junctions. The cap cells in the alveoli of the salivary gland connect to adjacent cells by gap junctions, hemidesmosomes and septate junctions into which microtubules are inserted.


An ultrastructural study of macrophage-mediated resorption of calcified tissue.

Ultrastructural observations on macrophage-mediated resorption of calcified tissue of killed fetal long bones are described and correlated with increased 45Ca release into the medium. Macrophages disrupt calcified tissue extracellularly and appear to engulf large fragments of mineralized matrix. Ruffled borders, which are common features of osteoclasts at sites of resorption of bone, do not develop in macrophages. However, clear zones are seen in macrophages as well as osteoclasts. These findings provide additional evidence for non-osteoclast-mediated resorption of calcified tissue.


Oogenesis in Campodea sp. (Diplura): the ultrastructure of the egg chamber during vitellogenesis.

The egg chamber of Campodea consists of a group of nurse cells and an oocyte, and is surrounded by a simple, markedly flattened follicular epithelium. Three types of yolk occur in the oocytes: type I appears within elements of the rough endoplasmic reticulum; type II is produced by specific complexes of endoplasmic reticulum and dictyosomes; type III is incorporated by micropinocytosis. Histochemical tests show that mature yolk spheres contain proteins and polysaccharides. The main function of the nurse cells is to synthesize RNA, but they also produce small amounts of type I yolk. Phylogenetic conclusions are drawn from this and other studies of oogenesis in apterygote insects.


Ultrastructural studies on oogenesis in Symphyla: previtellogenic and vitellogenic stages.

The paired ovaries of Symphyla are sac-shaped. Their interior is filled with synchronously developing oocytes surrounded by a simple follicular epithelium. Previtellogenic oocytes have spherical or oval nuclei, the latter containing a large number of small nucleoli. Part of the nuclear surface is covered by a thick "coat" made up of a granular substance of medium electron density. The "coat" is penetrated by cisternae of the smooth endoplasmic reticulum. Micropinocytosis is the principal source of yolk in Symphyla;a small amount of yolk material arises within the mitochondrial cristae. Vitellogenic oocytes are characterized by a highly active rough endoplasmic reticulum synthesizing a material, probably lipoprotein in nature, which is incorporated into lipid droplets.


The basiepithelial nerve plexus of the viscera and coelom of eleutherozoan Echinodermata.

The organisation of the basiepithelial nerve plexus in the alimentary canal of a starfish and the water vascular system of a sea-urchin is described. The plexus contains varicose aminergic neurones which terminate adjacent to the ciliated epithelial cells. It is proposed that the basiepithelial plexus innervates these cells and controls ciliary beating. The distribution of the basiepithelial plexus in various tissues described by other workers is discussed particularly in relation to whether it is the coelomic epithelium or the luminal epithelium which is innervated. It is concluded that where there is both an endothelium and a coelomic epithelium only one is innervated. The muscles, where present, of the viscera are innervated by a separate nervous system. The muscles are always on the opposite side of the non-cellular connective tissue sheath to the basiepithelial plexus.


Effects of fibroblast growth factor and bromocriptine on the mitotic activity of the anterior pituitary gland in organ culture.

Effects of fibroblast growth factor (FGF) and of bromocriptine, a dopaminergic receptor agonist, on the mitotic index in the organ-cultured anterior pituitary gland of the rat were investigated, using the colchicine metaphase-arrest technique. It has been found that FGF increases the mitotic index in the anterior pituitary explants. By contrast bromocriptine inhibits the mitogenic effect of FGF.


Isotocinergic neurons in the goldfish hypothalamus: physiological and morphological studies on chemically identified cells.

Isotocinergic (IT) neurons show physiological and morphological characteristcs that are similar to those of the other preoptic neuroendocrine cells in the goldfish. Preoptic IT cells show resting membrane potentials of 20-55 mV, action potentials of up to 100 mV, and physiological evidence of axonal branching. Dye-marked IT cells measure 14-56 micrometers, their dendrites projecting to the ependyma and into the hypothalamic neuropil, their multiple beaded axons projecting to the pituitary. Indirect immunofluorescence identifies these dye-marked cells as IT. By combining electrophysiological, dye-marking and immunocytochemical techniques we can now, for the first time, study single, antidromically-identified peptidergic neurons of a specific type in vertebrate and invertebrate species.


Effect of progesterone pretreatment on the uptake of estradiol-17 beta by the uterine epithelium of the rat.

Progesterone pretreatment of ovariectomised rats resulted in a moderate (44%) lowering of the level of nuclear estradiol receptors found in the uterine epithelium 2 h after a single injection of this estrogen.


Ultrastructural study of the follicle cells in the freshwater gastropod Viviparus viviparus L.

The morphology and the role of the follicle cells of Viviparus viviparus were examined by means of light and electron microscopy. The follicle cells appear to contain glycogen and fat, and often lysosomes or heterogeneous inclusions. Therefore, they seem to be active in phagocytosis and storage. They are probably involved in the nutrition of the oocyte. Their role in the formation of a selectively permeable barrier is discussed.


Bacteria-like structures in the endoplasm of Gregarina garnhami (Eugregarinida, Protozoa).

An investigation of bacteria-like structures, which are found in large numbers in the endoplasm of the eurgregarine, Gregarina garnhami, was carried out using light and electron microscopy. Gram staining indicates that these structures are gram negative; they show orange fluorescence when stained with acridine orange. Ultrastructural observations show that they resemble the endosymbiotic bacteria found in other protozoan species. Some of these structures appear to be dividing, and the possibility that these structures are in fact bacteria is discussed.


Cell junctions in the cyst envelope in the silkworm testis, Bombyx mori Linné.

The cell junctions of the cyst envelope in the testes of Bombyx mori were examined by electron miscroscopy utilizing a thin-sectioning technique following conventional fixation, tannic acid fixation and lanthanum tracer study, and also using a freeze-fracture technique. There are three kinds of junctions; septate junctions, gap junctions and tight junctions. Septate junctions are of the pleated type. Gap junctions are characterized by four electron-dense lines and three electron-lucent lines in the reduced intercellular spaces seen by thin-sectioning. They are of the "E" type, having clusters of intramembraneous particles on the E-fracture face. The most striking finding is the frequent presence of tight junctions on the fracture planes, while focally fused outer leaflets of the junctional unit membranes are rarely detected on thin-sectioned preparations. Tight junctions are characterized by branching zigzag ridges on the P-fracture face and complementary grooves on the E-fracture face. It is proposed that tight junctions are new morphological evidence of blood-germ cell barrier in an insect.


Epithelial differentiation at the mucogingival junction: a stereological comparison of the epithelia of the vestibular gingiva and alveolar mucosa.

The epithelial lining of normal human vestibular gingiva and the adjoining alveolar mucosa was subjected to a comparative stereological analysis. Five biopsies collected from 11 to 12 year-old males and females were selected from a total of 14 specimens and, under standardized conditions, processed for light- and electron microscopy. At two levels of magnification, electron micrographs were sampled from five strata in the oral-gingival, and from four strata in the alveolar-mucosal epithelium, mostly in regions of epithelial ridges. Standardized sterological point counting techniques were employed to analyze a total of 710 and 540 electron micrographs from the oral-gingival and the alveolar-mucosal epithelium, respectively. The two epithelia, although of similar thickness, show different differentiation patterns. The oral-gingival epithelium consists of four cytologically different strata, the major differentiation step occurring between the lower and upper stratum spinosum of epithelial ridges. Standardized stereological point counting techniques were alveolar-mucosal epithelium, consisting of two cytologically different cell compartments, displays a broad, superficial zone of differentiated flat cells, with 60% of the cytoplasm filled with a dense network of cytoplamic filaments. The major differentiation step occurs between basal and lower spinous layers. Differentiation phenomena in both epithelia are discussed and individual variations are interpreted in view of genetically determined factors.


Regeneration of the goldfish retina after exposure to different doses of ouabain.

After ouabain-induced degeneration, the retina of the goldfish shows a remarkable regeneration capacity. The extent of the damage depends on the dose of ouabain used in the experiment. After intraocular injection of 7 microliter 10(-5) M ouabain, the ganglion cells and the cells of the inner nuclear layer (INL) become necrotic except for most of the outer horizontal cells, some bipolar cells, and Müller cells. The outer nuclear layer (ONL) and the marginal growth zone at the ora serrata remain intact; the plexiform layers become spongy. The degenerated material is removed by the proliferated reactive macroglial cells and invading macrophages. The degenerated cellular elements of the retina are replaced by mitosis of neuroblasts in the marginal growth zone and of cells in the ONL. After intraocular injection of a 5-fold higher dose of ouabain (7 microliter 5 . 10(-5) M), the degeneration of the retina proceeds more rapidly and completely. In this experiment, the ONL is destroyed and the receptor outer segments are phagocytosed by cells of the pigment epithelium. In contrast to the regeneration of the amphibian retina, in the goldfish cells of the pigment epithelium do not participate by metaplastic transformation in the regeneration of the retina. The only source of cellular regeneration of the retina after complete destruction of its differentiated neural elements is the marginal growth zone, which is highly resistant to ouabain. The rate of mitoses in this region is strongly increased. The derivatives of these cells spread out tangentially over the entire fundus of the eye in a concentric manner. In this regenerate, mitotic processes continue in a radial direction, resulting in thickening and layering of the new retinal formation.


Morphology and behaviour of neural crest cells of chick embryo in vitro.

Neural primordia of chick embryos were cultured for three days and the behaviour of migrating neural crest cells studied. Somite cells were used as a comparison. Crest cells were actively multipolar with narrow projections which extended and retracted rapidly, contrasting to the gradual extension of somite-cell lamelleae. On losing cell contact, somite cells were also more directionally persistent. The rate of displacement of isolated crest cells was particularly low when calculated over a long time base. Both crest and somite cells were monolayered; contact paralysis occurred in somite cell collisions but was not ascertained for crest cells. However, crest cells in a population were far more directionally persistent than isolated cells. Contact duration between crest cells increased with time and they formed an open network. Eventually, retraction clumping occurred, initially and chiefly at the periphery of the crest outgrowth. Crest cells did not invade cultured embryonic mesenchymal or epithelial populations but endoderm underlapped them. No effects were observed on crest cells prior to direct contact. Substrate previously occupied by endoderm of ectoderm caused crest cells to flatten while substrate previously occupied by the neural tube caused them to round up and clump prematurely.


Neuronal connections in the ocellus of the wasp (Paravespula vulgaris L.).

Studies of the dorsal ocelli of the wasp Paravespula vulgaris (L.) led to the following results: Under a biconvex corneal lens, 150 microns in thickness, about 600 receptor cells are located. The rhabdomeres of two adjacent cells form a closed plate-like rhabdom (0.5--1.0 micron in thickness, 6 microns in width and 10--25 microns in depth or length). In the lateral ocellus the receptor cells synapse up to 8 ocellar nerve fibers, and in the median ocellus they synapse up to 16 (20--30 microns thick) ocellar nerve fibers. The ocellar synaptic plexus may display three types of synapses between the two types of neurons: (i) Receptor-cell axons are presynaptic to dendrites of the first-order interneurons. (ii) Dendrites of the first-order interneurons are presynaptic to receptor-cell axons. (iii) The subunits of a dendrite of first-order interneurons form synapses with each other.


Precocene-induced necrosis and haemocyte-mediated breakdown of corpora allata in nymps of the locust Locusta migratoria.

Light and electron microscopic studies, including the use of the vital dyes trypan blue and acridine orange, indicate that the topical application of precocene II rapidly triggers degenerative processes in the corpora allata (CA) leading within a few days to the virtual disappearance of the parenchymal cells of these glands. The following sequence of events was observed: 1) Within 2 h, many of the cells in fixed nymphal specimens showed increased electron density. During the next few hours, they decreased considerably in volume (shrinkage necrosis). Intercellular spaces increased simultaneously. Although a variable number of cells remained electron lucent, they showed nuclear and cytoplasmic degeneration later on (coagulative necrosis). 2) Haemocytes arrived at the CA sheath and invaded the CA in large numbers after 12 h. 3) CA cells became increasingly necrotic, and cell fragments were budded off and were phagocytosed most noticeably after 1 to 3 days. Thereafter no CA parenchyma remained. 4) Haemocytes dispersed.


Central projections of fibers in the auditory and tensor nerves of cicadas (Homoptera: Cicadidae).

The auditory and tensor nerves of cicadas are mixed nerves containing both afferent and efferent elements. In 17-year cicadas, and in Okanagana rimosa, the auditory nerve contains afferents from body hairs, from the detensor tympani-chordotonal organ, and some 1300--1500 afferents from the hearing organ. Within the fused metathoracic-abdominal ganglionic complex the receptors from both the auditory and tensor nerves form a neuropilar structure that reveals the metameric organization of this complex. A few fibers run anteriorly, projecting into the meso- and prothoracic ganglia. Within the ganglionic complex a division of auditory nerve afferents into a dense intermediate and a more diffuse ventral neuropile is observed. In addition, a dorsal motor neuropile is outlined by arborizations of the timbal motor neuron. This neuron is one of several efferent cell types associated with the auditory nerve, and there is an indication that several efferent fibers innervate the timbal muscle. There is anatomical evidence for a possible neuronal coupling between the bilaterally symmetrical large timbal motor neurons. In general, central projections from the auditory and tensor nerves support evidence of a structural "layering" within the CNS of insects.


Sensory projections from the wind-sensitive head hairs of the locust Schistocerca gregaria. Distribution in the central nervous system.

The neurones from the wind-sensitive hairs on the locust head have been filled with cobalt chloride and intensified with silver. All the neurones project through the brain to the suboesophageal ganglion, some continue to the prothoracic ganglion and a few as far as the mesothoracic ganglion. Three different types of projection are described and a regrouping is proposed of Weis-Fogh's five hair fields into three areas. The distribution of the neurones from these areas is described in relation to other structures in the ganglion and is discussed in relation to the function of the hair fields in stability control and grooming.


Ultrastructure of the muscles of the dorsal diaphragm in Locusta migratoria.

The alary muscles of Locusta migratoria adults make up the major tissue of the dorsal diaphragm which separates pericardial and perivisceral sinuses in the abdomen. The alary muscles are striated with a sarcomere at rest measuring about 9 microns. The Z-line has a staggered-beaded arrangement with A-bands and I-bands readily discernable. Thick myofilaments are surrounded by 10 or more thin filaments. The sarcoplasm has few mitochondria near the area of the Z-line, dyads are present and sarcoplasmic reticulum is poorly developed. Axons which innervate the alary muscle are either contained within invaginated folds of the sarcolemma of the muscle cells or the muscle cells send finger-like projections to envelop the axons. The synaptic terminals contain synaptic vesicles between 40 and 45 nm in diameter and a few electron-dense granules near or less than 170 nm in diameter. Away from synaptic terminals the axon profiles show few or no granules. The axons are accompanied everywhere by well-developed glial cells. This then is not typical neurosecretomotor innervation, however, the presence of electron-dense granules suggests the possibility of peptidergic neurotransmission.


DNA sequence variants in the G gamma-, A gamma-, delta- and beta-globin genes of man.

DNA prepared from 60 unrelated individuals was cleaved with one of eight different restriction endonucleases and the resulting DNA fragments were separated by agarose gel electrophoresis. DNA fragments containing G gamma-, A gamma-, delta- or beta-globin genes were detected by Southern blot hybridization, using as probe either a 32P-labeled cloned DNA copy of rabbit beta-globin messenger RNA or labeled human beta- and G gamma- globin cDNA plasmids. Three types of variant restriction enzyme patterns of globin DNA fragments were detected in otherwise normal individuals. One variant pattern, found in only one person, was caused by an additional restriction endonuclease Pst I cleavage site in the center of the delta- globin gene intervening sequence; the subject was heterozygous for the presence of this cleavage site and was shown to have inherited it from her mother. Another variant pattern resulted from the appearance of an endonuclease Hind III cleavage site in the intervening sequence of the A gamma-globin gene; this variant is polymorphic, with a gene frequency for the presence of the intragenic Hind III site of 0.23. This Hind III cleavage site polymorphism is also found in the G gamma-globin gene intervening sequence and thus the polymorphism itself appears to be duplicated over the pair of gamma-globin loci. These variants can be used to derive an approximate estimate of the total number of different DNA sequence variants in man.


Biogenesis of poxviruses: mirror-image deletions in vaccinia virus DNA.

Restriction endonuclease analysis of viral DNA extracted from wild-type and temperature-sensitive mutants of vaccinia IHD-W (Dales et al., 1978) revealed sequence alterations in approximately 20% of all ts clones examined. The rearrangements were due to deletions up to 250 nucleotide pairs long. Using Eco RI, Sal I, Bam I, Hpa I and Ava I, the deletions were always observed in the same fragments, while analysis with Hind III demonstrated deletions of identical size in the two terminal fragments. Since vaccinia virus contains inverted terminal repeats of more than 10 kb, these clones possess identical deletions of opposite orientation at both ends of the genome. Analysis of several revertants of the ts mutants demonstrated that the deletions probably arise as events independent from those producing ts lesions and are generated spontaneously at high frequency. This implies that a single event during replication caused the elimination of nonessential information, and suggests that circular intermediates must exist transiently during viral replication.


The integration sites of endogenous and exogenous Moloney murine leukemia virus.

Specific cDNA probes of Moloney and AKR murine leukemia viruses have been prepared to characterize the proviral integration sites of these viruses in the genomes of Balb/Mo and Balb/c mice. The genetically transmitted Moloney provirus of Balb/Mo mice was detected in a characteristic Eco RI DNA fragment of 16 x 10(6) daltons. No fragment of this size was detected in tissue DNAs from Balb/c mice infected as newborns with Moloney virus. We conclude that a viral integration site, occupied in preimplantation mouse embryos, is not necessarily occupied when virus infects cells in post-natal animals. Balb/Mo and Balb/c mice do carry the AkR structural gene in an Eco RI DNA fragment of 12 x 10(6) daltons. Further restriction analysis of this fragment indicated that both mouse lines carry one AKR-type provirus. Leukemogenesis in Balb/Mo and newborn infected Balb/c mice is accompanied by reintegration of Moloney viral sequences in new chromosomal sites of tumor tissues. Part of the reintegrated Moloney viral sequences are of subgenomic size. The AKR viral sequences, however, are not found in new sites. Further restriction analysis revealed that the development of Moloney virus-induced leukemia in Balb/Mo mice does not lead to detectable structural alteration of the genetically transmitted Moloney and AKR structural genes. Possible mechanisms of the reintegration process are also discussed.


Cytoplasmic regulation of two G1-specific temperature-sensitive functions.

tsAF8 and ts13 cells are temperature-sensitive (ts) mutants of BHK cells that specifically arrest, at nonpermissive temperature, in the G1 phase of the cell cycle. These two mutants can complement each other. Both cell lines can be made quiescent by serum deprivation (G0). When subsequently stimulated by serum, they can enter S phase at 34 degrees C but not at 39.5 degrees-40.6 degrees C. We have used these mutants to determine whether the nucleus is needed during the G0 leads to S transition for the expression of the G1 ts functions. For this purpose, we fused cytoplasts of G0-tsAF8 with whole ts13 cells in G0, and cytoplasts of G0-ts13 with whole tsAF8 cells in G0. Serum stimulation at the nonpermissive temperature induced DNA synthesis in both types of such fusion products. No DNA synthesis was induced by serum stimulation at the nonpermissive temperature in fusion products constructed between either G0-tsAF8 cytoplasts and whole G0-tsAF8 cells or G0-ts13 cytoplasts and whole G0-ts13 cells. These results demonstrate that the information for these two ts functions, which are required for entry of serum-stimulated cells into the S phase, are already present in the cytoplasm of G0 cells--that is, before serum stimulation commits them to the transition from the nonproliferating to the proliferating state.


Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation.

Serum-free media containing 10-50 ng insulin, glucagon and epidermal growth factor (EGF) ml-1 stimulate adult rat hepatocyte proliferation in 10-15 day old primary liver cell cultures. The kinetics of this response simulate hepatocellular transitions that accompnay liver regeneration after 67% hepatectomy. Amiloride, a Na+ influx inhibitor, reversibly blocks these transitions in vitro (ID50 approximately 0.02 mM) and in vivo (ID50 approximately 25 mg kg-1). Inhibition is observed with other cation flux modulators, including ouabain (ID50 approximately 0.2 mM), 0.2 microM monensin and 0.2 microM nigericin, but not with 0.3 mM furosemide or tetrodotoxin. The prereplicative interval in culture (0-12 hr) is characterized by preferential cellular responsiveness to EGF (0-3 hr) followed by insulin plus glucagon (3-12 hr). Parallel culture and animal studies show that the amiloride-sensitive and prereplicative intervals coincide. In culture, a "burst" of 22Na+ influx, stimulated by peptide-supplemented media within 1 min but decreased later at 12 hr, is retarded by amiloride. This drug also blocks delayed prereplicative events involving increased amino acid "A" transport system function at 4-8 hr, and 3H-uridine and 3H-leucine incorporation into RNA and protein, respectively, at 8-12 hr. These findings suggest that at least two time-ordered processes are necessary to initiate hepatic growth fully: first, activation of Na+ flux systems by peptides similar or identical to EGF; and second, potentiation of these and subsequent cellular events by the combined action of insulin plus glucagon. [Amiloride: N-amidino-3,5-diamino-6-chloropyrazinecarboxamide; furosemide: 4-chloro-N-furfuryl-5-sulfamoylanthranilic acid; AIB: alpha-aminoisobutyric acid; ID50: administered dose giving 50% inhibition of a maximal response; dFBS: dialyzed fetal bovine serum; L.I.: 3H-dT nuclear labeling index.]


Utilization of stored mRNA in Xenopus embryos and its replacement by newly synthesized transcripts: histone H1 synthesis using interspecies hybrids.

We studied H1 gene expression in hybrids of Xenopus laevis (female) x Xenopus borealis (male) and saw paternal H1 synthesis in mid-blastulae, which indicates that the H1 genes are active by this stage. The behavior of the maternal store of H1 histone mRNA was studied in androgenetic haploids. In which all stored mRNA is of the laevis type and all new transcripts are borealis. This showed that the initial activation of H1 synthesis occurs entirely by mobilizing maternal transcripts and that these are then unstable, disappearing in a few hours, by the early gastrula stage.


After fertilization, sperm surface components remain as a patch in sea urchin and mouse embryos.

Sea urchin and mouse sperm that are labeled on their surfaces with fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TMRTC) or 125I-diiodofluorescein isothiocyanate (125IFC) remain viable and can fertilize eggs. When sea urchin eggs were fertilized with 125IFC-labeled sperm, the radioactivity from the sperm was quantitatively transferred to the egg (at a ratio of one sperm equivalent per egg) and persisted in the embryo as it developed to the pluteus larval state (5 days at 12 degrees C). The radioactivity was acid-precipitable and was associated with the particulate fraction of embryo homogenates. In addition, FITC-labeled sea urchin sperm were used to fertilize eggs, and the labeled components were followed by fluorescence microscopy. In the embryo, labeled sperm components were present as a discrete patch that was partitioned unequally during early cleavages. In experiments using mouse sperm labeled with TMRTC, the labeled sperm components were also transferred to the embryo as a discrete patch that was again distributed unequally after cleavage. This physiological cell fusion system therefore has distinctive characteristics: there is limited lateral mobility of surface components, which have a low turnover rate unlike that see in other systems. In this paper, we discussed the possible morphogenetic role of this unusual behavior.


Glycoprotein synthesis and inhibition of glycosylation by tunicamycin in preimplantation mouse embryos: compaction and trophoblast adhesion.

The synthesis of glycoproteins and inhibition of protein glycosylation by tunicamycin were examined during development of preimplantation mouse embryos and trophoblast adhesion. Tunicamycin specifically inhibits glycosylation of asparaginyl residues of glycoproteins. Tunicamycin, 0.25-5.0 microgram/ml, had no effect on early cleavage or aggregation between embryos, but the embryos remained irreversibly uncompacted when control embryos developed to the blastocyst stage. Trophoblast adhesion and giant cell outgrowth were reversibly inhibited and the binding of Con A was also reduced. Incorporation of 3H-mannose into blastocysts was inhibited by 80%, but that of 3H-glucosamine and 3H-leucine by only 28 and 18%, respectively, in the presence of 1.0 microgram/ml tunicamycin. Qualitative analysis showed that the incorporation of the sugars was markedly reduced in the majority of the fractions, but the synthesis of these carbohydrate-deficient glycopeptides was essentially normal. However, protein-polysaccharide fractions with nearly 40% of the incorporated glucosamine and only 5% mannose and 1% leucine were insensitive to inhibition by tunicamycin. Membrane-bound N-glycosidically linked glycoproteins therefore evidently play an important role during compaction and in trophoblast adhesion of mouse embryos.


Structure analysis at the ends of the intervening DNA sequences in the chloroplast 23S ribosomal genes of C. reinhardii.

All of the chloroplast 23S ribosomal genes of C. reinhardii are interrupted by a 0.87 kb sequence (Rochaix and Malnoë, 1978). We have sequenced the DNA across the two ends of this intervening element. In parallel, we have examined the nucleotide sequences in the corresponding part of the 23S ribosomal RNA. This allowed us to locate precisely the boundaries between the coding (that is, transcribed into mature 23S rRNA) and the noncoding DNA. The results show that the intervening sequence is flanked by two identical sets of 3 bp (5'-CGT) oriented as direct repeats. In addition, a sequence of 5 bp (5'-CGTGA) lies exactly next to one end and is found very close (16 bp) to the other end, in the coding part of the gene. These two sets are also oriented as direct repeats. Finally, sequences near one end of the intervening element are found with a few alterations near the other end, but in an inverted orientation. Possible interpretations of these results are discussed.


Evolution of human influenza A viruses in nature: sequential mutations in the genomes of new H1N1.

The genetic variation of the new pandemic H1N1 influenza A viruses isolated in 1977 was analyzed by two-dimensional oligonucleotide fingerprinting and RNA sequencing. Differences were observed in the fingerprints of the RNAs of these viruses, and analysis of the changes suggested that sequential mutations occurred in their genomes. Based on these data, a scheme is presented which proposes divergent evolution of strains from a common ancestry. Furthermore, it was found that mutations were not restricted to the genes coding for the hemagglutinin and the neuraminidase, but were scattered throughout the genome, suggesting that selective antibody pressure is not solely responsible for the emergence of genetic variants. Our data also strengthen the hypothesis that the new H1N1 influenza virus strains are derived from strains circulating in 1950.


A detailed model of reverse transcription and tests of crucial aspects.

A model of reverse transcription has been devised by which the detailed architecture of ten molecular structures is predicted. The model includes a number of novel features for which experimental evidence is presented. First, growing minus DNA strand is copied from the viral RNA only up to a position about 150 nucleotides from the 5' end of the RNA. Second, plus-strand DNA, after being copied from approximately 600 nucleotides at the 5' end of the minus-strand DNA, then transcribes the first approximately 20 nucleotides of the tRNApro primer (which is covalently attaced to the 5' end of the minus DNA strand). The 3' ends of the minus and plus DNA probably form a hybrid through the homology conferred by the primer binding site sequences. Third, the minus and plus DNA strands are elongated in a continuous fashion resulting in a linear double-stranded DNA molecule containing a 600 nucleotide direct repeat at both ends. The most of the features of the model have experimental support, and it appears to provide a credible description of reverse transcription.


Transition probability and the division pattern of WI-38 Cells.

Analysis of the pedigrees of WI-38 clones reveals a division pattern in accord with the transition probability model of the cell cycle. This is in distinct contrast to a recent report by METS and VERDONK (1978).


The relationship between RNA content, cell volume and growth potential in ageing human embryonic mesenchymal cells.

Modal cell volume and cellular RNA content were meaume was found to correlate with both culture replication rate and cellular RNA content. This observation suggests that cell volume changes may be related to the rate of cell division during ageing in vitro, and that both cell volume and RNA content may be closely controlled. There was no change in the relative proportions of the major rRNA species during the cultures' lifespan.


Microflowfluorometric evaluation of homeostasis in cultured epidermal cells.

In isolating and culturing in vitro populations of basal cells from adult guinea pig skin, it has been possible to show that these cells are sensitive to both G1 and G2 inhibitions. Only a small fraction (10 percent or less) of the G1 blocked cell population would be governed by G1 inhibitory messages released by suprabasal, maturing keratinocytes. As regards the G2 block in vitro experiments confirm that basal cells produce a G2 blocker to which about 9 per cent or less are susceptible. In conclusion basal cells in culture are sensitive to homeostatic regulation as in vivo.


Increased prostaglandin F2 alpha and E2 production in late passage WI38 diploid fibroblasts.

The prostaglandin (PG) F2alpha and E2 concentrations in the culture medium of WI38 cells were determined at various passage levels. It was found that late passage cells produce significantly higher amounts of both PG F2alpha and PG E2.


Archenteron cells are responsible for the increase in ribosomal RNA synthesis in sea urchin gastrulae.

Paracentrotus lividus embryos were continuously labeled with P32 from hatching blastula to pluteus. The archenteron cells were then separated from the rest of the embryo and the radioactivity accumulated into the ribosomal RNA of the two cell groups measured. The results clearly indicate that the bulk of ribosomal RNA is mainly if not entirely, synthesized in this time interval by the archenteron cells.


alpha-Amanitin resistant RNA polymerase II from Aedes albopictus cell mutants resistant to alpha-amanitin.

Spontaneous and EMS-induced alpha-amanitin-resistant Aedes albopictus cells have been isolated and characterized. Two mutant sublines, one of intermediate resistance (alpha A2) and the other highly resistant (Ama18) contained RNA polymerase II activity, the resistance of which in vitro to alpha-amanitin correlated well with the resistance of these cells in vivo. The resistance of these cells to alpha-amanitin can likely be attributed to the presence of an altered RNA polymerase II.


Reversible retention of synchronized HeLa cells in G2 of the mammalian cell cycle.

The rate of progression of cycling HeLa cells from G2 into mitosis is greatly reduced by the amino acid analogue, p-fluorophenylalanine, but a small percentage of cells continually escapes into mitosis. Most of a presynchronized population can be held for several h in G2, thereby facilitating analyses of cells at this late stage of the cell cycle. They are reversed from the block by the addition of phenylalanine and resume progression within about 1 h with predictable kinetics but without a significantly enhanced synchrony. The mechanism of action of the analogue is consistent with the hypothesis that synthesis of false proteins interferes with the correct assembly of division-relevant structures.


A comparison of nuclear and nucleolar matrix proteins from rat liver.

The comparison of the gel electrophoresis patterns of nuclear and nucleolar matrix proteins reveals marked differences between these structures. The nucleolar matrix contains 5 prominent protein bands ( mol.wt.: 8.2; 7.0; 5.6; 4.0 and 3.0 x 10 4 ) which are not found in nuclei. It is suggested that the nucleolar matrix has a distinct structure participating in selective interactions.


Relationship between DNA alkylation and specific-locus mutation induction by N-methyl- and N-ethyl-N-nitrosourea in cultured Chinese hamster ovary cells (CHO/HGPRT system).

Chinese hamster ovary (CHO) cells in culture were utilized to determine the cytotoxicity, specific-locus mutation induction, and DNA alkylation which result from treatment of the cells with a range of concentrations of N-methyl-N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). With [3H]MNU over the concentration range 0.43--13.7 mM, methylation of DNA was found to increase linearly, with a mean value of 56.7 pmol residue per mumol nucleoside per mM. With [1-3H]ENU over the concentration range 1.7--26.8 mM, ethylation was linear, with a mean value of 3.8 pmol residue per mumol nucleotide per mM. Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus was quantified by determination of the frequency of resistance to 6-thioguanine under stringently-defined selection conditions. The mutation frequency increased linearly with MNU or ENU concentration (0.01--2.0 mM); mean values were 2800 and 840 mutants per 10(6) clonable cells per mM, respectively. At equal levels of DNA alkylation, ENU was found to be approx. 4.5 times as mutagenic as MNU.


Senescent changes in rodent hepatic epoxide metabolism.

Age-related alterations in epoxide metabolism were examined in subcellular fractions of liver from 3-, 12- and over 24-month-old male rats and mice. Using styrene oxide as the substrate, glutathione-S-transferase activity remained unchanged while the activity of epoxide hydrase increased with age in both species. The microsomally-mediated binding of benzo[a]pyrene to DNA was also increased in the old animals. Thus, senescent rodents retain or increase their ability to metabolize epoxides. The effect on epoxide metabolism of pretreatment of the senescent rodents with polychlorinated biphenyls was also examined. Glutathione-S-transferase activity was induced only in old animals. However, epoxide hydrase activity, while inducible in all age groups of rats, increased only in young mice. Therefore, there is an age-related difference in response of epoxide metabolizing enzymes to polychlorinated biphenyl treatments between rats and mice.


Aryl hydrocarbon hydroxylase induction in mouse liver cells--relationship to position in the cell cycle.

The inducibility of aryl hydrocarbon hydroxylase (AHH) by benzo[a]-pyrene (BaP) has been studied in synchronously grown cultures of mouse liver cells. These cells (NMuLi cl 8) have low basal levels of AHH which can be induced greater than 100-fold by BaP. Cells were synchronized in G1(G0) by serum starvation and in S by release from serum starvation in combination with excess thymidine. When released from G1(G0) by replating at lower cell density in fresh medium with 20% serum, cells began entering S with a lag of 12 h. Addition of BaP (1 microgram/ml) 8 h before serum stimulation, at the time of stimulation or 7.5 h after stimulation all gave similar induction kinetics: the AHH activity peaked as the cells began entering S regardless of when the BaP was added. Cells blocked in various parts of S by excess thymidine were inducible for AHH activity as efficiently as cells moving through S and into G2. These results indicate that the inducibility of AHH is greater when cells are actively proliferating and may be a contributing factor to why growing cells are more sensitive to mutagenesis and transformation than quiescent cells.


DNA damage and DNA repair in cultured human cells exposed to chromate.

DNA damage and DNA repair have been observed in cultured human skin fibroblasts exposed to potassium chromate but not to a chromic glycine complex. DNA repair synthesis (unscheduled incorporation of [3H]thymidine (TdR)) was measured in cells during or following exposure to chromate and was significant for chromate concentrations above 10(-6) M. Maximal DNA repair was observed at about 10(-4) M chromate. DNA repair capacity was found to be saturated at this concentration. Chromate was stable for at least 8 h in culture medium and produced approximately a linear increase in repair with duration of exposure. DNA damage as determined by alkaline sucrose gradient sedimentation was detected after treatment for 1.5 h with 5 . 10(-4) M chromate. Exposure to 10(-7) M chromate solution for 7 days inhibited colony formation while acute (1 h) treatment was toxic at 5 . 10(-6) M. The chromic glycine complex was toxic above 10(-3) M for a 1-week exposure but was not observably toxic after a 1-h treatment. These results indicate that chromate and not chromic compounds may be the carcinogenic form for man. The nature of the ultimate carcinogen is discussed. These findings illustrate the utility of the DNA repair technique to study the effects on human cells of inorganic carcinogens and mutagens.


Sister chromatid exchanges induced in cultured mammalian cells by chromate.

Chromate compounds induced sister chromatoid exchanges (SCEs) and chromosome aberrations in cultured mammalian cells. Similar increases in SCE frequency were observed in human fibroblasts exposed to the compounds K2Cr2O7 and K2CrO4. Marked increases in SCE frequency in cells exposed to chromate for a 48-h period were detected at concentrations between 10(-7) and 10(-6) M. Chromosome aberrations (primarily chromatid breaks) were also produced in human cells exposed to K2CrO4 at concentrations between 8 . 10(-7) and 3 . 10(-6) M. K2CrO4, but not the trivalent compound CrCl3, induced SCEs in Chinese hamster ovary (CHO) cells at low concentrations.


Quantitative and qualitative aspects of the binding of N-hydroxy-2-acetylaminofluorene to hepatic chromatin fractions.

The binding of a chemical carcinogen to components of hepatic chromatin in male rats was examined. After a single injection of N-[3H]hydroxy-2-acetylaminofluorene ([3H]OH-AAF) covalent binding to chromatin RNA, protein, and DNA occurs. The amount of carcinogen bound to RNA was approximately 5 times greater than to DNA, and 10 times that of the protein. However, loss of carcinogen from RNA with time was rapid, whereas a persistent binding to DNA equal to 15% of the initial values was observed. To localize the initial and persistent DNA-bound carcinogen, the genome was fractionated using two different chromatin fractionation procedures. The procedures used yielded 3 chromatin fractions based on physical characteristics, degree of association with nascent RNA and in vitro template capacity. Based on those parameters, these chromatin fractions have been tentatively classified as template expressed euchromatin, a repressed heterochromatin, and a highly condensed pelleted heterochromatin. With both the glycerol gradient chromatin fractionation procedure and the selective MgCl2 chromatin precipitation procedure, the initial (2 h) binding of carcinogen was greatest on the euchromatin DNA. Loss of carcinogen from the DNA, however, was also significantly faster from the euchromatin when compared to the heterochromatin and the pelleted heterochromatin. By 10 days after a single injection of the carcinogen, the largest amount of bound fluorene residues was located on the pelleted heterochromatin DNA, an apparently repressed portion of the genome, while less than 5% of the initial values were found on either the eu- or heterochromatin. When the rats were fed a 2-acetylaminofluorene-containing diet, loss of carcinogen from the pelleted heterochromatin DNA was enhanced, while loss from the euchromatin DNA was reduced. The covalent nature of the carcinogen modification of DNA was confirmed by thin-layer chromatography (TLC). These studies also demonstrated 2 separate carcinogen-purine base adducts which were identified as N-(guanin-8-yl)-N-AF and 3-(guanin-N2-yl)-N-AAF based on either co-chromatography with an authentic standard or on published Rf-values, respectively. The pelleted heterochromatin DNA had a significantly greater proportion of the 3-guanine-N2 adduct when compared to DNA from either the eu- or heterochromatin.


Effects of styrene oxide on chromosome aberrations, sister chromatid exchange and hepatic drug biotransformation in chinese hamsters in vivo.

In vivo inhalation exposure to styrene oxide (25, 50, 75 and 100 ppm) for 2, 4 or 20 days (25 ppm only) had no effects on chromosomal aberration rates or sister chromatid exchange (SCE) frequencies (BrdU/labelling performed in vitro) in the bone marrow cells of Chinese hamsters. The only positive response in aberration frequency was obtained when styrene oxide was injected in lethal concentration (500 mg/kg body weight, i.p.) into the animal. One animal out of six showed slightly elevated SCE values after this high dose. The response of the hepatic drug metabolizing enzymes to styrene oxide exposure was found to be rather weak, which may be due to rather high activity of epoxide hydratase in Chinese hamsters as compared to e.g. mouse.


The effects of bromosulphophthalein, indocyanine green and bilirubin on the biliary excretion of methylmercury.

The effects of three ligands for ligandin on the biliary excretion of methylmercury were investigated in male rats injected intravenously with 1.0 mg/kg Hg as Me203 HgCl. Bromosulphophthalein and indocyanine green inhibited the biliary excretion of methylmercury, while bilirubin had no such effect. None of the compounds tested which inhibited the biliary excretion of methylmercury decreased bile flow or changed the hepatic concentration of mercury of non-protein thiols. The possibility of the involvement of ligandin in the biliary excretion of methylmercury is discussed.


Correlation of biochemical and morphological changes induced by chemical injury to the lung.

Comparison has been made of injury to the rat pulmonary alveolar parenchyma evoked by intravenous injection of N-nitrosomethylurethane, intratracheal instillation of 3-methylcholanthrene or repeated inhalation for up to 15 days of carbon tetrachloride, trichloroethylene or gasoline vapour. Biochemical analyses, including assessment of rates of RNA and DNA synthesis and secretion of pulmonary surfactant, were correlated with morphological changes determined by electron microscopy. Single doses of N-nitrosomethylurethane or 3-methylcholanthrene inhibited incorporation of [14C] orotate into lung RNA 1--3 days after treatment. Daily exposure for 30 min to carbon tetrachloride or trichloroethylene vapour caused less marked reduction in orotate incorporation. Ultrastructural examination revealed that 3-methylcholanthrene toxicity was characterised by cytoplasmic change including disruption of surfactant lamellaie of Type 2 pneumocytes and variable degenerative changes Type 1 pneumocytes. Eight to ten days after treatment, the morphological evidence of hypertrophy/hyperplasia and transformation of Type 2 pneumocytes correlated well with biochemical evidence of stimulated incorporation of [3H]thymidine. Inhalation of carbon tetrachloride or trichloroethylene vapour produced milder responses including occasional degenerative changes in Type 1 pneumocytes, reduced numbers of surfactant lamellae in Type 2 pneumocytes and no change in [3H]thymidine incorporation. In contrast to the gradation of injury produced by the various chemicals, all procedures caused a marked and reproducible reduction in secretion of pulmonary surfactant as determined by endobronchial lavage. Following solvent inhalation, reduced recovery of surfactant was detected within 5 days of repeated exposure and thereafter no further change in this depressed level resulted from continued exposure for a further 10 days. The data are discussed in terms of a generalised pattern of response by pulmonary alveolar tissue to chemical injury and the apparent sensitivity of surfactant secretion as an indicator of damage to the lung.


Chromatin proteins as a possible target for antitumour agents: alterations of chromatin proteins in dibromodulcitol treated Yoshida tumors.

The effect of dibromodulcitol (DBD) on Yoshida sarcoma chromatin components has been investigated. Measurements on the radioactivity of nuclear components after in vivo treatment with [3H]DBD for 1 h indicated preferential drug binding to the high molecular weight component of the nuclear residual acidic protein (non-histones) and also to Histone 1 (H1) (very lysine rich, F1). Two-hour DBD treatment resulted in partial degradation and reduced [3H]leucine incorporation into the same fractions of chromatin. However, 6 h after DBD treatment, the synthesis of the degraded chromatin proteins began and by 24 h was completed. During the same treatment period the composition of chromatin showed a remarkable alteration; 2 h after DBD treatment the amount of the nuclear residual acidic proteins relative to DNA decreased by approx. 50%, but returned to control value 24 h after drug treatment. This in conjunction with the data on [3H]leucine incorporation suggests that certain chromatin proteins are degraded and subsequently newly synthesised after DND treatment resulting in an exchange of chromatin components. The formation of a nucleohistone complex between H1 and DNA was inhibited by pretreatment of H1 and DBD, dianhydrodulcitol (DAD) and bischloroethylnitrosourea (BCNU).