Reevaluation of the determinants of tyrosine sulfation.

The posttranslational sulfation of tyrosine has been thought to be initiated by the recognition of specific consensus features by the sulfating enzyme tyrosylprotein sulfotransferase (TPST). However, using these recognition features to identify new tyrosine sulfation sites misses recently characterized sites that lack these features. Rigorous analysis of the amino acids surrounding the target tyrosine using the position-specific scoring matrix (PSSM) demonstrates that a consensus sequence does not contain all the information necessary to predict tyrosine sulfation. Instead, accurate prediction requires consideration of all residues within five amino acids on either side of the target tyrosine. These results support the notion that secondary structure is the major determinant of sulfation and that other residues within the sulfation site can compensate for deviations from commonly observed features. This view implies that specific consensus features are not critical for TPST substrate recognition but that TPST may instead broadly recognize any sufficiently exposed tyrosine residue.

Tuberculosis testing. Physician attitudes and practice.

OBJECTIVE: To assess physician agreement with and adoption of American Academy of Pediatrics' (AAP) recommendations on tuberculosis screening in children. DESIGN AND PARTICIPANTS: Survey of a random sample of 1272 community pediatricians and family physicians (excluding academic institutions) in 4 mid-Atlantic states and the District of Columbia. RESULTS: The response rate was 66%. Seventy-five percent of the respondents were aware of the 1994 AAP screening recommendations. Most (64%) test children at low risk periodically (at age 1 year, preschool age, and adolescent age), and 81% test children at high risk annually. Eighty-one percent of the respondents estimated that 10% or less of their patients were at high risk for tuberculosis. Most use patient-specific factors, geographic or community prevalence, or both as principal criteria to determine risk. Eighty-seven percent used multiple-puncture tests in 1993; this declined to 55% in 1994. Multiple-puncture tests are still used exclusively by 29% of the respondents. Ninety-one percent of those using multiple-puncture tests and 19% of those using the Mantoux test allow parents to read the test reaction. Forty-six percent of the respondents believed that if a return visit was required for reading the test reaction, 50% or less of their patients would return. Only 22% of the respondents adhere to the 1994 AAP recommendations regarding frequency, testing method, and reading the test reaction. Factors associated with adherence to AAP recommendations included physician and practice characteristics and knowledge of AAP recommendations. CONCLUSIONS: Physician practice of tuberculosis testing varies widely; most do not adhere to the 1994 AAP recommendations. Most physicians vary the frequency and testing method based on a patient's risk status. The use of multiple-puncture tests has declined, although they are still widely used. Accurate reading of screening tests remains a major concern.

Evidence that CCK-58 has structure that influences its biological activity.

Many biologically active peptides exist in multiple molecular forms, but the functional significance of regions outside the region of bioactivity is unknown. The biological and immunological data presented in this study indicate that cholecystokinin-58 (CCK-58), unlike other forms of cholecystokinin, has structure that influences its bioactivity. CCK-58 was purified from acid extracts of canine intestinal mucosa until a single absorbance peak was obtained during reverse-phase chromatography. Amino acid analysis precisely determined the peptide concentrations of purified CCK-58 and synthetic CCK-8. Our hypothesis was that if the amino terminus of CCK-58 influences its bioactivity then its activity would be modified when this region was removed from the peptide. To evaluate the importance of the amino terminus of CCK-58 to influence its biological activity, the abilities of CCK-58 and CCK-8 to release amylase from pancreatic acini were compared before and after tryptic digestion. Tryptic digestion of CCK-58 decreased the half-maximal stimulation (EC50) for amylase release from 96 to 28 pM. The EC50 for digested CCK-58 was similar to that for CCK-8 (17 pM). These results suggest that CCK-58 has a structure that shields its bioactive carboxyl terminus. This is further supported by the finding that carboxyl fragments generated from CCK-58 by trypsin or by partial acid hydrolysis were greater than twofold more immunoreactive than the intact CCK-58. The diminished activity of CCK-58 SK shields the carboxyl terminus, which is important to its biological and immunological activities.

Diets causing taurine depletion in cats substantially elevate postprandial plasma cholecystokinin concentration.

Excessive secretion of the intestinal hormone cholecystokinin (CCK) was postulated to cause diet-related taurine depletion in cats. To test this hypothesis, plasma CCK-like immunoreactivity (CCK-LI) was measured in cats given four diets, two purified and two canned, that contained similar concentrations of protein, fat, moisture and taurine but produced variable rates of taurine depletion. Plasma CCK-LI was measured by RIA with a tyrosine-sulfate specific, C-terminal anti-serum, validated for use in cat plasma. As indicated by measurements of taurine in whole blood and urine, a purified diet containing casein maintained body taurine, whereas the same diet containing soybean protein and a commercial canned diet preserved either by freezing or cooking depleted body taurine. Preprandial and peak postprandial plasma CCK-LI in cats given the casein-containing diet were 10.6 +/- 1.4 and 27.6 +/- 4.8 pmol/L, respectively, approximately two- to tenfold greater than those reported in humans. Integrated postprandial plasma CCK-LI was less for cats given the casein diet than cats given both forms of the canned diet; it tended to be lower in cats given the casein diet than in cats given the soy protein diet. A negative linear correlation was observed between apparent nitrogen digestibilities of the diets and integrated plasma CCK-LI. The results indicated that diets that cause taurine depletion have lower protein digestibilities and cause greater endocrine secretion of CCK than diets that maintain body taurine status.

Elevation of plasma cholecystokinin (CCK) immunoreactivity by fat, protein, and amino acids in the cat, a carnivore.

The cat requires a diet high in protein and certain nutrients that are found only in animal tissue. It is possible that secretogogues of intestinal CCK in the cat may be different from those observed in non-carnivorous species. Plasma CCK concentrations were determined in cats (n = 6) given by oral-gastric tube either casein, whey protein, corn oil, or corn starch suspended in water. CCK was measured by RIA with a tyrosine sulfate-specific, C-terminal antibody, DINO. HPLC of plasma revealed that most CCK-immunoreactivity (CCK-LI) was associated with CCK-33 and a late eluting peak, presumably CCK-58. Casein, whey protein, and corn oil increased (P < 0.05) post-administration plasma CCK-LI, and at least for casein, the effect was dose related. An amino acid mixture approximating the residue composition of casein increased plasma CCK-LI (P < 0.05), however, the increase tended to be less than that caused by casein. Evaluation of post-administration levels of plasma amino acids indicated that intact protein and amino acids in the intestinal lumen affect CCK release by different mechanisms. Collectively, the results indicated that although cats are carnivores cats and humans secrete CCK in response to the same nutrients.

Cholecystokinin suppresses food intake by a nonendocrine mechanism in rats.

A cholecystokinin monoclonal antibody (CCK MAb) was used to immunoneutralize CCK to test the hypothesis that CCK produces satiety by an endocrine mechanism. We first characterized the effects of CCK MAb on pancreatic secretion. Conscious rats with jugular vein and bile-pancreatic duct cannulas received CCK MAb or control antibody intravenously 30 min before a 2-h maximal dose of CCK-8 (200 pmol.kg-1.h-1 i.v.) or access to food. CCK MAb caused dose-related inhibition of amylase secretion. CCK MAb (2 mg/kg) completely blocked the response to CCK-8 and inhibited the response to food by 89%. In feeding experiments, rats with free access to food received CCK MAb or control antibodies (2 mg/kg iv) 2 h after lights off. CCK MAb had no effect on 1.5- or 3.5-h food intake. Another group of rats received CCK MAb (4 mg/kg i.v.) or a combined injection of type A and type B CCK receptor antagonists devazepide and L-365,260 (1 mg/kg each i.v.). CCK MAb had no effect on feeding, whereas the receptor antagonists stimulated 1-, 2-, 3-, and 4-h intake by 62, 45, 43, and 29%. These results suggest that endogenous CCK stimulates pancreatic enzyme secretion at least partially by an endocrine mechanism and produces satiety by a nonendocrine mechanism.

Analysis of sequence requirements for protein tyrosine sulfation.

We analyzed sequences surrounding known tyrosine sulfation sites to determine the characteristics that distinguish these sites from those that do not undergo sulfation. Tests evaluated the number and position of acidic, basic, hydrophobic, and small amino acids, as well as disulfide and N-glycosylation (sugar) sites. We determined that composition-based tests that select close to 100% of known tyrosine sulfation sites reject 97% of the non-sulfated tyrosines. The acidic test, by far the most selective, eliminated 95% of the non-sulfated tyrosine residues and none of the sulfated tyrosines. Including the basic, hydrophobic, and disulfide tests increased the elimination rate to 97%. Whereas no position flanking the tyrosine residues had the same amino acid always present, imperfectly conserved amino acids found in some positions will improve the specificity of the tests.

Enzymatic sulfation of gastrin in rat gastric mucosa.

An enzyme which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to gastrin (G17) was identified in rat gastric mucosal cells. The enzyme activity was detected in the 105,000xg supernatant fraction. Formation of gastrin sulfate was shown by using 125I-gastrin and non-radioactive PAPS. The product was sensitive to acid hydrolysis, arylsulfatase treatment and removed by gastrin antibody, but not changed by treatments with chondro-4-sulfatase and chondro-6-sulfatase. The product had a molecular weight of 2050 daltons, close to the molecular weight of G17 sulfate, and, therefore, indicating the sulfated product is not APS derived from the degradation of PAPS. The enzyme activity showed a Km value of 5 microM for PAPS and a pH optimum of 6.0. The activity was not detected in the liver preparation.

Characterization of the major form of cholecystokinin in human intestine: CCK-58.

Acid extracts of human intestines obtained from surgical samples or from organ donors contain cholecystokinin (CCK) immunoreactivity. From surgical samples, extracted and eluted quickly, greater than 75% of the CCK immunoreactivity eluted in the same region as purified canine CCK-58 during analytical reverse-phase high-pressure liquid chromatography (HPLC). A major portion of the CCK immunoreactivity from donor intestinal extracts also eluted in this region. This immunoreactivity has been purified from human intestinal extracts by a series of several reverse-phase and cation-exchange chromatographies. Amino acid and microsequence analysis showed that this immunoreactivity is human CCK-58. Tryptic digestion of purified human CCK-58 produced another immunoreactive form that eluted in the position of CCK-8 during analytical reverse-phase HPLC. The immunoreactivity of the trypsin-digested material was 2.6-fold higher than that of an identical sample of CCK-58 incubated without trypsin. Thus the carboxyl-terminal antibody used for radioimmunoassay cross-reacts greater than twofold less with human CCK-58. This diminished cross-reactivity would lead to an underestimation of the relative proportions of CCK-58 in tissue and plasma extracts. If CCK-58 is the major circulating form this diminished cross-reactivity would also lead to underestimations of the circulating levels of total CCK. Determination of human CCK-58 structure confirms that one of the major components of human CCK that expresses biological activity is CCK-58.

Identification of serotonin from rabbit upper stomach as a stimulant of in vitro gallbladder contraction.

Using an in vitro rabbit gallbladder bioassay, the distribution and identification of bioactive substances in rabbit gastrointestinal tract were investigated. Comparison of the bioactivities of tissue extracts before and after cholecystokinin was removed by affinity chromatography demonstrated that the distributions of cholecystokinin and non-cholecystokinin substances were different. While cholecystokinin bioactivity per g of tissue was highest in the duodenum, non-cholecystokinin bioactivity was greatest in the upper stomach. The biochemical properties of the non-cholecystokinin substance in the upper stomach could not be distinguished from those of serotonin. These included molecular weights of 176, identical ultraviolet spectra, similar nuclear magnetic resonance spectra, and co-chromatography in HPLC. By weight, serotonin had 1/6th of the bioactivity of cholecystokinin octapeptide. We conclude that the principal gallbladder-contracting substance in rabbit upper stomach is serotonin.

Beta-endorphin is present in active and inactive forms in rat gastric antrum.

Lipotropin, beta-endorphin and a series of peptides related to beta-endorphin were extracted from rat antrum and resolved by gel filtration, ion exchange chromatography and high pressure liquid chromatography; the concentrations of the peptides were determined by radioimmunoassay. The major peptide with beta-endorphin immunoreactivity present in the antrum was lipotropin but it was accompanied by substantial quantities of beta-endorphin in its biologically active form; in addition there were minor quantities of a number of inactive beta-endorphin related peptides. The experiments demonstrate that in rat antrum gastrin can be accompanied by both active and inactive forms of beta-endorphin. The implications of post-translational processing mechanisms common to gastrin and beta-endorphin are discussed.

The effect of graded hypoxia on the embryonic chick heart.

Embryonic ventricular function in the chick was measured in response to graded levels of hypoxia. Myocardial contractility, as measured by cinephotoanalysis and expressed as shortening fraction, was significantly depressed after 1 hour of moderate hypoxia (6% O2) and after 5 hours of milder (16% O2 and 11% O2) levels of hypoxia (P less than .05). Microscopy confirmed associated myocyte damage with cell death noted after 5 hours of moderate hypoxic stress. Heart rate change was not related to the severity of hypoxia. The greatest level of tachycardia was noted with conditions of mildest hypoxia (16% O2). The data confirm that cardiac contractility, as measured by shortening fraction, is depressed on exposure to hypoxia, with impairment of function related to the severity of the hypoxic conditions.

Small left atrium and change in contour of the ventricular septum in total anomalous pulmonary venous connection: a morphometric analysis of 22 infant hearts.

Morphometric measurements of 22 hearts with total anomalous pulmonary venous connection (TAPVC) were compared with measurements of 8 matched control specimens without heart disease. Each of the TAPVC specimens had a shorter left atrium, smaller left atrial surface area and larger diameter of the fossa ovalis. In addition to increased length of the right ventricle and larger circumferences for tricuspid and pulmonary valve anuli, the left ventricular contour of the ventricular septum was flat or convex in 18 of the 22 hearts; the septum was significantly longer than normal in these specimens and wider at its midportion. Because mitral and aortic valve anuli were normal in circumference, the data suggest that left ventricular volume is not decreased despite change in ventricular shape.

Evidence of a noncholecystokinin stimulant of gallbladder contraction: comparison of fasting serum concentrations in healthy subjects and in patients with gallstones.

Some investigators have reported that patients with gallstones empty their gallbladders more rapidly than do healthy subjects. This may contribute to the formation of lithogenic bile. To date, cholecystokinin is considered the prime mediator of gallbladder contraction. Evidence exists that cholecystokinin may not be the major hormone accounting for gallbladder emptying. The purpose of this study was to demonstrate the existence of this noncholecystokinin substance in healthy persons and to compare its concentration with that in patients with cholesterol gallstones. Fasting serum levels from 15 healthy human subjects (8 women and 7 men, mean age 32 +/- 8 years) and 10 patients with cholesterol gallstones (5 women and 5 men, mean age 48 +/- 16 years) were studied. Using rabbit in vitro gallbladder bioassay and cholecystokinin-8 as standards, serum bioactivity was measured and expressed as cholecystokinin-8 equivalent bioactivity. The effectiveness of serum to contract the gallbladder was tested before and after removal of cholecystokinin from the serum. Cholecystokinin was removed from the serum samples by affinity chromatography with Sepharose 4B beads coated with cholecystokinin 5135 antibody. Gallbladder contractility from this treated serum thus reflects the action of a noncholecystokinin stimulant. The cholecystokinin-8 bioactivity equivalents in untreated samples from healthy subjects and from patients with gallstones were 2.9 +/- 0.3 and 7.6 +/- 0.7 ng/ml, respectively. The fact that bioactivity of serum persisted after removal of cholecystokinin in both groups of subjects provides evidence that a noncholecystokinin stimulant of gallbladder contraction exists. This substance is found in significantly higher concentrations in the fasting serum of patients with gallstones compared with healthy subjects. This finding may explain, at least in part, the increased gallbladder emptying rate in patients with gallstones and may account for the reduced bile salt pool size and, thus, formation of lithogenic bile.

Distribution and molecular heterogeneity of cholecystokinin-like immunoreactive peptides in the brain and gut of the rainbow trout, Salmo gairdneri.

Cholecystokinin-like immunoreactivity (CCK-li) was measured in extracts of various brain and gut regions of the rainbow trout, Salmo gairdneri. All regions of the brain except the cerebellum and pituitary contained detectable CCK-li. In the gut, the highest concentrations of CCK-li were found in the small intestine and pyloric caeca. Lesser amounts were found in rectum and gastric antrum extracts. In some fish, extracts of these regions contained no detectable CCK-li. Rainbow trout brain extracts contained CCK-li that co-eluted with CCK-8 in gel permeation chromatography whereas CCK-li extracted from the various gut regions exhibited marked molecular heterogeneity. Tissue distribution and apparent molecular size of CCK-li in trout is very similar to mammals for brain but distinct from the mammalian pattern in the gut.

Effect of conotruncal constriction on aortic-mitral valve continuity in the stage 18, 21 and 24 chick embryo.

The effect of conotruncal constriction on the development of aortic-mitral valve continuity in stage 18, 21 and 24 chick embryos was studied. A 10-0 nylon suture was tied around the conotruncus, constricting the outflow tract of the heart. The loop was removed after 4 or 24 hours or left permanently in place in 3 subgroups of the 236 experimental embryos. The embryo hearts were harvested at stages after completion of cardiac morphogenesis, fixed in end-diastole and microdissected. The distance between the mitral and aortic anuli was measured from the base of the heart with a calibrated filar micrometer eyepiece. This measurement was compared with the mitral-aortic separation in 72 normal and 132 control embryos. The mitral-aortic separation was similar among normal, control and 4- and 24-hour experimental embryo hearts. However, the mitral-aortic separation increased from 0.34 +/- 0.02 mm in normal hearts to 0.82 +/- 0.25 mm in stage 18, 1.11 +/- 0.36 mm in stage 21 and 0.75 +/- 0.33 mm in stage 24 permanent loop experimental hearts (p less than 0.01). In embryo hearts with an increased mitral-aortic separation, both great vessels arose from the right ventricle, the semilunar valves were at the same level and the ventricular septal defect was present beneath the aortic anulus. We conclude that conotruncal constriction modifies the relation of the aortic and the mitral valve. We speculate that conotruncal constriction alters the migration of mesenchymal tissue into the heart.