Total synthesis of a tyrosine suppressor tRNA gene. XV. Synthesis of the promoter region.

By use of polynucleotide kinase and polynucleotide ligase, the 10 deoxyoligonucleotide segments, whose syntheses have been described in accompanying papers, have been joined to form the 62-nucleotide-long DNA corresponding to the promoter region of an Escherichia coli suppressor tRNA gene. The following sequence in the joining reactions was used to obtain error-free and optimal yields of the products: 1) joining of Segment P-1 to P-3 in the presence of Segment P-2; 2) joining of Segments P-4 to P-7 to form Duplex [P4-7]; 3) joining of Segments P-8 to P-10 to Duplex [P4-7] to form Duplex [P4-10]; and finally, 4) joining of P-(1 + 3) and P-2 to Duplex [P4-10] to form the total promoter Duplex [P].     

Synthesis of A7,B7-dicarbainsulin, an analogue with a noncleavable bond between A- and B-chain. II. Synthesis of the A-chain segments

As part of the total synthesis of [A7,B7-L,L-2,7-diaminosuberoyl]-des-(B26-B30)-insulin B25-amide, an insulin analogue containing a non-cleavable bond between A- and B-chain, the chemical synthesis of the A-chain segments is described. The N-terminal sequence A(1-6), Boc-Gly-Ile-Val-Glu(OBut)-Gln-Cys(SBut)-NH-NH2, was synthesized in solution. The middle segment A(8-16), Ddz-Thr(But)-Ser(But)-Ile-Cys(SBut)-Ser(But)-Leu-Tyr- (But)-Gln-Leu-NH-NH2, was obtained by solid phase synthesis according to the Fmoc strategy. The C-terminal segment A(17-21), Bpoc-Glu(OBut)-Asn-Tyr-Cys(Acm)-Asn-OBut, was prepared in solution.     

Four new monomeric insulins obtained by alanine scanning the dimer-forming surface of the insulin molecule

The residues A21Asn, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyr and B27Thr, buried in the dimer of insulin, were identified by means of alanine-scanning mutagenesis. The receptor binding activity, in vivo biological potency and self-association properties of the seven single alanine human insulin mutants were determined. Four of the seven single alanine mutants, [B12Ala]human insulin, [B16Ala]human insulin, [B24Ala]human insulin and [B26Ala]human insulin, are monomeric insulin, which indicates that B12Val, B16Tyr, B24Phe and B26Tyr are crucial for the formation of insulin dimer. The monomeric [B16Ala]human insulin and [B26Ala]human insulin retain 27 and 54% receptor binding activity, respectively, and nearly the same in vivo biological potency compared with native insulin, so they could be developed as the fast-acting insulin.     

Chicken IgL gene rearrangement involves deletion of a circular episome and addition of single nonrandom nucleotides to both coding segments

Chicken immunoglobulin light chain (IgL) gene rearrangement has been characterized. Rearrangement of the single variable (VL) segment with the single joining (JL) segment within the chicken IgL locus results in the deletion of the DNA between VL and JL from the genome. This deletion is accomplished by a molecular mechanism in which a precise joining of the IgL recombination signal sequences leads to the formation of a circular episomal element. The circular episome is an unstable genetic element that fails to be propagated during B cell development. Evidence was obtained that the formation of the circular episome is accompanied by the addition of a single nonrandom base to both the VL and JL coding segments. The subsequent joining of the VL and JL segments appears to occur at random, as we observed at least 25 unique V-J junction sequences, 11 of which are out-of-frame. A novel recombination mechanism that accounts for the observed features of chicken IgL gene rearrangement is discussed.     

CXII. Total synthesis of the structural gene for an alanine transfer RNA from yeast. Enzymic joining of the chemically synthesized polydeoxynucleotides to form the DNA duplex representing nucleotide sequence 1 to 20

The DNA duplex (designated [A]) corresponding to the nucleotides 1 to 20 of the major yeast alanine transfer RNA (Fig. 1) has been synthesized. The first step involved the T4 ligase-catalyzed joining of d-(5′-³²P)-C-C-G-G-A-A-T-C (segment 4, Fig. 1) to the dodecanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T (segment 1, Fig. 1), in the presence of the complementary decanucleotide d-(5′-OH)-C-C-G-G-A-C-T-C-G-T (segment 3, Fig. 1). The resulting icosanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T-C-C-G-G-A-A-T-C, was isolated free from the decanucleotide (segment 3). The synthesis of [A] was then completed by the ligase-catalyzed joining of 5′-³²P or ³³P-labeled hexanucleotide d-(5′-P)-C-C-A-C-C-A (segment 2) to the 5′-³²P or ³³P-labeled decanucleotide, d-(5′-P)-C-C-G-G-A-C-T-C-G-T (segment 3), in the presence of the above icosanucleotide.     

Importance of the character and configuration of residues B24, B25, and B26 in insulin-receptor interactions

By use of isolated canine hepatocytes and insulin analogs prepared by trypsin-catalyzed semisynthesis, we have investigated the importance of the aromatic triplet PheB24-PheB25-TyrB26 of the COOH-terminal B-chain domain of insulin in directing the affinity of insulin-receptor interactions. Analysis of the receptor binding potencies of analogs bearing transpositions or replacements (by Tyr, D-Tyr or their corresponding 3,5-diiodo derivatives) in this region demonstrates a wide divergence in the acceptance both of configurational change (with [D-TyrB24,PheB26]insulin and [D-TyrB25,PheB26]insulin exhibiting 160 and 0.1% of the receptor binding potency of insulin, respectively) and of detailed side chain structure (with [TyrB24,PheB26]insulin and [TyrB25,PheB26]insulin exhibiting 2 and 80% of the receptor binding potency of insulin, respectively). Additional experiments addressed the solvent accessibilities of the 4 tyrosine residues of insulin and the insulin analogs at selected peptide concentrations by use of analytical radioiodination. Whereas two analogs ([TyrB25,PheB26]insulin and [D-TyrB24,PheB26]insulin) were found to undergo self aggregation, no strict correlation was found between the ability of an analog to aggregate and its potency for interaction with the insulin receptor. Related findings are discussed in terms of the interplay between side chain and main chain structure in the COOH-terminal domain of the insulin B-chain and the structural attributes of insulin that determine the affinity of insulin-receptor interactions.     

New segment synthesis of alpha-inhibin-92 by the acyl disulfide method

The thiocarboxyl group reacts with diaryl disulfides to give an unsymmetrical acyl disulfide in dimethylformamide (DMF) and a symmetrical diacyl disulfide in aqueous DMF. Both acyl disulfides react with the alpha-amino group to form the peptide bond. The method was used in a new segment synthesis of alpha-inhibin-92 (alpha-IB-92) with use of 2,2'-dipyridyl disulfide as activator. Thiocarboxyl peptides were synthesized by the solid-phase method on 4-[alpha-(Boc-Gly-S)benzyl]phenoxyacetamidomethyl-resin. The segments alpha-IB-92-(1-34)SH (I), Msc-alpha-IB-92-(35-65)SH (II), Msc-alpha-IB-92(66-92)OH (III), and Msc-alpha-IB-92-(35-92)OH (VI) were prepared in yields of 33, 36, 41, and 25%, respectively, with use of crystalline symmetrical anhydrides in double and triple coupling protocols. Segments I, II, and III were used in a 3-segment synthesis of alpha-IB-92 with an overall yield based on starting resin of about 8% while a 2-segment synthesis using I and IV gave 11%. An all stepwise synthesis of alpha-IB-92 gave 4.5%.     

Use of the T4 polynucleotide ligase in the joining of flush-ended DNA segments generated by restriction endonucleases.

Double-stranded DNA segments with completely base-paired ends were obtained by the action of various restriction endonucleases on phage and plasmid DNAs. These segments were joined covalently by the T4 polynucleotide ligase. The joining was monitored by the electron microscopy count of intramolecularly circularized segments. The highest extent of joining, close to 75%, was observed at 15-25 degrees C with the segments resulting from the action of the Bacillus subtilis (strain R) restriction endonuclease Bsu on the DNA of bacteriophage SPPI or of the plasmid pSC 101. The joining of double-stranded termini required about 10 times more enzyme than the short single-stranded termini produced by the Escherichia coli restriction endonuclease EcoRI. A shortened purification of the T4 ligase was found to give an enzyme devoid of interfacing nucleases.     

Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana

Metabolism of C26 bile alcohols in the bullfrog, Rana catesbeiana, was studied. [24-14C]-24-Dehydro-26-deoxy-5 beta-ranol (3 alpha,7 alpha,12 alpha-trihydroxy-27-nor-5 beta-cholestan-24-one) was chemically synthesized from [24-14C]cholic acid and incubated with bullfrog liver homogenate fortified with NADPH. 24-Dehydro-26-deoxy-5 beta-ranol was shown to be converted into both 26-deoxy-5 beta-ranol and 24-epi-26-deoxy-5 beta-ranol [(24S)- and (24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrols] in addition to 5 beta-ranol [(24R)-27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,26-pentol], which is the major bile alcohol of the bullfrog. [24-3H]-26-Deoxy-5 beta-ranol and [24-3H]-24-epi-26-deoxy-5 beta-ranol were prepared from 24-dehydro-26-deoxy-5 beta-ranol by reduction with sodium [3H] borohydride and administered respectively to two each of four bullfrogs by intraperitoneal injection. After 24 h, labeled 5 beta-ranol was isolated from the bile of the bullfrogs that received [24-3H]-26-deoxy-5 beta-ranol. In contrast little if any radioactivity could be detected in 5 beta-ranol or its 24-epimer after administration of [24-3H]-24-epi-26-deoxy-5 beta-ranol.     

Metabolism of bile alcohols in the perfused rabbit liver.

The mechanism and sequence of side chain hydroxylation of cholesterol in bile acid synthesis was studied in the isolated perfused rabbit liver. A comparison was made between the importance of 26- and 25-hydroxylation in cholic acid biosynthesis in the rabbit. The formation of [G-3H]cholic acid was observed when the liver was perfused with 5beta-[G-3H]cholestane-3alpha, 7alpha-diol, 5beta-[G-3H]cholestane-3alpha, 7alpha-12alpha-triol, and 5beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol. No [G-3H]chenodeoxycholic acid was detected in the bile. These findings indicate that potential precursors of chenodeoxycholic acid were hydroxylated at position 12alpha either subsequent to or before hydroxylation of the cholesterol side chain. In addition, no other intermediates (tetrahydroxy or pentahydroxy bile alcohols) were found in the bile when these compounds were perfused in the liver. Bile acid precursors were detected in bile when the rabbit liver was perfused with 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol. The 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol was hydroxylated in the liver at the 12alpha position to yield the corresponding 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol. The tetrol was further metabolized to a series of pentols (5beta-cholestane-3alpha, 7alpha, 12alpha, 22, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 23, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 24, 25-pentol; and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol). The major bile acid obtained from the perfusion of the 5beta-cholestane-3alpha, 7alpha, 25-triol was cholic acid. The experiments indicated that in the rabbit liver 12alpha-hydroxylation can occur after hydroxylation of the cholesterol side chain at either C-25 (5 beta-cholestane-3alpha, 7alpha, 25-triol) or C-26 (5beta-cholestane-3alpha, 7alpha-26-triol). Apparently, the rabbit can form cholic acid via the classical 26-hydroxylation pathway as well as via 25-hydroxylated intermediates.     

Somatic diversification of the chicken immunoglobulin light chain gene is limited to the rearranged variable gene segment

Previous studies have shown that the chicken lambda immunoglobulin light chain gene undergoes a single rearrangement that results in functional VJ joining of the unique variable (V lambda 1) and joining (J lambda) coding regions. The immunologic repertoire of lambda genes is created through extensive sequence diversification within the rearranged locus during B cell development in the bursa of Fabricius. This sequence diversification was detected only at the rearranged V lambda 1 segment and not within the 5' leader sequence, the J lambda segment, or the unrearranged V lambda 1 segment. The selective diversification of the rearranged V lambda 1 segment was associated with unique DNAase I-hypersensitive sites on the rearranged allele. While probes for V lambda 1 sequences detect multiple homologous V lambda segments, probes for both the 5' leader and J lambda segments fail to detect homologous sequences. Taken together, these results suggest that a highly selective process, possibly gene conversion, operates during B cell ontogeny to generate diversity within the lambda gene.     

Shortened insulin analogues: marked changes in biological activity resulting from replacement of TyrB26 and N-methylation of peptide bonds in the C-terminus of the B-chain

The role of three highly conserved insulin residues PheB24, PheB25, and TyrB26 was studied to better understand the subtleties of the structure-function relationship between insulin and its receptor. Ten shortened insulin analogues with modifications in the beta-strand of the B-chain were synthesized by trypsin-catalyzed coupling of des-octapeptide (B23-B30)-insulin with synthetic peptides. Insulin analogues with a single amino acid substitution in the position B26 and/or single N-methylation of the peptide bond at various positions were all shortened in the C-terminus of the B-chain by four amino acids. The effect of modifications was followed by two types of in vitro assays, i.e., by the binding to the receptor of rat adipose plasma membranes and by the stimulation of the glucose transport into the isolated rat adipocytes. From our results, we can deduce several conclusions: (i) the replacement of tyrosine in the position B26 by phenylalanine has no significant effect on the binding affinity and the stimulation of the glucose transport of shortened analogues, whereas the replacement of TyrB26 by histidine affects the potency highly positively; [HisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide and [NMeHisB26]-des-tetrapeptide (B27-B30)-insulin-B26-amide show binding affinity 529 and 5250%, respectively, of that of human insulin; (ii) N-methylation of the B24-B25 peptide bond exhibits a disruptive effect on the potency of analogues in both in vitro studies regardless the presence of amino acid in the position B26; (iii) N-methylation of the B23-B24 peptide bond markedly reduces the binding affinity and the glucose transport of respective analogue [NMePheB24]-des-tetrapeptide (B27-B30)-insulin-B26-amide.     

Bile acids. XLVII. 12alpha-Hydroxylation of precursors of allo bile acids by rabbit liver microsomes.

Rabbit liver microsomal preparations fortified with 0.1 mM NADPH effectively promote hydroxylation of [3beta-3H]- or [24-14C]allochenodeoxycholic acid or [5alpha,6alpha-3H2]5alpha-cholestane-3alpha,7alpha-diol to their respective 12alpha-hydroxyl derivatives in yields of about 25 or 65% in 60 min. Minor amounts of other products are formed from the diol. The requirements for activity of rabbit liver microsomal 12alpha-hydroxylase resemble those of rat liver microsomes. Of a number of enzyme inhibitors studied only p-chloromercuribenzoate demonstrated a marked ability to inhibit the reaction with either tritiated substrate. There was no difference in the quantity of product produced from the tritiated acid or the 14C-labeled acid. No clear sex difference was found in activity of the enzyme, nor was an appreciable difference noted in activity of the enzyme between mature and immature animals.     

Quantitative aspects of the conversion of 5 beta-cholestane intermediates to bile acids in man.

The in vivo conversion of several 5 beta-cholestane intermediates to primary bile acids was investigated in three patients with total biliary diversion. The following compounds were administered intravenously: 5 beta-[G-3H]-cholestane-3 alpha, 7 alpha-diol, 5 beta-[G-3H]cholestane-3 alpha, 7alpha, 26-triol, and 5 beta-[24-14C]cholestane-3 alpha, 7 alpha-25-triol. Bile was then collected quantitatively at frequent intervals for the next 21 to 28 h. The administered 5 beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol was found to be efficiently converted to cholic and chenodeoxycholic acids in two patients; 61 and 75% of the administered label was found in primary bile acids. The proportion of labeled cholic to chenodeoxycholic acid was 1.20 and 1.02 in the bile of these patients, indicating that the C-26 triol was efficiently converted to cholic acid. The ratio of cholic to chenodeoxycholic acid (mass) in the bile of these patients was 1.23 and 2.32. The 5 beta-cholestane-3alpha, 7alpha-diol intermediate was also efficiently converted (71%) to both primary bile acids. The cholic to chenodeoxycholic acid ratios by mass and label were similar (2.97 versus 2.23). By contrast, the 5beta-cholestane-3alpha, 7alpha, 25-triol was poorly converted to bile acids in three patients. Following the administration of this compound almost all of the administered radioactivity found in the bile acid fraction was in cholic acid (5 to 19%) and very little (less than 5%) was found in chenodeoxycholic acid. These findings indicate that ring hydroxylation at position 12 is not materially hindered by the presence of a hydroxyl group on the side chain at C-26 in patients with biliary diversion. The labeled C-26-triol which was efficiently converted to both primary bile acids in a proportion similar to that which was observed for the bile acids synthesized by the liver suggests that this 5beta-cholestane derivative may be a major intermediate in the synthesis of both cholic and chenodeoxycholic acids.     

Chemical synthesis of human beta-endorphin(1-27) analogs by peptide segment coupling. Leucine and glycine residues bearing thiocarboxyl functions as junctions for peptide segment coupling.

[Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2, two analogs of human beta-endorphin, were synthesized by both all-stepwise solid phase synthesis and peptide segment coupling. For the peptide segment coupling method, two thiocarboxyl peptides. Msc-[Gly8]beta hEP(1-8)SH and Msc-[L-Leu8]beta hEP(1-8)SH, were synthesized by standard solid phase method on 4-[alpha-(Boc-Gly-S)benzyl]phenoxyacetamidomethy-resin and 4-[alpha-(Boc-L-Leu-S)benzyl]phenoxyacetamidomethy-resin. These two thiocarboxyl peptides were coupled to H-[Lys(Cit)9,19,24]-beta hEP(9-27)NH2. [Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2 were obtained after removal of Msc groups and citraconyl groups from products of the segment coupling reaction. The yields of both [Gly8]beta hEP(1-27)NH2 and [L-Leu8]beta hEP(1-27)NH2 in the segment coupling reaction were approximately 18%. Less than 1% of racemization of Leu-8 occurred during coupling of Msc-[L-Leu8]beta hEP(1-8)SH to H-[Lys(Cit)9,19,24]-beta hEP(9-27)NH2. Results of amino acid composition analysis, analysis by reverse phase high pressure liquid chromatography and receptor binding activity assays of the analogs showed that peptide analogs prepared by segment coupling method and those prepared by all-stepwise solid phase synthesis were identical. Results of receptor binding activity assays suggested that the molecular charge properties of beta-endorphin(1-27) and its analogs influenced the receptor binding activity.     

Investigations into the possible regulation of negative gravitropic curvature in intact Avena sativa plants and in isolated stem segments by ethylene and gibberellins

Using Avena sativa L. cv. Victory oat seedlings and excised p-1 stem segments (including the p-1 and p-2 internodes) the effect of exogenously supplied ethylene and the removal of ethylene on internodal extension and gravitropic bending was assessed. Similarly, the ability of the excised system to respond to gravistimulation was assessed in the presence of inhibitors of ethylene action (AgNO₃) and ethylene synthesis (3,5-diiodo-4-hydroxybenzoic acid and benzyl isothiocyanate; BITC). The production of ethylene from both intact and excised systems was also measured from 0 to 48 h after gravistimulation, relative to vertical controls. Although gravitropic curvature is initiated, and indeed enters the most rapid phase of upward bending during the first 6 h, there is no difference in ethylene production between vertical and geostimulated plants during this period. The ethylene production of gravistimulated plants rises sharply to a maximum at 24 h, then decreases steeply to almost the control level by 48 h, at which time the rate of upward curvature is diminishing. Neither the addition nor removal of ethylene, nor the addition of inhibitors affecting ethylene-action (AgNO₃) or synthesis (DIHB) influence gravitropic bending or internodal extension in excised segments. Although the ethylene synthesis inhibitor BITC showed down the rate of upward bending, this effect could not be reversed by addition of ethylene. We conclude that the burst in ethylene production that develops in leaf-sheath bases (pulvini) after they have started to curve upwards is not primary to the induction of curvature. We further suggest that ethylene has no major effect or role in the induction of upward bending after gravistimulation. The metabolism of high specific activity gibberellin A₁ ([³H]-GA₁) in the excised system was assessed during 1, 2 and 4 h of gravistimulation. Changes in endogenous GAs and GA metabolism have been shown previously to be correlated (at the later stages) with gravistimulated bending in intact Avena shoots. The excised segments ‘leaked’ free [³H]-GAs and [³H]-GA glucosyl conjugate-like substances into the bathing medium, and this was a confounding factor. Nevertheless, gravistimulated stem segments, and especially the bottom half of the segment, were significantly less leaky then vertical segments. Thus, just 1 h after gravistimulation, bottom segment halves retained 22% more precursor [³H]-GA₁, 36% more free [³H]-GA-like metabolites, and 48% more [³H]-GA glucosyl conjugate-like metabolites than vertical segments. In contrast, the 1 h gravistimulated top halves retained slightly less (1–4%) precursor [³H]-GA and free [³H]-GA metabolites, but 21% more [³H]-GA glucosyl conjugate-like radioactivity than vertical segments.     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) in vitamin D receptor-ablated mice in vivo

The metabolism of 1alpha,25-dihydroxyvitamin D(3) was studied in vitamin D receptor-ablated mice following the administration of a physiological dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). The degradation of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) in the vitamin D receptor null mutant mice was substantially reduced compared to the wild-type control mice. At 24 h postadministration of radiolabeled 1alpha,25-dihydroxyvitamin D(3) more than 50% of the radioactivity was recovered unmetabolized, whereas in wild-type mice nearly all of the 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) was degraded. In wild-type mice three polar metabolites other than 1alpha,25-dihydroxyvitamin D(3) were detected and identified on straight-phase and reverse-phase high-performance liquid chromatography as 1alpha,24(R),25-trihydroxy-[26,27-(3)H]vitamin D(3), 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3), and (23S, 25R)-1alpha,25-dihydroxy-[(3)H]vitamin D(3)-26,23-lactone. Only one metabolite was detected in the plasma and kidneys of vitamin D receptor null mutant mice at 3 h following an intrajugular dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). This metabolite was clearly identified as 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3) by comigration on two HPLC systems and periodate cleavage reaction. At 6, 12, and 24 h postinjection 1alpha,24(R), 25-trihydroxy-[26,27-(3)H]vitamin D(3) was also detected at low levels in plasma, kidneys, and liver of some but not all mutant mice. The presence of 25-hydroxyvitamin D(3)-24-hydroxylase mRNA in the kidneys of these vitamin D receptor null mutant mice was confirmed by ribonuclease protection assay.     

Population biology of the clonal moss Hylocomium splendens in Norwegian boreal spruce forests. 5. Vertical dynamics of individual shoot segments

Demographic information was obtained for 12 528 mature segments (for which dry weight was estimated and vertical position in the bryophyte carpet recorded) and 3109 regenerated growing points for the perennial clonal moss Hylocomium splendens, recorded in Norwegian boreal spruce forests during a 6‐year period. Branching frequency varied with vertical position in the bryophyte carpet. Termination risk (probability of producing no offspring) was highest (44%) for buried segments, lowest (12%) for segments at intermediate vertical positions, and also high (26%) for emergent segments (due to increasing exposure to external mortality agents). Segment size increased from low levels in the bryophyte carpet to a maximum ca 2–10 mm below the top of the bryophyte carpet. This intermediate level was interpreted as the optimal compromise between incoming radiation (attenuating downwards) and microclimatic moisture conditions (improving downwards). Size‐corrected fitness, the number of offspring emerging from a mature segment within one year after maturation after allowance for differences in size, was lower for buried and emergent segments than for segments at intermediate positions. Small emergent segments were apparently liable to suffer from vitality reductions due to desiccation. The vertical position of a daughter segment depended on that of its parent segment, but also showed considerable stochastic variation. Burial acted as a strong sink for small segments regardless of vertical position. No evidence was found for species‐specific differences in the way pleurocarpous bryophytes interact, but reduced vertical mobility of H. splendens when growing among acrocarps indicated that growth‐form is an important determinant of bryophyte interactions. Evidence was found for vertical layering of the bryophyte carpet according to dominant type of interactions among individuals: none (environmental stress) above and at top, facilitation [a (+, +) interaction] at intermediate levels because of favourable water relationships in closed stands, and amensalism [a (0, ?) interaction] from higher‐situated segments that deprive lower‐situated segments access to light at lower relative levels. The intensity of amensalism increased downwards in the bryophyte carpet as indicated by a reinforced size hierarchy. The tendency for small H. splendens segments to become buried and lost from the population by amensalism is likely to represent a general mechanism for interactions between bryophyte species and succession in bryophyte‐dominated stands. Population effects of climatic and local environmental factors (favourability vs stress), disturbance and apparently random events are discussed with reference to their impact on the relative sizes of subpopulations acting as sources (due to facilitation) and sinks (due to amensalism).     

Solution-phase synthesis of Aib-containing cyclic hexapeptides

The 18-membered Aib-containing cyclohexapeptides, cyclo(-Gly-Aib-Aib-Gly-Aib-Phe-) (22), cyclo(-Gly-Aib-Phe(2Me)-Gly-Aib-Aib-) (24a), cyclo(-Gly-Phe(2Me)-Aib-Gly-Aib-Aib-) (24b), and cyclo(-Gly-Phe(2Me)-Aib-Gly-Aib-Phe-) (25), have efficiently been synthesized by solution-phase techniques. The linear precursors 1a-1d were prepared by combining the 'azirine/oxazolone method' for incorporation of alpha,alpha-disubstituted alpha-amino acids (Aib, Phe(2Me)) into the peptide chains by classical peptide coupling methods for segment condensations. Deprotection of the amino and carboxy termini of 1a-1d, followed by cyclization with DEPC as the coupling reagent, gave the above-mentioned cyclic hexapeptides 22, 24a, 24b, and 25 in good yields (26-57%). The solid-state conformations of the linear hexapeptides 1d, 16 and 27, and of the cyclohexapeptides 22 and 25 have been established by X-ray crystallography.