Grace L. Rosenquist

The morphology and distribution of peptide-containing neurons in the adult and developing visual cortex of the rat. I. Somatostatin

SummaryUsing conventional immunocytochemical techniques, we have examined the morphology and distribution of somatostatin-like immunoreactive neurons in the visual cortex of albino rats between the first postnatal day and maturity. In the adult, somatostatin-immunoreactive neurons were observed in layers II to VI but were concentrated in layers II and III. These cells displayed morphological features characteristic of the multipolar and bitufted varieties of cortical non-pyramidal neurons as described in Golgi preparations of rat visual cortex.On the first postnatal day and in the subsequent few days, immunoreactivity was confined to immature bipolar and multipolar neurons concentrated in layers V and VI. Labelled cells first appeared in the more superficial layers at the beginning of the second postnatal week and attained a distribution similar to that observed in adult animals at the end of this week. At this time they closely resembled their adult counterparts from which they appeared indistinguishable by the end of the third postnatal week. The late appearance of labelled cells in the superficial layers, where they are predominantly located in adult animals, suggests that the somatostatin immunoreactivity exhibited by most of these neurons develops several days after they have completed their migration and assumed their positions in the visual cortex.

Tyrosine sulfation is prevalent in human chemokine receptors important in lung disease.

Post-translational sulfation of tyrosines affects the affinity and binding of at least some chemokine receptors to their ligand(s) and has been hypothesized to be a feature in all chemokine receptors. This binding initiates downstream signaling cascades. By this mechanism, tyrosine sulfation can influence the cells involved in acute and chronic events of cellular immunity. These events include leukocyte trafficking and airway inflammation important in asthma and chronic obstructive pulmonary disease (COPD). We are using computational methods to convert the poorly defined hypothesis of more widespread sulfation of chemokine receptors to more specific assessments of how closely the sequence environment of each tyrosine residue resembles the sequence environment of tyrosine residues proven to be sulfated. Thus, we provide specific and readily tested hypotheses about the tyrosine residues in all of the chemokine receptors. Tyrosine sulfation was predicted with high scores in the N-terminus domain of 13 out of 18 human chemokine receptor proteins using a position-specific scoring matrix, which was determined to be 94.2% accurate based on Receiver Operating Characteristic analysis. The remaining chemokine receptors have sites exhibiting features of tyrosine sulfation. These putative sites demonstrate clustering in a manner consistent with known tyrosine sulfation sites and conservation both within the chemokine receptor family and across mammalian species. Human chemokine receptors important in asthma and COPD, such as CXCR1, CXCR2, CXCR3, CXCR4, CCR1, CCR2, CCR3, CCR4, CCR5, and CCR8, contain at least one known or predicted tyrosine sulfation site. Recognition that tyrosine sulfation is found in most clinically relevant chemokine receptors could help the development of specific receptor-ligand antagonists to modulate events important in airway diseases.

Geoepidemiology of COPD and idiopathic pulmonary fibrosis

Progress in improving patient outcomes and advancing therapeutics in chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) is hampered by phenotypic heterogeneity and variable responsiveness to clinical interventions that are not fully explained by currently held disease paradigms for COPD and IPF. Although these chronic lung diseases differ in their geoepidemiology and immunopathogenesis, emerging evidence suggest that organ-specific autoimmunity may underlie subphenotypes of COPD and IPF. In particular, the links to tobacco smoking, diet, gender, and environment are explored in this review. We also highlight potential mechanisms that could guide future investigations in both laboratory and clinical settings. A paradigm shift is needed in how we think about COPD and IPF, based on geoepidemiology and a broader understanding of disease pathogenesis that may ultimately lead to new therapies and improved patient outcomes.

Reevaluation of the determinants of tyrosine sulfation.

The posttranslational sulfation of tyrosine has been though 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 tyrosin 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.

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.

Cholecystokinin-like immunoreactivity in frog retina: Localization, characterization, and biosynthesis

We sought to localize and characterize cholecystokinin-like immunoreactivity (CCK-LI) in frog retinal tissues, CCK-LI was present in concentrations of 2.20 +/- 0.19 pmol/g wet weight (mean +/- SE, n = 6) in water extracts of frog retina and 0.23 +/- 0.04 pmol/g in acid extracts. The predominant form of CCK-LI co-eluted with the octapeptide of CCK on Sephadex G50 column chromatography. A minor form which eluted as a molecule larger than the 39 amino acid form of CCK was found only in the acid extracts. By immunohistochemistry, CCK-LI was localized to amacrine cells which give rise to processes ramifying in the distal 2/3 of the inner plexiform layer. Time dependent uptake of 35S-methionine into CCK-LI was observed in cultured frog retinas and this uptake was blocked with 0.2 mM cycloheximide. Our findings suggest that the retina may be a useful system for further characterization of the neural function of CCK-LI.

Prediction of tyrosine sulfation in seven-transmembrane peptide receptors

Posttranslational modification by tyrosine sulfation regulates many important protein-protein interactions and modulates the binding affinity and specificity of seven-transmembrane peptide receptors. We developed a log-odds position-specific-scoring-matrix (PSSM) to accurately predict tyrosine sulfation using 62 tyrosine sites known to be sulfated and 421 tyrosine sites known not to be sulfated. We predict that 49 tyrosines of 32 seven-transmembrane peptide receptors are sulfated. Although we did not incorporate characteristics of confirmed sulfation sites such as clustering and conservation across species into our PSSM, our predicted sites nevertheless exhibited these characteristics. The observed conservation suggests that there are strong evolutionary pressures to preserve selected biological activity of seven-transmembrane receptors. The predicted tyrosine sulfation sites predominantly occur in the extracellular tail and extracellular loop 2, regions consistent with their association with binding pockets of the receptor.

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.

Noncholecystokinin peptides in human serum which cause gallbladder contraction

In fasting human serum, cholecystokinin (CCK) is not the principal substance which causes in vitro rabbit gallbladder contraction. Removal of CCK by affinity chromatography from fasting sera from 8 healthy adults reduced bioactivity only by 18 +/- 4% (SEM). Unlike CCK, the bioactivity of serum was enhanced by 30 to 57% rather than destroyed by pronase and chymotrypsin respectively and was not inhibited by dibutyryl cGMP. Reduction of serum bioactivity by carboxypeptidase Y indicated that the bioactive substances in serum are peptides. On Sephadex G-50, bioactive substances eluted in positions different from any known form of CCK. Thus, the principal substances in fasting human serum causing in vitro gallbladder contraction are not CCK but are most likely small peptides which act at receptors different from the receptors for CCK.

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.