Biogenesis of matrix vesicles in cartilage growth plates.

Although the structure and function of cartilage matrix vesicles have been comprehensively documented in the literature, the method of vesicle production and export to the extra-cellular matrix is less well understood. The existing data are grouped into four general hypotheses of matrix vesicle biogenesis, with a consideration of the relative merits and weaknesses of each postulate. The hypotheses are: a) budding from cellular processes; b) extrusion of preformed structures; c) cellular degeneration and disintegration; and c)subunit secretion and extracellular assembly. Each of these possibilities offers conceptual advantages and disadvantages, so that it is difficult to adopt a monistic stance. Previous notions of vesicle biogenesis have derived from statis morphologic and analytical data, which are necessarily limiting when attempting to draw kinetic conclusions. Radiotracer studies of lipid synthesis and transport in cartilage lead to the conclusion that matrix vesicles in the growth plate are derived from chondrocytes; the present data favor the hypotheses of budding from cellular processes and/or subunit secretion with extracellular self-assembly.


Lipids of matrix vesicles.

The association of lipids with biological mineral formation was first indicated by histochemical observations. This was later confirmed by biochemical studies which revealed that calcium was bound to acidic lipids at sites of early mineral deposition. The morphological locus of initial mineral formation has now been shown to be membrane-enclosed vesicles in the extracellular matrix. Recent studies on isolated vesicles have documented that they are rich in lipids very similar to those previously found to be associated with newly forming mineral. The vesicle lipids are enriched in cholesterol, free fatty acids, sphingomyelin, glycolipids, lysophospholipids, and phosphatidylserine, and depleted in phosphatidylcholine and -ethanolamine. This composition is like that of the plasma membrane of cells and supports electron microscopic evidence that the vesicles arise by budding from the chondrocyte surface membrane. Metabolic studies indicate that formation of the vesicles occurs relatively rapidly (i.e., in less than 6 hours) by biochemical processes involving both lipid synthesis and degradation. In vitro studies suggest that the vesicle lipids may be involved in the initial binding and phase separation of calcium phosphate, as well as its conversion to crystalline apatite; however, final elucidation of the mechanism of action remains to be attained.


Modulation and mediation of the action of the renal kallikrein-kinin system by prostaglandins.

The coupling of the kallikrein-kinin and prostaglandin systems within the kidney may be unique: Prostaglandins mediate some of the actions of kinins and modulate others, while depending on the intrarenal generation of kinin to set their level and type of activity. Thus, not only production of prostaglandins but the functional consequences within the kidney of their enhanced production, as determined by the ratio of PGE2 to PGF2alpha, may be subject to regulation by kinins originating intrarenally.


The renal kallikrein-kinin system and the regulation of salt and water excretion.

Sodium excretion is correlated with kallikrein excretion in man, rabbits and rats on a free sodium and water intake, but not on a constant sodium or constant water intake. The correlation also exists during arterial infusion of angiotensin II, substance P and various vasodilators. During sodium depletion, the stimulation of the renin-angiotensin system causes increased drinking in rats and rabbits. The high angiotensin levels would stimulate kallikrein excretion. The excretion of water and dilution of urine are facilitated by the renal kallikrein-kinin system, even when antidiuretic hormone is high. This negative correlation between urinary osmolality and kallikrein excretion exists during arterial infusion of angiotensin or substance P and various vasodilators. During renal artery constriction, the kallikrein release per minute decreases, but over successive 10-minute periods, the kallikrein concentration in urine rises. This rise is correlated with some recovery in the clearance of rho-aminohippurate and inulin. Since kallikrein is released into renal lymph during saline infusion at a rate that correlates with its release into the urine, it is suggested that the renal kallikrein-kinin system protects the renal vasculature against the constricting action of the renin-angiotensin system. The decreased release of kallikrein (via the lymphatics into the circulation) during renal artery constriction, or decreased renal compliance, would potentiate the hypertensive effect of these procedures which cause increased renin release.


Interaction of mineralocorticoids, renal prostaglandins and the renal kallikrein-kinin system.

Kinins and prostaglandins of the E series (PGE) have the capacity to influence renal hemodynamic and excretory events and may interact intrarenally so as to reinforce one another. Thus, in the isolated Krebs-perfused rabbit kidney we showed that addition of either bradykinin or kininogen to the perfusing fluid augments the release of a PGE-like substance and that aprotinin, a kallikrein inhibitor, reduces the release of prostaglandins evoked by kininogen but not by bradykinin. Moreover, we have observed that deoxycorticosterone, an agent which increases urinary kallikrein, enhances the urinary excretion of PGE-like substance, and that this effect is prevented by simultaneous treatment with aprotinin. These observations and our demonstration that enhanced intrarenal activity of the kallikrein-kinin system, consequent to kininase II inhibition, is associated with renal vasodilation, diuresis, and natriuresis, suggest that a coupling of kinins and prostaglandins intrarenally may be directed towards the facilitation of salt-water excretion. The interdigitation of prostaglandins and the kallikrein-kinin system may thereby constitute the essential operation of a regulatory system in which the complementary actions of these hormones antagonize the sodium retaining effect of mineralocorticoids in those states in which salt-water balance is positive.


The effect of repeated injections of synthetic luteinizing hormone-releasing hormone on the response of plasma luteinizing hormone and follicle-stimulating hormone in young hypogonadotropic-hypogonadal patients.

Sixteen patients, ages 14 to 18, eleven with isolated gonadotropin deficiency and five with sporadic multiple pituitary hormone deficiency, were subjected to a course of five daily intramuscular injections of synthetic luteinizing hormone releasing hormone (LH-RH), 100 mug/day. Before and after the course of intramuscular injections, a rapid LH-RH test (by a one-bolus intravenous injection of 50 mug/sq m) was performed and the responses of plasma LH and follicle-stimulating hormone were measured by a radioimmunoassay method. The patients could be divided into three groups according to the response of the plasma LH to the second LH-RH test: group A, five patients with a significantly higher response of plasma LH to the second LH-RH test: group B, nine patients with a less significantly higher response of the plasma LH to the second LH-RH test; and group C, two patients with very low or no response to either stimulation used in this study. The patients in the three groups may represent different etiologic entities, namely that of a separate hypothalamic lesion, a "mixed" pituitary and hypothalamic lesion, and a "pure" pituitary lesion, respectively. It is concluded that the proposed procedure provides a useful tool for discriminating etiologic groups in patients with abnormal gonadotropic secretion. Recognition of tertiary hypogonadism (primary, pure, hypothalamic gonadotropin-releasing hormone deficiency) is of practical importance in selecting those patients who can benefit from long standing LH-RH therapy.


Circulating hormone levels in a case of granulosa cell tumor.

Hormone studies in a case of granulosa cell ovarian tumor were accomplished preoperatively by measuring the plasma steroids and the gonadotropin response to synthetic lutenizing hormone-releasing hormone. Plasma estradiol and estrone were elevated, but not to levels observed preovulation. The base line follicle-stimulating hormone level was markedly supressed and did not respond to luteinizing hormone-releasing hormone, whereas base line and peak luteinizing hormone values were nearly within the normal range. The clinical finding of copious cervical mucus suggested the diagnosis and prompted preoperative documentation of circulating hormone values.


Trends in return migration to the South.

The rate of return migration to the South rose by nearly 19 percent between the late 1950's and the late 1960's and was an important factor in changing the South's overall migration pattern. But an increase in the rate of return migration was somewhat less important in changing Southern migration than (1) a decline in the rate of out-migration of native Southerners and (2) an increase in the rate at which non-Southern-born persons move to the South. The probability of former migrants returning to the South was over four times greater for whites than for blacks in the 1955-1960 period and three and one-fourth times greater in the 1965-1970 period. Since 1970 the rate of return migration has apparently continued to rise at a faster rate for blacks, but the black rate of return migration is still below the white rate.


Regenerative techniques in periodontal therapy.

The remarkable ability of the periodontium to repair once the long-term etiologic factors have been eliminated is now belatedly being recognized as a key consideration in periodontal therapy. This will force many practitioners interested in comprehensive periodontal treatment to reconsider the classic resective approach to pocket elimination. Resective techniques have been and will probably remain the foundation of periodontal therapy for some time to come. However, the knowledge that is now available concerning the reparative potential of the periodontium opens new treatment avenues for selected patients. In addition, as more research and clinical information becomes available, we can anticipate that the philosophy of repair will play an ever-increasing role in the therapeutic approach to periodontal treatment. This fact should not make the practitioner who is secure with his present treatment philosophy uncomfortable, since the required therapeutic modifications can easily be incorporated into the office routine without confusion. If the clinician then completes each step of the treatment plan with close attention to detail, regeneration will occur in many cases. It should be reiterated that many of the concepts introduced in this paper are based on the authors' clinical observations and are applicable only to select patients. Admittedly, basic research on this subject is sparse, however, the efficacy of this treatment modality has been well substantiated clinically by many therapists. The authors estimate that regenerative therapy is utilized in 10 to 20 per cent of their patients. They believe it is the therapeutic program of choice in many cases showing extensive periodontal destruction, since an innovative and skilled therapist treating a well motivated patient can utilize regenerative periodontal therapy in conjunction with sophisticated restorative dentistry in order to avoid complete dentures.


Correction of periodontal abnormalities as a preliminary phase of oral rehabilitation.

The elimination of etiological factors and the correction of periodontal abnormalities are often a necessary preliminary phase of oral reconstruction. The development of optimal gingival and bony qualities using the surgical procedures outlined when indicated improves the prognosis of prospective abutment teeth and additionally may improve esthetics, retention, and maintenance of a fixed restoration.


Subpopulations of peripheral lymphocytes in juvenile diabetes.

Subpopulations of peripheral lymphocytes were studied in 26 children with insulin-treated juvenile diabetes and in 27 control children of comparable age. T-lymphocytes were quantitated by spontaneous rosette-formation with sheep erythrocytes and B-lymphocytes by indirect immunofluorescence with the use of monovalent, fluorescein-labeled rabbit antiserum specific to the heavy chains of human IgG IgM, or IgA. No significant quantitative difference in subpopulations of the peripheral lymphocytes, T-cells, and B-cells with IgG, IgA, or IgM markers found between children with juvenile diabetes and the control group, although the B-lymphocytes with IgG or IgA markers tended to be higher and those with IgM markers lower in the diabetic than in the control group.


The effect of acute hyperglycemia on gastric emptying in man.

Older work in man with meals of carbohydrates in water has indicated that such meals slow gastric emptying in proportion to their osomolarities. Nevertheless, different carbohydrates have been found to have differing efficacies per milliosmole. One possibility which would explain such discrepancies among carbohydrates is that hyperglycemia induced by carbohydrate absorption itself contributes to the slowing of gastric emptying. To test this possibility, normal subjects were made acutely hyperglycemic with intravenous loads of glucose during the ingestion of various liquid test meals, and rates of gastric emptying of these meals were compared in the same subjects during periods of induced hyperglycemia with rates of gastric emptying under euglycemia conditions. Induced hyperglycemia significantly slowed the rate of emptying of meals containing fat + protein, or protein, but did not significantly alter emptying of meals containing only NaCl. It is concluded that hyperglycemia does exert some effect on gastric emptying, but that these effects of hyperglycemia are variably expressed, depending on the presence of other factors which themselves slow gastric emptying.


Cholestatic effects of Escherichia coli endotoxin endotoxin on the isolated perfused rat liver.

The effects of Escherichia coli endotoxin on the function of the ex vivo perfused rat liver were examined in order to investigate the possible role of circulating endotoxin in the pathogenesis of cholestatic jaundice observed in humans with gram-negative bacterial infections. Endotoxin led to a dose-dependent impairment of bile flow and of sulfobromophthalein (BSP) excretion. The demonstration that indocyanine green excretion was also significantly decreased by endotoxin suggests that the impaired dye excretion was not due to an inhibition of BSP conjugation in the hepatocyte. Analysis of the kinetic data suggested that the effects of endotoxin were on the excretory mechanisms of the hepatocyte. These effects did not seem attributable to endotoxin-mediated changes in perfusate flow since a mechanical reduction in perfusate flow caused no decrease in the excretion of bile or BSP. The results of the present study provide supportive evidence for the hypothesis that circulating endotoxin can adversely affect hepatic function and may contribute to the production of intrahepatic cholestasis seen during bacterial infection. Hepatocyte injury by endotoxin, as judged by the leakage of enzymes from hepatocyte suspensions or from the perfused rat liver, could not be demonstrated.


[Treatment of the anovulatory sterility with LH-RH (author's transl)].

Thirthy one infertile women with different forms of anovulation were treated with LH-RH on various schemes of application: continuous intravenous (i.v.) infusion, intramuscular (i.m.) unique injection, continuous i.v. infusión plus i.m. unique injection, repeated i.v. injection, repeated i.m. injection and estrogens plus unique i.m. injection. On 46 cycles treated, ovulation was obtained in 18 (39.1%), 13 patients ovulated at least one cycle (41.8%) and six became pregnant (19.3%) three during the treatment and three during the first cycle post-treatment. The best results were obtained (63.2%) of ovulation) with the repeated i.v. injection scheme. Though the results obtained with LH-RH in relation to pregnancies, are lower than those obtained with other therapies of anovulation, the fact that we have been sucessful in cases on which other therapies of anovulation had been unsuccessful, the report up to now of only one case of mild ovary hyperestimulation, and the recent development of LH-RH analogs of more powerful and longer action, justifies the continuing of therpeutic assays with this hormone as to find the most effective scheme to induce ovulation.


Jejunal adenylate cyclase activity in human subjects during viral gastroenteritis.

The histopathological changes that occur in the jejunal mucosa of humans infected with the Norwalk or Hawaii agent of acute infectious nonbacterial gastroenteritis ("viral" gastroenteritis) have ben well characterized. The pathogenesis of diarrhea in this syndrome remains unknown; however, recent reports have suggested a possible role for the adenylate cyclase system. In this combined paper, two groups of investigators working independently and employing slightly different techniques report that: (1) there is marked interindividual variation in the apparent specific activity of adenylate cyclase in human jejunal biopsy tissue; (2) such variation can be minimized by expressing enzyme activity as a fraction of maximal that can be stimulated by 10(-2) M sodium fluoride; and (3) adenylate cyclase activity in jejunal mucosa is not increased during diarrhea or illness in human viral gastroenteritis, therefore suggesting no role for the adenylate cyclase system in the pathogenesis of diarrhea in this common clinical entity.


Colonic myoelectric activity in the irritable bowel syndrome.

Although the irritable bowel syndrome has been characterized as an abnormality in colonic motor activity occurring in response to certain stimuli, the etiology of this abnormality is unclear. The purpose of this study was to compare colonic myoelectric and motor activity in normal subjects and in patients with the irritable bowel syndrome. Myoelectric activity was recorded using a bipolar electrode clipped to the mucosa of the rectal and rectosigmoid areas. Basic electrical rhythm (BER), spike potential activity, and intraluminal pressure were recorded in both groups. Two types of BER were observed. The major component of the BER had a frequency of approximately 6 cycles per min, whereas the minor component had a frequency of approximately 3 cycles per min. Although both types of BER were recorded in the two groups, thitable bowel syndrome. The 3 cycles per min activity was present as 44.1 +/- 1.3% of the total BER in the irritable bowel syndrome, as compared with 10.0 +/- 1.6% in the normal group (P less than 0.001). Basal spike potential and motor activity were similiar in both groups. Because it had been demonstrated previously that colonic responsiveness to certain stimuli was increased during the slower frequency BER, it is suggested that the abnormalities in colonic motor response reported in the irritable bowel syndrome may be related to this difference in colonic BER.


Effect of oral metoclopramide on gastroesophageal reflux in the post-cibal state.

The effect of oral metoclopramide (15 mg), AlMgOH (30 ml), and placebo on the cumulative duration of gastroesophageal reflux induced by a protein-rich meal was compared in 15 patients with reflux esophagitis. Oral metoclopramide was found to be more effective than AlMgOH in reducing the cumulative duration of reflux after placebo over a 3-hr period. The same dose of oral metoclopramide increased resting lower esophageal sphincter pressures in all 15 patients for at least 1 hr and prevented gastroesophageal reflux after an intragastric acid load (300 ml of O.1 N HCl) in 8 of 10 of these patients. Oral metoclopramide, however, failed to increase the amplitude of esophageal contractions and acid clearing of the distal esophagus. These findings suggest that oral metoclopramide in the dose of 15 mg may be potentially valuable in the management of reflux esophagitis.


Intraluminal pressures during perfusion of the human colon in situ.

Nine healthy human volunteers underwent colonic perfusion and recording of the intraluminal pressure simultaneously in the right, transverse, and left colon. Isotonic saline was infused into the caecum at various flow rates from 10 to 30 ml per min. During colonic perfusions, pressure waves were simple and distorted only by respiratory artifacts. Their amplitude was large (10 to 44 cm of H2O), of long duration (8 to 68 sec), and of low frequency (0.2 to 1.8 wave per min). The frequency of the waves was very stable and this stability was uninfluenced by the site of recording and the rate of perfusion. A pressure wave in the right colon was always followed within 3 sec by a wave in the transverse and left colon. The frequency and amplitude of the waves increased in all subjects and at all rates of perfusion from right to left colon (P less than 0.001). The wave frequency increased with the perfusion flow rate (P less than 0.001). The relationship between the perfusion flow rate and the waves amplitude was curvilinear (P less than 0.01). It is concluded that in the human colon perfused in situ there is a gradient of motility preventing aboral propulsion. This study suggests that during perfusion, liquids are trapped in the right colon. Data on transport of water and electrolytes, obtained from whole colon perfusion, may in fact reflect events occurring in the proximal part of the large bowel.


Intestinal lipoprotein formation: effect of cholchicine.

The possibility that microtubules might be involved in intestinal lipoprotein formation or secretion was studied by determining the effect of colchicine, a known microtubule inhibitor, on intestinal lipid absorption. The effect of colchicine (0.5 mg per 100 g) in the lymphatic absorption of [14C]oleic acid was studied in rats with indwelling mesenteric lymph cannulas. Colchicine-treated animals showed a marked delay as well as a decrease in the lympatic absorption of [14C]oleic acid. Chylomicrons from colchicine-treated animals showed no difference in apoprotein content when examined on sodium dodecyl sulfate polyacrylamide gels. Micellar lipid absorption was next studied from in situ jejunal loops in animals pretreated with colchicine. Colchicine administration was associated with a 3-fold increase in residual mucosal lipid when compared with controls. Thin layer chromatography of residual lipid demonstrated that residual lipid was largely present as triglyceride, suggesting that the impairment in lipid transport induced by colchicine was at a site distal to triglyceride resynthesis. Electron microscopic examination of intestine from colchicine-treated animals revealed that most residual lipid was present within the endoplasmic reticulum and Golgi in numerous particles the size of chylomicrons (0.2 to 0.4 mu). These results suggest that the impairment in lipid transport induced by colchicine is distal and, in part, may represent an "exit block". These results suggest a possible role for microtubules in intestinal lipid transport. However, further studies are required to demonstrate directly the participation of microtubules in chylomicron secretion.


Effects of fasting and refeeding on secretory granules of the mouse gallbladder epithelium. A quantitative electron microscopic study.

Mouse gallbladder epithelial cells were studied with the electron microscope during fasting and refeeding. Morphometric data were obtained from randomly selected epithelial cells of normal starved (12, 24, and 48 hr) and refed (12 hr) mice. Deprivation of food significantly diminishes the volume density of the mucinous secretory granules by about 70% after 48 hr of fasting. Upon refeeding, this secretory granule parameter increases significantly ( 2.5 times). Stereological measurements were also performed on nuclei, mitochondria, and lysosomes, but no major morphometric changes were observed in these organelles. The findings suggest that a basal secretion of mucin granules occur in the mouse gallbladder, irrespective of the animal's nutritional state and that this discharge during starvation exceeds the formation of new granule material. The findings are discussed in relation to effects of fasting and refeeding on other secretory cell systems.


The clinical value of ascitic fluid culture and leukocyte count studies in alcoholic cirrhosis.

Because of the paucity of studies establishing the accepted leukocyte count is sterile ascites, less than 300 WBC per mm3 with 25% polymorphonuclear (PMN) leukocytes, peritoneal fluid WBC counts and bacterial cultures were studied in 63 consecutive hospitalized patients with alcoholic cirrhosis and ascities. In 58 culture-negative patients the ascitic fluid WBC count range was 28 to 1800 and 50% of counts were greater than 300 WBC per mm3. The percentage of PMN leukocytes ranged from 2 to 98%. Five patients with spontaneous bacterial peritonitis (SBP), proven by positive cultures, had an ascitic WBC count range of 300 to 6320 WBC per mm3 and a PMN leukocyte range of 20 to 76%. Physical findings did not allow clear separation of the two groups. Anaerobic organisms were not found. It is concluded: (1) differentiation of SBP from sterile ascities relies on prompt ascitic fluid bacteriology; (2) the improved survival of 40% of SBP patients in this study may be related to an increased awareness of the entity and early treatment.


Pasteurella multocida peritonitis in hepatic cirrhosis with ascites.

Two patients with spontaneous Pasteurella multocida peritonitis in association with cirrhosis and ascites are described. Both patients had close contact with domestic animals, but neither had been bitten. Both patients died, but only one as a result of his infection. We are aware of only one previous report of cirrhosis with spontaneous peritonitis caused by this organism. P. multocida should be considered as a possible cause of peritonitis in cirrhotic patients with a history of close contact with animals, particularly cats.


Spontaneous bacterial empyema in cirrhosis.

Spontaneous bacterial peritonitis in decompensated cirrhotic patients develops when bacteria invade preexising ascites. It is probable that other transudates such as pleural effusions can be afected in a similar manner. This paper describes a cirrhotic patient with long-standing pleural effusion in whom Escherichia coli caused a spontaneous bacterial empyema. The ascitic fluid was sterile.


Immunofluorescence studies of gastric heterotopia of the small intestine in Crohn's disease.

A patient with long-standing Crohn's disease of the large and small intestine was found to have extensive gastric metaplasia of the ileum. Most metaplastic glands were of the pyloric type, but numerous oxyntic glands with parietal and chief cells were also seen. By immunofluorescence the chief cells contained both the group I and group II pepsinogens, while the pyloric gland cells contained only the group II pepsinogens. Gastric-containing or other endocrine cells were not detected in the metaplastic pyloric and oxyntic glands. The latter findings are consistent with the concept expressed by Pearse that the endocrine and exocrine cells of the gastrointestinal mucosa may originate from different precursor elements during embryogenesis.


[Organization of mass-screening for early detection of cervical cancer and its precancerous conditions. I. report: Demonstration of a computer assisted mass-screening program (Model Rostock) (author's transl)].

Gynecological mass-screening leads to a decrease of prevalence and incidence of cervical cancer and precancerous conditions. In Rostock a program for early detection of cervical cancer was developed. Organization, application of computer in this model and the expenses of a computer assisted mass-screening-program are discussed. In order to facilitate comparison between various centers, a generally accepted classification and terminology of colposcopic, cytologic and histologic findings are essential. First epidemiological results of Rostock's women are demonstrated to characterize the screened population.


[Organization of mass-screening for early detection of cervical cancer and its precancerous conditions. II. report: Practical experience with a computer assisted mass-screening program (Model Rostock) (author's transl)].

Two years experiences with a computerassisted program for early detection of prestages and early cervical cancer in Rostock-city are reported. From 43.000 women invited to take part in the examination, 27.028 = 65% finally cooperated. Pathological Pap-smears were found in 134 cases (= 0,52%). Histological examination in 51 cases (= 0,2%) showed 1 Erosio vera (false positive), 9 severe dysplasia, 30 carcinomata in situ, 3 early invasions (microinvasive cancer), 3 microcarcinoma and 5 macrocarcinoma. Accessory findings were seen in 10,2%. A good organized mass-screening-system with unique nomenclature, diagnostic and treatment of cervical cancer and its prestages gives the best supposition to cut down its rate of incidence.


[Report on 21 uterine ruptures and the influence of single row uterotomy stitching on rupture prevention (author's transl)].

Wound closure by 1 row of interupted sutures provides optimal healing conditions. As a result scar tissue formation is cut down. This general surgical principle is valid in stitching an uterotomy as well. Increasing frequency of cesarian section could end up in a rising number of scar ruptures. This drawback for a subsequent pregnancy should be met by the single row technique. It was applied in 1434 women from 1962 to 1974. All of them had lower segment cesarian sections. Only 3 harmless ruptures occurred in this group. The clinical course of 12 scar ruptures after terraced uterotomy closure was less favourable. In 6 cases a pregnant uterus ruptured without previous operation. Clinical results are compared. This backs the technique practised. In 5 patients the ruptured uterus could be repaired in the same way as in cesarian section. Hysterectomy, however, was the usual treatment. The risk of rupture after classical cesarian section is lowered by the technique described. This applies to the surgical treatment of uterus duplex as well.


Integration, at hag or elsewhere, of H2 (phase-2 flagellin) genes transduced from Salmonella to Escherichia coli.

A fla mutant of E. coli K12 was given fla+ and H1-i by phage P1kc cotransduction from S. typhimurium, then made Fla- by transduction of ah1 from S. typhimurium. Motile clones expressing a Salmonella phase-2 antigen, e,n,x or 1,2, were obtained from the K12 i ah1 (therefore Fla-) line by P1kc transduction of flagellin-specifying genes, H2-e,n,x or H2-1,2, from Salmonella donors. Of eighteen such transductants sixteen failed to show phase variation, and on transduction back to Salmonella each structural gene for a phase-2 flagellin (or at least for its antigenically determinant part) now behaved as an allele of H1, presumably in consequence of incorporation in the hag region of the K12 recipient, in place of H1-i ah1. The e,n,x- and 1,2-specifying genes were shown to have been integrated in the K12 chromosome without the linked H1-repressor gene or the adjacent vh2 gene (controlling rate of phase-variation) and they responded to the repressing activity of an H2 allele elsewhere in the cell, in this respect resembling H1 alleles of Salmonella or hag alleles of E. coli. Two K12 e,n,x transductants had flagellin-specifying genes which when transduced back to Salmonella were integrated at H2; they are inferred to have resulted from integration of H2-e,n,x in the K12 chromosome elsewhere than the hag region. These two clones showed phase variation, between a Fla+ phase, with antigen e,n,x, and a Fla- phase (with e,n,x determinant in the nonactive state and the determinant of antigen i inactivated by ah1). The two integrated e,n,x genes when in the "active" state retained the ability to repress expression of exogenote H1 alleles, which indicates that the closely linked H1-repressor gene also was integrated. One of the two exceptional transductants derived its e,n,x gene from a Salmonella donor with the linked vh2- gene, which in Salmonella almost entirely prevents change of phase, and transduction of this e,n,x gene back to Salmonella recipients proved that vh2- had been incorporated into the E. coli chromosome along with the e,n,x determinant and the H1-repressor gene. The high frequency of change of phase (Fla+ in equilibrium Fla-) in the K12 e,n,x vh2- transductant concerned suggests that vh2- fails to prevent frequent change of state of the phase-determined part of H2 when vh2- and H2 are incorporated in the E. coli chromosome.


[Genetico-biochemical study of acid phosphatases in Saccharomyces cerevisiae yeast. V. Genetic control of regulation of acid phosphatase II synthesis].

Regulation of exocellular enzyme acid phosphatase 2 synthesis is studied. 21 mutants with consitutive synthesis of this enzyme are obtained by UV-irradiation. All mutants were recessive and were distributed among 3 complementation groups ACP80, ACP81, ACP82. Two groups, ACP80 and ACP81 corresponded to two different genes, which showed no linkage with ACP1, ACP2 and PHO1 genes. The type of synthesis of acid phosphatase 2 in strains acp1 acp80, acp1 acp81, acp2 acp80, acp2 acp81 is determined, and a conclusion is made about the participation of ACP2 gene in the regulation of acid phosphatase 2 synthesis. It is shown that some mutations in PHO1 gene, which block the activity of acid phosphatase 1, influence the activity and regulation of acid phosphatase 2.


[Veterinary genetics].

At the age of scientific and technical progress and of industrialization of animal husbandry neither theoretical nor applied science can dispense with the data of the veterinary genetics. Unfortunately this branch of science does not receive the attention it deserves. The following three problems have to be solved by the veterinary genetics: (1) the investigation of the relationship between the heredity and the pathology of animals; the examination of the mechanism of pathology at the molecular and organism genetic levels; (2) the elaboration of the methods of genetic diagnostics; (3) the search for scientifically-substantiated directions in breeding of animal breeds highly resistant to diseases. The main attention will be paid to the investigation of the mechanisms ensuring the natural resistance of animals to certain definite diseases. The establishment and development of veterinary genetics will exert a favourable influence on the progress of biological sciences. Its data will be indispensable both for the theory and the practice of agriculture.


Fetal circulation times and their implications for tissue oxygenation.

In an effort to understand O2 delivery to fetal tissues, we measured circulatory transit times using dye dilution methods in near-term fetuses of 19 ewes. Times from dye injection to peak response (+/-1 sec) were: femoral vein to carotid artery 2.2, to femoral arter 2.6, to umbilical artery 3.7; jugular vein to carotid artery 1.7, to femoral artery 2.6; umbilical vein to carotid artery 1.9, to femoral artery 3.4, to umbilical artery 5.1, and umbilical artery to umbilical vein 5.4 sec. These rapid transits suggest that changes in placenta and peripheral tissues will quickly affect one another. The time for a complete circuit of blood through the fetus was 12.6 (+/-1.0 SEM) sec. Following dye dilunjections into an umbilical vein double peaks, separated by 4.9 sec, were recorded in peripheral arteries (44 observations), and the divergence localized to the ductus venosus. Based on relative areas under the two peaks, ductus flow averaged 0.425 +/- 0.052 SEM of total flow returning from the placenta with the remainder taking a slow course through liver parenchyma.


[The hypermobile skin- and soft-tissue tube in thumb reconstruction].

Reconstruction of an amputated thumb using a bone graft covered by a tubed skin pedicle gives good results if sufficient vascular supply and sensory innervation is transferred to the reconstructed thumb by a neruovascular island pedicle flap and if the skin and soft tissue tube is fixed well enough to the bone graft. Hypermobility of the skin prevents firm and accurate gripping. Fixation of the skin tube to the bone is best achieved at the time of neurovascular pedicle transfer. At the margin of the skin incision a strip of dermis is left and screwed to the periosteum. This method has been described by CHASE. If hypermobility of the skin persists after the neurovascular pedicle has been transferred the skin can be fixed to the bone using strips of fascia, dermis or lyodura. The operative technique of this procedure is described in detail.


[The thenar flap].

In covering amputations of fingertips several methods, including free grafts and local flaps have proven useful. When the amputation is at a more proximal level near the cuticle closure of the wound is often accomplished by further shortening of the distal phalanx and eradication of the nail matrix in order to avoid a claw-like deformity of the nail. This results in a unpleasant stump of poor prehensile quality. Therefore attention is called to the distal thenar flap which offers all the advantages of the palmar flap e. g. adequate subcutaneous tissue for reconstruction of a fingertip, good texture and colour match and a great potential for recovery of sensation. To avoid permanent stiffness of the PIP joint the flap is fashioned well distally on the thenar eminence with the base on the radial side. Immobilisation is secured by means of a plaster cast with the thumb in full palmar abduction and the MP joint of the recipient finger in full flexion. This leaves the PIP joint in but moderate flexion. The base of the flap is severed at two weeks.


[Reestablishment of sensation in the distal phalanges using innervated flaps or grafts].

The authors describe a full thickness skin graft taken with its cutaneous nerve for the coverage of skin defects in the area of the distal phalanx for defects involving the pulp. The defect was covered by a cross-finger flap; its collateral nerve was raised with it and then anastomosed with the injured digit's nerve, using the operating microscope. Either of the above methods permitted improved sensory return.


[Central action of beta-phenylethylamine derivatives. (4) Effects on spontaneous motor activity and body temperature of beta phenylethyamine derivatives injected into the brain in reserpine pretreated mice].

Effects on spontaneous motor activity and body temperature of beta-phenylethlamine derivatives injected into the cerebral ventricles in reserpine or reserpine and isocarboxazide pretreated mice were investigated with the following results. 1) Each injection of tyramine (40 mug) and dopamine (40 mug) increased the spontaneous motor activity measured by the photo-cell counters method in reserpinized mice. 2) Each injection of tyramine (40 mug), dopamine (40 mug) and beta-phenylethylamine (40 mug) increased the spontaneous motor activity measured by both the wheel cage and photo-cell counters methods in reserpine and isocarboxazide-pretreated mice, but noradrenaline (20 mug) and isoproterenol (80 mug) did not increase the spontaneous motor activity as determined by both methods. 3) The injection of tyramine (40 and 80 mug), dopamine (10 and 40 mug) and p-octopamine (40 mug) increased the body temperature in reserpine and isocarboxazide pretreated mice. 4) Tyramine, dopamine and p-octopamine caused a marked increase in the body temperature as compared with control injection in reserpine and isocarboxazide-pretreated mice, whereas isoproterenol had no influence on body temperature. Our results suggest that beta-phenylethylamine derivatives have different effects in reserpinized and non-pretreated states.


Elimination of lethal and pre-mutational DNA lesions during the photoreactivation of UV-irradiated Escherichia coli.

The comparison of the frequency of trp+ revertants of Escherichia coli B/r Hcr+ thy trp after UV-irradiation on the one hand and after UV-irradiation plus photoreactivation on the other showed that both photoreversible pyrimidine dimers of the cyclobutane type and the non-photoreversible DNA lesions cause, at equal lethal effects, also trp+ reversions with the same efficiency. If lethal, the pyrimidine dimers may thus be conceived as primary pre-mutational lesions.


Biochemical aspects of cell death.

Studies of cell injury and death at the molecular and cell-biological level will help to provide methods for the reconstruction of the course of events between the occurrence of primary pathology and the time of investigation. The general role of component turnover, energy metabolism and lysosomes in cellular injury is briefly described.


Insulin secretion and glucose uptake by isolated islets of the hamster. Effect of insulin, proinsulin and C-peptide.

Isolated pancreatic islets of normal hamsters were perifused either in a closed or in a open system. When the buffer was recirculated and the endogenous insulin was allowed to accumulate, the islets secreted significantly less insulin than when the system was open and the endogenous insulin was washed away. The addition of monocomponent insulin or of proinsulin to the perifusion buffer significantly decreased insulin secretion. The inhibitory action of proinsulin was significantly greater than that of monocomponent insulin. C peptide had no effect. When pancreatic islets were incubated in a fixed volume of stationary buffer containing unlabeled glucose (1.0 mg or 3.0 mg/ml) and glucose-U-14C (1.0 muC/ml), the amount of insulin secreted and the 14CO2 produced by each islet decreased progressively as the number of islets in the sample increased. Under these conditions, the concentration of insulin required to inhibit insulin secretion increased with the concentration of glucose in the medium. Proinsulin did not alter the incorporation of leucine-4.5(-3). H into total extractable insulin (insulin + proinsulin). Thus, insulin and proinsulin appear to inhibit insulin release, but not insulin synthesis.


C- and N-terminal specific LH-releasing hormone (LRH) radioimmunoassay.

A highly sensitive and specific LRH radioimmunoassay is described. Three antisera were compared and characterized. High specificity was shown by 2 of them in that both C- and N-terminals were required for binding when testing LRH analogs. A lower detection limit of about 1 pg could be obtained using a simple organic solvent precipitation method for separation of bound/free antigen. Good correlation with the Ramirez-McCann bioassay was obtained, and rat median eminence extracts contained a material immunologically similar to synthetic LRH as indicated by parallel inhibition curves. Direct measurement of radioimmunoassayable LRH in plasma can lead to misleading results due to nonspecific interference of plasma factors. After employing a simple deproteinization method, rat plasma was found to contain less than 1-3 pg LRH/ml. Rat median eminence extracts contained approx. 7 ng LRH.


Interactions between the metabolism of L-leucine and D-glucose in the pancreatic beta-cells.

Beta-Cell-rich pancreatic islets microdissected from obese-hyperglycemic mice were used to study interactions between the metabolism of L-leucine and D-glucose. L-leucine reduced the islet content of aspartic acid whereas D-glucose, when added to L-leucine-incubated islets, increased the contents of aspartic acid and gamma-aminobutyric acid (GABA). D-glucose also increased the incorporation of L-leucine carbon into aspartic acid, GABA and glutamic acid suggesting stimulation of a malate shuttle mechanism. When expressed per mole of the individual amino acids, the incorporation of L-leucine carbon into GABA was 2.5-4 times higher than into glutamic acid indicating intracellular compartmentation of the latter amino acid. Both L-leucine and D-leucine stimulated 14CO2 production from 14C-labelled D-glucose. L-leucine did not affect 3H2O production from tritiated D-glucose. The present data do not indicate a role of other amino acids or D-glucose in L-leucine-stimulated insulin release.


Effects of somatostatin on exocrine and endocrine pancreatic function stimulated by intestinal hormones in man.

To evaluate the action of somatostatin on exocrine and endocrine pancreatic function, synthetic somatostatin (GIF) was administered (intravenous bolus of 300 mug followed by a constant 60-minute infusion, 5 mug/min) to 17 normal subjects. The secretin-induced volume and total bicarbonate contents of the duodenal aspirate were not affected whereas the bicarbonate concentration was significantly diminished. GIF reduced decisively the pancreatic enzyme secretion stimulated by pure (99%) cholecystokinin-pancreozymin. After the GIF infusion was stopped, a significant rise in enzyme secretion was observed. The secretin-induced insulin release was almost completely suppressed. Because GIF can be extracted in large quantities from pancreas, these data suggest that somatostatin may play a physiological role in the regulation of the secretory processes of this organ. Furthermore, GIF may be a useful adjunct in the treatment of acute pancreatitis.


Isolation and characterization of type III collagen from chick stain.

A collagen species was isolated from the skin of 8 week-old chicks by pepsin treatment at 4 degrees C and was identified as type III collagen by analogy to human and calf type III collagen. Like human type III collagen, the type III collagen molecule from chick skin is composed of three alpha1 (III) chains which are linked together by disulfide bridges. The amino acid composition of [alpha1 (III)]3 purified by CM-cellulose chromatography and agarose-gel chromatography includes two cysteine residues per chain, and the hydroxyproline/proline ratio is greater than 1.0. The cyanogen bromide peptide pattern of [alpha1 (III)]3 on sodium dodecylsulfate acrylamide gel electrophoresis differed from that of alpha1 (I), alpha1 (II) and alpha2 chains. Segment-long-spacing crystallites from chick skin type III collagen showed the same cross striation pattern as segment-long-spacing crystallites from calf skin type III collagen. Unlike human type III collagen, type III collagen from chick skin was highly soluble in 1.0M NaCl, 0.05M Tris/HCl buffer, pH 7.5, and could not be separated from type I collagen by fractional salt precipitation. On CM-cellulose chromatography, [alpha1 (III)]3 molecules from chick skin eluted in the same position as alpha2 chains, in contrast to human [alpha1 (III)]3, which elutes in the position of beta12.


Determination of the complete amino acid sequence of protein S21 from Escherichia coli ribosomes.

Protein S21 was digested with trypsin before and after maleylation, with chymotrypsin, thermolysin and a glutamyl-specific protease. The resulting peptides were isolated and their amino acid composition determined. The amino acid sequences of selected peptides were determined either by the manual subtractive Edman method or by the dansyl-Edman procedure. Additional information was obtained from the automatic Edman degradation of the whole protein in a modified Sequenator. All these results combined yielded the sequence shown in Fig. 1. Protein S21 consists of 70 amino acids (Asp1,Asn2,Thr3,Ser2,Glu8,Pro3,Gly1,Ala9,Val6,Cys1,Ile1,Leu4,Tyr2,Phe3,His1,Lys9 and Arg14). It contains neither Met nor Trp. The molecular weight amounts to 8359. Clustering of basic amino acids is observed in five regions. We also include a prediction for regions with alpha-helices and with beta-sheets.


Lipid-lipid and lipid-protein interactions as studied with a novel type of fluorescent fatty acid and phospholipid probes.

A novel fluorescent-labelled group of fatty acids and phospholipids has been applied to determine phase transitions in liposomes by fluorescence intensity and polarisation measurements. The chromophore of these amphiphilic lipids proved to be very suitable to demonstrate temperature-dependent lipid-lipid interactions. Liposomes from 1,2-dipalmitoyl-3-sn-glycero-phosphoethanolamine and from lipids isolated from membranes of E. coli K 1062 mutant grown on elaidic acid were used in these studies. These probes also made it possible to observe conformational changes in membrane proteins in isolated plasma membranes from this mutant. The changes in protein conformation were dependent on structural changes in the lipid phase.


Mechanisms of natural resistance to trypanosomal infection. Role of complement in avian resistance to Trypanosoma cruzi infection.

The natural resistance of chickens to Trypanosoma curzi infection and the capacity of their sera to lyse blood (trypomastigote) forms of the parasite in vitro were found to be complement-dependent phenomena. Parasites given intravenously to decomplemented chickens were detectable in their bloodstream for at least 24 h post-infection, whereas in untreated animals they became undetectable after 1 min (and destroyed flagellates were observed). One millilitre of serum had the capacity to lyse as many as 10-30 X 10(6) organisms. The lytic activity of serum in vitro was not impaired in chickens that had been immunosuppressed by four different procedures and was present in the absence of antibodies. In vitro lysis of T. cruzi by either normal or antibody-free chicken sera occurred in the absence of calcium ions but required magnesium ions, indicating that complement was activated via the alternative pathway. Administration of normal chicken serum to mice infected with T. cruzi provoked a marked decrease in their parasitaemias.


Hypoxanthine-guanine phosphoribosyl transferase deficiency.

In man congential lack of enzyme of the purine salvage system, hypoxanthineguanine phosphoribosyl transferase (HG-PRT E.C. 2.4.2.8), is mostly accompanied by a picture known as the Lesch-Nyhan snydrome.  The degree of deficiency may vary from zero to a few percent of normal activity but a correlation between the severity of HG-PRT deficiency and the clinical picture has not been observed, no more than a correlation HG-PRT deficiency and neurological dysfunction.  But individuals with undetectable HG-PRT activity but without the Lesch-Nyhan syndrome have been described.  Patients with partial HG-PRT defiency have clinically distinctive findings.  Sometimes mild neurological abnormalities are observed.  Because of marked overproduction of ric acid severe gouty arthritis and renal dysfunction are often encountered in both complete and partial deficiency.  There is considerable molecular heterogeneity in HG-PRT deficiency in man.  Mutant ebnzymes may exhibit different kinetic and electrophoretic properties, indicating that hterwe might be a mutation on the structural gene coding for HG-PRT.  Lack of HG-PRT disturbs purine interconversions profoundly.  In addition to an important function of HG-PRT in the uptake of the purine hypoxantine and guanine into the cell, the effective uptake of inosine, guanosine and adenosine also seems to be dependent on HG-PRT...


Antibody formation in mouse bone marrow. V. The response to the thymus-independent antigen Ecsherichia coli lipopolysaccharide.

The occurrence of plaque-forming cells (PFC) in mouse bone marrow was studied during primary and secondary responses to the thymus-independent antigen Escherichia coli lipopolysaccharide (LPS). Anti-LPS responses were induced by various doses of LPS. During the primary response, doses of 1 and 10 mug LPS intravenously (i.v.) were found to evoke a distinct PFC response in both spleen and bone marrow. The spleen contained the majority of PFC until about 5 days after immunization. During the course of the reaction the number of PFC in the bone marrow rose to a level which equalled or surpassed the level in the spleen. LPS doses of 0-001, 0-01 and 0-1 mug i.v. only induced a PFC response in the spleen. Apparently there is a minimal threshold dose of LPS of about 1 mug for PFC to appear in the bone marrow. The secondary response was studied in mice primed with 1 mug LPS i.v. and boosted with either 0-001, 0-1 or 10 mug LPS i.v. 3 months later. After each dose tested the PFC activity in the spleen was several times higher than during the primary response. As was observed in the primary response doses of 0-001 and 0-1 mug LPS i.v. did not evoke a PFC response in the bone marrow. After boosting with 10 mug of LPS i.v. a significant PFC response was found in spleen, bone marrow, thymus, lymph nodes, Peyer's patches and blood. From about 5 days after the booster injection the number of PFC in the bone marrow exceeded the total number found in all other lymphoid organs. The results are discussed in relation to the bone marrow PFC response to the thymus-dependent antigen sheep red blood cells. To this antigen a clear PFC response in the bone marrow is found only during the secondary response.


Partial characterization of an immunoenhancing factor from allogeneic human lymphocyte cell lines.

The mixing of two histoincompatible human lymphocyte cell lines generated the release of a soluble factor which was capable on non-specifically enhancing the in vitro immune response of normal mouse spleen cells against sheep erythrocytes. The mediator was secreted into the supernatant of the allogeneic cell cultures within 24 h of cultuvation. The human enhancing factor (HEF) must be added to assay cultures on day 2, of a 5-day culture period, for its activity to be manifest. HEF was resistant to DNase, RNase and heating at 56 degrees for 30 min, but was inactivated by exposure to protease or elevated temperature (80 degrees for 30 min). The molecular weight of HEF, purified by ammonium sulphate fractionation, followed by Sephadex gel filtration, DEAE-cellulose chromatography and SDS-polyacrylamide gel electrophoresis, was approximately 38,000 Daltons.


Experimental neonatal colibacillosis in cows: immunoglobin classes involved in protection.

Pregnant cows were vaccinated with one of four vaccine preparations to induce passive immunity in their offspring against a homologous oral challenge with Escherichia coli strain B-44. Quantitative assays of specific antibody in colostral whey from both immunized and nonimmunized dams revealed that immunoglobulin G, immunoglobulin A (IgA), and immunoglobulin M (IgM) with anti-O (somatic) activity were present in whey of all dams tested, whereas a marked deficiency of IgA and IgM anti-K immunoglobulin was noted in the whey from control dams only. The degree of scours (neonatal colibacillosis) induced by oral challenge was evaluated clinically and reported by a semiquantitative scour index as 0 to 4+. Calf scour indexes showed an inverse relationship to the frequency of occurrence and to the levels of IgA and IgM in whey of dams vaccinated with killed vaccine, live vaccine, and culture supernatant, and from nonvaccinated controls. The data strongly suggested that IgA and colostral IgM anti-K immunoglobulins were important in passive immunity in experimental neonatal bovine colibacillosis.


Indigenous microorganisms prevent reduction in cecal size induced by Salmonella typhimurium in vaccinated gnotobiotic mice.

Germfree CD-1 mice challenged by the oral route with Salmonella typhimurium had ceca that were abnormal in appearance and reduced in size compared to those of germfree controls. Similarly, germfree mice injected with heat-killed S. typhimurium or gnotobiotes associated with three indigenous microbes (Lactobacillus, Bacteroides, Clostridium), and subsequently challenged with S. typhimurium also had small ceca. By contrast, gnotobiotic mice that had been both injected with the heat-killed S. typhimurium and associated with the three indigenous microbes before challenge with S. typhimurium had ceca similar in size and appearance to germfree mice. Thus, indigenous microorganisms could interfere with the mechanism by which the pathogen induced the decrease in cecal size, but could do so only in mice injected with heat-killed bacteria. This phenomenon suggests synergism between the interference effected by the indigenous bacteria and the resistance mechanisms of the animal.


Adaptation of an osmotically fragile L-form of Streptococcus pyogenes to physiological osmotic conditions and its ability to destroy human heart cells in tissue culture.

An osmotically fragile L-form of Streptococcus pyogenes, type 12, was quickly rendered osmotically stable by decreasing the sodium chloride content of the growth medium and with the temporary use of oleic acid. The change from osmotic fragility to stability was accompanied by changes in cell yield, generation time, saturated/unsaturated fatty acid ratio of the membrane, and cytoplasmic protein composition. Finally, this resulting osmotically stable L-form survived and was capable of rapidly destroying Girardi human heart cells in tissue culture.


Specificity of secretory antibodies to bacterial immunogens.

The present investigation examined the specificity of the salivary immune response of axenic and conventional mice to topically administered Salmonella typhi, S. gallinarum, and S. typhimurium. Specific antibacterial antibodies were determined by passive hemagglutination and bacterial agglutination. Reciprocal antibody titers up to 320 were detected in saliva from mice immunized and assayed with homologous antigens. Antibodies to heterologous immunogens exhibited lower mean titers of 10 to 20 under identical conditions. High concentrations of specific antibodies to the somatic (O) antigen were detected in the saliva of mice administered these microorganisms; however, no significant differences in serum antibody levels were detectable after oral immunization. Only low levels of specific antiflagellar (H) antibodies were demonstrated in the saliva of immunized mice, whereas mean reciprocal titers of 20 were observed in the serum. Antibodies to the Vi antigen of S. typhi were detected in the saliva and serum of only those mice administered formalin-treated S. typhi. Examination of the classes of antibody elicited by these organisms indicated that immunoglobulin A (IgA) was the predominant class in saliva against the O antigens. The salivary response to the H antigens was comprised of both IgG and IgA, whereas the specific serum immunoglobulins were consistent with a primary humoral immune reaction. Local antibodies formed in response to the Vi antigen were exclusively IgG. Serum immunoglobulins produced after peroral administration of the somatic and virulence antigens were limited to the IgM class.


Experimental neonatal colibacillosis in cows: serological studies.

Serological studies of passive immunity in experimentally induced bovine colibacillosis was studied in a 41-cow university herd. Pregnant dams were antigenized prepartum with two injections administered by the subcutaneous and intrammamary routes with one of four vaccine preparations (killed bacteria, live bacteria, culture supernatant, or heart infusion broth [control]). The data indicate that 77% of the neonates born to vaccinated dams were strongly protected against oral challenge with Escherichia coli strain B-44. Bacterial agglutinin and passive hemagglutination titers of colostral whey directly reflected the efficacy of the vaccines. A notable decrease in the whey titers to somatic and capsular antigens occurred after heat treatment at 56 C for 30 min. Complicity of heat-liable immune factor(s) in protection from scouring was suggested. The nature of the protective antigen is not clearly defined by these studies but there is some evidence that the K antigen may play a vital role in this regard.


Human lymphocyte sub-populations and K cells.

Peripheral blood lymphocytes from 19 normal subjects were examined for surface Ig (SIg) and capacity to form rosettes with normal and neuraminidase-treated sheep erythrocytes and with chicken erythrocytes sensitised with IgG antibody. Information on the relationship between the presence of SIg and capacity to form rosettes was obtained by combined tests and depletion experiments. By these means, a population of lymphocytes with Fc receptors, but lacking SIg (mean 14.6%) was defined and shown to correlate closely with cytotoxic activity for antibody-sensitised target cells. Indirect evidence was also obtained that these lymphocytes, which are regarded as the major population of antibody-dependent cytotoxic cells, are capable of forming rosettes with normal and neuraminidase-treated sheep erythrocytes. The nature of these cells is briefly discussed.


Incomplete viral synthesis in Friend leukemia virus-induced reticulum cell sarcomas.

Tissue-culture-passaged, Friend leukemia virus (FV)-induced reticulum cell sarcomas from BALB/c mice (FVTCT-BALB) did not produce infectious FV, although retrieval of infectious FV occurred when these cells were co-cultivated with cell lines replicating non-defective murine leukemia viruses (MLVs). The level of FV expression in the FVTCT-BALB cell line was studied to understand better the process of FV retrieval. 3H-uridine labelling techniques and reverse transcriptase assays showed that FVTCT-BALB cells did not release C-type virus particles. Nucleic acid hybridization techniques demonstrated that the level of viral RNA synthesis in the FVTCT-BALB non-producer cell line was indistinguishable from that in cell lines productively infected with MLVs. These data suggest that in the FVTCT-BALB cell line the synthesis of FV is blocked in some late stage of virus assembly.


[Enzymatic quick determination of aminoglycoside antibiotics in serum].

The therapeutic range of aminoglycoside antibiotics is relatively narrow; therefore short-term controls of serum concentrations can be recommended, particularly for high-dosage therapy and for renal insufficiency. For the aminoglycosides Amicacin, Gentamycin, Sisomicin and Tobramycin an enzymatic quick-determination test within 2 hrs is being described, which is based on the pH-deviation of a medium containing urea by hydrolytic activity of proteus mirabilis-bacteriae. In more than 180 measurements of patients' sera as well as of sera with known concentrations the method was tried out and compared with the conventional agar diffusion procedure. In strict compliance with the methodical directions, reproducable results could be achieved at any time within the range of1.0-30.0mug/ml; the number of faults in the quick-determination test was with +/- 8.4% about the same as in the agar diffusion test. In comparison with other microbiological or biochemical quick-procedures, the simplicity of the described method must be emphasized, thus it can be performed, too, in non-bacteriological laboratories.


Double-bind comparison of maprotiline with amitriptyline in the treatment of depressive illness.

Thirty patients closely matched for age, sex and duration of illness were treated with either amitriptyline or maprotiline in a double-blind, between-patient trial. Within limits of the small number of patients in the trial, maprotiline produced a significantly quicker improvement during the first 2 weeks as judged by the scores on the Hamilton Rating Scale. Maprotiline showed better tolerability than amitriptyline overall, especially as far as trial drop-out rates were concerned.


A controlled comparison of cyclazocine and naloxone treatment of the paroled narcotic addict.

A controlled, double-blind study of the comparative effectiveness of the narcotic antagonists, cyclazocine and naloxone, was undertaken in a metropolitan narcotic clinic offering an abstinence program involving urine monitoring and ancillary counseling services. Seventy male addict parolees were randomly assigned to 6-month treatment with either cyclazocine, 4 mg administered on a daily basis, or naloxone, 500-2,000 mg administered on a locally developed and researched 'contingent' basis, i.e., whenever there was indication of narcotic drug use (daily and contingent placebos were utilized to preserve the double-blind). Criteria of treatment effectiveness included narcotic drug usage, clinic attendance, length of participation in the program, disposition at 6 months, and incidence of side effects. The two subsamples of 35 individuals were similar with respect to relevant demographic characteristics. Examination of comparative effects revealed little to no significant differences between the two groups in terms of measures of program adherence, treatment outcome, and personal and social adjustment. Side effects were more prevalent among cyclazocine patients. Typically, these included moderately severe somatic effects and perceptual and cognitive disturbances.


The genetic effects of elevated temperature in the yeast, Saccharomyces cerevisiae.

The growth of yeast cultures at supra-optimal temperatures up to 39 degrees C and exposure in non-nutrient solution up to 52 degrees C has been examined for the induction of genetic change. Both sets of conditions lead to the induction of mutation to antibiotic resistance, mitotic gene conversion, crossing-over and mitotic chromosomal non-disjunction.


On structural formulas of basic fuchsin and aldehyde-Schiff reaction products.

A variety of structural formulas has been suggested for the basic fuchsin moiety of aldehyde-Schiff reaction products. It was therefore deemed of interest to review the development of these different concepts of dye structure. Formulation of basic fuchsin as an ammonium salt preceded the quinonoid theory; however, chemists could not find such salts. The quinoid theory also could not be reconciled with chemical observations and spectroscopic data. In the 1920's the quinonoid formulas were superseded by benzenoid formulas with a positive charge at the central carbon atom. Using the resonance theory, basic fuchsin is often written with an apparently pentavalent nitrogen atom at a quinonoid ring. But such limiting structures do not exist in reality; the structure of the resonance hybrid is intermediate between the various contributing structures. The carbonium formula appears preferable to other limiting structures because it would remove temptations to endow basic fuchsin with a quinonoid ring, an imino or an ammonium group. Some formulas of aldehyde-Schiff reaction products carry two positive charges. But divalent basic fuchsin is very unstable. The divalent form of its derivatives formaldehyde- Schiff's reagent, aldehyde-fuchsin, and Crystal Violet is deep green; with decreasing H-ion concentration the divalent green compounds revert to the red or violet monovalent substance. It appears therefore highly unlikely that divalent basic fuchsin exists in PAS reaction products in washed and dehydrated sections.


Effect of supplemental dietary vitamin E on the serologic response of swine to an Escherichia coli bacterin.

Three groups of 10 pigs (6 to 8 weeks old) were fed a nutritionally complete ration (control ration, CR), CR plus 20,000 IU of vitamin E/ton (CR + recommended E), and CR plus 100,000 IU of vitamin E/ton (CR + high E), respectively. Each pig was given an intramuscular injection of an Escherichia coli bacterin at experimental days 0 and 35. Serums were collected 7 days prior to the first injection and at days 7, 14, 21, 35, 42, 49, and 56. Pigs fed the CR + high E ration developed anti-E coli serum antibody titers two- to threefold higher than those of the controls. Pigs fed the CR + recommended E ration developed serum antibody titers intermediate between those of pigs in the other 2 groups.


Histochemical and immunohistochemical properties of the CRF-granules and other "Gomori-positive" substances of the rat.

In adrenalectomized rats, histochemical and immunohistochemical properties of the following secretion products have been investigated: 1. CRF-granules in the outer layer of the median eminence; 2. neurosecretory material (NSM) in the supraoptico-hypophysial system of the hypothalamus; 3. secretory granules in the TSH-cells of the anterior lobe of the hypophysis; 4. secretory granules in the ependymal cells of the subcommissural organ (SCO); 5. beta-cell-granules in the islets of Langerhans in the pancreas. All these substances are characterized by their stainability with the so-called "Gomori method". The experiments have included studies into: a) the extractability of the substances by various solvents; b) the digestability of the substances by pepsin or trypsin; c) their histochemically detectable content of disulfide groups, arginine and periodic acid-Schiff (PAS) reactive carbohydrates; d) their reaction with porcine-neurophysin-II-antibodies. All substances exhibited a positive reaction for disulfide groups. Based on their solubility properties, their resistance to pepsin or trypsin, their respective content of PAS-reactive carbohydrates and their failure to react with anti-neurophysin serum the "Gomori-positive" granules in TSH-, SCO- and pancreatic beta-cells can be distinguished from one another and from CRF- and neurosecretory granules. In contrast, CRF-granules and NSM showed identical properties. Taking into consideration data from the biochemical and histochemical literature, the present findings suggest that CRF-granules and NSM consist of closely related biochemical substances.


Influence of acute hypertension on aortic valve competence.

The effect of acutely induced hypertension on aortic valve competence was studied in anesthetized dogs. Aortic pressure was increased by infusion of methoxamine or mechanically; aortic valve competence was evaluated by aortogrphy and by indicator dye. The aortic valve was normally competent; aortic insufficiency appeared with increase of mean pressure by as little as 20-50 mmHg; 6 of 9 animals showed aortic incompetence when mean aortic pressure was elevated  45-70 mmHg, but the valve remained competent in 2 of 9 animals with mean pressure increments of 60-90 mmHg. The aortic root was appreciably less distensible than was the proximal descending aorta; this factor may limit the degree of aortic insufficiency in response to acute hypertension in the dog.


Lactic acid removal rates during controlled and uncontrolled recovery exercise.

After a standardized 6-min bicycle ergometer exercise (89% VO2max) lactic acid removal rates were compared during recovery at rest and exercies at 29.7, 45.3, 61.8, and 80.8% VO2max, and twice while the subjects (N = 7) regulated their own recovery exercise. Blood samples were taken after the standardized exercise and every 5 min during the 30-min recovery periods. During the controlled recovery periods lactic acid removal rates were dependent on the intensity of the recovery (Y' = 0.103 + 0.218chi - 0.464 X 10(-2)chi2 + 0.252 X 10(-4)chi3). Optimal removal was predicted to occur at 32% VO2max. Removal rates during the self-regulated recoveries were not different (P greater than 0.05), but these removal rates were faster than during recovery at rest and exercise at 61.8 and 80.8% VO2max (P less than 0.01). Removal rates during the self-regulated recovery and recovery at 29.7 and 45.3% VO2max were not different (P greater than 0.05). The subjects were therefore able to remove lactic acid effectively when selecting their own recovery exercise.


Continuous positive-pressure ventilation and choledochoduodenal flow resistance.

Resistance to flow through the choledochoduodenal junction was measured during constant perfusion (0.8 ml saline/min). In eight dogs, intermittent positive-pressure ventilation (IPPV) and continuous positive-pressure ventilation (CPPV) were compared. Pressure in the common bile duct was always higher during JPPV than IPPV. With the first application of CPPV the rate of intravenous fluid was adjusted to maintain constant Hct. Mean hepatic venous pressure (Phv) increased from 6.6 to 11.5 cmH2O (P less than 0.001). Mean pressure in the common bile duct increased (P less than 0.001) from 11.6 to 14.1 cmH2O. The average increase in resistance was 21%. Changes reversed with return to IPPV. During the second application of CPPV, intravenous fluid was increased to maintain constant arterial pressure. Phv increased to 12.8 cmH2O and pressure in the common bile duct increased to 15.0 cmH2O (30% increase). In four additional dogs, choledochoduodenal resistance during continuous CPPV was reduced by intravenous vasopressin, intravenous norepinephrine and intraducta phenylephrine. CPPV increases resistance to flow through the choledochoduodenal junction, probably by vascular engorgement.


Fever: exchange of shivering by nonshivering pyrogenesis in cold-acclimated guinea pigs.

The pyrogenic response of adult, unanesthetized guinea pigs to 2 mug/kg iv of Salmonella enteritidis endotoxin was measured at 27 and 7 degrees C ambient temperatures, both before and after an 8-wk exposure to 7 degrees C. There were no significant differences between the onset, maximum height, and total duration of the fevers produced before and after cold acclimation in both thermal environments. However, in 27 degrees C, before cold acclimation, fever production was associated with vigorous shivering activity; two temperature maxima typically developed. After cold acclimation, visible shivering was not detectable during pyrogenesis; moreover, only a single maximum occurred, culminating during the interval between the two rises previously. In 7 degrees C, shivering occurred in both the non-cold- and cold-acclimated endotoxin-treated guinea pigs, but the increase in oxygen consumption was significantly greater in the latter. These results indicated, therefore, that nonshivering (NST) replaces shivering thermogenesis (ST) in a thermoneutral, while ST is added onto NST in a cold, environment in cold-acclimated guinea pigs in supplying the necessary heat for fever production, and that these effects involve alterations in the character of the febrile course.


Effect of propranolol on endotoxin-induced pyrogenesis in newborn and adult guinea pigs.

Previous studied have shown that nonshivering thermogenesis (NST) is the prevailing mechanism of fever production in both newborn (during the first 2-3 wk of life) and adult cold-acclimated guinea pigs. This study was undertaken to determine whether this process may be mediated by noradrenergic sympathetics. The temperatures in the interscapular brown fat pad (Tbat) and the colon (Tre), the rate of oxygen consumption (VO2), and shivering activity were measured continuously for 5 h at 27 degrees C ambient temperature in 8- and 16-day-old and adult cold-acclimated guinea pigs following 2 mug/kg iv of Salmonella enteritidis endotoxin, with and without 6.0 mg/kg of propranolol (a beta-adrenergic blocker) injected ip 2 min before the endotoxin. In the older animals, the increase in Tbat normally produced by endotoxin was reduced by propranolol administration, but shivering set in, maintaining both VO2 and Tre at their febrile levels. In the newborn animals, Tbat also was decreased by propranolol, but shivering did not set in, so that VO2 and Tre fell below their febrile values. It is concluded that endotoxin-induced NST is controlled by noradrenergic sympathetics. The failure of NST to be replaced in the present newborn guinea pigs by visible shivering might be related to other observations that the onset of shivering at this age occurs only when Tre is significantly reduced.


Measurement of cardiac output by electrical impedance at rest and during exercise.

A comparison was made between cardiac output values determined by the dye dilution and electrical impedance methods in ten subjects at rest and during graded exercise on a bicycle ergometer. The cardiac output values determined by the two methods were linearly related and significantly (P less than 0.001) correlated (r = 0.90). Movement artifact associated with exercise at maximum or near-maximum work loads caused severe distortion of the dZ/dt wave form and prevented calculation of impedance cardiac output at these levels of work. Use of the lowest value of L (distance between mean value of L in the impedance stroke volume equation (SV = p(L2/ZO2) (dZ/dt)mt), resulted in nearly identical values for the least-squares line and equalvalue line of impedance and dye cardiac outputs. Although absolute values of cardiac output determined by the two methods were not identical in all subjects the changes in cardiac output were nearly identical during the different levels of exercise. The data support the validity of the impedance method as a noninvasive, atraumatic measure of cardiac output at rest and during graded exercise.


On-line cardiac output by digital computer.

Many devices for fast cardiac output calculations employ special assumptions and departures from the Stewart-Hamilton method. These assumptions and departures can cause substantial errors. This publication describes an on-line computer system for evaluating dye-dilution curves according to the classical Stewart-Hamilton method. Dilution curves are smoothed, using a digital filter to eliminate heartbeat noise. Recirculation is removed using the constant ratio property of exponentials. The quality of the curves is evaluated by determining the length of the base line, the extent and the fit to an exponential downslope, and a figure of merit based on the five tanks-in-series model. Dilution curves, the logarithmic form, and the exponential ratios are displayed on a CRT for visual checks. Comparison of cardiac outputs obtained by this on-line system with results obtained by hand calculations showed a mean difference of 150 ml. Repeated evaluation of one electronically simulated dye curve resulted in SD = 10 ml for computer results and SD = 200 ml for hand calculations.


Automated gel permeation chromatographic cleanup of animal and plant extracts for pesticide residue determination.

Applications of an improved gel-solvent system for cleanup of pesticide residues by gel permeation chromatography (GPC) were investigated. Elution characteristics using Bio-Beads SX-3 gel and a toluene-ethyl acetate (1+3) elution solvent were determined for 16 nonionic chlorinated pesticides, 3 polychlorinated biphenyls (PCBs), 14 chlorophenoxy herbicide esters, and 7 organophosphate insecticides. Elution patterns for vegetable and animal lipids were also studied. Quantitative recoveries of the pesticides were achieved. No liquid-liquid partitioning cleanup steps were required with any type of nonionic chlorinated pesticide or sample matrix. Only GPC cleanup was required for the nonionic chlorinated pesticides, PCBs, and organophosphate pesticide residues in chicken and turkey fat samples. Electron capture and flame photometric detectors were used in the gas chromatographic method for the respective pesticides. Samples containing up to 0.5 g lipid each were processed at the rate of one every 30-40 min with the automated system. Results were in excellent agreement with those from accepted manual partitioning methods and were achieved with significant savings of both labor and chemicals.


Confirmation of vinyl chloride in foods by conversion to 1-chloro-1,2-dibromoethane.

A semiquantitative method has been developed for the confirmation of vinyl chloride (VC) in foodstuffs. The VC was brominated to give 1-chloro-1,2-dibromoethane which was analyzed by gas-liquid chromatography, using an electron capture detector. The yield of 1-chloro-1,2-dibromoethane varied between 40 and 55%, which permitted a lower limit of confirmation of VC of 15 ppb for vinegar and sherry and 50 ppb for peanut oil.


Collaborative study of an enzymatic digestion method for the isolation of light filth from ground beef or hamburger.

Collaborative results are presented for a proposed method for light filth extraction from ground beef or hamburger. The method involves enzymatic digestion, wet sieving, and extraction with light mineral oil from 40% isopropanol. Recoveries are good and filter papers are clean. This method has been adopted as official first action.


Pathogenicity of Escherichia coli recovered from food.

In Western nations, pathogenic biotypes are sporadically encountered in foodborne gastroentetis. Cholera, dysentery, and chronic ulcerative colitis syndromes are recognized. E. coli has been associated on the average with 2% of annual food outbreaks and 5% of total cases. In developing nations, the incidence may be greater. In contrast with Salmonella and Shigella, significance can be assessed only by fulfillment of Koch's postulates. Preliminary studies using vascular permeability reaction for the detection of heart-labile toxin and the Sereny keratoconjunctivities test for demonstration of invasiveness indicate limited incidence of these pathogenicity markers in cultures from foods. Two per cent of isolates from cheeses involved in recent outbreaks were toxigenic; 14% were invasive. Corresponding values for food isolates not associated with illness were 10 and 0, respectively. To facilitate examination of multiple isolates, 10 may be pooled for the detection of heat-labile toxin, and 5 for invasiveness. Present model pathogenicity systems require standardization, estimation of specificity and sensitivity limits, examination by collaborative study, and ascertainment of human equivalence. Supplemental tests include capacity for colonization of intestinal epithelium and intracellular growth. Stereotypes based upon serology, host range, and recognized toxic factors may require modification.


Renaturation of yeast inorganic pyrophosphatase denatured in urea and guanidine hydrochloride.

The renaturation of yeast inorganic pyrophosphatase [EC 3.6.1.1] (PPiase) denatured in guanidine-HCl and urea was studied. The molecular weight of PPiase was estimated to be ca. 63,000-70,000 by means of Sephadex G-75 column chromatography in 8 M urea and 6 M guanidine-HCl and by electrophoresis on polyacrylamide gel containing 8 M urea. The activities of PPiase denatured in various concentrations of denaturants were measured in the presence and absence of the denaturants. In the presence of the denaturants, enzymatic activity decreased as the denaturant concentration increased up to 1.5 M guanidine-HCl and 4 7 urea. The activities of PPiase denatured in these denaturants were not restored by dilution with buffer. However, the enzymatic activities of PPiase denatured at concentrations higher than 1.5 M guanidine-HCl and 4 M urea were restored by dilution with Tris-HCl buffer (pH 7.5). The recovery of the enzymatic activities of PPiase denatured in 3 to 6 M guanidine-HCl and 6 to 8 M urea was to a level of about 90% of the native enzyme. Irreversible denaturation of PPiase in lower denaturant concentrations was prevented in the presence of sulfhydryl reagents, dithiothreitol, glutathione, and 2-mercaptoethanol. In irreversibly denatured PPiase, the amount of free SH groups decreased markedly. These results indicated that in lower denaturant concentrations, SH groups in PPiase are very oxidizable and their oxidation may cause irreversible denaturation. In higher denaturant concentrations where PPiase was denatured completely, the SH groups became less reactive. The conformations of renatured PPiases was investigated by means of N-bromosuccinimide oxidation, fluorescence emission spectra and circular dichroism spectra. The PPiase denatured in 6 M guanidine-HCl showed fully restored native conformation, as checked by these methods, although renatured PPiase gave a trough in the 280 nm region of slightly less magnitude than that of PPiase. On the other hand, PPiase denatured in 8 M urea showed restored enzymatic activity, but restoration of its conformation was incomplete as compared to PPiase denatured in 6 M guanidine-HCl. PPiase renatured from material denatured in lower denaturant concentrations, such as 4 M urea and 1.5 M guanidine-HCl, had quite a different conformation from the native enzyme as judged from CD spectra, N-bromosuccinimide oxidation and fluorescence spectra. Differences in PPiases denatured in urea and guanidine-HCl were discussed in connection with the possible modification of amino groups in PPiase by cyanate ions.


Myosin aggregates as a requirement for contraction and a proposal to the mechanism of contraction of actomyosin systems.

Glycerinated fibers of rabbit psoas muscle showed no augmentation of tension development upon incubation with heavy meromyosin, irrespective of whether the fibers were of standard length, stretched, or extracted of their myosin content. The effect of heavy meromyosin was to suppress contraction. These observations are in disagreement with certain recent published reports (Oplatka et al., 1974) and do tend to support the current sliding filament theory of muscle contraction and the necessity of bipolar myosin filaments for contraction. A possible mechanism of contraction in protein systems, including tension generation in actomyosin fibers and superprecipitation, is described emphasizing the polarity of both myosin and actin filaments.


Transport of sugars and amino acids in bacteria. XVI. Theory and evaluation of a model for the membrane transport reaction mediated by a single carrier with three binding sites for substrate.

A novel model is presented for bacterial active transport reactions which show a curvilinear Eadie-Hofstee plot and negative homotropic cooperativity in the kinetics of substrate uptake. Various models of a single carrier with multi-binding sites for substrate were constructed and examined theoretically. The fit of these models with experimental data on the kinetics of branched chain amino acid transport reactions were tested by iterative computation using the non-linear least square method. The transport model which fitted the experimental data best consisted of a single carrier with three binding sites for substrate in which one of the substrate-carrier complexes, CSS, is not active in translocating substrate across the cytoplasmic membrane. The mechanism of homeostatic regulation of the intracellular concentration of amino acids by active transport systems is discussed on the basis of this transport model.


Uptake of radioactive D-glucose anomers by pancreatic islets.

Isolated rat islets were incubated in media containing either the alpha or beta anomer of D-[1-3H]glucose for 5 min at 37 degrees. The amounts of the two anomers incorporated were determined using L-[1-14C]glucose as an extracellular space marker. The incorporation of beta-D-glucose was about twice that of alpha-D-glucose. Our previous and present results suggest that the two anomers of D-glucose each have a preferential function in pancreatic beta cells; alpha-D-glucose stimulates insulin secretion, and beta-D-glucose is transported into the cells.


Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 1. General introduction.

With the ultimate objective of the total synthesis of a tRNA gene including its transcriptional signals, an Escherichia coli tyrosine suppressor tRNA gene was chosen. The arguments in favor of this choice are presented. A plan for the total synthesis of the 126-nucleotide-long DNA duplex corresponding to a precursor (Altman S., and Smith, J. D. (1971) Nature New Biol. 233, 35) to the above tRNA is formulated. The plan involves: (a) the chemical synthesis of 26 deoxyribooligonucleotide segments, (b) polynucleotide ligase-catalyzed joining of several segments at a time to form a total of four DNA duplexes with appropriate comlementary single-stranded ends, and (c) the joining of the duplexes to form the entire DNA duplex. Ten accompanying papers describe the experimental realization of this objective.


Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 2. Chemical synthesis of the deoxypolynucleotide segments corresponding to the nucleotide sequence 1-31.

Chemical synthesis of the undecanucleotides d(T-G-G-G-G-G-A-A-G-G-A), d(C-C-C-C-A-C-C-A-C-C-A), and d(T-C-G-A-A-T-C-C-T-T-C), and the nonanucleotides, d(T-T-C-G-A-A-C-C-T) and d(T-T-C-G-A-A-G-G-T) are described. The deoxyribopolynucleotides together represent the DNA duplex corresponding to the nucleotide sequence 1-31 (from the 3'-end) of the gene for the tyrosine suppressor tRNA. The synthesis, which basically used the previously developed chemical methods, started with 5' and N-protected deoxyribonucleosides. Successive condensations at the 3'-end were performed using suitably protected mononucleotides or preformed di- and trinucleotides. The condensing agents used were mesitylenesulfonyl chloride or triisopropyl benzenesulfonyl chloride. The required condensation products were isolated partly by solvent partition methods or, in the case of longer chains, by anion exchange chromatography. The completely deprotected deoxypolynucleotides were further purified by anion exchange chromatography in the presence of 7 M urea and characterized by chemical and enzymatic methods.


Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 4. Synthesis of deoxyribopolynucleotide segments corresponding to the nucleotide sequence 47-78.

The chemical syntheses of the dodecanucleotide, d(G-C-C-G-C-T-C-G-G-G-A-A), the decanucleotides, d(T-T-T-A-G-A-G-T-C-T), d(G-C-T-C-C-C-T-T-T-G), d(C-G-G-C-C-A-A-A-G-G), and the nonanucleotide, d(G-A-G-C-A-G-A-C-T), are described. The deoxypolynucleotides together represent the DNA duplex corresponding to the nucleotide sequence 47-78 of the gene for the tyrosine suppressor tRNA. Chemical syntheses used protected mono- and oligonucleotides and stepwise condensation methods. The detailed plans used are given in Diagrams 1 to 4 in the text. Two additional notable features in the present syntheses were: (a) solvent extraction procedures for the preparation of oligonucleotides as large as pentanucleotides and (b) the demonstration that a heptanucleotide containing 5'-phosphate group can be used successfully in condensation with the 3'-OH group of another oligonucleotide. The synthetic polynucleotides were purified and characterized at protected and unprotected levels.


Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 5. Synthesis of the deoxyribopolynucleotide segments representing the nucleotide sequence 71-103.

Chemical syntheses of the pentadecanucleotide, d(G-G-T-G-G-G-G-T-T-C-C-C-G-A-G), the undecanucleotides, d(G-G-T-G-G-G-G-T-T-C-C) and d(C-C-C-C-A-C-C-A-C-G-G), the decanucleotide, d(G-T-A-A-T-G-C-T-T-T), and the nonanucleotides, d(A-T-T-A-C-C-C-G-T) and d(A-G-T-A-A-A-A-G-C) are described. The deoxyribopolynucleotides together represent the DNA duplex corresponding to the nucleotide sequence 71-103 (from the 3'-end) of the gene for the tyrosine suppressor tRNA. Synthesis of the guanine-rich undecanucleotide d(G-G-T-G-G-G-G-T-T-C-C) was performed by the use of a new protecting group for the guanine ring, the methylbutyryl group. The heptanucleotide d[(MeOTr)mbG-mbG-T-mbG-mbG-mbG-mbG], prepared by the new method, was condensed with the tetranucleotide d[panC-anC-T-T(Ac)]. All of the condensations described followed previously developed chemical principles and started with the N- and 5'-protected deoxyribonucleosides. Successive condensations at the 3'-end with protected mononucleotides, preformed di-, tri-, or tetranucleotides gave products which were separated by anion exchange chromatography and characterized by chemical and enzymatic methods.


Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 6. Synthesis of the deoxyribopolynucleotide segments corresponding to the nucleotide sequence 100-126.

Chemical syntheses of the tridecanucleotide, d(G-C-T-T-C-C-C-G-A-T-A-A-G), the dodecanucleotide, d(G-C-T-C-C-C-T-T-A-T-C-G), the decanucleotide, d(G-G-A-G-C-A-G-G-C-C), the nonanucleotide, d(T-A-C-T-G-G-C-C-T), and the hexanucleotide, d(G-G-A-A-G-C), are described. Together, these syntheses represent the nucleotide sequence 100-126 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor. The synthesis of the dodecanucleotide d(G-C-T-C-C-C-T-T-A-T-C-G), was accomplished by the condensation of the previously described protected nonanucleotide, d[(MeOTr)ibG-anC-T-anC-anC-anC-T-T-bzA], with the trinucleotide block d[pT-anC-mbG(Ac)]. Synthesis of the other segments involved stepwise condensations to the 3'-OH group of growing oligonucleotide chains, starting with suitably protected deoxyribonucleosides and using protected mono-, di-, and trinucleotides as the incoming blocks. The final products, after deprotection, were purified by anion exchange chromatography and characterized. The synthesis of this part of the DNA was planned so that only a hexanucleotide segment is used to go up to the 3'-end (nucleotide 126) of the DNA and, therefore, it is amenable to elongation by chemical methods when the nucleotide sequence of the several nucleotides beyond this end becomes known.


Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 7. Enzymatic joining of the chemically synthesized segments to form a DNA duplex corresponding to the nucleotide sequence 1-26.

Duplex [I], which represents the nucleotide sequence 1-26 of the double-stranded DNA corresponding to the precursor for a tyrosine suppressor tRNA, has been synthesized by the enzymatic joining of five chemically synthesized deoxyribooligonucleotide segments. The synthesis was accomplished in two different ways. In a one-step synthesis, all of the five segments were used together: segments 2, 3, and 5 carried 5'-33P-labeled phosphate groups while segment 4 carried a 32P-phosphate group. An alternative, two-step method involved the joining of 5'-32P-phosphorylated segment 2 to segment 4 (carrying 5'-OH group or 5'-32P- or 33P-labeled phosphate group) in the presence of segment 3 followed by the joining of [5-32P]segment 5 in a second step. The duplex [I]' (segments 2 to 5) thus obtained was phosphorlated at the 5'-ends with polynucleotide kinase and then joined to segment 1 to give duplex [I] quantitatively. The preparative methods described have the desired flexibility for performing the subsequent operations necessary for the total synthesis of the structural gene for the tyrosine suppressor tRNA precursor.


Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 8. Enzymatic joining of the chemically synthesized segments to form DNA duplexes corresponding to nucleotide sequences 23-60 and 23-66.

Polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxyribopolynucleotide segments (Fig. 1) comprising the nucleotide sequence 23-66 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been systematically investigated. Joining was studied using all possible combinations of 3, 4, and 5 and larger numbers of segments at a time. The extent of joining varied widely (0 to about 90%) in three component systems. The "self-structure" of some of the components evidently inhibited the joining. Addition of a fourth segment in general enhanced the extent of joining and optimal yields were obtained in systems containing six or more segments. A comparison of the T4-induced ligase and the E. coli polynucleotide ligase for joining of the chemically synthesized segments showed the E. coli enzyme to be inferior to the T4-induced ligase. Satisfactory syntheses of the duplexes [IIa] and [IIb] comprising, respectively, eight and seven segments were achieved in single steps. Of the two terminal segments carrying 5'-OH groups in the duplexes, only one (segment 7) was used in the prephosphorylated form. The duplexes were isolated pure and characterized by enzymatic degradations and by electrophoresis.


Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 9. Enzymatic joining of chemically synthesized deoxyribopolynucleotide segments corresponding to nucleotide sequence 57-94.

The DNA duplexes representing nucleotide sequences 61-89 and 57-94 have been synthesized, isolated pure, and fully characterized. Synthesis of the duplex with the nucleotide sequence 61-89 involved the DNA ligase-catalyzed joining of chemically synthesized deoxyoligonucleotide segments 14 to 18 shown in Fig. 1A, while for the longer duplex (sequences 57-94) seven deoxyribooligonucleotides (segments 13 to 19, Fig. 1B) were used in one-step enzymatic joining. The joining of the short tetranucleotide (segment 16) to the segment 17 required the presence of the adjacent segment 14, even if the latter did not contain a 5'-phosphate group, to allow its joining to segment 16. However, in the synthesis of both of the DNA duplexes, the yields were comparatively low (30 to 40%) and could not be significantly increased although a variety of conditions was tried. The main cause in both cases evidently was the sluggish joining of segment 14 to 16 and of segment 16 to segment 17. Although the original plan for the total synthesis of this part of the gene for the tRNA precursor involved the DNA duplex consisting of segments 14 to 18, this duplex could not be quantitatively phosphorylated at the two 5'-OH ends for subsequent joining to the adjoining parts of the gene. The DNA duplex consisting of segments 13 to 19, which possesses both terminal 5'-OH groups at protruding single-stranded ends, was readily phosphorylated and used successfully in the total synthesis of the gene as described in an accompanying paper.