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The value of bone marrow biopsy (BMB) in advanced breast cancer at the time of first relapse was studied in a prospective manner. Bone marrow biopsy was performed in 142 consecutive unselected metastatic patients: 129 at the time of first recurrence, and 13 in patients with metastases at the time of first diagnosis. Overall, BMB was positive in 32 patients (23%). In the group with negative bone x-ray, it was positive in two patients of 84 (2%); both of them had doubtful scan. In the group with positive x-ray, BMB resulted positive in 30 of 58 (52%). There was a significant correlation between number of bone segments radiologically involved and BMB positivity rate, ranging from 15% in the patients with only one, to 68% in those with more than three sites involved (P = 0.02). Patients with x-ray evidence of metastases in the pelvis had significantly higher rate of BMB positivity (67% versus 32%; P = 0.02). The median survival time from the first relapse was 153 weeks in BMB-negative cases and 149 in positive ones. Considering only the patients with demonstration of bone invasion obtained with either or both x-ray and BMB, 34/62 patients had positive BMB (55%). In these cases BMB was found more often positive in patients 50 years or younger than in patients older than 50 years (80% versus 47%; P = 0.05); the median survival time was longer, but not significantly, in BMB-positive patients than in negative ones (149 weeks versus 119; P = 0.3). The authors conclude that BMB is not required in common restaging procedure when both bone survey and scan are negative. Bone marrow biopsy results are more often positive in younger patients and survival is not negatively affected by bone marrow invasion as diagnosed by BMB.

The pentapeptide H-Tyr-(Gln)3-Pro-OH has been recently postulated to be the basic repetitive unit of a sequential polypeptide contained in wheat bread alpha-gliadins, which are believed to be toxic factors in coeliac disease, gluten-dependent enteropathy. Solid-phase synthesis, purification and 1H n.m.r. characterization in water solution of this peptide are described.

A study of the modes of folding and self-association of Z-Aib-L-Pro-Aib-OMe (the protected 1-3 segment of the peptaibol antibiotics alamethicin and hypelcin) in the solid state was performed using i.r. absorption and X-ray diffraction. The stereochemically constrained tripeptide molecules adopt a 4 leads to 1 intramolecularly H-bonded form (beta-turn), where the single intramolecular H-bond is found between the peptide N-H group of the Aib3 residue and the urethane C = O group of the N-blocking benzyloxycarbonyl moiety. This folded structure is stabilized by an intermolecular H-bond between the urethane N-H group of the Aib1 residue and the peptide C = O group of the Pro2 residue of a symmetry related molecule. According to the i.r. absorption data, in CH2Cl2 and TMP solutions the same intramolecularly H-bonded form occurs as that found in solid state. Compared to the situation in the solid state, in CH2Cl2 and TMP solvation of the urethane N-H group replaces self-association (through the same N-H group). The results are also discussed in relation to those obtained for other protected -Aib-X-Aib- (X = Aib, L-Ala, L-Val) tripeptide segments of peptaibol antibiotics.

The crystal structure of Boc-(L-Phe-D-Phe)4-OMe has been determined by x-ray diffraction analysis. The peptide crystallizes in the triclinic system, space group P1 with a = 15.290 A, b = 15.163 A, c = 19.789 A, alpha = 102.49 degrees, beta = 96.59 degrees, gamma = 74.22 degrees, and Z = 2. The structure has been solved by coupling of the molecular replacement technique and expansion by tangent formula refinement of the set of known phases. Several cycles of Fourier calculations and least-squares refinement led to the location of 194 atoms of the two independent octapeptide chains and few molecules of cocrystallized solvent (chloroform, water, and methanol). The isotropic refinement converged to R = 0.13 for the 3077 "observed" reflections. The two independent octapeptide molecule form a dimer in the solid state: the two chains are associated by interstrand hydrogen bonds (12 of the type N-H ... O = C) with the formation of a double-stranded antiparallel right-handed -- beta 5.6-helix. These double helices can be represented as a cylinder with a hydrophilic inner core represented by the peptide units and an hydrophobic exterior constituted by the aromatic moieties. The dimensions of the cylinder are equal to those observed for Boc-(L-Val-D-Val)4-OMe. In the solid state the dimers pack with each other in an hexagonal fashion with the formation of layers; between the layers, solvent molecules fill empty spaces.

In a prospective randomized study, 53 patients with advanced gastric cancer previously untreated by chemotherapy were randomly assigned to treatment with 5-FU alone or combined with mitomycin and cytarabine (MFC). The response rate with MFC was higher (28%) than with 5-FU alone (21%). The difference, however, was not statistically significant. Median response duration was practically identical with 5-FU (16 weeks) and MFC (17 weeks). There was no difference in median survival between the two groups (39 and 43 weeks, respectively).

The infrared absorption and 1H nuclear magnetic resonance analyses of chloroform solutions of the terminally-blocked segment corresponding to the 2-9 sequence of emerimicins III and IV, -(Aib)3-L-Val-Gly-L-Leu-(Aib)2-, are consistent with the presence of a 3(10)-helical structure of high thermal stability. The crystal structure of the octapeptide, obtained by X-ray diffraction indicates the formation of a right-handed 3(10)-helix, stabilized by six consecutive intramolecular N-H....O:C H-bonds, slightly distorted at the level of the L-Leu residue.

The solid-state conformational analysis of t-AOC-L-Pro-OH has indicated that the molecules are not folded up to form an oxy-C7 peptide conformation, but rather that they are held together through intermolecular O-H .... 0 = C (urethane) hydrogen bonds. The tertiary amide bond is in the cis configuration. In solvents of high polarity strongly solvated species largely predominate. In cyclohexane solution non-associated and associated (involving the carboxyl C = O as the proton acceptor) species are simultaneously present. Obviously, the extent of association increases with increasing solute concentration. The amount of the oxy-C7 form, if any, should be extremely small. It is also demonstrated that CD measurements alone can lead to an incorrect picture of the conformational preferences of amino acid derivatives and small peptides in solution.

The crystal-state preferred conformation of the terminally blocked hepta- and octapeptides with the general formula -(Aib)n L-Leu-(Aib)2- (n = 4 and 5, respectively), determined by X-ray diffraction, was found to be a right-handed 3(10)-helix stabilized by five and six consecutive intramolecular NH...O = C H-bonds of the C(10)-III type, respectively. The octapeptide structure represents the first observation at atomic resolution of a regular, chiral 3(10)-helix larger than two complete turns. In both cases the right handed screw sense of the helix is dictated by the presence of the single, internal L-residue. This study confirms the propensity of short peptides rich in Aib, the prototype of the amino acid residues dialkylated at the alpha carbon, to adopt a 3(10)-helical structure and is expected to help our understanding of the conformational preferences of the membrane-active, channel-forming, ion-transporting peptaibol antibiotics.

The crystal-state preferred conformation of the terminally blocked homooctapeptide from the C(alpha,alpha)-dimethylated alpha-aminoisobutyric acid (Aib) residue, pBrBz-(Aib)(8)-OBu(t), in which pBrBz is para-bromobenzoyl and OBu(t) is tert-butoxy, determined by x-ray diffraction analysis using direct methods, was found to be a 3(10)-helix stabilized by six consecutive intramolecular N-H....O=C hydrogen bonds of the C(10)-III (or III') type. This is the first observation at atomic resolution of a regular 3(10)-helix longer than two complete turns. The solid-state structural analysis was extended to the terminally blocked, alpha-aminoisobutyric acid-rich octapeptide corresponding to the 2-9 sequence of the peptaibol antibiotics emerimicins III and IV, pBrBz-Aib(3)-L-Val-Gly-L-Leu-Aib(2)-OMe. Again, this peptide adopts a (right-handed) 3(10)-helical structure, although slightly distorted at the level of the L-leucine residue. The role of specific amino acid sequence and peptide main-chain length in stabilizing either the 3(10)- or the alpha-helical conformation and their possible implications on the nature of the channel formed by peptaibol antibiotics in the membrane are also briefly discussed.

The molecular structure of N-tert-butyloxycarbonyl-D-leucyl-L-phenylalanyl ethanolamide (t-Boc-D-Leu-L-Phe-EA), a protected analogue of the C-terminal dipeptide of the membrane-active linear antibiotic gramicidin A, has been determined by X-ray diffraction. One of the two independent molecules in the asymmetric unit is characterized by a chain reversal stabilized by an intramolecular, three-centre, double hydrogen bonding. It represents the first experimental evidence for a beta-turn conformation fused with the oxy-analogue of an alpha-turn.

Bone marrow biopsies (BMB) were performed in 173 consecutive unselected breast cancer patients at the time of diagnosis to define the value of this diagnostic tool in the initial staging of mammary carcinoma. In a group of 160 patients with a negative standard staging work-up, BMB was positive in two (1%). Both of them had negative x-ray but bone scan was positive in one and doubtful in the other. Bone marrow biopsy was positive in 31% of 13 additional patients with metastatic disease and in 44% of the nine among them with radiologically involved skeleton. These results exclude that BMB is able to discover micrometastatic foci of neoplastic disease. Its positivity appears strictly correlated with that of bone x-ray and scan. Based on the results of this prospective study, BMB is not required when both bone survey and scan are negative, but could be useful in clarifying diagnostic doubts of skeletal involvement.

The crystal structures of L-3,4-dehydroproline, t-butoxycarbonyl-L-3,4-dehydroproline amide, and acetyl-L-3,4-dehydroproline amide have been determined. L-3,4-Dehydroproline is orthorhombic with a = 16.756, b = 5.870, c = 5.275 Å, and Z = 4; t-butoxycarbonyl-L-3,4-dehydroproline amide is orthorhombic with a = 6.448, b = 8.602, c = 21.710 Å, and Z = 4; acetyl-L-3,4-dehydroproline amide is monoclinic with a = 4.788, b = 10.880, c = 7.785 Å, β = 105.25°, and Z = 2. The final R value for the L-3,4-dehydroproline is 0.046 based on 529 reflections; for t-butoxycarbonyl-L-3,4-dehydroproline amide, 0.050 based on 792 reflections; and for acetyl-L-3,4-dehydroproline amide, 0.058 based on 632 reflections. The structures clearly establish that the free amino acid exists in the zwitterionic form in the crystalline state. The molecular conformations of the t-Boc and acetyl derivatives consist of two planes: one involving the primary amide and the other the remaining atoms of the molecule. The acetyl-L-3,4-dehydroproline amide contains a tertiary amide bond in the cis conformation. To the best of our knowledge, this is the first example of a cis bond in an acetyl derivative of an amino acid or peptide. At variance with the previously reported proline amides, which present ϕ and ψ values corresponding to those of a right-handed α-helical conformation (conformation A), the t-Boc and acetyl derivatives both have ϕ and ψ values corresponding to a collagenlike conformation (conformation F)., (Copyright © 1981 John Wiley & Sons, Inc.)

From April 1982 to December 1987, 71 patients with small cell lung cancer entered a randomized clinical trial, and underwent bone marrow biopsy (BMB) as part of staging procedures. We identified 8 patients (11%) with bone marrow metastases, 6 with extensive disease independently of BMB, and 2 with extensive disease on the basis of the BMB only. BMB determined a change in the stage in only 3% (2/71) of the cases. No differences were found in the hematological parameters of the patients with or without bone marrow metastases. The median survival of the patients with bone marrow involvement was the same (41 weeks) as those with extensive disease but without bone marrow involvement. We conclude that unilateral BMB without aspiration detects a substantial proportion of bone marrow metastases in patients with extensive disease. This fact does not worsen the prognosis. A small proportion of patients with apparently limited disease has bone marrow involvement. The technique therefore contributes, to a small extent, to the definition of the clinical stage of the disease. However, bone marrow involvement is an important data of natural history, and therefore new methods to better assess this peculiar site of the disease are needed.

The amatoxins, highly toxic components of Amanita mushrooms, strongly inhibit the DNA-dependent RNA polymerase II (or B) in eukaryotic cell nuclei. For optimal binding to the enzyme a gamma-hydroxyisoleucine side chain in the 3-position is important as in gamma-amanitin (compound 1), where the OH-group is bound in the [S]-configuration. Amanullin, a non-toxic component, having an oxygen-free isoleucine side chain no. 3, exhibits an inhibitory effect on RNA polymerase II about two orders of magnitude smaller than that of gamma-amanitin. An equal, relatively weak, inhibitory effect has previously been found with the synthetically obtained Ile3-analog 7. In the present paper the synthesis of an analog (2) bearing a gamma-hydroxyl group in the isoleucine side chain is described. The compound was found to have about the same inhibitory effect on RNA polymerase II from Drosophila embryos as amanullin and the Ile3-analog 7. Structure analysis by X-ray diffraction revealed that the hydroxyl group at the -carbon atom of side chain-3 has the [R]-configuration, the new analog thus being -deoxo[( )-hydroxy-[Ile3]-amaninamide. It follows that the [S]-configuration of this chiral center is a prerequisite to maximal toxicity. Crystallographic data demonstrating great similarity between the peptide backbones of the new analog and those of natural amatoxins are given.

In the present paper we describe the single-crystal x-ray analyses of two cyclic hexapeptides containing an equal number of alternating L,D-residues as putative analogues of the metal binding compounds, enniatin and beauvericine. Both the molecules of c(L-Val-D-Val)3 and C(L-Phe-D-Phe)3 retain in the solid state the center of symmetry and crystallize with six and eight trifluoroacetic acid molecules, respectively. The peptides are strongly hydrogen bonded to the solvent molecules. We estimate, on the basis of the molecular geometry and spatial arrangement of the peptide carbonyl groups and in comparison with other metal binding cyclic peptides, the ability of these molecules to interact with metal ions as 1:1 complexes.