Application of the method of phage T4 DNA ligase-catalyzed ring-closure to the study of DNA structure. II. NaCl-dependence of DNA flexibility and helical repeat.

In this work, we demonstrate that it is possible to determine the molar cyclization factor jM from single ligation reactions in which both circular and linear dimer DNA species are formed concurrently from linear monomers. This approach represents a significant improvement over previous methods, in which jM is evaluated from the ratio of the rate constants for two separate processes; namely (1) the cyclization of linear DNA and (2) the association of two linear molecules to form linear dimers. Determination of jM for a 366 base-pair molecule yields 5.8 X 10(-8) M, in close agreement with the value of 5.6 X 10(-8) M determined by Shore et al. for the same molecule. Using the current approach for the determination of jM, we have investigated the dependence on NaCl concentration (0 to 162 mM-NaCl, 1 mM-MgCl2) of both the lateral and torsional flexibilities of DNA. The principal observation is that both quantities are essentially constant over the above range of NaCl concentrations, with the persistence length P approximately 450 (+/- 15) A, and the torsional elastic constant C approximately 2.0 (+/- 0.2) X 10(-19) erg cm. These observations are in accord with the previous theoretical prediction that P becomes essentially independent of NaCl concentration above 10 to 20 mM. We have examined the dependence of the helical repeat of DNA on NaCl concentration over the above range, and have found the value of 10.44 base-pairs per turn to be essentially constant over that range. This last result suggests that earlier studies have overestimated the dependence of DNA helical twist on salt concentration.

Five human gastric aspartic proteinases: N-terminal amino acid sequences and amino acid composition.

Human pepsin A consists of 4 or more isoenzymes (designated 1, 3a, 3b and 3c) one of which, pepsin 1, contains up to 50% carbohydrate moieties. The amino-acid composition and N-terminal sequence of pepsin 1 and the other isoforms have been determined and compared with data obtained for pepsin 3b and gastricsin (pepsin C or pepsin 5). Pepsins were isolated from penta-gastrin stimulated gastric juice using repetitive chromatography on DEAE-cellulose, or high performance ion-exchange chromatography. Sequencing was performed using automated solid-phase Edman degradation with a microsequence facility. The amino-acid compositions were similar for pepsins 1, 3a, b and c and the N-terminal sequences of pepsins 1, 3a and c, reported for the first time, were shown to be identical with that for pepsin 3b (the main component of pepsin A) although residue 28 was unassigned in pepsin 1. Residue 30 in all four isoenzymes is valine and we cannot confirm reports of major pepsins with leucine in this position. For gastricsin the sequence differed from the pepsin isoenzymes and in position 24 we find pro rather than ala as was first described. These observations suggest that pepsin 1 is identical to 3b or a mixture of 3a, 3b and 3c but not gastricsin. This data supports the hypotheses that the four pepsin isoenzymes are products of the same gene(s) but have undergone varying levels of post translational modification.

A general method for cloning DNA fragments in multiple copies.

A general method for the cloning of DNA fragments in tandem arrays is presented. Synthetic directional adapters are attached to the fragment ends to establish complete control over fragment orientation during ligation. Use of these directional adapters thereby ensures the production of direct repeats, an arrangement essential for clone stability. The technique involves a protocol that is both less complex and less time-consuming than previous methods. Using this technique, a 10-min ligation has yielded a plasmid containing 20 copies of a 151-bp fragment.

Limits to parathyroid imaging with thallium-201 confirmed by tissue uptake and phantom studies.

Correct location by 201TI imaging of 48 parathyroids in 35 patients was related to size; 25 out of 26 parathyroids of mass greater than 1.0 g were correctly located, none of ten parathyroids less than 0.3 g was correctly located. In seven patients previously imaged, 108 microCi (4.0 MBq) of 201TI was injected when the thyroid was first exposed surgically. Subsequently weighed and histologically confirmed samples of parathyroid, thyroid, and skeletal muscle were counted against a standard in a well counter. Thallium-201 uptake, as %/g, did not differ between hyperplastic and adenomatous parathyroids. Mean parathyroid uptake was 0.018%/g, thyroid 0.01%/g, muscle 0.0026%/g of administered dose. Lower limits for correct location lay between 0.006-0.0149% of administered dose and between 0.25-0.8 g. Studies using a 201TI phantom containing small aliquots of 201TI at higher concentrations suggested approximately 0.0075% of the usual patient imaging dose as a lower limit for correct location.

Effect of calcitonin treatment on osteoclast counts in Paget's disease of bone.

Histological and biochemical changes during calcitonin treatment have been studied in 15 patients with Paget's disease of bone. For each patient, osteoclast counts were made by the same observer on serial needle biopsies of diseased bone from the posterosuperior iliac spine. Serial estimations were also made of the serum alkaline phosphatase and urinary hydroxyproline excretion. A total of 66 biopsies was examined (ranging from two to seven per patient). Osteoclast populations and the biochemical measurements were log normally distributed. During calcitonin treatment there was a statistically significant decrease in: (1) the total osteoclast count per square millimetre; (2) the number per square millimetre of osteoclasts in resorption cavities on the trabecular surface; (3) the relative proportion of osteoclasts sited in resorption cavities compared with total osteoclasts; (4) the serum alkaline phosphatase level; (5) 24-hour urinary hydroxyproline excretion. On stopping treatment there was a statistically significant increase in all of these histological and biochemical values except that the proportion of osteoclasts in resorption cavities remained low. The trabecular cement line pattern remained abnormal during and after treatment in all biopsies examined, and complete suppression of osteoclast activity was not observed. One of the patients developed a Paget's osteosarcoma while on calcitonin therapy.

Radio-enzymatic determination of histamine: interference by fluoride and possible activation of histamine methyl transferase.

The reproducibility of a radio-enzymatic method for determining plasma histamine was found to be affected by the anti-coagulant used for collecting blood. Recovery experiments from whole blood indicated that heparin yielded values that were more accurate than with EDTA or sodium fluoride; fluoride gave a mean value which was +41% higher than with heparin (P=0.054). Addition of fluoride to a standard calibration curve, and of increasing amounts to aliquots of 5 ng histamine also yielded higher values than in controls, up to +15% (P<0.1) and +14.1% (P=0.03) respectively. Fluoride did not affect the detecting system and was not contaminated with histamine; nor did it breakdown the methyl donor, S-adenosyl-L-methionine. It is concluded that heparin is the anti-coagulant of choice and that fluoride may activate histamine methyl transferase from pig brain. Fluoride may possibly have a biological role as an enzyme-activator and a usefulness in the therapy of mastocytosis.

Interference in the measurement of red cell glycogen by glycogen from white cells.

An enzymatic method for determining red cell glycogen was developed and its recovery and precision ascertained. Initial red cell glycogen values in 24 healthy volunteers ranged from 11-60 micrograms/g haemoglobin which compares with 20-105 micrograms/g haemoglobin previously reported. More thorough removal of leucocytes suggested however that variations in the determined red cell glycogen were due to glycogen derived from leucocytes. In 34 red cell preparations with varying leucocyte numbers, the glycogen concentration correlated positively with leucocyte count (r = +0.81, p less than 0.001), whereas glycogen concentration showed poor correlation with red cell count (r = -0.30, 0.1 greater than p greater than 0.05). The results question the reported presence of glycogen in normal red cells and suggest that contamination by leucocytes has been responsible for the previously reported normal range.

Intracavernous anterior cerebral artery origin with associated arteriovenous malformations: a developmental analysis: case report.

OBJECTIVE AND IMPORTANCE: This case demonstrates an unusual association between arteriovenous malformations and an intracavernous anterior cerebral artery origin. To the best of our knowledge, this relationship has not been previously described. Identification and understanding of this relationship are important in pre-embolization and surgical planning and in offering some insight into neurovascular development. CLINICAL PRESENTATION: The patient presented with severe recurring headaches and an otherwise nonfocal neurological examination. He maintained a stable neurological course throughout evaluation and therapy. INTERVENTION: The patient underwent endovascular embolization of the arteriovenous malformations without consequence. He was then scheduled for radiosurgical treatment planning. CONCLUSION: This case demonstrates an unusual neurovascular anomaly with associated arteriovenous malformations. To the best of our knowledge, this is the first reported case of such an association. An understanding of anomalous angioarchitecture and neurovascular development is essential for prudent endov ascular and surgical planning.

Low serum magnesium concentration in Paget's disease of bone (osteitis deformans).

Approximately 25% of patients presenting with Paget's disease of bone have a low serum magnesium concentration (below 0.74 mmol/L). Metabolic balance studies of 12 normomagnesaemic and seven hypomagnesaemic subjects show that the latter have a significantly more positive balance and a significantly higher net absorption of magnesium than have the former. The urinary output of the two groups did not differ significantly. The serum magnesium and alkaline phosphatase (log) concentrations were significantly negatively correlated, as were the serum magnesium and the daily urinary hydroxyproline (log) output. Low serum magnesium concentrations are thus associated with highly active Paget's disease; they do not arise from increased urinary loss nor from a lowered net absorption of magnesium, but probably from increased uptake by bone. The balance data show that to avoid a negative magnesium balance, the magnesium intake in Paget's disease should be at least 8.0 mmol/day.

Determination of iron in cardiac and liver tissues by plasma emission spectroscopy.

A simple plasma emission spectroscopic method for the determination of iron in liver and cardiac tissue is described. Using this technique, iron was extracted quantitatively from liver tissue of mass 14.2 to 65.4 mg wet weight, and heart tissue of mass 5.9 to 27.4 mg wet weight. Iron added to liver as aqueous ferric nitrate was recovered in the range 93 to 108%. Reference ranges for liver and myocardial iron on post mortem tissue gave respectively mean values of 0.841 mg/g dry weight (Range 0.310 to 1.600, n = 37) and 0.340 mg/g dry weight (Range 0.290 to 0.470, n = 8). Data on patients with haemochromatosis and transfusion siderosis are also presented.

Plasma histamine in patients with tropical disorders and acute asthma who exhibit eosinophilia.

The mean venous plasma histamine of 14 patients with eosinophilia was 18.7 nmol/L (range 0-86.4) and significantly higher than that of 4.0 nmol/L for 29 normal subjects [range 0-10.8 (P < 0.01)]. The eosinophilia of schistosomiasis (two patients), strongyloidiasis (one patient), and asthma (four patients) was accompanied by raised plasma histamine concentrations. Normal levels occurred in onchocerciasis (four patients) and in two of three patients with non-specific eosinophilia. Plasma histamine concentrations in the tropical disorders are not thought to have been reported previously. The initial arterial and venous plasma histamine concentrations of 14 acute asthmatics, were significantly positively correlated with the respective arterial (P < 0.01) and venous (P < 0.05) eosinophil counts. The results in acute asthma support the hypothesis that increased plasma histamine may have a role in promoting eosinophil release from the bone-marrow. Eosinophilia is not dependent upon a raised plasma histamine in some disorders, but may be in others, such as schistosomiasis.

Ionic fluoride: a study of its physiological variation in man.

The serum ionic fluoride concentrations in 497 normal individuals, from areas with non-fluoridated water supplies, ranged from 0.25 to 2.20 micromol F-/1 and were positively correlated with age (r = 0.31, p less than 0.001). Distribution of the data with age, coupled with the distribution of serum calcium with age, suggests a possible change in bone metabolism between 26 and 35 years of age. Serum fluoride levels vary with the time of day with a mean minimum value at 0800 and a mean peak value at 2200. Renal clearance studies gave fluoride ion clearances ranging from 19.5 to 44.0 ml/min and tubular reabsorption of fluoride ion ranging from 61.5 to 86.5%. After oral ingestion of 0.48 mmol sodium fluoride, peak serum levels occurred at 60 to 90 minutes; the peak levels were significantly higher in women than in men.

Plasma emission spectrometry: measurement of calcium, phosphorus and chromium in metabolic balance studies.

Direct-current plasma emission spectrometry determines food calcium and phosphorus, faecal calcium and chromium, and urinary calcium and phosphorus accurately, precisely and rapidly without acid digestion or dry ashing. Food and faeces are homogenised, diluted and aspirated directly into the plasma. The method makes metabolic balances swifter and less labour-intensive; it enables the use of chromium sesquioxide as an inert marker, for perchlorate digestion is avoided. Both plasma emission (-9.9%) and a dry ashing procedure (-13.5%) give lower values for food phosphorus than those derived from food tables.

An improved radioenzymatic assay for histamine in human plasma, whole blood, urine, and gastric juice.

A radioenzymatic method suitable for the assay of histamine in human blood, urine, plasma, and gastric juice is described. It differs from earlier methods by use of a histamine methyltransferase preparation from pig brain, of high activity tritiated S-adenosylmethionine, and of a heat precipitation step to reduce the previously noted interference from plasma constituents. The method is simpler than those requiring solvent extraction and concentration of histamine, gives recoveries in the range 80-120%, and so sliminates the need for internal standardisation. The method is sensitive and precise with coefficients of variation for blood, urine, and plasma of 5%, 6%, and 13% respectively. The mean +/- standard deviation for normal human plasma histamine is 5 +/- 4 nmol/l, for whole blood 559 +/- 193 nmol/l, and for urine 229 +/- 128 nmol/24h.

Measurement of copper, zinc and magnesium in serum and urine by DC plasma emission spectrometry.

The application of a DC plasma emission spectrometer to the measurement of copper, zinc and magnesium in serum and urine is reported. Each assay requires only a simple dilution in 1% nitric acid. Comparisons with standard atomic absorption techniques showed good analytical agreement, especially for copper and magnesium. Analysis of quality control preparations for copper and zinc gave results ranging from -1.5% to +3.6% of the stated values for five copper and four zinc experiments, with an unexplained result of +7.6% for one zinc experiment. Reference ranges were compiled for serum zinc and urinary copper which agreed closely with those established by atomic absorption and neutron activation analysis. Assays by DC plasma emission are thus precise and reproducible, and simple enough to authenticate the method for use in a clinical laboratory.

Localization of enlarged parathyroid glands by thallium-201 and technetium-99m subtraction imaging. Gland mass and parathormone levels in primary hyperparathyroidism.

Twenty-two patients, all with surgically proven primary hyperparathyroidism, were studied by TI-201 thallous chloride and Tc-99m pertechnetate subtraction imaging. Fifteen parathyroid adenomata and one hyperplastic gland between 0.33 and 14.8 g were correctly localized in 16 patients. Two adenomata and seven hyperplastic or histologically normal parathyroids between 0.1 g and 1.4 g in seven patients were not localized. One patient had a correctly localized 13.0-g adenoma with a nonlocalized 0.3 g hyperplastic parathyroid gland and there were two false positive localizations. Sensitivity was 64% (glands), and 73% (patients). There was only fair correlation with parathormone (PTH) levels, but these were elevated in all but four of the patients with correctly localized parathyroids. The authors conclude that the imaging procedure is useful but its sensitivity is limited by difficulty in localizing correctly small glands, particularly those of less than 0.5 g, which comprised 29% of those excised.

The effect of dopamine on renal function during aortic cross clamping.

Eighteen male patients undergoing elective surgical reconstruction of the abdominal aorta were divided into two groups. Patients in Group I (nine) were given dopamine intravenously, in a dose of 2 micrograms/kg/min, during the first half of the period of cross-clamping, whilst those in Group II received dopamine during the second half. Each patient acted as his own control and for each, three periods were examined, namely: pre-clamp, clamping with dopamine and clamping without dopamine. Dopamine infusion during aortic clamping caused a significant rise in sodium output (P less than 0.01), potassium output (P less than 0.05), creatinine clearance (P less than 0.05) and urine output (P less than 0.05). We conclude that dopamine infusion during aortic clamping helps to protect the kidney from any deleterious effect of clamping.

The actions of carbenoxolone on enzymes and their relation to its therapeutic effect.

Carbenoxolone may bind to enzymes, inhibiting or activating them. Enzymes inhibited are human pepsins 1, 3 and 5, human pepsinogens 1, 3 and 5, swine pepsin, bovine trypsin, bovine chymotrypsin, porcine elastase, human gastric proteinase 2, human gastric prostaglandin 15-OH dehydrogenase and delta-5 reductase, and pronase. Enzymes activated are papain, bovine carboxypeptidase and gastric microsomal glycosyl transferase. Enzymes unaffected are human pancreatic amylase and porcine pancreatic lipase. Binding occurs away from the active site; inhibition thus occurs when binding impedes access of substrate to, or products from, the active site, and activation when access is facilitated. Carbenoxolone causes increased secretion of mucus; this action can be explained by activation of the gastric glycosyl transferases. Carbenoxolone also causes intraluminal loss of peptic activity and diminished secretion of pepsins; these actions are explained respectively by intraluminal inhibition of the pepsins and intramucosal inactivation of the pepsinogens, particularly of the peptic ulcer-associated enzyme, pepsin 1. The healing effect of carbenoxolone in peptic ulcer involves these actions together with a reduced turnover of gastric mucosal cells. Pepsins 1 and 3 have collagenolytic activity, causing release of alpha-chains from native collagens. Pepsin 1 is five-fold the more active. Carbenoxolone inhibits peptic collagenolysis.