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Biomedical subjects

R Nichols

Publications and source records attributed to R Nichols.

At least 91 records · Page 5Linked to original sources
PubMed1992

Isolation and structural characterization of Drosophila TDVDHVFLRFamide and FMRFamide-containing neural peptides.

An extract of adult Drosophila melanogaster was separated by gel exclusion, ion exchange, and reversed-phase chromatography. Four peptides, each with an -ArgPheNH2 C-terminal sequence, were identified by radioimmunoassay. The primary sequences were determined by Edman degradation and confirmed by mass spectrometry and sequence-specific radioimmunoassay. Three of the peptides are encoded by Drosophila proFMRFamide: AspProLysGlnAspPheMetArgPheNH2 (DPKQDFMRFamide), ThrProAlaGluAspPheMetArgPheNH2 (TPAEDFMRFamide), and SerAspAsnPheMetArgPheNH2 (SDNFMRFamide). A novel Drosophila peptide ThrAspValAspHisValPheLeuArgPheNH2 (TDVDHVFLRFamide) was also isolated. TDVDHVFLRFamide is structurally related to peptides isolated from chicken, cockroach, locust, and snail; the cockroach, fruitfly, and locust peptides differ only by the N-terminal amino acid residue. Two Drosophila neural genes, dsk and FMRFamide, are known to encode -ArgPheNH2-containing peptides; however, neither encodes TDVDHVFLRFamide, indicating that Drosophila contains another precursor encoding -ArgPheNH2 peptides.

Amino Acid Sequence↗
PubMed1991

Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein.

R67 dihydrofolate reductase (DHFR) is a novel protein that provides clinical resistance to the antibacterial drug trimethoprim. The crystal structure of a dimeric form of R67 DHFR indicates the first 16 amino acids are disordered [Matthews et al. (1986) Biochemistry 25, 4194-4204]. To investigate whether these amino acids are necessary for protein function, the first 16 N-terminal residues have been cleaved off by chymotrypsin. The truncated protein is fully active with kcat = 1.3 s-1, Km(NADPH) = 3.0 microM, and Km(dihydrofolate) = 5.8 microM. This result suggests the functional core of the protein resides in the beta-barrel structure defined by residues 27-78. To study this protein further, synthetic genes coding for full-length and truncated R67 DHFRs were constructed. Surprisingly, the gene coding for truncated R67 DHFR does not produce protein in vivo or confer trimethoprim resistance upon Escherichia coli. Therefore, the relative stabilities of native and truncated R67 DHFR were investigated by equilibrium unfolding studies. Unfolding of dimeric native R67 DHFR is protein concentration dependent and can be described by a two-state model involving native dimer and unfolded monomer. Using absorbance, fluorescence, and circular dichroism techniques, an average delta GH2O of 13.9 kcal mol-1 is found for native R67 DHFR. In contrast, an average delta GH2O of 11.3 kcal mol-1 is observed for truncated R67 DHFR. These results indicate native R67 DHFR is 2.6 kcal mol-1 more stable than truncated protein. This stability difference may be part of the reason why protein from the truncated gene is not found in vivo in E. coli.

Amino Acid Sequence↗
PubMed1991

Development and progression of aortic valve stenosis: atherosclerosis risk factors--a causal relationship? A clinical morphologic study.

Hospital records of 120 patients undergoing aortic valve replacement were retrospectively reviewed for risk factors associated with early aortic valve replacement. Patients were separated into four groups (rheumatic, congenital bicuspid, degenerative, and miscellaneous) based upon the morphologic etiology of aortic stenosis. Multiple regression analysis was performed using age at surgery as the dependent variable. Independent variables for the study were race, gender, systemic hypertension, total triglyceride level, total serum cholesterol level, tobacco smoking history, diabetes mellitus, and angiographic coronary artery disease. In the rheumatic valve group only race showed a statistically significant risk effect whereas in the congenital bicuspid valve group race, gender, and triglycerides were statistically correlated with age at surgery. In the degenerative valve group gender and smoking were found to be statistically significant risk factors. The establishment of aortic stenosis risk factors might be an indication for clinical trials of risk factor modification in patients with aortic stenosis.

Adult↗
PubMed1991

19-Nordeoxycorticosterone synthesis by rat kidney inner medullary collecting duct cells.

19-Nordeoxycorticosterone (19-nor-Doc), a potent mineralocorticoid, was found to be synthesized by the isolated rat kidney perfused by an adrenal precursor (19-oxo-Doc). To determine if this bioconversion is a function of renal tubular cells, various adrenal precursors of 19-nor-Doc were added separately to rat kidney inner medullary collecting duct cells culture media at a concentration of 10 nM. While 4.6% +/- 1.0% of 19-oxo-Doc (n = 3) and 14.4% +/- 1.4% of 19-oic-Doc (n = 3) were converted to 19-nor-Doc after 24 hours of incubation, Doc, and 19-OH-Doc were not converted. This represents further evidence that Doc has to be metabolized to 19-oxo-Doc or 19-oic-Doc (19-carboxy-Doc) before it can be converted by the kidney inner medullary collecting duct cells to 19-nor-Doc.

Analysis of Variance↗
PubMed1990

Serum thyroid hormone concentrations in clinically normal dogs after administration of freshly reconstituted vs previously frozen and stored thyrotropin.

Concentrations of serum thyroxine (T4) and 3,3',5-triiodothyronine (T3) were determined in 7 clinically healthy adult dogs before and after administration of freshly reconstituted thyrotropin (TSH) and TSH that had been previously reconstituted and frozen for 1, 2, and 3 months. The 4 TSH response tests were performed at 30-day intervals by collecting blood samples for serum T4 and T3 determinations before and 4 and 6 hours after IV administration of TSH (0.1 U/kg of body weight). Baseline serum concentrations of T4 and T3 were similar at each of the 4 sample collection times over the 3-month period of the study. Mean serum concentrations of T4 and T3 increased significantly (P less than 0.01) over baseline values after administration of freshly reconstituted TSH or TSH that had been previously frozen for 1, 2, or 3 months. Significant difference was not found in the mean post-TSH serum T4 or T3 concentration after injection of freshly reconstituted TSH or TSH that had been previously frozen for 1, 2, or 3 months. In 2 of the 7 dogs, mild reactions--mild ataxia and weakness--were observed during the last of the series of TSH response tests (ie, after IV administration of TSH that had been previously frozen for 3 months). Results of this study suggest that for use in dogs, reconstituted TSH stored at -20 C maintains adequate biological activity for at least 3 months. The ability to store reconstituted TSH for a longer period than the recommended 48 hours represents an economic advantage, because it allows clinicians to perform more TSH response tests per vial of TSH.

Animals↗
PubMed1990

Problems associated with medical therapy of canine hyperadrenocorticism.

The cause of hyperadrenocorticism determines treatment. Pituitary-dependent hyperadrenocorticism (PDH) can be treated surgically with bilateral adrenalectomy or hypophysectomy, or it can be managed medically with the adrenocorticolytic agent o,p'DDD or the cortisol synthesis inhibitor ketoconazole. Unilateral adrenocortical tumors should be surgically removed because of metastatic potential, although medical therapy has been used with some success. Regardless of the treatment method chosen, canine Cushing's syndrome cannot be treated easily, inexpensively, or without close monitoring and follow-up.

Adrenal Cortex Neoplasms↗
PubMed1990

Concurrent illness and complications associated with canine hyperadrenocorticism.

Spontaneous canine hyperadrenocorticism is a disorder caused by the excessive production of cortisol by the adrenal cortex. Without treatment, hyperadrenocorticism is generally progressive with an unfavorable prognosis. Many factors, however, influence prognosis, including rate of progression, severity, and underlying cause. In untreated hyperadrenocorticism, death may result from metastasis of an adrenocortical carcinoma, growth of a pituitary tumor, or most commonly from complications associated with sustained hypercortisolism itself such as hypertension, cardiovascular disease, thromboembolism, glucose intolerance, and increased susceptibility to infection. This chapter will discuss the origin, diagnosis and, in some cases, treatment of the many complications associated with hyperadrenocorticism in the dog.

Acute Disease↗
PubMed1990

Polyuria and polydipsia. Problems associated with patient evaluation.

Primary disorders of water balance (central diabetes insipidus, congenital nephrogenic diabetes insipidus, and psychogenic polydipsia) should always be considered in the differential diagnosis of polyuria and polydipsia. In general, animals with these disorders have only one laboratory abnormality, a low urine specific gravity. The more common causes of polyuria and polydipsia (eg, hypercalcemia, chronic renal insufficiency, pyelonephritis, hyperadrenocorticism), in most instances, have specific and obvious abnormalities associated with the complete blood count (CBC), serum chemistry profile, and urinalysis. However, in some cases, a low urine specific gravity may initially be the only abnormality in these more common ruleouts. The workup for polyuria and polydipsia, especially in those cases with normal or near normal blood work, can be tedious, time consuming, confusing, and not without significant patient morbidity. This chapter will focus on the problems associated with diagnostic testing used to evaluate animals with disorders of water balance.

Animals↗
PubMed1988

Identification and characterization of a Drosophila homologue to the vertebrate neuropeptide cholecystokinin.

Homologues to the cholecystokinin (CCK)-gastrin peptide family have been cloned from Drosophila. The CCK-like precursor found in Drosophila has been designated drosulfakinin (DSK). Genomic and cDNA clones corresponding to the Drosophila neuropeptide precursor encode for three putative peptides. The three peptides (DSK-0, Asn-Gln-Lys-Thr-Met-Ser-Phe-Gly; DSK-I, Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-Gly; DSK-II, Gly-Gly-Asp-Asp-Gln-Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-Gly) are flanked by prohormone processing sites and contain C-terminal glycyl residues, a potential amidation site. Two of the peptides, DSK-I and DSK-II, are homologous to CCK-gastrin peptides. Each of the two homologues include a CCK-gastrin-like C-terminal pentapeptide and a conserved sequence corresponding to the sulfated tyrosine in bioactive CCK. The third peptide encoded by the drosulfakinin precursor represents a novel peptide. In situ tissue hybridization indicates the presence of the transcript in the adult head. Chromosomal localization maps the gene to the third chromosome near 81F.

Amino Acid Sequence↗
PubMed1988

Rapid identification of clones using the same degenerate oligonucleotide mixture for both screening and sequencing.

A simple and rapid strategy for distinguishing between positively hybridizing colonies and false positive-hybridization signals is described. The isolation of a specific DNA sequence depends on the ability to distinguish between a clone that contains the correct sequence and a false hybridization-positive or background signal. This procedure utilizes the same oligonucleotide mixture both as a screening probe and as a sequencing primer. The mixture of oligonucleotides is used as a primer to obtain sequence information directly from double-stranded DNA. Conditions for sequencing with oligonucleotides having up to 64-fold degeneracy are described. Since the sequence information obtained is directly adjacent to the site of oligonucleotide:DNA hybridization, it is necessary to know only a minimal length of DNA or peptide sequence to both design oligonucleotide probes and confirm the authenticity of the hybridization positives. The advantages of the degenerate oligonucleotide sequencing method include the rapid, reliable identification of authentic versus false hybridization positives made directly without subcloning into single-stranded M13 phage, without sequencing large regions of DNA, or without synthesizing sequence-specific primers.

Animals↗
PubMed1988

Pancreatic proglucagon processing: isolation and structures of glucagon and glucagon-like peptide from gene I.

The anglerfish endocrine pancreas expresses two different genes for preproglucagon. The regions of the two proglucagons that correspond to glucagon have different sequences, as do the two glucagon-like peptides (GLPs). The products derived from processing the more abundant proglucagon-II have recently been determined. However, it was not known whether proglucagon-I was processed to similar products. The two major biologically active products of preproglucagon-I processing (glucagon-I and GLP-I) have now been purified to homogeneity. Their structures were determined using automated gas phase Edman degradation, tryptic mapping, and fast atom bombardment mass spectrometry. The preproglucagon-I-processing sites were identified. Glucagon-I represents residues 53-81, and GLP-I corresponds to preproglucagon-I-(91-124) (numbering from the initiator Met).

Amino Acid Sequence↗
PubMed1987

Characterization of primary translation products from ovine and rat salivary gland mRNAs.

Ovine and rat salivary gland mRNAs have been prepared and their translation products characterized. A 60 kD translation product from ovine submaxillary and sublingual gland mRNAs is identical in mass to the ovine apomucin. Two additional ovine translation products, 25 and 40 kD, are specific to mucin-producing salivary glands. Four rat mRNA translation products are encoded by mucin-producing salivary glands (38, 44, 67, 69 kD). These polypeptides were not detected in the parotid gland mRNAs, a serous gland. Each of these products has a high level of [3H]serine incorporation, a characteristic of mucins. The nature of these products suggests that they are mucins or mucin-like and that their molecular weights should approximate that of the corresponding apomucins.

Animals↗
PubMed1987

Post-translational processing of preprosomatostatin-II examined using fast atom bombardment mass spectrometry.

The products and an intermediate of preprosomatostatin-II processing in the anglerfish islet were purified and subjected to structural analysis. The peptides isolated identify the site of signal cleavage (between Ser-24 and Gln-25). The prohormone is further processed at Arg-97 and, to a lesser extent, at the two adjacent basic amino acid residues Lys-61 and Arg-62. A 28-residue somatostatin is also generated which can be hydroxylated at Lys-23. A proteolytic processing site which would form the 14-residue somatostatin does not appear to be used to a significant degree. Fast atom bombardment mass spectrometry (FABMS) was used to demonstrate that the amino-terminal residues of peptides 25-60, and 25-90 are pyroglutamic acid, a modification which precludes Edman degradation of these peptides. Analysis of the peptides and tryptic peptide maps by FABMS allowed confirmation of the sites of prohormone conversion and indicated that terminal basic residues were removed during processing. Three amino acid residues were also found to differ from the amino acid sequence deduced from the cDNA and were localized to specific regions by FABMS analysis. Residues found to differ from the cDNA (cDNA in parentheses) were: Asp-77 (Thr), Val-78 (Phe), and Gly-90 (Glu). Mass assignments were confirmed by running a single cycle of Edman degradation prior to FABMS. The peptides noted above were also examined by Edman sequence analysis. The sequence of a cDNA clone to preprosomatostatin-II was re-examined in light of the observed differences at the protein level. This study emphasizes the utility of FABMS in prohormone processing studies and in identification of post-translational processing events.

Amino Acid Sequence↗
PubMed1985

Characterization of Drosophila melanogaster rhodopsin.

A polypeptide present in Drosophila eye homogenates was identified as opsin. This polypeptide pI 7.8, with Mr 39,000 is a retina-specific protein. It has the spectral characteristics of rhodopsin contained in the R1-6 photoreceptors and decreases in amount with vitamin A deprivation. It contains a chromophore derived from vitamin A and linked to the protein moiety by a Schiff base. Moreover, the polypeptide identified corresponds to a retina-specific polypeptide that was shown previously to undergo light-dependent phosphorylation in living flies. These results indicate that many properties of Drosophila rhodopsin do not differ significantly from those reported for rhodopsins of other organisms. However, the isoelectric point of Drosophila opsin is considerably more basic than those reported for vertebrate rhodopsins.

Animals↗
PubMed1984

Metastases to the upper levels of the axillary nodes in carcinoma of the breast and its implications for nodal sampling procedures.

An axillary lymphadenectomy is important for the staging and treatment of metastatic disease in patients with carcinoma of the breast, especially since the nodal status is a primary criterion for using systemic adjuvant chemotherapy. As more conservative operations combined with radiation therapy have been increasingly used for selected patients with carcinoma of the breast, an axillary node sampling instead of a complete axillary dissection has been advocated by some oncologists. However, the possibility exists that node "sampling" understages patients who would otherwise have received adjuvant chemotherapy to improve their chances for cure. We retrospectively examined this hypothesis in a group of 72 patients with documented nodal metastases who had a radical mastectomy (modified or Halsted). Overall, 18 of 72 patients (25 per cent) had metastatic involvement confined to the upper axillary nodes (Levels II and III). Of the patients with no clinically palpable nodes, 32 per cent had metastatic nodal involvement confined to the upper nodes. Medial quadrant lesions exhibited this tendency more than lateral quadrant lesions (50 versus 20 per cent). Larger primary tumors were associated with an increasing likelihood of involved nodes; however, even 14 per cent of the smallest primary lesions of the breast (less than 2 centimeters) had metastases exclusively to the upper axillary region. Since approximately 40 per cent of the patients with carcinoma of the breast have nodal metastases and since 25 per cent of these metastases are confined to the upper portion of the axilla, it is estimated that at least 10 per cent of all women with carcinoma of the breast (25 X 40 per cent) would be understaged by an axillary node sampling procedure.

Axilla↗
PubMed1983

Cloning and biosynthetic studies of rat somatostatin.

The predicted amino acid sequence of rat preprosomatostatin has been obtained by cloning and subsequent DNA sequence analysis of a cDNA obtained from mRNA prepared from a rat medullary thyroid carcinoma (MTC). The predicted preprosomatostatin is 116 amino acid residues in length. Somatostatin-14 is located at the C-terminus of the preprohormone and the amino terminus contains a 'signal' peptide of 24 amino acids. A somatostatin amino terminal protein of 78 residues is found between the signal peptide and somatostatin-14. Both somatostatin-14 and somatostatin-28 are observed in the thyroid tumour C-cells. A comparison of the rat and human preprosomatostatin amino acid sequences shows only 4 substitutions observed in 116 amino acids. Patients with localized MTC often exhibit high serum calcitonin levels while patients showing cellular heterogeneity in the MTC appear to have lower calcitonin levels and a virulent neoplasia with a grave prognosis. Numerous rat MTCs have been examined by two dimensional gel electrophoresis. It is possible to distinguish characteristic differences in protein profiles of tumours producing high levels of calcitonin from those showing low calcitonin and high somatostatin levels. This analysis can be done with less than 1 mg of tissue and may represent a valuable prognostic tool in evaluating the clinical variability of MTC.

Amino Acid Sequence↗