1H-NMR studies of receptor-selective substance P analogues reveal distinct predominant conformations in DMSO-d6

Proton nmr parameters are reported for DMSO-d6 solutions of two receptor-selective substance P analogues: Ac[Arg6,Pro9]SP6-11, which is selective for the NK-1 (SP-P) receptor and [pGlu6,N-MePhe8]SP6-11, which selectively activates the NK-3 (SP-N) receptor. Full peak assignments of both analogues were obtained by COSY experiments. The chemical shifts, coupling constants, and temperature coefficients of amide proton chemical shifts as well as NOESY effects and calculated side-chain rotamer populations of Phe side chains are reported for both peptides. Analysis of coupling constants and temperature coefficients together with the nuclear Overhauser enhancement spectroscopy effects suggest that Ac[Arg6,Pro9]SP6-11 has a trans configuration about the Phe8-Pro9 amide bond and the preferred conformation of this analogue has a type I beta-turn. The nmr data for [pGlu6,N-MePhe8]SP6-11 suggest that this peptide exists as a mixture of cis-trans isomers in which the cis isomer can preferably adopt a type VI beta-turn conformation, and the trans isomer can adopt a gamma-turn conformation. There are indications that the two last turns are stabilized by a hydrogen bond between the syn carboxamide proton and the pGlu ring carbonyl.     

Three unique base pair changes in a family with Gaucher disease

Summary Single-stranded cDNA was prepared from RNA obtained from a patient with type 1 Gaucher disease. The cDNA was amplified in vitro and analyzed by sequencing. Three base-pair changes were identified which included a G to C transversion at nucleotide 3119 of the active gene (Asp¹⁴⁰His), an A to C transversion at nucleotide 3170 (Lys¹⁵⁷Gln) and a G to A change at nucleotide 5309 (Glu³²⁶Lys). To study the mode of inheritance of the three different base-pair changes, genomic DNA was prepared from blood or skin fibroblasts of several family members. Genomic glucocerebrosidase DNA sequences were amplified and subjected to hybridization with allele-specific oligonucleotides (ASOs). The hybridization profiles demonstrated that two of the basepair changes originated from the mother and were transmitted to her two affected sons and to a grandchild, while the third base-pair change, originating from the father, was transmitted to his two affected sons, a carrier daughter and a second grandchild. Tests of other patients with Gaucher disease failed to disclose the presence of the three base-changes. This is a unique family with three base-pair changes tightly linked to Gaucher disease.     

Oligosaccharides from placenta: early diagnosis of feline mannosidosis

High-pressure liquid chromatography analysis of oligosaccharides from placentas allowed the diagnosis of alpha-mannosidosis in three litters of kittens. The chromatography also afforded a detailed comparison of the oligosaccharide pattern and levels in placenta, liver, brain, urine and ocular fluid of the affected animals. In all cases, two series of compounds were observed, with one or two residues of N-acetylglucosamine at the reducing terminus, respectively, and between two and nine mannose residues. This pattern is unlike that of human mannosidosis, and resembles that of ruminants, except that the major oligosaccharide contains three mannose residues instead of two.     

Two abnormalities of hexosaminidase A in clinically normal individuals

Two abnormalities of beta-hexosaminidase A (HEX A) activity are described. One, found in two unrelated Jewish children, was characterized by the complete absence of HEX A activity in serum, but low levels of activity in leukocytes and fibroblasts using artificial substrate. The other, found in a non-Jewish man, was characterized by uniformly low levels of HEX A activity in leukocytes, fibroblasts, and serum against artificial substrate. In all cases, the pH optimum of HEX A was normal, there was no increased lability at 37 degrees C, and no inhibitor was detected to account for the deficiency of activity. Cultured fibroblasts of these individuals were capable of synthesizing and processing alpha- and beta-subunits of HEX A and capable of cleaving GM2 ganglioside. The patients, ranging in age from 6 to 30 years, are clinically normal. They are probably genetic compounds carrying the classical Tay-Sachs gene and a differently mutated allele that imparts the anomalous phenotypic features observed.     

Mannose metabolism in corn and its impact on leaf metabolites, photosynthetic gas exchange, and chlorophyll fluorescence

When intact corn leaves were provided millimolar concentrations of d-mannose through the transpiration stream photosynthesis was inhibited; 5.7 millimolar resulted in a 50% inhibition of the carbon exchange rate. This inhibition was partially reversible by the addition of orthophosphate to the feeding solution. Mannose metabolism by corn leaves was limited in that it did not act as a resource for sucrose or starch synthesis. Mannose 6-phosphate accumulated in the leaf tissues and was slowly metabolized by a pathway involving mannose 1-phosphate. Correlated with the mannose-6-phosphate accumulation were decreases in ATP, orthophosphate, sucrose, and phosphoenolpyruvate and increases in starch and maltose. When provided in the transpiration stream mannose had access to both mesophyll and bundle sheath cells. Mannose feeding led to oscillations in steady state chlorophyll fluorescence emission (680 nanometers) and an elimination of the Kautsky effect during fluorescence induction. Pyridoxal 5-phosphate and 2,4-dinitrophenol were found to be inhibitors of CO₂ exchange when provided in the transpiration stream of intact corn leaves. However, Pyridoxal 5-phosphate induced a quenching of steady state fluorescence while 2,4-dinitrophenol led to an increase in fluorescence emission.     

Prevalence of nine mutations among Jewish and non-Jewish Gaucher disease patients.

The frequency of nine different mutated alleles known to occur in the glucocerebrosidase gene was determined in 247 Gaucher patients, of whom 176 were of Jewish extraction, 2 were Jewish with one converted parent, and 69 were of non-Jewish origin. DNA was prepared from peripheral blood, active glucocerebrosidase sequences were amplified by using the PCR technique, and the mutations were identified by using the allele-specific oligonucleotide hybridization method. The N37OS mutation appeared in 69.77% of the mutated alleles in Jewish patients and in 22.86% of the mutated alleles in non-Jews. The 84GG mutation, which has not been found so far among non-Jewish patients, existed in 10.17% of the disease alleles among Jewish patients. The IVS + 1 mutation constituted 2.26% of the disease alleles among Jewish patients and 1.43% among the non-Jewish patients. RecTL, a complex allele containing four single-base-pair changes, occurred in 2.26% of the alleles in Jewish patients and was found in two (1.43%) of the patients of non-Jewish extraction. Another complex allele, designated "RecNciI" and containing three single-point mutations, appeared in 7.8% of alleles of non-Jewish patients and in only two (0.56%) of the Jewish families. The prevalence of the L444P mutation among non-Jewish Gaucher patients was 31.43%, while its prevalence among Jewish patients was only 4.24%. The prevalence of two other point mutations--D409H and R463C--was 5.00% and 3.57%, respectively, among non-Jewish patients and was not found among the Jewish Gaucher patient population. The prevalence of the R496H mutation, found so far only among Jewish patients, was 1.13%. The results presented demonstrate that seven mutations identify 90.40% of the mutations among Jewish patients and that these seven mutations allow diagnosis of only 73.52% of the non-Jewish patients. Identification of additional mutant alleles will enhance the accuracy of carrier detection.     

Insulin receptors in 3T3 fibroblasts : Relationship to growth phase, transformation and differentiation into new cell types

The amount of [¹²⁵I]insulin binding per 2 × 10⁶ cells is measured in three lines of mouse embryonic 3T3 fibroblast at different growth stages. Insulin binding is found to be lowest in growing cells of all three types, increasing as cells reach stationary phase. Binding in 3T3-M cells approaches a plateau as cells become stationary. Insulin binding in 3T3-L cells, many of which differentiate into adipocytes following cessation of growth, show further increase in insulin binding post-confluence, in parallel with their differentiation into adipocytes. Binding of insulin in spontaneously transformed cells is higher at all phases of growth than the other two lines, rising to a much higher eventual plateau at approx. 17 days post-confluence. Scatchard plots of insulin binding tend to reflect the same degree of relative insulin binding in these three cell lines. Previously starved cells of all three types exhibit a drop in insulin binding following their first feeding, which corresponds with a second growth spurt in response to nutrients in fresh serum. These results suggest that insulin, as reflected by binding per cell, may play only a minor role in actively growing adequately fed cells of all three types, its major role developing as these cells approach confluence. It is also suggested that higher insulin binding in transformed vs non-transformed cells may indicate a special role for insulin in the loss of contact inhibition, by preserving transport of limiting nutrients in dense, nutrient-depleted transformed cultures.     

Evidence for the emergence of β-trefoils by ‘Peptide Budding’ from an IgG-like β-sandwich

As sequence and structure comparison algorithms gain sensitivity, the intrinsic interconnectedness of the protein universe has become increasingly apparent. Despite this general trend, β-trefoils have emerged as an uncommon counterexample: They are an isolated protein lineage for which few, if any, sequence or structure associations to other lineages have been identified. If β-trefoils are, in fact, remote islands in sequence-structure space, it implies that the oligomerizing peptide that founded the β-trefoil lineage itself arose de novo. To better understand β-trefoil evolution, and to probe the limits of fragment sharing across the protein universe, we identified both ‘β-trefoil bridging themes’ (evolutionarily-related sequence segments) and ‘β-trefoil-like motifs’ (structure motifs with a hallmark feature of the β-trefoil architecture) in multiple, ostensibly unrelated, protein lineages. The success of the present approach stems, in part, from considering β-trefoil sequence segments or structure motifs rather than the β-trefoil architecture as a whole, as has been done previously. The newly uncovered inter-lineage connections presented here suggest a novel hypothesis about the origins of the β-trefoil fold itself–namely, that it is a derived fold formed by ‘budding’ from an Immunoglobulin-like β-sandwich protein. These results demonstrate how the evolution of a folded domain from a peptide need not be a signature of antiquity and underpin an emerging truth: few protein lineages escape nature’s sewing table.