Comparison of flexible sigmoidoscopy with other diagnostic techniques in the diagnosis of rectocolon neoplasia.

Flexible sigmoidoscopy was compared to rigid sigmoidoscopy in the detection of colorectal neoplasia in a select group of patients. A distance of 30 cm or greater was obtained by flexible sigmoidoscopy in 94% of patients and a distance of 50 cm or greater in 46% of patients. A significant number of cancers and adenomas detected by flexible sigmoidoscopy were not detected by rigid sigmoidoscopy. Flexible sigmoidoscopy was tolerated better than rigid sigmoidoscopy but required twice the time. Flexible sigmoidoscopy could be combined with air-contrast barium enema the same day with the one preparation and did not interfere with the x-ray examination. All cancers and a significant number of adenomas detected subsequently on colonoscopy were detected by the combination of flexible sigmoidoscopy and air-contrast barium enema. Although the combination of flexible sigmoidoscopy and air-contrast barium enema is not adequate for thorough diagnostic evaluation of patients with a positive screening test, it may be of value in other select clinical situations. Flexible sigmoidoscopy has the potential for higher yield and better patient tolerance as compared to rigid sigmoidoscopy. This warrants further evaluation.


Autoimmune hemolytic anemia in ulcerative colitis: report of three cases, review of the literature, and evaluation of modes of therapy.

Three cases of autoimmune hemolytic anemia in association with idiopathic ulcerative colitis are reported; the literature is reviewed; and therapeutic modalities are considered. Successful treatment is shown to have resulted with steroids alone, steroids and immunosuppressives, splenectomy, and colectomy. It is concluded that moderate or severe hemolysis should be treated first with high-dose corticosteriods; if unsuccessful, immunosuppressive therapy may be added or a splenectomy performed. Finally, total colectomy should be reserved for fulminant colitis and its complications and is not indicated solely for hemolysis.


Lack of supportive evidence for an autosomal recessive inheritance in chronic hepatitis B infections.

Two families with persistent hepatitis B infections are described. The patterns of HBsAG positivity in the parents and offspring are not compatible with the theory that susceptibility to chronic hepatitis B infection is inherited as an autosomal recessive trait.


Relationship of hiatal hernia to endoscopically proved reflux esophagitis.

The relationship between reflux esophagitis and hiatal hernia was studied using fiberoptic endoscopy. Of 293 patients having upper-gastrointestinal endoscopy during an 18-month period, 64 (22%) had a hiatal hernia and 38 (13%) had esophagitis. Half the patients with a hiatal hernia had esophagitis, whereas the vast majority (84%) of patients with esophagitis had a concomitant hiatal hernia. The association between esophagitis and hiatal hernia was highly significant (P less than 0.0001). There was no statistically significant association between the size of the hiatal hernia and the degree of esophagitis on endoscopy. A permissive role of hiatal hernia in the genesis of reflux esophagitis is suggested.


Instrumental esophageal perforation: a possible complication of bougienage following Quinton-Rubin esophageal biopsy.

A patient with a perforation following dilatation of the esophagus is presented. The temporal relationship between earlier Quinton-Rubin esophageal biopsies obtained by the use of the multi-purpose Quinton-Rubin tube and subsequent perforation is noted. The possibility of these events being causally related is discussed.


Nasogastric suction in alcoholic pancreatitis.

In order to assess the usefulness of nasogastric suction in acute alcoholic pancreatitis, 37 patients with alcoholic pancreatitis were prospectively investigated. The study failed to demonstrate efficacy of nasogastric suction in those patients with mild disease. Application of a system of prognostic signs proved useful in discriminating between mild and severe disease. Routine use of ultrasound examinations detected three pancreatic pseudocysts before they became clinically apparent. In instituting appropriate therapy in mild pancreatitis, factors such as patient comfort should be considered in the absence of proven significant value of nasogastric suction.


[67Ga]citrate scintiscanning in active inflammatory bowel disease.

Twenty-five hospitalized patients were studied prospectively with [67Ga]citrate (GA) abdominal scintillation scanning in an attempt to define its role in the evaluation of patients with active inflammatory bowel disease (IBD). There were nine patients with ulcerative colitis (UC), seven with Crohn's disease (CD), and nine controls. In four patients, two with UC and two with CD, a tissue/plasma radioactivity ratio was obtained and compared to normals. All the UC patients had positive GA scans and only one of seven of the CD patients had a positive scan. There were no false positive scans. Scans performed after a 3- or 5-day delay were more accurate than 6-hr scans alone. Well-delineated colinic radioactivity 6 hr after injection which persists for 3 to 5 days indicates the presence of UC in patients with IBD, while a negative scan is more consistent with active CD. Colonic uptake at 6 hr which clears by 48 or 72 hr is not indicative of UC. This procedure aided in following the course of UC, delineating the extent of disease, and in differentiating active CD from an intraabdominal abscess. Tissues from UC patients had increased tissue/plasma ratioactivity ratios while tissues from CD patients had normal or decreased ratios which were consistent with the imaging data.


Scanning electron microscopy of rectal mucosa in Crohn's disease.

Rectal biopsies from healthy control subjects, and from patients with Crohn's disease whose rectal mucosa appeared normal by sigmoidoscopy and light microscopy, were examined by scanning electron microscopy. Appearances within both groups were quite variable, and no clear differences between the groups could be distinguished.


The effect of 15(R)-15-methyl prostaglandin E2 on meal-stimulated gastric acid secretion, serum gastrin, and pancreatic polypeptide in duodenal ulcer patients.

The effects of 100-microgram of 15(R)-15-methyl prostaglandin E2 on meal-stimulated acid secretion, serum gastrin, and pancreatic polypeptide concentrations were measured in patients with duodenal ulcer. The drug given in encapsulated or unencapsulated form significantly reduced gastric acid secretion by 59% or 70%, respectively. Rises in serum gastrin and pancreatic polypeptide concentrations after the meal were significantly blunted by 15(R)-15-methyl prostaglandin E2. This dose of prostaglandin led to no side effects and merits clinical evaluation in the treatment of peptic ulcer disease.


Cholestatic and hepatocellular injury associated with erythromycin esters: report of nine cases.

A combined cholestatic and hepatocellular injury occurred in nine patients, following therapy with erythromycin estolate (EE) or other erythromycin derivatives. Eight of the nine patients developed jaundice within three weeks after initiation of treatment; pain was one of the main symptoms in five patients while fever and itching were noted in four patients. Symptoms and signs subsided and abnormal tests of liver function returned to normal after withdrawal of the drug. The major histologic finding was cholestasis, but the majority of cases also had evidence of hepatocellular injury of variable severity; one biopsy specimen showed centrilobular necrosis. Ultrastructural findings in one case included changes related to cholestasis as well as hepatocellular injury with striking mitochondrial abnormalities. Our data are compared with those of the literature, with special reference to morphologic features.


Pancreatic duct arteriovenous fistula and the metastatic fat necrosis syndrome.

This report summarizes the course of a patient with asymptomatic chronic pancreatitis associated with hemorrhage into the pancreatic duct and metastatic fat necrosis. Retrograde cannulation of the pancreatic duct and superior mesenteric arteriography established the presence of a pseudocyst with a pancreatic duct-arteriovenous (DAV) fistula as the cause of the syndrome. Ligation of feeder vessels with external drainage of the cyst as the initial surgical procedure stopped the bleeding but failed to prevent recurrence of the pancreatic duct-venous fistula. A pancreaticoduodenectomy with resection of the cyst and fistula was required to arrest destruction of distant tissues. Although serum and urine amylase concentrations were markedly elevated, serum lipase levels were normal throughout the patient's course. Elevation of serum lipase does not seem to be a necessary condition for the development of the metastatic fat necrosis syndrome.


Quantitative effect of oral feeding on gastrointestinal myoelectric activity in the conscious dog.

Gastrointestinal myoelectric activity was recorded in seven studies in five dogs during two hours of fasting immediately followed by feeding and subsequent recording for four hours. In four studies serial plasma samples were taken for radioimmunoassay of insulin and gastrin. In all animals there was a significant reduction (P less than 0.01) in gastric basic electrical rhythm (BER) frequency on feeding which was sustained throughout the postprandial period. There was no change in the duodenal BER. Feeding induced a significant (P less than 0.01) increase in overall jejunal and ileal (but not duodenal) spike activity. Ileal (but not jejunal) spike activity again increased significantly (P less than 0.05) after the first two post-prandial hours. The changes in serum gastrin or in serum insulin did not appear to account for most of the observed changes in myoelectric activity, suggesting that other humoral and/or neural factors mediate the response to food.


Transport kinetics of D-glucose in human small intestinal mucosa: rate constants in histologically normal and abnormal mucosal biopsies.

Using D-glucose as the probe molecule, we analysed conditions which must be fulfilled in mucosal biopsy studies before the kinetic nature of the transport process can be established. Mucosal biopsies were obtained from the region of the ligament of Treitz from four healthy volunteers and from 47 patients: 29 of the 47 had histologically normal mucosa; seven had mild abnormalities; and 11 had moderate or severe ("flat") abnormalities in villus architecture. The rate of uptake of 40 mM glucose was constant only between 4 and 10 min, extrapolating through zero uptake at zero time with a constant adherent mucosal fluid volume. Incubation for shorter or longer periods was associated with over- or underestimation of the rate of uptake. Failure to use a nonabsorbable marker was also associated with overestimation of the rate of uptake. When biopsies were incubated for 6 min, a curvilinear relationship was observed between uptake rate and concentration. In biopsies with moderate and severe abnormalities, there was a marked reduction in the magnitude of the maximal transport rate and the apparent passive permeability coefficient, with little change in the magnitude of the apparent affinity constant. When human mucosal biopsies are used to study nutrient absorption, certain criteria must be fulfilled to establish valid rate constants and to make comparisons between normal and abnormal mucosa.


The effects of a prostaglandin endoperoxide analogue on canine gastric acid and mucus secretion.

The effects of U-46619, a stable analogue of the prostaglandin endoperoxide PGH2, were studied on canine gastric acid secretion, gastric mucosal blood flow, and secretion of mucus into gastric juice and compared to those of PGE2. U-46619 was approximately four and three times as potent as PGE2 in inhibiting acid secretion and stimulating mucus secretion, respectively. When infused at a low dose, U-46619 inhibited acid secretion directly without causing a decrease in the ratio of mucosal blood flow to volume rate of secretion (R), this effect being similar to that observed for PGE2. However, unlike PGE2, U-46619 when administered in a higher dose caused a decline in R while decreasing acid secretion and mucosal blood flow, suggesting a primary restriction of blood flow. The antisecretory effects of arachidonic acid may be due in part to the endogenous formation of prostaglandin endoperoxides as opposed solely to prostaglandin formation. Considering the antisecretory and mucogenic actions of U-46619, nontoxic analogues of prostaglandin endoperoxides may be of value as antiulcer agents.


Efficacy of the morphine-prostigmin test for evaluating patients with suspected papillary stenosis.

The morphine-Prostigmin test (MPT) has been advocated as a diagnostic test for identifying patients with papillary stenosis. We studied 14 patients referred for possible papillary stenosis with the MPT and ERCP sphincter of Oddi manometry. The response to the MPT (symptoms, serum amylase/lipase values) was evaluated by multiple criteria. The MPT was also done on 6 volunteer control subjects. Operative evaluation of the sphincter of Oddi was obtained in 8 of the 14 patients. There was no correlation between the results of the MPT and ERCP manometry or operative findings. There was good correlation, however, between ERCP manometric findings and operative assessment of the sphincter of Oddi. Fifty percent of the volunteer subjects had a significantly positive enzyme response to the MPT. In our experience the MPT does not reliably detect patients with papillary stenosis.


Effects of long-term cimetidine on serum gastrin in duodenal ulcer.

Basal and food-stimulated gastrin were measured in 16 patients with duodenal ulcer before and during long-term maintenance therapy with 400 mg cimetidine twice daily. Basal gastrin (mean +/- SE) rose significantly from 27.5 +/- 3.1 pmol/liter precimetidine to 32.8 +/- 2.1, 37.2 +/- 2.6, and 38.5 +/- 3.3 pmol/liter at 1, 3, and 6 months, respectively. The total integrated gastrin response to a protein meal was 1.67 +/- 0.18 nmol/liter/120 min pre-, and 2.54 +/- 0.35, 3.29 +/- 0.3, and 4.36 +/- 0.4 nmol/liter/120 min at 1, 3, and 6 months, respectively. These increases were significantly higher at each time period. This study has thus demonstrated a progressive increase in both basal and food-stimulated gastrin during cimetidine therapy, and this increase could theoretically lead to an increase in gastric acid secretion following cessation of cimetidine.


Zollinger-Ellison syndrome with pneumopericardium.

A 55-year-old white male was found to have the Zollinger-Ellison syndrome in 1971. Supposed total gastrectomy was performed at that time. When an esophageal ulcer was found, six years later, esophagoscopic biopsy revealed residual gastric mucosa. The patient was given cimetidine 300 mg qid because it was felt he could not tolerate further surgery. After eight months of cimetidine therapy, the patient was admitted to the hospital because of retrosternal pain. Pneumopericardium was discovered, and at autopsy a large penetrating gastrojejunal ulcer was demonstrated.


Peyronie's disease and sclerosing cholangitis in a patient with ulcerative colitis.

A 51-year-old patient with a 30-year history of ulcerative colitis developed sclerosing cholangitis and later Peyronie's disease. The occurrence of these diseases in the same patient supports the view of an immunological origin of these associated disorders.


Motilin and the interdigestive migrating motor complex in man.

In order to assess the possible role of the new candidate gut hormone, motilin, in controlling the interdigestive migrating motor complex (MMC) in man, 14 normal subjects were studied after an overnight fast by means of three pressure-recording catheters with orifices 25 cm apart in the upper small intestine. The typical aboral progressing bursts of pressure waves occurred at a mean interval of 137 minutes and were preceded by a peak motilin level 25 pmol/liter higher than the lowest level in the postactivity-front quiescent period. To study the effect of exogenous motilin, an infusion of pure porcine motilin at various dose levels was given to 16 normal volunteers shortly after the onset of the phase I quiescent period. Motilin infusion induced an activity front in 12 of the 16 subjects. The mean activity front interval was reduced to 46 min (P less than 0.001). This effect could be obtained even at the low dose level of 0.4 pmol/kg/min, which produced an increase in plasma motilin level of only 57 pmol/liter. These data suggest that a cyclic rise in plasma motilin levels is one of the factors involved in the production of the activity front of the migrating motor complex in man.


Glucagon effects on the human small intestine.

In healthy volunteers, the effects of intravenously administered glucagon on small intestinal function was investigated. Bolus doses resulting in plasma glucagon concentrations of greater than 800 pg/ml (5 min after injection) abolished jejunal contractions for 4.4 +/- 0.4 (SEM) min after a latency period of 49 +/- 4 sec. During continuous intravenous glucagon infusion, jejunal dilatation and increase in mean transit time (MTT) occurred at plasma levels greater than 720 pg/ml, while inhibition of water and electrolyte absorption was observed only with plasma glucagon concentrations of 1760 +/- 114 pg/ml. Under these conditions, the propulsion of fasting intestinal contents was slowed without change in flow rate. The observed effects cannot be attributed to the simultaneously occurring rise in plasma insulin and glucose concentrations. Short-term increases in circulating glucagon concentration inhibit intestinal tone, contractions, and propulsion with only a minor effect on water and electrolyte absorption limited to a narrow concentration range of plasma glucagon. Neither effect occurs at glucagon levels likely to occur under physiologic concentrations. The latency period preceding the abolition of jejunal contractions suggests that glucagon does not act directly on intestinal smooth muscle cells.


Active E rosette-forming cells in the peripheral blood of patients with chronic active hepatitis.

E rosette-forming cells (E-RFC) and active E-RFC were studied in the peripheral blood in patients with chronic active hepatitis (CAH), chronic persistent hepatitis (CPH), and in control subjects. A significant reduction in active E-RFC (a subpopulation of T lymphocytes considered actively involved in cell-mediated immune reactions) was found in patients with untreated CAH, both HBSAg positive or negative. In contrast, patients with immunosuppressive-treated CAH showed an increased relative number of active E-RFC. A significant increase in active E-RFC was demonstrable in other immunosuppressive-treated chronic inflammatory disorders. In CPH active E-RFC were not different from normal controls. The number of E-RFC showed a relative and absolute decrease in both CPH and untreated CAH. These studies demonstrate an increase in active E-RFC in immunosuppressive-treated CAH and suggest that prednisone and azathioprine may have a differential effect on T lymphocyte subpopulations.


A new animal model with hyperbilirubinemia: the indigo snake.

An unconjugated hyperbilirubinemia has been observed in all species of normal indigo snakes. The plasma clearance of large organic anions such as sulfobromophthalein and unconjugated bilirubin was markedly delayed when compared to other snake species. Endogenous bile flow and biliverdin and bilirubin excretory rates and the excretion of bile pigments after a bilirubin load were measured in various snakes. The indigo snake represents a new animal model in which to study mechanisms important to hepatic anion uptake and biliary transport.


A syndrome of acute self-limiting ulcerative esophagitis in young adults probably due to herpes simplex virus.

Five healthy young adults developed an acute self-limiting ulcerative esophagitis. Two had definite evidence of herpes virus being present and a third one had appropriate changes in herpes simplex viral titer. All cases followed a characteristic and similar course consisting of sudden onset of odynophagia, multiple discrete small ulcers in the esophagus and herpetiform lesions elsewhere in the skin or mouth. Although most previous reports of herpes esophagitis indicate that it is an "opportunistic" infection in debilitated hosts, the present report indicates that this infection, in patients who are otherwise well, may occur more frequently than one would have previously suspected. Therefore, herpes simplex virus should be considered as a possible etiological agent in the differential diagnosis in patients presenting with a sudden onset of odynophagia.


Colonic absorption of unconjugated bile acids: perfusion studies in man.

Colonic absorption of three major unconjugated bile acids--cholate, chenodeoxycholate, and deoxycholate--was measured under steady-state conditions using a technique of colonic perfusion in healthy volunteers. Aqueous solutions at pH 8.0 and varying in concentration from 1 mM to 10 mM were used. The rate of chenodeoxycholate absorption averaged nine times that of cholate absorption; deoxycholate absorption was somewhat less than that of chenodeoxycholate absorption, averaging six times that of cholate. At concentrations below 5 mM, the rate of absorption of bile acids was directly proportional to concentration, so that "clearance" could be calculated. Clearance values for a 1-mM solution (ml/min/colon, mean +/- SE) were: chenodeoxycholate, 9.84 +/- 1.0; deoxycholate, 7.0 +/- 1; and cholate, 0.82 +/- 0.10. Since absorption was proportional to concentration in the lumen, and was more rapid for the dihydroxy acids, the major mechanism of absorption was thought to be passive nonionic diffusion. Maximal rates of bile acid absorption were calculated from a 1-mM solution and found to be as high as 4.2 g/day for chenodeoxycholate, 3.2 g/day for deoxycholate, and 0.5 g/day for cholate, and the rate would be still greater for more concentrated solutions. Colonic absorption may contribute significantly to conservation of the dihydroxy bile acid pool, especially in conditions of bile acid malabsorption.


Hepaticoportal fistula and portal hypertension.

A large intrahepatic fistula between the hepatic artery and portal vein was detected at angiography in a patient with portal hypertension and bleeding esophageal varices. Hemodynamic studies demonstrated that increased vascular resistance in the liver rather than increased flow through the fistula was responsible for the portal hypertension and a portocaval shunt was performed. A repeat angiogram two months after the operation showed that the fistula had closed spontaneously.


[Gardner's syndrome (author's transl)].

An exact history was taken and clinical, endoscopic and histologic studies were performed in 14 family members of a case of Gardner's syndrome with triple symptomatology documented with biopsy and autopsy findings. Among the symptom-free probands five cases of colonic polyposis and three cases with gastric polyps were found. All polyps were histologically adenomatous. Dysplasias grade I and II were found repeatedly, in one 16-year-old adolescent there was already severe dysplasia within the gastric mucosal polyps. A warning is given against sub-classification of familial colonic polyposis and other syndromes within the definition of Gardner's syndrome. In future during diagnostic investigations for cases of Gardner's syndrome isolated gastric polyposis should be sought for as well as monosymptomatic colonic polyposis. Diagnostic procedures and treatment should depend on endoscopic and histological findings. Regular follow-up with endoscopic biopsies are to be encouraged not only in the diseased cases but also in all family members available.


[Combined chemotherapy and radiotherapy of localized and metastasizing Ewing's sarcoma (author's transl)].

Between 1973 and 1978 combined radiotherapy and chemotherapy were given to 22 patients with histologically proven Ewing's sarcoma. The combined chemotherapy consisted of cyclophosphamide, vincristin, adriamycin, as well as dacarbazine in some cases. The neoplasm was a localized one at the beginning of treatment in 14 of the 22. These patients received high-voltage radiotherapy to the primary focus at a focal dose between 42 and 55 Gray (4200-5500 rad), followed by chemotherapy. After 6--8 treatment cycles, adriamycin was replaced by methotrexate. Nine of the 14 patients survived without recurrence for 12 to over 59 months. Eight patients had extensive metastases at the beginning of treatment: they at first received only chemotherapy, followed by radiotherapy or operation, as indicated. Full clinical remission was achieved in five of them: in three this remission has now lasted for more than 18, 40 and 44 months, respectively. These results indicate that (1) additional chemotherapy improves the prognosis of localized Ewing's sarcoma, and (2) even in far-progressed cases the combination of chemotherapy and radiotherapy can achieve lasting remission, which may in fact be a true cure.


[Glucose tolerance in the course of treating endogenous hypertriglyceridaemia (author's transl)].

Abnormal glucose tolerance was found in 30 of 100 patients with endogenous hypertriglyceridaemia (74 males, 26 females, age 45.9 +/- 11.4 years, Broca index 116 +/- 25%; 44 type II b, 7 type III and 49 type IV hyperlipoproteinaemia). In 19 cases the abnormal glucose tolerance was discovered during treatment with diet and, if necessary, lipid-lowering substances for an average of 5.8 +/- 2.8 years. Compared with the normal population there was in these patients an 8- to 10-fold increase in the incidence of abnormal glucose tolerance. Therapy-induced lowering of the serum triglyceride concentration was followed by an improvement of the abnormal glucose tolerance. The "antidiabetic" effect of hyperlipidaemia treatment was only slightly improved by concurrent weight reduction. These results indicate that the treatment of hyperlipidaemia in patients with endogenous hypertriglyceridaemia is of importance in the prevention of diabetes.


[Sniffing addiction: chronic solvent abuse with neurotoxic effects in children and juveniles (author's transl)].

25 cases of clinically severe toxic polyneuropathy were observed among young people in Berlin who were addicted to sniffing methylethylketone-containing solvents. The peripheral motor defects took 2 1/2 to 3 years to regress. In severe cases there were additional spastic signs. This form of neurotoxic effect can be reproduced in animals. It is due to a disorder of axonal transport which destroys peripheral and central axons.


[Pharmacokinetics and thyrotoxicity of the sodium nitroprusside metabolite thiocyanate (author's transl)].

Thiocyanate (SCN) serum levels were measured in relation to renal function and SCN-induced changes in thyroid function in 17 patients treated with sodium nitroprusside. SCN serum levels rose linearly, dose-dependent, after the distribution phase. If there was creatinine retention the rise was much steeper. Delayed SCN elimination in renal failure, previously shown experimentally, was also demonstrated clinically. Suppression of thyroid function, to a not precisely predictable extent, is likely to occur at a SCN serum level from 18 mg/l upward.


[Telephone transmission of ECGs for checking cardiac pacemaker function (author's transl)].

A single-channel system is described for transmitting by telephone and checking the ECG of patients with implanted pacemakers, and at the same time diagnosing a complex arrhythmias.


[Risk of a second malignant neoplasm after successful treatment of a malignant tumour in children (author's transl)].

One boy, given radiotherapy and cytostatic drugs for a rhabdomyosarcoma died nine years later of acute leukaemia. A girl, who had received radiotherapy for an inoperable suprasellar tumour and also given cytostatic drugs, fell ill seven years later with a glioblastoma. Among a total of 750 children with malignant neoplasm observed by the authors, a permanent cure rate of about 50% is to be expected among about 300 children treated recently. If this rate applies to the entire Federal Republic of Germany, about 1,000 children are likely to be cured annually. In these circumstances it is likely that 80-160 children treated in any one year will develop a second neoplasm or leukaemia 20 years later, most commonly as a late sequela of the treatment.


[Absorption and excretion of g-strophanthin after intravenous or sublingual administration (author's transl)].

Tritiated g-strophanthin was administered to six volunteer patients in a dose of 0.25 mg intravenously and 6.0 mg sublingually 1-3 weeks apart. Radioactivity in plasma and urine was measured afterwards for 48 or 96 hours, respectively. Plasma concentration fell to about a fifth of the maximal concentration within 6 min after intravenous administration, total excretion within three days being about 50%, with a half-time of 11 hours. Measurable plasma concentration after sublingual administration was demonstrable only after 15 min in some and after 2 hours in all the six patients. Radioactivity counts increased up to the 24th hour, in three patients up to the 48th hour. Maximal plasma concentration ranged from 4.7 to 0.7 ng/ml, corresponding to levels reached 15 min or 4 hours, respectively, after intravenous administration. Urinary radioactivity reached its maximum after 24-48 hours and then decreased. Within three days 0.6 and 2.5% of the administered radioactivity was recovered, corresponding to the g-strophanthin doses of 0.072 to 0.3 mg. The findings once again confirm the low and irregular absorption of g-strophanthin when administered sublingually or orally.


[A new concept in the surgery of chronic pancreatitis (author's transl)].

There is an obvious advantage to partial duodenopancreatectomy with occlusion of the pancreatic duct by prolamine (a rapidly solidifying aminoacid solution) for the prevention of recurrences, while preserving the stomach and proximal duodenum, in the treatment of severe destruction of the pancreas, localised to the head or generalised. The death rate of partial duodenopancreactectomy has been lowered from 8.5 to 2.3% since using this method. Compared with total duodenopancreatectomy which--according to the authors' earlier results carried a postoperative mortality of 20%--the operative risk has been reduced to a tenth. Taking into account the benign nature of chronic pancreatitis, on the other hand, and the extent of the operative operation, on the other, the present death-rate of 2.3% would appear reasonable. Occlusion of the pancreatic duct with prolamine in order to exclude any still present excretory pancreatic function in the residual pancreatic tissue has made all patients pain-free. No early recurrence has been observed.


[Treatment of tachy-arrhythmias by implanted pacemaker (author's transl)].

A recently developed antitachycardic pacemaker was implanted into seven patients with drug-resistant tachycardias. The implantation was made after diagnosis of a tachy-arrhythmia by invasive techniques and after experimental determination of the effective site, optimal mode and effective frequency of stimulation, as well as the regulation and duration of impulse release. Thus the pacemakers were individually adapted to the particular arrhythmia. High-frequency stimulation and competitive stimulation were used as stimulation modes. The systems work automatically, triggered either by the ECG, the patient or externally. The frequency of attacks was significantly reduced in five patients, the duration of attacks in all.


[Intracutaneous desensitization in a case of insulin allergy with anaphylactic shock (author's transl)].

In a 72-year-old woman with keto-acidaemia due to out-of-control diabetes, anaphylactic reaction occurred immediately after intravenous administration of 8 units of insulin. Extensive intracutaneous testing with highly purified insulins, including sheep and human insulin, revealed an allergy of the immediate type against the insulin molecule itself. Desensitization with subcutaneous insulin administration failed. Intracutaneous insulin injection three hours apart, starting at a dilution of 1:100, produced good subcutaneous tolerance of insulin within seven days.


[Metastasising squamous-cell carcinoma of the skin in a ten-year-old girl].

Because of recurrent paronychia of the right thumb "granulation" tissue was removed from the nail-bed of a ten-year-old girl. Histological examination revealed a cornified squamous-cell carcinoma. About five months later a lymph-node metastasis was found in the right axilla. After radical dissection of the axillary region further lymph-node metastases were found, as well as a lymphangitic carcinosis with tumour infiltration in the veins. Despite telecobalt radiation there were recurrent retastases which could no longer be removed by operation. Two years after the diagnosis the girl died. Necrospy revealed diffuse tumour infiltration of the soft tissue of the axilla, upper arm, shoulder, neck and thoracic wall on the right, including the breast tissue, with thoracic para-aortic-abdominal and left-sided axillary lymph-node metastases, and lung and pleural metastases bilaterally.


[Instrumental perforation of the intrathoracic oesophagus (author's transl)].

Due to a wide iatrogenic perforation of the oesophagus situated at the level of the carina a 90-year-old woman was admitted to hospital. Prompt recognition and immediate surgery were successful. The operation consisted of a right-sided thoracotomy, wide mediastinotomy, over-sewing of the perforation and pleural drainage. Oesophagostomy and gastrostomy were performed to relieve the oesophagus. A tube was inserted into the duodenum for artificial feeding. This method has been successful in previous cases.


[Medicine in the contemporary world].

The author begins with the premise that, contrary to what has been attained to date, greater order and higher justice are both possible and desirable in medical care and, therefore, that it is worthwhile to consider the shortcomings of the past and present and the improvements that can be carried out in the future. He analyzes the various meanings given to the "health-sickness" complex and such current social phenomena as the rapid spread of the view that health care is a social right, the charge against medical practice and training of being dominated by "simplistic empiricism" and of relegating the importance of of the critical faculty, and widespread specialization. He expands on the accessibility of medical care to broad segments on the population, the delegation in modern society of the responsibility for health care to the institutional medical apparatus, and the contradictions generated by overemphasis on technology and specialization and by the pressure of demands from the community. He contends that the dilemma today es twofold: how to offer the entire population satisfactory medical services through comprehensive system consistent with national financial capabilities, and what these services should be so that their social function is based on sound scientific foundations. In the closing section of the article, the author makes several points about the future, with emphasis on humanizing medicine and of basic care services of the population--that is, on shifting medical practice toward the general community.


[Design of a course in physiology for medical training].

This article describes a course in general physiology and neurophysiology being offered at the School of Medicine of the Catholic University in Chile since 1971. The course was designed to provide the student skills in the planning, performance, and analysis of experimental problems. An account is given of the changes made in the course in response to annual evaluations over seven years. The procedures for grading student progress in developing critical judgment, handling information, and solving problems are analyzed. The authors are of the view that a course of this kind helps improve the ability to judge problems beyond the course curriculum. The describe the advantage of such courses for the training of physicians and recommend that activities that could be continued throughout medical training be designed and evaluated to stimulate the development of the students' critical faculties.


[Education and training of the general physician].

The authors of this paper, part of which was presented at the Seventh Pan American Conference on Medical Education (New Orleans, October 1978), recognize that training programs for general physicians should relate to the particular socioeconomic conditions and characteristics of the individual country and region. They propose that the general physician in developing countries be prepared in graduate courses to provide primary care, in order to give service immediately after graduation and because opportunities elsewhere for recent graduates are limited. After outlining new curriculum proposals for the general physicians, they argue against the establishment of departments of family medicine in Latin American medical schools. It is suggested that the general physician's training take place in outpatient clinics, health centers, and community and teaching hospitals, to make possible the integration of the medical education and medical care. Until such time when integration is achieved, the teaching hospital should afford medical education and care on a small scale, offering outpatient general clinics, nonspecialized services, and community health activities.


[Manpower training at the Institute of Nutrition and Food Technology].

The Institute of Nutrition and Food Technology (INTA) of the University of Chile was established in 1976. Its functions are, among others, to study the country's nutritional problems, conduct research into measures for improving the nutritional status of the community, and train researchers and professionals, technical, and auxiliary staff in the nutrition field. Since 1977 the Institute has operated as an associated center of the United Nations University (UNU) in the area of human needs and, as such, carries out research in food and nutrition problems, provides training and education to UNU fellows, collaborates in the establishment of a worldwide network of institutions interested in the problem of world hunger, and advises UNU on the conduct of occupational training and research programs. This article refers more specifically to the teaching functions of INTA and describes its undergraduate and graduate activities and pedagogical training for nutrition instructors.


[Ultrasonic diagnosis of abdominal diseases (author's transl)].

Ultrasonic examination is a modern method used to visualize intraabdominalorgans and pathological processes affecting their organisation. This method of examination is safe and gentle as regards the patient and suitable for estimation of extraluminar pathological conditions. The report concerns experiences gained with abdominal ultrasonography effected with Echoview VI Picker (Transducer 2,25 MHz, diameter 13 mm) at the department of internal medicine of the University Policlinic in Prague in 1975--1977. There was a total of 400 examinations of the abdominal cavity effected in 358 subjects. The principal indications for examination included a clinical finding or suspicion of a disease affecting the pancreas (180X), liver (79X), spleen (30X), gall-bladder (24X), kidney (21X), or undefined palpation findings in the abdomen (74X). In 41,75% of cases sonography confirmed the abdominal finding, in 44,75% of cases the finding was not confirmed, in 13,5% of cases the finding could not be estimated dependably. As the frequency of positive findings achieved by ultrasonic examination in pancreatic pseudocysts is high, we effect sonography in the instance of suspection of this affection prior to the planned endoscopic retrograde cholangiopancreaticography as orientation examination.


[Prepyloric, cystic adenomatous hamartia in a newborn infant. Contribution to the morphogenesis of gastric duplications (author's transl)].

The so-called adenomyomas of the stomach in the antric region can often be seen. However, cystic alterations including duplications are a rarity. The author reports on a case of a duplication of the stomach in connection with a so-called adenomyoma of the pyloric channel. On the basis of a case report he describes morphogenesis, clinical aspects, complications and therapy with due consideration of the literature.


[X-ray diagnosis and the clinical aspects after pancreatectomy].

11 patients (three women) were examined 7 month to 12 years after total pancreatectomy. There were found differences of--34% to +23% between bodyweight and ideaweight. Three patients with a bodyweight less than 75% of the idealweight died, steatorrhea (8--19 g/die), bloodsugar oscillation in the 24 h-profile (40--820 mg%) and an intrinsic-factor-non-responsive vitamin B12-malabsorption without megaloblastic anemia. The roentgenexamination permitted a classification of the upper gastrointestinaltract in a type similar to B II or B I resection and a type with Y- Roux anastomosis for bilarydrainage.


Evidence favoring the existence of two high molecular weight precursor forms of dog kidney renin.

Extraction of canine renal cortical tissue at pH 7.4 in the presence of the protease inhibitors diisopropylfluorophosphate (0.2 mM), Na2EDTA (7.8 mM), sodium tetrathionate (7.8 mM). N-ethyl maleimide (7.8 mM) yielded renin activity in two high molecular weight (HMW) forms, 65,000 (65K) and 55,000 (55K). Serial gel filtration chromatography of such extracts stored at 4 C showed that over the course of 2 days, activity at both 65,000 and 55,000 decreased almost entirely, while low molecular weight (LMW) activity at 41,000 (41K), not present immediately after extraction, had appeared in the extracts, The renin activity of the extract doubled over the first 24 h of storage and remained stable over the next 24 h. The activity of all three renin forms was comparably inhibited by antirenin antibodies. Our results support the concept that HMW renin(s) is a biological precursor of 41K renin. The new finding of a renin form intermediate in apparent molecular weight between 65K and 41K renin suggests that proteolytic processing of HMW to LMW renin may involve more than one step. The fact that in vitro conversion of HMW to LMW renin will occur under these conditions but takes place slowly may provide a technique for the future study of the precise manner in which HMW is converted to LMW renin.


Cholinergic involvement in osmotic control of vasopressin release by the organ-cultured rat hypothalamo-neurohypophyseal system.

A nicotinic-cholinergic receptor appears to mediate osmotic stimulation of vasopressin (VP) release by the hypothalamo-neurohypophyseal explant. Nicotinic blocking agents, hexamethonium, tetraethylammonium chloride, and trimethaphan, blocked VP release in response to the addition of sufficient NaCl to yield a 10 mosm/kg H2O increase in culture medium osmolality. Atropine at a similar molar concentration was ineffective in blocking VP release in response to the same osmotic stimulus. Tetraethylammonium chloride and trimethaphan also blocked acetylcholine-stimulated VP release. These findings support the hypothesis that the osmoreceptive element responsible for controlling VP release resides in a separate cell and communicates with the VP cell by way of a nicotiniccholinergic receptor.


Bioassay and radioimmunoassay of serum luteinizing hormone in the male rat.

Measurements of immunoactive and bioactive serum LH in the rat were performed after optimization of both RIA and rat interstitial cell-testosterone (RICT) assay to measure the low circulating LH levels of intact male rats. The effective sensitivity of the RIA was increased 3- to 5-fold to 6.2 or 4.7 ng RP1/ml by extraction of 1--1.5 ml serum, respectively. Serum LH levels of adult male rats were 48.5 +/- 16.3 ng RP-1/ml (n = 50), became undetectable after hypophysectomy, and were suppressed 50% after estrogen treatment. For RICT assay of serum rat LH, sensitivity was increased 5-fold by reduction of the incubation volume to 0.35 ml containing 2--4 x 10(6) interstitial cells/tube, with a detection limit of 3 ng RP-1 or 0.1 ng pure LH. The within-assay coefficient of variation for measurement of a pool of control male rat serum (126 +/- 19l6 ng RP-1) WAS +/- 12.5% and the between-assay variation was +/- 15%. Bioactive serum LH levels in adult male rats were 124.8 +/- 32.3 ng RP-1/ml or 2.74 +/- 0.71 ng rat LH/ml, with biopotency to immunopotency (B:I) ratios of 2.8 +/- 0.2 and 1.1 +/- 0.1, respectively, that were unchanged during elevations of serum LH by LHRH administration. Pituitary LH content was 726 +/- 183 micrograms RP-1 or 16.4 micrograms rat LH/gland, with B:I ratios of 2.3 +/- 0.4 and 1.0 +/- 0.2, respectively. The difference between B:I ratios in assays employing pure rat LH and assays using the RP-1 preparation was attributable to the presence of alpha-subunit and biologically inactive material in the RP-1 standard. Precise measurements of immunoactive and bioactive rat LH in male rat plasma can not be performed by these modified RICT and RIA procedures. The sensitive RICT assay can also be applied to the measurement of low levels of LH in the serum of primates and other species.


Ovarian responses of pregnant mare serum gonadotropin- and human chorionic gondotropin-primed rats: desensitizing, luteolytic, and ovulatory effects of a single dose of human chorionic gonadotropin.

We conducted a study to determine the morphological appearance and functional responsiveness of ovarian tissues after administration of hCG to 28-day-old rats primed 65 h earlier with PMS gonadotropin (PMSG) and after administration of a second dose of hCG 5 days later, i.e. to 33-day-old rats containing heavily luteinized ovaries. Sixty-five hours after the administration of 50 IU PMSG sc to 25-day-old rats, ovaries already contained an abundance of luteinized follicles and an adenylyl cyclase (AC) system that was responsive to LH, epinephrine, and NaF. The administration of 50 IU hCG sc at this time initially resulted in a loss of LH-responsive ovarian AC. Within 4 days of the hCG injection, the ovaries of the now 32-day-old rats were heavily luteinized, and ovarian AC was highly responsive to LH, epinephrine, and NaF. The administration of a single sc dose of 200 IU hCG to 33-day-old PMSC- and hCG-primed rats with luteinized ovaries resulted in a rapid desensitization of the ovarian AC to LH and a drop in serum progesterone levels, During the subsequent 7 days, serum progesterone levels continued to decline, while total ovarian AC reacquired responsiveness to LH by days 4--5 after the densensitizing dose of hCG. Dissection of ovarian components revealed, however, that the AC system of the corpora lutea originally present at the time of the second hCG injection remained permanently refractory to LH and that the AC in corpora lutea newly formed from freshly ovulated follicles exhibited a significant responsiveness to LH, epinephrine, and NaF. However, these new corpora lutea were not fully active, since serum progesterone never rose. Subcutaneous administration of 50 IU hCG to 33-day-old PMSG- and hCG-primed rats also promoted a rapid loss of AC responsiveness to LH. This lower concentration of hCG was not sufficient to promote follicular development or ovulation, and the ovarian AC remained refractory to LH for at least 7 days. Intravenous administration of 75 IU hCG to 33-day-old PMSG- and hCG-primed rats similarly promoted a rapid and permanent loss of luteal AC responsiveness to LH; again, follicles did not mature to a preovulatory state and, in fact, appeared to undergo atresia rather than ovulation. These results indicate that in heavily luteinized ovaries 1) hCG promotes desensitization of rat luteal AC to LH, 2) Desensitization of AC to LH stimulation in corpora lutea is permanent and irreversible, and 3) only under conditions where follicles mature and ovulate and new corpora lutea are formed does total ovarian AC reacqure responsiveness during the subsequent week.


Acute effects of hypothalamic ablation on plasma thyrotropin and prolactin concentrations in the suckling rat: evidence that early postnatal pituitary-thyroid regulation is independent of hypothalamic control.

Hypothalamic ablation was performed at various periods postnatally in animals previously administered propylthiouracil to raise plasma TSH concentrations. There was no significant change in plasma Tsh up to 8 h after hypothalamic ablation in pups 1--4 days old, whereas hypophysectomy of such pups produced a 60% fall in plasma TSH within 4 h. By the 5th postnatal day, hypothalamic ablation produced a 30% fall in plasma TSH within 4 h (P less than 0.05). By the 12th postnatal day and thereafter, the fall in plasma TSH after hypothalamic ablation was not significantly different from that seen in adults, except in 30-day-old rats in which there was a lesser effect of hypothalamic ablation on plasma TSH (P less than 0.01 in comparison to 23-day-old and adult groups). The greatest effect of hypothalamic ablation on plasma TSH was in 45-day-old animals (P less than 0.01 in comparison to adults). No significant change was produced in plasma PRL within 4 h postoperatively at any age. Our data indicate that regulation of TSH secretion in the rat is independent of hypothalamic control until after the 5th postnatal day and is fully developed by day 12. This corresponds temporally with the postnatal rise of plasma TSH, T4, and T3 and hypothalamic TRH to adult concentrations and indicates maturation of the hypothalamic regulation of TSH secretion.


Serum melanocyte-stimulating hormone levels and pituitary melanocyte-stimulating hormone content during pseudopregnancy in the rat.

Pseudopregnancy (PSP) was induced in rats by vaginal stimulation and the levels of MSH in serum and pituitary were examined. One hour after vaginal stimulation, the serum MSH level was increased, and a cyclical variation was observed by the second day of PSP. There were two surges of about 50 pg/ml, one in the morning and the other at night. On the sixth day of PSP, the pattern of serum levels still showed two peaks, but while the nocturnal peak remained at 50 pg/ml, the diurnal value was halved. The pituitary MSH content also underwent cyclical variations; the lowest levels coincided with the peak levels in serum. These observations raise the possibility that MSH is involved in PSP.


Involvement of adenohypophysial dopamine in the regulation of prolactin release during suckling.

Dopamine (DA) was measured in the anterior lobe of the pituitary and median eminence from lactating rats. The effect of pup separation and suckling was studied in order to correlate changes in DA levels with changes in serum PRL. In lactating rats separated from their pups, low levels of circulating PRL were found at 2,4, and 8 h. DA levels in the median eminence showed a decline at 2h; at 4 and 8 h of separation, a significant increase was observed. In the pars distalis, the concentration of DA increased with the length of the nonsuckling interval. Suckling induced a rapid rise in serum PRL levels in rats that were separated from their pups 4 h earlier. Under these conditions, a significant decrease in DA levels in the median eminence and pars distalis was observed as early as 5 min after the onset of suckling; at 30 min, the DA levels were still low. In the situations studied (suckling and pup separation), a negative correlation between serum PRL and DA levels in both the median eminence and pars distalis was always found.


Focal cerebellar hyperthermia: effects on cerebral paroxysmal afterdischarges.

Small area hyperthermia is used to selectively increase cerebellar activity, and, as shown by electroencephalographic tracings of sensorimotor area, focal cerebellar hyperthermia with temperatures limited between 39.5 degrees and 41 degrees C effectively reduces the duration of electrically induced afterdischarges. Additional observations on paroxysms induced in the caudate nucleus which involve the sensorimotor area indicate that these also show shortened durations during focal cerebellar hyperthermia. A comparison of effects on these two forebrain structures is given and some similarities are noted between these results and those reported from previous studies in which electrical stimuli were applied directly to the cerebellar cortex.


An epilepsy workshop for professionals.

This experimental study evaluated the impact of an epilepsy workshop format designed for community professionals who may be involved in the treatment plan and follow-up of patients. The goals of the workshop were to increase the professional's knowledge and comfort in the presence of epilepsy and to effect better delivery of services to patients. To achieve this, a didactic approach to learning was modified to include patient contact and participant involvement in the learning process. A pre- and posttest experimental design was utilized. Those who attended the workshop improved significantly more than the two control groups in their knowledge of and comfort with epilepsy. Following the workshop, there was a marked increase in the referral of school children for possible neurological problems by teachers who had attended. There was a subsequent improvement in the delivery of health care services to such children in this county.


Neurophysiological and neurochemical studies on the action of the anticonvulsant gamma-hydroxy, gamma-ethyl, gamma-phenyl-butyramide.

The effect of gamma-hydroxy, gamma-ethyl, gamma-phenyl-butyramide (HEPB) on afterdischarges produced by hippocampal stimulation in cats was studied. HEPB notably diminished the duration of afterdischarges and in some cats blocked their propagation into the substantia nigra and the amygdala. HEPB treatment also antagonized the enhancement of afterdischarge duration produced by subconvulsive doses of bicuculline, whereas treatment with diphenylhydantoin strongly potentiated this effect of bicuculline. The intracisternal injection of HEPB or gamma-aminobutyric acid (GABA) in mice resulted in a potentiation of strychnine-induced convulsions. On the other hand, neurochemical experiments in mouse brain cortex slices and in synaptosomes demonstrated that HEPB did not affect the high affinity uptake of [3H] GABA, its spontaneous or Ca2+ dependent release stimulated by depolarizing K+ concentrations, and its Na+ independent binding to synaptic plasma membranes.


Evidence for the existence of phosphoenolpyruvate carboxykinase ferroactivator activity in adult and fetal guinea pigs.

Newborn (24--72 h) guinea pig liver cytosolic phosphoenolpyruvate carboxykinase (PEPCK) activity is increased by incubation of the cytosol with the metal salts FeCl2, MnCl2, CoCl2 and CdCl2. FeCl2 at 30 micromol/l concentration is the most effective activator causing a 3.5-fold increase in activity. Purified rat liver cytosolic PEPCK is activated by 30 mumol/l FeCl2 in the presence of liver cytosol of fetal and newborn guinea pigs. These results confirm the existence of PEPCK ferroactivator in the guinea pig which has properties similar to the one found in rat liver. The tissue distribution of ferroactivator activity parallels that of cytosolic PEPCK, being highest in the gluconeogenic organs liver and kidney. Hepatic PEPCK ferroactivator activity can be demonstrated by day 45 of gestation, increasing linearly in specific activity to adult levels at term (65 days). The distribution and development of the ferroactivator is consistent with the hypothesis that it may play a role in the physiologic control of PEPCK.


Lymphatic transport of cellular enzymes from muscle into the intravascular compartment.

In an experimental study, employing anaesthetized dogs, it was investigated whether cellular enzymes from peripheral skeletal muscle get into the circulating blood by diffusion across capillary membranes or by lymphatic transport. In the experimental group 1, the animals were anaesthetized only. The plasma activities of the four enzymes measured--lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, creatine kinase--did not show any mentionable change during a time period of 6 h. In group 2 one hind limb of each animal was moved passively for 1 h. Alanine aminotransferase remained unchanged in plasma, the activities of the three other enzymes increased significantly. In group 3 one hind limb was made hypoxic by clamping the femoral blood vessels for 1 h. No activity changes were observed. When the period of hypoxia was followed by a 1-hour period of passive movement in group 4, the alterations in plasma activities were almost identical to those observed in group 2. In group 5 the experimental procedure was as in group 4, in addition the lymph from the thoracic duct was quantitatively withdrawn. The enzyme activities in plasma revealed a tendency to decrease rather than increase. Lymph flow increased significantly as well as the lymphatic activities of those enzymes which have high intracellular activities in muscle. The results prove, that enzymes from muscle are transported from the interstitial into the intravascular compartment mainly by lymphatic transport. Indications were found that the interruption of blood flow in one hind limb did not result in an enzyme release from muscle cells. It is discussed how changes in lymph flow, occurring during physical exercise for example, affect enzyme activities in plasma.


Glycogen phosphorylase activities in skeletal muscle and heart of genetically dystrophic Syrian hamster.

A simple, rapid and reliable procedure of tissue preparation was devised to estimate glycogen phosphorylase activity in cardiac and skeletal muscle of normal and genetically dystrophic Syrian hamsters of various ages. Total phosphorylase activities of dystrophic skeletal muscle, compared to normal, were reduced. Except for the case of heart from the younger dystrophic animals (45 days old), in which higher phosphorylase activity was noted, hearts from dystrophic hamsters, compared to normal, also showed reduced phosphorylase activities. There were, however, no significances in the ratios of phosphorylase alpha to total phosphorylase between the normal and dystrophic tissues.


A double-labelling radioassay for the determination of 5'-nucleotidase activity.

For the determination of 5'-ribonucleotide phosphohydrolase (EC 3.1.3.5;5'-Nase) in rat liver, a radiochemical double-labelling assay was developed. [14C]-labelled AMP which is hydrolyzed to [14C]-adenosine by 5'-Nase activity is added to crude liver homogenates. After 30 min, the process is stopped and [2-3H]-adenosine added to estimate the loss of [14C]-adenosine during separation by ion exchange column chromatography. The enzymatic reaction was found to be linear in correlation with the enzyme content and the incubation time. The specificity of the reaction was evaluated by addition of beta-glycerophosphate which acts as a competitive inhibitor to eliminate the catalytic effect of non-specific phosphatases, and addition of alpha, beta-methylene adenosine 5'-diphosphate, a specific inhibitor of 5'-Nase; both cause an almost complete suppression of enzyme activity.


Ceruloplasmin elevation and synthesis in rats with transplantable tumors.

In rats with transplantable mammary or hepatic tumors, plasma ceruloplasmin oxidase activity was increased 50--200%. This occurred progressively with tumors weighing 0.3% of body weight of more, and did not occur upon sham operation or implantation of normal tissue. Incorporation of [3H]-leucine indicated a specific enhancement of ceruloplasmin synthesis in the tumor-bearing rats, and a greater state of activation of the enzyme was also observed. The mechanism of the increase in ceruloplasmin levels in rats and humans with cancer thus appears to involve increased synthesis and activation of the enzyme.


Turnover studies of human intestinal brush border membrane glycoproteins in organ culture.

Turnover in organ culture of human small intestinal membrane glycoproteins was measured by the pulse-chase technique, using 14C-glucosamine, 14C-fucose or 14C-leucine as tracers. Apparently, low degradation rates were found for the major high-molecular-weight proteins which co-migrated on SDS-polyacrylamide gels with maltase-glucoamylase, lactase-phlorizin-hydrolase and sucrase-isomaltase enzymic activities. In contrast, an unidentified glycoprotein appearing on gels next to alkaline phosphatase exhibited a higher degradation rate with an apparent half-life of about 30 h, this being similar to the half-life of total glycoprotein as measured in mucosal homogenates. The results obtained with the pulse-chase technique were confirmed by double isotope experiments using 14C-leucine and 3H-leucine as tracers. These findings indicate that in organ culture there is a low basic turnover of human intestinal membrane glycoproteins which co-migrate on gels with known glycosidase enzymic activities.


A method for designing peptide substrates for proteases. Tripeptidyl-p-nitroanilide substrates for subtilisin Carlsberg.

1. The kinetic parameters of 25 peptidyl-p-nitroanilide substrates were investigated with subtilisin Carlsberg as model enzyme. 2. For a series of 12 substrates, the contribution of various side chains to the affinity constant was computed by regression analysis. From these contributions the sequence of a new and better substrate, N-benzyloxycarbonyl-arginyl-norleucyl-norleucyl-p-nitroanilide (Z-Arg-Nle-Nle-Nan) was predicted. The compound was synthesized and assayed. Its calculated 1/Km value, 43.5 mM-1, was in a good agreement with the value of 40.0 mM-1 that was determined experimentally. 3. On expanding the series to 19 substrates, it was found that the productivity of enzyme-substrate binding is influenced primarily by those subsites which have a significantly greater contribution to the affinity constants than others. 4. The additivity principle applied reasonably well for the contribution of individual side chains to the kinetic parameters. This fact suggests that regression analysis can be used for the prediction of the amino acid sequence of better substrates than those already tested, probably not only for subtilisin but also for other proteolytic enzymes.


Protein--non-ionic detergent interaction. Interaction of bovine serum albumin with alkyl glucosides studied by equilibrium dialysis and infrared spectroscopy.

The binding isotherms of bovine serum albumin with octylglucoside and decyl glucoside were determined at 7 degrees C and 25 degrees C at pH 7.4 and ionic strength 0.1 M. The average number of detergent molecules bound was found to increase with increasing hydrocarbon chain length. Competitive binding indicates that alkylglycosides combine with the same sites as alkyl sulphates. Native bovine serum albumin has about 12 and 10 sites for non-ionic ligands at 7 degrees C and about 15 and 13 sites at 25 degrees C for octyl and decyl glucosides respectively. The values for standard free energy changes--delta G0, were calculated from the intrinsic association constants. Fourier-transformed infrared spectroscopy was used to study the effects of alkyl glucosides on the conformation of albumin. The results obtained indicate that there are no significant changes in protein structure.


Norepinephrine-stimulated fatty-acid release and oxygen consumption in isolated hamster brown-fat cells. Influence of buffers, albumin, insulin and mitochondrial inhibitors.

Brown fat cells isolated from adult golden hamsters have earlier been found to respond to addition of the physiological agonist norepinephrine with an increased rate of oxygen consumption and with fatty acid release. Working with these cells, we found the following. 1. The presence of albumin in the incubation medium (phosphate buffer) increases norepinephrine-induced fatty acid release and tends to stabilize the rate of oxygen consumption; bubbling of phosphate buffer with 5% CO2 in air has only a slight effect on fatty acid release. 2. In the presence of albumin, the norepinephrine-induced rate of oxygen consumption is also stable in bicarbonate buffer; it is higher than in the phosphate + CO2 buffer and the brown fat cells have a higher sensitivity to norepinephrine. 3. 20 mM phosphate (as e.g. present in a phosphate buffer) inhibits both fatty acid release and oxygen consumption. 4. Insulin inhibits the rate of oxygen consumption, but only at suboptimal concentrations of norepinephrine. 5. Atractylate inhibits submaximal norepinephrine-induced respiration, indicating that some oxidative phosphorylation takes place in norepinephrine-stimulated brown fat cells. 6. Fatty acid export from brown fat should be regarded as physiologically important.


Conformational energy calculation on the peptide part of murein.

Conformational energy calculations have been carried out on N-acetyl-L-alanyl-D-gamma-glutamyl-L-lysyl-D-alanyl-D-alanine as a model of the peptide moiety of peptidoglycan. Although many conformations were of comparable energy, particular favoured conformations were selected by assuming conformational similarity between the pentapeptide and the tetrapeptide found during biosynthesis subsequent to the cross-linking of the peptide chains in murein. The common feature of these conformations, which include the global minimum of the pentapeptide, is a ring-shaped backbone. The global minimum is stabilised by a hydrogen bond between the -NH group of L-alanine and the -CO group of the penultimate D-alanine. The distance between the D-lactyl group and the side-chain of the diamino acid is about 1.5 nm. The ring-like structures will accomodate chemical modifications that have been observed in peptidoglycan. The present ring-like structure differs considerably from the models proposed as yet. Energetically beta-pleated sheet conformations and a flat 2.2(7) helical structure are not favoured. Furthermore, an alpha helix cannot occur. The suggested new model exhibits no significant relationship to the solid state conformation of beta-lactam antibiotics.


(Photo)chemistry of 5-deazaflavin. A clue to the mechanism of flavin-dependent (de)hydrogenation.

The catalytic action of 5-deazaflavin in the photochemical reduction of flavin and iron proteins [Massey, V. and Hemmerich, P. (1978) Biochemistry, 17, 9--17] is shown to be due to the highly reactive 5-deazaflavosemiquinone. This radical is generated in a complex sequence of reactions, which involves (a) covalent photoaddition of the substrate residue to the deazaflavin, (b) fast secondary photoreaction of this adduct with starting deazaflavin to yield a covalent radical dimer, accompanied by the liberation of the oxidized substrate, and (c) deazaflavin-sensitized cleavage of the radical dimer to the monomers. The structure and properties of this radical (redimerisation or dismutation) and the precursor intermediates as well as the mechanism of the photoreaction are described. Deazaflavins and their natural parent compounds are compared with respect to their different redox behavior and radical stability. The syntheses of 5-deuterated deazaflavins are described and their redox reactions are compared with those of normal deazaflavins.


The complete amino-acid sequence of a trout-testis non-histone protein, H6, localized in a subset of nucleosomes and its similarity to calf-thymus non-histone proteins HMG-14 and HMG-17.

The complete amino acid sequence of a basic non-histone protein, H6, isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells, has been determined. Protein H6, first described by D. T. Wigle and G. H. Dixon [J. Biol. Chem. 246, 5636--5644 (1971)] was extracted with 5% trichloracetic acid and purified by ion-exchange chromatography on carboxymethyl-cellulose (CM-52). Sequence analysis was performed by automatic Edman degradation of the amino terminus of the intact protein and a series of large fragments derived by cleavage with chymotrypsin, staphylococcal protease and with mild acid to cleave at aspartic acid residues. Protein H6 possesses 69 residues and shows considerable similarities to the 89-residue calf thymus HMG-17 protein previously sequenced [Walker, J. M., Hastings, J. R. B. & Johns, E. W. (1977) Eur. J. Biochem. 76, 461--468]. B. Levy W. and G. H. Dixon [Proc. Natl Acad. Sci. U.S.A. 74, 2810--2814 (1977)] have shown that H6 is selectively solubilized when trout testis nuclei (or chromatin) are digested with DNase I under conditions which preferentially hydrolyze that portion of DNA enriched in transcribed sequences [Levy, W. B. & Dixon, G. H. (1977) Nucleic Acids Res. 4, 883--898]. Recently H6 has been located as a stoichiometric component of a distinct subset of trout testis nucleosomes that are complexed with a core nucleosome comprising 140 base pairs of DNA and the inner histones H2A, H2B, H3 and H4 [Levy, W. B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem., in the press].


Globin mRNA precursor. Cross-linking in situ of double-stranded segments with aminomethyltrioxalen.

The globin mRNA sequences present in duck erythroblast nuclei appear under non-denaturing conditions to be associated with heterogeneous nuclear RNA (hnRNA) molecules of various sizes. Under denaturing conditions, however, the bulk of the globin mRNA sequences associated with hnRNA are released as molecules of size close to that of the active globin mRNA. To find out whether hydrogen-bonded structures occur in situ or arise after RNA extraction, nuclei were treated with aminomethyltrioxalen and exposed to ultraviolet light. This treatment generates covalent links between opposite strands of double-stranded nuclei acids, which were visualised by electron microscopy. It appears that, after cross-linking, a fraction of the globin mRNA sequences present in nuclei is associated with high-molecular-weight hnRNA molecules by a link found associated with a band of 0.9 x 10(6) molecular weight approximately. It is suggested that within the erythroblast nucleus, globin mRNA sequences are associated by hydrogen bonds with RNA of high molecular weight. These structures may represent intermediate steps in globin mRNA processing.


Expression of external-surface membrane proteins in differentiated and undifferentiated mouse neuroblastoma cells.

Murine neuroblastoma cultures were labeled externally with the cationic reagent N,N,N-[3H]-trimethylamino-beta-alanyl-N-hydroxy-succinimide ester ([3H]Me3N-beta Ala-NSuc) or with 125I/lactoperoxidase. The cells were labeled in the logarithmic and confluent growth phases as well as in a highly differentiated state following treatment with 2% dimethylsulfoxide. The labeled exterior membrane proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Major changes in the exterior membrane proteins were observed during maturation and differentiation of the cells. Most of these changes were clonal-specific, while others were common to several clones. Two proteins of Mr 55,000 and 65,000 were labeled by both 125I/lactoperoxidase and Me3N-[3H]-beta Ala-NSuc. The level of labeling was dependent on the clonal lines used and the state of the cell maturation. A group of proteins displaying a molecular weight between 150,000 and 200,000 was found to be related to the transition of a culture from logarithmic to confluent growth phases. An additional protein, with an apparent molecular weight of 95,000, was common to differentiated cells of the two inducible clones used. In general the maturation of logarithmic  phase cells into confluent cells resulted in a less complex electrophoretic distribution of the pattern of labeling. After dimethyl-sulfoxide treatment, further reduction in the complexity of the externally labeled proteins was observed.


Induction of the phosphoenolpyruvate: hexose phosphotransferase system associated with relative anaerobiosis in an obligate aerobe.

Arthrobacter pyridinolis possesses alternative transport systems for D-fructose: a respiration-coupled transport system whereby D-fructose transport occurs with concomitant oxidation of L-malate, and a phosphoenolpyruvate: D-fructose phosphotransferase system. Studies of D-fructose uptake by whole cells in the presence and absence of cyanide demonstrate that respiration-coupled transport is used almost exclusively during the first half of logarithmic growth, after which it accounts for only 15-20% of D-fructose uptake. Phosphotransferase levels are low during log phase, peak during late log, and then slowly decline. In a mutant of A. pyridinolis which requires delta-aminolevulinic acid for growth, the growth rate, cell cytochrome content, and activity of the respiration-coupled transport system increased with increasing concentrations of delta-aminolevulinic acid up to 50 microgram/ml. By contrast, phosphotransferase activity was highest in cells grown on limiting delta-aminolevulinic acid. L-Malate, which stimulates respiration-coupled transport, repressed the phosphotransferase system. The respiratory activity and the ability to release CO2 from internalized  d-fructose was consistently low in D-fructose-grown cells. A cyanide-resistant cytochrome, tentatively identified as cytochrome d, appeared in the late exponential phase of growth. Isocitrate lyase activity, required for aerobic growth of this organism, declined markedly during the late exponential phase. Thus the phosphotransferase system is maximally induced, in this obligate aerobe, under conditions of relative anaerobiosis during which metabolism is primarily fermentative.


Hepatocyte chromosomal non-histone proteins in developing rats.

Rat hepatocytes taken a different stages of the perinatal period were partially purified. On sodium dodecylsulphate acrylamide electrophoresis chromosomal non-histone proteins showed important variations in complexity during development. Chromosomal phosvitin kinase strongly increased during the last days of fetal life; it strongly decreased just after birth and increased again for a short time, while the cytosol phosvitin kinase increased more significantly after birth. Chromosomal non-histone proteins prepared at varoius stages were incubated with [gamma-32P]ATP and resolved on polyacrylamide gel. The incorporation was very low in sample taken at the 15th say of the fetal life. A dramatic increase was observed at the 17th day. This incorporation strongly decreased in the samples taken thereafter and it was negligible in proteins from adult rats. The variations in protein kinase and in 32P incorporation into non-histone proteins were correlated with the pattern of appearance of enzymes in this period of life, with cell growth and with the hormone-induced maturation.


Isolation and characterization of a native placental basement-membrane collagen and its component alpha chains.

Native type IV collagen was isolated from human placenta using pepsin solubilisation followed by fractional salt precipitation and chromatogarphic purification. The native preparation was characterised using amino acid analyses, disc gel electrophoresis, segment-long-spacing crystallites and immunological methods. Two component alpha chains were isolated with molecular weights of approximately 95000 and 70000. Cyanogen bromide digests of these chains indicated that they are not related to any of the known alpha chains of interstitial collagens or to the recently described collagen containing alphaA and alphaB chains. They are also not related to one another and are therefore probably fragments of two genetically distinct type IV collagen alpha chains.


Construction in vitro of hybrid plasmids carrying all the EcoRI fragments from lambdarifd18 DNA.

The cloning of all the eleven fragments obtained by degrading the phage lambdarifd18 by the restriction enzyme EcoRI into the plasmid pSF2124 has been achieved: nine of these fragments have been cloned individually, whereas two others have been cloned jointly in the same plasmid. These fragments harbor, in addition of lambda genes, the genes for ribosomal proteins, the elongation factor Tu, the beta and beta' subunits of RNA polymerase and the ribosomal RNAs. The clones carrying the ribosomal RNA genes have been constructed to provide convenient plasmids to determine the primary structure of ribosomal RNAs. Some further genetic manipulations in vitro have been performed on two of them to remove extraneous non-ribosomal RNA gene sequences; the ribosomal genes purified this way have been subcloned into the plasmid pBR322. Other clones of interest have been obtained which carry the genes for the elongation factor Tu, a number of 50-S ribosomal proteins and the beta subunit of RNA polymerase.


Dissociation and reassembly of Limulus polyphemus hemocyanin.

Limulus polyphemus hemocyanin is a 3.3 x 10(6)-Mr protein containing 48 subunits in an assemblage of eight hexamers. The molecule can be dissociated into monomers and dimers at pH 8.9 in the presence of 0.01 M EDTA. These subunits are heterogeneous and can be separated into five zones (I--V) by DEAE-Sephadex chromatography. Reassembly experiments were carried out with varied subunit mixtures, based on different combinations of the five chromatographic zones, in order to study the structural role of the diverse subunits in the eight-hexamer molecule. The reassembly products were analysed by electron microscopy and ultracentrifugation. No structural role for zone I could be found. Zone V and possibly zone II are needed to form structures larger than hexamers. Absence of zone III causes irregular aggregation of hexamers. Zone IV and perhaps zone II are needed to make eight-hexamer molecules from four-hexamer molecules. From these results we conclude that there is a high degree of subunit specificity in the inter-subunit contacts in the native Limulus hemocyanin molecule.


Synthesis of albumin and malic enzyme in wheat-germ lysates and Xenopus laevis oocytes programmed with chicken-liver messenger RNA.

Undegraded, biologically-active, polyadenylated RNA was isolated from chicken liver by a rapid, simple procedure. Liver cells were dispersed mechanically and then broken gently by controlled Dounce homogenization in the absence of detergent or ribonuclease inhibitors. After removing lysosomes and mitochondria by centrifugation, RNA was precipitated at pH 5.2. Polyadenylated mRNA was isolated directly from the detergent-solubilized precipitate by oligo(dT)-cellulose chromatography. The resulting RNA was translated into liver-specific peptides in both the wheat germ lysate and Xenopus laevis oocytes. Translatable albumin mRNA was detected in the liver cytoplasm of both fed 3-week-old chicks and unfed, day-old chicks. Translatable malic enzyme mRNA was only detected in the livers from the fed chicks.


Cobra venom acetylcholinesterase. Purification and molecular properties.

Acetylcholinesterase from cobra (Naja naja oxiana) venom has been purified by affinity chromatography to an homogeneous state, as ascertained by sodium dodecylsulfate/polyacrylamide gel electrophoresis and sedimentation analysis. The specific activity of the preparation was 5000 IU/mg with acetylcholine as substrate. Unlike acetylcholinesterases from insoluble cell structures, the native molecule of the cobra venom enzyme consists of a single polypeptide chain of molecular weight 67,000 +/- 2000. At high enzyme concentrations (greater than 0.2 mg/ml, greater than 1 microM) and ionic strength 0.1 M, it reversibly tends to form  higher-molecular-weight 7.1-S aggregates. Despite the apparent structural simplicity of the venom acetylcholinesterase, the disc electrophoresis and isoelectric focusing experiments revealed that the enzyme exists in a number of forms with a common molecular weight but with different isoelectric points. Neuraminidase treatment did not reduce the number of the forms.


Analysis of the Vicia faba genome by use of restriction endonucleases.

DNA of the broad bean, Vicia faba, was cleaved by the restriction endonucleases endoR . EcoRI, endoR . HindIII, endoR . HincII, endoR . BamI, and endoR . BspRI. Separation in agarose gels of the resulting fragments revealed, in addition to the bulk DNA, an enzyme-specific pattern of bands composed of restriction fragments of 300 to more than 30,000 base pairs in length. Bulk DNA was characterized by an unusual size distribution which significantly deviated from that expected according to the random fragmentation theory. It is argued that the observed distribution is due to the high proportion of repetitive DNA within this species (approximately equal to 75%). In all digests, a class of high-molecular-weight restriction fragments of more than 30,000 base pairs in length was observed which comprised 5-8% of the genome. It showed hybridization with highly repetitive DNA (c0t less than or equal to 2 x 10(-2) M . s) and included a fraction (2-3% of the genome) highly resistant to the activity of all the enzymes tested. The buoyant density in CsCl of this resistant DNA was not different from that of the total DNA (36% dG + dC). In endoR . EcoRI digests, the high-molecular-weight fragment class contained, in addition to the resistant DNA, a fraction of relatively high buoyant density (calculated dG + dC content: 61%) containing cleavage sites for the other enzymes used.


Physarum thymidine kinase. A step or a peak enzyme depending upon temperature of growth.

The variations of thymidine kinase or ATP:thymidine 5'-phosphotransferase (EC 2.7.1.21) during the cell cycle of Physarum polycephalum plasmodia have been studied at two extreme physiological temperatures: 22 degrees C and 32 degrees C. At 22 degrees C the enzyme activity increases near mitosis and stays constant during late S and G2 phases, exhibiting the typical pattern of a 'step enzyme'. But at 32 degrees C thymidine kinase activity goes through a maximum 1 h 30 min after mitosis and decreases during the subsequent phases as expected for a 'peak enzyme'. The rate of enzyme degradation and/or inactivation, measured in the presence of metabolic poisons (cycloheximide or dinitrophenol), appears to follow a simple exponential function with a half-life of approximately 3 h and 1 h at 22 degrees C and 32 degrees C respectively. The effect of growth temperature on the decrease of thymidine kinase activity can account entirely for the differences in the pattern of enzyme activity at the two extreme temperatures. Tentative calculations indicate that the rate of enzyme synthesis is nearly constant during the cell cycle except near mitosis, where it is temporarily increased. The results suggest the existence of a regulatory mechanism able to modulate the rate of synthesis of thymidine kinase during the cell cycle.


The complete amino-acid sequence of the sweet protein thaumatin I.

The primary structure of the sweet-tasting protein thaumatin has been elucidated. The protein consists of a single polypeptide chain of 207 residues. The sequence of the N-terminal part of the chain was determined by sequenator analysis. As the protein contains only one methionine residue, it was possible to deduce the N-terminal sequence of the C-terminal cyanogen bromide fragment by automatic sequencing of the cyanogen-bromide-cleaved, succinylated protein. To arrive at the sequence of the whole protein tryptic and Staphylococcus protease peptides, together with chymotryptic peptides and a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine (BNPS-skatole) fragment were also sequenced. Comparing the amino acid sequence of thaumatin with that of the other sweet-tasting protein, monellin, we have located five sets of identical tripeptides. Since immunological cross-reactivity of thaumatin antibodies with monellin has recently been described, one or more of these tripeptides might be part of a common antibody recombination site and possibly be involved in the interaction with the sweet-taste receptor.


Differential effects of two interferon-induced translational inhibitors on initiation of protein synthesis.

At least two different mechanisms for the inhibition of mRNA translation operate in extracts of interferon-treated L cells. One is mediated by an interferon-induced protein kinase which, when activated by double-stranded RNA and ATP, phosphorylates the small subunit of initiation factor eIF-2. Addition of the purified interferon-induced protein kinase to L cell extracts, strongly reduces the amount of methionyl-tRNA bound to 40-S ribosomal subunits. The second translational inhibition is due to the synthesis of (2'-5')oligo(adenylate) by interferon-induced enzyme E. The oligonucleotide in turn activates a ribonuclease F constitutively present in L cells. Addition of the purified nuclease with its oligonucleotide activator to L cell extracts produces a strong decrease in polyribosome formation and an accumulation of initiation complex. These experiments differentiate the effects of the two interferon-induced inhibitors on mRNA translation.


The phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. Complete tyrosine assignments in the 1H nuclear-magnetic-resonance spectrum of the phosphocarrier protein HPr.

Upon nitration of the phosphocarrier protein HPr three nitrated derivatives of the protein were isolated: mononitrated HPr, dinitrated HPr and trinitrated HPr. Tryptic digestion of the derivatives leads to nitrotyrosine-containing peptides which were isolated and characterized by amino acid analysis. This resulted in the determination of the positions of the nitrated tyrosyl residues in the amino acid sequence. In mononitrated HPr only Tyr-56 was modified, in dinitrated HPr both Tyr-56 and Tyr-37 had reacted with the nitrating agent; modification of all three tyrosyl residues in trinitrated HPr required more drastic reaction conditions. The nuclear magnetic resonance spectra of the three derivatives allowed the assignments of the tyrosine resonances as follows: Tyr-A and Tyr-B with pK values of 10.5 and 11.5 were designated Tyr-56 and Tyr-37 whereas Tyr-C, whose protons are not titratable before denaturation of the protein, was assigned to Tyr-6 in the amino acid sequence. The nitration studies, together with the titration behaviour of the three tyrosines, indicate the topology of the tyrosyl residues to be as follows: Tyr-56 is located at the surface, Tyr-37 is slightly buried, Tyr-6 is deeply buried. The nitrotyrosyl derivatives retain their biological activity.


Base crystallization and base stacking in water.

2H2O solutions of 6-methylpurine and N6,N9-dimethyladenine have been examined by neutron diffraction. Comparison of the scattering from various molar solutions of 6-methylpurine with the scattering from its crystalline precipitates indicated base stacking in solution with a separation of approximately 0.34 nm. In particular at 1.75 M the effective stacking number in 6-methylpurine, as seen by neutron diffraction, was found to be approximately nine. Attempts to observe similar scattering from N6, N9-dimethyladenine were hampered because solutions with a molarity greater than or approximately 0.02 tended to form filamentary crystallites as a function of time. An analysis of the autocatalytic reaction involved in the formation of these dendrites is given. The rate constant is found to be 2.12 +/- 0.05 M-1 h-1. By comparing the diffraction from wet and dry crystallites it is concluded that hydrophilic interactions mediate the aggregation of both bases in water.


DNA-protein interactions in nucleosomes and in chromatin. Structural studies of chromatin stabilized by ultraviolet-light induced crosslinking.

Crosslinking induced by ultraviolet light irradiation at 254 nm has been utilized to investigate the structure of chromatin and isolated nucleosomes. The results presented here imply that the four core histones, as well as histone H1, have reactive groups within a bond length of the DNA bases. In nucleosomes depleted of H1, all of the core histones react similarly with the DNA and form crosslinks. In chromatin, the rate of crosslinking of all histones to DNA is essentially similar. Comparison of mononucleosomes, dinucleosomes and whole chromatin shows that the rate of crosslinking increases significantly with increasing number of connected nucleosomes. These differences in the rate of crosslinking are interpreted in terms of interactions between neighbouring nucleosomes on the chromatin fiber, which are absent in an isolated mononucleosome.


Helix-coil transition of the self-complementary dG-dG-dA-dA-dT-dT-dC-dC duplex.

The helix-coil transition of the octanucleotide self-complementary duplex dG-dG-dA-dA-dT-dT-dC-dC has been monitored at the Watson-Crick protons, the base and sugar nonexchangeable protons and the backbone phosphates by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The melting transition of the octanucleotide monitored by ultraviolet absorbance spectroscopy is characterized by the thermodynamic parameters delta H degree = -216.7 kJ/mol and delta S degree (25 degrees C) = -0.632 KJ mol-1 K-1 in 0.1 M NaCl, 10 mM phosphate solution. Correlation of the transition midpoint values monitored by the ultraviolet absorbance studies at strand concentrations below 0.2 mM and by NMR studies at 5.3 mM suggest that both methods are monitoring the octanucleotide duplex-to-strand transition. The NMR spectra of the Watson-Crick ring NH protons of the octanucleotide duplex have been followed as a function of temperature. The resonance from the terminal dG.dC base pairs broadens out at room temperature while the resonances from the other base pairs broaden simultaneously with the onset of the melting transition. The nonexchangeable base and sugar H-1' protons are resolved in the duplex and strand states and shift as average peaks through the melting transition. The experimental shifts on duplex formation have been compared with calculated values based on ring-current and atomic diamagnetic anisotropy contributions for a B-DNA base-pair-overlap geometry in solution. Several nonexchangeable proton resonances broaden in the fast-exchange region during the duplex-to-strand transition and the excess widths yield a duplex dissociation rate constant for the octanucleotide of 1.9 x 10(3) s-1 at 32 degrees C (fraction of duplex = 0.86) in 0.1 M NaCl, 10 mM phosphate buffer. The 31P resonances of the seven internucleotide phosphates are distributed over 0.6 ppm in the duplex state, shift downfield during the duplex-to-strand transition and undergo additional downfield shifts during the stacked-to-unstacked strand transition with increasing temperature.


Identification of initiation factors and ribosome-associated phosphoproteins by two-dimensional polyacrylamide gel electrophoresis.

A two-dimensional polyacrylamide gel electrophoresis procedure has been used to identify initiation factors rapidly in the high-salt-wash fraction from reticulocyte ribosomes. Initiation factors are identified by relative mobility and by co-electrophoresis with purified factors. A creatine phosphate/ATP/GTP/Pi exchange system is described which has been used to maintain [gamma-32P]ATP and [gamma-32P]GTP at constant specific activity in the cell-free protein-synthesizing system. Phosphorylated proteins associated with the protein-synthesizing complex have been identified using a combination of the two procedures. The salt-wash fraction contains eight major phosphorylated proteins and a number of minor ones. Two phosphorylated proteins are observed to comigrate with two of the three subunits of eukaryotic initiation factor 2 (eIF-2), the initiation factor involved in binding Met-tRNAf onto the 40-S subunit and promoting dissociation of 80-S ribosomes. eIF-4B, one of the proteins involved in binding mRNA to 40-S subunits is also phosphorylated. The remainder of phosphorylated proteins in the high-salt-wash fraction are not previously characterized initiation factors and have not been identified further. Two of the six phosphoproteins associated with the salt-washed ribosomes comigrate with ribosomal proteins; one is the major phosphorylated protein in 40-S ribosomal subunits, the other is an acidic protein.


Quantitative fluorimetric determination of cell-surface glycoconjugates with fluorescein-substituted lectins.

Fluorescein-substituted lectins, which can be used to visualize cell surface glycoconjugates, are shown to be usable in the quantitative determination of the number of receptor sites and of their association constant. The fluorescence measurements of the fluorescein-substituted lectins released from the cell surface with the related inhibitor, give quantitative data in a large range of fluorescein-substituted lectin concentration (0.1 to 100 microgram/ml). Using fluorescein-substituted concanavalin A or [3H]acetyl-concanavalin A, it was found that baby hamster kidney cells (BHK 21, wild-type) bind 10 +/- 2 x 10(6) lectin molecules per cell with an apparent association constant of 1.8 or 1.7 x 10(6) 1 x mol-1, respectively. Using the fluoresceinyl and [3H]acetyl-substituted wheat germ agglutinin, we found 40 +/- 5 x 10(6) sites per cell with an apparent binding constant of 1 and 1.3 +/- 0.3 x 10(6) 1 x mol-1, respectively. When fluorescein-substituted succinyl wheat germ agglutinin was used instead of the unsuccinylated wheat germ agglutinin derivatives, the number of binding sites was reduced 7 times, while the binding constant was very slightly lowered. Concanavalin A derivatives gave monotonic Scatchard plots; on the opposite, wheat germ agglutinin derivatives gave biphasic Scatchard plots suggesting that wheat germ agglutinin binds to two classes of receptors.


The synthesis of chloroplast high-molecular-weight ribosomal ribonucleic acid in spinach.

Illuminated suspensions of chloroplasts isolated from young spinach leaves show incorporation of [3H]uridine into several species of RNA. One such RNA species of Mr 2.7 x 10(6) shows sequence homology with both the chloroplast 23-S rRNA (Mr = 1.05 x 10(6)) and 16-S rRNA (Mr = 0.56 x 10(6)), as judged by DNA/RNA competition hybridization. Leaves labelled in vivo with [32P]orthophosphate in the presence of chloramphenicol accumulate labelled RNAs of Mr 1.28 x 10(6), 0.71/0.75 x 10(6) and 0.47 x 10(6). The 1.28 x 10(6)-Mr RNA shows 80.5% sequence homology with the 1.05 x 10(6)-Mr rRNA and the 0.71/0.75 x 10(6)-Mr RNA mixture shows 76% sequence homology with the 0.56 x 10(6)-Mr rRNA. We conclude that the pathway of rRNA maturation in spinach chloroplasts is similar to that of Escherichia coli.


The synthesis and origin of chloroplast low-molecular-weight ribosomal ribonucleic acid in spinach.

Chloroplasts isolated from young spinach leaves incorporate [3H]uridine into RNA species which co-electrophorese with 5-S rRNA and tRNA, but show very little incorporation into 4.5-S rRNA. Chloroplast 4.5-S rRNA is labelled in vivo after a distinct lag period relative to 5-S rRNA and tRNA. The kinetics of labelling in vivo of chloroplast 5-S rRNA are similar to those of the immediate precursors to the 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs, whereas the kinetics of labelling of the 4.5-S rRNAare similar to those of mature 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs. Chloramphenicol inhibits the labelling of chloroplast 4.5-S rRNA in vivo, and concomitantly inhibits the processing of the immediate precursors to the 1.05 x 10(6)-Mr and 0.56 x 10(6)-Mr rRNAs, but has little effect on the appearance of label in chloroplast 5-S rRNA. DNA/RNA hybridization using 125I-labelled RNAs suggests that chloroplast DNA contains a 2--3-fold excess of 4.5-S and 5-S rRNA genes relative to the high-molecular-weight rRNA genes. Competition hybridization experiments show that the immediate precursor to the 1.05 x 10(6)-Mr rRNA effectively competes with 125I-labelled 4.5-S rRNA for hybridization with chloroplast DNA, and is therefore a likely candidate for  a common precursor to both the 1.05 x 10(6)-Mr and 4.5-S rRNAs.


RNA metabolism and membrane-bound polysomes in relation to globulin biosynthesis in cotyledons of developing field beans (Vicia faba L.).

In cotyledon cells of developing field beans the RNA content per cell does not change in the second half of developmental period 2, whereas globulin biosynthesis continues. The constant RNA content per cell results from an equilibrium between RNA synthesis and degradation. All types of RNA are synthesized until the end of globulin biosynthesis, but poly(A)-containing RNA was preferentially labelled during maximum globulin formation. During stage 2 of seed development of poly(A)-containing RNA fraction represents a discrete peak in the 12--18-S region on agarose gels and corresponds to the peak of poly(A)-containing RNA isolated from polysomes. alpha-Amanitin inhibits selectively the labelling of poly(A)-containing RNA and concomitantly globulin formation. Translation of total poly(A)-containing RNA, free and membrane-bound polysomes in a cell-free wheat germs demonstrates that the globulins are preferentially produced on membrane-bound polysomes and that poly(A)-containing RNA includes the mRNA for both vicilin and legumin.


Immunochemistry of Ii-active glycosphingolipids of erythrocytes.

Fractions of complex glycosphingolipids were prepared from adult, cord, and i phenotype erythrocytes by the method elaborated for the isolation of poly(glycosyl)ceramides. In contrast to poly(glycosyl)ceramides which comprise on the average 30 glycosyl units and about 5 branching points, i.e. 3,6-di-O-substituted galactopyranosyl residues, per mole of glucose, complex glycosphingolipids from cord and i erythrocytes comprise 6 and 15 glycosyl units respectively and only 0.7 branching points. The latter substances exhibited also a high i activity which was not detected in poly(glycosyl)ceramides. Erythrocyte membranes were labeled with radioactive N-acetylgalactosamine (GalNAc) from UDP-GalNAc using a purified A-blood-group gene-specified transfered of GalNAc. It was found that electrophoretic mobilities in dodecylsulfate-gel electrophoresis of all glycoconjugates which accepted GalNAc were increased in i as compared to I membranes. We conclude that the absence of highly branched glycosphingolipids in cord and i erythrocytes as well as the reduction of apparent molecular weights of the glycoconjugates, which are substrates for A-gene-specified transferase of GalNAc, result from a single cause, that is an inadequacy of the biosynthetic process which is responsible for the formation of GlcNAc1 leads to 6Gal structures.